Detection of Antibiotic Resistance Genes in Vibrio cholerae isolated from Human Fecal samples during a Diarrheal Outbreak in Peshawar, Pakistan

Abstract Vibrio cholerae , the etiological agent of cholera, remains a significant public health concern in several regions of Khyber Pakhtunkhwa, Pakistan. Cholera is characterized by acute watery diarrhea, which can lead to severe dehydration and death if left untreated. Contributing factors to outbreaks in Khyber Pakhtunkhwa include poor sanitation, limited access to clean drinking water, and overcrowded living conditions. This study aimed to determine the prevalence, antimicrobial susceptibility patterns, and molecular characterization of V. cholerae during a diarrheal outbreak in Peshawar, Pakistan, in July 2023. A total of 45 stool samples were collected from patients suffering from diarrhea. Samples were cultured on Thiosulfate-Citrate-Bile-Sucrose agar. Isolates were identified using standard biochemical tests, followed by molecular confirmation through PCR amplification of the 16S rRNA gene. Biochemical tests confirmed 34 (75.55%) isolates as V. cholerae . The highest infection rate was observed in the age group of ≤ 18 years (64.70%), with a higher frequency in male patients (79.41%). Antibiotic susceptibility test using the Kirby-Bauer disc diffusion method revealed multidrug resistance in 13 isolates, showing resistance to at least three distinct antibiotic classes. Sequencing and phylogenetic analysis of the 16S rRNA gene confirmed the multidrug resistant isolates as V. cholerae . The isolates exhibited high resistance to ERY (100%), LVX (100%), AZM (92.85%), TET (88.89%), SXT (80%), AMP (75%), and CRO (75%). Conversely, CIP (53.84%), AMK (61.53%), and MEM (81%) showed the highest susceptibility. Statistical analysis revealed no significant association between V. cholerae infection and age groups. The molecular analysis of Vibrio cholerae isolates revealed a significant prevalence of antibiotic resistance genes linked to various antibiotics classes, emphasizing the multi-drug resistant characteristics of Vibrio cholerae . PCR-based molecular characterization of multi-drug resistant isolates demonstrated higher prevalence of resistance genes including dfrA1 (92.3%), sul1 (92.3%), sul2 (76.9%), tetA (76.9%), blaTEM (84.6%), blaCTX-M (69.2%), blaOXA (61.5%), blaSHV (38.5%), and mcr-1 (53.8%). The identification of mcr-1 is especially alarming because of its correlation with COL resistance. The findings highlight the alarming pattern of multidrug resistance in V. cholerae isolates from Peshawar, emphasizing the need for effective surveillance and control measures to combat the spread of antibiotic resistant strains.