Virulence-determinants and antibiotic-resistance genes of MDR- *E. coli* isolated from secondary infections following FMD-outbreak in cattle

Foot-and-mouth disease (FMD) is a highly contagious disease of ruminants causing significant economic losses globally. In Egypt, serotypes A, O, and SAT2 are endemic and outbreaks remain frequent despite vaccination. Vaccination can be associated with immunosuppression, predisposing animals to secondary bacterial infections. Escherichia coli is an opportunistic pathogen of the intestinal tract implicated in various secondary infections in animals. Its pathogenicity is mediated by virulence factors including hemolysins, enterotoxins (LT, ST), Shiga toxins, intimin, adhesins (fimH), alkaline phosphatase (phoA), and temperature-sensitive hemagglutinin (tsh), encoded by specific genes (hly, lt, sta, stx1/2, eaeA, fimH, phoA, tsh). Pathotypes include enterotoxigenic, enteropathogenic, enteroinvasive, enteroaggregative, and Shiga-toxigenic E. coli with distinct mechanisms of disease. Widespread use of β-lactam antibiotics (penicillins, cephalosporins, carbapenems) has selected for ESBL-producing E. coli carrying genes such as blaTEM, blaCTX, and blaKPC, contributing to multidrug resistance (MDR). This study investigated the prevalence, antimicrobial resistance, and PCR detection of key virulence and β-lactam resistance genes in E. coli isolated from secondary infections following an FMD outbreak in cattle.

The paper contextualizes E. coli pathogenesis and pathotypes, detailing how virulence factors—hemolysins (hly), enterotoxins LT and ST (lt, sta), Shiga toxins (stx1, stx2), intimin (eaeA), fimbrial adhesins (fimH), alkaline phosphatase (phoA), and temperature-sensitive hemagglutinin (tsh)—govern disease. It distinguishes intestinal and extra-intestinal pathotypes and their mechanisms (adhesion, invasion, toxin production). It also reviews the global predominance of β-lactams and the role of ESBLs in resistance, highlighting resistance genes blaTEM (penicillins), blaCTX (cephalosporins), and blaKPC (carbapenems). Prior reports have documented emergence of virulent MDR E. coli in animal settings, underscoring public health relevance.

Ethics: Procedures approved by the Animal Ethics Review Committee of Suez Canal University (AERC-SCU), Egypt, with trained personnel handling animals per guidelines. Sampling and clinical examination: From March–August 2019, 160 specimens (milk n=40 from clinical mastitis; blood n=40 from febrile animals; fecal swabs n=40 from diarrheic animals; nasal swabs n=40 from animals with respiratory signs) were collected aseptically from two nearby private cattle farms in Damietta Province, Egypt, with history of FMD outbreak. Animals had prior unsuccessful treatment with trimethoprim and amoxicillin. Samples were transported/processed same day in tryptic soy broth. Isolation and identification: Swabs enriched in MacConkey’s broth (24 h, 37°C), then streaked on MacConkey’s agar and EMB agar. Identification based on colonial morphology (lactose-fermenting pink colonies on MacConkey’s; metallic sheen on EMB), hemolysis on blood agar, Gram stain (Gram-negative rods), motility, and biochemical tests (catalase+, indole+, methyl red+; oxidase−, VP−, citrate−, H2S−, urease−). Other pathogens were isolated on nutrient, blood, mannitol salt, cetrimide, and MacConkey’s agars and identified by standard methods. Serotyping: O-antigen serogrouping via slide agglutination with polyvalent/monovalent antisera (Denka Seiken) at Animal Health Research Institute, Dokki, Egypt. Congo red binding assay: Growth on trypticase agar containing 0.03% Congo red at 37°C for 24 h; plates held 48 h at room temperature; red colonies interpreted as positive (pathogenic/invasive phenotype). Antimicrobial susceptibility testing: Disc diffusion on Mueller–Hinton agar per CLSI using AMP (10 µg), AMX (10 µg), AMC (30 µg), CTX (30 µg), CAZ (30 µg), IMP (10 µg), MEM (10 µg), AK (30 µg), LEV (5 µg), CT (10 µg), SXT (19:1 µg). E. coli ATCC 25922 as control. Classification into MDR/XDR/PDR per Magiorakos et al. PCR detection: Genomic DNA extracted (QIAamp DNA Mini Kit). PCR assays targeted virulence genes (tsh, phoA, hly, eaeA, sta, lt) and β-lactam resistance genes (blaTEM, blaCTX, blaKPC) using published primers and cycling conditions (amplicon sizes: lt 605 bp; sta 299 bp; eaeA 248 bp; tsh 620 bp; phoA 720 bp; hly 1177 bp; blaKPC 882 bp; blaCTX 593 bp; blaTEM 516 bp). Positive (A.H.R.I., Egypt) and negative (no template) controls included. Products resolved on 1.5% agarose with ethidium bromide. Statistics: Chi-square for prevalence and resistance comparisons (SAS 9.4), significance P<0.05. Correlation analyses using R (corrplot) for relationships among antibiotics, genes, and MDR phenotypes.

