This is the accepted version of the journal article: Argente Viñals, Marc; Miró, Elisenda; Martí, Carmina; [et al.]. «Molecular characterization of OXA-48 carbapenemase-producing Klebsiella pneumoniae strains after a carbapenem resistance increase in Catalonia». Enfermedades infecciosas y microbiología clínica, Vol. 37, Issue 2 (February 2019), p. 82-88. 7 pàg. DOI 10.1016/j.eimc.2018.02.003 This version is available at https://ddd.uab.cat/record/285200 under the terms of the license *Manuscrito revisado 1 EIMC-D-17-00387R1. REVISADO 1 2 2 3 4 3 5 6 4 7 8 9 5 Molecular characterization of OXA-48 carbapenemase-producing Klebsiella 10 11 6 pneumoniae strains after a carbapenem resistance increase in Catalonia. 12 13 7 14 15 8 16 17 18 9 Abstract 19 20 10 21 22 23 11 Purpose: To characterise OXA-48 carbapenemase-producing Klebsiella pneumoniae 24 25 12 strains isolated after an increase in carbapenem resistance in Catalonia. 26 27 28 13 Methodology: K. pneumoniae identification, antimicrobial susceptibility studies, the 29 30 31 14 Modified Hodge Test method, amplification of antimicrobial resistance genes (against 32 33 15 β-lactamases, quinolones and aminoglycosides), molecular typing (by PFGE and 34 35 16 MLST), conjugation assays, plasmid characterisation (PBRT-PCR and Southern blot), 36 37 17 a description of mobile genetic elements and statistical analysis were done. 38 39 40 18 Results: OXA-48 was the only carbapenemase detected, with a prevalence of 1.9%. 41 42 43 19 The blaOXA-48 gene was located in an IncL conjugative plasmid of 62 kb and integrated 44 45 20 into the transposons Tn1999.2 (91.7%) or Tn1999.1. Five PFGE profiles (A to E) were 46 47 21 found, which exactly matched the MLST: ST101, ST17, ST1233, ST14 and ST405, 48 49 22 respectively. ST1233 is described here for the first time. K. pneumoniae OXA-48- 50 51 52 23 producing strains were also CTX-M-15 carriers, some producing OXA-1 and TEM-1 53 54 24 penicillinases. The acquired qnrB66 and qnrB1 and aac(3’)-IIa, aac(6’)-Ib genes were 55 56 25 also identified. 57 58 59 60 1 61 62 63 64 65 26 Conclusion: The K. pneumoniae ST405 clone has played an important role in the 1 2 27 growing prevalence of OXA-48 in Catalonia. All clones described preserved the blaOXA- 3 4 28 48 genetic environment and mobile genetic elements (Tn1999). Notably, the three 5 6 7 29 strains with minor sequence types in this study are not multiresistant strains. These 8 9 30 strains are expanding in elderly patients (average age of 76 years) with serious 10 11 31 underlying diseases, mainly women (61.2%).. 12 13 14 32 15 16 17 33 18 19 20 34 Caracterización molecular de las cepas de Klebsiella pneumoniae productoras 21 22 23 35 de carbapenemasa OXA-48 tras un aumento de la resistencia a los 24 25 36 carbapenemas en Cataluña. 26 27 28 37 29 30 31 38 Introducción: el objetivo de este estudio fue caracterizar las cepas de Klebsiella 32 33 39 pneumoniae productoras de carbapenemasas OXA-48 aisladas tras observar un 34 35 36 40 aumento de estos aislados resistentes a los carbapenémicos en Cataluña 37 38 39 41 Métodos: se realizó la identificación de K. pneumoniae, estudios de sensibilidad 40 41 42 antimicrobiana, el test de Hodge modificado, amplificación de genes de resistencia 42 43 43 antimicrobiana (contra β-lactamasas, quinolonas y aminoglucósidos), tipificación 44 45 44 molecular (por PFGE y MLST), ensayos de conjugación, caracterización de plásmidos 46 47 48 45 (PBRT-PCR y Southern blot), descripción de los elementos genéticos móviles y el 49 50 46 análisis estadístico. 51 52 53 47 Resultados: OXA-48 fue la única carbapenemasa presente, con una prevalencia del 54 55 48 1.9%. El gen blaOXA-48 se localizó en un plásmido conjugativo IncL de 62 kb e integrado 56 57 49 en los transposones Tn1999.2 (91.7%) o Tn1999.1. Se encontraron cinco perfiles 58 59 60 2 61 62 63 64 65 50 diferentes de PFGE (A a E), que tenían una concordancia exacta con MLST: ST101, 1 2 51 ST17, ST1233, ST14 y ST405, respectivamente. El ST1233 se describe aquí por 3 4 52 primera vez. Las cepas productoras de K. pneumoniae OXA-48 también fueron 5 6 7 53 portadoras de CTX-M-15 y algunas de ellas producen penicilinasas OXA-1 y TEM-1. 8 9 54 Los genes adquiridos qnrB66 y qnrB1 y aac(3')-IIa, aac(6')-Ib también se identificaron. 10 11 55 Conclusión: el clon K. pneumoniae ST405 tiene un papel importante en la creciente 12 13 56 prevalencia de OXA-48 en Cataluña. Todos los clones descritos preservaron el 14 15 57 entorno genético de blaOXA-48, así como los elementos genéticos móviles (Tn1999). 