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Phenotypic and genotypic detection of both mecA- and blaZ-genes mediated β-lactam resistance in staphylococcus strains isolated from bovine mastitis
Abstract
In this study, 25 (52%) and 118 (67%) Staphylococcus strains were isolated from milk samples collected from cows suffering from clinical and subclinical mastitis, respectively. Identification of these isolates was performed using combined phenotypic and genotypic methods. A novel multiplex PCR assay was developed and used for both genotypic identification of the isolated Staphylococci and for detection of β-lactam-mediated resistance genes. Out of 25 Staphylococcal isolates recovered from clinical mastitic cases, one (4%) was S.aureus, 10 (40%) were coagulase positive staphylococci (CPS) other than S.aureus and 14 (56%) were coagulase negative staphylococci (CNS). Among the 118 isolates recovered from cases of subclinical mastitis, 32 (27%) were S. aureus, 30 (25%) were other CPS and 56 (48%) were CNS. Phenotypic prediction of mecA gene presence (historically referred to methicillin resistance, MR) was conducted by both cefoxitin and oxacillin disk diffusion tests, while prediction of blaZ gene presence was monitored by penicillin and amoxicillin/clavulinic acid (AMC) disk diffusion tests. Out of 25 Staphylococcal isolates recovered from clinical mastitic cases, 15, 19, 25 and 20 isolates were resistant to cefoxitin, oxacillin, penicillin and AMC, respectively. In subclinical mastitis, out of 118 isolates recovered, 43, 36, 43 and 22 isolates were resistant to cefoxitin, oxacillin, penicillin and AMC, respectively. According to PCR results, among the 25 Staphylococci isolated from clinical mastitis, 20 (80%) were methicillin resistant (MR) carrying the mecA gene, including 8 MRCPS and 12 MRCNS. Also, 17(68%) were β-lactamase producers harboring the 6/oZgene, including 1 S.aureus, 8 CPS and 8 CNS. On the other hand, among the 118 Staphylococci isolated from cases of subclinical mastitis, 27 (22.9%) were MR including 5 MR 5. aureus (MRSA), 7 MRCPS and 15 MRCNS. Also, 27 (22.9%) were β-lactamase producers including 12 S.aureus, 3 CPS and 12 CNS. The results showed higher incidence for MR and β-lactamase production in Staphylococci isolated from clinical mastitis versus those of subclinical mastitis (80 versus 22.9% and 68 versus 22.9%, respectively). Incidence of MRSA was found to be 15.6% of S.aureus caused subclinical mastitis. Close correspondence between phenotypic and genotypic tests was observed for the majority of isolates. Resistance to antimicrobials other than (3-lactams was determined. In general, multi-drug resistance was more common among blaZ &/or mecA positive isolates (P< 0.05) in comparison to blaZ & mecA negative isolates. In conclusion, high incidence of staphylococci causing bovine mastitis was observed in this study. Most importantly, it is ascertained that CPS other than S. aureus and CNS are also deeply implicated in both clinical and subclinical bovine mastitis. Considering β-lactams resistance, it was found to be widely spreading among staphylococcal isolates. Additionally, association between blaZ &/or mecA mediated β-lactam resistance genes and resistance to other antibiotics was also declared.
... Mastitis is an economically important disease responsible for several problems in dairy production worldwide (Asfour and Darwish, 2011). Staphylococci are identified to be the most prevalent mastitis-causing pathogens in dairy cows (Pitkala et al., 2004;Wald et al., 2019) and they are classified accordingly as coagulasepositive and coagulase-negative staphylococci (CoNS) (Taponen and Pyorala, 2009). ...
... Overall, the sensitivity and specificity of the phenotypic test used in the study were undefined and thus, unsuitable for comparison. Regardless, other studies (Reischl et al., 2000;Asfour and Darwish, 2011) find phenotypic methods to be time-consuming and labor-intensive, and disadvantageous due to Table 2. Absolute and relative (%) frequencies of beta-lactamase production detection among Staphylococcus aureus isolates (n = 9) in mastitic milk. ...
