Prevalence of blaKPC and its occurrence with other beta-lactamases in Klebsiella pneumoniae

Introduction

Klebsiella pneumoniae (K. pneumoniae) is one of the most clinically important Gram-negative bacilli, with high degree of multidrug resistance.[12] Carbapenems are the last resort of antibiotics used to treat serious infections caused by them. Carbapenems are dipolar and rapidly enter Gram-negative bacterial cell wall through outer membrane proteins (or porins) and target bacterial penicillin-binding proteins.[3] Carbapenemases are enzymes that inhibit almost all beta-lactam antibiotics including carbapenems and belong to the Ambler Class A, B, and D beta-lactamases.[45]

K. pneumoniae carbapenemase (KPC) is plasmid encoded and belongs to Ambler Class A enzymes.[6] Their occurrence in K. pneumoniae was first reported in 1996 from the USA. Within few years, KPC producers spread worldwide and are now encountered in many Gram-negative bacteria.[4] Of late, the coexistence of KPC with other carbapenemases in K. pneumoniae is also being encountered.[7]

From India, KPC in K. pneumoniae was first reported in 2011.[8] Since then, there has been scarcely any publication on the prevalence of KPC.[91011] Hence, this study was undertaken to detect the presence of KPC in clinical isolates of K. pneumoniae in a tertiary care hospital.

Materials and Methods

Detection of beta-lactamase-encoding genes

blaKPC gene by polymerase chain reaction

Template DNA was extracted from the isolates by boiling method.[14] All the isolates were tested for blaKPC gene using the previously described primers.[15] The primers were KPC forward-CGTCTAGTTCTGCTGTCTTG and KPC reverse-CTTGTCATCCTTGTTAGGCG. Amplicon size of KPC gene was 798 bp. Polymerase chain reaction (PCR) was performed with a final volume of 25 μl. Each reaction contained 10 pmol of each primer (Sigma-Aldrich, India), 10 mM of dNTP mixture (Takara, India), and 5U Taq polymerase (Takara, India) in 2.5 μl of 10X Taq polymerase buffer (Mg2+plus). About 2 μl of template DNA was added to 23 μl of the master mixture. Negative controls were PCR mixture with water (instead of template DNA) was included in every PCR run.

Amplification reactions were performed under the following conditions: initial denaturation 10 min at 94°C, followed by 36 cycles of amplification for 30 s at 94°C, annealing at 52°C for 40 s, and 50 s at 72°C with final extension for 5 min at 72°C. DNA fragments were analyzed by electrophoresis in a 2% agarose gel containing ethidium bromide at 100 V in IX TBE buffer.

Other beta-lactamase genes

Some of the other commonly occurring beta-lactamases were also looked for employing PCR. They were plasmid-mediated AmpC beta-lactamases and metallo-beta-lactamases such as IMP, VIM, and NDM. Class D carbapenemase such as OXA-48 and extended-spectrum beta-lactamases (ESBLs) such as SHV, TEM, and CTX-M were also tested using primers described previously.[141516] The primers used are given in Table 1.

Table 1 Other beta-lactamase primers used in this study

Results

Of the 370 study isolates, 41 (11.1%) were resistant to imipenem by both disc diffusion method and minimum inhibitory concentration (MIC) determination. MIC₅₀ and MIC₉₀ were 0.125 μg/mL and 4 μg/mL, respectively.

Susceptibility to other classes of antimicrobials were amikacin (82.16%), piperacillin/tazobactam (80.27%), ciprofloxacin (62.97%), ceftazidime (50.54%), and cefotaxime (41.62%).

Screening for carbapenemase production by MHT was positive in 90 out of 370 isolates. Of the total isolates, KPC screening test using ertapenem and boronic acid was showed positive in 14 isolates.

blaKPC was detected in one isolate only [Figure 1]. The MIC of this isolate to imipenem was 64 μg/mL. MHT and KPC screen test were also positive. The source of the isolate was urine. This isolate also harbored blaSHV and blaCTX-M. Notably, the other beta-lactamases looked for, namely, TEM, AmpC, and MBL including NDM, IMP, and VIM, OXA-48 were not detected in this KPC-harboring isolate.

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Figure 1

Gel picture of amplified KPC gene. Lane 1: 100 bp DNA ladder, Lane 2: Test positive for blaKPC at 798 bp, Lane 3: Negative control

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Discussion

The Ambler Class A carbapenemase was named after the organism from which it was first identified.[4] KPC beta-lactamase can hydrolyze all beta-lactams, including carbapenems, cephalosporins, cephamycins, monobactams, and clavulanic acid combinations.[17] In addition, they exhibit resistance to aminoglycosides and fluoroquinolones.[18] KPCs may not confer frank resistance to carbapenems but may impart reduced susceptibility to carbapenems. For a full blank resistance to carbapenems, impaired outer membrane permeability is often required.[19] KPC has been detected in other Gram-negative bacteria such as the family Enterobacteriaceae (E. coli, Klebsiella oxytoca, Enterobacter spp., Serratia spp., and Salmonella spp., and Citrobacter freundii) as well as in glucose nonfermenters (Acinetobacter spp. and Pseudomonas aeruginosa).[620]

Following its first detection in 1996, KPC has been reported globally. The endemic spread of KPC-producing K. pneumoniae has been reported from the USA, China, Italy, Poland, Greece, Israel, Brazil, Argentina, Colombia, and Taiwan. Sporadic occurrence has also been observed in many European countries and in several countries in the Asia-Pacific region, including India, South Korea, and Australia.[7]

The coexistence of KPC with other carbapenemase such as NDM, IMP, and VIM is common.[7] In India, the first report of KPC along with NDM-1, CTX-M-15, and SHV-12 was published in 2011.[8] Since then, there are only a few reports of KPC occurring along with other beta-lactamases.[1021] In Chennai, India, it has been observed that of the 46 carbapenem-resistant Enterobacteriaceae, 31 isolates carry blaKPC.[9] Elsewhere among 100 carbapenem-resistant Enterobacteriaceae, KPC along with NDM was detected in 14%, while KPC alone was detected in 1%.[10] Co-carriage of blaKPC along with blaNDM-1 has been reported in P. aeruginosa also.[21]

In a study conducted in Manipal, India, KPC was observed in 3.7% of 54 carbapenem-resistant K. pneumoniae.[11]

Most of the KPC-associated infections are either systemic infections, occurring in patients with multiple invasive devices or urinary tract infections without an indwelling catheter, particularly in immunocompromised patients.[19]

In this study, even though there were 14 isolates which exhibited a positive KPC screen test using boronic acid, only one isolate among them harbored blaKPC gene. The false-positive results may be due to the production of AmpC beta-lactamases or some CTX-M beta-lactamases because boronic acid derivatives are potent inhibitors of these enzymes also.[22] Thus far, there is hardly any report of KPC-2 along with SHV-12 and CTX-M-15 in a single isolate.[8]

KPC along with ESBLs limits the therapeutic options to patients, and the death rates attributed to KPC infections are high (≥50%).[23] Antibiotic options are generally limited to tigecycline and polymyxins for KPC-producing isolates.

Conclusion

Detection of KPCs using PCR remains the gold standard, since all other phenotypic tests are prone to give false-positive results. A single strain harboring multiple resistant genes is really challenging in the clinical laboratory in terms of detection and management.