OXA-48-Like-Producing Klebsiella pneumoniae in Southern Spain in 2014-2015

OXA-48-like-producing Klebsiella pneumoniae in southern Spain 1 in 2014-2015. 2 3 Jesús MACHUCA1,2,3, 4 Lorena LOPEZ-CERERO*,1,2,3,4, 5 Felipe FERNANDEZ-CUENCA1,2,3, 6 Laura MORA-NAVAS5, 7 Concepción MEDIAVILLA-GRADOLPH6, 8 Inmaculada LÓPEZ-RODRÍGUEZ7, 9 Álvaro PASCUAL1,2,3,4 10 11 1Unidad de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital 12 Universitario Virgen Macarena, Sevilla, Spain; 13 2Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen 14 Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain. 15 3 Red Española de Investigación en Patología Infecciosa (REIPI RD16/0016), Instituto de 16 Salud Carlos III, Madrid, Spain; 17 4Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain; 18 5Hospital Clínico Universitario Virgen de la Victoria, Malaga, Spain; 19 6 Hospital Regional Universitario Carlos Haya Malaga, Spain; 20 7 Hospital Costa del Sol, Marbella, Spain 21 22 Keywords: K. pneumoniae, OXA-48, Southern Spain 23 Running title: OXA-48-producing K. pneumoniae, Southern Spain 24 *Corresponding author: Lorena López-Cerero 25 Mailing address: Unidad de Enfermedades Infecciosas y Microbiología, Hospital 26 Universitario Virgen Macarena, Dr Fedriani s/n. 41009 Spain. 27 Phone: +34 954 55 28 63. Fax: +34 954 37 74 13 28 E-mail: [email protected] 29 Text length: 2862 30 Abstract length: 242 31 Table/Figure/Supplementary data number: 2/0/4 32 33 ABSTRACT 34 The aim of this study was to characterize the population structure of 56 OXA-48-like- 35 producing K. pneumoniae isolates, as well as ESBLs and carbapenemases genes, 36 recovered in 2014 and 2015 from 16 hospitals in southern Spain. XbaI PFGE and MLST 37 were performed to assess clonal relatedness. Representative isolates belonging to OXA- 38 48-like-producing and CTX-M-15-co-producing pulsotypes were selected for 39 characterization of blaOXA-48-like- and blaCTX-M-15-carrying plasmids by PCR-based 40 replicon typing, IncF subtyping, WGS analysis and typing of Tn1999 structures. Forty- 41 three (77%) OXA-48-producing isolates were recovered from clinical samples and 13 42 from rectal swabs. All isolates showed ertapenem MIC values ≥ 1 mg/L, although 70% 43 remained susceptible to imipenem and meropenem. Forty-nine isolates (88%) produced 44 OXA-48, 5 produced OXA-245, and 2 produced OXA-181. Twenty-eight different 45 pulsotypes (5 detected in more than one hospital) and 16 sequencetypes were found. 46 The most prevalent clones were ST15 (29 isolates, 52%) and ST11 (7 isolates, 13%). 47 Forty-five (80%) isolates were also blaCTX-M-15 carriers. The blaCTX-M-15 gene was 48 mostly (82%) located on IncR plasmids, although ST15 and ST11 isolates also carried 49 this gene on IncF plasmids. The composite transposon variant Tn1999.2-like was the 50 most frequent. Among ST15 and ST11 isolates, different transposon variants were 51 observed. The blaOXA-48 gene was mainly located on IncL plasmids, although IncM 52 plasmids were also observed. The spread of OXA-48-like-producing K. pneumoniae in 53 southern Spain is mainly due to ST15 and ST11 clones. Variation within clonal lineages 54 could indicate different acquisition events for both ESBL and carbapenemase traits. 55 56 INTRODUCTION 57 The most important carbapenemases in Enterobacteriaceae are the KPC (class 58 A), NDM, IMP, VIM (class B) and OXA-48 (class D) types.(1) Among carbapenemase- 59 producing microorganisms, the increasing number of OXA-48-producing 60 Enterobacteriaceae, mainly Klebsiella pneumoniae, is becoming a serious threat, 61 particularly in Europe.(2) OXA-48 was first described in a clinical isolate of K. 62 pneumoniae in Turkey in 2001.(3) Since that time it has spread across North Africa and 63 Europe.(1) OXA-48-type carbapenemases hydrolyse penicillins and carbapenems, but 64 show weak activity against extended-spectrum cephalosporins.