The study was conducted to compare phenotypic and genotypic methods of
detection of two beta-lactam resistant genes in Staphylococcus aureus isolates from
mastitic milk. Nine (9/92) S. aureus isolates were revived and used. The isolates
were subjected to phenotypic prediction of blaZ gene using the penicillin disk
diffusion method and nitrocefin test while prediction of mecA gene was observed
using the cefoxitin disk diffusion method. Both the penicillin disk diffusion method
and nitrocefin test detected three (33.33%) isolates to be producing beta-lactamase
while PCR detected two (22.22%) isolates positive for blaZ gene. Kappa coefficient
revealed perfect agreement (1.00) between phenotypic methods and substantial
agreement (0.7270) between the phenotypic methods and PCR. In the detection of
mecA-mediated methicillin resistance, none of the isolates resulted positive for
mecA gene for both tests hence, no methicillin-resistant S. aureus (MRSA) was
detected. The results of the study show that there are S. aureus isolates from
mastitic milk that expresses beta-lactam resistance through beta-lactamase
production however, while there was no MRSA detected, the study highly suggests
the monitoring of the antimicrobial usage in the farms and their management
practices to avoid further development of resistance and possible transfer of
resistance from animals to humans and/or humans to animals.
... Out of total Staphylococcus spp. isolates, only 75% were positive for nuc (nuclease) gene, which is an important virulence factor and a unique marker for S. aureus (20,21). The confirmed strains were further subjected to the toxin gene analysis using PCR; seb gene (enterotoxin B) and hla (hemolysin-A). ...
Mastitis is a multi-etiological complex disease of dairy cows and negatively affects the quality and quantity of milk. Milk is a nutritious food for human being; milk quality is negatively affected by intramammary infection of dairy cows. A total of 300 milk samples were collected from mastitis dairy cows irrespective of parity and stage of lactation, 235 (78.33%) samples were culturally positive and yielded 1,100 bacterial isolates. Staphylococcus aureus was found to be the prime etiological agent involved in the mastitis of dairy cows, followed by Escherichia coli and other environmental pathogens. On the molecular characterization of isolates obtained from the milk culture, various toxic genes such as nuc, seb, hla, stx1, stx2, hly, and Sagl were found on different isolated bacteria. Milk somatic cell counts (SCC) were found to be directly related to the severity of mastitis. On drawing the SCC correlation with milk components, it was found that SCC had a significant negative correlation with fat, lactose, solid not fat (SNF), and ash. It was concluded that mastitis-affected milk contains numerous pathogenic bacteria, toxins, and reduced milk quality, which is unfit for human consumption.
... Igualmente, con el agar DNasa (composición en g/L: triptosa: 20; ácido desoxirribonucleico: 2; NaCl: 5, y agar: 12), se evalúa la presencia de la enzima termoestable DNasa, que causa hidrólisis del adn. El ácido clorhídrico (HCl 1N) se utiliza para determinar la actividad enzimática, y su resultado muestra una zona transparente alrededor de la colonia (Asfour & Darwish, 2011;El-Hadedy & El-Nour, 2012). En algunos casos, cuando se requiere la confirmación de identidad de una accesión, se realiza un análisis bioquímico mediante el uso de tarjetas Vitek, un sistema automatizado que se basa en la inoculación de una suspensión de microorganismos en tarjetas con determinados paneles de reacciones bioquímicas. ...
Esta publicación hace parte de las acciones de promoción y divulgación del Banco de Germoplasma de Microorganismos en su interés de fortalecer capacidades técnicas, científicas y metodológicas en conservación, caracterización y uso de las colecciones a su cargo, además de promover condiciones para la interconectividad e interoperabilidad de estas. El conocimiento plasmado en este libro es producto de una trayectoria de años de investigación por parte de cada uno de los curadores, que han dejado su legado y sus enseñanzas en una acción de relevo generacional que esperamos que sea de gran ayuda tanto para los próximos curadores como para quienes buscan, en su hacer diario, aumentar y conservar la diversidad microbiana. Entregamos este libro como parte de una estrategia de gestión de conocimiento para garantizar y mantener un uso sostenible de la agrobiodiversidad y contribuir de forma eficiente al crecimiento económico del país.
