Evaluation of bla SHV, bla TEM and bla OXA encoding Clinical Isolates from Chronic Tonsillitis using Phenotypic and Molecular Technique: First report from Pakistan

Objective: To evaluate bla SHV, bla TEM and bla OXA encoding clinical isolates in chronic tonsillitis using phenotypic and molecular techniques.Place and Duration:  The study was conducted in Department of Microbiology and Molecular Genetics, University of Punjab, Lahore from August 2016 to August 2017.Methodology: Sample processing, identification and characterization of isolates was done by using (CLSI, 2016) criteria. Antibiotic susceptibility testing by using disc diffusion assay and biofilm forming ability was analyzed by ring test and slime production test. Combination disc test was used for phenotypic detection of antibiotic resistance genes. Multiplex-PCR assay was used to check the presence of bla SHV, bla TEM and bla OXA genes. 16S rRNA sequencing and phylogenetic analysis was performed.Results: Here, variable resistance pattern was observed against applied antibiotics. 100 % resistance towards azotreonam and penicillin was observed. While 60-85 % resistances were observed against cephalosporins. Biofilm formation increased with the passage of time. 77% strains indicated positive combination disc test. Multiplex-PCR indicated 60% strains harbored tested genes. 40 % bla SHV genes, 30 % bla TEM genes and 60% bla OXA genes were observed among selected isolates. GenBank Accession number obtained for Klebsiella pneumoniae was KY810693 and for S. aureus was KY810692.Conclusion: In conclusion, K. pneumoniae and S. aureus came out to be common causative agents of tonsillitis in the current study. Resistance towards multiple classes of antibiotics and strong biofilms of these micro-organisms explain the chronicity and recurrent nature of the infection. bla OXA genes were frequent among genes tested

Distribution of Extended-spectrum β-lactamase and Metallo-β-lactamase-producing Pseudomonas aeruginosa in Tertiary Care Hospitals of Lahore, Pakistan

Background: Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen most frequently associated with nosocomial infections, especially in immuno-compromised patients. Early detection of these life threatening, β-lactamase producing bacteria is essential for infection control and to prevent their dissemination. The aim of our study was to detect the presence of Extended-Spectrum β-Lactamase (ESBL) and Metallo-β-Lactamase (MBL) strains of Pseudomonas aeruginosa.Material and Methods: Eighty-eight identified strains of P. aeruginosa were collected from Chughtai Laboratories, Combined Military Hospital and Children Hospital, Lahore. These strains were sub-cultured and after confirming the cultural characteristics by Gram staining and colony morphology, manual biochemical identification was done. Susceptibility to various antibiotics and production of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were determined using modified Kirby Bauer disk diffusion method, double disk synergy test, combined disk synergy test (CDST) and inhibitor-potentiated disk diffusion test (IPD) respectively.Results: Out of eighty-eight strains tested, three were ESBL producers (3.4%) and eleven strains (12.5%) were found to be resistant to carbapenems. Of these, eight were MBL producers (72.7%). All these β-lactamase producing strains (14 strains) were multidrug-resistant (MDR). Piperacillin and piperacillin/tazobactam proved to be the most effective antibiotics in both types of β-lactamase producing strains.Conclusion: Our study shows noticeable emergence of β-lactamases (ESBLs & MBLs) in P. aeruginosa. All of these strains were MDR. It reveals a correlation of these β-lactamases with multidrug resistant genes

Evaluation of bla SHV, bla TEM and bla OXA encoding Clinical Isolates from Chronic Tonsillitis using Phenotypic and Molecular Technique: First report from Pakistan

Objective: To evaluate bla SHV, bla TEM and bla OXA encoding clinical isolates in chronic tonsillitis using phenotypic and molecular techniques.Place and Duration:  The study was conducted in Department of Microbiology and Molecular Genetics, University of Punjab, Lahore from August 2016 to August 2017.Methodology: Sample processing, identification and characterization of isolates was done by using (CLSI, 2016) criteria. Antibiotic susceptibility testing by using disc diffusion assay and biofilm forming ability was analyzed by ring test and slime production test. Combination disc test was used for phenotypic detection of antibiotic resistance genes. Multiplex-PCR assay was used to check the presence of bla SHV, bla TEM and bla OXA genes. 16S rRNA sequencing and phylogenetic analysis was performed.Results: Here, variable resistance pattern was observed against applied antibiotics. 100 % resistance towards azotreonam and penicillin was observed. While 60-85 % resistances were observed against cephalosporins. Biofilm formation increased with the passage of time. 77% strains indicated positive combination disc test. Multiplex-PCR indicated 60% strains harbored tested genes. 40 % bla SHV genes, 30 % bla TEM genes and 60% bla OXA genes were observed among selected isolates. GenBank Accession number obtained for Klebsiella pneumoniae was KY810693 and for S. aureus was KY810692.Conclusion: In conclusion, K. pneumoniae and S. aureus came out to be common causative agents of tonsillitis in the current study. Resistance towards multiple classes of antibiotics and strong biofilms of these micro-organisms explain the chronicity and recurrent nature of the infection. bla OXA genes were frequent among genes tested

Prevalence of extended-spectrum-β-lactamase-producing Enterobacteriaceae: first systematic meta-analysis report from Pakistan

Abstract Background South-Asia is known as a hub for multidrug-resistant (MDR) bacteria. Unfortunately, proper surveillance and documentation of MDR pathogens is lacking in Pakistan. The alarming increase in the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a serious problem. From this perspective, we analysed published data regarding ESBL-producing Enterobacteriaceae in different regions of Pakistan. Methods A meta-analysis was performed to determine the prevalence of ESBL-producing Enterobacteriaceae in Pakistan. A Web-based search was conducted in electronic databases, including PubMed, Scopus and PakMedi Net (for non-indexed Pakistani journals). Articles published (in either indexed or non-indexed journals) between January 2002 and July 2016 were included in the study. Relevant data were extracted, and statistical analysis was performed using the Metaprop command of STATA version 14.1. Results A total of 68 studies were identified from the electronic data base search, and 55 of these studies met our inclusion criteria. Pakistan’s overall pooled proportion of ESBL-producers was 0.40 (95% CI: 0.34–0.47). The overall heterogeneity was significant (I2 = 99.75%, p < 0.001), and significant ES = 0 (Z = 18.41, p < 0.001) was found. OXA, SHV, TEM and CTX-M were the most commonly found gene variants for ESBLs in these studies. Conclusion The prevalence of ESBL-producing Enterobacteriaceae is high in Pakistan. Little is known about the annual frequency of ESBLs and their prevalence in different provinces of Pakistan. No data are available regarding ESBL frequency in Baluchistan. This underscores an urgent demand for regular surveillance to address this antimicrobial resistance problem. Surveillance to better understand the annual ESBL burden is crucial to improve national and regional guidelines

High frequency and molecular epidemiology of metallo-β-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan

Abstract 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