Detection of linezolid resistance cfr gene among MRSA isolates.

BACKGROUND: Linezolid (Oxazolidinones) is commonly used against a variety of Gram-positive infections, especially methicillin-resistant Staphylococcus aureus (MRSA). The emerging resistance to linezolid curtail the treatment of infections caused by MRSA and other Gram-positive bacteria. Presence of cfr gene plays a crucial role in Linezolid resistance. OBJECTIVE: Present study was aimed to detect cfr gene among clinical MRSA isolates. MATERIALS AND METHODS: The suspected Staphylococcus aureus isolates were processed through Kirby Bauer disc diffusion methods for the confirmation of MRSA strains. Phenotypic Linezolid resistance was determined through broth micro-dilution method. The plasmid and DNA of Linezolid resistant isolates were subjected to molecular characterization for the presence of cfr gene. RESULTS: Among 100 Staphylococcus aureus isolates, 85 of them were confirmed as MRSA isolates. Categorically, 65% MRSA isolates were sensitive to linezolid with MIC lower than 8 µg/ml, whereas, 35% of them were resistant to linezolid having MIC greater than 8 µg/ml. MIC level of 128 µg/ml was observed among 3.5% of the resistant isolates. Similarly, MIC level of 64 µg/ml, 32 µg/ml, 16 µg/ml and 8 µg/ml were noted for 3.5%, 4.7%, 8.2% and 15.3% isolates respectively. Linezolid resistance cfr gene was detected only in 9.4% of the resistant isolates. CONCLUSION: Multi drug resistance among MRSA isolates is keenly attributed to the presence of cfr gene as evident in the present study, and horizontal dissemination of cfr gene among MRSA strains is accredited to cfr-carrying transposons and plasmids.

Detection of blaNDM-1 gene in ESBL producing Escherichia coli and Klebsiella pneumoniae isolated from urine samples.

INTRODUCTION: Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant blaNDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates. METHODOLOGY: A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumß-lactamase (ESBL), carbapenemase and metallo ß-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The blaNDM-1 gene was detected by PCR and confirmed via Sanger sequencing method. RESULTS: Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured blaNDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the blaNDM-1 gene. CONCLUSIONS: The blaNDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the blaNDM-1 spread in our population.

Molecular Epidemiology of mcr-1, bla KPC-2, and bla NDM-1 Harboring Clinically Isolated Escherichia coli from Pakistan.

PURPOSE: The multiple-drug resistant Escherichia coli are among the deadliest pathogens causing life-threatening infections. This study was planned to determine the molecular epidemiology of mcr-1, bla KPC-2, and bla NDM-1 harboring clinically isolated E. coli from Pakistan. METHODS: In total, 545 strains of E. coli from clinical samples were collected from June 2018 to September 2019. All the isolates were screened for colistin-resistance, extended-spectrum-ß-lactamases (ESBL), and carbapenemases through the micro-dilution method, Double-Disk-Synergy-Test (DDST), and Modified-Hodge-Test (MHT). The detection, sequence-typing, conjugal transfer, S1-PFGE, plasmid-replicon-typing, and southern-blotting for mcr, ESBL, and carbapenemase-encoding genes were performed. FINDINGS: A total of four (0.73%) colistin-resistant strains carrying alongside mcr-1 and bla CTX-M-15 genes, three of these strains also had the bla TEM-1 gene. The presence of ESBL genes was detected in 139 (25.5%) isolates harboring bla CTXM-15 (74.82%), bla TEM (34.53%), bla SHV (28.06%) and bla OXA-1 (28.78%). In 129 carbapenemase-producers, 35.83% possessed bla NDM-1, 26.67% bla KPC-2, 8.3% bla OXA-48, 25% bla VIM-1, and 20.83% bla IMP-1 genes. The sequence typing revealed that mcr-1 harboring isolates belonged to ST405, ST117, and ST156. Fifty percent of bla KPC-2 and 48.83% of bla NDM-1 were found on ST131 and ST1196, respectively. Two rare types of STs, ST7584, and ST8671 were also identified in this study. The mcr-1 gene was located on Incl2 (60-kb) plasmid. The bla KPC-2 was present on (140-kb) IncH12, (100-kb) IncN, (90-kb) Incl1, while bla NDM-1 was located on (70-kb) IncFIIK, (140-kb) IncH12, (100-kb) IncN, (60-kb) IncA/C, and (45-kb) IncFII plasmids, which were successfully trans-conjugated. Among the plasmid types, the Incl1 carrying bla KPC-2, IncH12 harboring bla KPC-2 and bla NDM-1, and IncFIIK carrying bla NDM-1 were for the first time detected in Pakistan. CONCLUSION: The mcr-1, bla KPC-2, and bla NDM-1 genes finding in various clonal and plasmids types indicate that a substantial selection of the resistance genes had occurred in our clinical strains.

