Integrons and multidrug resistance across phylogenetic groups of clinical isolates of Escherichia coli.

Objective: This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings. Methods: A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays. Results: Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-ß-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively. Conclusion: Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against ß-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.

First Detection of Extensively Drug-Resistant Salmonella Typhi Isolates Harboring VIM and GES Genes for Carbapenem Resistance from Faisalabad, Pakistan.

Rapid emergence of resistance in Salmonella enterica serovar Typhi (Salmonella Typhi) against most of the available therapeutic options for typhoid has rendered its treatment more difficult. This study sought to determine the current scenario of antimicrobial resistance in local isolates of Faisalabad following several treatment failure reports. Out of 300 clinical specimens collected in 2018, 45 isolates were identified as Salmonella Typhi. To assess changes, we compared their antibiogram profile with 31 Salmonella Typhi strains isolated in 2005. The isolates collected during 2018 showed a significant rise in antimicrobial drug resistance as compared with isolates revived from the cultures of 2005, including 15 multidrug-resistant (MDR), 20 extensively drug-resistant, and 14 pan drug-resistant isolates compared with only 8 MDRs from 2005. Notably, in 2018 isolates, resistance to azithromycin was seen in 75% of the isolates. Extended-spectrum beta-lactamase production was detected in 47% of Salmonella Typhi isolates and 18% isolates showed resistance against carbapenems. The sequences of two carbapenemase genes, VIM and GES, found in Salmonella Typhi were submitted in NCBI. The carbapenem resistance is rare in Enterobacteriaceae and probably first time reported in Salmonella Typhi. H58 haplotype was identified in the 2018 Salmonella Typhi isolates and PCR-restriction fragment length polymorphism method identified 16.7% of H58 strains that belonged to lineage I, 19.4% of H58 strains that belonged to lineage II, and the remaining 63.9% that belonged to the node. The regional difference in the antimicrobial resistance trend needs effective epidemiological studies.

Level of biofilm production by Staphylococcus aureus isolates is critical for resistance against most but not all antimicrobial drugs.

Background and Objective: Staphylococcal biofilms cause a wide range of acute and chronic infections, both in hospital and community settings across the world. This study explores biofilm forming propensity among Staphylococcus aureus clinical isolates from Faisalabad, Pakistan and their association with antimicrobial drug resistance. Methods: The study was conducted during July to December 2020. The biofilm forming ability of S. aureus isolates was assessed by crystal violet staining in 96 well plates. Antimicrobial susceptibility was determined by disk diffusion method against ten antimicrobials representing whole spectrum of antimicrobial drugs. Results: All the isolates (n=22) produced biofilm; 14 (63.6%) were strong, and 8 (36.4%) moderate biofilm producers. Comparative data were obtained for moderate and strong biofilm producers. Increased biofilm production did not affect azithromycin, clindamycin and mupirocin. However, stronger biofilm production significantly increased resistant isolates in case of augmentin (23.2%), cefoxitin (17.9%), levofloxacin (26.8%), tetracycline (23.2%), vancomycin (14.3%) and trimethoprim (21.4%). Conclusions: Our findings indicate that the ability to produce large amount of biofilm is an important factor, and S. aureus isolates with this ability, do not require acquisition of drug resistance genes from other bacteria. Our study also provides a guideline for selection of antimicrobials which are not adversely affected by level of biofilm production by various strains of S. aureus.

Immunological characterization of chitosan adjuvanted outer membrane proteins of Salmonella enterica serovar Typhi as multi-epitope typhoid vaccine candidate.

BACKGROUND: Outer membrane proteins (OMPs) of Gram-negative bacteria have been known as potential vaccine targets due to their antigenic properties and host specificity. Here, we focused on the exploration of the immunogenic potential and protective efficacy of total OMPs of Salmonella enterica serovar Typhi due to their multi epitope properties, adjuvanted with nanoporous chitosan particles (NPCPs). The study was designed to extrapolate an effective, low cost prophylactic approach for typhoid fever being getting uncontrolled in Pakistan due to emergence of extensively drug resistant (XDR) strains. METHODS & RESULTS: The OMPs of two S. Typhi variants (with and without Vi capsule) alone and with nanoporous chitosan particles as adjuvant were comparatively analyzed for immunogenic potential in mice. Adaptive immunity was evaluated by ELISA and relative quantification of cytokine gene expression (IL4, IL6, IL9, IL17, IL10, TNF, INF and PPIA as house keeping gene) using RT-qPCR. Statistical analysis was done using Welch's test. The protection was recorded by challenging the immunized mice with 50% ×LD50 of S. Typhi. The Vi + ve-OMPs of S. Typhi showed the most promising results by ELISA and significantly high expression of IL-6, IL-10 and IL-17 and 92.5% protective efficacy with no detectable side effects. CONCLUSION: We can conclude that the OMPs of Vi + ve S. Typhi are the most promising candidates for future typhoid vaccines because of cost effective preparation without expensive purification steps and multi-epitope properties. Chitosan adjuvant may have applications for oral protein based vaccines but found less effective in injectable preparations.

