Short- and Long-Term Implications of Human Milk Microbiota on Maternal and Child Health

International Journal of Molecular Sciences Review Short- and Long-Term Implications of Human Milk Microbiota on Maternal and Child Health María García-Ricobaraza 1,2,3,†, JoséAntonio García-Santos 1,2,3,†, Mireia Escudero-Marín1,2,3, Estefanía Diéguez 1,2,3 , Tomás Cerdó1,4,*,‡ and Cristina Campoy 1,2,3,5,*,‡   Citation: García-Ricobaraza, M.; García-Santos, J.A.; Escudero-Marín, M.; Diéguez, E.; Cerdó, T.; Campoy, C. Short- and Long-Term Implications of Human Milk Microbiota on Maternal and Child Health. Int. J. Mol. Sci. 2021,22, 11866. https://doi.org/ 10.3390/ijms222111866 Academic Editor: Maria Santagati Received: 30 August 2021 Accepted: 29 October 2021 Published: 1 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1EURISTIKOS Excellence Centre for Paediatric Research, Biomedical Research Centre, University of Granada, 18016 Granada, Spain; [email protected] (M.G.-R.); [email protected] (J.A.G.-S.); [email protected] (M.E.-M.); [email protected] (E.D.) 2Department of Paediatrics, School of Medicine, University of Granada, Avda. Investigación 11, 18016 Granada, Spain 3Instituto de Investigación Biosanitaria Ibs-GRANADA, Health Sciences Technological Park, 18012 Granada, Spain 4Carlos III Health Institute, Avda. Monforte de Lemos 5, 28028 Madrid, Spain 5Spanish Network of Biomedical Research in Epidemiology and Public Health (CIBERESP), Granada’s Node, Carlos III Health Institute, Avda. Monforte de Lemos 5, 28028 Madrid, Spain *Correspondence: [email protected] (T.C.); [email protected] (C.C.); Tel.: +34-627-385-755 (T.C.); +34-629-308-695 (C.C.) † These authors share the first authorship. ‡ These authors share the senior authorship. Abstract: Human milk (HM) is considered the most complete food for infants as its nutritional composition is specifically designed to meet infant nutritional requirements during early life. HM also provides numerous biologically active components, such as polyunsaturated fatty acids, milk fat globules, IgA, gangliosides or polyamines, among others; in addition, HM has a “bifidogenic effect”, a prebiotic effect, as a result of the low concentration of proteins and phosphates, as well as the presence of lactoferrin, lactose, nucleotides and oligosaccharides. Recently, has been a growing interest in HM as a potential source of probiotics and commensal bacteria to the infant gut, which might, in turn, influence both the gut colonization and maturation of infant immune system. Our review aims to address practical approaches to the detection of microbial communities in human breast milk samples, delving into their origin, composition and functions. Furthermore, we will summarize the current knowledge of how HM microbiota dysbiosis acts as a short- and long-term predictor of maternal and infant health. Finally, we also provide a critical view of the role of breast milk-related bacteria as a novel probiotic strategy in the prevention and treatment of maternal and offspring diseases. Keywords: human milk; microbiota; health programming; probiotics 1. Introduction Human milk (HM) represents the gold standard, providing protective and functional nutrients for the newborn, ensuring healthy growth and development [ 1 , 2 ]. Accordingly, the World Health Organization (WHO) and other international organizations, such as the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), recommend that infants should be exclusively breastfed during their first 6 months of life, with breastfeeding continuing until two years of age, combined with complementary feeding [ 3 , 4 ]. However, it is necessary to point out that exclusively breastfed infants present a particular risk of vitamin D deficiency, due to its low concentration in breast milk [ 5 ], low maternal vitamin D levels and daily intake, as well as the lack of exposure of newborns and suckling infants to sunlight [ 6 ]. Therefore, associations such as the American Academy of Int. J. Mol. Sci. 2021,22, 11866. https://doi.org/10.3390/ijms222111866 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 11866 2 of 19 Pediatrics and ESPGHAN recommend vitamin D supplementation at doses of 400 IU/day in infants who are exclusively or partially breastfed [7,8]. Unlike its traditional consideration as a sterile fluid, HM is now recognized as an interesting source of potentially probiotic and commensal bacteria such as Bifidobacterium spp., Lactobacillus spp., Clostridiales spp., viral organisms, among others, which may lead to healthy infant gastrointestinal (GI) microbiota and immune system maturation [ 9 – 11 ]. Although studies provide strong evidence of the existence of microbes in human milk, their origin and potential role in breastfed infants’ health need to be studied in greater depth, particularly concerning the contribution of HM microbes to the growth and development of infant gut microbiota. In fact, some authors reported that HM microbiota contributes less than previously expected to gut microbiota composition during early life, and virtually nothing at 6 months of age [ 12 , 13 ]. Nevertheless, as human microbiota is essential for optimal host physiology, there is currently great interest in better understanding how HM microbiota dysbiosis could be related to the development of non-communicable diseases in both mother and child [ 14 ]. Consequently, methodological advances and more consistent findings are still needed to better understand the uncertain aspects related to HM microbiota origin, composition and function [ 15 ], and its potential interaction with other bioactive components. This review aims to summarize and evaluate the current knowledge of HM microbiota, highlighting avenues for future research and potential therapeutic implications for both maternal and child health. 