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

This study aimed to evaluate the prevalence, multidrug-resistance traits, PCR-detection of virulence, and antibiotic-resistance genes of E. coli isolated from secondary infections following FMD-outbreak in cattle. A total of 160 random samples were gathered from private dairy farms in Damietta Province, Egypt. The specimens were subjected to bacteriological examination, serotyping, congo-red binding assay, antibiogram-testing, and PCR-monitoring of virulence-determinant genes (tsh, phoA, hly, eaeA, sta, and lt) as well as the antibiotic-resistance genes (blaTEM, blaKPC, and blaCTX). The prevalence of E. coli was 30% (n = 48) distributed in 8 serogroups (40/48, 83.3%), while 8 isolates (8/48, 16.6%) were untypable. Besides, 83.3% of the examined isolates were positive for CR-binding. The tested strains harbored the virulence genes phoA, hly, tsh, eaeA, sta, and lt with a prevalence of 100% and 50%, 45.8%, 25%, 8.4%, and 6.2%, respectively. Furthermore, 50% of the recovered strains were multidrug-resistant (MDR) to penicillins, cephalosporins, and carbapenems, and are harboring the blaTEM, blaCTX, and blaKPC genes. Moreover, 25% of the examined strains are resistant to penicillins, and cephalosporins, and are harboring the blaTEM and blaCTX genes. To the best of our knowledge, this is the first report concerning the E. coli secondary bacterial infections following the FMD-outbreak. The emergence of MDR strains is considered a public health threat and indicates complicated treatment and bad prognosis of infections caused by such strains. Colistin sulfate and levofloxacin have a promising in vitro activity against MDR-E. coli.

Sampling and clinical examination. One hundred and sixty specimens; milk (n = 40), blood (n = 40), fecal swabs (n = 40), and nasal swabs (n = 40) were randomly collected under complete aseptic conditions from two private cattle farms (native breeds cows of both sexes with average two years old age and with a history of FMD-outbreak) at Damietta Province, Egypt (From March 2019 to August 2019). The sampling was carried out after FMD-outbreak. The examined farms are very close to each other and sharing the same management practices, nutrition, and water supply. The sampling was performed according to the clinical signs. Blood specimens were gathered from animals suffering from fever, milk specimens were collected from clinically mastitic animals, fecal swabs were collected from diarrheic animals, and nasal swabs were collected from animals that exhibited respiratory manifestations. The examined animals were previously treated with trimethoprim and amoxicillin without improvement. The obtained specimens were processed as soon as possible at the same day of collection and were collected on tryptic soy broth (Oxoid, Hampshire, UK).

Isolation and identification of E. coli and other pathogens.
For isolation of E. coli, swabs from the obtained specimens were inoculated in McConkey's broth (Oxoid, Hampshire, UK), followed by incubation for 24 h at 37 °C. A loopful of broth-culture was streaked onto MacConkey's agar, and eosin methylene blue agar (Oxoid, Hampshire, UK). The suspected colonies were identified according to their colonial characters, hemolytic activity, microscopical examination using Gram's staining, motility test, hemolytic activity on blood agar, and biochemical reactions (oxidase, catalase, indole, lactose fermentation, methyl-red, citrate-utilization, H 2 S, Voges-Proskauer, and urease tests) as described by Quinn 19 .
For isolation of other bacterial pathogens, swabs from the processed specimens were inoculated on nutrient agar, blood agar, mannitol salt agar, cetrimide agar, and MacConkey's agar (Oxoid, Hampshire, UK), then the inoculated plates were incubated for 24-48 h at 37 °C. The obtained pure colonies were identified according to their colonial characters, morphological characters, and biochemically as described by Quinn 19 . E. coli serotyping. The retrieved isolates were serotyped for somatic antigen (O-antigen) by the aid of slide agglutination test using standard polyvalent and monovalent commercial E. coli antisera (Denka Seiken-Co., Ltd., Tokyo, Japan) at the Animal-Health Research-Institute, Dokki, Egypt as described by Starr 20 . Congo-red binding. To emphasize the pathogenicity and the invasiveness of the isolated strains, the assessment of congo-red binding was performed on trypticase agar (containing 0.03% CR dye) (Oxoid, UK). The tested strains were inoculated on trypticase agar and then incubated at 37 °C for 24 h. Then plates were preserved at room temperature (for 48 h). The positive result is indicated by the appearance of red colonies as previously reported by Panigrahy and Yushen 21 .
