Background Frequency of extended-spectrum- β -lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-β-lactamase-producing Clinical isolates in Pakistan. Methods A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data. Results A total of 497/995 (50%) isolates including Escherichia coli 65% ( n = 321), Klebsiella spp. 25% ( n = 124) and Pseudomonas. 5% ( n = 24), Enterobacter spp. 4% ( n = 20) and Acinetobacter spp. 2% ( n = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% ( n = 278) followed by pus 20% ( n = 99) and wound swab 6% ( n = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% ( n = 392). PCR revealed bla CTX − M (76%) gene followed by bla OXA (52%), bla TEM (28%) and bla SHV (21%) were most prevalent among ESBL-producers detected by CDST. bla CTX − M − 1 (65%), bla OXA (78%) and bla TEM (57%) genes were carried on plasmids. Amplicon sequencing revealed bla CTX − M − 15 (75%), bla OXA − 1 (49%) and bla TEM − 1 B (34%) and 21 ( n = 28) isolates carried three genes in them. Conclusion Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isol...

BackgroundMetallo-β-lactamase (MBL)-producing isolates have a strong impact on diagnostic and therapeutic decisions. A high frequency of MBL-producing gram-negative bacilli has been reported worldwide. The current study was based on determining the incidence of MBL-producing imipenem-resistant clinical isolates and investigating the β-lactamase gene variants in strains conferring resistance to a carbapenem drug (imipenem).MethodsA total of 924 gram negative isolates were recovered from a tertiary care hospital in Lahore, Pakistan, during a two-year period (July 2015 to February 2017). The initial selection of bacterial isolates was based on antibiotic susceptibility testing. Strains resistant to imipenem were processed for the molecular screening of β-lactamase genes. Statistical analysis for risk factor determination was based on age, gender, clinical specimen and type of infection.ResultsThe rate of imipenem resistance was calculated to be 56.51%. Among the 142 strains processed, the phenotypic tests revealed that the incidence of MBLs was 63.38% and 86.61% based on the combination disc test and the modified Hodge test, respectively. The frequencies of blaTEM, blaSHV, blaOXA, blaIMP-1, and blaVIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of blaMBL (blaIMP and blaVIM) and blaESBL (blaTEM, blaSHV, blaOXA) was also detected through multiplex and singleplex PCR. blaOXA, blaTEM and blaSHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates.ConclusionTo our knowledge, this is the first report from Pakistan presenting the concomitant expression of blaOXA, blaTEM and blaSHV with blaIMP-1 and blaVIM in MBL-producing gram-negative bacilli.

Extensively drug-resistant (XDR) Salmonella Typhi has been reported in Sindh province of Pakistan since 2016. The potential for further spread is of serious concern as remaining treatment options are severely limited. We report the phenotypic and genotypic characterization of 27 XDR S. Typhi isolated from patients attending Jinnah Hospital, Lahore, Pakistan. Isolates were identified by biochemical profiling; antimicrobial susceptibility was determined by a modified Kirby–Bauer method. These findings were confirmed using Illumina whole genome nucleotide sequence data. All sequences were compared to the outbreak strain from Southern Pakistan and typed using the S. Typhi genotyping scheme. All isolates were confirmed by a sequence analysis to harbor an IncY plasmid and the CTX-M-15 ceftriaxone resistance determinant. All isolates were of the same genotypic background as the outbreak strain from Sindh province. We report the first emergence of XDR S. Typhi in Punjab province of Pakistan confirmed by whole genome sequencing.