2. Human Milk Microbiota: Practical Approaches to Detect Microbial Communities in Human Breast Milk Samples Culture-dependent approaches were initially used to demonstrate the existence of specific microbiota in HM samples. In this regard, Martin et al. [ 16 ] reported the presence of potential probiotic and commensal lactic acid bacteria (LAB), mainly Lactobacillus gasseri and Enterococcus faecium, in aseptically collected HM samples from healthy women. These bacteria can inhibit the growth of pathogenic bacteria through acetate and lactate production, suggesting that both strains may have a preventive role in neonatal infectious diseases. This experimental methodology has been widely used to identify other bacterial species naturally present in HM, including different LAB strains (Lactobacillus,Lactococcus, Leuconostoc,Weissella, etc.), bifidobacteria, facultative anaerobic bacteria, and novel bacterial species as Streptococcus lactarius [ 17 – 19 ]. However, it fails to detect both strictly anaerobic and non-culturable bacteria, and new experimental approaches have been required to discover and describe the whole HM microbiota. Thus, the use of culture-independent molecular techniques (qPCR, denaturing/ temperature gradient gel electrophoresis, etc.), and Next Generation Sequencing (NGS) technology, have made it possible to overcome these limitations, identifying strictly anaerobic bacteria (Bifidobacterium spp., Bacteriodes spp., and Clostridia) in HM samples from healthy women [ 18 ] (Table 1). Hunt et al. [ 20 ], using 16S-ribosomal RNA gene pyrosequencing, showed that central node of HM microbiota was mostly formed by Streptococcus, Staphylococcus,Serratia and Corynebacteria. Other bacteria, including Lactobacillus,Bifidobacterium,Propionibacterium,Pseudomonas,Bradyrhizobium,Sphingomonas and Ralstonia, were also found to a greater or lesser extent in HM samples. Similar results were obtained by Jost et al. [ 21 ] using the above-mentioned experimental approach in combination with culture-dependent methods. Interestingly, gut-associated obligate anaerobes bacteria, such as Bacteroides and Clostridia were also found, which could explain the potential beneficial role of HM and its microbiota on colonic health. Moreover, culture-independent molecular techniques, such as qPCR plus pyrosequencing and 16S rRNA sequencing, have been crucial for understanding how populations of HM microbiota change over lactation or due to maternal factors during pregnancy or at delivery [ 22 , 23 ]. These findings were further expanded using metagenomic approaches, identifying archaea, viruses, fungi and protozoa as other common members of HM microbial metagenome [ 24 , 25 ]. Although their practical limitations, including the over- or underestimation of certain bacterial groups, dead bacteria detection or targeting the in- Int. J. Mol. Sci. 2021,22, 11866 3 of 19 herent properties of 16S rRNA region, these methodological approaches have allowed the diversity and complexity of HM microbial communities to be evaluated [ 18 ]. However, further studies using novel experimental approaches, such as NGS and omics technologies, are still needed, not only to better understand the detailed HM microbiota composition and functions, but also to understand their potential interactions with other milk bioactive compounds. Table 1. Main features of experimental procedures in analysis of bacterial communities. Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose–response study of antibiotics effects on bacteria growth 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] To provide estimates of microbes capable of replicating under experimental conditions. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Low-cost method. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Limited selective media for potential strains of interest. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Inaccurate representation of whole species present in a sample. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Subjective identification. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] To identify unlimited number of species present in the sample. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] To quantify both abundance and expression of taxonomic and functional gene markers. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Safe and fast method. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Artifacts generation by unequal amplification of PCR products (PCR bias). 