Targeting Antimicrobial Resistance in Salmonella Enteritidis: Synergistic Action of N-acetylcysteine with Florfenicol and CRISPR-Cas System Involvement

Salmonella enterica subsp.enterica serovar Enteritidis (SE) is one of the significant public health threats, mainly due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains.Along with several known factors, such as irrational antibiotic use, horizontal gene transfer, and the possible involvement of the CRISPR-Cas system, these have necessitated the exploration of alternative treatment strategies.The present study aimed to identify the antibiogram of SE isolates from poultry and evaluate the synergistic effects of florfenicol (FF) and N-acetylcysteine (NAC) against MDR SE as well as explore the interplay of the CRISPR-Cas system in the emergence of AMR.Two hundred and seventy-one samples were collected.The samples included both cloacal and fecal samples from poultry birds in the Bahawalpur and Lahore Regions of Punjab, Pakistan.Identification and isolation were performed through conventional bacteriological methods followed by PCR.Phenotypic assays, including minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), mutant prevention concentration (MPC), and fractional inhibitory concentration index (FICI), were performed.Genotypic confirmation was performed by identifying AMR genes.Out of 271 samples, thirty (n=30) were confirmed positive for SE with IE gene-based PCR.Results revealed that the isolates were resistant against most of the antibiotics including, (86.67%),AMP (96.67%),AMX (96.67%),CTX (83.33%),GEN (83.33%),CH (60%), CLI (90%), TET (96.67%),SMZ/TMP (66.67%),VAN (56.67%),ENR (63.33%),CIP (53.33%), and CO (96.67%).Conversely, FF was effective against 56.67% of isolates.All of the resistant isolates (n = 30) exhibited the presence of antimicrobial resistance genes, including mphA, aac(3)-IIa, tetA, Sul1, gyrA, floR, qnrA, and mcr-1.The co-administration of NAC with FF demonstrated a significant synergistic effect against XDR SE isolate and the MIC of FF was notably reduced when combined with NAC (FICI; 0.2 to 0.5).Additionally, a significant (P<0.05)downregulation of CRISPR-Cas 3 genes was also recorded through qRT-PCR in both resistant and ATCC13076 strains after treatment with co-administration of FF and NAC compared with isolates treated with FF alone.It was concluded that the combination of NAC enhances the antimicrobial activity of FF, reduces biofilm formation, and potentially mitigates the antimicrobial resistance associated with the CRISPR-Cas system.