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Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.

The aim of this work was to evaluate the relative gene expression levels of the cytokines IL- 1B, IL-8, IL-12, IFN-γ, IL-4, IL-10 and TGF-β in somatic milk cells of French Alpine breed, anestrous goats that were experimentally infected in the left mammary gland with Staphylococcus chromogenes during the lactation peak. Milk samples were obtained from both glands for 21 consecutive days post infection. Total RNA was extracted, and real-time PCR was conducted using primers specific to each cytokine. The relative RNA expression of the evaluated cytokines was determined by the comparative method 2-ΔΔCT, using milk from the right gland of the goats as a reference (control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. According to the Wilcoxon test results, IL-1B and IL-12 expression levels showed significant differences compared to those in the control group (p⟨0.05) from 24 hours post infection until the end of lactation; on day three, IL1β, IL8, IL12 and TGF-β had a statistically significant change in expression with respect to those in the control group (p⟨0.05); closer to the end of the lactation period, there is no overexpression of the anti-inflammatory interleukins (IL-4 and TGF-β) which may reflect the effort of the host immune system to eradicate the microorganism from the mammary gland., (Copyright© by the Polish Academy of Sciences.)

The present study aimed to provide a detailed characterization of coagulase-negative staphylococci (CoNS) isolated from cows and buffaloes with mastitis. The study included seventy-five CoNS isolates (60 came from cattle and 15 from buffaloes) originating from 68 individual quarters of 67 dairy cows (53 cattle and 14 buffaloes). The animals belonged to five different small holding dairy herds (n = 140 cows) that show clinical or subclinical mastitis. CoNS isolates were phenotypically characterized using MALDI-TOF-MS and were further genotypically characterized by microarray-based assays. Furthermore, the antimicrobial susceptibility of CoNS strains which carried the mecA gene was examined by broth microdilution. The occurrence of CoNS in the respective five herds was 10.5%, 14.7%, 14.8%, 12.8%, and 9.9%, with an average of 12.4%. Six different CoNS species were identified: S. sciuri (n = 37; 30 from cattle and 7 from buffaloes), S. chromogenes (n = 14; 8 from cattle and 6 from buffaloes), S. haemolyticus (n = 10; nine from cattle and one buffalo), S. xylosus (n = 10; nine from cattle and one buffalo), S. hyicus (n = 2), S. warneri (n = 1), and unidentified CoNS (n = 1). Twenty percent (20%) of CoNS isolates (17.3% of cattle origin) carried at least one antimicrobial resistance gene, while 4% of the isolate including two isolates of S. haemolyticus and one S. warneri of cattle origin carried the mecA gene and were phenotypically identified as methicillin-resistant strains. The genes detected were blaZ (16%), followed by tet(K) (8%), aacA-aphD (4%), aphA3 (2.6%), msr(A) (2.6%), [far1 (2.6%), and fusC (2.6%)], sat (2.6%), and cat (1.3%) conferring resistance to penicillin, tetracycline, gentamicin, neomycin/kanamycin, erythromycin, fusidic acid, streptothricin, and chloramphenicol, respectively. The majority of investigated CoNS strains displayed considerably low prevalence of resistance genes, while resistance to more than three antibiotics was found in S. haemolyticus and S. warneri. Implementing effective preventive measures is, therefore, important for limiting the transmission of CoNS, rather than using antibiotics to control mastitis in bovines.

The study described in this Research Communication investigated the genotypic and phenotypic profiles of resistance to beta-lactams and other antimicrobials in non-aureus Staphylococci (NAS) isolated from buffalo, goat and sheep mastitis in the Northeast region of Brazil. A total of 190 isolates were analyzed and 42.3, 43.9 and 23.6% of them were positive for blaZ gene in buffalo, goat and sheep, respectively. Regarding the animal groups, in goats, amoxicillin was the antimicrobial with highest resistance index (72.7%), followed by penicillin G in buffaloes (51.9%) and ampicillin in sheep (43.1%). With regard to multiple antimicrobial resistance, 30.8% of NAS isolates from buffalo milk samples, 25.8% from goats and 25.0% from sheep presented multidrug-resistance. In the minimum inhibitory concentration (MIC) technique, amoxicillin MIC50 and MIC90 were 64 and 128 μg/ml, respectively, among isolates of the three animal species. In conclusion, high rates of resistance to beta-lactams are presented among NAS isolated from mastitis cases in buffaloes, goats and sheep in Northeast region of Brazil. These results provide an alert to animal and human health researchers, suggesting that the frequency of NAS needs to be reduced because they carry resistance genes which might increase the existing levels of antimicrobial resistance.