... β-lactamase was investigated by PCR assay using primers which are F: AAG AGA TTT GCC TAT GCT TC and R:GCT TGA CCA CTT TTA TCA GC (14) to amplify theblaZ gene with size 517bp( blaZgene is responsible for β-Lactamase enzyme) . The amplification was performed under the conditions: Temperature 94 ºC as initial denaturation for 4 minutes ,35 cycles in order to denaturation with 94ºC , annealing (55ºC) , extension(72 ºC) and each one of these stages is accompanied with one minute , final extension (72 ºC) for 7 minutes. ...
Background: Staphylococcus aureus is constantly evolving and possesses new mechanisms of resistance to antibiotics. Antibiotics that unable to eliminate bacteria may cause them to become more virulent.Effort should be continuous to find new antibiotics, and accurate diagnostic techniques and tests that are absolutely relied upon in knowing the characteristics of the diagnosed bacteria to find the appropriate treatment. Methods:Staphylococcus aureus isolates were diagnosed by morphological, biochemical, and molecular methods. S. aureus sensitivity to antibiotics was tested by Disk Diffusion Method and Vitek®2 system, . Also, D-test was performed for both food and clinical samples. Result: The differences were found between the two tests (disk diffusion method and Vitek®2 system) which were used to sensitivity test. Food isolateshad an inducible MLSB phenotype with 80% and clinical isolates had it with 33.3%.All S. aureus isolates hadblaZ gene. The largest percentage of isolates were having the mecA gene. Conclusion: There is no test can be considered absolutely accurate. Food sample isolates are the most tolerant to the altered conditions . The resistance to each one of antibiotics is controlled by many and different genes anddifferent materials then, if one of these genes became inactive, the other genes or materials can do its action
This study aimed to assess the antimicrobial susceptibility of 52 Staphylococcus aureus (S. aureus) bovine mastitis isolates obtained from 37 dairy herds from the northwest of Portugal against antibiotics belonging to the β-lactam family, evaluate the detection of blaZ and mecA resistance genes, and study the diversity of positive isolates. The antimicrobial susceptibility tests were performed by the disk diffusion method. The detection of blaZ and mecA genes was performed using specific polymerase chain reaction (PCR) and the diversity of blaZ was evaluated by phylogenetic analysis. The antimicrobial susceptibility test showed a prevalence of phenotypic resistance by S. aureus of 52.0% against penicillin and ampicillin, 34.6% against oxacillin, 17.3% against amoxicillin plus clavulanic acid, and 21.1% against cefazolin. A prevalence of phenotypic intermediate resistance of 5.7% against penicillin, 9.6% against amoxicillin plus clavulanic acid, and 1.9% against ampicillin and cefazolin, respectively, was demonstrated. A 100.0% phenotypic susceptibility was found against piperacillin. Of the 52 S. aureus isolates, 35 (67.3%) were PCR positive for blaZ gene, and isolate 25 was positive for mecA gene. The phylogenetic analysis of partial blaZ gene S. aureus isolates consensus sequences were placed in two different clades, clade A (cluster A, A.1) and B (cluster B), all closely related to animal and/or human S. aureus strains. Isolate 2 appeared in the phylogenetic tree as the most divergent. This study indicates that blaZ resistance gene plays a role in β-lactam resistance in the tested bovine mastitis S. aureus isolates within dairy herds in the northwest of Portugal, especially in case of penicillin and ampicillin antibiotics that have shown a high phenotypic prevalence. Indeed, the proportion of bovine mastitis isolates with phenotypic resistance did not agree with the proportion of those identified for blaZ, as isolates with 100.0% of phenotypic susceptibility for all tested antibiotics also harbored blaZ. BlaZ phylogenetic analysis from S. aureus isolates showed diversity inside or between different herds in the northwest of Portugal. Piperacillin, as a suggestion, could be tested for S. aureus bovine mastitis treatment in the future to evaluate this new possibility of therapy.
