Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil

ORIGINAL ARTICLE

Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil

Thiago Pavoni Gomes Chagas^I; Ronaldo Mendes Alves^II; Deyse Christina Vallim^III; Liliane Miyuki Seki^IV; Leila Carvalho Campos^V; Marise Dutra Asensi^VI

^IGraduated in Biological Sciences, Universidade do Estado do Rio de Janeiro (UERJ); MSc Student in Tropical Medicine, Instituto Oswaldo Cruz (IOC)/Fiocruz, Rio de Janeiro, RJ, Brazil

^IIGraduated in Biological Sciences; Technologist, IOC/FIOCRUZ, Rio de Janeiro, RJ, Brazil

^IIIPhD, Microbiology, Universidade Federal do Rio de Janeiro (UFRJ); Technologist, IOC/Fiocruz, Rio de Janeiro, RJ, Brazil

^IVMSc, Microbiology, Universidade Federal Rural do Rio de Janeiro (UFRRJ); Technician, IOC/Fiocruz, Rio de Janeiro, RJ, Brazil

^VPhD, Microbiology, Researcher, CPqGM/Fiocruz, Rio de Janeiro, RJ, Brazil

^VIPhD in Microbiology; Chief, Hospital Infection Research Laboratory, IOC/Fiocruz, Rio de Janeiro, RJ, Brazil

^{Correspondence to}

ABSTRACT

OBJECTIVE: The present study was undertaken to characterize CTX-M ESBL-producing Klebsiella pneumoniae collected from hospitals in different cities of Brazil.

MATERIAL AND METHODS: Eighty-five K. pneumoniae strains isolated from hospitalized patients in six different hospitals of three cities of Brazil were analyzed. ESBL production was confirmed by the standard double-disk synergy test and the Etest®. The MIC₅₀ and MIC₉₀ for ESBL-producing isolates were determined by the Etest^® method. The antimicrobial susceptibilities of bacterial isolates were determined using the agar diffusion method according to the CLSI. Screening for bla_TEM, bla_SHV, bla_CTX-M genes and class 1 integron was performed by PCR amplification. To determine the genomic diversity of CTX-M-producers, isolates were analyzed by macrorestriction profile analysis following PFGE.

RESULTS AND DISCUSSION: Seventy-one K. pneumoniae isolates were ESBL-producing. PCR and sequencing experiments detected 38 CTX-M-producing K. pneumoniae belonged to groups CTX-M 1, CTX-M 2, CTX-M 8 and CTX-M 9. The association of different types ESBL (CTX-M, SHV and TEM) was frequent. All K. pneumoniae isolates carried class 1 integron. PFGE analysis revealed thirty-one clonal types among CTX-M-producing isolates. The data presented herein illustrate the diversity of genotypes of CTX-M producing K. pneumoniae among Brazilians hospitals.

Keywords:Klebsiella pneumoniae; β-lactamases; genotype.

INTRODUCTION

Klebsiella pneumoniae is an opportunistic pathogen that has emerged as an important cause of hospital-acquired infections, especially in hospitalized immunocompromised patients with severe underlying diseases or admitted to neonatal intensive-care units.¹ The worldwide spread of plasmidencoded extended-spectrum β-lactamases (ESBLs)-producing Klebsiella strains which are resistant to the bactericidal activity of all cephalosporins is also considered a great threat.² Various types of ESBL have been described worldwide.³ In particular, CTX-M-type ESBLs have become the prevailing non-TEM, non-SHV ESBL among Enterobacteriaceae and is recognized as a rapidly growing family of ESBLs that preferentially hydrolyze cefotaxime rather than ceftazidime.^2,4,5

In recent studies, high rates of CTX-M enzymes among ESBL-producing K. pneumoniae isolates have been reported from South America, Asia and Europe.^5-8 In some of those settings, rates of CTX-M-production as high as 58.5% in K. pneumoniae have been reported,⁷ and South America appears as an important source of CTX-M type ESBL.^9,10

In the present study we describe the molecular characterization of CTX-M-producing K. pneumoniae clinical strains isolated from six different hospitals in three cities of Brazil.