Staphylococcus xylosus (S. xylosus) is one of the emerging pathogens causing bovine mastitis with high rate of isolation in most of the reported clinical and field cases. To verify the role of glutamine synthetase (GS) in the pathogenesis of S. xylosus, we evaluated the virulence level of the wild-type strain and its glnA mutant strain in biofilm assays in vitro and murine infection model in vivo. From the results, it was observed that the glnA mutant strain was attenuated and could reduce tissue damage. 1-Hydroxyanthraquinone (1-HAQ) is a kind of anthraquinones, it exhibited a significant inhibitory effect on the growth of S. xylosus and biofilm formation in vitro and provided anti-inflammatory effects in vivo. In addition, the rate at which it inhibits the biofilm, inflammatory factors, and CFU of wild-type strains were significantly higher than that of the mutant strains, indicating that 1-hAQ might have pharmacological effects against S. xylosus through the regulation of GS protein. The effect of 1-hAQ on GS was further confirmed by the down-regulation of glnA expression, reduced GS activity, Gln content and the results of molecular docking. Taken together, these findings suggest that 1-hAQ facilitated a significant attenuation of S. xylosus pathogenicity by regulating the GS protein: a vital virulence factor. Therefore, it can be inferred that 1-hAQ may serve as a potential source of organic compound for the development of novel alternative drugs in mitigating the menace of bovine mastitis., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Numerous factors affect milk somatic cell score (SCS) in dairy goats including physiologic factors such as stage of lactation, and pathological factors such as bacterial intramammary infection (IMI), e.g. staphylococcal IMI. The association between IMI and udder inflammation and especially differences in the inflammatory response between the different staphylococcal species are yet to be identified. The objectives of this study were to use data from a recently published longitudinal study that characterized IMI status over time to evaluate the species-specific effect of staphylococcal IMI on udder half-level milk SCS, and to evaluate the effect of time since IMI diagnosis on this relationship. Half-level milk samples for culture and determination of SCS were collected from 171 goats starting within 10 days in milk (DIM), and then at various intervals until ≥ 120 DIM. A mixed linear model was used to assess the effect of species-specific staphylococcal IMI on SCS, accounting for confounding by lentivirus infection, parity, DIM, and the contralateral half IMI status, and for repeated measurements within udder half and for clustering of halves within goat. A second model was used to evaluate the effect of time since first IMI diagnosis and its interaction with staphylococcal IMI status on the relationship between staphylococcal IMI status and SCS. Lentivirus seropositivity and contralateral half IMI status were associated with SCS. Staphylococcal IMI status was associated with SCS, with differences between species. Staphyloccocus caprae, Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus xylosus were associated with higher SCS than a number of other species. Time since first diagnosis did not modify the relationship between IMI status and SCS., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mastitis in ruminants is an important disease with major effects on both the economy and animal welfare. It is caused by major pathogens such as Staphylococcus aureus and minor pathogens such as coagulase-negative staphylococci. The objective of this study was to identify and characterize staphylococci as a cause of sheep mastitis in Algeria. In this study, 123 milk samples were collected directly from the udder of sheep suffering from clinical mastitis in 2 provinces in Algeria. Recovered isolates were identified using MALDI-TOF mass spectrometry. Virulence-associated and antimicrobial resistance genes as well as clonal complexes (CC) of S. aureus were determined using microarray-based analysis. A total of 45 staphylococci isolates were cultivated from sheep milk samples, and 28 S. aureus were identified as methicillin susceptible (62.2%). Seventeen other Staphylococcus isolates of different species were identified using MALDI-TOF mass spectrometry. Subsequent microarray analysis typed the methicillin-susceptible S. aureus to 6 CC: CC8-MSSA, CC97-MSSA, CC130/521-MSSA, CC479-MSSA, CC522-MSSA, and CC705-MSSA. The accessory gene regulator agrIII and the ruminant leukocidin genes lukF-P83 and lukM were found in all isolates of CC130/521, CC479, CC522, and CC705. The toxic shock syndrome toxin gene tst1 was detected exclusively in CC130/521. Additionally, virulence-associated genes (sea, sed, sak, hld, hlgA, edinB, and others) were detected. The presence of antibiotic resistance genes [blaZ, erm(B), and tet(K)] was detected in small numbers of staphylococci. Staphylococci possessing these genes are considered potential hazards for farm animals, farmers, and consumers. Data concerning the prevalence and diversity of staphylococci causing mastitis in sheep from Algeria are lacking. Presented results on different aspects about staphylococci in Algerian sheep populations should at least partially close that gap. However, further extensive studies covering more geographical regions are needed to assess the epidemiological risk., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This work aimed to evaluate the effects of CasuL on growth and viability of 15 mastitis isolates from cows and goats, to determine the synergistic potential between CasuL and antibiotics, and to investigate the effects on bacterial ultrastructure and antibiofilm activity. The lectin inhibited the growth of Staphylococcus isolates from either bovine (Ssp6PD and Sa) or caprine (Ssp5D and Ssp01) mastitis. The minimal inhibitory concentrations were ranged from 3.75 to 15 µg/ml. Synergistic effect was observed for CasuL-tetracycline against Sa and Ssp6PD and CasuL-ampicillin against Ssp01. No structural damage was observed under the scanning electron microscope in CasuL treatments. Flow cytometry analysis using thiazol orange and propidium iodide demonstrated that CasuL was unable to reduce the cell viability of the isolates tested. At sub-inhibitory concentrations, CasuL reduced biofilm formation by the isolates Sa and Ssp5D. However, CasuL-tetracycline and CasuL-ampicillin combinations inhibited biofilm formation by Ssp6PD and Ssp01, respectively. In conclusion, CasuL is a bacteriostatic and antibiofilm agent against some mastitis isolates and displayed a synergistic potential when used in combination with either ampicillin (against one isolate) or tetracycline (against two isolates). The results stimulate the evaluation of CasuL for the treatment of mastitis, particularly when used in conjunction with antibiotics., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

A single-tube multiplex real-time PCR targeting the nuclease (nuc) gene and subsequent high-resolution melting analysis (HRMA) were used to identify 13 different Staphylococcus species. The nuc gene was targeted due to its low intraspecies variation and the greater interspecies variation than the 16S rRNA gene in Staphylococcus. We used HRMA software that can store and compare HRMA profiles from different runs as long as the runs contain the same reference reaction. Thus, we reduced the 14 PCRs to 2 different PCRs, one targeting the unknown sequence and the other targeting the reference sequences to screen 13 different Staphylococcus species. The specificity of the developed method was tested on 16 different Staphylococcus reference strains and 115 different field strains that were isolated from the milk of cattle with subclinical mastitis. We conclude that the method can be used to quickly and cost-effectively differentiate Staphylococcus aureus (S. aureus) from other Staphylococcus species (S. epidermidis, S. lugdunensis, S. schleiferi, S. hyicus, S. chromogenes, S. lentus, S. haemolyticus, S. xylosus, S. saprophyticus, S. warneri, S. simulans and S. hominis)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Staphylococci have been isolated from various sites of the body of healthy sheep, as well as from many infections of those animals, the main one being mastitis. The objective of this review is to appraise the importance and significance of staphylococci in causing mastitis in ewes. The review includes a brief classification and taxonomy of staphylococci and describes the procedures for their isolation and identification, as well as their virulence determinants and the mechanisms of resistance to antibacterial agents. Various staphylococcal species have been implicated in staphylococcal mastitis and the characteristics of isolates are discussed with regards to potential virulence factors. Staphylococcal mastitis is explicitly described, with reference to sources of infection, the course of the disease and the relevant control measures. Finally, the potential significance of staphylococci present in ewes' milk for public health is discussed briefly.

The objectives of the research described here were to describe the persistence of intramammary infections (IMI) caused by coagulase negative staphylococci (CNS) in goats using strain-typing, and to evaluate the relationship between species-specific CNS IMI and somatic cell score (SCS) at the udder-half level. Udder-half milk samples were collected from all 909 lactating goats (1817 halves; 1 blind half) in a single herd. Milk samples were cultured on Columbia blood agar, and 220 goats with at least one half yielding a single colony type CNS were enrolled for two additional half-level samplings at approximately 1-month intervals. Isolates were identified to the species level by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry or PCR amplification and partial sequencing of tuf or rpoB. An IMI was defined as persistent when ≥1 follow-up sample yielded the same species and strain as on Day 0 based on pulsed-field gel electrophoresis. A generalised mixed linear model was used to evaluate the odds of persistence as a function of CNS species. A mixed linear model was used to evaluate the relationship between IMI status on a given day and SCS. Among 192 IMI, 69.8% were persistent based on species and strain-type. Staphylococcus simulans IMI had higher odds of persistence than Staphylococcus arlettae IMI. In primiparous goats, Staphylococcus epidermidis IMI was associated with higher SCS than S. arlettae, Staphylococcus xylosus and 'other CNS' IMI. The differences detected in the present study between CNS species, with regard to persistence of IMI and association with SCS, highlight the need to study CNS at the species and strain level to understand the pathogenicity and epidemiology of CNS in goats.

