Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil

Genes de multirresistência, incluindo blaKPC e blaCTX-M-2, em Klebsiella pneumoniae isoladas em Recife, Brasil

MAJOR ARTICLE

Multidrug resistance genes, including bla_KPC and bla_CTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil

Genes de multirresistência, incluindo bla_KPC e bla_CTX-M-2, em Klebsiella pneumoniae isoladas em Recife, Brasil

Adriane Borges Cabral; Rita de Cássia de Andrade Melo; Maria Amélia Vieira Maciel; Ana Catarina Souza Lopes

Departamento de Medicina Tropical, Universidade Federal de Pernambuco, Recife, PE

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ABSTRACT

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care.

METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (bla_TEM, bla_SHV,bla_CTX-Mbla_KPC, bla_VIM, bla_IMP, and bla_SPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction).

RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented bla_SHV, 62.5% presented bla_CTX-M-2, 29% bla_TEM, and 41.7% bla_KPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried bla_SHVbla_TEM, bla_CTX-M-2 and bla_KPC simultaneously.

CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.

Keywords:Klebsiella pneumoniae. bla_KPC. bla_CTX-M-2. ERIC-PCR.

RESUMO

INTRODUÇÃO: A prevalência de cepas de Klebsiella pneumoniae resistentes a cefalosporinas e carbapenêmicos está aumentando no Brasil, com sérias consequências em termos de desfechos dos pacientes e cuidados gerais.

MÉTODOS: Este estudo caracterizou 24 isolados clínicos de K. pneumoniae provenientes de dois hospitais de Recife, Brasil, através do perfil de susceptibilidade a antimicrobianos, análise de genes de β-lactamase (bla_TEM,bla_SHV,bla_CTX-Mbla_KPC,bla_VIM, bla_IMP,and bla_SPM), perfil plasmidial e ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction).

RESULTADOS: A análise da ERIC-PCR e do perfil plasmidial agrupou os isolados em 17 e 19 perfis, respectivamente. Seis isolados de um hospital apresentaram o mesmo padrão de ERIC-PCR, indicando disseminação clonal. Todos os isolados apresentaram bla_SHV, 62,5% apresentaram bla_CTX-M-2, 29% bla_TEM e 41,7% bla_KPC. Genes de metalo-β-lactamase bla_VIM, bla_IMP e bla_SPM não foram detectados. Onze isolados foram identificados carreando, pelo menos, três dos genes de β-lactamase estudados, dentre estes, dois isolados continham bla_SHV,bla_TEM, bla_CTX-M-2 e bla_KPC simultaneamente.

CONCLUSÕES: O acúmulo de genes de resistência em algumas cepas, observado nesse estudo, impõem limitações nas opções terapêuticas disponíveis para o tratamento de infecções causadas por K. pneumoniae em Recife, Brasil. Estes resultados devem alertar as autoridades médicas brasileiras para estabelecer rigorosos métodos para controlar eficientemente a disseminação de genes de resistência a antimicrobianos no ambiente hospitalar.

Palavras-chaves:Klebsiella pneumoniae. bla_KPC. bla_CTX-M-2. ERIC-PCR.

INTRODUCTION

Klebsiella pneumoniae is an opportunistic pathogen responsible for many nosocomial infections including septicemia, urinary tract infections, and pneumonia, especially in immunocompromised individuals¹. Nosocomial infections are caused by multidrug resistant K. pneumoniae strains, mainly extended-spectrum β-lactamases (ESBLs) producers. Extended-spectrum β-lactamases are enzymes often clavulanate-susceptible that can hydrolyze oxyimino-cephalosporins (cefotaxime, ceftazidime, ceftriaxone, cefepime) and monobactams, but not cephamycins and carbapenems². Most of ESBLs are derived from the TEM (Temoniera) and SHV (sulphydryl variable) groups, however, since around 2008 CTX-M (cefotaximase) ESBLs have become dominant in many countries, including Brazil, mainly in K. pneumoniae nosocomial isolates^3-5.