16 17 18 58 Notablemente, las tres cepas con tipos de secuencia menos prevalentes en este 19 20 59 estudio no son cepas multirresistentes. Además, la expansión de estas cepas con 21 22 60 blaOXA-48 se está produciendo en pacientes de edad avanzada (promedio de edad de 23 24 61 76 años), la mayoría mujeres (61,2%) con enfermedades subyacentes graves. 25 26 27 28 62 29 30 31 63 Keywords: Antibiotic resistance, OXA-48, carbapenemase, PFGE, MLST 32 33 34 64 35 36 65 37 38 66 39 40 67 41 42 43 68 44 45 69 46 47 70 Introduction 48 49 71 The emergence and spread of carbapenem-resistant Enterobacteriaceae due to 50 51 52 72 carbapenemase production is a serious public health problem worldwide. Colistin or 53 54 73 tigecycline are last-resort antibiotics against these carbapenemase-producing 55 56 74 Enterobacteriaceae1, but some resistant strains have been described. 57 58 59 60 3 61 62 63 64 65 75 Class A, B and D carbapenemases have been reported in Enterobacteriaceae 1 2 76 worldwide since 19932,3, but were not described in Spain until 20034. VIM and IMP 3 4 77 (class B) were initially the most frequent, but they have since declined and OXA-48 5 6 7 78 (class D), first documented in Spain in 2009, is currently the most prevalent5. 8 9 10 79 The OXA-48 carbapenemase exhibits strong penicillin-hydrolysing activity and weak 11 12 80 activity against carbapenems. Derivatives such as OXA-163 (first described in 13 14 81 Klebsiella pneumoniae), OXA-247 (K. pneumoniae) and OXA-405 (Serratia 15 16 82 marcescens) hydrolyse penicillins, ceftazidime and cefotaxime, but as their 17 18 19 83 carbapenem-hydrolysing activity is far lower than OXA-48 they are barely considered 20 21 84 as carbapenemases1–3. 22 23 24 85 An OXA-48-producing K. pneumoniae strain was first identified in 2001 in Istanbul, with 25 26 86 reservoirs becoming established in North Africa and the Mediterranean area. It has 27 28 87 now spread through the rest of Europe, Asia and America6,7. The dissemination of this 29 30 31 88 enzyme could be explained by two factors. First, blaOXA-48 has been described in an 32 33 89 IncL plasmid in Enterobacteriaceae as well as in both non-fermenters 34 35 90 (Acinetobacter baumannii and Pseudomonas aeruginosa)1. Second, blaOXA-48 gene is 36 37 91 part of the transposon Tn1999, which is inserted in the tir gene of the plasmid IncL. 38 39 40 92 Different Tn1999 variants are known (Tn1999.2, Tn1999.3 and Tn1999.4)8, varying in 41 42 93 the presence or not of the insertion sequence IS1R1 or the transposon Tn2015, which 43 44 94 also contains blaCTX-M-15. On the other hand, the insertion of Tn1999 in the tir gene of 45 46 95 this plasmid has been associated with an enhanced conjugation performance and 47 48 96 could contribute to the high diffusion of this plasmid type and its resistance genes. 49 50 51 52 97 Enterobacteriaceae strains carrying the OXA-48-carbapenemase can also co-express 53 54 98 extended-spectrum β-lactamases (ESBLs) such as CTX-M-15 and SHV-129, or 55 56 99 acquired AmpC β-lactamases (acAmpC) such as DHA-14, mainly in different plasmids; 57 58 59 60 4 61 62 63 64 65 100 strains resistant to quinolones, cotrimoxazole and aminoglycosides have also been 1 2 101 described2,10. 3 4 5 102 In a national study conducted in 20094, we observed a very low prevalence of 6 7 103 carbapenemase-producing Enterobacteriaceae in Catalonia (0.04%). However, in 8 9 10 104 2012, hospitals in the Barcelona area reported an increase in K. pneumoniae strains 11 12 105 resistant to third generation cephalosporins and ertapenem and suspected of 13 14 106 expressing carbapenemases. The aim of this study was to explain this growing 15 16 107 prevalence by characterizing the β-lactamases involved in this resistance phenotype 17 18 19 108 and establishing the genetic relationships between strains. 20 21 22 109 Material and Methods 23 24 25 110 Ethics 26 27 28 111 The study was approved by the Ethical Review Committee of the Institut de Recerca de 29 30 112 l’Hospital de la Santa Creu i Sant Pau. 31 32 33 113 Strains and patients 34 35 36 114 In a prospective study in 2012 (January to December) involving 12 hospitals in 37 38 39 115 Catalonia, K. pneumoniae isolates resistant to β-lactams were collected, excluding 40 41 116 those with a natural resistance pattern. Epidemiological data on patient gender, age, 42 43 117 chronic diseases and treatment were collected in parallel. Only one strain per patient 44 45 118 was included. The selected strains were resistant to any of the following β-lactams: 46 47 48 119 cephalotin, cefoxitin, cefuroxime, cefotaxime, ceftazidime, cefepime, ertapenem, 49 50 120 imipenem, aztreonam, amoxicillin/clavulanic acid and/or piperacillin/tazobactam. 