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotin-labelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Simultaneous detection of thousands of genes or target DNA sequences. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Fast method. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] High cost. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Large number of probe designs based on low-specificity sequences. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Lack of control over the pool of analyzed transcripts. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze bacteria solution based on their fluorescent or light scattering characteristics. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Rapid assay times and data generation. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] High numbers of cells analyzed per sample with minimal volume. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] High performance. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence In Situ Hybridization) Target DNA is hybridized using specific DNA probe for further fluorescence microscopy analysis 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Extensive philogenetic identification. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] No PCR bias 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Unable to identify unknown species. [32] Next-generation DNA sequencing Identification of nucleotides order in whole genome or targeted DNA/RNA regions using sequencing technology characterized by ultra-high-throughput, scalability and speed. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Detect “unculturable” bacteria. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] High precision when exploring the phylogenetic composition of microbial populations. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Detailed analysis of individual genome stretches. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Precise analysis of RNA transcripts for gene expression. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Unable to analyze metabolic activity and bacterial viability. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Measurement of the relative abundance of bacterial populations rather than absolute abundance. 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] High cost 1 Method Description Advantages Disadvantages References Culture-Dependent Approaches Isolation media Isolation of target bacteria using general or selective culture media; dose– response study of antibiotics effects on bacteria growth  To provide estimates o f microbes capable of replicating under experimental conditions.  Low-cost method.  Limited selective media for potential strains of interest.  Inaccurate representation of whole species present in a sample.  Subjective identification.  Need for trained personnel to obtain reliable results. [26,27] Culture-Independent Approaches Quantitative Polymerase Chain Reaction (q-PCR) “Classic” target template detection PCR plus fluorescent detection methods to record product formation during each cycle of PCR amplification. Quantification of gene (or transcript) numbers is determined during the exponential phase.  To identify unlimited number of species present in the sample.  To quantify both abundance and expression of taxonomic and functional gene markers.  Safe and fast method.  Artifacts generation by unequal amplification of PCR products (PCR bias).  Unable to identify unknown species. [28] DNA microarray Synthesis of complementary DNA (cDNA) chains from mRNA and subsequent amplification to biotinlabelled complementary RNA (cRNA). Once these fragments are obtained, they will be hybridized with microarray probes and stained for analysis.  Simultaneous detection of thousands of genes or target DNA sequences.  Fast method.  High cost.  Large number of probe designs based on lowspecificity sequences.  Lack of control over the pool of analyzed transcripts.  PCR bias. [29,30] Flow cytometry Liquid bacteria suspension is moved into liquid stream (sheath fluid) and then subjected to the effects of a laser, which scatters light in two major directions: Forward angle light scatter (Forward scatter or FSC) or Side-angle light scatter (Side scatter or SSC). Both light signals are converted into electronic signals to analyze b acteria solution based on their fluorescent or light scattering characteristics.  Rapid assay times and data generation.  High numbers of cells analyzed per sample with minimal volume.  High performance.  Low number of fluorescent stains available to analyze bacterial cells’ viability, structure and/or metabolism [31] FISH (Fluorescence Target DNA is hybridized using specific DNA probe  Extensive philogenetic identification.  