The carbapenems are often used for the treatment of infections caused by K. pneumoniae ESBLs producers. However, this species has presented an efficient mechanism of resistance to carbapenems, known as Klebsiella pneumoniae carbapenemase (KPC), which was initially detected in the United States and Israel and later in other countries⁶. In Brazil, KPC enzymes have been described since 2009⁷ when originally reported in isolates of K. pneumoniae from Recife⁸ . Considering that Monteiro et al.⁸, reported the one-time identification of KPC enzymes in Recife and analyzed only 4 K. pneumoniae isolates and 3 of them were clones, is of utmost importance the study of the occurrence of this resistance genes in a larger number of isolates from Recife, with no clonal relationship. Therefore, the objective of this study was to genetically characterize clinical isolates of K. pneumoniae from patients in two hospitals, in the City of Recife, Brazil, through the investigation o bla_TEM,bla_,SHV, bla_CTX-M, bla_KPC,bla_VIM, bla_IMP, and bla_SPM and through molecular typing by ERIC-PCR (enterobacterial repetitive intergenic consensus-polymerase chain reaction) and plasmidial profile.

METHODS

Bacterial isolates

Twenty-four clinical isolates of K. pneumoniae from different patients from the City of Recife, State of Pernambuco (PE), Brazil, were analyzed. Ten isolates were from different units of a public hospital (Hospital A) and 14 were from the intensive care unit of a private hospital (Hospital P), from 2007 and 2008, respectively (Table 1). All isolates were identified by Vitek 2 (bioMérieux, Marcy I'Etoile, France).

Antimicrobial susceptibility and phenotypic detection of ESBL and KPC

The antimicrobial susceptibility was tested on Mueller-Hinton agar by the disk diffusion method⁹. Commercially available disks (Oxoid) loaded with the following antimicrobial were used: ampicillin (AMP), amoxicillin (AMO), amoxicillin/clavulanate (AMC), ampicillin/ sulbactam (APS), aztreonam (ATM), cefepime (CPM), cefoxitin (CFO), cefotaxime (CTX), ceftazidime (CAZ), cefalotin (CFL), ciprofloxacin (CIP), gentamicin (GEN), imipenem (IMP), meropenem (MPM), piperacilina/ tazobactam (PIT), trimethoprim/sulfamethoxazole (TSU), amikacin (AMI), nalidixic acid (NAL), chloramphenicol (CLO), streptomycin (EST) and tetracycline (TET). The isolates were further examined by the double-disk synergy test (DDST) and Modified Hodge Test (MHT) for ESBLs and KPC detection, respectively, as previously described by Vercauteren et al.¹⁰ and Anderson et al.¹¹.

DNA extraction and PCR for β -lactamase genes

Genomic deoxyribonucleic acid (DNA) of the K. pneumoniae isolates was extracted according to Maniatis et al.¹² and quantified by comparison with known amounts of lambda DNA digested with Hind III in 1% agarose gel. The β-lactamase genes were investigated by PCR using specific primers^13-17 (Table 2). The amplification reactions were prepared in a total volume of 25µl, containing 5ng of genomic DNA, 2.0U of Taq DNA polymerase (Promega, USA), 200µM of each dNTP (Promega, USA), 1.5mM of MgCl₂, 1µM of each primer and 1X reaction buffer (final concentration). The PCR amplifications were performed in a thermocycler (Biosystems) as follows: 95ºC for 5min and 35 cycles of 1min at 95ºC, 1min at specific temperature for each primer (Table 2) and 1min at 72ºC. A final extension step of 10min at 72ºC was performed (genes bla_SHV, bla_TEM, bla_CTX-M e bla_KPC) and 5min at 68ºC (genes bla_VIM, bla_IMP e bla_SPM). In each PCR (Polymerase chain reaction) were included positive control strains for genes: bla_SPM-1 (P. aeruginosa PSA319) supplyed by Laboratório Alerta, Universidade Federal de São Paulo (UNIFESP); and bla_IMP (P. aeruginosa 48-1997A), bla_VIM (P. aeruginosa VIM-1), bla_SHV, bla_TEM, bla_CTX-M (K. pneumoniae K16R) and bla_KPC (K. pneumoniae K1P), from the culture collection of the Laboratório de Microbiologia, Departamento de Medicina Tropical, Universidade Federal de Pernambuco (UFPE). The amplifications products were analyzed by electrophoresis in 1% agarose gels in TBE buffer.

