Characterization of an outbreak due to CTX-M-15-producing Klebsiella pneumoniae lacking the bla(OXA-48) gene belonging to clone ST405 in a neonatal unit in southern Spain

While international CREC isolates primarily harboured metallo-b-lactamases and OXA-48, 4 our data support a growing role for bla KPC -mediated CREC in the USA. At least three different bla KPC -encoded carbapenemases have been described in ST171 alone. 8 This points to the efficiency of E. cloacae in acquiring a variety of resistance plasmids, potentiating the emergence of new resistant strains. Moreover, E. cloacae ST171 and to a lesser extent ST78 appear to be able to acquire and maintain bla KPC -harbouring plasmids and persist as hospital-associated CRE strains. This was a single-centre, retrospective study, and thus our findings may have limited generalizability. Notably, the increased incidence was unlikely to result from changes in CLSI breakpoints as only revised guidelines were applied, or from a system-wide breakdown of infection control as it was restricted to E. cloacae. The alarming increase in CREC infections was driven by diverse clones, suggesting frequent acquisition of bla KPC -encoded resistance as well as an increase in ST78 and ST171. The emergence of bla KPC -harbouring ST171 across the USA suggests its potential as an epidemic CREC clone. Future molecular surveillance studies are urgently needed to further assess the spread of CREC including ST171. Funding This work was supported in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (1R01AI116939 and 1R01AI116939-S01 to A.-C. U. and 5T32AI100852 to A. G.-S.) and the Columbia University Irving scholarship (A.-C. U.). Transparency declarations None to declare. Supplementary data Table S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/). References 1CDC. Antibiotic Resistance Threats in the United States, 2013. http://www. cdc.gov/drugresistance/threat-report-2013/. 2Doumith M, Ellington MJ, Livermore DM et al. Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 2009; 63:659–67. 3Nordmann P, Naas T, Poirel L. Global spread of carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 2011; 17: 1791–8. 4Girlich D, Poirel L, Nordmann P. Clonal distribution of multidrug-resistant Enterobacter cloacae.Diagn Microbiol Infect Dis 2015; 81: 264–8. 5Marchaim D, Chopra T, Perez F et al. Outcomes and genetic relatedness of carbapenem-resistant Enterobacteriaceae at Detroit Medical Center. Infect Control Hosp Epidemiol 2011; 32: 861–71. 6Kiedrowski LM, Guerrero DM, Perez F et al. Carbapenem-resistant Enterobacter cloacae isolates producing KPC-3, North Dakota, USA. Emerg Infect Dis 2014; 20: 1583–5. 7AhnC,SyedA,HuFet al. Microbiological features of KPC-producing Enterobacter isolates identified in a U.S. hospital system. Diagn Microbiol Infect Dis 2014; 80: 154–8. 8Pecora ND, Li N, Allard M et al. Genomically informed surveillance for carbapenem-resistant Enterobacteriaceae in a health care system. MBio 2015; 6: e01030. 9Hargreaves ML, Shaw KM, Dobbins G et al.Clonaldisseminationof Enterobacter cloacae harboring bla KPC-3 in the upper midwestern United States. Antimicrob Agents Chemother 2015; 59: 7723–34. 10 Miyoshi-Akiyama T, Hayakawa K, Ohmagari N et al.Multilocus sequence typing (MLST) for characterization of Enterobacter cloacae. PLoS One 2013; 8: e66358. 11 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-fifth Informational Supplement M100-S25. CLSI,Wayne, PA, 2015. 12 Voets GM, Fluit AC, Scharringa J et al. A set of multiplex PCRs for genotypic detection of extended-spectrum b-lactamases, carbapenemases, plasmid-mediated AmpC b-lactamases and OXA b-lactamases. Int J Antimicrob Agents 2011; 37: 356–9. J Antimicrob Chemother 2016 doi:10.1093/jac/dkw137 Advance Access publication 26 April 2016 Characterization of an outbreak due to CTX-M-15-producing Klebsiella pneumoniae lacking the bla OXA-48 gene belonging to clone ST405 in a neonatal unit in southern Spain Jesu ´s Machuca1, Lorena Lo ´pez-Cerero1*, Felipe Ferna ´ndez-Cuenca1, Irene Gracia-Ahufinger2, Guillermo Ruiz-Carrascoso3, Fernando Rodrı ´guez-Lo ´pez2 and A ´lvaro Pascual1,4 1 Unidad intercentros de Enfermedades Infecciosas, Microbiologı ´ay Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain; 2 Unidad de Microbiologı ´a Clı ´nica, Hospital Universitario Reina Sofı ´a-IMIBIC-Universidad de Cordoba, Cordoba, Spain; 3 Unidad de Microbiologı ´a Clı ´nica, Hospital Universitario La Paz-IdiPaz, Madrid, Spain; 4 Department of Microbiology, University of Seville, Seville, Spain *Corresponding author. Tel: +34-954-55-28-63; Fax: +34-954-37-74-13; E-mail: [email protected] Sir, The dissemination of high-risk clones presents a major challenge for the worldwide health system because of their ability to overcome control measures and cross institutional and national borders and so provide a stable platform for the spread of resistance determinants. The traceability of such successful clones is important for the design of control strategies. 