animal production; antibiotic; dairy cattle; disease
Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic bacterium that causes many human and animal infections worldwide. MRSA infections are classified as priority infections owing to their high morbidity and mortality, with a significant risk of zoonotic transmission. This study aimed to determine the pooled prevalence of MRSA in dairy cattle farms and its heterogeneity. Relevant studies were retrieved from three databases: PubMed, Web of Science, and Scopus. The pooled prevalence of MRSA in dairy farms was estimated using a random-effects model. Subgroup and meta-regression analyses were used to assess the probable sources of heterogeneity. Sensitivity and publication bias analyses were also performed. A total of 94 articles were eligible for inclusion in this meta-analysis. The pooled prevalence of MRSA was estimated to be 3.81% [95% confidence interval (95% CI) = 2.61–5.20] with significantly high heterogeneity (I² = 96.6%, p = 0.00). For the subgroup analysis among continents, the prevalence was highest in Asia (4.89%; 95% CI = 2.88–7.35) and lowest in South America (1.33%, 95% CI = 0.00–5.49). As for the year of publication, MRSA prevalence was highest in reports published from 2015 to 2018 (4.36%, 95% CI = 2.41–6.80) and lowest in reports published before 2015 (2.65%, 95% CI = 0.75–5.52). As for sample type, the prevalence of MRSA in cattle milk (3.91%, 95% CI = 2.64–5.39) was higher than that in other sample types (1.19%, 95% CI = 0.05–3.24). These three factors were not significantly associated with the pooled prevalence of MRSA (p > 0.05). Therefore, the findings of this study indicate that the prevalence of MRSA has been minimal and consistent in dairy cattle farms over time.
Aims:
This meta-analysis aims to assess the point prevalence of methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine mastitis cases at the global level.
Methods and results:
Several electronic databases were searched for relevant publications (PubMed, EMBASE, Web of Knowledge and Cochrane Library). Heterogeneity between studies was assessed using the Cochrane Q test and I2 test statistics based on the random-effect model. The potential sources of between-study heterogeneity were evaluated using subgroup analysis and meta-regression. Sensitivity and publication bias analyses were performed. Sixty-six studies with a total of 77,644 mastitis cases were eligible and included in the analysis. The overall pooled prevalence of MRSA was 4·30% (95% CI: 3·24-5·50) with a significant heterogeneity (I2 = 97·48%, p < 0·001). In the subgroup analysis by region, the highest prevalence was found in Asia (6·47%, 95% CI: 4·33-8·97), and the lowest prevalence was reported in Europe (1·18%, 95% CI: 0·18-2·83). The pooled prevalence was significantly higher in clinical mastitis and cases published during 2016-2020.
Conclusions:
The results of this study suggest that there is a lower prevalence of MRSA in bovine mastitis. However, its prevalence increased in the past 4 years. Therefore, continuous surveillance is urgently required for monitoring the dissemination of these clinically important bacteria.
Significance of the study:
To our knowledge, this is the first meta-analysis to estimate the global prevalence of MRSA isolated from bovine mastitis cases.
In this study, it was aimed to investigate the prevalence of Staphylococcus aureus in poultry meats (32 chickens, 9 turkeys, 9 quails) sold in Isparta and Antalya provinces, and to determine the presence of enterotoxin genes as well as antibiotic resistance profiles and resistance genes. Baird Parker agar medium was used to isolate presumptive S. aureus from poultry meats. The species-level identification of isolates was done by polymerase chain reaction (PCR) using primer pair specific to the thermostable nuclease gene (nuc) in S. aureus. As a result of the PCR analysis, amplicons of 458 bp specific to the nuc gene were obtained in 16 of 130 presumptive S. aureus isolates. The frequency of S. aureus in poultry meat samples was found to be 20% (10/50). Coagulase test showed that all the S. aureus isolates were coagulase positive. Disk diffusion test showed that all of the isolates (100 %) were susceptible to chloramphenicol and teicoplanin and resistant to penicillin G. It was determined that 81.25 % (13/16) of the isolates were methicillin resistant S. aureus (MRSA). The most common antibiotic resistance gene in S. aureus strains was found to be blaZ (62.5 %, 10/16) as a result of PCR. The presence of sea, seb, sec, sed and see genes was not detected in any of the S. aureus strains.
