Tetracycline resistance phenotypes and genotypes of coagulase-negative staphylococcal isolates from bubaline mastitis in Egypt.

AIM: This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes' milk in Egypt. MATERIALS AND METHODS: A total of 81 milk samples from 46 normal buffalo milk samples and 35 SCM buffalo milk samples at private dairy farms of Egypt were used in this study. CNS were identified using phenotypic and molecular methods (polymerase chain reaction [PCR]). CNS isolates were tested for tetracycline resistance using routine methods and multiplex PCR targeting tetracycline (tet) resistance genes followed by sequencing of positive PCR products and phylogenetic analysis. RESULTS: Isolation and identification of 28 (34.5%) CNS from normal and SCM buffaloes' milk, namely, Staphylococcus intermedius (39.2%), Staphylococcus xylosus (25.0%), Staphylococcus epidermidis (10.7%), Staphylococcus hominis (10.7%), and 3.5% to each of Staphylococcus sciuri, Staphylococcus hyicus, Staphylococcus lugdunensis, and Staphylococcus simulans. Using nested PCR, all the 28 CNS isolates revealed positive for 16srRNA gene specific for genus staphylococci and negative for thermonuclease (nuc) gene specific for Staphylococcus aureus species. The presence of tetracycline resistance-encoding genes (tetK, tetL, tetM, and tetO) was detected by multiplex PCR. All isolates were negative for tetL, M, and O genes while 14 (50%) CNS isolates were positive for tetK gene, namely, S. lugdunensis (100%), S. hominis (100%), S. epidermidis (66.6%), S. intermedius (45.4%), and S. xylosus (42.8%). Nucleotide sequencing of tetK gene followed by phylogenetic analysis showed the high homology between our CNS isolates genes of tetracycline resistance with S. aureus isolates including Egyptian ones. This proves the transfer of the tetracycline resistance encoding genes between coagulase-negative and coagulase positive Staphylococcus spp. CONCLUSION: CNS isolates have distinguishingly high resistance to tetracycline. Abundant tetracycline usage for mastitis treatment leads to the spread of genetic resistance mechanisms inside CNS strains and among all Staphylococcus spp. Consequently, tetracycline is not effective anymore.

Monitoring of Brucella reactor does following milk examination using different techniques.

Milk samples from 129 does were collected and monitored for Brucella antibodies using immunological tests such as Milk Ring Test (MRT), Whey Agglutination Test (WAT), Whey Antiglobulin Coombs Test (WCT) and milk ELISA (m ELISA) using Brucella Periplasmic protein antigen. Results obtained from these tests were compared to PCR and bacterial isolation. The highest incidence of positive reactors was given by Whey Antiglobulin and Whey Agglutination Test (9.3%) while the lowest incidence was given by bacterial isolation (Br. melitensis biovars 3, 3.8%). PCR showed the highest agreement with the bacterial isolation, while WAT and WCT showed the lowest one. PCR showed a high sensitivity of 1 x 10 B. melitensis CFU mL(-1) of milk. The results of mELISA here suggests its efficiency to be used as a screening test and/or confirmatory test, while the modified MRT still need more investigations to diagnosis caprine brucellosis.

Investigations on brucellosis in Egyptian Baladi Does with emphasis on evaluation of diagnostic techniques.

Investigations were carried out on caprine brucellosis in a costal area in Egypt. A total number of 577 Baladi Does was examined for Brucella infection using different serological tests. Specimens were taken from seropositive obligatory slaughtered Does (No = 33) for Brucella isolation, histopathological examination, Polymerase Chain Reaction (PCR) assay and determination of serum copper (Cu), zinc(Zn) and iron (Fe) concentrations. Results indicated that the incidence of brucellosis was 3.0-5.0%, by using the different serological tests. Buffered Acidified Plate Antigen Test (BAPAT) is of the highest sensitivity followed by Rose Bengal Plate Test (RBPT), L-ELISA, Complement Fixation Test (CFT), P-ELISA, Rivanol test (RVT) and Tube Agglutination Test (TAT). In seropostive Does, Brucella melitensis biovar 3 was isolated from 78.78% and PCR yielded expected products in 81.81%. Moreover, granulomatous endometritis, lymphocytic mastitis and lymphoid depletion in both lymph nodes and spleen were evident together with significant (p<0.001) decreases in serum Cu, Zn and Fe concentrations. In conclusion, more attention should be paid to goat in brucellosis epidemiology in the application of national program of brucella control and eradication.