Foot-and-mouth disease (FMD) is a highly contagious disease impacting ruminants, causing significant economic losses globally due to treatment, vaccination, and production losses. In Egypt, despite vaccination programs, FMD outbreaks remain prevalent. FMD vaccination can induce immunosuppression, increasing susceptibility to secondary bacterial infections. *Escherichia coli* (*E. coli*), an opportunistic pathogen inhabiting the intestinal tracts of humans and animals, is frequently implicated in these secondary infections. *E. coli* pathogenicity is determined by several virulence factors, including hemolysins, enterotoxins, Shiga toxins, intimin, fimbriae, alkaline phosphatase, and Temperature Sensitive Hemagglutinin (Tsh-protein), encoded by genes such as *hly*, *lt*, *sta*, *stx1*, *stx2*, *eaeA*, *fimH*, *phoA*, and *tsh*, respectively. *E. coli* is categorized into intestinal and extraintestinal pathotypes, with virulent strains exhibiting various pathotypes (enterotoxigenic, enteropathogenic, enteroinvasive, enteroaggregative, and Shiga-toxigenic). β-lactam antibiotics (cephalosporins, carbapenems, and penicillins) constitute a significant portion of antimicrobial agents used globally, and the emergence of multidrug-resistant (MDR) *E. coli* poses a serious public health threat, often mediated by Extended-Spectrum Beta-Lactamases (ESBLs) encoded by genes like *bla_TEM*, *bla_KPC*, and *bla_CTX*. This study aimed to investigate the prevalence, antibiogram, and the presence of virulence genes (*tsh*, *phoA*, *hly*, *eaeA*, *sta*, *lt*) and antibiotic resistance genes (*bla_TEM*, *bla_KPC*, *bla_CTX*) in *E. coli* isolates from secondary bacterial infections following an FMD outbreak in cattle.

The literature review section extensively cites previous research on FMD outbreaks, *E. coli* pathogenicity and its various pathotypes, the role of virulence factors and their associated genes, the global prevalence of β-lactam antibiotic resistance in *E. coli*, and the public health implications of MDR strains. Studies on the specific virulence genes (*tsh*, *phoA*, *hly*, *eaeA*, *sta*, *lt*) and antibiotic resistance genes (*bla_TEM*, *bla_KPC*, *bla_CTX*) in *E. coli* are referenced to justify their selection in this study. The review also notes existing data on the prevalence of various secondary bacterial pathogens, including those identified in this study, in different geographical locations and animal populations. This provides a comprehensive background for understanding the significance and context of the current study.

This study involved collecting 160 samples (milk, blood, fecal, and nasal swabs) from two private cattle farms in Damietta Province, Egypt, experiencing an FMD outbreak. Samples were cultured on McConkey's broth and agar, and eosin methylene blue agar for *E. coli* isolation. Identification was confirmed through colonial morphology, Gram staining, hemolytic activity, and biochemical tests. *E. coli* serotyping was conducted using slide agglutination with commercial antisera. The Congo-red binding assay determined invasiveness. Antimicrobial susceptibility testing (disc diffusion method) was performed using various antibiotics commonly used in Egypt's veterinary and human health sectors. PCR was used to detect virulence genes (*tsh*, *phoA*, *hly*, *eaeA*, *sta*, *lt*) and antibiotic resistance genes (*bla_TEM*, *bla_KPC*, *bla_CTX*) using specific primers (Table 1). Genomic DNA extraction was performed using the QIAamp DNA Mini Kit. Statistical analyses (Chi-square test and correlation analysis using SAS and R software) were employed to assess statistical significance. Animal handling and procedures were approved by the Animal Ethics Review Committee of Suez Canal University.

*E. coli* was detected in 30% (48/160) of the samples, with prevalence varying slightly between the two farms (no statistically significant difference). Other bacterial pathogens were also isolated from 70% (112/160) of the diseased animals, including *S. uberis*, *S. bovis*, *E. faecalis*, *P. aeruginosa*, *M. hemolytica*, *P. multocida*, and *P. mirabilis*. The *E. coli* isolates belonged to 8 O-serogroups (O1, O114, O111, O18, O26, O55, O86a, O158), with 16.6% untypable. 83.3% of *E. coli* isolates were positive for Congo-red binding. High resistance was observed to penicillins (100%), cephalosporins (83.3%), and carbapenems (50%). Colistin sulfate (100%) and levofloxacin (93.8%) showed high susceptibility. All isolates harbored the *phoA* gene, while other virulence genes (*hly*, *tsh*, *eaeA*, *sta*, *lt*) showed varying prevalences (Table 5). Antibiotic resistance genes (*bla_TEM*, *bla_CTX*, *bla_KPC*) were also detected (Table 5). Significant correlations were found between various virulence genes and antibiotic resistance genes. 50% of isolates were MDR, exhibiting resistance to penicillins, cephalosporins, and carbapenems and harboring *bla_TEM*, *bla_CTX*, and *bla_KPC* genes (Table 7).

This study is the first to report on *E. coli* secondary infections following FMD outbreaks, indicating the potential contribution of immunosuppression from FMD vaccination failure to increased susceptibility. The findings highlight a significant prevalence of MDR *E. coli*, particularly resistant to penicillins, cephalosporins, and carbapenems. The high prevalence of virulence genes underscores the potential for severe infections. The observed correlations between virulence genes and antibiotic resistance genes suggest a potential link between pathogenicity and resistance. The in vitro efficacy of colistin sulfate and levofloxacin against MDR *E. coli* provides valuable insights for treatment strategies. However, the widespread use of antibiotics is implicated in the development of these MDR strains, necessitating prudent antibiotic use and routine antimicrobial susceptibility testing. The predominance of specific *E. coli* serogroups in different infection types warrants further investigation into their specific pathogenic mechanisms.

This study provides the first evidence of *E. coli* secondary infections following FMD outbreaks, emphasizing the importance of considering this opportunistic pathogen during FMD control strategies. The high prevalence of MDR *E. coli* with multiple virulence genes represents a significant threat to cattle health and necessitates responsible antibiotic stewardship. Future research should investigate the phylogenetic relationships of the isolates, explore the molecular mechanisms underlying the observed correlations between virulence and resistance genes, and evaluate in vivo treatment effectiveness of the promising antibiotics.

The study is limited to two farms with similar management practices, potentially limiting the generalizability of findings to other farms or regions. The study utilized in vitro antimicrobial susceptibility testing, which may not perfectly reflect in vivo efficacy. Further studies are needed to assess the long-term consequences of these infections and to further evaluate the impact of various virulence determinants on the development of multidrug resistance. The study did not include genetic analysis to establish the clonal relatedness of the isolates, which could provide more detailed epidemiological information.