Escherichia coli are important pathogens causing bovine mastitis, inflammation of mammary gland, in certain environmental conditions (Ali et al., Reference Ali, ur Rahman, Zhang, Shahid, Zhang, Liu, Gao and Han2016; Lan et al., Reference Lan, Liu, Meng, Xing, Dong, Gu and Zheng2020; Zhang et al., Reference Zhang, Zhang, Huang, Gao, Wang, Liu and Liu2018). E. coli have been categorized into phylo-groups A, B1, B2, and D (Clermont et al., Reference Clermont, Bonacorsi and Bingen2000). The occurrence and severity of mastitis depends on the immune response and the genetic makeup of the host (cow factors) and the virulence of the causative bacterial strains. Virulence determinants in E. coli include afimbrial adhesins, fimbrial proteins, resistance to the serum complement, outer membrane proteins and cytotoxic necrotizing factor (Kaipainen et al., Reference Kaipainen, Pohjanvirta, Shpigel, Shwimmer, Pyörälä and Pelkonen2002; Cruz-Soto et al., Reference Cruz-Soto, Toro-Castillo, Munguía-Magdaleno, Torres-Flores, Flores-Pantoja, Loeza-Lara and Jiménez-Mejía2020). No particular strain, genotype or virulence factor in E. coli had yet been commonly associated with bovine mastitis infection (Cruz-Soto et al., Reference Cruz-Soto, Toro-Castillo, Munguía-Magdaleno, Torres-Flores, Flores-Pantoja, Loeza-Lara and Jiménez-Mejía2020). The emergence of antimicrobial resistance in mastitis-causing E. coli has been reported (Ali et al., Reference Ali, ur Rahman, Zhang, Shahid, Zhang, Liu, Gao and Han2016; Zhang et al., Reference Zhang, Zhang, Huang, Gao, Wang, Liu and Liu2018). Several studies have been carried out across the world for the characterization of E. coli isolated from bovine mastitis. However, such studies are lacking in Peshawar region of Khyber Pakhtunkhwa, Pakistan, therefore, the current study was designed. The aims of this study were to determine any phylogenetic groupings, virulence genes and to determine antimicrobial susceptibility profiles of E. coli isolated from cases of bovine mastitis.
Material and methods
Milk samples (n = 385) from suspected cases of mastitis were brought to laboratory either by local farmers having 1–3 dairy animals or from small dairy farms (20–50 cattle) of Peshawar and surrounding rural areas. The California mastitis test (CMT) was used to confirm mastitis using recommendations of the kit supplier (Techni. Vet., Inc. USA). Milk samples tested positive for mastitis were processed for the isolation of bacteria and biochemical characterization according to standard protocols (Ali et al., Reference Ali, ur Rahman, Zhang, Shahid, Zhang, Liu, Gao and Han2016).
Genomic DNA was extracted from E. coli isolates by boiling water. Triplex PCR was conducted to identify the phylo-groups of E. coli on the basis of presence or absence of the chuA and, yjaA genes and TspE4.C2 DNA fragments (Clermont et al., Reference Clermont, Bonacorsi and Bingen2000). The virulence genes intimin (eae), shiga toxin producing E. coli (STEC) agglutinating adhesion (saa), S Fimbriae (sfa), cytotoxic necrotizing factors (cnf), aerobactin biosynthesis (iucD), P fimbriae (papC), shiga toxin 1 and 2 (stx1, stx2), fimbriae 41 (F41), fimbriae 17-A (f17A) and serum resistance gene (traT) were detected as described by Paton and Paton (Reference Paton and Paton2002), Kaipainen et al. (Reference Kaipainen, Pohjanvirta, Shpigel, Shwimmer, Pyörälä and Pelkonen2002), Suojala et al. (Reference Suojala, Pohjanvirta, Simojoki, Myllyniemi, Pitkälä, Pelkonen and Pyörälä2011), Zhang et al. (Reference Zhang, Zhang, Huang, Gao, Wang, Liu and Liu2018) and Cruz-Soto et al. (Reference Cruz-Soto, Toro-Castillo, Munguía-Magdaleno, Torres-Flores, Flores-Pantoja, Loeza-Lara and Jiménez-Mejía2020).
Antibiotic susceptibility profiles of all the E. coli isolates were tested on Muller−Hinton agar (OxoidTM, Thermo Scientific Inc. US) by the Kirby−Bauer disk diffusion method. A panel of 11 different antimicrobial agents (OxoidTM, Thermo Scientific Inc.) was used; ampicillin, amoxicillin with clavulanic acid, cefotaxime, erythromycin, enrofloxacin, gentamicin, kanamycin, meropenem, norfloxacin, pipracillin and streptomycin. Interpretation of the results was done according to the Clinical and Laboratory Standard Institute method (Ali et al., Reference Ali, ur Rahman, Zhang, Shahid, Zhang, Liu, Gao and Han2016).
Results
A total of 30 E. coli isolates (7.8%) were recovered from 385 milk samples collected from mastitic cows. The majority of isolated E. coli strains belonged to group A (n = 24; 80%), followed by two isolates from group B1 (6.7%), group B2 (6.7%) and group D (6.7%). Nineteen isolates (63.3%) did not carry any virulence gene. Only 11 (37%) isolates were positive for either the traT gene (n = 2; 6.7%) or the iucD gene (n = 3; 10%) or both (n = 6, 20%). None of the isolates contained eae, saa, sfa, cnf, papC, stx1, stx2, F41 or f17A.