(3) Isolates harbouring 65 blaOXA-48 also carry other β-lactamases, such as blaCTX-M-15 and blaOXA-1, resulting in 66 resistance to cephalosporins and amoxicillin/clavulanate.(4) In Spain, increasing 67 numbers of cases of carbapenemase-producing Enterobacteriaceae (CPE) have been 68 reported in recent years. In a Spanish multicentre study performed in 2012, the most 69 common CPE detected as a cause of nosocomial outbreaks and individual infection was 70 OXA-48-producing K. pneumoniae.(5) 71 Dissemination of OXA-48 is principally related to the spread of a highly 72 transferable IncL/M plasmid carrying only blaOXA-48-like genes as resistance 73 determinant.(6) The blaOXA-48 gene is carried by the Tn1999.1 transposon or its variants, 74 Tn1999.2 and Tn1999.3 (with one or two copies of IS1R respectively inserted into 75 IS1999).(7) Two new variants of Tn1999.2 have recently been reported: Tn1999.4 and 76 Tn1999.5, in which the lysR gene is truncated by Tn2015 and ISKpn19, respectively.(8, 77 9) Despite the growing prevalence of OXA-48 producers, little is known of the 78 prevalence or genetic environment of blaOXA-48 and its accompanying resistance 79 determinants in Spain. 80 During 2014 and 2015, 16 hospital laboratories in Andalusia (southern Spain) 81 submitted 276 isolates of non-wild carbapenem susecptibility type K. pneumoniae 82 isolates, (10) both when they were single or clustered in time, to the reference 83 laboratory of the PIRASOA program (Hospital Universitario Virgen Macarena, Seville, 84 Spain). One hundred and seventy-seven (64%) were confirmed to be carbapenemase 85 producers: 56 (32%) isolates were OXA-48-like producers and the remainder were 86 KPC-3 producers. The resistance determinants (blaOXA-48-like and blaCTX-M-15 genes), 87 plasmids, Tn1999-type composite transposons, and the population structure of OXA-48- 88 like-producing K. pneumoniae isolates were characterized. 89 90 MATERIAL AND METHODS 91 Bacterial isolates 92 Between March 2014 and December 2015, 56 OXA-48-like-producing isolates 93 were analyzed. The OXA-48- producing isolates came from 16 hospitals located in 7 of 94 the 8 provinces of Andalusia (1–17 isolates/hospital, arbitrarily assigned numbered 95 names H1-H16) (Figure S1). 96 Bacterial identification and drug susceptibility testing 97 The isolates were identified using Matrix-Assisted Laser Desorption/Ionization 98 Time-of Flight mass spectrometry (MALDI-TOF Biotyper 3.1; Microflex BRUKER, 99 Madrid, Spain). Susceptibility testing was performed on commercial microdilution 100 panels (MicroScan, Beckamn Coulter, Madrid, Spain), using EUCAST breakpoints.(11) 101 Screening for ESBL and plasmid-encoded AmpC (pAmpC 102 ) production was performed with the double disc synergy test on Mueller Hinton (MH) 103 agar and MH agar supplemented with 200 mg/L of cloxacillin. The β-CARBATM test 104 (Bio-Rad, Madrid, Spain) was used to check for imipenem hydrolysis.(12) Initial 105 carbapenemase characterization was performed with combined-disk tests employing 106 meropenem plus inhibitors and temocillin susceptibility disks, according to EUCAST 107 guidelines.(10) 108 Characterization of ESBL and carbapenemase genes and genetic platforms of 109 OXA-48-like-encoding genes 110 The presence of genes encoding ESBLs (blaTEM, blaSHV, blaCTX-M-1 group and 111 blaCTX-M-9 group), pAmpC (blaMOX, blaCIT, blaCMY, blaDHA, blaFOX, blaACC and blaEBC) and 112 carbapenemases (blaOXA-48, blaKPC, blaIMP, blaVIM and blaNDM) was determined using 113 group-specific PCR primers and further sequencing.(13, 14) 114 The structure of Tn1999 of blaOXA-48 and blaOXA-245 genes from selected isolates 115 was determined by PCR using specific primers (Table S1) and further sequencing. The 116 sequences obtained were compared with those in the GenBank database. The primers 117 used did not differentiate between Tn1999.2, Tn1999.4 or Tn1999.5 variants. 