No PCR bias  Unable to identify unknown species. [32] Need for qualified personnel and massive amount of work to analyze the obtained results. [31,33] 3. Composition and Potential Origin of the HM Microbiota Different studies have reported the existence of “core” HM microbiota, mostly formed by Staphylococcus,Streptococcus and Propioninacterium [ 21 , 34 – 36 ], although its composition can vary between 2 and 18 taxa, including other members at lower abundances such as Bifidobacterium,Veillonella,Rothia,Lactobacillus,Corynebacterium,Ralstonia,Acinetobacter, Acidovorax,Pseudomonas,Bacteroides,Clostridium,Escherichia/Shigella,Gemella, and Enterococcus [ 37 ]. This lack of consensus in HM microbiota composition seems to be due to differences in both methodologies (sample collection and stored, DNA extraction proto- Int. J. Mol. Sci. 2021,22, 11866 4 of 19 cols, 16S rRNA sequenced region) [ 38 ] and study populations (geographical and lifestyle factors) [ 39 ]. However, it seems clear that “core” HM microbiota acquisition occurs gradually over lactation, and this could potentially drive healthy gastrointestinal microbiota development and subsequent optimal later-life health [37,40]. There are different questions about the origin of HM microbiota. Scientific evidence supports a pre-existing mammary gland microbiota formed before pregnancy and lactation, thus acting as a basis for HM microbiota establishment [ 41 – 43 ]. Once set, the existence of multiple microbial sources, such as maternal skin and GI tract, as well as infant oral cavity, could direct the development of HM microbiota. This fact would not only explain the differences in composition between mammary gland and HM microbiota, but also its dynamic composition during breastfeeding [ 37 ]. Nevertheless, to date, this is still under discussion; in fact, some studies have detected human skin bacteria, mainly genus Staphylococcus,Propionibacterium and Corynebacterium, in HM samples, suggesting the ability of maternal skin microbiota to colonize the mammary gland [ 20 ]. However, other findings fail to identify this microbial source. Thus, although LAB were found in both HM and mammary areola, these bacteria showed different DNA profiles according to the analyzed source [ 16 ]. Similarly, there were also significant differences in Streptococcus and Propionibacteria abundance between microbial communities present in the HM and maternal skin [ 21 ]. It is also important to highlight that the bacterial genera shared between HM and maternal skin also seem to be related to GI tract and mouth [ 34 ], thus reinforcing the need for further studies to clarify the real contribution of maternal skin to HM microbiota. Interestingly, HM microbiota origin could be also explained by bacterial retrograde flow between breast milk and infant oral cavity. This assumption is not only suggested by the presence of typical oral bacteria species, mainly Streptococcus,Rothia and Gemella, in HM samples [ 44 ], but also by the significant differences in oral microbiota composition found between formula-fed and breastfed infants [45]. Finally, there is growing interest in entero-mammary pathway to explain the potential HM microbiota origin, which is founded by the presence of typical GI bacteria (Bifidobacterium,Veillonella,Bacteroides, and Clostridium, among others) in breast milk [ 12 , 21 , 34 , 46 , 47 ]. This hypothesis is also supported by studies performed in mice models, which suggest that dendritic cells (DC) can regularly reach the intestinal epithelium and selectively recognize commensal gut bacteria to move them to the mammary gland through lymphatic system [ 36 ]. Zhou et al. [ 48 ] also found that DC shared bacterial signatures with those reported in the intestine, lymph nodes and breast milk in lactating mice. Furthermore, interventional studies performed in lactating women have reported that HM microbiota composition can be selectively modified by diet or pro- and prebiotic treatment, emphasizing the potential maternal GI origin of HM microbiota [ 49 , 50 ]. Recent studies also suggest that the entero-mammary pathway is involved in immune cell circulation, which may partially explain the immunomodulatory role of HM, which is discussed in more detail below. In this sense, Ikebuchi et al. observed a higher proportion of CD4+ and CD8+ T cells in milk compared to mammary gland in mouse model [ 51 ]. Moreover, CD8+ T cells showed increased expression of claudin polymerization-associated genes (cldn3 and cldn7), as well as the tjp1 gene, which is related to the biosynthesis of tight junction protein ZO-1. This expression profile, observed in CD8+ T cells (and probably in CD4+ T, γδ T, and NK cells), allows immune cells to selectively translocate through the tight junction region to milk. Consequently, when the infant is breastfed, T cell populations could migrate to lymphoid tissues, thus reinforcing neonatal-cell-mediated immunity [ 52 ]. Therefore, CD4+ T-cell-mediated adaptive immune response is crucial to maintain intestinal immune homeostasis against food molecules and non-harmful microbial components [ 53 ], while CD8+ T-cell-mediated response plays a key role in protection against intracellular pathogens, thereby maintaining an optimal balance in the gut microbial community [54]. Taken together, further studies are not only needed to better evaluate the potential contributions of different sources to HM microbiota composition, but also to clarify whether the bacterial communities