The lowest antimicrobial susceptibility was against ampicillin (10%), followed by cefotaxime (16.7%), streptomycin (23.3%), amoxicillin plus clavulanic acid (30%), erythromycin (33.4%) and kanamycin (50%). The highest susceptibility of E. coli isolates was against meropenem (80%), gentamicin (73.4%), piperacillin (73.3%), norfloxacin (56.7%) and enrofloxacin (53.4%).
Discussion
E. coli associated with bovine mastitis are belonging mainly to the phylogenetic groups A and B1 (Suojala et al., Reference Suojala, Pohjanvirta, Simojoki, Myllyniemi, Pitkälä, Pelkonen and Pyörälä2011; Zhang et al., Reference Zhang, Zhang, Huang, Gao, Wang, Liu and Liu2018; Cruz-Soto et al., Reference Cruz-Soto, Toro-Castillo, Munguía-Magdaleno, Torres-Flores, Flores-Pantoja, Loeza-Lara and Jiménez-Mejía2020). The present study also found that 80% of E. coli isolates belonged to group A. Previous studies have assigned the commensal and diarrheagenic strains mainly to group A and B1, whereas extra-intestinal E. coli strains were assigned to group B2 and D (Clermont et al., Reference Clermont, Bonacorsi and Bingen2000). Our results are in agreement with the previous studies in which the majority of the isolates belonged to group A, which belongs to commensal E. coli strains.
No particular virulence-associated genes in E. coli have so far been linked to mastitis pathogenesis (Cruz-Soto et al., Reference Cruz-Soto, Toro-Castillo, Munguía-Magdaleno, Torres-Flores, Flores-Pantoja, Loeza-Lara and Jiménez-Mejía2020). Our results showed no virulence gene in 63% isolates despite testing for 11 genes. Suojala et al. (Reference Suojala, Pohjanvirta, Simojoki, Myllyniemi, Pitkälä, Pelkonen and Pyörälä2011) also reported a lack of virulence genes in E. coli isolates. The lack of virulence genes in the majority of isolates may indicate that mastitis associated E. coli may originate from commensal flora already fin the host's environment (Marashifard et al., Reference Marashifard, Aliabad, Hosseini, Darban-Sarokhalil, Mirzaii and Khoramrooz2019).
In the present study, only two virulence genes, traT and iucD were detected and only in 37% of isolates. The genes iucABCD produce siderophores that can uptake iron from extra-intestinal niches and concentrate it in bacterial cytoplasm, this ability is fundamental to survive in iron poor sites of the host and iron uptake systems are common in extra-intestinal strains. Avian pathogenic E. coli (APEC) and uropathogenic E. coli (UPEC) are the two main subsets of extraintestinal pathogenic E. coli (ExPEC) that harbor various genes for iron acquisition. The gene traT is known for serum resistance, the ability to resist the bactericidal activity of host serum. The traT is an outer membrane protein that provides resistance to bacteria against killing action of host serum. This characteristic enables bacteria to survive in body fluids such as urine. The traT is also one of the important virulence genes of UPEC isolates. Although the E. coli tested probably originated from commensal flora, they contained iron uptake and serum resistance genes that are commonly found in UPEC isolates (subset of ExPEC). This could be due to the heterogeneous genome of mastitis E. coli isolates. This was described by Cruz-Soto et al. (Reference Cruz-Soto, Toro-Castillo, Munguía-Magdaleno, Torres-Flores, Flores-Pantoja, Loeza-Lara and Jiménez-Mejía2020), who suggested bovine mastitis E. coli might form a new putative pathotype using the same pathogenic mechanisms as ExPEC. Marashifard et al. (Reference Marashifard, Aliabad, Hosseini, Darban-Sarokhalil, Mirzaii and Khoramrooz2019) questioned if bovine mastitis E. coli are commensal in origin or if mastitis E. coli resemble ExPEC because of the presence of virulence genes. The grouping could be called mammary pathogenic E. coli (MPEC) isolates or, better, mammary associated E. coli (MAEC) as the isolates are facultative and opportunistic pathogens from the greatly varying bovine gastrointestinal microbiota.
Antibiotics are frequently used to treat mastitis and their extensive use has given rise to the emergence of antimicrobial resistance in E. coli (Ali et al., Reference Ali, ur Rahman, Zhang, Shahid, Zhang, Liu, Gao and Han2016; Lan et al., Reference Lan, Liu, Meng, Xing, Dong, Gu and Zheng2020; Zhang et al., Reference Zhang, Zhang, Huang, Gao, Wang, Liu and Liu2018). This despite the fact that routine use of antimicrobial treatment is not recommended for coliform mastitis (Suojala et al., Reference Suojala, Pohjanvirta, Simojoki, Myllyniemi, Pitkälä, Pelkonen and Pyörälä2011). Our study identified the highest resistance in E. coli isolates against ampicillin, followed by cefotaxime and streptomycin. This is consistent with the high resistance to ampicillin reported by Zhang et al. (Reference Zhang, Zhang, Huang, Gao, Wang, Liu and Liu2018) and to both ampicillin and streptomycin by Suojala et al. (Reference Suojala, Pohjanvirta, Simojoki, Myllyniemi, Pitkälä, Pelkonen and Pyörälä2011).
In conclusion, this study suggests that the E. coli isolated from mastitis cases may have originated from commensal flora. Only traT and iucD genes were detected in E. coli isolates, which are reported to be commonly harbored by ExPEC strains. The virulence genes detected in E. coli in this study could be an attribute of high genetic variability of E. coli.