118 Molecular epidemiology 119 PFGE analysis of XbaI-digested DNA (http://www.cdc.gov/pulsenet) was used 120 to determine the degree of genetic relatedness between isolates. Isolates differing by 121 two or more bands by XbaI-PFGE assay were assigned to different pulsotypes. A 122 dendrogram was created with Fingerprinting 3.0 software (Bio-Rad, Madrid, Spain), 123 using the Dice coefficient with position tolerance settings: 1% optimization and 1.2% 124 band position tolerance. The Institut Pasteur MLST scheme was used to characterize a 125 subset of isolates representing different PFGE clusters 126 (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). All pulsotypes assigned to the same 127 MLST were considered to belong to the same clone. 128 Transformation assays and characterization of OXA-48 and CTX-M-15 plasmids. 129 A representative isolate of each pulsotype was selected for transformation 130 assays. Plasmid DNA was extracted using the Kieser method (15) and transformed by 131 electroporation into E. coli DH10B (streptomycin-resistant). Transformants were 132 selected on MacConkey agar (Becton Dickinson France SA, Le Pont de Claix, France) 133 supplemented with 0.125 mg/L ertapenem or 2 mg/L cefotaxime to select transformants 134 with blaOXA-48-like or blaCTX-M-15 genes, respectively. The presence of blaOXA-48-like in 135 ertapenem resistant transformants and, similarly, blaCTX-M-15 in cefotaxime resistant 136 transformants was confirmed by phenotypic and genotypic methods, as described 137 above, and the presence of only one plasmid was confirmed after an extraction using the 138 Kieser method. 139 more than one pulsotype in six hospitals. The number of isolates belonging to the ST15 228 clone in our area decreased from 22 (69%) in 2014 to 7 (29%) in 2015. 229 Only one isolate belonging to ST405 was detected. This clone was previously 230 responsible for a major nosocomial outbreak of OXA-48-producing K. pneumoniae in a 231 hospital in Madrid (centre of Spain), as well as in other Spanish regions.(32) In 232 Andalusia, the same clone was responsible for an outbreak due to CTX-M-15-producing 233 K. pneumoniae lacking the blaOXA-48 gene.(33) 234 Genetic context of OXA-48-like genes and characterization of OXA-48 and CTX- 235 M-15 plasmids 236 OXA-48-like enzymes are frequently carried on the epidemic IncL-type plasmid, 237 responsible for the worldwide dissemination of blaOXA-48-like genes. The blaOXA-181 gene 238 has been located on IncT and IncX3 plasmids.(34) In order to gain some insight into the 239 type of transmission of blaOXA-48 and blaCTX-M-15 genes, plasmids recovered from 240 transformants were analyzed. E. coli DH10B transformants producing OXA-48-like 241 carbapenemases were obtained from 24 of 28 (79%) selected isolates representative of 242 the pulsotypes detected. No transformants were obtained from OXA-181 producers. 243 Using the PBRT scheme, twenty-two pulsotypes (48 isolates) carrying blaOXA-48-like 244 plasmids belonged to the IncL compatibility group (Table 1, Table S2) which is 245 consistent with the recognized IncL plasmid pOXA-48, responsible for the international 246 dissemination of OXA-48.(17) In two pulsotypes (P16 and P17), the blaOXA-48 gene was 247 located on an IncM plasmid, a result that was confirmed by WGS, indicating a new 248 incRNAI respect to M1 and M2, M3. To our knowledge, this is the first description of 249 the blaOXA-48 gene located on a new IncM3 plasmid. These two pulsotypes belonged 250 to ST392. 251 Twenty-six representative isolates of every pulsotype were selected for 252 characterization of transposon Tn1999. OXA-181 producers were not included because 253 blaOXA-181 is generally located on Tn2013.