Antimicrobial and virulence associated characteristics of E. coli isolated from mastitis and endometritis cases in sheep and goats

Research Abstract

The results of clinical examination of the udders of lactating ewes (n=90; 180 udder halves) and does (n=32; 64 udder halves) revealed that out of 244 examined udder haves, 158 halves were apparently normal;115 in ewes and 43 in she-goats, while 86 halves showed complete loss of function;58 in ewes and 28 in she-goats. A total number of 125 bacterial isolates were recovered from 122 milk samples from sheep and goats including 73 (58.4 %) Gram negative and 52 (41.6 %) Gram positive bacterial isolates. Gram negative bacterial isolates (n=73) were identified as 68 E. coli (54.4 %), 3P. multocida (2.4%) and 2M. haemolytica (1.6%). On the other hand, Gram positive bacterial isolates (n=52) as 20 Streptococci (16% including S. agalactiae 13 (10.4%), S. dysgalactiae 4 (3.2%) and S. uberis 3 (2.4%) and 32 S. aureus (25.6 %). Regarding animals, results of endometritis followed by collection of uterine swabs from sheep and goats showed that out of 84 apparently healthy examined animals, a total of 80 animals (68.97%) were positive (subclinical endometritis), while 4 animals (3.44%) were negative isolates from the samples of endometritis (116 samples). A total number of 113 bacterial isolates were recovered from 116 examined sheep and goats and identified as follow; 89 (78.76 %) Gram negative and 24 (21.23 %) Gram positive bacterial isolates. Gram negative bacterial isolates (n=89) were identified as 69 E. coli (61.06 %), 4 P. multocida (3.5%), 13 P. aeuginosa (11.5%) and 3M. haemolytica (2.65%). On the other hand, Gram positive bacterial isolates (n=24) as 6 E. faecalis (5.3 %), 5 E. faecium (4.44%), 4 Streptococci (3.53 %) and 9 S. aureus (7.96%). The results of haemolytic activity of bacterial isolates (selected representatively) from mastitis milk samples on sheep blood agar revealed that 70.5% of the bacterial isolates were haemolytic; 47.6% were β-haemolytic while 22.9 % were α-haemolytic. Haemolysis was represented as 100%, 90%, 85.71% and 56% in M. haemolytica, Streptococcus spp., Staphylococcus spp. and E. coli, respectively. They were mostly β-haemolytic 29 isolates (47.6%) while, 18 isolates (29.5%) were γ-haemolysis. The results of haemolytic activity of bacterial isolates from endometritis samples (selected representatively) on sheep blood agar of different bacterial pathogens revealed that 56.8% of the bacterial isolates were haemolytic; 20.3% were α-haemolytic and 36.5% were β-haemolytic. Haemolysis was represented as 100%, 87.5%, 85%, 61.1% and 40% in M. haemolytica, Streptococcus spp., Staphylococus spp., E. coli and P. aeruginosa, respectively. In addition, 27 isolates were β -haemolytic with a percentage of 36.5%. Whereas, 32 (43.2%) of the isolates were γ-haemolysis. Biofilm production was represented as 39 E. coli isolates (65%) (18 isolates from mastitis 30% and 21isolates from endometritis 35%), 20 Staphylococci isolates (66.67%) (16 isolates from mastitis 53.33% and 4 isolates from endometritis 13.34%), 7 Streptococci isolates (70%) (5 isolates from mastitis 40%) and 3solates from endometritis (30%), 3P. aeruginosa isolates (42.85%), 3 P. multocida (50%) (1 isolates from mastitis 16.67% and 2 isolates from endometritis 33.33%) and two isolates from endometritis 66.67% and 2 Enterococci isolates (one isolate of E. fecalis from goat and one isolate from E. faecium from sheep) (40%) from uterine samples. The results of in-vitro antimicrobial susceptibility of mastitis milk samples against 17 antimicrobial agents showed high susceptibility against ofloxacin (76%), followed by co-trimethoprim (73.6%), sulfamethoxazole-trimethoprim (71.2%), ciprofloxacin (70.4%), fosfomycin (68.8%), chloramphenicol (67.2 %) and doxycycline (65.6%). On the contrary, most of the isolates were resistant to amoxicillin-clavulanic acid (75.2 %), cefotaxime (72.8 %), amoxicillin (69.6 %), cephalexine (68.8 %), and cefotriaxone (64.8%). All bacterial isolates recovered from uterine samples were tested for in-vitro antimicrobial susceptibility against 17 antimicrobial agents of 9 different antimicrobial classes. The results showed high susceptibility against co-trimethoprim (76.11%) followed by ofloxacin (73.45%), fosfomycin (72.57%), doxycycline (71.68 %), ciprofloxacin (69.91%), sulfamethoxazole-trimethoprim (69.03%), tetracycline (69.02%) and chloramphenicol (67.26%). On the contrary, most of the isolates were resistant to amoxicillin-clavulanic acid (76.11%), cefotaxime (71.68 %), amoxicillin (70.79 %), cephalexine (69.02 %) and cefotriaxone (58.41 %). Using PCR to assess biofilm associated genes and MDR genes in different bacterial isolates as E. coli isolates harbored blaTEM genes (100%), while, qnrA gene was detected in 50% of the tested isolates of ovine endometritis. Meanwhile, all tested isolates harbored fimH virulence genes (100%). The iutA was detected in 25% of the tested isolates of endometritis of sheep only. P. aeruginosa harbored blaTEM, tetA, toxA and las1 with a prevalence rate of 100%. S. aureus isolates harbored mecA and coa (100%). The tetK and icaA genes were detected in 75% of the tested isolates. Streptococci isolates harbored aac(6')aph(2'') gene (100%). The tetO, cylE and cfb were detected in 75% of the tested isolates. P. multocida isolates isolates harbored nanB, toxA and omp87 with a prevalence rate of 100%. M. haemolytica isolates harbored gcp, itkC and ssa with a prevalence rate of 75%. The results concluded that there is a strong correlation between the phenotypes and genotypes of AMR in different bacteria. Moreover, biofilm is a main problem for dairy farms affecting the udder health, uterine tissue and is considered as a risk for milk quality, fertility and consumer health. Also, biofilm formation, in turn, is associated with acquiring MDR by a high prevalence of bacterial pathogens recovered in this study. .

Research Keywords

virulence, antimicrobial sensitivity, sheep, goats, mastitis, endometritis