1 The dissemination of different high-risk MDR Klebsiella pneumoniae clones belonging to clonal complex 258/340 (ST11, ST258, ST340, ST437 and ST512) has been a key factor in the propagation of carbapenemase genes, especially bla KPC . 2 The role played by other clones is less well known. In 2012, a nosocomial large-hospital outbreak Research letters 2353 JA C Downloaded from https://academic.oup.com/jac/article/71/8/2353/2238523 by guest on 07 April 2025 caused by ST405 K. pneumoniae was described in Madrid (the central region of Spain). 3 This clone and its allelic variant, ST663, were responsible for several outbreaks of OXA-48 producers in other regions of Spain between 2010 and 2012, 4 although little is known of non-carbapenemase-producing ST405 isolates. The isolates from the nosocomial outbreak in Madrid were found to harbour two plasmids: an IncF plasmid carrying qnrB,aac(6′)- Ib-cr and bla CTX-M-15 genes and an IncL plasmid harbouring the bla OXA-48 gene. 5 Here, we characterize the first nosocomial outbreak caused by MDR CTX-M-15-producing K. pneumoniae ST405 lacking the bla OXA-48 gene in a neonatal unit of a hospital in Cordoba (southern Spain). Twenty-nine ESBL-producing K. pneumoniae recovered from 18 patients were collected in the neonatal unit of the Hospital Universitario Reina Sofia (Cordoba, Spain) between June and October 2013. All patients were colonized rectally and six patients were also infected (clinical samples were five blood samples, two bronchial aspirate samples, two catheter samples and two urine samples). Only isolates from surveillance studies were available and one isolate was selected from each patient for the following studies. Susceptibility tests were performed using commercial microdilution plates (Wider system, Francisco Soria Melguizo, Madrid, Spain). The isolates showed an ESBL phenotype along with resistance to ciprofloxacin (MIC .2 mg/L) and were susceptible to nalidixic acid (MIC ≤16 mg/L) and carbapenems (ertapenem, meropenem and imipenem MICs were ≤0.5 mg/L) according to CLSI breakpoints. ESBL production was confirmed by the doubledisc synergy test. By using specific PCRs and further sequencing, 6,7 bla CTX-M-15 ,bla TEM-1 ,qnrB1 and aac(6′)-Ib-cr genes were identified. XbaI PFGE analysis revealed that all isolates belonged to a single pulsotype (one band of difference) (Figure S1, available as Supplementary data at JAC Online). PFGE showed that the isolates described here had similarity of 94% (two or three bands of difference) with the OXA-48-producing K. pneumoniae ST405 isolates that caused the outbreak in Madrid. 3 One isolate was selected for MLSTanalysis, according to the Pasteur Institute MLSTscheme, and was assigned to clone ST405. Two isolates (which differed by one band by PFGE) from Cordoba and one isolate from Madrid were selected for plasmid analysis. Plasmid DNA was extracted by the method of Kieser 8 and was transformed by electroporation into Escherichia coli DH10B using MacConkey agar (Becton Dickinson France SA, Le Pont de Claix, France) supplemented with 2 mg/L cefotaxime. Specific PCRs using plasmid DNA from transformants and clinical isolates were positive for bla CTX-M-15 ,bla TEM-1 ,qnrB1 and aac(6′)-Ib-cr genes 6,7 and negative for bla OXA-48 . 9 Plasmids from transformants were classified into incompatibility groups using PCR-based Inc/rep typing. 10 IncF subtyping was performed with a replicon sequence typing scheme using the nomenclature of the plasmid MLST (pMLST) web site (http://www.pubmlst.org/ plasmid/). 11 A single 180 kb IncFIIK plasmid with the FAB formula K6:A2:B2was observed in the three transformants; the 70 kb RepP-positive plasmid from the Madrid isolate was not detected. No epidemiological links between the hospitalized patients in Cordoba and Madrid or other Spanish hospitals were found, although the index case was not located. The main risk factor was very preterm or extremely preterm; all infected patients were treated with meropenem with favourable outcome. TheK.pneumoniae ST405 clone has emerged asa new successful clone capable of disseminating different quinolone and b-lactam resistance determinants (including ESBL and carbapenemase) between different hospitals in Spain and other European countries. 