Ninety-five randomly ESBL-producing E. coli were selected in this study for further genotypic characterization for the major ESBL-encoding genes (CTX, SHV, and TEM) and COL-resistant genes mcr -(1-9). We found that CTX-M type was the most prevalent 90.52% (n =86), followed by TEM 33.68% (n =32) and SHV 31.57% (n =30). While in COL-resistant genes only mcr-1 was identified and prevalent 78.94% (n =75) in these isolates. This indicates thatbla CTX-M genes are present on most dairy cows suffering from mastitis. In our findings CTX-M-1 was the most common group (87%), followed by CTX-M-9 (16.27%) were found in CTX-M PositiveE. coli isolates. Among CTX-M-1 (CTX-M-28 and CTX-M-66) were the most common determinants found in 33 and 12 E. coli isolates respectively. While in CTX-M-9 group the most dominant genotype found was CTX-M-14 found in 15 isolates. Our findings of higher prevalence of CTX-M genotypes in bla CTX-M-1 group, and sporadically detection of CTX-M genes belonging to other groups are consistent with the previous studies from Chinese mainland and other parts of the world (Ali et al., 2017; Coque, Baquero, & Canton, 2008; Geser, Stephan, & Hächler, 2012; Yu et al., 2015).
In our study, the objective was to investigate the most common phylo-groups of E. coli strains isolated from bovine mastitis, according to the new phylo-grouping method of Clermont et al. (Clermont et al., 2013). Therefore, phylogenetic analysis was carried out in this study, and the results confirmed that group A was the most prevalent group with 54.73%, followed by group B1 and B2 (24.21%, 10.52%) respectively. Our findings were also in consistent with the earlier studies on bovine mastitis in which phylo-groups A and/or B1 were found to be the most predominant phylo-groups (Ali et al., 2017; Müştak et al., 2015; Zhang et al., 2018). Similarly, several other studies indicated that both pathogenic and nonpathogenic E. coli from bovine origin were mainly assigned to phylo-group A and B1 (Henriques, Silva, Lemsaddek, Lopes-da-Costa, & Mateus, 2014; Silva et al., 2009; Zhang et al., 2018). We also studied the occurrence of the virulence factors, and the results confirmed that phylogenetic group A exhibited higher prevalence of various combination of virulence factors. The targeted virulent factors found in 97.89% (n =93) isolates. The most predominant virulent gene detected was ompC , found in 97.89% (n =93). Sixteen different gene patterns were observed for virulence factors, and the most prevalent pattern of four different virulence genes ompC, ompF, fimH, and ECs3703 was detected in thirteen different isolates. One recent study from China (Zhang et al., 2018), also found similar phylo-grouping and incidence circulation of virulence genes in E. coli from dairy herds with mastitis. Our results are also in accordance to these findings. This is troublesome, as these commensals may serve as reservoir for the spread of both resistant and virulent factors, and could embrace virulent traits in the future.
MLST made it possible to type and establish clonal relationship of our selected isolates and MLSA further expedited us to accomplish phylogenetic association amongst the isolates and the E. coliisolates species. MLST of 22 randomly ESBL- and mcr-1 E. coliisolates were organized into a total of 20 sequence types (STs). Sequence types 58 and ST410 were the most predominant comprising 2 each (2/20=20%). All other STs were present found only once. The geoBURST analysis of about 7256 ST available in the PubMLST database including all 20 STs mentioned in this report revealed that majority of the STs were founders such as ST10 and ST58. Unpredictably, geoBURST analysis revealed that majority of the isolates under study were belong to founders, (ST58, ST10, ST410, and ST178 etc.), while the remaining such as ST2108, ST2113 etc . were demarcated as co-founders. Interestingly, all the above founders STs found in this study were positive for mcr-1 which is troublesome, and may in the future hold resistant traits in the commensal E. coli. Similarly, one recent study from China also found ST410 from mastitic cows in Inner Mongolia (Ali et al., 2017). The major carrier of different STs in these 22 E. coli isolates were detected in CTX-M-1 group. Of particular concern is that the bla CTX-M-28 andbla CTX-M-14 co-harboring mcr-1 carried major of STs in these isolates. One recent study also found thatbla CTX-M-14 allele spread in different clonal populations and are now widely disseminated in China (Zheng et al., 2019). The STs found in our study were different from the previously identified STs of mcr-1- positive E. coli strains from food producing animals and humans in China (Kuo et al., 2016). However, ST10 identified in mcr-1 positive E. coli isolate was similar to the previous finding of ST10 in mcr-1 isolate from cattle in Japan and Egypt (Suzuki, Ohnishi, Kawanishi, Akiba, & Kuroda, 2016) and sequence type 58 (ST58) identify here were similar to the previous report in mcr-1 E. coli isolate in Brazil (Sacramento et al., 2018). In our findings, two E. coli isolates EC-021 and EC-022 were identified positive for ST410 in bovine mastitis isolates and harbored CTX-M and mcr-1 , which is in consistent with the previous reports identified in Netherland and in China (Skov & Monnet, 2016). These findings highlight that clonal isolates of ST410 have been identified from diverse environments, animals, and humans (Falgenhauer et al., 2016; Schaufler et al., 2016) and, as we demonstrate here, in bovine mastitis milk isolates. Hence, the simultaneous spreading of the CTX-M and mcr-1 genes suggests that mcr-1 is already present in the diverse reservoirs.
Xbal -PFGE of the 22 E. coli isolates was determined and analyzed successfully. All the representative E. coli isolates selected for clonal relatedness yielded clear fingerprint patterns. There were 16 different clusters sharing ≥ 80% similarity in PFGE pattern. Majority of the E. coli isolates carrying ESBL andmcr-1 isolates were clonally unrelated. Only four isolatesE. coli strains EC (002, 003) and EC (021,022) from the same farms, which showed 100% clonal similarity and sequence types identified were ST58 and ST410 respectively. PCR-based replicon typing (PBRT) for these twenty representative E. coli isolates which are positive for ESBL and/or mcr-1 carrying different plasmid types, the most prevalent Inc. types found were IncHI2 (n=11), FIB (09), FIC (07), and X4 (05). A previous study, analyzed the mcr-1 carrying plasmids available in the database at GenBank and found variety of plasmid types involved in the dissemination of mcr-1 in E. coli isolates, the major plasmid types were IncI2, IncX4, IncHI2, IncFIB and IncFII (Gao et al., 2016).