Results
Isolation and prevalence of ESBL- and COL-resistant E. coli
A total of 430 (80.37%) E. coli isolates were recovered from five-hundred and thirty-five clinical mastitis milk samples based on colony morphology and growth on MacConkey agar plates. Among these isolates, 325 (75.58%) were confirmed as ESBL-producing E. coli. The maximum percentage of ESBL-producing E. coli isolates was detected in Jiangsu province (147/165, 89.09%), followed by Shanghai (103/137, 75.18%), whereas the detection rate in Zhejiang province was (75/128, 58.59%). Furthermore, COL-resistance in 325 ESBL-producing E. coli isolates in Jiangsu were more prevalent (119/165, 72.12%), followed by Shanghai (97/137, 70.80%) and Zhejiang (86/128, 67.18%), as shown in Table 1.
Antimicrobial susceptibility
All the isolated E. coli isolates were tested against 13 commonly used antibiotics Figure 1. MIC testing (μg/mL) for all ESBL-producing E. coli was against all used antibiotics. Of the 325 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%), SXT (86.76%), CHL (76.30%), COL (71.07%), POL (69.84%), KEN (69.84%), GEN (66.15%), and CIP (49.53%). All these isolates were found susceptible to MEM. MDR was observed in more than 85% of isolates (resistance to three or more than three antibiotics classes) by MIC.
Genotyping of ESBL-producing and COL-resistance genes
For further molecular characterization, 95 isolates were randomly selected among 325 ESBL-producing E. coli isolates. Genotyping of these isolates by PCR assay showed that among ESBL determinants, blaCTX-M was the most common bla group detected (n=86), followed by blaTEM (n=32) and blaSHV (n=30). While in COL-resistant genes, only mcr-1 was identified and prevalent (n=75) in these isolates. The blaTEM and blaSHV genes were most commonly detected in combination with blaCTX-M, and mcr-1 Figure 2A.
Detection of blaCTX-M groups using PCR
All blaCTX-M positive MDR E. coli isolates (n=86) were further tested for the detection of major blaCTX-M groups, i.e., blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, and blaCTX-M-25, using a multiplex PCR approach. 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. Both blaCTX-M-2 and blaCTX-M-8/25 groups were absent. Further sequence analysis of blaCTX-M genotypes showed that blaCTX-M-28 was predominant allele, which was carried by 33 (38.37%) isolates, followed by blaCTX-M-14 (n=15, 17.44%), blaCTX-M-66 (n=12, 13.95%), blaCTX-M-55 (n=9, 10.46%), blaCTX-M-69 (n=6, 6.97%), blaCTX-M-15 (n=4, 4.65%), blaCTX-M-177 (n=4, 4.65%), and blaCTX-M-148 (n=3, 3.48%). Figure 2B shows the frequency (%) of various ESBL-encoding genes among 86 positive blaCTX-M E. coli isolates.
Phylogenetic analysis of E. coli isolates
Out of 95 E. coli isolates, phylogenetic group A was the most dominant group with 54.73% (n=52), followed by group B1 24.21% (n=23), group B2 10.52% (n=10), group D 5.26% (n=5), group C 2.10% (n=2), and one isolate was positive 1.05% (n=1) which were assigned to phylogenetic group E. None of the strains were found to assign to group F. The remaining two isolates were negative for all the genes. Details of each phylogenetic group presenting, unlike gene patterns, are given in Table 2.
Frequency and combination patterns of virulence genes
The targeted virulence genes were found in 97.89% (n=93) isolates, while two isolates were found negative for the virulent genes. The majority of the isolates belong to groups A, B1, B2, C, D, and E showed virulence factors. Of these 95 isolates the most predominant gene was ompC, detected in 97.89% (n=93) isolates followed by fimH in 61.05% (n=58), ECs3703 57.89% (n=55), ompF 50.52% (n=48), irp2 31.57% (n=30), fyuA 21.05% (n=20), iucD 15.78% (n=15), and 2.10% (n=2) isolates were positive for colV. Two virulence genes eaeA and ler were not detected in any of these isolates. The incidence of virulence genes among E. coli isolated from bovine clinical mastitis is shown in Table 3.
Sixteen different gene patterns were observed in ninety-five E. coli isolates, which are shown in Table 4. The most common pattern of four different virulence genes ompC, ompF, fimH, and ECs3703 was detected in thirteen different isolates, followed by the combination of six different virulent factors ompC, ompF, ECs3703, fimH, irp2, fyuA, which was detected in eight isolates.
MLST and population structure
A total of 22 isolates were organized into 20 sequence types (STs) with seven groups and 15 edges. Sites from where the strains were got, antibiotic resistance genes, plasmid replicon, STs, and antibiogram are stated in Table 5. Moreover, STs found along with the allelic profile of 22 typeable E. coli isolates are given in Table 6.
The total epidemiological facts of the dominant STs were based on the MLST data of the selected isolates (n=22) revealed 20 STs. Sequence types ST58 and ST410 were the most predominant, comprising two each (2/20=20%). All other STs were present found only once. Details of distribution and abundance of STs are labeled in Figure 3A and 3B. The most prevalent STs, ST58, and ST410 were originated from Zhejiang and Jiangsu, respectively.
All 20 STs originated in this study were grouped into 7 BURST group (BG) and 17 based on goeBURST analysis. Interestingly, no double locus variants (DLV) were identified among all 20STs; however, 4 single locus variants (SLVs) were identified among all the seven groups. The major BG  contained six isolates with 6 STs (ST354, ST2737, ST2113, ST4355, ST3901, and ST3736) with a predicted founder of ST354 (Figure 3.3.C) comprised of 3 SLVs. A second BG consisted of 5 isolates and 5 STs (ST58, ST2108, ST4024, ST865, and ST3599) with ST58 as predicted founder Figure 3.3.D. The burst analysis with all 7256 STs accessible in the database, including all 20 STs mentioned in this report, revealed that the majority of the STs were founders such as ST10 and ST58 Figure 3C, 3D, and 3E. Unpredictably, geoBURST analysis revealed that most of the STs found in the isolates under study (ST58, ST10, ST410, and ST178, etc.) were all founders, while other such as ST2108 and ST2113 were delineated as co-founders.
Multi-locus sequence analysis (MLSA)
To precisely categorize the investigated E. coli species with MDR features, we applied MLSA, based on the phylogenetic analysis of the nucleotide sequences of the alleles used in the MLST. Therefore, by implicating the nucleotide sequences of the typed strains (n=22), the phylogenetic analysis was performed using the strains' MLST information. The maximum probability tree was created using nucleotide sequences of seven loci. The study strains were clustered into two main sub-clusters with a more massive cluster of a different lineage, and the majority of isolates (n=13) were grouped into a major cluster Figure 4. A small cluster comprised of 7 isolates were originated from random places. Two of the isolates were grouped separately into an out-group, indicating out liars. Furthermore, the strains were not clustered based on the source or location. The clustering together of isolates from different regions more likely suggests that the correlation between the sample's genetic background and origin could not be established.
Genetic relatedness of E. coli isolates
Xbal-PFGE successfully analyzed the clonal relatedness of the 22 representative E. coli isolates. All the selected E. coli isolates were typable and yielded clear fingerprint patterns for 22 isolates Figure 5. There were 16 different clusters based on the cutoff value of 80% N. Most of the isolates carrying ESBL and mcr-1 isolates were clonally unrelated, except for a small proportion of E. coli strains EC (02, 03) and EC (021, 022) from the same farms, which showed 100% clonal similarity Figure. 3.5. PCR-based replicon typing (PBRT) 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).