Rapid emergence of ESBL producers in E. coli causing urinary and wound infections in Pakistan.

OBJECTIVES: Production of extended spectrum beta -lactamases (ESBLs) by clinical isolates of pathogenic E. coli is a very serious therapeutic threat. This study was aimed to investigate the prevalence of ESBLs and associated drug resistance in E. coli isolates from urine and pus, and to report the drift from 2005 to 2009-10. METHODOLOGY: Among 173 E. coli isolates, 82 were phenotypically detected as ESBL producers by standard cefotaxime / clavulanic acid and ceftazidime / clavulanic acid disc diffusion tests. Antimicrobial resistance of all ESBL producers was assessed by disc diffusion method. Presence of CTX-M, TEM, SHV and OXA groups was investigated by PCR. RESULTS: The prevalence of ESBL producing E. coli increased significantly from 33.7% in 2005 to 60.0% in 2009-10 (urine: 31.8% to 62.9%; pus: 41.1% to 55.5%). Resistance to cefotaxime, ceftazidime, ciprofloxacin, gentamicin, nalidixic acid, ticarcillin-clavulanic acid, and trimethoprim-sulfamethoxazole was above 85% in both sets of isolates. Imipenem and Fosfomycin resistance was non-existent in 2005 but ranged from 3-15% in 2009-10. Remarkable increase from 9.5% to 64.7% in urinary tract isolates and from 0 to 55% in pus isolates was observed in colistin sulphate resistance. The dissemination of genes encoding ESBLs was: CTX-M 3.5%; TEM 10.7%; both CTX-M and TEM 3.5% in 2005, and CTX-M 42.5%; TEM 48.1%; both CTX-M and TEM 29.6% in 2009-10. CONCLUSIONS: Our results showed very rapid emergence of multidrug resistant ESBL producing E. coli in Pakistan posing a very serious threat in the treatment of nosocomial and community acquired infections.

Molecular profiling of antimicrobial resistance and integron association of multidrug-resistant clinical isolates of Shigella species from Faisalabad, Pakistan.

Bacillary dysentery, common in developing countries, is usually caused by Shigella species. A major problem in shigellosis is the rapid emergence of multidrug-resistant strains. This is the first detailed molecular study on drug resistance of Shigella isolates from the Faisalabad region of Pakistan. Ninety-five Shigella isolates obtained after screening of 2500 stool samples were evaluated for in vitro resistance to commonly used antimicrobial agents; the presence or absence of 20 of the most relevant drug resistance genes; and the prevalence of integrons 1, 2, and 3. Shigella flexneri was found to be the most prevalent and most resistant species. Collectively, high resistance was found towards ampicillin (96.84%), tetracycline (93.68%), streptomycin (77.89%), and chloramphenicol (72.63%). Significant emerging resistance was detected towards the modern frontline drugs ciprofloxacin (12.63%), cefradine (17.89%), ceftriaxone (20.00%), cefoperazone (22.10%), and cefixime (28.42%). Prevalence rates for bla(TEM), bla(CTX-M), gyrA, gyrB, qnrS, aadA1, strAB, tetA, tetB, catA, and catP were 78.94%, 12.63%, 20.00%, 21.05%, 21.05%, 67.36%, 42.10%, 12.63%, 53.68%, 33.68%, and 25.26%, respectively. Class 2 integrons (42.10%) were more common in the local isolates. Simultaneous detection of class 1 and 2 integrons in some isolates and a rapidly emerging resistance to modern frontline drugs are the major findings of this study.

High prevalence of 16S rRNA methylase RmtB among CTX-M extended-spectrum ß-lactamase-producing Klebsiella pneumoniae from Islamabad, Pakistan.

The aim of this study was to characterise extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and wound infections from Pakistan (n=25). Isolates were subjected to commercially available microarray analysis to determine the presence of ESBLs and acquired AmpC enzymes. The genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid replicon typing and capsular serotyping were conducted by PCR. Finally, screening for virulence genes, plasmid-mediated quinolone resistance (PMQR) genes, and genes encoding 16S rRNA methylases was done using PCR. All K. pneumoniae isolates hosted blaCTX-M genes and all strains belonged to phylogroup CTX-M-1. Acquired AmpC ß-lactamases (ACT/MIR and CIT group) were found in 16% of isolates. Two clusters were observed with ≥80% similarity among profiles obtained by PFGE, and two sequence types (STs) by MLST, namely ST215 and ST307, were observed in these clusters. Three ST215 isolates carried virulence factor wcaG and three ST215 isolates had capsular type K20. IncFIA, IncFIB, IncFIIK and FrepB replicons were most commonly found in this collection. Among the PMQR determinants, aac(6')-lb-cr was present in 96% (24/25) of the isolates, qnrB was found in 88% (22/25) and qepA was found in 4% (1/25). The 16S rRNA methylase-encoding gene rmtB was found in 60% (15/25) of the isolates. In conclusion, CTX-M-producing ST215 and ST307 K. pneumoniae were the two major clones detected. Of particular concern was the high prevalence of 16S rRNA methylases conferring resistance to all aminoglycosides.

Molecular evaluation of drug resistance in clinical isolates of Salmonella enterica serovar Typhi from Pakistan.

INTRODUCTION: This study aimed to determine the drug susceptibility patterns and genetic elements related to drug resistance in isolates of Salmonella enterica serovar Typhi (S. Typhi) from the Faisalabad region of Pakistan. METHODOLOGY: The drug resistance status of 80 isolates were evaluated by determining antimicrobial susceptibility, MICs, drug resistance genes involved, and the presence of integrons. Nalidixic acid resistance and reduced susceptibility to ciprofloxacin were also investigated by mutation screening of the gyrA, gyrB, parC, and parE genes. RESULTS: Forty-seven (58.7%) isolates were multidrug resistant (MDR). Among the different resistance (R) types, the most commonly observed (13/80) was AmChStrTeSxtSmzTmp, which is the most frequent type observed in India and Pakistan. The most common drug resistant genes were blaTEM-1, cat, strA-strB, tetB, sul1, sul2, and dfrA7. Among the detected genes, only dfrA7 was found to be associated in the form of a single gene cassette within the class 1 integrons. CONCLUSIONS: MIC determination of currently used drugs revealed fourth-generation gatifloxacin as an effective drug against multidrug-resistant S. Typhi, but its clinical use is controversial. The Ser83→Phe substitution in gyrA was the predominant alteration in nalidixic acid-resistant isolates, exhibiting reduced susceptibility and increased MICs against ciprofloxacin. No mutations in gyrB, parC, or parE were detected in any isolate.