The purpose of this study was to isolate and characterize Staphylococcus aureus from cattle milk in Sokoto metropolis. A total of three hundred mastitic milk samples were collected from dairy farms, abattoir and livestock market (Kara). Phenotypic identification of the S. aureus isolates by culture, microscopy and biochemical reactions were performed. Two hundred and nineteen (73%) of the samples were positive for species of mastitis causing bacteria. The isolates were S. aureus (47.9%), Streptococcus spp. (24.7%), Corynebacterium spp. (14.6%), Escherichia coli (5.9%), Bacillus spp (5.0%) and Klebsiella spp. (1.8%).The result obtained shows that S. aureus was the principal pathogen responsible for bovine mastitis in the study area with 35.0% prevalence. The need for strict hygienic and improved milking techniques as preventive measures against the disease was emphasized.
In this work, we investigated the phenotypic profile of Staphylococcus spp. isolates recovered from raw milk and artisanal cheese, and their enterotoxigenic potential through the detection of classical enterotoxin genes (sea, seb, sec, sed and see). A total of 104 isolates (58 coagulase-positive Staphylococcus – CoPS; and 46 coagulase-negative Staphylococcus- CoNS) were used, of which 33 were retrieved from raw milk and 71 from artisanal cheese produced in the Serrana region of Santa Catarina. Identification of CoPS was conducted via biochemical tests. Detection of the genes sea, seb, sec, sed, and see was carried out by multiplex PCR technique. Among the 58 CoPS analyzed, 64% were identified as S. aureus, 22% as S. scheiferi coagulans, 12% as S. hyicus and as 2% S. intermedius. In the present study was noted that 40% of CoPS isolates retrieved from milk harbored seb gene, while only one from artisanal cheese was positive for gene sea. In this study all CoNS samples investigated were negative for enterotoxins genes. The enterotoxigenic potential of CoPS, is an issue of great importance for public health. For that reason, it is necessary that cheese factories strictly follow the safety processes involved in manufacturing.
The objective of this work was to study the protein patterns, plasmid profiles, and antibiotic susceptibility of Staphylococcus intermedius and Staphylococcus aureus isolates originating from mastitic mammary glands of dairy cattle in different parts of Konya province. A total of 114 Staphylococcus species were isolated and identified by conventional bacteriological methods from bovine mastitis. Of the total isolates 77 were identified as S. aureus and 37 as S. intermedius. Intra- and inter-species diversities in the coagulase-positive staphylococci were investigated by analysis of whole-cell protein profiles using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Plasmid profiling also demonstrated that 75 S. aureus isolates and 36 S. intermedius isolates contained plasmid. In addition, 88.3% of S. aureus and 59.4% of S. intermedius isolates were resistant to penicillin. Sixty-six of the 77 S. aureus isolates were also resistant to amoxicillin+clavulanic acid (85.7%). The corresponding number for S. intermedius was 17 (45.9%). Only 1 S. aureus isolate was resistant to danofloxacin. One of each of the Staphylococcus isolates was resistant to methicillin. Results from the study showed that the susceptibility of S. intermedius isolates to antibiotics used widely in mastitis therapy is a matter of concern.
The purpose of this study was to determine the minimum number of tests that could be used to differentiate between the coagulase-positive strains of staphylococcus: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius. Eighty coagulase-positive strains of each of the three species were examined. The five tests conducted were growth on modified Baird-Parker agar, growth on P agar supplemented with acriflavin, production of acetoin, anaerobic fermentation of mannitol, and presence of beta-galactosidase. Positive test percentages for S. aureus were 100% for growth on modified Baird-Parker agar, 100% for growth on P agar supplemented with acriflavin, 94% for production of acetoin, 99% for anaerobic fermentation of mannitol, and 0% for presence of beta-galactosidase. Positive test percentages for S. intermedius were 0% for growth on modified Baird-Parker agar, 0% for growth on P agar supplemented with acriflavin, 1% for production of acetoin, 0% for anaerobic fermentation of mannitol, and 100% for presence of beta-galactosidase. S. hyicus isolates were negative in all five tests. Results from the 240 coagulase-positive staphylococcus strains tested would suggest correct identification of coagulase-positive staphylococci with P agar supplemented with acriflavin and the beta-galactosidase test. These two tests are simple to conduct and result in quick and easy differentiation of the three coagulase-positive staphylococcal species.