present in mammary gland are a true permanent microbiota or Int. J. Mol. Sci. 2021,22, 11866 5 of 19 constantly provided by external sources. It will be also of interest to explore the probable interactions between HM microbiota and immune components present in HM. 4. Potential Factors That Influence HM Microbiota Composition In recent years, it has been established that HM microbiota composition is highly influenced by genetic, maternal and early-life factors (gestational age, delivery mode, maternal nutrition and body composition, time of day and stage of lactation, intake of antibiotics, and geographic location), which cause substantial inter-individual variation in its composition [55] (Figure 1). Figure 1. Potential factors that influence breast milk microbiota composition. In this line, both gestational age and mode of delivery can modify the abundance of certain bacteria in HM microbial community. Khodayar-Pardo et al. [ 56 ] observed a lower abundance of Lactobacillus spp. (L. fermentum and L. salivarius) and Bifidobacterium spp. in HM samples from women who had a cesarean delivery with respect to those who gave birth vaginally. Moreover, human milk Bifidobacterium concentrations were increased in mothers who had a term delivery compared to those who had a preterm birth. On the other hand, Cortés-Macías et al. [ 57 ] reported that C-section delivery and antibiotic exposure caused compositional changes in the HM microbial community, in terms of lower abundances of Lactobacillus,Bacteroides, and Sediminibacterium genera. Regarding the influence of maternal diet and body composition on HM microbiota composition, the results achieved to date are limited and indicate a need for further research. Padilha et al. [ 58 ] observed that HM microbiota diversity was associated with vitamin C intake during pregnancy, while human milk Bifidobacterium concentration was positively correlated to polyunsaturated and linoleic fatty acids intake during lactation. Interestingly, maternal BMI seems to have an impact, either positive or negative, on the diversity of certain bacteria phylum, but not on overall α -diversity [ 59 ]. However, the potential influence of maternal diet and body composition on HM microbiota composition is still controversial. In this line, studies conducted to date suggest that both the aforementioned factors seem to be associated with changes in the HM profiles of oligosaccharides Int. J. Mol. Sci. 2021,22, 11866 6 of 19 (HMOs), fatty acids, proteins, hormones, immune cells and antibodies [ 60 – 62 ]. These changes could alter the milk microenvironment and, ultimately, microbial community composition [59]. Recently, scientific research has focused on the role of circadian rhythms in HM microbiota composition. Corona-Cervantes et al. [ 63 ] showed changes in Shannon diversity index during the day, as well as a predominance of Proteobacteria,Actinobacteria and Firmicutes at night, in HM samples from healthy women who had vaginal deliveries. Despite the limitations of this study, the obtained findings seem to suggest that HM microbiota could follow a circadian rhythm, with dynamic changes in its composition depending on the time of day. Lactation stages have been also identified as a novel factor that can contribute to the remodeling of HM microbiota composition. Gonzalez et al. [ 64 ] found that HM samples collected during early lactation (6–46 days post-partum) were rich in Staphylococcus and Streptococcus spp., both related to infant oral and intestinal tract, while aromatic compound degradation-related species such as Sphingobium and Pseudomonas were prevalent in HM samples collected at the late stage (109–184 days post-partum). Likewise, Moossavi et al. [ 59 ] showed that HM microbiota composition, but not α -diversity, was related to lactation stage within the period of study (17 ± 5 weeks). Moreover, they also showed that indirect breastfeeding decreased the relative abundance of Bifidobacterium but increased relative abundance of potential pathogens, including Enterobacteriaceae and Pseudomonas. Similar to other studies [ 21 , 65 ], these authors also suggest that the collection method is an extrinsic factor influencing HM composition analysis. It is known that geographic location influences HM composition, including levels of micro- and macronutrients, bioactive compounds, and immunological factors, and thus ultimately also affects HM microbiota composition [ 66 , 67 ]. In fact, after analyzing HM samples belonging to 117 Chinese mothers from three different geographic locations, Wan et al. [68] reported that microbial diversity and richness during lactation were potentially related to maternal geographic location. 