51 52 121 Each hospital carried out identification and antibiotic susceptibility tests and provided 53 54 122 epidemiological patient data. The participating hospitals were: Hospital Municipal de 55 56 123 Badalona (HMB), Hospital de Barcelona (HB), Corporació de Salut del Maresme I la 57 58 59 124 Selva (HC), Hospital General de Granollers (HGG), Hospital General Universitari de 60 5 61 62 63 64 65 125 Catalunya at Sant Cugat del Vallès (HGC), Hospital General de L’Hospitalet (HGH), 1 2 126 Hospital Sant Joan de Déu at Manresa (HSJDD), Hospital Sant Joan Martorell (HMLL), 3 4 127 Hospital de Mataró (HM), Hospital Universitari de Vic (HGV), Hospital Universitari de 5 6 7 128 Sant Joan de Reus (HUSJR) and Hospital de la Santa Creu i Sant Pau (HSCSP) 8 9 129 (covering a population of approximately 250,000 inhabitants). 10 11 12 130 The patients were classified in three categories: nosocomial (the infection occurring 13 14 131 after 48h of hospital admission), healthcare centre (resident in a healthcare centre) and 15 16 132 community (no recent contact with a medical environment). 17 18 19 133 Bacterial identification and antimicrobial susceptibility testing 20 21 22 23 134 K. pneumoniae identification and antimicrobial susceptibility tests were performed in 24 25 135 each hospital following routine laboratory methods, either manual or automated 26 27 136 [MicroScan WalkAway (Siemens) and Vitek system (bioMérieux, Marcy l'Etoile, 28 29 137 France)]. The susceptibility pattern to β-lactams, quinolones and aminoglycosides was 30 31 138 obtained by the disc-diffusion method (Rosco Diagnostica A/S, Taastrup, Denmark) 32 33 34 139 following following Clinical and Laboratory Standards Institute (CLSI) criteria, as used 35 36 140 routinely in the laboratory11. The antibiotics used were: Ampicillin (AMP), Piperacillin 37 38 141 (PIP), Amoxicillin/clavulanic acid (AMC), Piperacillin/tazobactam (TZP), Cephalotin 39 40 142 (CEF), Cefoxitin (FOX), Cefuroxime (CXM), Cefotaxime (CTX), Ceftazidime (CAZ), 41 42 43 143 Aztreonam (ATM), Cefepime (FEP), Ertapenem (ERT), Imipenem (IMP), Kanamycin 44 45 144 (K), Gentamicin (G), Tobramycin (T), Amikacin (A), Netilmicin (Nt), Neomycin (Nm), 46 47 145 Nalidixic acid (NAL), Ciprofloxacin (CIP), and Cotrimoxazole (SXT). 48 49 146 The then recommended Modified Hodge Test (MHT) was performed to detect 50 51 52 147 carbapenemase activity, using imipenem according to CLSI criteria11. The strains 53 54 148 selected for analysis were resistant to any of the studied β-lactams. Strains with 55 56 149 positive or weakly positive MHT results were included for the molecular 57 58 150 characterization of carbapenemase resistance mechanisms. 59 60 6 61 62 63 64 65 151 Amplification of antimicrobial resistance genes 1 2 3 152 The Polymerase Chain Reaction (PCR) was used to detect the following genes in all 4 5 153 studied strains according to their resistance phenotype: carbapenemases (blaOXA-48, 6 7 154 blaVIM, blaSPM, blaIMP, blaGIM, blaSME, blaNMC, blaKPC, blaIMI and blaGES, blaNDM), ESBLs (blaTEM, 8 9 10 155 blaSHV, blaCTX-M), acquired AmpC genes (acAmpC) (blaACC, blaCIT, blaEBC, blaDHA, blaFOX, 11 12 156 blaMOX) and the penicillinase blaOXA-1, quinolones (qepA and qnrA, qnrB, qnrS, qnrC, 13 14 157 qnrD) and aminoglycoside modifying enzymes (AME) (aac(3’)-IIa, aac(6)-Ib, aph(3’)-Ia, 15 16 158 ant(2’)-Ia and aac(2’)-Ia) 4,12,13,. 17 18 19 159 The blaOXA-48 genetic platform was determined by PCR using specific primers for 20 21 22 160 IS1999, lysR and IS1R of Tn1999 and Tn1999.2 6,14 in all blaOXA-48-carrying strains. 23 24 25 161 All amplicon sequences were obtained by Sanger sequencing in an external genome 26 27 162 service enterprise (Macrogen Europe, Amsterdam, The Netherlands). 28 29 30 163 Molecular typing of blaOXA-48-carrying K. pneumoniae strains 31 32 33 164 Pulsed-field gel electrophoresis (PFGE) with genomic DNA digestion by the enzyme 34 35 165 XbaI was performed with the CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UK) 36 37 38 166 and analysed by BioNumerics 6.6 (Applied-Maths, Ghent, Belgium)2,12,15. The 39 40 167 relatedness was calculated by the unweighted-pair group method using an average 41 42 168 linkage (UPGMA) algorithm, with band similarity calculated using the Dice coefficient 43 44 169 with a 2% optimization value and 1% tolerance. PFGE patterns were interpreted 45 46 47 170 as previously described11,12. MultiLocus Sequence Typing (MLST) was performed 48 49 171 according to the Pasteur Institute website 50 51 172 (www.pasteur.fr/recherche/genopole/P8/mlst)15 in 11 strains selected according to their 52 53 173 PFGE pattern (A-E). For cluster E, we selected the majority and minority patterns 54 55 56 174 consulting the resistance pattern. 