Characteristics of advanced methods used for typing bacterial isolates from mastitis with particular reference to Staphylococci.

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.

Clustering of Staphylococcus aureus bovine mastitis strains from regions of Central-Eastern Poland based on their biochemical and genetic characteristics.

Staphylococcus aureus strains were isolated from mastitic milk of cows with infected mammary glands. The animals were living in 12 different farms near Lublin, in Central-Eastern Poland. A biochemical identification method based on enzymatic assay was performed, followed by haemolytic and proteolytic tests. PCR-RFLP targeted on the gap gene allowed the genetic identification of strains at the species level and verified phenotypic identification results. A molecular typing method using triplex PCR was performed to recognize the genetic similarity of the analyzed strains. DNA microarray hybridization (StaphyType, Alere Technologies) was used for detection of antibiotic resistance and virulence associated markers. The results obtained indicate high genetic similarity in strains isolated from the same sites. High genetic similarities were also detected between strains isolated from cows from different farms of the same region. A slightly lower similarity was noted however, in strains from various regions indicating that the strains are herd specific and that the cow's infections caused by S. aureus were of a clonal character. In 21 representative isolates selected for DNA-microarray testing, only fosfomycin (fosB) and penicillin resistance markers (blaZ, blaI, blaR) were detected. The presence of genes coding for haemolysins (lukF, lukS, hlgA, hla, hld, hlb), proteases (aur, sspA, sspB, sspP), enterotoxins (entA, entD, entG, entI, entJ, entM, entN, entO, entR, entU, egc-cluster), adhesins (icaA, icaC, icaD, bbp, clfA, clfB, fib, fnbA, map, vwb) or immune evasion proteins (scn, chp, sak) was common and, with exceptions, matched triplex PCR-defined clusters.

Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland.

Staphylococcus aureus is the predominant causative agent of bovine mastitis, a disease that remains a major economic burden for the dairy industry worldwide. In this study, the antimicrobial resistance patterns and the genetic composition of 80 S. aureus mastitis isolates collected from 14 dairy farms in Eastern Poland were determined. Of the 10 antimicrobial agents evaluated, only testing for penicillin G produced drug resistance. As 41% of the S. aureus isolates were penicillin resistant, this drug along with other ß-lactamase-sensitive ß-lactams, should rather not be considered for the treatment of bovine mastitis caused by S. aureus. Upon genotyping, with a triplex PCR method, a total of 11 distinct PCR types were produced. The population structure of S. aureus isolates was highly clonal, with 1 predominant genotype circulating on each farm. The observed similarities in the genotype composition of S. aureus populations from geographically distant farms underscore the significance of interfarm transmission of S. aureus in Poland. This, in turn, argues for the establishment of a nationwide surveillance program for bovine mastitis due to this pathogen.

Proteolytic activity of Staphylococcus aureus strains isolated from the colonized skin of patients with acute-phase atopic dermatitis.

Staphylococcus aureus strains isolated from the colonized skin lesions of 26 patients with acute-phase atopic dermatitis were reported to produce various extracellular proteolytic enzymes. Using the skim-milk-agar culture plating method, it was shown that 97% of the strains (65 of 67 examined) produced proteolytic activity, with 61% (42 strains) producing activity comparable to that of the proteolytically hyperactive reference strain Staphylococcus aureus V8. This observation was confirmed by azocasein degradation with culture supernatants, which indicated that 91% of the strains produced extracellular proteinases and 43% exceeded the 2% activity threshold of the reference strain. Control strains were isolated from the nose vestibules of 18 healthy carriers; the proteolytic activity of these strains never exceeded 2.5% of the activity of the reference strain. In 54% of the patients examined ( n=14), the activity of the strains was higher than that determined for the isolates from the control group. The combined use of assays incorporating azocasein and a synthetic chromogenic substrate, N-CBZ-Phe-Leu-Glu- pNA, showed that two staphylococcal enzymes, Staphylococcus aureus metalloproteinase (SAMP) and Staphylococcus aureus serine proteinase (SASP), contributed to the total proteolytic activity released by the strains examined. The contribution of each of the two enzymes varied greatly between different isolates. The undamaged skin of the patients was not colonized with Staphylococcus aureus. The presence of several strains with atypical proteinase characteristics was also reported, suggesting the possible involvement of enzymes other than serine- and metallo-proteinases in the proteolytic activity of Staphylococcus aureus. Taken together, the results of the study imply that staphylococcal proteinases may contribute to the pathogenicity of atopic dermatitis.

[Selected parameters of the cellular and humoral immunity in atopic dermatitis. Relationship to the severity of the disease]. / Charakterystyka wybranych parametrów odpornosci komórkowej i humoralnej u chorych na atopowe zapalenie skóry. Zwiazek z zaawansowaniem klinicznym choroby.

