Sir,
The Philippines, an archipelago in the Western Pacific region with a population of ∼89 million, has a tradition of robust emigration and thus large traffic in international travel with countries such as China, India and the USA. However, data on the epidemiology of antimicrobial resistance genes among Enterobacteriaceae in the Philippines are limited. Here we report an analysis of plasmid-mediated antimicrobial resistance determinants present among extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae identified at a tertiary hospital in Manila, Philippines.
Three hundred non-duplicate Enterobacteriaceae that were identified from clinical specimens at the Central Microbiology Laboratory of the Philippine General Hospital were randomly collected between September and December 2007. Clinical isolates that were suspected to produce ESBL based on the disc diffusion method1 were subjected to Etest ESBL (bioMérieux, Durham, NC, USA) for confirmatory testing. A reduction in the MIC of ceftazidime of at least three dilutions in the presence of clavulanate was interpreted as a positive test. As a result, a total of 39 ESBL-producing isolates were identified. The species were then further verified with the API20E system (bioMérieux). They included 15 Escherichia coli, 15 Klebsiella pneumoniae, 5 Enterobacter cloacae, 3 Citrobacter freundii and 1 Proteus mirabilis. PFGE was performed for all K. pneumoniae and E. coli isolates using XbaI as the restriction enzyme (New England Biolabs, Ipswich, MA, USA). For E. coli, phylogenetic typing and PCR analysis for identification of the sequence type (ST) 131 international epidemic clone were also performed.2,3 PCR analyses were performed to identify various resistance genes in all study isolates. They included: β-lactamase genes blaTEM, blaSHV and blaCTX-M (including blaCTX-M-1, blaCTX-M-2 and blaCTX-M-9 groups);4 plasmid-mediated AmpC β-lactamase genes blaCMY-1, blaCMY-2, blaDHA, blaACC, blaACT and blaFOX;5 16S rRNA methylase genes armA, rmtB and rmtC;6 pentapeptide repeat protein genes qnrA, qnrB, qnrC and qnrS; the fluoroquinolone-modifying aminoglycoside acetyltransferase gene aac(6′)-Ib-cr; and the plasmid-mediated fluoroquinolone efflux pump gene qepA.7 PCR products were sequenced on both strands using an ABI 3100 instrument (Applied Biosystems, Foster City, CA, USA).
Of the 39 study isolates, 30 (77%), 39 (100%), 19 (49%) and 34 (87%) were non-susceptible to ceftazidime, cefotaxime, cefepime and aztreonam, respectively. All were susceptible to ertapenem. As with non-β-lactam agents, non-susceptibility to ciprofloxacin and gentamicin was very common [37 isolates (95%) for both], whereas susceptibility to amikacin was better maintained [7 isolates (18%) non-susceptible].
No or little clonal relationship was detected among the 15 E. coli and 15 K. pneumoniae isolates. Of the E. coli isolates, three, five, one and six belonged to phylogenetic groups A, B1, B2 and D, respectively. The only isolate belonging to phylogenetic group B2 was identified as ST131, which represents the international epidemic clone.
Table 1 summarizes various resistance genes present in the study isolates. An ESBL gene was identified in all 39 isolates. Thirty-seven (95%) possessed blaCTX-M. Of them, the blaCTX-M-1 group was the most common, followed by the blaCTX-M-9 group. The blaCTX-M-2 group was not identified. The two blaCTX-M-negative isolates had blaSHV-12. In addition, six isolates had blaSHV known to exhibit the ESBL phenotype in addition to blaCTX-M (Table 1). All blaTEM were identified as blaTEM-1 encoding a non-ESBL. Four isolates carried blaDHA-1, a plasmid-mediated AmpC β-lactamase gene, in addition to blaCTX-M-15 and were resistant to cefoxitin.
Table 1.