Objective was to recognise areas potentially of high risk for increased frequency of subclinical mastitis in ewes. Milk samples were collected, for bacteriological and cytological examination, from 2198 clinically healthy ewes in 111 farms in all administrative regions of Greece. Data on farms were located in the field using hand-held Global Positioning System Garmin units. Collected data were analysed by an Ecological Niche Model under the framework of a geographic information system. Two separate analyses were performed: one for subclinical mastitis independently of causal agent (prevalence in population sampled: 0.260) and one for subclinical mastitis caused specifically by slime-producing staphylococci (prevalence in population sampled: 0.153). A model was constructed in which sheep farms were divided into two clusters, according to prevalence of subclinical mastitis: farms in the upper three quartiles of prevalence were used as occurrence points for the Ecological niche modelling procedure ('infected farms'); farms in the lower quartile of prevalence within each category were (pseudo)negative points. Significant differences in environmental parametres prevailing in locations of farms into the study, were identified for up to 13 parametres between locations of farms according to management system applied in farms. When farms in each management system were considered separately, differences became evident between farms in each management system, as well as between the two infections. The factor with the highest relative contribution in the analyses was the distance from other sheep farms; other factors also of importance in the predictive models were the altitude, the maximum temperature of warmest month and the total precipitation of driest month. Verification of the model revealed that ≥ 0.760 of infected farms' were located in areas predicted as high risk for prevalence of subclinical mastitis or slime staphylococcal subclinical mastitis. The paper describes for the first time potential association of mastitis with environmental factors and presents predictive models for mastitis in ewes taking into account environmental parametres., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hitherto, research work in slime production from staphylococcal strains of mastitis origin has focused in laboratory properties of these organisms. Objective of present work was to study subclinical mastitis in sheep, caused specifically by slime-producing staphylococci: to investigate its frequency and to identify potential factors playing a role therein. Slime production was evaluated in 708 staphylococcal isolates recovered from cases of subclinical mastitis in a field study in 2198 ewes performed in an extensive countrywide field investigation across Greece. Isolates were studied by means of microbiological and molecular methods. Of these strains, 262 were characterised as slime-producing, 227 as weak slime-producing and 219 as non slime-producing. Most frequently detected genes were eno and icaB; Staphylococcus aureus possessed more genes than coagulase-negative strains; greater number of genes was detected in slime-producing than in weak slime-producing or non-slime-producing strains. Subclinical mastitis caused specifically by slime-producing staphylococci was detected in 337 ewes: prevalence in population sampled was 0.153. A multivariable mixed-effects model revealed that milking mode (highest prevalence in hand-milked flocks) and flock management system (highest prevalence in semi-intensive flocks) were the two factors associated with increased prevalence of mastitis in flocks. The results confirmed the significance of slime producing staphylococcal strains of importance in the aetiology of subclinical mastitis of sheep. Hand-milking was identified as the most important factor predisposing to that infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mastitis in goats is mainly caused by coagulase-negative Staphylococcus (CNS). The identification methods for this group are based on evaluating the expression of phenotypic characteristics such as the ability to metabolize various substrates; however, this is disadvantageous as these methods are dependent on gene expression. In recent years, genotyping methods such as the Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and gene identification have been useful for epidemiological study of several bacterial species. To develop a genotyping method, the genome sequence of Staphylococcus chromogenes MU970 was analysed. The analysis showed nine virulence genes described in Staphylococcus aureus. The MLVA was developed using four loci identified in the genome of S. chromogenes MU970. This genotyping method was examined in 23 strains of CNS isolated from goat mastitis. The rate of discrimination for MLVA was 0.8893, and the highest rates of discrimination per the index of Simpson and Hunter-Gaston were 0.926 and 0.968 for the locus 346_06, respectively. The virulence genes were present in all strains of S. chromogenes but not in other CNS. The genotyping method presented in this paper is a viable and easy method for typifying CNS isolates from mastitis cases in different regions and is an ideal mean of tracking this disease.

Objective: Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes., Results: From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in cases of subclinical mastitis in dairy cows. CNS species may differ in their pathogenicity, but very little is known about their virulence factors or their immune response in intramammary infections. To our knowledge, no experimental studies into the mastitis pathogenesis caused by CNS have been described in lactating goats. The aim of this study was to induce an experimentally Staphylococcus chromogenes mastitis in lactating goats aimed at verifying if the model can be used to evaluate the inflammatory response, the dynamics of infection and the pathological findings within the first hours of intramammary inoculation. Six Saanen goats in mid-lactation were inoculated with 1 × 10 7 colony forming units of S. chromogenes. Bacterial growth peaked in milk from the challenged right halves of the mammary glands (RMG) at 4 h post inoculation (PI). Shedding of viable bacteria showed a marked decrease at 12 h PI. An increase in mean somatic cell counts was observed in the milk samples from 8 h PI onwards. Mild clinical signs were evoked by intramammary inoculation. Staphylococcus chromogenes could be isolated in tissue from all RMG. Histological examination of specimens of the RMG and lymph nodes of the goats showed an increased inflammatory response throughout the experiment with respect to control halves. In conclusion, the experimental inoculation of S. chromogenes in lactating goats is capable of eliciting an inflammatory response and capable of causing pathological changes. This research represents a preliminary study for a better knowledge of the mastitis pathogenesis caused by S. chromogenes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Staphylococci are very common human and animal pathogens. A variety of staphylococcal virulence determinates leads to vast range of infections. One of them is mastitis which is a common disease of the mammary glands. The incidence of this disease is widespread all over the world and depends on bacterial virulence and on prevention programs. The influence of mastitis on human health is not globally evaluated, however, in veterinary fields loses in milk production caused by bovine mastitis are a constant economic problem. One of the most important parts of the mastitis control programs is accurate diagnosis of the inflammation and characterization of the etiological factors which leads to reduction of mastitis spread. Recent reports show that staphylococci are common bacterial etiological factors of mastitis, and this paper is an overview of the diagnostic typing methods used for characterization of staphylococcal isolates. A number of different techniques available to applicate is described. Phenotypic methods to identify and to differentiate isolates or discriminate virulence factors are still in use, however, some advanced genetic methods offering higher discriminatory power are reported as more accurate. In fact, nowadays the most powerful tool on that field is next generation sequencing (NGS) of the whole genome, but its high cost and requirement of special laboratory equipment makes it hard to use for routine diagnostics. That is why standard PCR techniques-based methods, and the sequencing of particular genes, are mostly used for typing bacterial isolates. Most of these techniques are characterized by a high discriminatory power, big epidemiological concordance, and repeatable results. The presented report describes the techniques used most frequent in mastitis diagnostics related to staphylococci typing and shows their advantages and disadvantages., (Copyright© by the Polish Academy of Sciences.)

Aims: Mastitis causes economic losses and antimicrobials are frequently used for mastitis treatment. Antimicrobial resistance surveys are still rare in the ovine field and characterization of strains is important in order to acquire information about resistance and for optimization of therapy., Methods and Results: Bacterial pathogens recovered in milk samples from mastitis-affected ewes were characterized for resistance to tetracyclines and aminoglycosides, members of which are frequently used antimicrobials in small ruminants. A total of 185 strains of staphylococci, streptococci, and enterococci, common mastitis pathogens, were tested for minimal inhibitory concentration (MIC) to tetracycline, doxycycline, minocycline, gentamicin, kanamycin, streptomycin, and for resistance genes by PCR. Effects of different tet genes arrangements on MICs were also investigated. Staphylococci expressed the lowest MIC for tetracycline and tet(K) was the most common gene recovered; tet(M) and tet(O) were also found. Gene content was shown to influence the tetracycline MIC values. Enterococci and streptococci showed higher MICs to tetracyclines and nonsusceptible strains always harboured at least one ribosomal protection gene (MIC above 8 μg ml(-1) ). Streptococci often harboured two or more tet determinants. As regards the resistance to aminoglycosides, staphylococci showed the lowest gentamicin and kanamycin median MIC along with streptomycin high level resistant (HLR) strains (MIC >1024 μg ml(-1) ) all harbouring str gene. The resistance determinant aac(6')-Ie-aph(2″)-Ia was present in few strains. Streptococci were basically nonsusceptible to aminoglycosides but neither HLR isolates nor resistance genes were detected. Enterococci revealed the highest MICs for gentamicin; two str harbouring isolates were shown to be HLR to streptomycin., Conclusion: Evidence was obtained for the circulation of antimicrobial-resistant strains and genes in sheep dairy farming., Significance and Impact of the Study: Tetracycline MIC of 64 μg ml(-1) and high-level resistance were detected for streptomycin (MIC >1024 μg ml(-1) ), so that effectiveness of common treatments may be at risk., (© 2016 The Society for Applied Microbiology.)

