Acquisition of carbapenem resistance in multiresistant Klebsiella pneumoniae strains harbouring bla CTX-M-15, qnrs1 and aac(69)-ib-cr genes

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1 Journal of Medical Microbiology (2012), 61, DOI /jmm Acquisition of carbapenem resistance in multiresistant Klebsiella pneumoniae strains harbouring bla CTX-M-15, qnrs1 and aac(69)-ib-cr genes Elena Ruiz, 1 Alain A. Ocampo-Sosa, 2 Antonio Rezusta, 3 María José Revillo, 3 Elena Román, 2 Carmen Torres 1 and Luis Martínez-Martínez 2,4 Correspondence Carmen Torres carmen.torres@unirioja.es 1 Área Bioquímica y Biología Molecular, Univ. La Rioja, Logroño, Spain 2 Service of Microbiology, Hosp. Univ. Marqués de Valdecilla-IFIMAV, Santander, Spain 3 Hosp. Univ. Miguel Servet, Zaragoza, Spain 4 Department of Molecular Biology, University of Cantabria, Santander, Spain Received 10 September 2011 Accepted 26 January 2012 Three closely related Klebsiella pneumoniae strains isolated from the same patient harboured bla CTX-M-15, bla OXA-1, bla SHV-11, qnrs1, aac(69)-ib-cr, oqxab, aac(3)-ii and aph(39)-ia genes. Two of the isolates were recovered after treatment with meropenem and showed resistance to carbapenems. Sequencing of ompk35 and ompk36 porin genes of the carbapenem-resistant strains revealed the presence of premature stop codons in both, and OmpK35 and OmpK36 porins were not detected by SDS-PAGE. One carbepenem-resistant strain showed a high amount of LamB protein and did not express OmpK26 porin whereas the other strain expressed OmpK26 but not LamB. The lack of major porins apparently causes changes in the expression of other, specific, porins. INTRODUCTION Treatment of nosocomial infections due to multidrugresistant Gram-negative bacteria is becoming a complex clinical challenge. In enterobacteria, one of the most prevalent resistance mechanisms is extended-spectrum b- lactamases (ESBLs), especially CTX-M enzymes, which are frequently associated with quinolone, aminoglycoside, tetracycline and/or sulfonamide resistance mechanisms. Carbapenems remain one of the main therapeutic options to treat severe infections caused by those resistant pathogens. Nevertheless, carbapenem resistance can be acquired through different mechanisms. Firstly, metallo-b-lactamases such as VIM, IMP or NDM or other types of carbapenemases of the IMI/NMC, SME, OXA or KPC families can be acquired (Doumith et al., 2009). Secondly, production of b-lactamase with (very) weak hydrolytic activity against carbapenems (such as AmpC-type enzymes or even some ESBLs) associated with a decreased permeability of the outer membrane as a result of the loss of porins can also cause resistance to carbapenems (Doumith et al., 2009; Martínez-Martínez et al., 2000). Porin loss may also slightly affect susceptibility to fluoroquinolones, Abbreviations: ESBL, extended-spectrum b-lactamase; OMP, outermembrane protein. chloramphenicol and tetracycline when other mechanisms of resistance are present (Martínez-Martínez et al., 2002). In Klebsiella pneumoniae, the complete loss of the two major porins, OmpK35 and OmpK36, is usually required to cause clinically relevant carbapenem resistance. Loss of OmpK36 alone has also been reported to cause intermediate resistance to ertapenem and decreased susceptibility to meropenem (Findlay et al., 2012). Loss of OmpK36 can be selected by meropenem in strains overexpressing the rama transcriptional activator causing inhibition of ompk35 (Findlay et al., 2012). The role of the minor porin OmpK37, and of other outer-membrane proteins (OMPs), including the specific PhoE and LamB porins and the monomeric oligogalacturonate-specific OmpK26 porin (García-Sureda et al., 2011a, b; Kaczmarek et al., 2006), has also been reported. The aim of this work was to characterize the mechanisms of resistance to carbapenems acquired by a K. pneumoniae strain recovered from a patient treated with carbapenems. METHODS Bacterial strains and antibiotic susceptibility testing. Three K. pneumoniae strains were recovered from the same patient (a preterm female newborn) admitted to the Hospital Universitario Miguel G 2012 SGM Printed in Great Britain

