Coagulase-negative Staphylococci Samples Research Articles

Influence of subclinical mastitis and intramammary infection by coagulase-negative staphylococci on the cow milk peptidome

Coagulase-negative staphylococci (CNS) are the most prevalent microorganisms isolated from cow milk and are associated with subclinical mastitis and persistent increases in the bulk milk somatic cell count (BMSCC) of low BMSCC herds. By combining peptide enrichment, LC-ESI-MS/MS, and statistical analysis, we investigated the influence of subclinical mastitis and CNS infection on the milk peptidome. Quarter milk samples from clinically healthy Holstein cows were subjected to bacteriological culture (BC) and somatic cell counting (SCC) for two consecutive samplings and 28 (including 11 negatives and 17 positives) were selected for peptidomic analysis.The study identified 1363 different endogenous peptides and highlighted a significant increase of peptides in CNS-positive milk, mainly represented by casein fragments. Milk peptidome changes increased with the SCC, as also demonstrated by protein electrophoresis and densitometry. Peptides significantly different in CNS or CONTROL samples were identified and characterized. Our results indicate that subclinical mastitis by CNS can induce significant changes in the milk peptidome, opening the way to future studies for the identification of a biomarker panel as well as for the understanding of their consequences for the technological and sensorial characteristics of cow milk and dairy products. SignificanceThis is the first investigation on the impact of subclinical CNS mastitis on the bovine milk peptidome. The peptide enrichment strategy combined with a highly sensitive MS/MS analysis enabled the compilation of a very large peptide dataset for healthy and mastitic milk. The comparison of CNS and Control samples, also considering SCC classes, highlighted several peptides with potential for understanding milk protein and peptide dynamics in subclinical mastitis, with possible implications for its detection.

Characteristics of resistance and virulence factors in different species of coagulase-negative staphylococci isolated from milk of healthy sheep and animals with subclinical mastitis

Coagulase-negative staphylococci (CNS) are among the main responsible agents for mastitis in sheep. Cure rates can be reduced due to several causes, such as those related to virulence factors presented by microorganisms. This study aims at characterizing the virulence and resistance factors to antimicrobial agents in different CNS species isolated from sheep milk. After collecting milk samples, the samples were analyzed and the CNS species were identified. After identification, the susceptibility-sensitivity profile was examined using the disk diffusion technique for 10 antimicrobial agents. The DNA was extracted to detect the presence of the mecA gene, biofilm (icaADBC, bap, and bhp) and toxin genes (sea, seb, sec, sed, tst, and luk-PV) by PCR. Samples carrying toxin genes had their expression assessed using the reverse-transcription PCR technique. The biofilm production was assessed using the adherence method on a polystyrene plate. One hundred twelve CNS samples were isolated, 53 (47.3%) from animals with subclinical mastitis and 59 (52.7%) from healthy animals. Drugs tested have shown to be efficient for most CNS samples. The largest resistance percentage of CNS was found for the penicillin (17.0%) and tetracycline (10.7%) and 4 samples carried the mecA gene. As for the biofilm genes, the icaADBC operon was found in 10 (8.9%) samples, the bap gene was found in 16 (14.3%), and the bhp gene was found in 3 (2.7%). In addition, 69 (61.6%) samples produced biofilm. The survey of toxin genes has shown that 70 (62.5%) samples showed some toxin-encoding gene. However, none of the samples has expressed any of the genes from those toxins studied.

Determination of toxigenic capacity by reverse transcription polymerase chain reaction in coagulase-negative staphylococci and Staphylococcus aureus isolated from newborns in Brazil

Staphylococcus spp. are frequently found in hospital environments and are associated with a wide variety of infections. Various virulence factors are responsible for the pathogenicity of staphylococci, among which staphylococcal enterotoxins and TSST-1 (toxic-shock syndrome) are noteworthy. In this study, 90 samples of Staphylococcus aureus and 90 samples of coagulase-negative staphylococci (CNS) isolated from different clinical materials were investigated by polymerase chain reaction (PCR) in order to study the genes encoding staphylococcal toxins A (sea), B (seb), C (sec-1), D (sed) and TSST-1(tst). The samples shown to be positive for the presence of one or more genes were tested for their capacity to express mRNA encoding the respective toxins by reverse transcription-PCR (RT-PCR). As regards the CNS species, S. epidermidis was the most frequently isolated, corresponding to 71.1% of the total number of samples of CNS investigated. One hundred and eight samples were positive according to PCR, of which 59 (54.6%) were S. aureus and 49 (45.4%) were CNS. S. aureus showed toxigenic genes for all classes of toxins investigated whereas CNS showed all genes except for that of toxin D. Assessment of mRNA expression by RT-PCR showed 43 positive samples, 37 (86.0%) S. aureus samples producing SEA, SEB, SEC, SED and/or TSST-1 and six (14.0%) CNS samples producing SEA and SEC. RT-PCR and sequencing of PCR products confirmed the toxigenic capacity of S. epidermidis and S. lugdunensis, indicating the need for greater attention to such microorganisms when they are isolated from infectious processes.

