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Relationship between Phenotypic and Genotypic Antimicrobial Resistance of Escherichia coli Isolates from Mastitic Milk

| Bovine mastitis is the predominant problem in dairy farms worldwide which caused mainly by Escherichia coli (E. coli) as one of the main causes of what is called “environmental mastitis”. A total of 68 E. coli isolates from 205 raw milk samples of Holstein cows with mastitis in different dairy farms from different governorates by bacteriological isolation and 63 by PCR were investigated for the E. coli 16S rRNA and rfbEO157 encoding gene as Shiga toxin-producing E. coli (STEC). The occurrence of E. coli O157 in mastitic cows was 3% within E. coli isolates. Molecular investigation of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (PABLs) encoding genes reported in all of the isolates (100%) encoded TEM-type ESBLs, none of which (0%) encoded OXAtype ESBLs, on the other hand, CTX-M-type ESBLs and SHV-type β-lactamases were encoded in 34/63 (53.9%) and 3/63 (4.7%) of the ESBL isolates, respectively and 27% exhibited CMYІІ-type PABLs. Plasmid-mediated colistin resistance encoding gene (mcr-1) was expressed in 1.6% of E. coli isolates. All E. coli isolates exhibited antibiotic multiresistances with higher resistance to tetracycline and Trimethoprim-Sulfamethoxazole (45.7% and 37.3%, respectively), while the lowest resistance was observed for Amoxicillin/clavulanate (10.1%). Phenotypic resistance to extendedspectrum cephalosporins (ESCs) revealed that 42.3% of these strains were resistant to (cefotaxime and cefquinome), 15% resistant to Cefoxitin, while 32.2% were resistant to ceftazidime. Conclusively, E. coli was found to be the major cause of bovine mastitis treatment failure due to the multidrug resistance to most newly developed cephalosporins (third and fourth generations).

Clinical mastitis can be caused mainly by coliform infections (De Vliegher et al., 2012). A wide range of systemic disease severity, from mild to severe with systemic signs including dehydration, shock, and even death (Wenz et al., 2001). As well as zoonotic public health impact on human especially Shiga-toxigenic E. coli (STEC) strains including O157 causing bovine mastitis (Lin et al., 2011).
The misuse of antibiotics caused drug resistance and treatment failures in many cases, especially for multidrugresistant bacteria (Suojala et al., 2013;Sweeney et al., 2018). Carattoli (2008) has announced the antimicrobial resistant E. coli strains increase within animals and claimed these animals to be a reservoir of such strains for humans and the environment. Potential transmission of resistant E. coli within animals and humans can occur through various pathways, as the food chain (Poirel et al., 2018).
Extended-spectrum β-lactamases (ESBLs) producing E. coli, which shows resistance to penicillins, aminopenicillins, and cephalosporins, including the third (ceftiofur) and fourth (cefquinome) generations, has been commonly isolated from food-producing animals with global veterinary and public health issues (Seiffert et al., 2013;Poirel et al., 2018). ESBLs that inactivates ESCs were graded as class A (TEM, SHV and CTXM) and class D (OXA) β-lactamases, While plasmid AmpC β-lactamases (PABLs) belonged to class C (CMYII) confer resistance to a wide variety of β-lactams, primarily 7-a-methoxycephalosporins (Cephamycins) such as cefoxitin (Livermore and Woodford, 2006;Jacoby, 2009). Antibiotics used for humans and animals are closely related, abuse of these drugs resulted in the development of multidrug-resistant bacteria (Cantas et al., 2013;Walther et al., 2017). So, for efficient control and treatment of mastitis; the causative agents of IMI in dairy herds need to be well-identified. Antimicrobial susceptibility determined in vitro has been considered as a pre-requisite for treatment. However, in vitro activity does not guarantee in vivo effectiveness in bovine mastitis treatment (Pyörälä, 2009).
Colistin, a member of polymyxins (polymyxin E), is the main drug for E. coli (Kempf et al., 2013;Poirel et al., 2017). But, Colistin resistance was identified due to the emergence of highly transmissible plasmid-mediated colistin-resistant (mcr-1) gene in E. coli strains obtained from animals, food, and patients from China (Liu et al., 2016). This resistance has created global issues due to the high transmission rate of the mcr-1 gene to epidemic strains of Enterobacteriaceae and thus hinders the effectiveness of colistin in humans (Rebelo et al., 2018).
The objectives of this study were to identify the impact of multidrug resistance development of E. coli strains isolated from mastitic dairy cow's milk, evaluate phenotypic antibiotic resistance profile of isolated strains and their association to genotypic antimicrobial resistance to provide efficient treatment.

SamplE CollECtion
205 pooled milk samples were collected using the California mastitis test (CMT) from 205 mastitic dairy cows from five dairy farms located in Fayoum, Ismailia, El-sharkia, Alexandria and Giza governorates between November 2019 and October 2020. Milk samples (approximately 15 ml) were aseptically drawn from each cow immediately according to the National Mastitis Council, 1990 then samples were transferred to the laboratory for further examination.

phEnotypiC idEntiFiCation
Milk samples were cultured in Eosin Methylene Blue agar media (EMB) (Oxoid). Agar plates were incubated at 37ºC, and the bacterial growth was evaluated after 24 and 48 hrs. Using phenotypic differentiation of bacterial species presumptively based on colony morphology and Gram's staining (David, 2011).