Staphylococcus caprae is an emerging microorganism in human bone and joint infections (BJI). The aim of this study is to describe the features of S. caprae isolates involved in BJI (H for human) compared with those of isolates recovered in goat mastitis (A for animal). Fourteen isolates of each origin were included. Identifications were performed using a Vitek 2 GP ID card, tuf gene sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Vitek MS. Molecular typing was carried out using pulsed-field gel electrophoresis (PFGE) and DiversiLab technology. The crystal violet method was used to determine biofilm-forming ability. Virulence factors were searched by PCR. Vitek MS technology provides an accurate identification for the two types of isolates compared to that of gold-standard sequencing (sensitivity, 96.4%), whereas the Vitek 2 GP ID card was more effective for H isolates. Molecular typing methods revealed two distinct lineages corresponding to the origin despite few overlaps: H and A. In our experimental conditions, no significant difference was observed in biofilm production ability between H and A isolates. Nine isolates (5 H isolates and 4 A isolates) behaved as weak producers while one A isolate was a strong producer. Concerning virulence factors, the autolysin atlC and the serine aspartate adhesin (sdrZ) genes were detected in 24 isolates (86%), whereas the lipase gene was always detected, except in one H isolate (96%). The ica operon was present in 23 isolates (82%). Fibrinogen-binding (fbe) or collagen-binding (cna) genes were not detected by using primers designed for Staphylococcus aureus or Staphylococcus epidermidis, even in low stringency conditions. Although S. caprae probably remains underestimated in human infections, further studies are needed to better understand the evolution and the adaptation of this species to its host., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The aim of this study was to investigate genes involved in adhesion expression, biofilm formation, and enterotoxin production in isolates of Staphylococcus spp. from goats with subclinical mastitis and associate these results with the staphylococcal species. One hundred and twenty-four isolates were identified and polymerase chain reaction (PCR) was performed to detect the following genes: cna, ebpS, eno, fib, fnbA, fnbB, bap, sea, seb, sec, sed and see. The most commonly Staphylococcus species included S. epidermidis, S. lugdunensis, S. chromogenes, S. capitis ss capitis and S. intermedius. With the exception of fnbB, the genes were detected in different frequencies of occurrence in 86.3% of the Staphylococcus spp. isolates. Eno (73.2%) and bap (94.8%) were more frequently detected in coagulase-negative staphylococci (CNS); ebpS (76%), fib (90.9%) and fnbA (87%) were the most frequent genes in coagulase-positive staphylococci (CPS). Regarding enterotoxins, genes sed (28.2%) and see (24.2%) had a higher frequency of occurrence; sec gene was more frequently detected in CPS (58.8%). There was no association between the presence of the genes and the Staphylococcus species. Different virulence factors genes can be detected in caprine subclinical mastitis caused by CNS and CPS. The knowledge of the occurrence of these virulence factors is important for the development of effective control and prevention measures of subclinical mastitis caused by CNS and CPS in goats., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Coagulase-negative staphylococci (CNS) are a group of bacteria classified as either minor mastitis pathogens or commensal microbiota. Recent research suggests species- and even strain-related epidemiological and genetic differences within the large CNS group. The current pilot study investigated in 2 experiments whether a mouse mastitis model validated for bovine Staphylococcus aureus can be used to explore further differences between CNS species and strains. In a first dose titration experiment, a low inoculum dose of S. aureus Newbould 305 (positive control) was compared with increasing inoculum doses of a Staphylococcus chromogenes strain originating from a chronic bovine intramammary infection to a sham-inoculated mammary glands (negative control). In contrast to the high bacterial growth following inoculation with S. aureus, S. chromogenes was retrieved in very low levels at 24 h postinduction (p.i.). In a second experiment, the inflammation inflicted by 3 CNS strains was studied in mice. The host immune response induced by the S. chromogenes intramammary strain was compared with the one induced by a Staphylococcus fleurettii strain originating from cow bedding sawdust and by a S. chromogenes strain originating from a teat apex of a heifer. As expected, at 28 and 48 h p.i., low bacterial growth and local neutrophil influx in the mammary gland were induced by all CNS strains. As hypothesized, bacterial growth p.i. was the lowest for S. fleurettii compared with that induced by the 2 S. chromogenes strains, and the overall immune response established by the 3 CNS strains was less pronounced compared with the one induced by S. aureus. Proinflammatory cytokine profiling revealed that S. aureus locally induced IL-6 and IL-1β but not TNF-α, whereas, overall, CNS-inoculated glands lacked a strong cytokine host response but also induced IL-1β locally. Compared with both other CNS strains, S. chromogenes from the teat apex inflicted a more variable IL-1β response characterized by a more intense local reaction in several mice. This pilot study suggests that an intraductal mouse model can mimic bovine CNS mastitis and has potential as a complementary in vivo tool for future CNS mastitis research. Furthermore, it indicates that epidemiologically different bovine CNS species or strains induce a differential host innate immune response in the murine mammary gland., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular identification based on gene sequencing or fingerprinting techniques have been developed. In addition to culture-based detection of isolates, culture-independent methods may be of interest. On the basis of molecular studies, the five CNS species commonly causing intramammary infections (IMI) are Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Current knowledge suggests that S. chromogenes is a bovine-adapted species, with most cases of IMI due to this bacterium being opportunistic. S. haemolyticus also appears to be an opportunistic pathogen, but this bacterium occupies a variety of habitats, the importance of which as a source of IMI remains to be elucidated. S. xylosus appears to be a versatile species, but little is known of its epidemiology. S. epidermidis is considered to be a human-adapted species and most cases of IMI appear to arise from human sources, but the organism is capable of residing in other habitats. S. simulans typically causes contagious IMI, but opportunistic cases also occur and the ecology of this bacterium requires further study. Further studies of the ecology and epidemiology of CNS as a cause of IMI in cattle are required, along with careful attention to classification of these bacteria and the diseases they cause., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The aim of this study was to verify the existence of a nitric oxide (NO) cycle in goat milk and to study how changes in it affect milk composition during subclinical mastitis. Fifteen lactating dairy goats in which one udder-half was free from bacterial infection and the contra-lateral one was naturally infected with various species of coagulase-negative staphylococci were used. In comparison to uninfected glands, subclinical mastitis was associated with a decrease in milk yield, lactose concentration, and curd yield and an increase in nitrite and nitrate concentrations and with measurements reflecting increased formation of NO-derived free-radical nitrogen dioxide. The occurrence of NO cycling in goat milk was largely confirmed. The increase in the NO-derived stress during subclinical infection was not associated with significant increase in oxidatively modified substances, 3-nitrotyrosine, and carbonyls on proteins, but with increased levels of peroxides on fat. However, the relatively modest nitrosative stress in subclinically infected glands was associated with significant reduction in total antioxidant capacity and vitamin C levels in milk. We concluded that subclinical mastitis in goats caused by coagulase-negative staphylococci imposes negative changes in milk yield, milk quality for cheese production, and negatively affects the nutritional value of milk as food. Thus, subclinical mastitis in goats should be considered as a serious economic burden both by farmers and by the dairy industry., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant cause of intramammary infections (IMI) in dairy cows and in water buffaloes, as well. A longitudinal field study was carried out on one well-managed dairy water buffalo herd to determine the prevalence and distribution of CNS and a recently described CNS-species, Staphylococcus rostri, in milk samples to explore its relevance for buffaloes' udder health throughout lactation, and to gain insight into the susceptibility of the latter species toward commonly used antimicrobials. Twice weekly quarter milk samples from a cohort of 11 lactating water buffaloes were collected over an 8-mo period. The CNS (n=109; 76.2% of all culture-positive samples) were the predominant pathogens causing IMI, followed by Corynebacterium bovis (n=11; 7.6%) and Streptococcus spp. (n=9; 6.2%) other than Stretococcus uberis (n=2; 1.4%). Thirty-seven hemolytic staphylococci suspected to be Staphylococcus aureus were further differentiated using transfer DNA-intergenic spacer-PCR and rpoB-gene sequencing because they were coagulase-negative. Thirty-three of those isolates were identified as Staph. rostri, whereas 2 others were identified as Staphylococcus epidermidis. None of the Staph. rostri isolates displayed resistance to the antimicrobial agents tested. Mean quarter milk somatic cell count (qSCC) of all samples collected throughout lactation was 20,970 cells/mL. The qSCC at sampling of quarters infected with Staph. rostri (34,466 cells/mL) and CNS other than Staph. rostri (34,813 cells/mL) were significantly higher than the qSCC of noninfected quarters (20,287 cells/mL), yet not significantly different from each other. These findings provide novel insight into the prevalence and distribution, antimicrobial susceptibility, and relevance of Staph. rostri compared with other CNS species causing IMI in water buffaloes. Further studies are needed to pinpoint the relevance, niches, and transmission routes of Staph. rostri, as well as other CNS in water buffaloes., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Coagulase-negative staphylococci (CNS) are the most commonly isolated bacteria from goat milk. The goal of this study was to explore and describe differences between CNS species in persistence of intramammary infection (IMI) and in effect on somatic cell count (SCC) and milk yield (MY). Milk samples were collected from 530 does from 5 Dutch dairy goat herds on 3 occasions during 1 lactation. Coagulase-negative staphylococci species were identified at the species level by transfer RNA-intergenic spacer PCR (tDNA-PCR) followed by capillary electrophoresis. The most prevalent CNS species were Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus xylosus, but large differences were seen in species distribution between herds. Staphylococcus caprae and Staph. xylosus appeared to be more persistent than other species, but confidence intervals were overlapping. The effect of IMI caused by the 4 most prevalent CNS species on SCC and on MY was determined with linear regression models, and Staph. aureus and Corynebacterium bovis were included in the analyses as reference organisms. Most species were associated with a significantly higher SCC than noninfected udder halves, but the effect of CNS species on SCC was much smaller than the effect of Staph. aureus on SCC. We found a significant positive association between infection with Staph. caprae and MY. Intramammary infection caused by Staph. xylosus, on the other hand, had a negative association with milk yield, comparable to the effect of Staph. aureus, but these effects were not significantly different from zero. Intramammary infections with CNS species have a high prevalence in goats and are persistent, but have a limited effect on SCC compared with IMI with Staph. aureus. The effect of CNS species on MY differed between species, but differences were nonsignificant because limited numbers per species were available for analysis. Therefore, CNS species appear to behave as minor pathogens in goats, but larger studies are needed to give better estimates for the effect on MY., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A cross-sectional study was conducted in desert environment of Jhang (Pakistan) from November 2008 to October 2009 on she-camels kept under pastoralist conditions to determine the prevalence of mastitis, impact of risk factors, and isolate the dominant mastitis-causing bacteria on total of 150 lactating she-camels by using clinical examination and surf field mastitis test. From the 150 she-camels examined, 69 (46%) were positive for mastitis at animal level, 12 (8%) clinical, and 57 (38%) subclinical. Age, parity number, stage of lactation, breed, production system, hygiene of milking process, and presence of lesion on udder/teat were found significantly associated (p<0.05) with the prevalence of mastitis in she-camels. There was the lowest prevalence (33.33%; 15 of 45) of mastitis in she-camels of 5-7 years of age, while the highest (80%; 12 of 15) in the animals aged between 14 to 16 years. Stage of lactation significantly affected (p<0.05) and was found to be associated with the prevalence of mastitis being the highest (54.55%; 18 of 33) during the initial stage of lactation (0 to 1 month) followed by last 2 months (10-12 months) as 54.17% and mid-stages (1-3 and 3-10 months) of lactation as 28.57% (6 of 21) and 37.50% (9 of 24), respectively. According to breed of camels, it was noted that the prevalence of mastitis affected significantly (p<0.05) being the highest in crossbred (Desi × Mareecha) as 51.39% (37 of 72) followed in order by Mareecha and Desi as 43.14% and 37.04%, respectively. Staphylococcus (42.19%) and Streptococcus (15.63%) genera were the dominant isolates identified. Good hygiene in milking process, milking clinically infected she-camels at last, culling chronic mastitis carriers, treating clinically infected she-camels, and dry period therapy could reduce the prevalence of contagious mastitis in the study area.