Plasmid analysis and ERIC-PCR

Plasmid DNA was extracted by the UltraClean Endotoxin-Free Mini Plasmid Prep Kit (Mo Bio Lab, USA). The primers described by Duan et al.¹⁸ were used for ERIC-PCR. The amplification reactions were prepared in a total volume of 25µl, containing 100ng of genomic DNA, 1.0U of Taq DNA polymerase (Promega, USA), 200µM of each dNTP (Promega, USA), 1.52mM of MgCl₂, 0,4µM of each primer and 1X reaction buffer (final concentration). The PCR amplifications were performed in a thermocycler (Biosystems) as follows: 95ºC for 3min and 40 cycles of 1min at 92ºC, 1min at 36ºC and 8min at 72ºC. A final extension step of 16min at 72ºC was performed. The products of plasmid extraction and ERIC-PCR were analyzed by electrophoresis on agarose gel 0.7% and 1.5%, respectively. The band patterns generated by ERIC-PCR were analyzed according to Tenover et al.¹⁹ and the software DARwin 5.0 was used to generate dendrogram.

RESULTS

Antimicrobial susceptibility profile

The antimicrobial susceptibility profiles of the 10 K. pneumoniae isolates from the public hospital (Hospital A) showed that the carbapenems presented the highest levels of activity; imipenem inhibited all isolates and meropenem inhibited 9 (90%) isolates. The aminoglycosides, amikacin and streptomycin inhibited 80% of the isolates, and gentamicin inhibited 50% of the isolates. The other antimicrobials inhibited less than 50% of the isolates. The aminoglycosides were the most effective antimicrobials against the isolates from the private hospital (Hospital P), followed by the carbapenems. Interestingly, the isolates K2A and K7A, and K8P and K12P showed pair wise identical antimicrobial resistance profiles and were not clones according to the ERIC-PCR results.

ERIC-PCR

Six K. pneumoniae isolates from Hospital P (K10P, K12P, K13P, K15P, K16P, and K22P) presented the same band pattern by ERIC-PCR (profile E11) indicating clonality (Figure 1). All other isolates from both hospitals showed distinct genetic profiles (Table 3), with a maximum of 60% similarity (Figure 1). These other isolates were considered genetically unrelated by differing in at least seven bands, which indicates changes that are consistent with the occurrence of three or more independent genetic events.

β -lactamase phenotypic and genotypic detection

Out of the 24 K. pneumoniae clinical isolates studied, 9 (37.5%) were ESBL producers by the DDST (double-disc synergy test). The non-ESBL producers by DDST presented at least one of the three studied genes, bla_SHV, bla_TEM, or bla_CTX-M. All isolates that were positive by PCR for bla_CTX-MA were positive for bla_CTX-M-2. The gene bla_CTX-M-1 was not detected. All 24 K. pneumoniae isolates presented the bla_SHV, 15 (62.5%) presented the bla_CTX-M-2, and 7 (29%) had the bla_TEM(Table 3). Based on two characteristics, the presence of the bla_CTX-M-2 gene (15 isolates) and a positive DDST (K3P), 67% (16/24) of the isolates were identified as ESBL producers. The most frequently observed combination of genes was bla_SHV and bla_CTX-M-2 (42%), followed by bla_SHV and bla_TEM (8%); the bla_CTX-M-2 and bla_TEM combination was not observed. The three β-lactamase genes studied were identified simultaneously in five isolates (21%): K1A, K5A, K10A, K1P, and K14P (Table 3). The ERIC-PCR results showed that these isolates were not clones.