2 Carbapenem-resistant K. pneumoniae Servet (Zaragoza, Spain). The first isolate (C2613) was recovered from a pharyngeal exudate obtained when the patient was 4 months and 2 days old. The other two isolates (C2615 and C2616) were recovered 14 days later from a rectal swab from the patient, who was treated for 10 days with meropenem (100 mg every 8 h). The patient also received other antimicrobials (amoxicillin clavulanic acid, ceftazidime, cefotaxime, amikacin and vancomycin). Susceptibility testing for a set of antimicrobial agents was determined by disc diffusion and agar dilution methods. In addition, susceptibility to cefotaxime, cefotaxime+clavulanic acid, ceftazidime, ceftazidime+clavulanic acid, cefepime, cefepime+clavulanic acid, imipenem and meropenem was determined by agar dilution in the presence and absence of 250 mg cloxacillin l 21 (Sigma). The breakpoints recommended by EUCAST ( were used, except for nalidixic acid, cefotetan, tetracycline, fosfomycin and sulfamides, for which Clinical and Laboratory Standards Institute criteria were used (CLSI, 2011). The modified Hodge test, according to the CLSI, was performed using meropenem, imipenem, cefoxitin and cefotetan discs. Molecular typing and phylogenetic characterization. PFGE analysis of XbaI-digested genomic DNA was performed. The phylogenetic group of isolates was determined by amplification of the gyra gene and consequently digestion with TaqI andhaeiii restriction enzymes (Ruiz et al., 2010). MLST was carried out according to the strategies of the Institut Pasteur scheme for K. pneumoniae ( Kpneumoniae.html). Characterization of antimicrobial resistance genes. The following types of b-lactamases were studied by PCR and sequencing: (1) CTX-M, OXA-1, SHV and TEM b-lactamases (Ruiz et al., 2010); (2) metallo-b-lactamases and carbapenemases of class A (Ellington et al., 2007; Hong et al., 2010); and (3) plasmid-encoded AmpC b- lactamases (Pérez-Pérez & Hanson, 2002). The presence of inti1 and inti2 genes, which encode the integrases of class 1 and class 2 integrons, respectively, was determined by PCR (Ruiz et al., 2010). Several genes conferring resistance to quinolones and/or aminoglycosides were tested for: qnra, qnrb, qnrs, aac(69)-ib-cr, qepa, oqxab, aac(3)-i, aac(3)-ii, aac(3)-iii, aac(3)-iv, aph(39)-ia, aph(39)-ii and ant(299). Mutations in gyra and parc genes were analysed by amplification and sequencing of the quinolone resistance-determining region (Ruiz et al., 2010). The genetic environment of b-lactamase and quinolone resistance genes was studied by PCR mapping based on genetic environment regions previously described (Ruiz et al., 2010). Analysis of OMPs. Mutations in ompk35, ompk36 and ompk37 porin genes were analysed by PCR and sequencing (Lee et al., 2007; Doménech-Sánchez et al., 2003). OMPs were obtained from the three strains from the patient (C2613, C2615 and C2616) and also from three K. pneumoniae control strains [CSUB10R, which lacks OmpK35 and OmpK36 (Ardanuy et al., 1998), a transformant of CSUB10R expressing OmpK35 and a transformant of CSUB10S (a CSUB10R-related isolate expressing OmpK36) producing OmpK35, thus resulting in a strain producing both OmpK35 and OmpK36) (Doménech-Sánchez et al., 2003)]. Bacteria were grown in cation adjusted Mueller Hinton broth, Nutrient broth or Nutrient broth supplemented with 20 % sorbitol. Cell envelopes from sonicated cells were obtained by centrifugation ( g, 45 min, 4 uc). OMPs were prepared after treatment of cell envelopes with sodium lauryl sarcosinate and visualized by SDS- PAGE as previously described (Martínez-Martínez et al., 2000), using gels with different concentrations of acrylamide (10 %, 11 %, 12 %, 4 20 %). Plasmid characterization. The plasmids of strains were classified according to their incompatibility group using the PCR-based replicon-typing method (Carattoli et al., 2005). Moreover, a PFGE assay was carried out with the total DNA of all the strains digested with S1 nuclease in order to analyse the number and size of the plasmids they contained. S1-PFGE fragments were then transferred onto membranes by Southern blotting and hybridized with specific probes for the bla CTX-M-15, qnrs1 and aac(69)-ib-cr resistance genes (Ruiz et al., 2010). Hybridization was performed with the DIG-High Prime DNA Labelling and Detection Starter kit I (Roche Applied Science). RESULTS AND DISCUSSION The three K. pneumoniae isolates showed a similar resistance phenotype including b-lactams, fluoroquinolones and aminoglycosides, although only the two isolates obtained after meropenem treatment (C2615 and C2616) also exhibited resistance to carbapenems and cefoxitin, a phenotype that in K. pneumoniae is compatible with porin loss (Ardanuy et al., 1998; Doménech-Sánchez et al., 2003) (Table 1). No significant differences were observed in the MICs of the b-lactams tested in the presence and absence of cloxacillin, and negative results were obtained in the modified Hodge test with either carbapenems or cephamycins (cefoxitin and cefotetan). The three isolates presented an indistinguishable or closely related PFGE pattern and belonged to the KpI phylogenetic group. K. pneumoniae C2613 and C2616 were typed by MLST as a new sequence type registered as ST433. This new sequence type is a single locus variant of the recently reported ST341 found in a K. pneumoniae strain which harboured bla CTX-M-15, qnrs1 and aac(69)-ib-cr resistance genes (Ruiz et al., 2010). Antibiotic resistance genes harboured by the K. pneumoniae strains tested here are shown in Table 1. The gyra and parc genes carried by these strains were all wild-type. In addition, they showed the same genetic environment of bla CTX-M-15, aac(69)-ib-cr and qnrs1 genes: the ISEcpI insertion sequence was found upstream of bla CTX-M-15 and IS26 was upstream of qnrs1 truncating the ISEcl2 insertion sequence. Two structures were detected surrounding the aac(69)-ib-cr gene: aac(3)-ii+is26+aac(69)-ib-cr+ bla OXA-1 ;andaac(3)-ii+is26+dcatb3+bla OXA-1 +aac(69)- Ib-cr. The genetic environments of the aac(69)-ib-cr and qnrs1 genes were the same as those recently described in K. pneumoniae strains belonging to the ST341 mentioned above (Ruiz et al., 2010). The three strains from the patient harboured six detectable plasmids, in which IncR and ColE replicon types were identified. A plasmid of 33.5 kb hybridized with specific probes of IncR, bla CTX-M-15, aac(69)-ib-cr and qnrs1. K. pneumoniae C2613 showed a wild-type ompk35 gene and two mutations at the ompk36 gene, while both K. pneumoniae C2615 and C2616 showed the same sequence of ompk35 and ompk36 genes as strain C2613 but with stop codons in both genes. The sequence of the ompk37 gene of the three K. pneumoniae strains was identical except 673