We report the antibiotic treatments administered to a female dog with mastitis and successive pyoderma. Microbiological investigations allowed the identification of Staphylococcus pseudintermedius after 54 days of various antibiotic treatments. The isolate carried the mecA gene and was resistant to 9 of 15 tested antibiotics. Consistent antibiotic treatment of the infection was possible only after accurate microbiological diagnosis.

Persistent buffalo mastitis caused by Staphylococcus spp. gives rise to economic losses and may be resistant to antimicrobial therapy. The aim of the present study was to determine resistance patterns and the presence of mecA, blaZ, and efflux pump in Staphylococcus spp. isolated from cases of mastitis in Brazilian buffalo herds. Susceptibility to antimicrobials was determined by the disk diffusion test and detection of the mecA and blaZ genes by polymerase chain reaction. The efflux pump screening test was performed by growing samples in Muller-Hinton agar containing ethidium bromide. The percentages for resistance to the drugs tested were: 71.8% to penicillin, 49.2% to amoxicillin, 65.8% to oxacillin, 62.3% to cefquinome, 44.7% to cephalonium, 45.2% to ciprofloxacin, 32.6% to enrofloxacin, 58.7% to erythromycin, 42.7% to florfenicol, 34.6% to gentamicin, 35.1% to trimethoprim-sulfamethoxazole, 8.5% to tetracycline + neomycin + bacitracin, 43.2% to cephalothin, 38.1% to streptomycin, 58.7% to tetracycline, 31.6% to norfloxacin, 45.2% to ceftriaxone, 43.2% to nitrofurantoin, 57.7% to doxycycline, and 53.7% to cephalexin. Simultaneous resistance to 4 or more antimicrobial drug groups was observed in 112 isolates, using the mecA (11) and blaZ (79) genes, and efflux pump (47). It is concluded that Staphylococcus spp. isolates from cases of mastitis in Brazilian buffalo show varying levels of resistance to antibiotics, and caution should be exercised in choosing therapeutics in order to minimize the risk to public health.