KPC phenotypic and genotypic detection

The gene bla_KPC was identified in 10 (71%) isolates from the Hospital P, on the other hand, this gene was not detected in the isolates from the hospital A. The isolate K14P did not show resistance to carbapenems, was negative in the MHT, however, it carried the bla_KPC gene (Table 3). The K8P, K12P, K16P, and K20P isolates showed negative MHT results but presented resistance to carbapenems and contained the bla_KPC gene.The isolate K5P was resistant to carbapenems, however, it was MHT negative and did not carry the bla_KPC gene. The bla_VIM,bla_IMP, and bla_SPM genes were not detected in any of the isolates analyzed in this study.

Plasmidial profile analysis

All isolates harboured 1 to 10 plasmids of different sizes (Table 4). Plasmids of >150 kb, 150kb, and <3.4kb were identified in 50%, 54%, and 83% of the isolates, respectively. Plasmids of 100kb, 80kb, and 12kb were identified in 57%, 78%, and 50% of the isolates from the Hospital P, suggesting a dissemination of these plasmids. It should be noted that the isolates K4A and K10A; K12P and K13P; K14P and K19P; and K15P and K16P, presented the same plasmidial profile, defined by the number and molecular weight of the bands (Table 4).

DISCUSSION

Multidrug resistance among ESBL-producer isolates is predictable because the genes that encode for ESBLs are usually located in self-transferable plasmids, which can also carry other antimicrobial resistance genes20. All isolates were tested against 9 classes of antimicrobials, however, for the majority, the therapeutic options were restricted to a maximum of 4 classes, evidencing the problematic aspects of treatment of multidrug resistant K. pneumoniae isolates. It was possible to establish an association between antimicrobial resistance profiles and β-lactamases genes because the isolates K2A and K7A, and K8P and K12P showed the same β-lactamase genes and the same resistance profile. All isolates from the Hospital A that had two or three genes (bla _SHV, bla_TEM, and bla_CTX-M-2) were resistant to the oxyiminocephalosporins tested (ceftazidime, cefotaxime and cefepime) and to aztreonam, whereas isolates that carried only bla_SHV were resistant to only one of these antimicrobials. On the other hand, this association was not observed between the isolates from the Hospital P because they were resistant to all tested cephalosporins and monobactam, regardless of the number of genes carried.

The DDST was effective in detecting the ESBL producers among all isolates from the Hospital A (PCR positive for bla_CTX-M-2) that were KPC negative. On the other hand, this test failed to detect 7 ESBL producers from the Hospital P. The failure of DDST to detect ESBLs in the K1P, K10P, K13P, K14P, K15P K16P Hospital P isolates can be explained by the concomitant presence of the KPC carbapenemase. Tsakris et al.²¹ showed that the phenotypic detection of ESBLs is hampered when the isolates also produce KPC; therefore, these authors suggest the addition of boronic acid to the test to inhibit the KPC enzymes, thus facilitating the detection of the ESBL enzymes.

The negative result in the ESBL detection through the DDST for the isolate K5P, which was positive for the bla_CTX-M and negative for the bla_KPC, can be explained by the fact that: I) this is not a confirmatory test for the detection of ESBLs⁹ because it relies on the bacterial phenotype that can be altered by the conditions of in vitro cultivation; II) additionally, the test only detects ESBLs that are inhibited by the clavulanic acid (36 of 135 TEM enzymes, and 5 of 72 SHV enzymes have currently demonstrated resistance to the clavulanic acid and related inhibitors, however, no CTX-M has demonstrated this feature so far)²; and III) the molecular methods present higher sensitivity for the detection of ESBLs compared to the phenotypic methods.

The MHT did not detect carbapenemases in five isolates carrying the bla_KPC gene and resistant to carbapenems. In Brazil, it is more common to encounter false positive results for carbapenemase production, for example, in isolates of K. pneumoniae cefotaximases producers that simultaneously present loss of porins²². The problematic detection of KPC in Enterobacteriaceae has been reported by other authors, supporting the suggestion of phenotypic detection of KPC using boronic acid²³.