Detection of synergistic antimicrobial resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from post-operative wound infections.

Infections caused by carbapenem-resistant Pseudomonas aeruginosa are life-threatening due to its synergistic resistance mechanisms resulting in the ineffectiveness of the used antimicrobials. This study aimed to characterize P. aeruginosa isolates for antimicrobial susceptibility, biofilm formation virulence genes, and molecular mechanisms responsible for resistance against various antimicrobials. Out of 700 samples, 91 isolates were confirmed as P. aeruginosa which were further classified into 19 non-multidrug-resistant (non-MDR), 7 multidrug-resistant (MDR), 19 extensively drug-resistant (XDR), and 8 pan drug-resistant (PDR) pulsotypes based on standard Kirby Bauer disc diffusion test and pulse field gel electrophoresis. In M9 minimal media, strong biofilms were formed by the XDR and PDR pulsotypes as compared to the non-MDR pulsotypes. The virulence genes, responsible for the worsening of wounds including LasB, plcH, toxA, and exoU, were detected among all MDR, XDR, and PDR pulsotypes. Carbapenemase activity was phenotypically detected in 45% pulsotypes and the responsible genes were found as blaGES (100%), blaVIM (58%), blaIMP (4%), and blaNDM (4%). Real-time polymerase chain reaction showed the concomitant use of multiple mechanisms such as oprD under-expression, enhanced efflux pump activity, and ampC overexpression in the resistant isolates. Polymyxin is found as the only class left with more than 80% susceptibility among the isolates which is an alarming situation suggesting appropriate measures to be taken including alternative therapies. KEY POINTS: • Multidrug-resistant P. aeruginosa isolates formed stronger biofilms in minimal media. • Only polymyxin antimicrobial was found effective against MDR P. aeruginosa isolates. • Under-expression of oprD and overexpression of ampC were found in resistant isolates.

Association of biofilm formation and cytotoxic potential with multidrug resistance in clinical isolates of Pseudomonas aeruginosa.

Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity. In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.

Multiple Drug Resistance and Virulence Profiling of Salmonella enterica Serovars Typhimurium and Enteritidis from Poultry Farms of Faisalabad, Pakistan.

The zoonotic serovars of Salmonella enterica particularly Typhimurium and Enteritidis pose a continuous global threat to poultry industry and public health. We report the prevalence of Salmonella Typhimurium and Salmonella Enteritidis serovars in local poultry, phenotypic antimicrobial resistance profiling, and molecular detection of antimicrobial resistance and virulence genes. A total of 340 clinical samples were collected and 239 carried Salmonella, which were identified by genus-specific PCR (invA gene) and by matrix-assisted laser desorption/ionization time-of-flight. The 68 and 22 isolates were confirmed as Salmonella Typhimurium (stm gene) and Salmonella Enteritidis (sdfI gene) respectively. Pulsed-field gel electrophoresis (PFGE) revealed 27 and 9 PFGE types of Salmonella Typhimurium and Salmonella Enteritidis respectively. Among 24 antimicrobials tested, highest resistance was observed against pefloxacin while highest susceptibility was found for ertapenem in Salmonella Typhimurium and aztreonam in Salmonella Enteritidis. All isolates were found multiple drug resistant, 98.8% as motile and 8.8% as extended spectrum beta lactamase producers. Most prevalent resistance gene in Salmonella Typhimurium was parE (69.1%) while in Salmonella Enteritidis blaTEM-1 (72.7%). High prevalence of SopE gene in Salmonella Typhimurium (91.1%) and Salmonella Enteritidis (81.8%) indicated their zoonotic potential. The study is first of its kind from this region and highlights the emerging trends of antimicrobial resistance of global concern.