3 674 Journal of Medical Microbiology 61 Table 1. Resistance phenotype and genotype of K. pneumoniae strains NAL, Nalidixic acid; CIP, ciprofloxacin; LEV, levofloxacin; NOR, norfloxacin; TOB, tobramycin; AMK, amikacin; KAN, kanamycin; GEN, gentamicin; AMP, ampicillin; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; CTT, cefotetan; ETP, ertapenem; IMP, imipenem; MER, meropenem; DOR, doripenem; AMC, amoxicillin clavulanic acid; AZT, aztreonam; SUL, sulfamethoxazole; FF, fosfomicin. The breakpoints of the CLSI were used for NAL, CTT, FF and SUL because EUCAST does not include them. Strain* Sample origin MIC (mg l 1 ) Resistance phenotype C2613 Pharyngeal exudate NAL CIP LEV NOR TOB AMK KAN GEN AMP CTX CAZ FOX CTT ETP IMP MER DOR CIP, NOR, TOB, KAN, GEN, AMP, CTX, AMCD, CAZ, AZT, FFD C2615 Rectal swab CIP, NOR, TOB, KAN, GEN, AMKD, AMP, CTX, AMC, FOX, CAZ, AZT, ETP, IMPD, MERD, DOR, SULD, FF C2616 Rectal swab CIP, NOR, GEN, TOB, AMK, KAN, AMP, CTX, AMC, FOX, CAZ, AZT, ETP, IMPD, MERD, DOR *PFGE patterns: A1 (C2613 and C2615); A2 (C2616). DIntermediate resistance. E. Ruiz and others

Carbapenem-resistant K. pneumoniae Table 2. Analysis of OmpK35, OmpK36 and OmpK37 porin sequences of K. pneumoniae strains GenBank accession numbers for the porin sequences for the K.

