Extended-spectrum β-lactamase producing Klebsiella pneumoniae from blood cultures in Puducherry, India.

Sir,

Extended-spectrum β-lactamases (ESBLs) are plasmid-mediated enzymes that confer resistance to all penicillins and cephalosporins, including the sulbactam and clavulanic acid combinations and monobactams such as aztreonam1. ESBLs are most commonly detected in Klebsiella pneumoniae, which is an opportunistic pathogen associated with severe infections in hospitalized patients, including immunocompromised hosts with severe underlying diseases2. ESBL producing K. pneumoniae was first reported in 1983 from Germany, with a steady worldwide increase in K. pneumoniae-mediated resistance against cephalosporins in the subsequent decades3.

Bloodstream infections associated with K. pneumoniae may arise as a consequence of pneumonia (community- and ventilator-acquired), the urinary tract, intra-abdominal pathologies, and central venous line-related infections4. However, though evidence shows that this pathogen is associated with nosocomial infections worldwide, relatively little information is currently available regarding ESBL producing K. pneumoniae isolates from southern India, and Puducherry in particular. Thus a molecular characterization study was performed on blood isolates of ESBL producing K. pneumoniae collected from a tertiary care hospital in Puducherry, India.

In this retrospective study, 39 non-repeat blood culture isolates of K. pneumoniae were collected during a 3 month period (June-August) in 2008. Isolates were obtained from patients admitted to 8 different wards at JIPMER (Jawaharlal Institute of Postgraduate Medical Education & Research), Puducherry, south India (Table). Blood culture was performed using biphasic medium consisting of Brain Heart Infusion (BHI) agar and BHI broth with sodium polyanethol sulphonate as an anticoagulant. K. pneumoniae was identified using standard microbiological procedures5. The antimicrobial susceptibility profiles of ampicillin (10 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin (100 μg), piperacillin/tazobactum (100/10 μg), cefoperazone/sulbactum (75/10 μg), cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg) and meropenem (10 μg) (Hi-Media, Mumbai) were tested by disk diffusion methods as described by the Clinical and Laboratory Standards Institute (CLSI formerly NCCLS)6. Phenotypic evidence of ESBL production was tested by the combination disk method6. K. pneumoniae ATCC 700603 and Escherichia coli ATCC 25922 were used as controls. These controls were available from the Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry. Isolates were stabbed into semi-solid nutrient agar butts and were stored at 4°C before retrieval for further investigation.

Table

Isolate number, patient age, ward of isolation and diagnosis of 39 patients presenting with K. pneumoniae blood stream infections in Puducherry between June and August 2008

All 39 isolates were subjected to molecular analysis, with PCR screening and sequencing being performed to identify the β-lactamase resistance genes; blaTEM, blaSHV, blaOXA-1 group and blaCTX-M, as previously described7–10. Additional sequencing primers were required for blaTEM PCR product sequencing (‘Lagging strand 7’ 5’-TTACTGTCATGCCATCC-3’ and ‘Lagging strand 3’ 5‘-AGAGAATTATGCAGTGC-3’). PCR primers corresponding to sequences downstream of blaCTX-M genes were also used (‘M3 int upp’ 5’-TCACCCAGCCTCAACCTAAG-3’ and ‘ORF1 pol M3’ 5’-GCACCGACACCCTCACACCT-3’)11. PCR products of blaCTX-M positive isolates were subjected to sequencing using primers ‘CTX-M-1 fw multi’ 5’-AAAAATCACTGCGCCAGTTC-3’, ‘CTX-M-1 multi (REV)F seq’ 5’-AACGTGGCGATGAATAAGCT-3’ and ‘ORF1 pol M3’ 5’-GCACCGACACCCTCACACCT-3’. A PCR-based replicon typing method was performed to study the relationship between the resistance plasmids present, with the individual plasmid types FIA, FIB, FIIs, A/C and I1 replicons being screened12. These replicon types are representative of the plasmid incompatibility groups circulating among Enterobacteriaceae12. Isolate genotyping was performed using pulsed field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Cluster analysis was performed using the method of Dice and the unweighted pair group method with arithmetic mean (UPGMA; ).

Of the 39 isolates investigated, 37 (94.8%) were found to be resistant to at least one of the third generation cephalosporins. Among these 37 isolates, 36 (97.2%) were found to be ESBL positive by phenotypic testing. Antibiotic susceptibility testing revealed that the majority of these 36 isolates were multidrug resistant exhibiting 95, 87 and 92 per cent resistance to gentamicin, ciprofloxacin and ceftriaxone, respectively. Of the 39 isolates, 21 per cent showed resistance to amikacin and only 5 per cent to meropenem. By PCR, of the 39 isolates, 32 (82%) were positive for blaTEM, 18 (46%) for blaSHV, 36 (92%) for blaCTX-M, and 32 (82%) for blaOXA-1group, respectively. The sequenced amplicons of the isolates positive for blaCTX-M revealed the presence of blaCTX-M-15 in all isolates. PCR-based replicon typing revealed that only a single isolate harboured both FIA and FIB replicons carrying blaCTX-M-15. Plasmids with FIIs, A/C and I1 replicons types were not detected. PFGE analysis showed that the 39 isolates belonged to 3 (non-clonal) major genotypic clusters with no obvious association between genotype and ward.

In recent years, a significant increase in ESBL producing Klebsiella spp. has been reported in India mostly identified using phenotypic methods13–16. Further, according to our earlier report (January- July, 2006), 130 patients with K. pneumoniae blood stream infections were identified with a very high proportion of these, (126 or 97%), producing ESBLs17. From our current study, 44 per cent of K. pneumoniae isolates carried both blaTEM and blaSHV genes, 41 per cent a blaTEM gene only, and only 5 per cent a blaSHV gene. In the past 15 years, CTX-M-type ESBLs have become more prevalent worldwide1819. Among our blood culture isolates, a very high incidence of multiple ESBL-gene carriage was detected, with the most notable result being the presence of CTX-M-15 in 92 per cent of isolates, as well as the combination of CTX-M-15 resistance and OXA-1 resistance in 82 per cent and 36 per cent of isolates possessing TEM, SHV, CTX-M and OXA-1 resistance combined. Two isolates (5%) were also meropenem resistant, with carbapenems currently being considered the preferred antimicrobial agent for the treatment of serious infections caused by ESBL-producing K. pneumoniae in our hospital. This finding seriously limits treatment options, and causes great concern with respect to the adequate treatment and spread of ESBL resistant K. pneumoniae isolates both within hospitals and from the hospital environment to the community.

The spread of antimicrobial resistance in K. pneumoniae isolates is complicating the treatment of serious nosocomial infection worldwide, not least because resistance in K. pneumoniae is typically caused by the acquisition of plasmids containing multiple antimicrobial resistances (including genes coding for ESBL resistance)20. Molecular characterization of such ESBL-carrying isolates is essential in allowing hospitals to identify the source of these pathogenic bacteria, whilst providing useful information regarding the distribution of clonally related (‘outbreak’ strains) or non-related ESBL genotypes. Further, monitoring of the spread of individual β-lactamase genes and their associated genetic platforms (in particular plasmids), provides a means to monitor for the appearance of new ESBL enzymes and genotypes, as well as establishing the dominance of older established ESBL enzymes/ genotype combinations.

In conclusion, this study emphasizes the major role that CTX-M-15 plays in facilitating ESBL-mediated antimicrobial resistance in Puducherry, India, and reiterates its association with multiple antibiotic resistance determinants, including carbapenem resistance.