In spite of an early dissemination of KPC-2-producing K. pneumoniae strains in Brazil, bla_KPC gene was not detected in the isolates from Hospital A. This fact may indicate a good clinical practice in Hospital A. The bla_KPC gene occurrence was 71% in the isolates from the Hospital P, which is equal to the occurrence of the ESBL producers in this hospital. Probably, this fact can limit the therapeutic options and increase the mortality rates. Another relevant finding was the lack of expression of resistance to carbapenems by K14P isolate that was positive for bla_KPC. Similar results were reported by Peleg et al.²⁴, showing that only 5 out of 19 isolates carrying carbapenemases genes expressed resistance to carbapenems. The acquisition of genes that encode for carbapenemases is not always associated with high levels of resistance to carbapenems²⁵. This variable susceptibility can be explained by a number of factors such as: I) co-presence of other mechanisms of resistance²⁶; II) genetic suppression leading to a silenced gene; or III) dosage of the gene that is dependent on the plasmid copy number²⁴.

The presence of identical plasmid profile in different isolates from Hospital P suggests that plasmids with the same molecular weight may have disseminated among these isolates. Aktas et al.²⁷, considered that K. pneumoniae is a nosocomial reservoir and the source for the transmission of resistance plasmids. According to Sharma et al.²⁸, there is a correlation between the number of plasmids harbored by an isolate and the multidrug resistance. This association was not observed in this study; the isolates showing resistance to the greatest number of drugs (K8P and K12P) did not carry the greatest number of plasmids, and the isolates resistant to fewer drugs (K3A and K4A) were not the ones carrying the smallest number of plasmids. Additionally, the isolates that presented the same antimicrobial resistance profile did not present the same plasmidial profile.

No correlation was observed in a quantitative analysis between the plasmids and the bla_SVH, bla_TEM, and bla_CTX-M genes, because some isolates were positive for only one gene, but carried one to nine plasmids. Other isolates presented two genes with varying numbers of plasmids. K1P was positive for all β-lactamases genes studied (bla_SHV, bla_TEM,bla_CTX-M, and bla_KPC) and carried only two plasmids (100kb and 8kb). According to Wei et al.²⁹, the number of antibiotic resistance genes in plasmids of multidrug-resistant K. pneumoniae can reach up to five. The presence of high molecular weight plasmids (60 to 180Kb) in clinical isolates of K. pneumoniae is quite common and has often been associated with the production of ESBLs^29,30.

The ERIC-PCR revealed that 6 isolates from the Hospital P showed clonal relationship even when presenting different antimicrobial resistance phenotypes, contents of resistance genes, and plasmidial profiles. Molecular typing data reported in the literature corroborate these results. Ben-Hamouda et al.³¹, reported isolates with identical PFGE or ERIC-PCR profiles and different drugs susceptibility profiles. Bennett et al.³² did not observe any correlation between PFGE profiles and resistance genes. Souza Lopes et al.³⁰ identified RAPD subtypes correlated with different plamidial profiles. These discrepancies in the typing techniques result from the fact that, the evidences for clonality are best considered as relative instead of absolute, because of the potential of genetic changes detectable only by DNA sequencing or other specific analyses.

The finding that 11 (46%) isolates were carriers of at least 3 of the β-lactamase studied genes is worrying. It should be noted that the isolates K1P and K14P presented the bla_SHV, bla_TEM, bla_CTX-M, and bla_KPC genes and were not clones. The simultaneous production of three β-lactamases by K. pneumoniae deserves to be highlighted³³.

The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. The presence of intra-hospital clones was another remarkable observation. These results should alert the Brazilian medical authorities to establish rigorous methods for detection and thus, more efficiently control the dissemination of antibiotic resistance genes in the hospital environment. Moreover, measures to control the excessive use of antimicrobials in hospitals must also be taken.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FINANCIAL SUPPORT

Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE).

Received in 09/02/2012

Accepted in 20/04/2012

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Publication Dates

• Publication in this collection
  12 Nov 2012
• Date of issue
  Oct 2012

History

• Received
  09 Feb 2012
• Accepted
  20 Apr 2012