Strain Porins Mutations in genes (amino acid change) Amino acid changes in protein ompk35* ompk36* OmpK37 C2613 Wild GCT219ATCG, AAC220ACAC (A219S, N220H) Insertions: TERY after H237 and SSTNGG after

4 Carbapenem-resistant K. pneumoniae Table 2. Analysis of OmpK35, OmpK36 and OmpK37 porin sequences of K. pneumoniae strains GenBank accession numbers for the porin sequences for the K. pneumoniae strains used as reference for OmpK35, OmpK36 and OmpK37 were AJ011501, Z33506 and AJ011502, respectively. Strain Porins Mutations in genes (amino acid change) Amino acid changes in protein ompk35* ompk36* OmpK37 C2613 Wild GCT219ATCG, AAC220ACAC (A219S, N220H) Insertions: TERY after H237 and SSTNGG after T275 Mutations: I70M, I128M, N230G, M233Q, T234H, Q235Y, N237H, R239K, E244D, N274S, D275T, V277I C2615 CAG174ATAG (Q174stop) AAA103ATAA (K103stop) Same sequence as in strain C2613 with additional Y311H mutation C2616 CAG174ATAG (Q174stop) AAA103ATAA (K103stop) Same sequence as in strain C2613 with additional Y311H mutation *K. pneumoniae C2615 and C2616 showed the same sequence (including mutations) as in K. pneumoniae C2613 in addition to stop codons. for the presence of the mutation Y311H in both K. pneumoniae C2615 and C2616 (Table 2). The optimal resolution of OMPs was obtained on resolving SDS-PAGE gels containing 11 % or 12 % (Fig. 1) of acrylamide. K. pneumoniae C2613 consistently expressed OmpA and one single major porin, independently of whether the organism was grown in high-osmolarity media, such as Mueller Hinton or Nutrient broth supplemented with sorbitol, or in the low-osmolarity medium Nutrient broth. This expression pattern indicates that the observed protein is OmpK36, in agreement with previous reports indicating that this porin is both not regulated by differences in osmolarity (Hernández-Allés et al., 1999), and produced by most ESBL-producing isolates (Martínez- Martínez et al., 2002). The absence of OmpK35 in this isolate may be the consequence of transcriptional or posttranscriptional regulation (Findlay et al., 2012). In any case, the relevant fact is that C2613 produces a porin in its outer membrane: this allows efficient carbapenem penetration into the organism and explains its susceptibility to carbapenems (Doménech-Sánchez et al., 2003). However, isolates C2615 and C2616 lacked both OmpK35 and OmpK36, a situation directly related to carbapenem resistance, as previously observed in several studies (Doumith et al., 2009; Kaczmarek et al., 2006; Lee et al., 2007; Elliott et al., 2006). Some differences were also observed among the carbapenem-susceptible isolate and the resistant strains in the expression of other OMPs (Fig. 1). The three isolates in this study produced LamB, not only in Mueller Hinton broth (containing starch, a maltose polymer), but also in the maltose-deficient nutrient broth. This protein was overproduced in the carbapenem-resistant isolate C2615. It has been recently reported (García-Sureda et al., 2011a) that this overexpression may compensate for the loss of the major porins OmpK35 and OmpK36, as in our case * + kda Fig. 1. SDS-PAGE analysis of OMPs from K. pneumoniae C2613 (lane 2), C2615 (lane 3) and C2616 (lane 4) strains obtained after growing in Nutrient broth. Molecular mass marker (lanes 1 and 8) and K. pneumoniae control strains harbouring OmpK35 (lane 5), OmpK35 and OmpK36 (lane 6) and neither OmpK35 nor OmpK36 (lane 7) are also shown. The positions of OmpK35 (black arrowhead) and OmpK36 (white arrowhead) are indicated to the right of the lane 6, and positions of LamB and OmpK26 are indicated with an asterisk and a cross, respectively