- Overall E. coli prevalence: 30% (48/160). Farm-level prevalence: 28.75% (23/80) vs 31.25% (25/80), not significantly different (P>0.05). - By sample type: milk 42.5% (17/40), blood 27.5% (11/40), nasal swabs 17.5% (7/40), fecal swabs 32.5% (13/40). - Co-infections: 70% (112/160) of diseased animals yielded other bacterial pathogens (e.g., Streptococcus uberis 25% of mastitis samples, P. aeruginosa 25% in fever and 10% in respiratory cases, Proteus mirabilis 42.5% in diarrheic samples, etc.). - Serogroups (40/48 typed; 83.3%): O1 (18.7%), O114 (14.6%), O111 (10.4%), O18 (8.4%), O26 (8.4%), O55 (8.4%), O86a (8.4%), O158 (6.2%); untypable 16.6%. Distribution by syndrome: respiratory O86a; diarrhea O114; fever O111; mastitis O1 (P<0.05 across sample types). - Congo red binding: 83.3% (40/48) positive; all typed serovars positive; untyped strains negative. - Antimicrobial susceptibility: 100% resistance to ampicillin and amoxicillin; 60.4% resistance to amoxicillin–clavulanic acid; 83.3% resistance to cefotaxime and ceftazidime; 50% resistance to imipenem and meropenem; high susceptibility to colistin (100%), levofloxacin (93.8%), and amikacin (56.2% susceptible). Significant differences across antibiotics (P<0.0001). Positive correlations observed among several antibiotic responses (e.g., AK–LEV r=0.83; AK–CT r=0.86; IMP/MEM with multiple β-lactams r≈0.5–0.54). - Virulence genes: phoA 100% (48/48), hly 50% (24/48), tsh 45.8% (22/48), eaeA 25% (12/48), sta 8.4% (4/48), lt 6.2% (3/48). Significant differences in prevalence (P<0.0001). - Resistance genes: blaTEM 100% (48/48), blaCTX 83.3% (40/48), blaKPC 50% (24/48) (P<0.0001). - Gene correlations: strong positive associations including blaCTX/blaTEM with phoA (r=0.95), tsh with sta (r=0.72), eaeA with blaTEM and phoA (r=0.69), and hly with blaKPC (r=0.6) and blaCTX (r=0.59). - MDR phenotypes: 50% (24/48) were MDR to penicillins, cephalosporins, and carbapenems and carried blaTEM, blaCTX, blaKPC; 25% resistant to penicillins and cephalosporins carrying blaTEM and blaCTX; 8.3% MDR including aminoglycoside (amikacin) resistance with blaTEM and blaCTX. Strong correlations between phenotypic resistance patterns and corresponding genes (e.g., blaKPC with MEM/IMP r=1; blaTEM with AMX/AMP/AMC r=1; blaCTX with CAZ/CTX r=0.99). - Therapeutic implication: colistin sulfate and levofloxacin showed promising in vitro activity against MDR E. coli.

The study demonstrates that cattle affected during/after an FMD outbreak are frequently secondarily infected with E. coli, likely facilitated by immunosuppression associated with FMD and its vaccination context. The isolates showed high rates of resistance to β-lactams, including cephalosporins and carbapenems, consistent with carriage of ESBL and carbapenemase genes (blaTEM, blaCTX, blaKPC). The significant presence of virulence determinants (e.g., phoA universally, hly, tsh, eaeA) suggests pathogenic potential across different clinical syndromes (mastitis, diarrhea, respiratory disease, septicemia). The serogroup distribution aligns with syndrome-specific patterns (e.g., O1 with mastitis, O114 with diarrhea). The strong genotype–phenotype correlations confirm that the resistance genes measured largely explain the observed β-lactam resistance profiles. Clinically, the susceptibility to colistin and high susceptibility to levofloxacin indicate potential treatment options for MDR infections, though stewardship and caution are warranted. These findings underscore the public health implications of MDR E. coli in livestock and the need for prudent antibiotic use and routine susceptibility testing.

This first report of E. coli secondary bacterial infections following an FMD outbreak in cattle in Egypt shows a 30% prevalence of E. coli with diverse serogroups and high virulence gene carriage. Half of isolates were MDR to penicillins, cephalosporins, and carbapenems, carrying blaTEM, blaCTX, and blaKPC, posing treatment challenges and public health concerns. Colistin sulfate and levofloxacin retained strong in vitro efficacy against MDR isolates. The study highlights the necessity of antimicrobial stewardship, regular susceptibility testing, and improved farm management to mitigate secondary infections during FMD outbreaks. Future work should include molecular epidemiology (e.g., MLST or PFGE) to elucidate clonal relatedness and transmission dynamics.

The study did not perform phylogenetic or clonal relatedness analyses (e.g., MLST, PFGE) of the E. coli isolates, limiting insights into transmission and population structure. Additionally, the work is limited to two farms in one province and a defined post-outbreak period, which may affect generalizability. In vivo treatment outcomes were not evaluated; susceptibility results are in vitro.