57 58 59 175 Conjugation Assay 60 7 61 62 63 64 65 176 Conjugation was performed in the aforementioned 11 isolates. Rifampicin-resistant- 1 2 177 GFP E. coli VA6190 was used as a recipient11, which also expresses a green 3 4 178 fluorescent protein (GFP) marker. Luria-Bertani broth (LB) was used for the conjugation 5 6 7 179 or, if LB results were negative, solid mating with HA 45µm-pore-size filters (Millipore, 8 9 180 Billerica, MA). Putative transconjugants were selected on LB agar supplemented with 10 11 181 ceftazidime (10 mg/L) and rifampicin (100 mg/L), and exposed to UV illumination to 12 13 182 check for GFP fluorescence. The transfer frequency was expressed as the ratio of 14 15 183 transconjugants to total recipient cells. The presence of blaOXA-48 in the transconjugants 16 17 18 184 was confirmed by PCR. 19 20 21 185 Plasmid Typing 22 23 24 186 Plasmid Identification was performed in 37 strains by a homemade PCR-based 25 26 187 replicon typing (PBRT-PCR) method based on Carattoli et al. (2009)16. The studied 27 28 188 strains represented each PFGE subcluster, adding some strains with the same PFGE 29 30 31 189 pattern but different resistance patterns. Poirel et al. (2012)17 observed that PBRT-PCR 32 33 190 did not detect a blaOXA-48-carrying IncL/M replicon. Carattoli et al. (2015)18 described 34 35 191 specific primers to detect the blaOXA-48-carrying IncL plasmid, which distinguished IncL 36 37 192 from IncM. We used these primers to describe the plasmid in our OXA-48-producing K. 38 39 40 193 pneumoniae strains. 41 42 43 194 The plasmid localization of the bla genes was determined in the same 11 strains by 44 45 195 PFGE using S1 nuclease and Southern blot hybridization methods, as previously 46 47 196 described12. The probes were obtained by the DIG Probe Synthesis Kit (SigmaAldrich) 48 49 197 using primers corresponding to the bla and replicon genes. 50 51 52 198 Statistical analysis 53 54 55 56 199 Categorical variables were compared by the Х2 test and continuous variables by the 57 58 200 Student’s t-test or Mann–Whitney test. P-values of <0.005 were considered statistically 59 60 8 61 62 63 64 65 201 significant. The software GraphPad Prism (GraphPad Software, Inc. CA. USA) was 1 2 202 used for the analyses. 3 4 5 203 RESULTS 6 7 8 204 Bacterial isolates and susceptibility data 9 10 11 205 Between January and December 2012, we collected a total of 3,901 K. pneumoniae 12 13 14 206 isolates resistant to β-lactams, not including those with a natural resistance pattern. 15 16 207 Among these, 171 (4.38%) gave positive MHT results, including strains with only a 17 18 208 weak growth around the streak. And among these171 strains, 98 (57.3%) were 19 20 209 resistant to ertapenem and 22 (12.8%) to imipenem. After the PCR and sequencing, 85 21 22 23 210 strains gave positive results for blaOXA-48, nine of them from faecal carriers. These 85 24 25 211 strains were from eight out of the 12 participating hospitals. Thus, the prevalence of 26 27 212 OXA-48-producing K. pneumoniae strains in Catalonia was 1.9% (subtracting the nine 28 29 213 faecal carriers). No other targeted carbapenemases were detected. The 85 OXA-48- 30 31 214 producing strains were resistant to ampicillin, piperacillin and the association 32 33 34 215 amoxicillin/clavulanic acid. Only 23.5% were resistant to imipenem, whereas 95.3% 35 36 216 were resistant to ertapenem, which is the most sensitive drug for OXA-48-producing 37 38 217 strain detection. Most strains were resistant to cephalosporins, in some cases 39 40 218 remaining susceptible to cefoxitin and cefepime (Table 1). These different phenotypes 41 42 43 219 were due to the presence of an ESBL, as 89.4% co-expressed the CTX-M-15, together 44 45 220 with the penicillinases OXA-1 (94.1%) and TEM-1 (85.4%). All 85 isolates also 46 47 221 expressed the chromosomal penicillinase SHV characteristic of this species: in 77.64% 48 49 222 we detected blaSHV-76, 20% blaSHV-1, 1.1% blaSHV-11 and 1.1% blaSHV-42. 50 51 52 223 These strains were also resistant to aminoglycosides (90.5%), cotrimoxazole (89.4%) 53 54 55 224 and ciprofloxacin (83.5%). Only four strains remained susceptible to all 56 57 225 aminoglycosides and quinolones tested. Regarding aminoglycoside resistance, we 58 59 226 found nine different phenotype patterns: KTG (64.7%), KTGN (14.1%), KTGNm (4.7%), 60 9 61 62 63 64 65 227 KTA (1.1%), G (2.3%), KTAGN (1.1%), KTAN (3.5%), KT (2.3%) and KTN (1.1%). The 1 2 228 AMEs present in these strains were AAC(3’)-IIa (83.5%), AAC(6’)-Ib (81.2%), APH(3’)- 3 4 229 Ia (3.5%), AAC(2’)-Ia (2.4%) and ANT (2’)-Ia (1.2%). Finally, 88.2% carried QnrB and in 5 6 7 230 one case QnrS (Table 2). 