The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR-RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR-RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR-RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Streptococcus gallolyticus subsp. macedonicus ST91KM produces a bacteriocin (macedocin ST91KM) active against Streptococcus agalactiae, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Staphylococcus epidermidis. Macedocin ST91KM is, according to tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. Antimicrobial activity remained unchanged after 2 h of incubation at pH 2.0-10.0 and after 100 min at 100 degrees C. The peptide was inactivated after 20 min at 121 degrees C and when treated with proteolytic enzymes. Treatment with alpha-amylase had no effect on activity, suggesting that the mode of action does not depend on glycosylation. Amplification of the genome of strain ST91KM with primers designed from the macedocin precursor gene (mcdA) produced 2 fragments (approximately 375 and 220 bp) instead of one 150-bp fragment, as recorded for macedocin produced by Streptococcus gallolyticus subsp. macedonicus ACA-DC 198. Strain ACA-DC 198 was not available. However, DNA amplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACA-DC 198, revealed 99% homology to the mcdA of strain ACA-DC 198 (accession No. DQ835394). Macedocin ST91KM may thus be a second putative bacteriocin described for Streptococcus gallolyticus subsp. macedonicus.

We aimed (i) to determine differences in bacterial flora of teat duct and mammary gland of ewes before and after suckling, (ii) to evaluate factors potentially affecting those. We collected samples of teat duct material and mammary secretion from 11 ewes immediately before and after sucking by lambs, as well as 120 min later. We processed samples bacteriologically and compared changes in infection by the Sign Test. We isolated bacteria from 3.5% duct and 1.5% secretion samples before suckling. Respective figures post-suckling were 10.6% and 2.0%, and 120 min later 6.8% and 1.5%. We recorded differences in infection of duct samples before and after suckling in 40 cases; bacteria were isolated before suckling from six samples, whereas after it from 34 (p < 0.001). Also, we recorded differences in samples collected after suckling and 120 min later in 12 cases; bacteria were isolated immediately post-suckling from eight samples, whereas 120 min later from four (p = 0.375). No significant changes were seen for secretion. We found neither difference between ewes with single or twin lambs, nor among stages of lactation. Mostly, we isolated staphylococci: 70% of isolates before suckling, 80% of isolates after it, 91% of isolates 120 min later. After suckling we also isolated two Mannheimia haemolytica strains. Suckling predisposes to entrance of bacteria into the teat; however, increased teat infections did not result in mammary infections. Isolation of M. haemolytica post-suckling indicates that lambs act as source of infection for this pathogen.

The use of antimicrobial substances in human and veterinary medicine inevitably results in a selection pressure for drug resistance in exposed bacteria. Preventive measures, apt to avoid the consequent development of new resistances and selection for existing ones, respectively, have to be elaborated. Moreover, it has to be ensured that neither resistant bacteria nor resistance genes are spread to and consequently via the food chain. Respiratory diseases as well as mastitis in dairy cattle belong to the most frequently occurring diseases in food-producing animals. For the first time in Germany, a comprehensive, cross-sectional study into the antimicrobial susceptibility of bacteria associated with these disease patterns in food-producing animals was conducted by the Federal Office of Consumer Protection and Food Safety (BVL) in 2001. The selection of examined bacterial species comprised Pasteurella multocida and Mannheimia haemolytica associated with respiratory disease in pigs, and Escherichia coli, Streptococcus spp. and Staphylococcus spp. causing mastitis in dairy cattle. Bacterial strains were collected following a representative sampling scheme, taking into account the total number of animals in the individual German federal Länder. In an analogous study conducted in 2002/2003, this selection was extended by the indication respiratory disease in juvenile cattle, caused by P. multocida and M. haemolytica, respectively. In comparison with data from 2001, MIC values determined in 2002/2003 suggested significantly lower or higher degrees of drug susceptibility only for a few antimicrobial agents. Comparison was carried out on the basis of bacterial species and individual federal Länder, respectively. Overall, the data raised in both studies revealed substantially lower resistance rates than published for Germany so far. This is particularly true for results from those Länder, whose animal health services had implemented preventive strategies to control infectious diseases. No correlation could be established between differing animal population densities and differences in the prevalence of resistance in corresponding Lander. However, the geographical distribution of occurrence of resistance against beta-lactam antimicrobial agents suggests different therapeutic strategies employed in different sized animal flocks. In federal Länder marked by large-scale livestock farming, significantly higher resistance values could be measured for cephalosporins than for penicillins, whereas in Länder with rather traditional farming structures, resistance to penicillins was predominant. Assuming otherwise similar factors of influence on the emergence of resistance, this pattern suggests that cephalosporins are preferably used in large enterprises and penicillins in smaller farms, respectively. Currently, mechanisms effecting changes in antimicrobial resistance are being further investigated in a successive study.

Methicillin-resistant Staphylococcus aureus (MRSA) is defined by the presence of the mec A gene, which is considered to have been transferred horizontally from unknown bacterial species to S. aureus. As a candidate of evolutionary precursor of the mec A, the mec A-like gene (mec A homologue), which is ubiquitously present in Staphylococcus sciuri has been proposed. In this study, sequences of the mec A homologue in four S. sciuri strains (SCBM 1-SCBM 4) derived from dairy cows were determined to analyze their genetic characteristics and relatedness to mec A and the mec A homologue reported so far. The mec A-like gene sequences of the four S. sciuri strains were identical with each other and were considered to encode a product comprising 665 amino acids that is one amino acid smaller in size than products of mec A-like gene reported previously for S. sciuri strains K1, K1 1, and K3 (mec A1). The mec A homologue of a representative strain SCBM 1 showed 79.3--79.8% sequence identity to MRSA mec A and 93.4--94.4% identity to mec A homologues reported for the three S. sciuri strains. Between S. sciuri strain SCBM 1 and strains K1, K1 1, or K3, amino acid sequence identities in transpeptidase domain of the mec A-like gene product (98.2--98.5%) were higher than those in the transglycosylase domain (92.1--94.3%). In addition, SCBM 1 showed extremely high sequence identities of hsp 60, sodA, and rpoB genes (more than 98.7%) to S. sciuri strains, while showing 70.3--94.2% identity of these genes to other staphylococcal species. These findings indicated that mec A homologues in S. sciuri may be genetically more divergent than mec A in MRSA and methicillin-resistant coagulase-negative staphylococci.

