Discussion
Nowadays, AMR is a worldwide concern. A global effort is needed to focus on the emergence of bacterial resistance to find new epidemiological patterns and apply suitable approaches to eradicate the infection source and improve the treatment success rate. In the present study, four-hundred and thirty (80.37%) E. coli isolates were recovered from 535 mastitic milk samples in three provinces. The frequency of E. coli isolates in the previous, and recent studies differ among herds in different countries, different regions of one country, due to smaller sample size or even caused by using different detection and identification methods (Marashifard et al. 2019). In this study, the high isolation rate of E. coli suggests that farm management should be improved to lower the risks of bovine mastitis, like E. coli and K. pneumoniae are two environmental pathogens frequently linked with poor hygiene (Munoz et al. 2008; Breen et al. 2009). In particular, this would include the strict inspection of the udder, milking procedures, where contamination with environmental pathogens occurs in dairy herds.
AMR in Gram-negative bacteria is rising globally, especially for E. coli. More troubling are the increasing reports of MDR pathogenic E. coli from food-producing animals, raising worries about animal and public health (Seiffert et al. 2013). This study determines an overall 75.58% of ESBL-producing E. coli from bovine mastitis, which is higher than the previous reports of ESBL from bovine mastitis in China and other parts of the world (Kar et al. 2015; Ali et al. 2017; Heuvelink et al. 2019; Zheng et al. 2019). The overall incidence of ESBL-producing E. coli isolates was much higher in Jiangsu province (89.09%), followed by Shanghai (75.18%) and Zhejiang (58.59%). E. coli isolates, characterized in the present study, showed high resistance to colistin 302/430 (70.23%). Indeed, the consistent selective pressure produced by the third generation of cephalosporins and colistin in animal production stimulated this rise of ESBL resistance. Berge et al. observed high resistance in dairy cows not exposed to antibiotics (Berge et al. 2005). They determined that an individual antimicrobial therapy could lead to selective pressure to establish a resistant gene-pool in the herd-level bacterial population. Among ESBL-producing E. coli isolates, the highest proportions of resistance were found against CFX (98.15%), followed by CTX (96%), CEF (94.76%), AMP (93.84%), TET (92.61%). Interestingly, none of the E. coli isolates were resistant to carbapenems, which is inconsistent with our previous report in pigs (Shafiq et al. 2019).
Ninety-five randomly ESBL-producing E. coli were selected in this study for further genotypic characterization for the major ESBL-encoding genes (blaCTX, blaSHV, and blaTEM) and COL-resistant genes mcr-1 to mcr-9. We found that blaCTX-M type was the most prevalent, 90.52% (n=86), followed by blaTEM, 33.68% (n=32) and blaSHV, 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 that blaCTX-M genes are present in most dairy cows suffering from mastitis. In our findings, blaCTX-M-1 was the most common group (87%), followed by blaCTX-M-9 (16.27%) were found in blaCTX-M positive E. coli isolates. Among blaCTX-M-1 (blaCTX-M-28 and blaCTX-M-66) were the most common determinants found in 33 and 12 E. coli isolates, respectively. While in the blaCTX-M-9 group, the most dominant genotype found was blaCTX-M-14 found in 15 isolates. Our findings of higher prevalence of blaCTX-M genotypes in the blaCTX-M-1 group and sporadically detection of blaCTX-M genes belonging to other groups are consistent with the previous studies from the Chinese mainland and other parts of the world (Coque et al. 2008; Geser et al. 2012; Yu et al. 2015; Ali et al. 2017).
In our study, the objective was to investigate the most common phylogroups of E. coli strains isolated from bovine mastitis, according to the new phyllo-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 (24.21%) and B2 (10.52%). Our findings were also inconsistent with the earlier studies on bovine mastitis in which phylogroups A and B1 were found to be the most predominant phylogroups (Müştak et al. 2015; Ali et al. 2017; Zhang et al. 2018). Similarly, several other studies indicated that both pathogenic and nonpathogenic E. coli from bovine origin were mainly assigned to phylogroup A and B1 (Silva et al. 2009; Henriques et al. 2014; Zhang et al. 2018). We also studied virulence factors, and the results confirmed that phylogenetic group A exhibited a higher prevalence of various combinations of virulence factors. The targeted virulent factors were found in 97.89% (n=93) E. coli 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 common 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 phyllo-grouping and incidence circulation of virulence genes in E. coli from dairy herds with mastitis. Our results are also in accordance with these findings. This is troublesome, as these commensals may serve as a 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 the clonal relationship of our selected isolates, and MLSA further expedited us to accomplish phylogenetic association amongst the E. coli species. MLST of 22 randomly ESBL-producing and mcr-1-positive E. coli isolates were organized into a total of 20 STs. ST58 and ST410 were the most predominant, comprising two each (2/20=20%). All other STs were present found only once. The burst analysis of about 7256 STs available in the PubMLST database, including all 20 STs mentioned in this report, revealed that the majority of the STs were founders such as ST10 and ST58. Unpredictably, geoBURST analysis revealed that the majority of the isolates under study belonged 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 founder's 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 dominant carrier of different STs in these 22 E. coli isolates was detected in the blaCTX-M-1 group. Of particular concern is that the blaCTX-M-28 and blaCTX-M-14 co-harboring mcr-1 carried major of STs in these isolates. One recent study also found that the blaCTX-M-14 allele spread in different clonal populations and is 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 et al. 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 blaCTX-M and mcr-1, which is inconsistent with the previous reports identified in Netherland and in China (Skov and 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 blaCTX-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 the PFGE pattern. The majority of the E. coli isolates carrying ESBL and mcr-1 isolates were clonally unrelated. Only four isolates E. coli strains EC (02, 03) and EC (021,022) from the same province showed 100% clonal similarity, and sequence types identified were ST58 and ST410, respectively. PCR-based replicon typing for these twenty representative E. coli isolates, which are positive for ESBL and mcr-1 carrying different plasmid types, the most prevalent Inc. types found were IncHI2 (n=11), IncFIB (n=09), IncFIC (n=07), and IncX4 (n=05). A previous study analyzed the mcr-1 carrying plasmids available in the database at GenBank and found various plasmid types involved in disseminating mcr-1 in E. coli isolates; the major plasmid types were IncI2, IncX4, IncHI2, IncFIB, and IncFII (Gao et al. 2016).