Antimicrobial susceptibility testing. The recovered E. coli strains were assessed for their antimicrobial resistance using the disc diffusion method on Mueller-Hinton agar (Oxoid, UK). The following antimicrobial agents were involved; ampicillin (AMP) (10 μg), meropenem (MEM) (10 μg), amikacin (AK) ( Molecular typing of virulence-determinant genes and antibiotic-resistance genes. PCR-monitoring of virulence-determinant genes (tsh, phoA, hly, eaeA, sta, and lt) and the antibiotic-resistance genes (bla TEM , bla KPC , and bla CTX ) was carried out. The selection of these antibiotic-resistance genes was based upon the results of the antimicrobial susceptibility testing, moreover, the selection of the current virulence genes is based upon their significant role in the pathogenesis of the disease as described in previous studies 9, 10,18 . Genomic DNA of the examined strains was extracted regarding the manufacturer's guidelines of the QIAamp DNA Mini Kit (Qiagen, GmbH, Germany/Catalogue No.51304). The reaction volume was adjusted at 25-μl (3 μl of genomic-DNA, 5 μl of 5 × Master Mix, and 20 pmol of each prime, the reaction volume was completed by adding distilled H 2 O). Positive controls (provided by A.H.R.I, Egypt) and negative controls (DNA-free) were used in all reactions. The sequences of the used primers (Metabion International AG, Germany) and the PCR-cycling conditions are illustrated in Table 1. Finally, the separation of the obtained products was performed using the agar gel electrophoresis (1.5% agarose stained with ethidium bromide 0.5 μg/ml), and the gel was photographed.
Statistical analyses. The Chi-square test was performed to analyse the obtained results (SAS software, version 9.4, SAS Institute, Cary, NC, USA) (significance level; P < 0.05). Furthermore, the correlation analysis was conducted using R software (version 4.0.2; https ://www.r-proje ct.org/), it was calculated using the "cor" function and visualization using the "corrplot" functions from the "corrplot" package.

Results
Prevalence of E. coli and other bacterial pathogens in the examined animals. Regarding the phenotypic characteristics of the retrieved E. coli; isolates were identified as E. coli based on their morphology and biochemical characteristics. Microscopically, the bacteria appeared as Gram-negative moderate size, motile, and non-sporulated rods. The bacteria grew well on MacConkeys's agar and gave characteristic pink colonies due to lactose fermentation. On blood agar, the colonies are hemolytic, moreover on EMB; the bacteria gave characteristic metallic sheen colonies. Biochemically, all isolates were positive for catalase, lactose fermentation, indole, and methyl-red, tests. Simultaneously, they were negative for cytochrome oxidase, Voges-Proskauer, citrate-utilization, H 2 S production, and urease tests. The bacteriological inspection proved that the total prevalence of E. coli was 30% (48/160); the prevalence of E. coli was 28.75% (23/80) in the farm (1), while it was 31.25% (25/80) in the farm (2) as described in Table 2.

The in-vitro multidrug-resistance patterns and the distribution of antibiotic-resistance genes.
