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Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt
Mastitis caused by multi- or pan-drug resistant bacteria is a growing health concern. A total of 110 milk samples were collected:
Staphylococcus aureus
,
Streptococcus agalactiae
,
Streptococcus dysgalactiae
,
Enterococcus faecalis
, and
Escherichia coli
were present in 54/110 (49.09%), 37/110 (33.63%), 25/110 (22.72%), 7/110 (6.36%), and 50/110 (45.45%) samples, respectively. A total of 20 methicillin-resistant
S. aureus
(MRSA) isolates, 19
Streptococcus
sp. isolates, and 15
E. coli
isolates were selected, and 100% were positive for (coagulase and hemolysins), streptokinase, and hemolytic activity, respectively. A number of 11
E. coli
isolates were serotyped, and the serotypes were: O26, O55, O111, O119, O124, O125, O127, and O158. The antimicrobial resistance index ranges for MRSA,
Streptococcus
sp., and
E. coli
were 0.49–0.83, 0.39–0.83, and 0.56–1, respectively. The most effective antimicrobials on Gram-positive isolates were cephradine, ciprofloxacin, doxycycline, norfloxacin, and vancomycin, while doxycycline and norfloxacin were effective on
E. coli
serotypes. All of the selected isolates exhibited slime layer production. The efflux pumps of the 12 MRSA, 12
Streptococcus
sp., and 11
E. coli
isolates exhibited activity with ethidium bromide concentrations of 1, 1.5, and 0.5 µg/ml, respectively. There was a simultaneous antimicrobial activity of the efflux pump inhibitor chlorpromazine with amoxicillin/clavulanic acid, erythromycin, and oxacillin, to which the isolates were resistant. The 12 MRSA isolates harboured the methicillin resistance genes
mec
(A,A1, and A2),
mec
A1, and
mec
C at frequencies of 9/12 (75%), 9/12 (75%), and 8/12 (66.7%), respectively, and the penicillin resistance gene
Bla
Z was present at a frequency of 5/12 (41.7%). The distributions of
erm
(A),
erm
(B),
erm
(C),
erm
(F),
erm
(G), and
erm
(Q) were 8/12 (66.7%), 5/12 (41.7%), 12/12 (100%), 2/12 (16.7%), 0/12 (0.0%), and 8/12 (66.7%), respectively. The 12
Streptococcus
sp. isolates harboured
mec
(A, A1, and A2),
mec
A1,
mec
C, and
bla
Z at rates of 4/12 (33.33%), 4/12 (33.33%), 5/12 (41.7%), and 4/12 (33.33%), respectively. The frequencies of
erm
(A) and
erm
(F) were 4/12 (33.33%), and 9/12 (75%), respectively. The 11
E. coli
isolates harboured the extended-spectrum β-lactamases integrase1, integrase2,
bla
CTX-M,
bla
CTX-M-1, and
bla
TEM at frequencies of 10/11 (90.90%), 11/11 (100%), 9/11 (81.81%), 6/11 (54.54%), and 10/11 (90.90%), respectively. Moreover, the frequencies of
erm
(A),
erm
(B),
erm
(C),
erm
(F),
erm
(G), and
erm
(Q) were 7/11 (63.63%), 4/11 (36.36%), 4/11 (36.36%), 5/11 (45.45%), 10/11 (90.90%), and 10/11 (90.90%), respectively. Our results demonstrated the high antimicrobial resistance of the investigated isolates and confirmed the existence of multiple mechanisms underlying multidrug resistance.