Most methods currently available to clinical microbiology laboratories for phenotypic detection of oxacillin resistance in Staphylococcus aureus, e.g., MIC and disk diffusion methods, perform well if the tests are inoculated and incubated properly. However, in rare circumstances, additional testing for confirmation of susceptibility or resistance may be needed. These circumstances include the occurrence of extremely heterogeneous mecA-determined resistance where conventional methods may categorize the strain as susceptible. In addition, confirmation of borderline resistance determined by mechanisms other than the presence of the mecA gene may need to be verified because treatment with oxacillin may be effective. Several new methods that detect either the mecA gene or the gene product (e.g., penicillin-binding protein 2a, or PBP 2a) have been cleared by the Food and Drug Administration and are now available. These methods, including an easy-to-perform latex agglutination test, may be considered for use by many clinical laboratories when more sophisticated molecular techniques (i.e., PCR assay) are not available.
Minimum inhibitory concentrations were determined for selected antimicrobial agents against 872 bacteria isolated from intramammary infections in heifers in New Zealand (n = 401) and Denmark (n = 471). These values were reported in micrograms per milliliters. Antimicrobial agents tested against isolates from New Zealand were penicillin, cloxacillin, cephapirin, ceftiofur, novobiocin, enrofloxacin, erythromycin, and pirlimycin. The minimum inhibitory concentrations that inhibit 90% of the strains tested for these antimicrobial agents with Staphylococcus aureus were 4.0, 0.5, 0.5, 2.0, 1.0, 0.25, 0.5, and 1.0, respectively. The minimum inhibitory concentration values that inhibit 90% of the strains tested against the Staphylococcus spp. ranged from 0.5 to 1.0 for all antimicrobics. The minimum inhibitory concentrations against streptococci were < or = 0.06, 0.5, 0.13, 0.13, 4.0, 1.0, 0.13, and < or = 0.06, respectively. Antimicrobial agents tested against isolates from Denmark included penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin plus novobiocin, erythromycin, and pirlimycin. Against S. aureus, the minimum inhibitory concentrations were 0.13, 0.5, 0.5, 0.5, 1.0, 0.25, 0.5, and 0.5, respectively. The minimum inhibitory concentrations against Staphylococcus spp. were 0.25, 0.25, 0.5, 0.5, 1.0, < or = 0.06, 0.13, 1.0, and 0.5, respectively. The minimum inhibitory concentrations against the streptococci were < or = 0.06, 0.13, 0.5, 0.5, 1.0, < or = 0.06, 0.13, 0.5, and 0.5, respectively. Minimum inhibitory concentration values for staphylococci from New Zealand and Denmark were similar to values reported for US isolates. Streptococci from New Zealand and Denmark had lower minimum inhibitory concentration values than did US isolates. Only ceftiofur and enrofloxacin were active against the Gram-negative bacilli.
Three distinctly different mechanisms of methicillin resistance have been described in Staphylococcus aureus. The best-documented and probably most important mechanism is production of a unique, low affinity penicillin-binding protein, PBP 2a. Strains possessing PBP 2a are resistant to methicillin, oxacillin, and probably all other currently available beta-lactam antibiotics. Two additional mechanisms of reduced susceptibility to methicillin have been described. Borderline resistance (BORSA) to the semi-synthetic penicillins has been attributed to the hyperproduction of normal staphylococcal beta-lactamase. A third mechanism has recently been advanced that describes an intermediate level of resistance to methicillin due to production of modified, normal PBPs with reduced affinity for beta-lactams (MODSA). Little is known regarding the prevalence or clinical significance of the BORSA and MODSA strains. The most reliable in vitro susceptibility test methods for detecting MRSA (strains possessing PBP 2a) include the microdilution minimum inhibitory concentration (MIC) test (with 2% NaCl supplemented broth), the oxacillin agar screen plate test (incorporating 6 micrograms/ml oxacillin in 4% NaCl supplemented agar), and the National Committee for Clinical Laboratory Standards (NCCLS) disk diffusion test with oxacillin. All three methods use direct inoculum preparation and incubation of tests at 35 degrees C for a full 24 hours.