MATERIAL AND METHODS

Bacterial isolates

We analyzed 85 non-replicate K. pneumoniae strains isolated from hospitalized patients in six different hospitals of three cities of Brazil (Niterói, Rio de Janeiro, and São Paulo), from May 2003 to September 2006. The isolates were recovered from urine (n = 25), blood (n = 25), pulmonary secretion (n = 5), catheter (n = 5), and other sites (n = 25). Preliminary identification of the isolates was accomplished using the Vitek^® (bioMérieux) automated system and established biochemical procedures.

Antimicrobial susceptibility testing

ESBL production was confirmed by the standard doubledisk synergy test and the Etest^® (ceftazidime/ceftazidime + clavulanic acid) (AB Biodisk, Solna, Sweden). The MIC50 (minimum concentration capable to inhibit 50% of the isolates) and MIC₉₀ (minimum concentration capable to inhibit 90% of the isolates) values of five antimicrobial agents (aztreonam, cefepime, ceftazidime, cefotaxime, and imipinem) for ESBL-producing isolates were determined by the Etest^® me715t5ttthod. All K. pneumoniae isolates were also tested against the following antimicrobial agents: gentamicin (CN); amikacin (AK); norfloxacin (NOR); ciprofloxacin (CIP); aztreonam (ATM); cefepime (FEP); imipinem (IPM); and trimethoprim-sulphametoxazole (SXT) by the disk diffusion method, and the results were interpreted based on the CLSI guidelines.¹¹ Quality control was carried out using standard strains of Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27953), Staphylococcus aureus (ATCC 27953), and Klebsiella pneumoniae (ATCC 700803).

Characterization of β-lactamase-encoding genes

Screening for resistance genes was performed by PCR amplification using previously reported conditions and primer sets for detection of bla_TEM, bla_SHV,bla_CTX-M and class 1 integron.¹⁴ PCR products were sequenced with the ABI PRISM Dye Terminator Cycle Sequecing Ready Reaction Kit on a 3730 DNA Sequence Analyzer (Applied Biosystem). Obtained sequences were aligned and compared with those in GenBank (http://www.ncbi.nlm.nih.gov/BLAST).

Pulsed-field gel electrophoresis (PFGE) typing

Clonal relationships were studied by pulsed field gel electrophoresis (PFGE) of SpeI-digested genomic DNA with a CHEF DRII apparatus (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom).¹⁵ Band patterns were compared visually and interpreted according to the criteria established by Tenover et al.,¹⁶ and analyzed with BioNumerics v.4.0 software (Applied Maths, Sint-Martins-Latem, Belgium). Isolate clustering were performed by the unweighted pair group method using arithmetic averages (UPGMA) in combination with Dice similarity coefficient.

RESULTS

Of 85 K. pneumoniae isolates, 71 (84%) were ESBL-producing bacteria as determined by the doubledisk synergy test and the Etest^®. In 71 ESBL-producing isolates, the MIC50/MIC90 values (µg/mL) of aztreonam, cefepime, ceftazidime, cefotaxime and imipinem were 16/256, 6/16, 32/256, 32/256 and 0.125/0.19, respectively. In this study, a multiresistant pattern was observed in ESBL-producing K. pneumoniae isolates and we detected the co-resistance of gentamicin (70%), amikacin (36%), norfloxacin (46%), ciprofloxacin (49%), aztreonam (68%), cefepime (41%), and trimethoprim-sulphametoxazole (71%).

Sixty-five (92%) of the isolates were positive for blaTEM and 32 (45%) were positive for blaSHV. All ESBL-producing isolates had class 1 integron. The genetic analysis of these isolates by PCR revealed that 38 of 71 (54%), from four different hospitals (Niterói and Rio de Janeiro cities), were positive for the CTX-M gene. The remaining 33 isolates (46%) were negative for this gene and these CTX-M gene-negative isolates showed other ESBL type (SHV and TEM) (Table 1). The association of different types of ESBL was frequent.