8 9 10 231 As mentioned above, 86 out of 171 positive MHT strains (50.3%) were non- 11 12 232 carbapenemase-producing. Among these isolates, 17 (19.7%) were resistant to 13 14 233 ertapenem and 1 (1%) to imipenem. To explain the positive MHT results, we checked 15 16 234 for the presence of ESBLs and acAmpC. The PCR results revealed that 47 strains 17 18 19 235 (54.6%) carried an acAmpC (80.8% DHA, 10.6% ACC and 8.6% CMY), 21 (24.4 %) 20 21 236 carried an ESBL (CTX-M-1-type) and 14 (16.3%) co-expressed both ESBL and 22 23 237 acAmpC (64.3% CTX-M-1-type+DHA, 14.3% CTX-M-9-type+DHA, 14.3% CTX-M-1- 24 25 238 type+ACC and 7.1% CTX-M-1-type with CMY). Finally, four strains (4.6%) did not show 26 27 239 any studied acAmpC or ESBL. The 86 strains without carbapenemase production were 28 29 30 240 excluded from further studies. 31 32 33 241 Clinical and molecular epidemiological data 34 35 36 242 The origin of the 85 OXA-48-producing strains was: 47 (55.3%) from urine samples, 13 37 38 243 (15.3%) from respiratory tract samples, six (7%) from blood, four (4.7%) from surgical 39 40 244 wounds, six (7%) from other samples (vaginal discharge, peritoneal fluid, sore, 41 42 43 245 cellulitis, bile, post-operative ulcer) and nine (10.6%) were isolated from faeces (Table 44 45 246 3). 46 47 48 247 The demographic data collected from the 85 patients with OXA-48-producing K. 49 50 248 pneumoniae infection showed that 52 were female (61.2%) of an average age of 76 51 52 249 years (range 26-98 years). There was no significant difference between genders 53 54 55 250 (P>0.005). In 41 (48.2%) cases the infection had a nosocomial origin, 30 (35.3%) were 56 57 251 related to a healthcare centre, and the remaining 14 (16.5%) were acquired in the 58 59 252 community (Table 3). 60 10 61 62 63 64 65 253 The PFGE analysis revealed five well-defined clusters (A-E), which were subdivided in 1 2 254 multiple subclusters, with 76% homology between the main clusters. Cluster A, B, C, D 3 4 255 and E represented 18.8%, 1.1%, 1.1%, 1.1% and 77.6% of all isolates, respectively. 5 6 7 256 Clusters A and E showed subclusters A1-3 and E1-18. The cluster distribution is depicted 8 9 257 in Figure 1. 10 11 12 258 By MLST, the five PFGE clusters corresponded to five different K. pneumoniae 13 14 259 sequence types (STs) as follows: cluster A belonged to ST101 (n=16); B to ST17 15 16 260 (n=1); C to ST1233 (n=1); D to ST14 (n=1) and E to ST405 (n=66). ST1233 is 17 18 19 261 described here for the first time and is a single-locus variant (SLV) of ST540 in the 20 21 262 gapA gene. Cluster E, which corresponds with ST405, was isolated in six of the eight 22 23 263 OXA-48-detecting hospitals; cluster A, all ST101, was present in three of the eight 24 25 264 hospitals. The minor clusters B (ST17), C (ST1233) and D (ST14), with one strain 26 27 265 each, were from HGG, HM and HGV, and were isolated in different hospitals together 28 29 30 266 with isolates belonging to major clones (Supplementary Figure 1). 31 32 33 267 We observed that all OXA-48-producing K. pneumoniae strains, except those 34 35 268 belonging to ST14, ST17 and ST1233, were resistant to multiple antimicrobial drugs 36 37 269 (mainly aminoglycosides and quinolones) by acquiring resistance genes. blaOXA-48 38 39 40 270 seemed to be associated with blaOXA-1, aac(3’)-IIa, aac(6’)-Ib and qnrB, and in most 41 42 271 cases with blaCTX-M-15 and blaTEM-1 (Table 2). 43 44 45 272 Plasmid characterization and the blaOXA-48-carrying genetic platform 46 47 48 273 The PBRT-PCR data show that the 37 selected strains carried a plasmid with an IncL 49 50 274 plasmid, 96% out of the 37 a plasmid replicon FIIK and 33.3% a plasmid replicon ColE 51 52 275 (Table 4). Two cluster A-ST101 strains also carried one plasmid replicon R and the 53 54 55 276 strain of cluster E16-ST405 showed the plasmid replicon FIA. The strains belonging to 56 57 277 ST17 and ST14 were the only ones without plasmid replicons other than IncL. 58 59 60 11 61 62 63 64 65 278 PFGE and Southern blot studies revealed that that blaOXA-48 was present in all 85 OXA- 1 2 279 48-producing strains in an approx. 62kb plasmid belonging to the IncL incompatibility 3 4 280 group. These data were confirmed by conjugation assays. In all 11 conjugated strains 5 6 7 281 we found OXA-48-producing E. coli transconjugants with a conjugation frequency 8 9 282 between 1.3 x10-5 and 5x10-7. All transconjugants with blaOXA-48 were resistant to 10 11 283 penicillins, remaining susceptible to cefotaxime and ceftazidime and with a reduction in 12 13 284 their susceptibility to ertapenem. In all cases the carbapenemase had transferred 14 15 285 alone, as confirmed by PCR. 16 17 18 19 286 Southern blot experiments revealed that blaCTX-M-15 and blaOXA-1 were carried in FIIK 20 21 287 plasmids of approx. 