5. Human Milk Microbioma Functions and Activity The recent technological advances discussed above have allowed the potential functions of human-milk-associated bacteria in promoting host health to be identified. In this line, the results obtained from diverse studies suggest that HM microbiota may influence infant gut microbial colonization through vertical mother-child bacterial transmission [ 34 , 69 – 71 ]. In support of this hypothesis, both HM and breastfed infants’ stool samples seem to share specific bacteria patterns (mainly Lactobacillus spp., Lactobacillus plantarum,Bifidobacterium breve and Bifidobacterium longum subsp. longum), with significant differences also seen compared to strains obtained in faeces from formula-fed infants [ 34 , 69 – 72 ]. Interestingly, this similarity in microbial composition remains throughout the first year of life [ 73 ]. The vertical transmission of HM microbes to infant gut would not only be facilitated by milk’s own characteristics, but also by specific microbial features that could allow them to survive the GI environment. On the one hand, the neutral pH of breast milk counteracts the acidity of the GI tract during lactation. Moreover, HM is also rich in non-digestible carbohydrates (HMOs) that facilitate commensal bacterial growth [ 74 ]. On the other hand, members of the Bifidobacteria genus present in HM might colonize and persist in the GI tract due to the specific structural features present in its surface combined, with crucial molecular tools specifically designed to respond to environmental changes [ 75 , 76 ]. Among them, Bifidobacteria has the ability to resist the acidic environment of GI tract through its F0F1-ATPase activity, which is responsible for active proton extrusion and the maintenance of pH homeostasis [77]. Nevertheless, despite this evidence, the potential influence of HM bacteria on infant gut colonization is still under review as studies carried out to date show inconsistent findings. In fact, Pannaraj et al. [ 12 ] reported that the contribution of HM to infant gut microbiota in breastfed infants ranged between 15 and 20%, while Williams et al. [ 13 ] found that HM bacteria represented only about 5% to the infant gut microbiome on day 2 of life, but virtually nothing at 6 months of Int. J. Mol. Sci. 2021,22, 11866 7 of 19 age. Consequently, further studies are still needed to better understand the potential role of HM bacteria on infant gut colonization. It is also important to highlight that HM microbiota might be involved in the microbiota establishment in other niches. For instance, HM microbiota might drive oral microbiota development, as suggested by the higher abundances of HM-related bacteria in oral samples from breastfed infants compared to those who received infant formula [ 13 , 78 , 79 ]. Likewise, HM microbiota seems to influence upper respiratory tract (URT) microbiota establishment during early life, thus supporting the protective effects of HM against infants URT infections. In fact, URT microbiota from breastfed infants is mainly formed by LAB, but there is a low abundance of Staphylococcus and anaerobic bacteria, with the latter being predominant in URT microbiota from formula-fed infants [80,81]. Due to its hypothetical role in GI microbiota establishment, HM microbial may have other potential functions in the infant gut. Studies suggest that HM-related bacteria protect against gastrointestinal infections through different mechanisms including: (i) growth inhibition of pathogenic bacteria by competitive exclusion; (ii) production of antimicrobial compounds such as hydrogen peroxide (H 2 O 2 ); (iii) enhancing intestinal barrier protection via increased mucine production, lower intestinal permeability, and upregulation of detoxifiying enzymes [ 82 ]. Consequently, HM-related bacteria strains belonging to Lactobacillus (L. salivarius CECT5713 and L. fermentum CECT5716) and Staphylococcus epidermidis, have emerged as promising therapeutic agents in the treatment of child gastrointestinal infections [82–84]. Scientific evidence also suggests a potential metabolic role of HM microbiota in infants; as an example, both lactobacilli and bifidobacteria have the ability to break down nondigestible HMOs into butyrate, which is not only used for colonocytes as energy source, but also has modulatory effects on intestinal health [ 85 , 86 ]. Additionally, HM microbiota seems to be involved in nutrient metabolism and synthesis, as suggested by the fact that stool samples from breastfed infants showed a significant increase in carbohydrates, amino acids and nitrogen metabolism, as well as cobalamin synthesis, compared to those obtained from formula-fed infants [87]. Certain bacteria strains provided by breast milk, along with other HM bioactive compounds (nutritional components, hormones, growth factors, neuropeptides, cytokines and nucleotides) can maturate and modulate immune responses in neonates and infants, thus protecting them against asthma, allergies and other non-communicable diseases [ 88 ]. In vitro models suggest that Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 could have potent immunomodulatory effects by regulating the activation of NK cells, CD4+/CD8+ T cells and regulatory T cells [ 89 ]. Moreover, compared to formula-fed infants, the immune response observed in breastfed infants is largely based on regulatory T cells and TH1/TH2 balanced responses [ 90 ]. Other human-milk-related bacteria strains, including Lactobacillus gasseri CECT5714 and viridans streptococci, also seem to have a protective role in allergic conditions, mainly cow´s milk protein allergy/intolerance [91] and atopic eczema [ 92 ], respectively. Overall, these studies suggest the possible immunomodulatory role of HM microbiota in infants. 