A total of 156 dairy cows was randomly assigned to one of four groups at drying off over an 18-mo period: untreated control or intramammary treatment of each mammary quarter with either 400 mg novobiocin, 300 mg cephapirin, or 1 g dihydrostreptomycin with 1 million units penicillin. Quarter foremilk samples were aseptically collected from each cow within 1 mo of drying off and within 1 mo after parturition for bacteriological analysis. Prior to drying off, 28.7% of quarters were positive of which 46.7% were Corynebacterium bovis and 45.5% were coagulase-negative staphylococci. Reductions in infection prevalence from drying off to postpartum samplings for control, novobiocin, cephapirin, and streptomycin-penicillin groups were 13.6, 60.5, 74.4, and 35.3% of quarters. Recovery rates for C. bovis infections were 47.6, 100, 100, and 94.1%; for coagulase-negative staphylococci infections they were 72.7, 86.4, 80.0, and 100%. The cephapirin group showed the lowest new infection rate (1.3%) with coagulase-negative staphylococci compared with control (6.9%). There were no significant differences in lactation milk production among groups following dry period therapy. Results suggest that dry treatment reduces the prevalence of infections by the minor mastitis pathogens.
Contemporary clinical isolates of Staphylococcus aureus possess a formidable array of determinants for resistance to antimicrobial agents, a number of which are plasmid encoded. The plasmids detected in such multiresistant isolates from Australia, Europe and the USA are compared and some of the potential genetic rearrangements involved in their evolution discussed.
The continuously high prevalence of methicillin-resistant staphylococci (MRS) throughout the world is a constant threat to public health, owing to the multiresistant characteristics of these bacteria. Methicillin resistance is phenotypically associated with the presence of the penicillin-binding protein 2a (PBP2a) not present in susceptible staphylococci. This protein has a low binding affinity for beta-lactam antibiotics. It is a transpeptidase which may take over cell wall synthesis during antibiotic treatment when normally occurring PBPs are inactivated by ligating beta-lactams. PBP2a is encoded by the mecA gene, which is located in mec, a foreign DNA region. Expression of PBP2a is regulated by proteins encoded by the plasmid-borne blaR1-bla1 inducer-repressor system and the corresponding genomic mecRl-mecl system. The blaRl-blal products are important both for the regulation of beta-lactamase and for mecA expression. Methicillin resistance is influenced by a number of additional factors, e.g. the products of the chromosomal fem genes which are important in the synthesis of normal peptidoglycan precursor molecules. Inactivation of fem-genes results in structurally deficient precursors which are not accepted as cell wall building blocks by the ligating PBP2a transpeptidase during antibiotic treatment. This may result in reduced resistance to beta-lactam antibiotics. Inactivation of genes affecting autolysis has shown that autolytic enzymes are also of importance in the expression of methicillin resistance. Methicillin resistance has evolved among earth microorganisms for protection against exogenous or endogenous antibiotics. Presumably the mec region was originally transferred from coagulase negative staphylococci (CNS) to Staphylococcus aureus (SA). A single or a few events of this kind with little subsequent interspecies transfer had been anticipated. However, recent data suggest a continuous horizontal acquisition by S. aureus of mec, being unidirectional from CNS to SA. Methicillin resistance may also be associated with mechanisms independent of mecA, resulting in borderline methicillin resistance. These mechanisms include beta-lactamase hyperproduction, production of methicillinases, acquisition of structurally modified normal PBPs, or the appearance of small colony variants of SA. Most MRS are multiresistant, and the mec region may harbour several resistance determinants, resulting in a clustering of resistance genes within this region.