5 E. Ruiz and others Interestingly, in the other carbapenem-resistant isolate, C2616, loss of both OmpK35 and OmpK36 was associated with overexpression of OmpK26, a small monomeric porin allowing permeation of oligogalacturonate and homologous to NanC (from Escherichia coli) and KdgM (from Dickeya dadantii) (García-Sureda et al., 2011b). OmpK26 can also compensate for loss of the major porins in K. pneumoniae (García-Sureda et al., 2011b). Interestingly, the level of carbapenem susceptibility in the two isolates expressing higher amounts of either LamB or OmpK26 was similar (Table 1), in support of the central role of the loss of major porins in this resistance phenotype. Additional studies on the regulatory mechanisms of OMP production in the in vivo generated carbapenem-resistant C2615 and C2616 will provide new clues for our understanding of the role of porins and other OMPs in the resistance of K. pneumoniae to carbapenems and other drugs. Some recent studies reported in vivo development of carbapenem resistance in CTX-M-producing K. pneumoniae and E. coli strains in Spain (Elliott et al., 2006). In our study, we detected that the acquisition of ertapenem resistance was related to the association of the production of CTX-M-15 enzyme with the loss of OmpK35 and OmpK36 porins in a carbapenemase-negative K. pneumoniae strain. The loss of these porins was previously reported after a 6 day treatment with ertapenem, although porins were apparently restored when ertapenem was replaced with imipenem (Elliott et al., 2006). Also, it has been recently observed that meropenem therapy resulted in in vivo development of carbapenem resistance in a K. pneumoniae strain producing CTX-M-15 and lacking OmpK35 after additional loss of OmpK36 (Findlay et al., 2012). In our study, we have observed the loss of porins after 10 days of treatment with meropenem, which seems to exert a selective pressure that favours the acquisition of carbapenem resistance. A previous report also indicated that porin loss has more relevance for increased resistance to meropenem than to imipenem (Martínez-Martínez et al., 2000). In conclusion, our K. pneumoniae strains acquired resistance to ertapenem as a result of the loss of the major porins, and presumably because of the additional production of CTX-M-15; they also presented qnrs1, aac(69)-ib-cr and oqxab genes conferring resistance to quinolones as well as aac(3)-ii and aph(39)-ia genes conferring resistance to aminoglycosides. Since carbapenems are frequently the unique therapeutic options available for treatment of multiresistant members of the Enterobacteriaceae, the combination of such a number of resistance mechanisms, involving the widespread CTX-M-15 enzyme, poses a serious challenge in the selection of a suitable treatment. ACKNOWLEDGEMENTS This work was supported by the Gobierno de la Rioja of Spain, which awarded E. R. a predoctoral fellowship, and by the Ministerio de Ciencia e Innovación of Spain (Project SAF ). A. A. O.-S. and L. M.-M. are supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008). This work was presented at the 21st European Congress of Clinical Microbiology and Infectious Diseases, Milan, Italy, May 2011, abstract 594. REFERENCES Ardanuy, C., Liñares, J., Domínguez, M. A., Hernández-Allés, S., Benedí, V. J. & Martínez-Martínez, L. (1998). 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6 Carbapenem-resistant K. pneumoniae Lee, C. H., Chu, C., Liu, J. W., Chen, Y. S., Chiu, C. J. & Su, L. H. (2007). Collateral damage of flomoxef therapy: in vivo development of porin deficiency and acquisition of bla DHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 b-lactamases. J Antimicrob Chemother 60, Martínez-Martínez, L., Conejo, M. C., Pascual, A., Hernández-Allés, S., Ballesta, S., Ramírez De Arellano-Ramos, E., Benedí, V. J.& Perea, E. J. (2000). Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal b-lactamase and showing altered porin profiles. Antimicrob Agents Chemother 44, Martínez-Martínez, L., Pascual, A., Conejo, M. C., García, I., Joyanes, P., Doménech-Sánchez, A. & Benedí, V. J. (2002). Energy-dependent accumulation of norfloxacin and porin expression in clinical isolates of Klebsiella pneumoniae and relationship to extended-spectrum b-lactamase production. Antimicrob Agents Chemother 46, Pérez-Pérez, F. J. & Hanson, N. D. (2002). Detection of plasmidmediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40, Ruiz, E., Rojo-Bezares, B., Sáenz, Y., Olarte, I., Esteban, I., Rocha- Gracia, R., Zarazaga, M. & Torres, C. (2010). Outbreak caused by a multi-resistant Klebsiella pneumoniae strain of new sequence type ST341 carrying new genetic environments of aac(69)-ib-cr and qnrs1 genes in a neonatal intensive care unit in Spain. Int J Med Microbiol 300,

SUMMARY. Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M,

SUMMARY. Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M, SUMMARY Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M, The doctoral thesis entitled Prevalence of Enterobacteriaceae producing extendedspectrum beta-lactamases (ESBL) isolated from broilers