12 The isolates described here did not harbour OXA-48 and this is the first time that this clone has been described without this carbapenemase and also the first time that it has been detected as causing an outbreak and not harbouring a carbapenemase. The reporting of successful clones enables us to trace their behaviour independently of the resistance determinants harboured. 1 Our results showed that K. pneumoniae ST405 can overwhelm single institutions because we found very similar pulsotypes in two different geographically distant hospitals (two or three bands of difference). The actual dissemination of K. pneumoniae ST405 has barely been explored beyond carbapenemase producers, found in Spain and France among OXA-48and CTX-M15-producing isolates and in Yemen among NDM and CTX-M-15 producers. 4,12,13 This clone was not detected in a multicentre Spanish study that characterized the ESBL-producing K. pneumoniae population. 14 This raises the question of whether this particular clone was disseminating bla CTX-M-15 before the OXA-48 plasmid capture. International clones such as ST15 and ST11 were well known in the past as bearing bla CTX-M-15 , 15 and now these clones are found to be dominant among CTX-M-15 and OXA-48 producers. Acquisition of the bla OXA-48 gene may be associated with clones already carrying bla CTX-M-15 ,qnrB and aac(6′)-Ib-cr plasmids, and these determinants may lead to the spread of the clone. Further studies are needed to explore in greater depth the genetic characteristics and epidemiological behaviour of CTX-M-15 producers and the contribution of these isolates to the current OXA-48 dissemination. Acknowledgements We would like to thank the collaboration of the Reference Laboratory, Program of Prevention and Control of Infections Associated to Health Assistance and Antimicrobial Stewardship of Andalucı ´a(PIRASOA, Servicio Andaluz de Salud). Funding This work was supported by the Instituto de Salud Carlos III, Subdireccio ´n General de Redes y Centros de Investigacio ´n Cooperativa, Ministerio de Economı ´a y Competitividad, the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF. Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/). References 1Mathers AJ, Peirano G, Pitout JDD. The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin Microbiol Rev 2015; 28:565–91. 2Lo ´pez-Cerero L, Almirante B. Epidemiology of infections caused by carbapenemase-producing Enterobacteriaceae: reservoirs and transmission mechanisms. Enferm Infecc Microbiol Clin 2014; 32 Suppl 4: 10–6. Research letters 2354 Downloaded from https://academic.oup.com/jac/article/71/8/2353/2238523 by guest on 07 April 2025 3Pan˜o-Pardo JR, Ruiz-Carrascoso G, Navarro-San Francisco C et al. Infections caused by OXA-48-producing Klebsiella pneumoniae in a tertiary hospital in Spain in the setting of a prolonged, hospital-wide outbreak. J Antimicrob Chemother 2013; 68: 89–96. 4Oteo J, Herna ´ndez JM, Espasa M et al. Emergence of OXA-48-producing Klebsiella pneumoniae and the novel carbapenemases OXA-244 and OXA-245 in Spain. J Antimicrob Chemother 2013; 68: 317–21. 5Rodrı ´guez Martı ´nez JM, Dı ´az-de Alba P, Lopez-Cerero et al. Presence of quinolone resistance to qnrB1 genes and bla OXA-48 carbapenemase in clinical isolates of Klebsiella pneumoniae in Spain. Enferm Infecc Microbiol Clin 32: 441–2. 6Rodrı ´guez-Ban˜o J, Lo ´pez-Cerero L, Navarro MD et al. Faecal carriage of extended-spectrum b-lactamase-producing Escherichia coli:prevalence,risk factorsandmolecularepidemiology.JAntimicrobChemother2008;62:1142–9. 7Rodrı ´guez-Martı ´nez JM, Cano ME, Velasco C et al. Plasmid-mediated quinolone resistance: an update. J Infect Chemother 2011; 17: 149–82. 8Kieser T. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli.Plasmid 1984; 12: 19–36. 9Poirel L, Walsh TR, Cuvillier V et al. Multiplex PCR for detection of acquiredcarbapenemasegenes.DiagnMicrobiolInfectDis2011;70:119–23. 10 Carattoli A, Bertini A, Villa L et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–28. 11 Villa L, Garcı ´a-Ferna ´ndez A, Fortini D et al. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J Antimicrob Chemother 2010; 65: 2518–29. 12 Liapis E, Pantel A, Robert J et al. Molecular epidemiology of OXA-48-producing Klebsiella pneumoniae in France. Clin Microbiol Infect 2014; 20: O1121–3. 