Considering the CTX-M-producers, 53% of these co-produced TEM and SHV, and 47% co-produced only TEM. It was possible to identify 4 CTX-M clusters. The genotyping of 38 CTX-M gene-positive isolates showed that 1 (3%), 23 (61%), 7 (18%) and 4 (11%) isolates belonged to groups CTX-M 1, CTX-M 2, CTX-M 8 and CTX-M 9, respectively. The remaining three isolates were not classified into these groups.

The PFGE analysis of CTX-M-producing K. pneumoniae isolates showed 31 clonal types, A - EE, considering the genetic relatedness strain (defined as Dice coefficients of < 85%) (Figure 1). Based on these data, a diversity of clones within the hospitals was found. However, two hospitals (HU1 ad HU5) presented the same clonal group of CTX-M-2, characterized as genotype O. All CTX-M-producers genotypes were characterized as multidrug-resistant (Table 2).

DISCUSSION

The prevalence of K. pneumoniae producing ESBL varies between different countries and regions. The clinical relevance of multidrug resistance among ESBL-producing Klebsiella spp. is of great concern due to the limited therapeutic options and increased risk of treatment failure in patients infected with such strains.¹⁷ In Brazil, there has been some reports of multi-drug resistant hospital-acquired K. Pneumoniae.^18,19 In our study, 71 (84%) were ESBL positive by the double-disk synergy test and the Etest^® and this value is considered high. This highlights the importance of investigating resistance mechanisms in hospitals.

Among the antimicrobials evaluated for minimum inhibitory concentration, the carbapenemics presented the best potency (MIC50), with imipenem (0.125 µg/mL). Carbapenems such as imipenem and meropenem are recommended as therapy of choice for severe infections caused by CTX-M- and other types ESBL-producing bacteria. Most CTX-M positive isolates exhibited resistance to non-β-lactam antibiotics displaying co-resistance for gentamicin, amikacin, norfloxacin, ciprofloxacin, aztreonam, cefepime, and trimethoprim-sulphametoxazole.

In clinical strains, CTX-M-coding genes have been commonly located on plasmids and these plasmids can also carry genes for resistance to multiple other antibiotics, including aminoglycosides, chloramphenicol, sulfonamide, trimethoprim, and tetracycline. Resistance of ESBL-producing isolates to other classes of antimicrobial agents shown in this report has been confirmed by other studies.^5,20 Among Enterobacteriaceae, these resistance pattern was commonly associated with a few types of integrons.

In this study, all isolates that carried the gene bla_CTX-M also contained class 1 integrons. Insertion sequences (IS) might be involved in the mobilization of blaCTX-M genes.²¹ The bla_CTX-M genes have also been associated with ISCR1, which is often found downstream of complex class 1 integrons.²²

CTX-M-2, CTX-M-8 and CTX-M-9 groups were the most frequently detected CTX-M-type enzymes among ESBL-producing Enterobacteriaceae isolates from South American countries.^4,9,23 In the present study, CTX-M 2 (61%) was the predominant cluster. CTX-M-2 was first characterized and appears to be dominant in Argentina.²⁰ Studies carried out in Europe have shown that the occurrence of the blaCTX-M-2 group is rare. Recent studies in Southeast Brazil have demonstrated the presence of bla_CTX-M-2 gene in K. Pneumoniae.^24,25 Dissemination of CTX-M-9 and CTX-M-8 clusters has also been reported in Brazil.^4,26,27

Many of the CTX-M positive isolates harbored other β-lactam resistance enzymes and the association of types TEM, SHV and CTX-M (n = 20) was more frequent. Previous studies have shown that ESBL mediating plasmids may carry more than one β-lactamase gene and that they may be responsible for high-level β-lactamase resistance phenotypes.²⁸ In our study, the resulting PFGE gel of CTX-M-producing K. pneumoniae isolates showed genotypic diversity. However, two hospitals presented the same clonal group, characterized as genotype O, CTX-M-2 producers, suggesting intrahospital dissemination.