GEnotypiC idEntiFiCation
The genomic DNA of all E. coli strains was extracted (Kang et al., 2004) and stored at -20°C for detection of genes encoding for 16srRNA, rfbEO157 encoding virulence gene and antibiotic resistance genes of E. coli strains isolated from mastitic milk samples (Table 1).
PCR amplification of 16srRNA encoding gene was performed according to Wang et al. (2002) as illustrated in Table 1. The reaction was performed in a volume of 25 μl containing 12.5 μl of 2X Qiagen Multiplex PCR Master Mix (Qiagen GmbH, Hilden, Germany), 0.5 µl (10pmol/ µl) concentrations of each primer, and 3 μl of DNA template. The amplified PCR products were subjected to electrophoresis using 1.5% agarose gel.

Advances in Animal and Veterinary Sciences
August 2021 | Volume 9 | Issue 8 | Page 1225

ESC RESiStant E. Coli iSolatES idEntiFiCation
ESC E. coli isolates were determined by resistance to one or more third and fourth generation cephalosporins (CDC, 2020).

antimiCRobial SuSCEptibility tESt
Antibiotic susceptibility test of E. coli isolates against nine different antibiotics was performed according to the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (Bauer et al., 1966). The susceptibility of the E. coli isolates against each antimicrobial agent was measured and readings have been noted and compared with the Clinical and Laboratory Standards Institute guidelines (CLSI, 2020) ( Table 2). The proved 63 E. coli strains were then subjected for detection of E. coli O157 virulence gene, where only two STEC strains having rfbEO157 encoding gene had been detected using uniplex PCR at a percentage of 3.2% (Figure 1 & 2). Shiga toxin-producing E. coli (STEC) strains considered to be the most important pathogens of a recently emerged group of food-borne strains in the milk of infected cows. This type of strain has been associated with outbreaks of diarrhoea, gastroenteritis and hemorrhagic colitis (HC) or the hemolytic uremic syndrome (HUS) in humans (Karmali, 1989;Paton and Paton, 1998;Beutin et al., 2004). It is agreed with Hassan et al. (2012) who recorded that STEC strains can induce bovine mastitis and reduce milk quality for human consumption because some of the mastitis cases are subclinical and the diagnosis is based solely on accurate diagnostic tests.

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August 2021 | Volume 9 | Issue 8 | Page 1226     21 E. coli isolates demonstrated phenotypic resistance to cefotaxime (CTX) and were encoding for the bla CTX gene. But, only 4 isolates showed phenotypic resistance although they lack bla CTX resistance gene (Table 3 & 4) & ( Figure  3 & 4). The phenotypic resistance to ESCs antibiotics as (Cefotaxime, Cefquinome and Ceftazidime) was increased, due to their extensive and widespread use in veterinary medicine as mentioned by Ahmed and Shimamoto, (2015) when they declared that ESCs (3 rd and 4 th generation Cephalosporins) are necessary antibiotics used in vet

Advances in Animal and Veterinary Sciences
August 2021 | Volume 9 | Issue 8 | Page 1227    (Figure 3 & 4). Chirila et al. (2017) and Poirel et al. (2018) declared that E. coli may develop resistance to antimicrobials by chromosomal genes mutation or by horizontal gene transfer of resistance genes within commensal and pathogenic E. coli strains, rendering E. coli as a major reservoir of resistant genes that could be responsible for human and veterinary treatment failure.
There was a significant increase in isolates with resistance genes and exhibit ESC resistance as isolates carried bla TEM, bla TEM + bla CTXM and bla TEM + bla CTXM + bla CMYІІ . In addition, ESC susceptible isolates also bear resistance genes such as bla TEM +, bla TEM + bla CTXM + bla CMYІІ , bla TEM + bla CTXM and bla TEM + bla CMYІІ (Table 6). ESC resistant E. coli strains were determined according to their resistance to one or more of

Advances in Animal and Veterinary Sciences
August 2021 | Volume 9 | Issue 8 | Page 1229   Livermore et al. (2007) and Seiffert et al. (2013) when it was noted that the worldwide evolution of β-lactamase CTX (Cefotaximase) has been identified and is known to be the most common cause of ESC resistance in the Enterobacteriaceae.
Concerning the antimicrobial susceptibility profile; Seven E. coli isolates (11.8%) were susceptible to all antimicrobials and only two isolates (3.4%) were resistant to all antimicrobials, eleven isolates (18.6%) expressed resistance to a single compound, and 36 isolates (61%) showed resistance to more than one antimicrobial agent. Whereas, twelve isolates (20.3%) were expressing resistance to 3 related compounds (i.e., Cefotaxime, Cefquinome and Ceftazidime) as extended spectrum cephalosporins (

CoNCLUSIoN
The emergence of antimicrobial resistance in particular to the recently introduced antimicrobials such as 3 rd and 4 th generations of cephalosporins in E. coli strains attributed to antimicrobial misuse in dairy farms for bovine mastitis therapy. E. coli strains acquire antimicrobial resistance through plasmid-mediated transfer leading to a widespread of multidrug resistance to ESCs, β-lactams and non-βlactams antibiotics that can induce treatment failure in dairy farms.

ACKNoWLEDGMENTS
The authors would like to thank Dr. Mohamed Ibrahim, farm managers, staff and all helped with this work.

CoNFLICT oF INTEREST
There are no conflicts of interest in this report, according to both of the contributors.

AUTHoR CoNTRIBUTIoNS
Adel Abdel-Azim Fayed approved of and arranged the tests. Soliman Mohamed Soliman created the hypothesis, computed it, and analyzed the results. The experiment was carried out by Samah El-sayed Mahmoud, and the manuscript was written with input and help from all contributors. Adel Abdel-Azim Fayed and Samia Abd El-Hamid Ahmed created the model and nearly all of the technical data. All writers discussed the conclusions, offered critical input, assisted in the creation of the study, and collaborated on the final manuscript.

ETHICS STATEMENT
All research procedures were carried out in accordance with the Animal Ethics Committee of APRI, ARC, Egypt's recommendations for the treatment and use of lab animals.