13 Gharout-Sait A, Alsharapy S-A, Brasme L et al. Enterobacteriaceae isolates carrying the New Delhi metallo-b-lactamase gene in Yemen. J Med Microbiol 2014; 63: 1316–23. 14 Oteo J, Cuevas O, Lopez-Rodriguez I et al. Emergence of CTX-M-15producing Klebsiella pneumoniae of multilocus sequence types 1, 11, 14, 17, 20, 35 and 36 as pathogens and colonizers in newborns and adults. J Antimicrob Chemother 2009; 64: 524–8. 15 Damjanova I, To ´th A, Pa ´szti J et al. Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type b-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? J Antimicrob Chemother 2008; 62: 978–85. J Antimicrob Chemother 2016 doi:10.1093/jac/dkw131 Advance Access publication 18 April 2016 High prevalence of Neisseria gonorrhoeae with high-level resistance to azithromycin in Hangzhou, China Chunshan Ni1†, Juan Xue1†, Chuanling Zhang2, Haiyong Zhou3and Stijn van der Veen4* 1 Department of Microbiology and Parasitology, School of Medicine, Zhejiang University, Hangzhou, China; 2 Clinical Laboratory, Zhejiang Xiaoshan Hospital, Hangzhou, China; 3 Urology Department, Zhejiang Xiaoshan Hospital, Hangzhou, China; 4 Department of Microbiology and Parasitology, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Zhejiang University, Hangzhou, China *Corresponding author. Tel: +86-571-88206684; Fax: +86-571-88208022; E-mail: stijnvanderv[email protected] †Contributed equally to this work. Sir, In China, and most other countries, the first-line empirical monoantimicrobial therapy against gonorrhoea consists of extendedspectrum cephalosporins, such as ceftriaxone or cefixime. 1 Due to the recent rise in resistance and confirmed treatment failures, Europe and the USA now recommend a combination of ceftriaxone and azithromycin as the standard treatment for gonorrhoea. 2 Resistance to azithromycin is, however, common in many countries, particularly in settings where this antimicrobial is frequently used and widely available. High-level azithromycin-resistant Neisseria gonorrhoeae isolates with MICs ≥256 mg/L have now also emerged in several countries. 3–5 Furthermore, an outbreak with eight cases of high-level azithromycin-resistant gonorrhoea infections has been reported in Leeds, England. 6 These high-level azithromycin-resistant isolates are a significant threat to the efficacy of ceftriaxone and azithromycin dual-antimicrobial therapy and limit its success as a first-line therapy. Recently, we identified and characterized two high-level azithromycin-resistant N. gonorrhoeae strains that were isolated from patients attending the Zhejiang Xiaoshan Hospital in Hangzhou, China. 7 In the current study, we performed a retrospective analysis of the prevalence of high-level azithromycin resistance in all N. gonorrhoeae isolates (n¼118) that were collected at this hospital in 2011–12. After 2012, N. gonorrhoeae isolates were no longer stored and available for antimicrobial susceptibility testing. The susceptibility of N. gonorrhoeae isolates to antimicrobials was determined by agar dilution. 4 Breakpoints for antimicrobial resistance were adopted from the latest recommendations of EUCAST (http://www.eucast.org). Azithromycin resistance was observed for 25 of the isolates (21%), but, more importantly, 21 isolates (18%) showed high-level resistance (Table 1). All 118 isolates were susceptible to ceftriaxone, cefixime and spectinomycin, but resistance was observed to ciprofloxacin (100%), tetracycline (64%) and penicillin (71%). Of the high-level azithromycin-resistant isolates, 20 (95%) were resistant to tetracycline and 18 (86%) were resistant to penicillin. Molecular analysis of the clinical isolates was performed using the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method. 8 The 118 N. gonorrhoeae isolates were assigned to 79 different STs, among which 45 were new. The 21 high-level azithromycin-resistant N. gonorrhoeae isolates belonged to seven STs, namely ST3102 (7 isolates), ST1866 (6 isolates), ST5309 (3 isolates), ST12732 (2 isolates), ST12731 (1 isolate), ST12736 (1 isolate) and ST304 (1 isolate) (Table 1). Interestingly, one of the isolates belonged to ST1866, but did not display high-level azithromycin resistance. These results indicate that the high-level azithromycin-resistant ST1866 isolates might be a local derivative of a non-resistant ST1866 isolate. The NG-MAST method assigns an ST based on a combination of the two highly variable por Research letters 2355 JA C Downloaded from https://academic.oup.com/jac/article/71/8/2353/2238523 by guest on 07 April 2025