The high levels of CTX-M ESBL detected are worrisome and warrant special attention by both the clinician and the microbiology laboratory. While the former has to re-evaluate the antibiotic policies, the laboratory must be capable to readily identify these isolates. Given the degree to which these CTX-M-producing microorganisms have spread, they should be seen as a city public health issue instead of a problem of each hospital. Widespread use of antimicrobial therapy has often been held responsible for the occurrence of multiresistant Klebsiel la strains in hospitals.²⁹ Our data corroborate the importance of antibiotic use restriction and implementation of preventive measures.

CONCLUSION

In conclusion, CTX-M enzymes have emerged in our hospitals. The intensive use of broad-spectrum cephalosporins such as cefotaxime could account for the emergence of the CTX-M plasmid-mediated enzymes among pathogens. Several groups of bla genes were detected in clinical samples in the studied hospitals, but the bla_{CTX-M 2} was the predominant. The data presented herein illustrate the diversity of genotypes of CTX-M producing K. pneumoniae among hospitals and the intrahospital dissemination of these genotypes was uncommon. Thus, the high prevalence of CTX-M K. pneumoniae in our hospitals is probably not a consequence of the transmission of a common strain between patients. Diversity of genotypes was also described in other studies suggested that the increase of ESBL K. pneumoniae was mainly due to horizontal dissemination of gene transfer between isolates.³⁰ In contrast, it is important to note that currently there is a worldwide spread of ESBL and KPC-producing K. pneumoniae, which seems to occur due to the dissemination of specific clones ST258, ST11 and ST437 in Brazil.^31,32

ACKNOWLEDGEMENTS

We thank all hospitals who collected isolates for the study and PDTIS-IOC platform for DNA sequencing.