Distribution of antimicrobial resistance genes among the Enterobacteriaceae clinical isolates
| No. (%) of strains positive for the genes |
|||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| β-lactamase resistance genes |
quinolone and aminoglycoside resistance genes |
||||||||||||||||||||
| AmpC |
16S rRNA methylase |
qepA | Qnr |
||||||||||||||||||
| Species | No. of strains | blaCTX-M-1 groupa | blaCTX-M-2 group | blaCTX-M-9 groupb | blaSHVc | blaTEM | blaCMY-1 | blaCMY-2 | blaDHA | blaACC | blaACT | blaFOX | aac(6′)-Ib-cr | armA | rmtB | rmtC | qnrA | qnrB | qnrC | qnrS | |
| E. coli | 15 | 12 | 0 | 3 | 1 | 13 | 0 | 0 | 2 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 2 |
| K. pneumoniae | 15 | 14 | 0 | 0 | 13 | 14 | 0 | 0 | 2 | 0 | 0 | 0 | 9 | 3 | 0 | 0 | 0 | 0 | 5 | 0 | 9 |
| E. cloacae | 5 | 4 | 0 | 0 | 4 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 2 | 0 | 0 | 0 | 0 | 2 | 0 | 4 |
| C. freundii | 3 | 3 | 0 | 0 | 1 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 |
| P. mirabilis | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| Total | 39 | 34 (87) | 0 (0) | 3 (8) | 19 (49) | 35 (90) | 0 (0) | 0 (0) | 4 (10) | 0 (0) | 0 (0) | 0 (0) | 18 (46) | 5 (13) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 12 (31) | 0 (0) | 16d (41) |
aTwenty-four isolates with blaCTX-M-15, six isolates with blaCTX-M-3 and four isolates with blaCTX-M-55.
bTwo isolates with blaCTX-M-14 and one isolate with blaCTX-M-24.
cSix isolates with blaSHV-12, one isolate each with blaSHV-28, blaSHV-32 and blaSHV-11, respectively, and 10 isolates with blaSHV-1.
dFour isolates also positive for qnrB.
Eight and 12 isolates carried the qnrB and qnrS alleles, respectively. Four isolates possessed both. Three of the eight qnrB were qnrB4, which occurred in the presence of blaDHA-1. Two of the qnrS-positive isolates were susceptible to ciprofloxacin, whereas all qnrB-positive isolates were non-susceptible to this agent. None of the isolates gave positive PCR results for qnrA, qnrC or qepA. Twenty-six isolates were positive for aac(6′)-Ib. The deduced amino acid sequences for 18 of them were consistent with AAC(6′)-Ib-cr, which is a variant of aminoglycoside acetyltransferase AAC(6′)-Ib and has been implicated in low-level resistance to fluoroquinolones.8 Of six isolates that were resistant to amikacin, five isolates possessed armA encoding 16S rRNA methylase.
To the best of our knowledge, the only previous report which studied ESBL-producing Enterobacteriaceae in the Philippines was conducted at the same hospital between 2001 and 2002 and found blaSHV-12 to be the sole ESBL gene across species.9 No blaCTX-M was found in that study. Our results indicate that the ESBL gene contents of Enterobacteriaceae have shifted dramatically from blaSHV-12 to blaCTX-M-15 in less than a decade in the same hospital in the Philippines, as has been observed in many other parts of the world.10 They further underscore the urgent need for effective infection control measures and antimicrobial stewardship especially in countries such as the Philippines, where the costs of medical care related to infections due to multidrug-resistant organisms often need to be paid for by the patients themselves.
Funding
G.-B. T. was supported in part by the China Scholarship Council (CSC). Y. D. was supported by grants from the National Institutes of Health (1K22AI080584-01 and 1R03AI079296-01A1).
Transparency declarations
Y. D. receives research support from Merck. All other authors: none to declare.
Acknowledgements
We thank Hanna E. Sidjabat for technical assistance. We also thank Peter J. Veldkamp and Bernard J. Macatangay for facilitating the collaboration.
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