240Kb in ST101strains and approx. 290Kb in ST405. 22 23 24 288 The blaOXA-48 -carrying genetic platform was related with the transposon Tn1999 (Table 25 26 289 4). Seventy-eight strains (91.8%) showed Tn1999.2 and seven the intact Tn1999. We 27 28 290 sequenced the blaOXA-48 genetic surroundings in eleven randomly selected strains 29 30 31 291 (Figure 2) (GenBank accession numbers: KT265174, KT265175, KT265176, 32 33 292 KT265177, KT265178, KT265179, KT265180, KT265181, KT265182, KT265183 and 34 35 293 KT265173). In 10 cases a unique sequence between the IS1R element, upstream of 36 37 294 blaOXA-48 and lysR, was found, with 100% homology with the Tn1999.2 sequence from 38 39 40 295 pKpn-E1.Nr7 (KM406491.1). The remaining strain had 100% homology with K. 41 42 296 pneumoniae E71T (KC335143.1) and pKPoxa-48N1 (KC757416.2). The only 43 44 297 difference between the two groups was due to a transversion T-G at position 911, 45 46 298 ºwhere the K. pneumoniae E71T (KC335143.1) and pKPoxa-48N1 (KC757416.2) 47 48 299 sequences have a guanine. 49 50 51 52 300 Discussion 53 54 55 301 Determining OXA-48-producing strain prevalence is hampered by the lack of a 56 57 302 standard method19. The MHT, in 2012 recommended by the CLSI for carbapenemase 58 59 303 detection1,11, is now considered unsuitable as a single screening method, being too 60 12 61 62 63 64 65 304 unspecific and insensitive for metallo-β-lactamase detection. In this study, 51% of 1 2 305 positive MHT strains did not express any carbapenemase. These false positives were 3 4 306 due to the presence of ESBLs such as CTX-M-15, acAmpCs such as DHA or porin 5 6 7 307 alterations, which may be involved in reduced susceptibility to carbapenems12. A low 8 9 308 affinity for carbapenems of some ESBLs has been described, specifically for the CTX- 10 11 309 M-15 enzyme, which can hydrolyse ertapenem when highly expressed20. The three 12 13 310 OXA-48-producing K. pneumoniae strains without additional β-lactamases were only 14 15 311 resistant to ampicillin, piperacillin and amoxicillin/clavulanic acid, remaining susceptible 16 17 18 312 to ertapenem and imipenem. In contrast, all the OXA-48- and ESBL- or acAmpC- 19 20 313 producing strains were resistant to penicillins, cephalosporins and ertapenem, and 21 22 314 23.5% also to imipenem. 23 24 25 315 Therefore, assuming that the phenotypic method might not be accurate to determine 26 27 316 the prevalence of oxa-48 producing strains, prevalence was determined according to 28 29 30 317 PCR results. The prevalence of OXA-48-producing-K. pneumoniae strains in 2012 in 31 32 318 Catalonia was 1.9%, matching previous studies in some Spanish tertiary hospitals2,9 a 33 34 319 0.04% and 5.3% prevalence was described. A recent European survey of 35 36 320 carbapenemase-producing Enterobacteriaceae (EuSCAPE)21,found a 69.8% 37 38 321 prevalence of OXA-48-producing Klebsiella pneumoniae strains among 136 strains 39 40 322 collected by ISCIII. 41 42 43 323 The lack of standardization in detection methods affects not only prevalence data, but 44 45 324 also the implementation of a policy to avoid the establishment of multiresistant 46 47 325 strains22, although new chromogenic plate methods have been described. 48 49 50 326 Like ESBL- or acAmpC-producing strains4,12,13, OXA-48-producing K. pneumoniae 51 52 327 isolates are also resistant to aminoglycosides, cotrimoxazole and ciprofloxacin. 53 54 328 Nevertheless, the blaOXA-48-carrying IncL plasmids described to date (such as pkPoxa- 55 56 57 329 48N118), responsible for OXA-48 dissemination, do not include any associated 58 59 330 antimicrobial resistance genes. Our results show that blaCTX-M-15 is present in an IncFIIk 60 13 61 62 63 64 65 331 plasmid, in agreement with other authors23, together with quinolone and 1 2 332 aminoglycoside resistance genes24. 3 4 5 333 The worldwide expansion of blaOXA-48 could be due to its association with Tn1999, 6 7 334 located on a characteristic IncL plasmid1. In turn, Tn1999 is inserted into the tir gene 8 9 10 335 responsible for plasmid transfer inhibition. We found the increasing OXA-48 prevalence 11 12 336 was also due to the expansion of the ST405 clone, present in all hospitals with OXA- 13 14 337 48-producing K. pneumoniae except two. This major clone was originally identified in 15 16 338 Casablanca (Morocco)25 in a K. pneumoniae strain carrying blaCTX-M-15, blaOXA-1 and 17 18 19 339 blaTEM-1, but not blaOXA-48, and has been widely described in Europe, including Spain9 As 20 21 340 described by Baquero et al.26, this expansion could be due to colonization and 22 23 341 transmission between particular hosts, which acquire antibiotic resistance and 24 25 342 enhanced survival capacity. In this study, the ST405 clone was found in six of the eight 26 27 28 343 hospitals with OXA-48 carbapenemase. 