Routine examination of milk was performed on five herds of lactating goats in northern Italy as part of a milk quality-monitoring program in the year 2000. As part of the study, aseptic samples of foremilk were collected monthly from both half udders during the entire lactation for 305 goats, resulting in a total of 4571 samples. The samples were tested with cytological and bacteriological analyses to evaluate the relationship between mammary infections and somatic-cell count (SCC; Fossomatic (TM) method). Prevalence of intramammary infection (IMI) was 40.2% (n = 1837) of all udder-half samples examined. The most-prevalent mastitis agents were coagulase-negative Staphylococci (CNS), 80% (n = 1474 udder-half samples); within this group, Staphylococcus epidermidis was the most-prevalent species (38%). Other prevalence were Staphylococcus aureus 6% (n = 112 udder-half samples) and environmental pathogens 14% of infected udder-half samples (n = 251) with a diverse mixture of species, none of which had a frequency of > 4%. Enterococcus faecalis was the most-frequently isolated among this group. Neither Salmonella spp. nor Listeria monocytogenes were detected. The risk (sample level) of infection differed across herds, parities, and stage of lactation according to results from logistic multiple regression. Infection was more common among goats in third and fourth parities and during the later stages of lactation. Of the 2734 samples from uninfected udder halves, the mean log2 SCC was 3.9 cell/ml; of the 1837 bacteriological positive samples, the mean log2 SCC was 5.6 cell/ml. According to results from a linear mixed model, concentrations of somatic cells tended to increase with increasing age and days in milk and with the presence of bacteria. Infection with S. aureus was associated with the highest SCS.

A total of 156 goats from 2 commercial dairy goat farms were monitored for intramammary infections during an entire lactation. Most of the infections (80.7%) observed were due to coagulase-negative staphylococci (CNS) species. In herd 1, nearly all of the infections (96%) were due to CNS species, with Staphylococcus caprae (SCAP) being the most common specific pathogen observed, accounting for about 43% of the infections. In herd 2, the proportion of the infections due to CNS was 67% and Staphylococcus epidermidis (SEPI) was the most common pathogen (48% of infections) and SCAP was not present. Linear somatic cell scores (SCS) were greater in milk from infected udder halves, with an average difference of 0.78 SCS. The SCS for infected udder halves was greater than noninfected for all CNS species, although differences among species were observed. The ranking across CNS species was SCAP > other CNS > SEPI > no infection. However, infections by SEPI tended to be more persistent. Increased SCS was associated with a statistically significant decrease in milk yield, but no effect was observed for intramammary infections (IMI). Ninety-seven isolates of CNS (53 SCAP and 44 SEPI) were investigated for in vitro susceptibility to several antimicrobial agents. Benzylpenicillin was the most effective antimicrobial agent against SCAP and SEPI. A concentration of 0.05 microg/mL was sufficient to inhibit growth of 90% of SCAP colonies, and 0.10 microg/mL yielded a similar effectiveness for SEPI. Amoxicillin and the combination of amoxicillin and clavulanic acid were only slightly less effective. Tetracycline (62.5 microg/mL) and tilmicosin (500 microg/mL) were the least effective treatments for SEPI and SCAP, respectively.

The antibiotic susceptibility of 70 strains of coagulase-negative staphylococci (CNS) isolated during the 2001 lactating period from the milk of dairy goats, was evaluated. The antibiotics tested were benzylpenicillin, cloxacillin, amoxicillin, amoxicillin plus clavulanic acid, cephalonium and cefoperazone, erythromycin and tylmicosin, kanamycin and tetracycline. Minimum inhibitory concentration (MIC) measurements showed that all beta-lactams (except cefoperazone) were effective against Staphylococcus epidermidis and Staphylococcus caprae, whereas the other antibiotics were either less effective or showed no activity. Other CNS species showed very variable sensitivity to the antibiotics; testing would be required before therapy for the clinical control of goat mammary infections., (Copyright 2004 Elsevier B.V.)

A strain of Staphylococcus chromogenes was introduced into the teat cistern of five ewes, teat inflammation and stenosis being the primary consequences. Initially, the inoculated teats were swollen and warm; later, a hard structure was palpated running lengthwise inside the teat, with a thick ring above the tip of the teat, which interfered with expression of milk. Mastitis, confirmed by clinical, cytological, bacteriological and histological findings, was evident 4 days after infection. Ultrasonographically, a hyperechoic line under the mucosa of the teat cistern was observed. At necropsy, the duct wall of the inoculated teats was found to be thickened. Histopathological features included leucocytic infiltration, especially under the mucosa of the teat, and extensive fibrosis in the subcutaneous tissues. S. chromogenes was recovered from scrapings from the duct and the cistern of the inoculated teats.

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.

One hundred ovine milk samples were subjected to bacteriological analysis to detect staphylococci. Twenty-four staphylococcal strains isolated were characterised for methicillin resistance with disk diffusion test (DDT) after incubation at 24 and 48 h, oxacillin agar screen test, Minimal Inhibitory Concentration (MIC), nitrocefin test for beta-lactamase production and PCR for the mecA gene. Nine staphylococcal strains resulted resistant in DDT; some differences in the halo diameter at double incubation period were noted; eight of these strains were resistant in MIC test; just one strain was positive to oxacillin agar screen test. All strains were mecA negative by PCR and positive by nitrocefin test. On the basis of these results methicillin-resistant strains can be classified as beta-lactamase hyperproducers.

Coagulase-negative staphylococci producing cell-damaging toxins were isolated from the milk of sheep with subclinical mastitis. The haemolytic activity of Coagulase-negative staphylococcal strains was assessed on solid and liquid culture media. More than 61% and 76% of the tested strains on solid media produced evidence of alpha- and delta- haemolysins and more than 78% produced synergistic haemolysis. However almost all isolates producing haemolysin in liquid culture media produced only very few units of haemolysin compared to the positive control of five Coagulase-positive strains of staphylococci. It was concluded that solid media are better for classifying Coagulase-negative staphylococci as producers or not of haemolysins, and liquid media for measuring the size of this activity within the first few hours of intramammary infection.

For control of the udder health status of milk sheep the minimal inhibitory concentration of mastitis causing pathogens to 12 different chemotherapeutics was determined by means of Sensititre microplate method. 73 staphylococci- and 8 streptococci-strains was examined. Following staphylococci-strains were found: S. aureus (11), S. epidermidis (34), S. chromogenes and S. xylosus (6 each), S. hyicus and S. warneri (each 5) as well as S. sciuri and S. simulans (3 each). MIC-values to penicillin, amoxycillin and ampicillin of 2 penicillinase-positive S. aureus-strains were more than 4.0 micrograms/ml. The remaining isolates and the major part of coagulase-negative staphylococci displayed MIC-values up to 1.0 microgram/ml to beta-lactamantibiotics except cloxacillin. Against cephalosporines, cephalexin excluded, and aminoglycosides very low MIC-values concerning staphylococci were observed. In case of spiramycin MIC-levels more or less than 8.0 micrograms/ml were determined. Streptococci exhibited MIC-values in the range of 0.06 to 0.5 microgram/ml against beta-lactamantibiotics with exclusion of cloxacillin, cefoperazone and cefquinome and spiramycin as well.