Submitted on: 02/07/2011

Approved on: 02/18/2011

We declare no conflict of interest

Financial Support: FAPERJ CNPq

• 1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11(4):589-603.
• 2. Paterson DL, Bonomo, RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev 2005;18(4):657-86.
• 3. Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14(4):933-51.
• 4. Cantón R, Coque TM. The CTX-M β-lactamase pandemic. Curr Opinion in Microbiol 2006;9(5):466-75.
• 5. Bonnet R. Growing group of extended-spectrum β-lactamase: the CTX-M enzymes. Antimicrob Agents Chemother 2004;48(1):1-14.
• 6. Quinteros M, Radice M, Gardella N et al. Extended-Spectrum β-Lactamases in Enterobacteriaceae in Buenos Aires, Argentina, Public Hospitals. Antimicrob Agents Chemother 2003;47(9):2864-67.
• 7. Yan JJ, Hsueh PR, Lu JJ et al. Extended-Spectrum β-Lactamases and Plasmid-Mediated AmpC Enzymes among Clinical Isolates of Escherichia coli and Klebsiella pneumoniae from Seven Medical Centers in Taiwan. Antimicrob Agents Chemother 2006;50(5):1861-64.
• 8. Livermore DM, Hawkey PM. CTX-M: changing the face of ESBLs in the UK. J Antimicrob Chemother 2005;56(3):451-54.
• 9. Villegas MV, Kattan JN, Quinteros MG et al. Prevalence of extended-spectrum β-lactamases in South America. Clin Microbiol Infect 2008;14:154-8.
• 10. Rossolini GM, D'Andrea MM, Mugnaioli C. The spread of CTX-M-type extended-spectrum β-lactamases. Clin Microbiol Infect 2008;14:33-41.
• ¹¹
  CLSI. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. CLSI/NCCLS M100-S19. Wayne, PA:CLSI; 2009.
• 12. Hasman H, Mevius D, Veldman K et al. β-lactamases among extended-spectrum β-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands. J Antimicrob Chemother 2005;56(1):115-21.
• 13. Mulvey MR, Soule G, Boyd D et al. The Multi-Provincial Salmonella Typhimurium Case Control Study Group. Characterization of the first extended-spectrum β-lactamaseproducing Salmonella isolate identified in Canada. J Clin Microbiol 2003;41(1):460-462.
• 14. Sandvang D, Aarestrup FM, Jensen LB. Characterization of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhmurium DT104. FEMS Microbiol Lett 1997;157:177-181.
• 15. Seifert H, Gerner-Smidt P. Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Aci netobacter isolates. J Clin Microbiol 1995;33(5):1402-07.
• 16.Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33(9):2233-9.
• 17. Hyle EP, Lipworth AD, Zaoutis TE et al. Risk Factors for Increasing Multidrug Resistance among Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella Species. Clin Infect Dis 2005;40(9):1317-24.
• 18. Mendes C, Kiffer C, Segura A et al. Klebsiella pneumoniae with multiple antimicrobial resistance. Braz J Infect Dis 2004;8(1):109-11.
• 19. Lincopan N, Mcculloch JA, Reinert C et al. First isolation of metallo-β-lactamase-producing multiresistant Klebsiella pneumoniae from a patient in Brazil. J Clin Microbiol 2005;43(1):516-9.
• 20. Valverde A, Coque TM, Sanchez-Moreno MP et al. Dramatic increase in prevalence of fecal carriage of extendedspectrum β-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain. J Clin Microbiol 2004;42(10):4769-75.
• 21. Bonnet R, Sampaio JLM, Labia R et al. A novel CTX-M β-lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil. Antimicrob Agents Chemother 2000;44(7):1936-42.
• 22. Partridge SR, Hall RM. In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob Agents Chemother 2003;47(1):342-49.
• 23. Bauernfeind A, Casellas JM, Goldberg M et al. A new plasmidic cefotaximase from patients infected with Salmonella typhimurium Infection 1992;20(3):158-63.
• 24. Do Carmo FJR, Silva RM, Castanheira M et al. Prevalence and genetic characterization of blaCTX-M among Klebsiella pneumoniae isolates collected in an intesive care unit in Brazil. J Chemother 2008;20(5):600-3.
• 25. Garcia DO, Doi Y, Szabo D et al. Multiclonal outbreak of Kleb siella pneumoniae producing extended-spectrum β-lactamase CTX-M-2 and novel variant CTX-M-59 in a neonatal intensive care unit in Brazil. Antimicrob Agents Chemother 2008;52(5):1790-3.
• 26. Clímaco EC, Minarini LA, Da Costa Darini AL. CTX-M-producing Klebsiella spp. in a Brazilian hospital: what has changed in 6 years? Diagn Microbiol Infect Dis 2010;68(2):186-9.
• 27. Minarini LA, Poirel L, Trevisani NA et al. Predominance of CTX-M-type extended-spectrum β-lactamase genes among enterobacterial isolates from outpatients in Brazil. Diagn Microbiol Infect Dis 2009;65(2):202-6.
• 28. Kiratisin P, Apisarnthanarak A, Laesripa C et al. Molecular characterization and epidemiology of extended spectrum-βlactamase-producing Escherichia coli and Klebsiella pneumo niae isolates causing health care-associated infection in Thailand, where the CTX-M family is endemic. Antimicrob Agents Chemother 2008;52(8):2818-24.
• 29. Tullus K, Berglund B, Fryklund B et al. Epidemiology of fecal strains of the family Enterobacteriaceae in 22 neonatal wards and influence of antibiotic policy. J Clin Microbiol 1998;26(6):1166-70.
• 30. Weller TM, MacKenzie FM, Forbes, KJ. Molecular epidemiology of a large outbreak of multiresistant Klebsiella pneumoniae J Med Microbiol 1997;46(11):921-6.
• 31. Cuzon G, Naas T, Truong H et al. Worldwide diversity of Kleb siella pneumoniae that produce β-lactamase blaKPC-2 gene. Emerg Infect Dis 2010;16(9):1349-56.
• 32. Pereira, PS, Seki LM, Figueira et al. Epidemiologia molecular de cepas de K. pneumoniae produtoras de KPC-2 do Rio de Janeiro: disseminação do ST 437 (abstract MH-003). In: Abstracts: II Simpósio Internacional de Microbiologia Clínica (Florianópolis) Florianópolis: SBM, 2010.

Publication Dates

• Publication in this collection
  17 Oct 2011
• Date of issue
  Oct 2011

History

• Received
  02 July 2011
• Accepted
  18 Feb 2011