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. 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. 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 blaCTX − M (76%) gene followed by blaOXA (52%), blaTEM (28%) and blaSHV (21%) were most prevalent among ESBL-producers detected by CDST. blaCTX − M − 1(65%), blaOXA (78%) and blaTEM (57%) genes were carried on plasmids. Amplicon sequencing revealed blaCTX − M − 15 (75%), blaOXA − 1 (49%) and blaTEM − 1B (34%) and 21 (n = 28) isolates carried three genes in them. Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding blaCTXM − 15 gene and narrow spectrum blaOXA − 1 and blaTEM − 1B were also prevalent.

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.

Introduction: The multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates have been increasingly reported from the Asian and African countries. The emergence of isolates with decreased susceptibility to fluoroquinolones and cephalosporins has worsened the situation. Recently, an outbreak from Sindh, Pakistan was reported caused by extensively drug-resistant (XDR) S. Typhi strains. Methodology: In the present study, a total of 82 cases of typhoid have been investigated during 2018 from the febrile children referred to a tertiary care hospital in the population-wise largest province (Punjab) of Pakistan. S. Typhi was identified by standard microbiological techniques and isolates were characterized for antimicrobial resistance profiling and minimum inhibitory concentrations were determined. The presence of various ESBL genes in S. Typhi was confirmed by the PCR. Results: Out of the 82 isolates tested, 35 (43%) were found to be XDR; resistant to the first-line drugs. The resistance to third-generation cephalosporins was mainly mediated by extended-spectrum beta-lactamases i.e. blaTEM and blaCTX-M genes. Conclusions: The higher prevalence of ESBL producing Salmonella typhi clinical strains raises the concern about transmission prevention and infection management in the community as well as clinical settings. Moreover, the study highlights the problem concerning the declining antibiotic arsenal for the therapeutic management of typhoid fever and the emergence and spread of XDR strains in Pakistan.

Background: Metallo-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). Methods: A 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. Results: The 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 bla_TEM, bla_SHV,bla_OXA,bla_IMP-1, and bla_VIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of bla_MBL (bla_IMP and bla_VIM) and bla_ESBL (bla_TEM, bla_SHV,bla_OXA) was also detected through multiplex and singleplex PCR. bla_OXA, bla_TEM and bla_SHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates. Conclusion: To our knowledge, this is the first report from Pakistan presenting the concomitant expression of bla_OXA, bla_TEM and bla_SHV with bla_IMP-1 and bla_VIM in MBL-producing gram-negative bacilli.

Introduction: The aminoglycosides are widely used for the therapeutic management of infections caused by gram-negative bacteria, including the Acinetobacter baumannii strains. However, the resistance to the members of the aminoglycoside family, such as amikacin, gentamicin, and tobramycin, is increasingly being common among the clinical isolates. Purpose: This study aimed to investigate the presence of 16SrRNA methylases and aminoglycoside modifying enzymes (AMEs) genes among aminoglycoside resistant A. baumannii isolates and to study the genetic diversity of the clinical population of A. baumannii in local hospitals. Material and Methods: The 143 A. baumannii clinical strains were analyzed for antimicrobial susceptibility, genetic screening for enzymes conferring aminoglycosides resistance followed by the multilocus sequence typing. Results: The 133/143 (93%) isolates were non-susceptible to at least one of the tested aminoglycosides, including amikacin, gentamicin, and tobramycin. The MIC distribution has shown that 87.486.7% strains were resistant to amikacin and gentamicin, respectively. The aphA 6, aadB, aacC 1, and aphA 1 were found in 74.1%, 59.4%, 16.1%, and 11.2% isolates, respectively, whereas the armA was found in 28% of the strains having a higher MIC value (MIC; ≥ 256μg/mL). The MLST data have shown that the ST589 and ST2 were the most common STs and corresponded to 51 (35.7%) and 38 (26.6%) isolates, respectively, and few of the isolates corresponding to these STs were found to harbor the armA gene with a variable genotypic profile for AMEs. Discussion: The study has reported the incidence of various enzymes conferring aminoglycoside resistance among the A. baumannii clones for the first time from Pakistan. The findings suggest the possibility of transmission of aminoglycoside resistance determinants through the lateral gene transfer as well as clonal dissemination.

Prevalence of Metallo-β-Lactamase IMP and VIM Producing Gram Negative Bacteria in Different Hospitals of Lahore, Pakistan