(35) The composite transposon variant 254 Tn1999.2 (the left IS1999 is truncated by IS1R) (36) or its variants Tn1999.4 and 255 Tn1999.5 was detected in 19 pulsotypes (65.5%) (Table 1). Sixteen and 3 pulsotypes 256 carryed OXA-48 and OXA-245, respectively. The Tn1999.1 variant (IS1999 intact and 257 IS1R absent) (37) was less prevalent and was detected in 8 pulsotypes (27.5%). This 258 isoform was found in 6 pulsotypes linked to OXA-48 and in 2 pulsotypes associated to 259 OXA-245. Tn1999.3 (with both upstream and downstream IS1999 truncated by IS1R) 260 (7) was not found. Different isoforms of Tn1999 were detected in three clones: ST15, 261 ST11 and ST392. In ST15 clone, Tn1999.2-like was found in 4 pulsotypes (15 isolates, 262 52 %) and Tn1999.1 in 3 pulsotypes (14 isolates, 48%). In clone ST11, the Tn1999.1 263 variant was detected in only one pulsotype (one isolate, 14%), while Tn1999.2 or its 264 variants was detected in 3 pulsotypes (6 isolates, 86%). In clone ST392, Tn1999.1 was 265 detected in one pulsotype and Tn1999.2-like in the other pulsotype of this clone. These 266 findings are consistent with different lineages of the same clone acquiring the blaOXA-48 267 gene on separate occasions and with other plasmids spreading this determinant. 268 Among the 28 pulsotypes detected in our set of isolates, only 21 included CTX- 269 M-15-producing isolates. CTX-M-15-producing E. coli DH10B transformants were 270 obtained from 18 pulsotypes (none were obtained from P12, P14 and P15). Only 6 271 plasmids could be typed using PBRT (Table 1),(16, 38) and all were IncFII-type: five 272 pulsotypes yielded the FIIK7:A-:B- formula (including P06, an ST15 pulsotype) and 273 one pulsotype, the FIIK1:A-:B- formula. As the PBRT scheme targets only replicons of 274 the major plasmid families in Enterobacteriaceae, WGS was used to characterize the 275 CTX-M-15 plasmid of the other twelve pulsotypes. These 12 pulsotypes carried the 276 blaCTX-M-15 gene on IncR-type plasmids (Table 1), which is different from what has been 277 published previously in the literature, in which blaCTX-M-15 is located principally on IncF 278 group plasmids.(39) Nevertheless, the association between the blaCTX-M-15 gene and the 279 IncR plasmid has been described previously in K. pneumoniae isolates belonging to 280 successful clones (ST15, ST11 and ST147) in Europe.(40, 41) In our study, most ST15 281 isolates co-harbouring blaCTX-M-15- and blaOXA-48 (25 isolates from six pulsotypes) 282 carried the blaCTX-M-15 gene on an IncR plasmid, and only the isolate with pulsotype P06 283 carried it on an IncFII-type plasmid. With respect to ST11 isolates, blaCTX-M-15 was 284 located on IncR plasmids in 3 pulsotypes (5 isolates) and on IncFII group plasmids in 285 one pulsotype (2 isolates). As was the case with blaOXA-48 acquisition, the blaCTX-M-15 286 gene was acquired by different lineages of each clone on separate occasions. 287 IncR plasmids carrying the blaCTX-M-15 gene co-harboured very diverse resistance 288 genes (beta-lactamases TEM-1 and OXA-1, Qnr proteins B1 and B66, 3´´-O- 289 phosphotransferases type Ib, 6´-O-phosphotransferases type Id, 6´-N-acetyltransferases 290 type Ib-cr, 3´-N-acetyltransferases type IIa, etc). An IncR plasmid from pulsotypes P02 291 and P04 (both belonging to ST15) harboured the same resistance genes, and IncR 292 plasmids from P10 and P13 similarly shared the same resistance determinants. The 293 other eight IncR plasmids analyzed however carried different resistance genes (Table 2). 294 These results reinforce the idea that different resistance determinants were captured 295 separately over time. 296 The major limitation of our study is that the collection studied was based on the 297 estimated OXA-48-producing K. pneumoniae population in our region in 2014 and 298 2015. It was not obligatory to send isolates to the PIRASOA program laboratory, 299 although in fact most isolates obtained from nosocomial outbreaks or clusters were sent. 300 On the other hand, centralized analysis of isolates was able to demonstrate the spread of 301 the same lineage of OXA-48 producers between hospitals in the same province, and 302 between different provinces in our region, which was not previously suspected