Characterisation of Bacteriocins Produced by Lactobacillus spp. Isolated from the Traditional Pakistani Yoghurt and Their Antimicrobial Activity against Common Foodborne Pathogens.

Lactic acid bacteria (LAB) are widely known for their probiotic activities for centuries. These bacteria synthesise some secretory proteinaceous toxins, bacteriocins, which help destroy similar or interrelated bacterial strains. This study was aimed at characterising bacteriocins extracted from Lactobacillus spp. found in yoghurt and assessing their bactericidal effect on foodborne bacteria. Twelve isolated Lactobacillus spp. were examined to produce bacteriocins by the organic solvent extraction method. Bacteriocins produced by two of these strains, Lactobacillus helveticus (BLh) and Lactobacillus plantarum (BLp), showed the most significant antimicrobial activity, especially against Staphylococcus aureus and Acinetobacter baumannii. Analysis of SDS-PAGE showed that L. plantarum and L. helveticus bacteriocins have a molecular weight of ~10 kDa and ~15 kDa, respectively. L. plantarum (BLp) bacteriocin was heat stable while L. helveticus (BLh) bacteriocin was heat labile. Both bacteriocins have shown activity at acidic pH. Exposure to a UV light enhances the activity of the BLh; however, it had negligible effects on the BLp. Different proteolytic enzymes confirmed the proteinaceous nature of both the bacteriocins. From this study, it was concluded that bacteriocin extracts from L. helveticus (BLh) can be considered a preferable candidate against foodborne pathogens as compared to L. plantarum (BLp). These partially purified bacteriocins should be further processed to attain purified product that could be useful for food spoilage and preservation purposes.

Phylogeny of hospital acquired MRSA, and its comparative phenotypic clinico-epidemiology with vancomycin resistant S. aureus (VRSA).

Staphylococcus aureus is emerging as complicated pathogen because of its wide-ranging origin, multiple variants, and compromised antibiotic susceptibilities. Current study was planned to find lineage of hospital acquired methicillin resistant Staphylococcus aureus (HA-MRSA), and its comparative phenotypic clinico-epidemiology with vancomycin resistant S. aureus (VRSA). A total of (n = 200) samples were aseptically collected from wound, nose, and cerebrospinal fluid of patients from metropolitan and rural background hospitals along with on spot filling in of questionnaire. Phylogenetic analysis of HA-MRSA was identified by targeting mecA gene in S. aureus. At optimal tree branch length of 1.91 and evolutionary distance 0.1, high level sequence similarity (97%-99%) was observed with different strains of S. aureus isolated from both human and animal. Non-descriptive statistics at 5% probability found 61% S. aureus, while 43.44% of them were HA-MRSA, 92.62% VRSA, and 42.62% were both MRSA and VRSA. Among assumed risk factors, use of antibiotics, venous catheterization, chronic disease, pre-hospital visits, and ICU admitted patients showed significant association (p<0.05) with pathogen. HA-MRSA was 37.50%, 80%, and 37.50% sensitive to chloramphenicol, gentamicin, and oxacillin, respectively. While <50% of VRSA were sensitive against oxacillin, enoxacin, and chloramphenicol. A significant difference (p<0.05) of percentage responses of MRSA and VRSA at resistant, intermediate, and sensitive cadre against all antibiotics except chloramphenicol was obvious in this study. The Current study concluded higher prevalence of MRSA & VRSA, significant association of risk factors, limiting antibiotic susceptibility profile, and genetic transfer at animal-human interface which suggests further studies cum preventive strategies to be planned.

Plasmid-mediated mcr-1 gene in Acinetobacter baumannii and Pseudomonas aeruginosa: first report from Pakistan.

INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.

Plasmid-mediated mcr-1 gene in acinetobacter baumannii and pseudomonas aeruginosa: first report from pakistan

Abstract INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.