Concerning the occurrence of multidrug-resistance phenomena, in the present study, 50% of the recovered strains are multidrug-resistant (MDR) (MDR: non-susceptible to ≥ one agent in ≥ three antimicrobial classes); to penicillins: ampicillin, amoxicillin, and amoxicillin-clavulanic acid; cephalosporins: ceftazidime and cefotaxime; carbapenems: meropenem and imipenem, and are harboring the bla TEM , bla CTX , and bla KPC genes. Moreover, 25% of the examined strains are resistant to penicillins: ampicillin, amoxicillin, and cephalosporins: ceftazidime, and cefotaxime, and are harboring the bla TEM and bla CTX genes. Furthermore, 8.3% of the recovered strains were multidrug-resistant (MDR) to penicillins: ampicillin, and amoxicillin; cephalosprins: ceftazidime, cefotaxime, and aminoglycosides: amikacin, and possessed the bla TEM and bla CTX resistance genes ( Table 7). The correlation analysis performed between various phenotypic multidrug-resistance patterns and the antibioticresistance genes. The obtained results revealed strong positive correlations between: bla CTX gene, CAZ, and CTX (r = 0.99); bla TEM gene, AMX, AMP, and AMC (r = 1); bla KPC gene, MEM, and IMP (r = 1) (Fig. 8).    Table 7. The frequency of the phenotypic multidrug-resistance and the antibiotic-resistance genes among the retrieved strains (n = 48). Characteristics of multidrug resistance (MDR), extensively drug-resistance (XDR), and pandrug-resistance (PDR) in E. coli: PDR non-susceptible to all antimicrobial agents listed, XDR nonsusceptible to ≥ one agent in all but ≤ two antimicrobial classes, MDR non-susceptible to ≥ one agent in ≥ three antimicrobial classes. www.nature.com/scientificreports/

Discussion
Globally, cattle are representing the main supply of high-quality meat and milk. However, few reports explained the role of pathogenic E. coli as a secondary bacterial pathogen following the FMD-outbreaks. The current study was conducted to inspect the prevalence, antibiogram, PCR detection of virulence-determinant genes (tsh, phoA, hly, eaeA, sta, and lt) and the antibiotic-resistance genes (bla TEM , bla KPC , and bla CTX ) of E. coli isolated from secondary bacterial infections following FMD-outbreak in cattle.
The bacteriological assay proved that E. coli was detected in 30% of the examined samples. Besides, other bacterial pathogens were isolated from 112 (70%) examined diseased animals. There is no significant difference in the prevalence of E. coli between the surveyed farms (P > 0.05), as the inspected farms are very close to each other and sharing the same management practices, nutrition, and water supply. E. coli is a common opportunistic microorganism that incriminated in several infections, especially diarrhea, mastitis, septicemia, and respiratory manifestations 11,18,23 . In Nigeria, S. uberis and S. bovis clinical mastitis are also reported by Amosun 31 . In China, the emergence of P. mirabilis as a causative agent of diarrhea was reported by Gong 32 . Moreover, in Nepal, E. faecalis diarrhea was recorded in immune-compromised persons by Sah 33 . El-Seedy 34 reported that P. multocida and M. hemolytica are major pathogens of calf pneumonia in Egypt, while Algammal 11 categorized P. aeruginosa as a common pathogen of pneumonia in calves. In Egypt, although the available FMD-vaccine is efficient to minimize the mortality rate, the vaccination-failure may happen that results in the occurrence of FMD-outbreak and the emergence of secondary bacterial infections due to the immunosuppression 35 . A previous study in Cambodia reported the occurrence of FMD-vaccination failure in more than 50% of the vaccinated animals. The vaccination failure is mainly attributed to improper technique, insufficient dose, immunological factors, and vaccine coldchain miscarriage 36 . Several causes are implicated in the existence of E. coli secondary infection, including; bad sanitation, intensive-breeding management, bad environmental conditions, stress, and weak animal immunity 18 .
Concerning the E. coli serovars, the most prevalent E. coli serovar accompanied the respiratory infection was 086a (n = 4), diarrhea: O114 (n = 7), fever: O111 (n = 5), mastitis: O1 (n = 9). The investigation of E. coli O-serogroups has a major public health concern. The recovered serovars are analogous to those reported by previous studies, which concerned the E. coli infections [37][38][39] . In the present study, the CR-binding assay proved that 83.3% of the examined isolates (40/48) were CR-binding positive. All the tested serovars were positive. Moreover, the untypable strains were negative (8/48). The current results agreed with Algammal 18 , who reported that 89.8% of the tested strains are invasive by congo-red binding assay, which confirms the pathogenicity of these isolates.
Regarding the in-vitro antimicrobial susceptibility testing, the retrieved strains exhibited a remarkable resistance to penicillins, cephalosporins, and carbapenems which gave a public health alarm. The current findings nearly agreed with those reported by Shahrani 40 ,Gupta 41 , and Touwendsida 42 . The uncontrolled widespread use of antibiotics in veterinary and health sectors as well as the bacterial antibiotic-resistant genes are incriminated in the development of such multidrug-resistant strains 43,44 . Regrettably, E. coli is capable to resist various antibioticclasses due to possessing resistant genes and/ or R-plasmids 45 .