even for 303 sporadic cases. It should be borne in mind however that detection of the same lineage in 304 different centres could also be due to recent transmission phenomena between 305 institutions or to unknown long-term transmission in the community. Another limitation 306 of our study consists that all isolates belonging to the same pulsotype were considered 307 to share the same OXA-48 plasmid and genetic plattform. The individual location of the 308 gene was not studied for every isolate, but they were very close genetically (only 1 band 309 of difference) and were found in the same area. 310 311 CONCLUSIONS 312 In summary, the spread of OXA-48-producing K. pneumoniae in the south of 313 Spain is mainly due to inter-hospital dissemination of ST15 and ST11 clones. Our 314 results suggest that lineages of the same clone captured blaOXA-48, blaCTX-M-15 and 315 resistance genes on separate occasions. The greater diversity of the genetic platform of 316 blaOXA-48 compared with the CTX-M-15 plasmids could indicate that successful clones 317 acquired CTX-M-15 first, and then OXA-48. Transmission of OXA-48 producers 318 should lead to a review of the considerations for regional patient transfer, as well as 319 local hospital measures, including both patient tracking and rapid detection of 320 successful clones. 321 322 FUNDING 323 This work was supported by the Plan Nacional de I+D+i 2013‐2016 and Instituto 324 de Salud Carlos III, Subdirección General de Redes y Centros de Investigación 325 Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for 326 Research in Infectious Diseases (RD16/0016/0001), co-financed by the European 327 Development Regional Fund “A way to achieve Europe”, Operational Programme 328 Intelligent Growth 2014–2020. 329 330 ACKNOWLEDGEMENTS 331 The authors would like to thank the Reference Laboratory, Program for the 332 Prevention and Control of Healthcare-Associated Infections and Antimicrobial 333 Stewardship in Andalucía (PIRASOA, Servicio Andaluz de Salud) for their 334 collaboration. 335 336 TRANSPARENCY DECLARATIONS 337 None to declare. 338 339 REFERENCES 340 1. 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Antibiotic resistance genes located on sequenced IncR plasmids. 522 MLST PFG E Isolate blaTEM-1B blaOXA-1 blaSHV-28 strA-strB aac(6')Ib-cr aac(3)-IIa sul2 catA2 catB4 dfrA14 qnrB1 qnrB66 tetA ST15 P01 2014081 + - - + - - + - - - - - - ST15 P02 2014127 + + - + + - + - + + - - - ST15 P03 2014028* - + + - + - - - - - - - - ST15 P04 2015254 + + - + + - + - + + - - - ST15 P05 2014142 + + - + + + + - + + - - - ST15 P07 2014090* - - - - - - - - - - - - - ST11 P08 2014126 - + - - + - - + + - + - - ST11 P09 2015083 + + - + + - + + + - + - - ST11 P10 2015013 + + - + + - + + + - + - - ST1 P13 2015017 + + - + + - + + + - + - - ST391 P23 2015141 + - - + - - - - - + - + + ST836 P26 2014139 - + - - + + - - + + - + + 523 All plasmids carried blaCTX-M-15 gene. blaTEM-1B, gene encoding the beta-lactamase TEM-1B; blaOXA-1, gene encoding the beta-lactamase OXA-1; blaSHV-28, gene encoding the 524 beta-lactamase SHV-28; strA-strB, pair of genes encoding 3´´-O-phosphotransferase type Ib and 6´-O-phosphotransferase type Id, respectively; aac (6´)Ib-cr, gene encoding 525 the 6´-N-aminoglycoside acetyltransferase type Ib-cr; aac(3)-IIa, gene encoding the 3´-N-acetyltransferase type IIa; sul2, gene encoding the dihydroteroate synthetase; catA2, 526 gene encoding the cloramphenicol acetyltransferase type A2; catB4, gene encoding the cloramphenicol acetyltransferase type B4; dfrA14, gene encoding the dihydrolate 527 reductase type II14; qnrB1, gene encoding the Qnr B1 protein; qnrB66, gene encoding the Qnr B66 protein; tetA, gene encoding the efflux protein type A. *From these 2 528 pulsotypes , a transformant could not be obtained and both IncR and resistance determinants were obtained from whole genome secuencing of clinical isolates bearing only 529 one plasmid. 530 531