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; bla
TEM, bla
SHV, bla
OXA-1 group and bla
CTX-M, as previously described7–10. Additional sequencing primers were required for
bla
TEM PCR product sequencing (‘Lagging strand 7’ 5’-TTACTGTCATGCCATCC-3’ and ‘Lagging
strand 3’ 5‘-AGAGAATTATGCAGTGC-3’). PCR primers corresponding to sequences downstream
of bla
CTX-M genes were also used (‘M3 int upp’ 5’-TCACCCAGCCTCAACCTAAG-3’ and ‘ORF1 pol
M3’ 5’-GCACCGACACCCTCACACCT-3’)11. PCR products of bla
CTX-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 Enterobacteriaceae
12. 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;
http://en.wikipedia.org/wiki/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 bla
TEM, 18 (46%) for bla
SHV, 36 (92%) for bla
CTX-M, and 32 (82%) for blaOXA-1group, respectively. The sequenced amplicons of the
isolates positive for blaCTX-M revealed the presence of bla
CTX-M-15 in all isolates. PCR-based replicon typing revealed that only a single isolate
harboured both FIA and FIB replicons carrying bla
CTX-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 bla
TEM and bla
SHV genes, 41 per cent a bla
TEM gene only, and only 5 per cent a bla
SHV gene. In the past 15 years, CTX-M-type ESBLs have become more prevalent worldwide18
19. 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.