In the current study, the PCR proved that the recovered E. coli strains were found to posse 2-5 virulence genes. The most prevalent virulence genes accompanied the respiratory infections are phoA and hly genes, in diarrhea: phoA, sta, lt, eaeA, and hly genes, in fever and mastitis: phoA, tsh, and hly genes. These findings agreed with those obtained by previously reported by Algammal 18 , Andrade 46 , and Whitelegge 47 . The pathogenesis of virulent E. coli is controlled by multiple virulence determinants that vary among different pathotypes. The most common virulence determinants that accompanied the E. coli-pathotypes are enterotoxins, hemolysins, www.nature.com/scientificreports/ siderophores, intimin, fimbria-mannose binding type1-H adhesion, alkaline phosphatase, and temperaturesensitive haemagglutinin (Tsh-protein). Furthermore, the production of these virulence-determinants is regulated by the expression of specific virulence genes 48,49 .
In the present study, 50% of the recovered strains are MDR to penicillins, cephalosporins, and carbapenems, and are harboring the bla TEM , bla CTX , and bla KPC genes. Furthermore, 25% of the examined strains are resistant to penicillins and cephalosporins, and are harboring the bla TEM and bla CTX genes. The Extended Spectrum β-lactamases (ESBLs) produced by E. coli incriminated in the β-lactam-antibiotic resistance. The heavy use of penicillin, cephalosporins, and carbapenems-antibiotics in medications is resulting in the evolution of multidrug-resistant strains. The resistance to the β-lactam-antibiotics is mainly mediated by the ESBL-genes; bla TEM , bla CTX , and bla KPC which are encoded for penicillin, cephalosporins, and carbapenem-resistance, respectively [50][51][52][53] . Different mechanisms explain the emergence of MDR-E. coli strains include: 1-Shared resistance mechanisms; occur especially for the antimicrobial agents in the same category due to penicillin-binding protein mutations as well as the β-lactamases. Furthermore, it could happen for different antibiotics in various classes due to the efflux pumps acting on numerous drugs in different species. 2-Linkage among the antibiotic resistance genes, this mechanism plays a significant role in association links between various resistances and to differentiate between resistance mechanisms (either the resistance arise due to alterations in the target protein of the antibiotic or due to a resistance gene encoded for an enzyme that destroys the antibiotic). 3-Correlated drug exposure of the host, it mainly occurs due to routine use of combination therapy and the repeated treatment failure [54][55][56][57][58][59] .
Limitations and future recommendations: Future work is recommended to perform phylogenetic analysis either by MLST or PFGE to understand the clonal relatedness of the obtained strains.
In conclusion, to the best of our knowledge, this is the first report concerning the E. coli secondary bacterial infections following the FMD-outbreak. The immunosuppression due to the FMD increases the animal susceptibility to E. coli secondary infections. The most prevalent E. coli serovar associated the respiratory infections was O86a, in diarrhea: O114, in fever: O111, and in mastitis: O1. Furthermore, the most predominant virulencedeterminant genes accompanied the E. coli respiratory infections were phoA and hly genes, diarrhea: phoA, sta, lt, eaeA, and hly genes, fever, and mastitis: phoA, tsh, and hly genes. A high percentage of the isolated E. coli strains were multidrug resistant (MDR) to penicillins: ampicillin, amoxicillin, and amoxicillin-clavulanic acid; cephalosporins: ceftazidime and cefotaxime; carbapenems: meropenem and imipenem, and are harboring the bla TEM , bla CTX , and bla KPC genes. In-vitro, colistin sulfate and levofloxacin have promising activity against MDR-E. coli. The emergence of highly pathogenic MDR-E. coli strains constitutes a significant threat to the cattle health resulting in multiple severe infections and huge economic losses in the livestock production. Furthermore, the evolution of penicillins, cephalosporins, and carbapenems-resistant strains is reflecting a public health alarm and specifies the convoluted treatment of the infections caused by these strains. Moreover, it recommends the proper use of antimicrobial agents in the veterinary and health sectors as well as the routine application of the antimicrobial susceptibility testing.