6. Short- and Long-Term Implications of HM Microbiota on Maternal and Child Health Regardless of its origin, the existence of commensal microbes in HM might have a beneficial role in the health of mothers and their newborn infants. In fact, research carried to date points to a potential influence of HM microbiota on health outcomes [ 93 , 94 ]. However, further studies are still needed to obtain stronger scientific evidence, particularly considering that HM not only contains commensal bacteria, but also many other immune, nutritional and bioactive factors that may influence maternal and child health. 6.1. Maternal Pathologies and Human Milk Microbiota Dysbiosis It is established that breastfeeding provides short- and long-term positive effects on maternal health, including better postpartum recovery, lower risk of breast and ovarian Int. J. Mol. Sci. 2021,22, 11866 8 of 19 cancer, and a reduced incidence of cardiovascular and autoimmune diseases [ 95 , 96 ]. Interestingly, clinical and scientific evidence also suggests a bidirectional interaction through which maternal health can modify HM microbiota composition. Mastitis is a common inflammatory condition that affects 33% of lactating women and causes pain during lactation, redness of the breast and fever, ultimately leading to decreased milk production and subsequent early suppression of breastfeeding [ 97 ]. There is evidence suggesting an association between this inflammatory condition and HM microbiota dysbiosis in terms of low microbial diversity, decreased relative abundances of commensal bacteria (Lactococcus and Lactobacillus) and the establishment of opportunistic pathogens such as Staphylococcus,Streptococcus and Corynebacterium [ 98 , 99 ]. In fact, acute mastitis is widely caused by S. aureus, reaching concentrations of 4.0 log 10 colony-forming cells (cfu)/mL in milk of acute mastitis-suffering women compared to concentrations from 1.5 to 3 log 10 cfu/mL reported in the milk of healthy women. However, other potentially pathogenic strains, including coagulase-negative Staphylococci,S. epidermidis and Corynebacterium, also lead to subacute, chronic or granulomatous mastitis in lactating women, respectively [ 100 , 101 ]. As these bacteria are often resistant to antibiotic therapy, promising strategies for mastitis treatment are currently based on the use of Lactobacillus strains isolated from the human milk of healthy women. Clinical trials published to date indicate that oral intake of different Lactobacillus strains isolated from human milk, either alone, such as L. fermentum CECT5716 and L. salivarius CECT5713 [ 102 ], or combined (L. salivarius CECT5713 plus L. gasseri CECT5714) [ 49 ], reduces the counts of pathogenic bacteria and improves mastitis symptoms after 14–21 days of treatment, thereby emerging as promising treatment for lactational infectious mastitis when antibiotic treatment fails. Interestingly, both L. fermentum CECT5716 and L. salivarius CECT5713 were also found in HM samples of treated women, suggesting that both strains are able to recolonize the mammary gland to reduce and reverse mastitis-associated dysbiosis [ 102 ]. Further studies have been performed to better understand the key biomarkers and potential mechanisms involved in this probiotic effect. Overall, these studies showed that Lactobacillus-based probiotic treatment did not affect milk macronutrient composition, but was associated with specific microbiological, immunological and metabolomic markers related to the improved integrity of mammary gland epithelia [ 103 , 104 ]. Moreover, a recent study also suggest that probiotic treatment might act on specific genes involved in inflammatory and cell-growth signaling pathways, thus opening new avenues for research based on specific responsive molecular targets [ 105 ]. Finally, daily oral intake of HM-related L. salivarius PS2 between week 30 of gestation and delivery, significantly reduced the incidence of mastitis in women who suffered this pathology in previous pregnancies, compared to those who received a placebo during the same period [98]. The potential protective role of HM microbiota in breast health is also indirectly suggested by the close link between the microbial communities present in mammary tissues and risk of breast cancer. In this line, Urbaniak et al. [ 106 ] reported cancer-related dysbiosis characterized by a significantly lower abundance of LAB, but increased abundance of Bacillus spp., Staphylococcus epidermidis, family Comamonadaceae and Enterobacteria such as Escherichia coli. However, the potential mechanisms through which microbiota dysbiosis could contribute to breast cancer are still unknown. On the one hand, this effect could be explained due to the ability of S. epidermidis and E. coli to induce DNA damage by double-strand breaks [106]. Moreover, breast-cancer-associated microbial dysbiosis could downregulate the host immune system, which, in turn, leads to