Avaliação da colonização nasal por Staphylococcus spp. resistenteà oxacilina em alunos de enfermagem

INTRODUÇÃO: O gênero Staphylococcus é responsável por um grande número de infecções bacterianas em humanos, principalmente no ambiente hospitalar. OBJETIVO: Diante dessas considerações, da importância do cuidado de enfermagem e do controle de infecções hospitalares, este trabalho verificou a taxa de portadores de S. aureus e estafilococos coagulase-negativa (ECN) resistentes a oxacilina em alunos do curso de enfermagem durante a formação profissional. MATERIAS E METÓDOS: Foram coletadas amostras das fossas nasais de alunos do curso de enfermagem da Faculdade de Medicina de Botucatu (FMB). RESULTADOS: Das 109 amostras de Staphylococcus encontradas, 30 (27,5%) foram de Staphylococcus aureus e 79 (72,5%), de ECN. Das 79 amostras de ECN submetidas às provas de identificação, 63 (79,7%) foram da espécie S. epidermidis, nove (11,4%), S. warneri, três (3,8%), S. haemolyticus, duas (2,5%), S. capitis, uma (1,3%), S. simulans e uma (1,3%) S. lugdunensis. O teste de suscetibilidade a antibióticos demonstrou 100% de sensibilidade às drogas nas amostras de S. aureus e, entre as 79 amostras de ECN, 10 (12,6%) foram resistentes a oxacilina. A técnica de reação em cadeia da polimerase (PCR) demonstrou resultado negativo para o gene mecA nas amostras de S. aureus e 11 amostras positivas entre as espécies de ECN. DISCUSSÃO: Não houve relação entre a taxa de portadores de S. aureus e o envolvimento hospitalar durante a graduação, os resultados também demonstraram maior incidência de resistência nas amostras de ECN, semelhante ao encontrado na literatura científica.

Study of virulence factors in coagulase-negative staphylococci isolated from newborns

Coagulase-negative staphylococci (CNS) have been identified as the etiological agent in various infections and are currently the microorganisms most frequently isolated in nosocomial infections. However, little is known about the virulence factors produced by CNS that contribute to the pathogenesis of infections caused by these microorganisms. The study of CNS isolated from infectious processes of newborns hospitalized in the Neonatal Unit of the Hospital of the Botucatu Medical School, UNESP, indicated Staphylococcus epidermidis as the most frequently isolated species (77.8%), which was also associated with clinically significant situations. The analysis of virulence factors revealed the production of slime in 20 (17.1%) of all CNS samples isolated and the synthesis of a broad spectrum of enzymes and toxins, including hemolysins (19.6%), lipase (17.1%), lecithinase (3.4%), DNAse (15.4%), thermonuclease (7.7%), and enterotoxin A, B or C (37.6%). Taking into consideration that the etiological importance of CNS has often been neglected, the present investigation confirmed that these microorganisms should not be ignored or classified as mere contaminants.

Selecting linear-score distributions for modelling milk-culture results

The data for this cross-sectional retrospective study are from surveys of 65 dairy-cattle herds in central New York, USA sampled between February, 1993 and March, 1995. The objective was to identify probability distributions of logarithmically transformed somatic-cell counts (linear score) for use in a simulation model of mastitis and milk quality. Probability density functions were estimated using maximum-likelihood estimators for the linear score of individual-cow compositemilk samples culture negative and culture positive for the pathogens Streptococcus agalactiae, Streptococcus non- agalactiae, Staphylococcus aureus, and coagulase-negative staphylococci for the complete dataset and by bulk-tank somatic-cell count group (< 500 000, ≥ 500 000 SCC/ml). Based on the rankings of three goodness-of-fit tests (Anderson-Darling, Kolmogorov-Smirnov and x 2), the Weibull distribution (among the three top-ranking distributions for 14 out of 15 cases) may be used to model the individual-cow linear-score response by culture-result-specific bulk-tank somatic-cell count group. A β distribution was among the three top-ranking distributions for nine out of 15 culture-result-specific bulk-tank somatic-cell count groups and has a logical relationship to linear score because it is defined on a fixed interval. On the other hand, the normal distribution had a poorer fit than the Weibull and at least two other distributions for all culture negative and coagulase-negative staphylococci samples. We do not assume that the underlying biological processes are fully explained by either Weibull or β distribution—but modelling the linear score for the above culture results with these distributions provided an adequate fit to the survey data, reduced the need for two-sided truncation that open intervals needed, and had errors that did not appear to be systematically positive or negative.

Reduction of bacterial contamination during mechanical autotransfusion by uv irradiation--initial results

To investigate the effects of UV-radiation of autologous blood on bacteria and red blood cells when using intraoperative sampling of autologous blood using a cell separator--an established method for reducing the need for donor blood during surgery--which is reported to have a bacterial contamination rate of 5-75%, due mainly to coagulase-negative staphylococci (CNS). Cell-separator blood was diluted to a haematocrit level permitting transmission of 1% of the UV-radiation used in this study (lambda 254 nm, coat thickness 1 mm). CNS samples were irradiated for 2, 4, 10, 20 and 30 seconds. Free haemoglobin and methaemoglobin levels were measured, and erythrocytes examined microscopically at the end of the procedure. Blood samples had to be diluted to a haematocrit of 1% to permit transmission of 1% of the UV light. The optimal irradiation duration was 4 seconds, when bacteria were completely eliminated. Longer irradiation durations were associated with increasing levels of free haemoglobin and methaemoglobin, the levels of which at 4 seconds exposure were 12.5 mg/L and 15.5%, respectively. It is possible to prevent CNS contamination of cell-separator blood by irradiation with UV light. Prior to clinical application, however, the method will need to be modified to minimize side effects and increase its decontamination efficacy.