Various species of coagulase-negative staphylococci (C-NS) are reported to be common in milk and on the teat skin of domestic ruminants. The commonest C-NS species in mastitic milk of cows varies between reports, with Staphylococcus simulans (Jarp, 1991) in one and Staph. hyicus in another (Watts & Washburn, 1991). The teat skin of heifers may be colonized by Staph. xylosus or Staph. chromogenes, while Staph. chromogenes and Staph. warneri are reported as frequent isolates from teat canals and secretion (Boddie & Nickerson, 1986; White et al. 1989). Staph. haemolyticus was isolated frequently from the nares, the teat skin and the milk of goats (Valle et al. 1991), although others reported Staph. xylosus (Bedidi-Madani et al. 1992) or Staph. epidermidis and Staph. capitis (Kalogridou-Vassiliadou, 1991) as the most predominant C-NS in goats' milk. Staph. simulans has been found experimentally to be pathogenic for the mammary gland of meat ewes (Fthenakis & Jones, 1990), but little is known about the prevalence of this species in ewes' milk collected from cases of naturally occurring subclinical mastitis (SCM). The aim of the present investigation was the identification of the commonest C-NS species in ewes' milk collected from field cases of SCM or predominating in the ewes' environment.

Milk from mastitis-free goats from French herds was examined for the presence of coagulase-negative staphylococci (CNS), and 165 positive isolates were evaluated for their capacity to produce exoproteins. Most isolates were identified as Staphylococcus caprae (N = 91) or S. xylosus (N = 36), but members of at least nine other species were present. Overall, some 57% of isolates produced toxins with phenotypic properties of alpha-hemolysin, and 75% produced toxins resembling S. aureus beta and/or delta-hemolysins. Thermostable desoxyribonuclease (TNAse) was secreted by 29% of isolates and thermolabile DNAse by 66%. Slime was produced by 42% of our cultures and, although none of them showed activity to insoluble elastase, over 70% lysed the soluble substrate. No clinical consequences were observed to correlate with exoprotein production, which proved to be inconstant within individual CNS species.

Three strains of coagulase-negative staphylococci identified as Staphilococcus warnery, Staphylococcus simulans and Staphylococcus haemolyticus were examine for evidence of capsular polysaccharide, in vitro by negative staining with India ink, and in vivo by transmission electron microscopy. In vitro, unstained materials surrounded clusters or single cocci. In vivo, capsula materials were surrounding phagocytosed bacteria cells. These capsular materials were either closely or loosely bound to bacterial cells, or projecting like "pili" from the surface of the cell wall. The evidence suggests that capsular polysaccharide is a common characteristic of coagulase-negative staphylococci causing prolonged intramammary infection of sheep.

Virulence factors such as hydrophobicity of cell surface, capsule formation and slime production were investigated in 393 CNS strains, 190 isolated from bovine mastitis and 203 from human infections; in addition a selected number of CNS species were experimentally infused into the teat cistern of lactating rabbits for pathogenicity tests. Most human (95%) and bovine (82.6%) strains showed cell hydrophobicity properties, however only 11.3% and 7.9% were capsulated strains, respectively. Slime production was detected in 10.5% of bovine strains but only in 0.9% of human strains. Using the lactating rabbit model it was shown that S. chromogenes, S. epidermidis, S. saprophyticus and S. haemolyticus were the most pathogenic species.

A total of 359 Micrococcaceae strains isolated from goat milk samples were differentiated with the commercially available ATB 32 Staph differentiation system. Of these strains, 303 (84.4%) were identified. Six strains were sensitive in the bacitracin resistance test, and accordingly classified as Micrococcus spp. Staphylococcal species isolated of goat milk were S. epidermidis, S. aureus, S. caprae, S. lentus, S. simulans, S. capitis, S. lugdunensis, S. xylosus, S. chromogenes, S. hominis, S. arlettae, S. warneri, S. sciuri, and S. saprophyticus. Highest somatic cell count (SCC) in milk and the highest prevalence of clinical udder alterations were associated with coagulase-positive S. aureus. Increases in milk SCC as well as pathological udder findings were observed in infections with coagulase-negative staphylococci such as novobiocin-sensitive S. epidermidis, S. simulans, S. lugdunensis, S. chromogenes, and S. warneri.

Of 41 coagulase-negative staphylococcal isolates from cases of ovine mastitis, 80% were speciated by the 'API-Staph SYSTEM' and 90% by a combination of biochemical tests. Staphylococcus simulans and Staph. xylosus were the two most prevalent species.

Experiments on the development of a vaccine against staphylococcal mastitis were carried out in ewes. The vaccine (Spanish patent no. 9200223) has the following components: (i) inactivated (formalinized) bacteria (Staphylococcus aureus and a coagulase-negative staphylococcal species. Staphylococcus simulans) and S. aureus toxoid in presence of an adjuvant (dextran sulfate, Mw 500,000); and (ii) S. aureus exopolysaccharide included within liposomes. High serum antibody titres were obtained against whole cells from Staphylococcus aureus, Staphylococcus simulans, Staphylococcus hyicus and Staphylococcus epidermidis strains. However, there was no response to cells from Staphylococcus warneri and Staphylococcus chromogenes strains. An immune response (serum IgG) against the inoculated exopolysaccharide was obtained when > or = 20 micrograms of exopolysaccharide were included in liposomes and when > or = 20 mg of exopolysaccharide were adjuvanted with dextran sulfate instead of liposomes. For experimental infection assays, ewes were vaccinated during pregnancy and challenged either with a low virulence S. simulans strain or with a highly virulent S. aureus strain. In these assays, the incidence of S. simulans subclinical mastitis and of S. aureus acute mastitis was significantly lower in vaccinated animals than in unvaccinated controls. Specifically, on challenge with S. simulans, two out of 14 glands became infected among the vaccinated animals and nine out of ten glands in the unvaccinated group (p < 0.001). On challenge with S. aureus, no protection was detected when component (ii) was omitted from the vaccine; nine out of ten animals developed mastitis (two mild, two moderate and five severe).(ABSTRACT TRUNCATED AT 250 WORDS)

A total of 447 micrococcaceae strains isolated from 88 ewe and 359 goat milk samples from cases of chronic mastitis were differentiated by means of the ATB 32 STAPH-test. Of these strains 389 (= 87%) could be identified. Fourteen strains were sensitive in the bacitracin-resistance-test and therefore classified as Micrococcus spp. In ewe milk following Staphylococcus spp. were found: S. epidermidis, S. aureus, S. lentus, S. xylosus, S. warneri, S. equorum, S. haemolyticus, S. simulans, S. hominis and S. saprophyticus. Staphylococcus spp. identified in goat milk samples were: S. epidermidis, S. aureus, S. caprae, S. lentus, S. simulans, S. capitis, S. lugdunensis, S. xylosus, S. chromogenes, S. hominis, S. arlettae, S. warneri, S. sciuri, and S. saprophyticus. Highest cell counts in the milk of both animals species, and the highest incidence of clinical udder alterations were caused by S. aureus. Increases in milk cell counts as well as pathological udder findings were observed in coagulase-negative staphylococcal infections for novobiocin-sensitive Staphylococcus spp. (S. epidermidis, S. warneri, S. simulans, S. lugdunensis, and S. chromogenes) and several S. lentus strains.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S. xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.