a permissive environment for breast tumorigenesis [ 107 ]. Xuan et al. [ 108 ] found a lower abundance of Sphingomonas yanoikuyae in breast tumor tissue, a gram-negative bacteria involved in immune cell activation and the inhibition of tumor growth. In a similar study, nipple aspirate fluid from breast cancer women was rich in genus Alistipes and other bacteria with β -glucuronidase enzymatic activity, which is associated with profound changes in estrogen metabolism and an increased risk of breast cancer [ 41 ]. It is also important to note that the use of chemotherapy to treat breast cancer might alter the bacterial communities present in HM Int. J. Mol. Sci. 2021,22, 11866 9 of 19 and mammary tissues, reducing these potentially beneficial bacteria for mother and infant health [ 43 ]. Despite this evidence, further studies are still needed to clarify whether these bacteria strains could grow into a tumorigenic environment or whether they are a direct cause of breast cancer. Other maternal pathologies also seem to be accompanied by HM microbiota dysbiosis. For instance, González et al. [ 109 ] showed that breast milk of human immunodeficiency virus (HIV)-infected women presented increased bacterial diversity and Lactobacilllus spp. frequency, but its content in S. hominis and S. aureus was lower compared to breast milk of healthy women. Nevertheless, no evidence for HIV-related microbial dysbiosis was found in other studies [ 110 ]. These contradictory findings may be explained by methodological and population differences between studies, making it difficult to identify whether changes in HM microbiota composition were a response to maternal disease or vice versa. Similar conclusions have been obtained in studies that analyzed HM microbiota composition in women who suffer from allergies or celiac disease. In both cases, lower relative levels of Bifidobacterium and Bacteroides were found in breast milk samples, but there were significant differences in dietary habits between healthy and unhealthy women [111,112]. Special mention should be made to the potential relationship between HM and coronavirus disease 2019 (COVID-19). Although HM microbiota dysbiosis has not yet been found in women positive for SARS-CoV-2, recent evidence suggest that GI microbiota is profoundly altered in COVID-19 patients, particularly in terms of reduced bacterial diversity and lower levels of commensal bacteria with immunomodulatory role (Faecalibacterium prausnitzii,Eubacterium rectale and Bifidobacterium), as well as increased growth of potentially pathogenic Enterococcus strains [ 113 , 114 ]. These changes in GI microbiota composition seem to be positively related to cytokine storm intensity and subsequent disease severity [ 114 ], and preliminary results suggest that therapeutic strategies focused on GI microbiota modulation using pro- and synbiotics (mainly Lactobacillus spp. and Bifidobacterium spp.) could be effective in the prevention and treatment of severe COVID-19 [ 115 ]. Interestingly, noting the entero-mammary origin, it is plausible that COVID-19 disease also involves dynamic changes in HM microbiota composition. Moreover, due to the hypothetical role of HM microbiota in the establishment of infant GI microbiota, breastfeeding could have a potential protective effect on severe COVID-19-related dysbiosis in infants. While both assumptions require further research, there are different approaches, emphasizing that mothers infected by SARS-CoV-2 can breastfeed their infants, with the necessary precautions, in order to transmit HM’s protective properties against COVID-19 disease [ 116 – 119 ]. In fact, unlike other virusessuch as HIV and human cytomegalovirus, which can be transmitted to infants via breast milk, Bäuer et al. reported that HM samples obtained from mothers with SARS-CoV-2 infection and/or those who have recovered from COVID-19, showed no presence of SARS-CoV-2 RNA [ 120 ]. Interestingly, these authors also observed that HM could provide passive immunity to breastfed infants via the transfer of SARS-CoV-2 spike-protein-specific antibodies. Similarly, Demers-Mathieu et al. found a positive correlation between antigens and secretory antibodies in breast milk samples from mothers with confirmed COVID-19 PCR, characterized by higher levels of S2 subunit SARS-CoV-2-specific IgG, while SIgA and SIgM were polyreactive and cross-reactive to S1 or S2 subunit SARS-CoV-2 [ 121 ]. In conclusion, the data discussed here seem not only to support the breastfeeding recommendations during the COVID-19 pandemic, but also its potential beneficial role for mothers and their offspring in the prevention of severe COVID-19 disease [ 122 ]. However, as mentioned above, further studies are required to better understand the role of both HM and GI microbiota in the physiopathology and management of COVID-19. Lastly, there is growing interest in evaluating the effects of maternal metabolic conditions during pregnancy on the composition and activity of HM microbiota, as well as its potential associations with later maternal and child health status. 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