29 30 31 344 The expansion of all these clones is taking place in the elderly population and is related 32 33 345 to the healthcare system. These results are similar to those obtained from patients 34 35 346 infected by ESBL- or acAmpC-producing strains27. 36 37 38 347 Like ST405, the sequence types ST101, ST14 and ST17 are also described as 39 40 348 multiresistant clones present in various European and American countries7,28–31. 41 42 43 349 Nevertheless, in our case, ST14 and ST17 were only detected exceptionally and did 44 45 350 not carry additional resistances. Finally, ST1233, described here for the first time, was 46 47 351 a single locus variant of ST540, and did not show resistance to aminoglycosides or 48 49 352 quinolones. All these clones were isolated in a timely manner. 50 51 52 353 Taken together, our results show that the increasing prevalence of carbapenemase 53 54 55 354 OXA-48 in Catalonia is due to the expansion of the K. pneumoniae ST405 clone. All 56 57 355 clones described preserved the blaOXA-48 genetic environment as well as the mobile 58 59 356 genetic elements (Tn1999). Curiously, the three strains with minority ST were not 60 14 61 62 63 64 65 357 resistant to multiple drugs, perhaps because of an absence of selection pressure due 1 2 358 to their infrequency and therefore low exposure to antibiotics. 3 4 5 359 Acknowledgements 6 7 8 360 We thank all participating health centers for their willingness to enter the study and also 9 10 11 361 the group of microbiologists of Country Hospitals in Catalonia and the Balearic Islands 12 13 362 (http://www.scmimc.org/grupstreball02.php) for collecting the strains and 14 15 363 epidemiological data. 16 17 18 364 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 15 61 62 63 64 65 365 References 1 2 3 366 1. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: The phantom 4 5 367 menace. J Antimicrob Chemother 2012;67:1597–1606. 6 7 8 368 2. 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Frasson I, Lavezzo E, Franchin E, Toppo S, Barzon L, Cavallaro A, et al. 8 9 10 466 Antimicrobial treatment and containment measures for an extremely drug- 11 12 467 resistant Klebsiella pneumoniae ST101 isolate carrying pKPN101-IT, a novel 13 14 468 fully sequenced bla(KPC-2) plasmid. J Clin Microbiol 2012;50:3768-3772. 15 16 17 469 31. Peirano G, Hung King Sang J, Pitondo-silva A, Laupland KB, Pitout JDD. 18 19 470 Molecular epidemiology of extended-spectrum-β-lactamase-producing Klebsiella 20 21 22 471 pneumoniae over a 10 year period in Calgary, Canada. J Antimicrob Chemother 23 24 472 2012;67:1114-1120. 25 26 27 473 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20 61 62 63 64 65 Table 1. Antibiotic resistance in OXA-48-producing K. pneumoniae strains. 1 2 Antibiotic Resistance (%) 3 4 Ampicillin (AMP) 100 5 6 7 Piperacillin (PIP) 100 8 9 Amoxicillin/clavulanic acid (AMC) 100 10 11 Piperacillin/tazobactam (TZP) 95.2 12 13 Cephalotin (CEF) 97.6 14 15 Cefoxitin (FOX) 28.2 16 17 18 Cefuroxime (CXM) 94.1 19 20 Cefotaxime (CTX) 90.5 21 22 Ceftazidime (CAZ) 82.3 23 24 Aztreonam (ATM) 87 25 26 27 Cefepime (FEP) 42.5 28 29 Ertapenem (ERT) 95.3 30 31 Imipenem (IMP) 23.5 32 33 Kanamycin (K) 90.5 34 35 36 Gentamicin (G) 84.7 37 38 Tobramycin (T) 90.5 39 40 Amikacin (A) 8.3 41 42 Netilmicin (Nt) 20 43 44 45 Neomycin (Nm) 4.7 46 47 Nalidixic acid (NAL) 85.8 48 49 Ciprofloxacin (CIP) 83.5 50 51 Cotrimoxazole (SXT) 89.4 52 53 54 55 56 57 58 59 60 21 61 62 63 64 65 1 2 3 4 5 6 7 Table 2. Antimicrobial resistance genes present in the different MLST and PFGE profiles of the 85 OXA-48-producing K. pneumoniae 8 9 strains. 10 11 ST PFGE n β-lactamase genes AME Qnr 12 13 14 ST101 A1 6 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-1 (100%*) aac(3’)-IIa, aac(6’)-Ib (66.6%) qnrB66 15 16 aac(3’)-IIa, aac(6’)-Ib, aph(3’)-Ia (33.3%) qnrS (50%) 17 18 A2 9 blaCTX-M-15, blaOXA-1, blaSHV-1 (55.5%) aac(3’)-IIa, aac(6’)-Ib (66.6%) qnrB66 19 20 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-1 (44.4%) aac(3’)-IIa, aac(6’)-Ib, aac(2’)-Ia (11.1%) (45.5%) 21 22 23 aac(3’)-IIa, aac(6’)-Ib, ant(2’)-Ia, aac(2’)-Ia (11.1%) 24 25 aac(3’)-IIa, aac(6’)-Ib, aph(3’)-Ia (11.1%) 26 27 28 29 A3 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-1 aac(3’)-IIa, aac(6’)-Ib, aph(3’)-Ia qnrB1 30 31 32 ST17 B 1 blaSHV-11 - - 33 34 ST1233 C 1 blaSHV-42 - - 35 36 ST14 D 1 blaSHV-1 - - 37 38 ST405 E1 11 blaOXA-1, blaTEM-1, blaSHV-76 (9%) aac(3’)-IIa, aac(6’)-Ib (36.3%) qnrB66 39 40 41 blaOXA-1, blaSHV-76 (9%) aac(3’)-IIa (36.3%) (91%) 42 43 44 45 22 46 47 48 49 1 2 3 4 5 6 7 blaCTX-M-15, blaOXA-1, blaSHV-76 (9%) aac(6’)-Ib (18.1%) 8 9 blaCTX-M-15 , blaTEM-1 , blaSHV-76 (18%) 10 11 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 (55%) 12 13 14 E2 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 15 16 E3 1 blaOXA-1, blaSHV-76 aac(6’)-Ib qnrB66 17 18 E4 3 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 19 20 E5 30 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib (90%) qnrB66 21 22 23 aac(3’)-IIa (10%) (96%) 24 25 E6 2 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 (50%) aac(3’)-IIa, aac(6’)-Ib (50%) qnrB66 26 27 blaOXA-1, blaSHV-76 (50%) aac(6’)-Ib (50%) 28 29 E7 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 30 31 32 E8 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 33 34 E9 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 35 36 E10 1 blaOXA-1, blaTEM-1, blaSHV-76 aac(6’)-Ib qnrB66 37 38 E11 1 blaCTX-M-15, blaOXA-1, blaTEM-1,blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 39 40 E12 5 blaCTX-M-15, blaOXA-1, blaTEM-1,blaSHV-76 (100%) aac(3’)-IIa, aac(6’)-Ib qnrB66 41 42 43 44 45 23 46 47 48 49 1 2 3 4 5 6 7 E13 1 blaCTX-M-15, blaOXA-1, blaTEM-1,blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 8 9 E14 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 10 11 E15 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 12 13 14 E16 1 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 15 16 E17 1 blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 17 18 E18 3 blaCTX-M-15, blaOXA-1, blaTEM-1, blaSHV-76 aac(3’)-IIa, aac(6’)-Ib qnrB66 19 20 21 22 23 *The percentages represent the total of strains with a genetic endowment in each subcluster. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 24 46 47 48 49 Table 3. Clinical data of the 85 patients with OXA-48-producing K. pneumoniae. 1 n % 2 3 Origin of samples 4 5 Urinary tract 47 55.3 6 7 8 Respiratory tract 13 15.3 9 10 Blood 6 7 11 12 Surgical wound 4 5 13 14 Faeces 9 10.6 15 16 Others 6 7 17 18 19 Gender 20 21 Men / Women, 33/52 39/61 22 23 Average age 24 25 Age (range) 76 (26-96) years 26 27 28 Men/Women 72/79 years 29 30 Men/Women over 65 years old 20/46 31 32 Origin of Infection 33 34 Nosocomial 41 48.2 35 36 37 Residential healthcare centre 30 35.3 38 39 Community 14 16.4 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 25 61 62 63 64 65 Table 4. Plasmid typing (PBRT-PCR) in 37 OXA-48-producing K. pneumoniae 1 strains. 2 3 4 ST Plasmid replicons (%) Genetic platform Subtypes (n) 5 6 L, FIIK, ColE, R Tn1999.2 A1(1) 7 8 101 L, FIIK, ColE, R Tn1999.2 A2(1) 9 10 11 L, FIIK, ColE Tn1999.2 A3(1) 12 13 17 L Tn1999.2 B(1) 14 15 122 L, ColE Tn1999.2 C(1) 16 17 14 L Tn1999.2 D(1) 18 19 20 L, FIIK (100%) ColE (50%) Tn1999.2 E1(8) 21 22 L, FIIK Tn1999.2 E2(1) 23 24 L, FIIK Tn1999.2 E3(1) 25 26 L, FIIK Tn1999 E4(1) 27 28 29 L, FIIK (100%) ColE (50%) Tn1999.2 E5(4) 30 31 L, FIIK Tn1999 E6(1) 32 33 L, FIIK Tn1999.2 E7(1) 34 35 L, FIIK Tn1999.2 E8(1) 36 37 38 L, FIIK Tn1999.2 E9(1) 39 405 40 L, FIIK Tn1999.2 E10(1) 41 42 L, FIIK Tn1999 E11(1) 43 44 L, FIIK (100%) Tn1999 E12(2) 45 46 47 L, FIIK Tn1999.2 E13(1) 48 49 L, FIIK Tn1999.2 E14(1) 50 51 L, FIIK Tn1999.2 E15(1) 52 53 L, FIIK, FIA Tn1999 E16(1) 54 55 56 L, FIIK, ColE Tn1999.2 E17(1) 57 58 L, FIIK (100%) ColE (50%) Tn1999.2/ Tn1999 E18(2) 59 60 26 61 62 63 64 65 1 2 3 4 5 Figure 1. Distribution of the different PFGE clusters by hospital 6 7 8 9 10 Figure 2. Genetic backgrounds of the blaOXA-48 gene in our K. pneumoniae 11 12 carbapenemase-producing strains. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 27 61 62 63 64 65 Figura 1 Figure 1. Distribution of the different PFGE clusters by hospital 30 25 20 A B 15 C D 10 E 5 0 HGV HGH HM HGG HSJDD HSCSP HC HGC HC (Consorci de salut del Maresme), HGC (Hospital General Universitari de Catalunya), HGG (Hospital General de Granollers), HGH (Hospital General de l’Hospitalet), HGV (Consorci hospitalari de Vic), HM (Hospital de Mataró), HSCSP (Hospital de la Santa Creu i Sant Pau) i HSJDD (Hospital Sant Joan de Déu de Manresa). Figuras 2 Figure 2. Genetic backgrounds of the blaOXA-48 gene in our K. pneumoniae carbapenemase-producing strains. (Genbank Access Numbers of fragment sequenced: KT265173, KT265174, KT265175, KT265176, KT265177, KT265178, KT265179, KT265180, KT265181, KT265182 and KT265183).