UNLABELLED: Despite the great progress, our understanding of the pathogenesis of atopic dermatitis (AD) is still incomplete. In particular, the clinical importance of various changes of the immune system parameters is unclear. Accordingly we have undertaken the study to compare selected parameters of cellular and humoral immunity between AD subjects (n = 26) and healthy controls (n = 10). These parameters included immunoglobulin levels (IgE in particular), neutrophil respiratory, oxygen burst, peripheral blood lymphocyte phenotype and response to mitogens. We also analysed the relationship between these parameters and clinical severity of skin lesions. RESULTS: Mean total immunoglobulin E levels were very significantly increased in the AD group (1563 +/- 459 vs 35.5 +/- 12.1 IU/ml; p = 0.001). Simultaneously total serum IgE levels varied extensively between individual subjects with AD and were significantly correlated to clinical severity of the disease (Rs = 0.44; p = 0.02). Atopic dermatitis was also associated with the increase in the number of CD4+ and simultaneous decrease in the CD8+ lymphocytes causing statistically significant difference in CD4:CD8 ratio compared to the control group. We also observed changes of proliferation indices to phytohaemagglutinin (decrease) and increase of responses to anti CD3 mAb (OKT-3) and staphylococcal enterotoxin B. None of these immune parameters however, appeared to be statistically correlated to clinical status. CONCLUSIONS: We find that atopic dermatitis is associated with significant changes of several important indices of cellular and humoral immunity including increased IgE levels and altered peripheral lymphocyte proliferation capacity and phenotype. Change of total IgE levels appears to be the most important from clinical point of view.

Effect of Staphylococcus aureus serine proteinase on the respiratory burst in phagocytic cells in vitro.

The in vitro effects of the Staphylococcus aureus serine proteinase (SASP) on the respiratory burst of human blood mononuclear phagocytes and rat lung macrophages were investigated. The generation of reactive oxygen species (ROS), determined by means of luminol-based chemiluminescence, was stimulated by treatment with SASP in both types of the defense cells. Cell activation depended on the concentration of the enzyme and the response of monocytes was an order of magnitude stronger relative to macrophages. The chemiluminescence emission kinetics were different for both cell types and the maximum signal was achieved in approximately 3 and 17 min, respectively. In experiments involving further cell activation by latex particles, macrophages pretreated with various SASP concentrations reacted with enhanced ROS generation whereas for monocytes, the latex-induced chemiluminescence was weakened by the enzyme. The results concerning the modification of the phagocytic host cell activity by SASP suggest that this enzyme might play an important role in pathomechanisms of staphylococcal infections in vivo.

Characteristics of Staphylococcus aureus isolates from atopic dermatitis with reference to proteolytic activity.

Staphylococcus aureus strains from atopic dermatitis (AD) patients were investigated. Diversities of biological properties, strain relationships and/or group tendencies between strains were analysed. Fifty four S. aureus strains were divided into seven biotypes using a standard biochemical API Staph system. The largest population (twenty two isolates, 40.7%) belonged to biotype A No. 6716153. Using a standard phage set S. aureus isolates were typed into three groups: I, II and III. However, twenty seven (50.0%) isolates belonged to group No. III. Production of proteolytic enzymes was expressed by all isolates, and 87.0% showed high or moderate proteolytic activity. Also alpha or beta haemolysins production by 83.0% of the strains was demonstrated. Antimicrobial susceptibility for each strain was analysed using fifteen antibiotics. Most isolates were sensitive to chloramphenicol (98.0%), neomycin (98.0%) and fucidin (88.0%) and were resistant to ampicillin, oxacillin and rondomycin (< 20.0%). No isolate was sensitive to all antibiotics of our study. Obvious correlations were not observed between biochemical types, phage types, haeomolysin production and antibiotic resistance pattern but proteolytic activity was demonstrated by most strains in each test.

Staphylococcal serine proteinase increases intracellular free calcium concentration in human polymorphonuclear leukocytes.

Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 micrograms/ml. The highest Ca2+ rise (104 +/- 47 nM) was observed for 10 micrograms/ml SSP. It was mainly dependent (81 +/- 11%) on extracellular calcium influx, however, SSP mobilized 68 +/- 7% of Ca2+ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca2+ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca2+ concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.

[Staphylococcus strains isolated from cases of osteomyelitic patients qualifying for autovaccine therapy]. / Szczepy Staphylococcus aureus izolowane z przypadków zapalen kosci u pacjentów kwalifikowanych do leczenia autoszczepionka.

Staphylococcus aureus strains isolated from osteomyelitic were analysed biochemically and phage-typed. All examined strains were classified to the following biochemical groups: biotype I 6,332,153, biotype II 6,732,151, biotype III 6,732,151, biotype IV 6,732,150. Among the strains the following types were frequent: 3A, 3C, 55, 71 and 96.

[Regulatory influence of Staphylococcal serine protease on selected functions of lung macrophages in vitro. Introduction of cellular carbonic paramagnetic probe method for evaluation of cellular phagocytic activity]. / Regulacyjny wplyw gronkowcowej proteinazy serynowej na wybrane funkcje makrofagów pucnych in vitro. Wprowadzenie metody komórkowej sondy paramagnetycznej weglowej dla oceny aktywnosci fagocytarnej komórek.

The study was aimed at evaluation of properties of serine protease produced by Staphylococcus aureus V8. Influence of enzyme on chemotactic and phagocytic activity of rat lung macrophages was investigated. Chemotactic activity of rat lung macrophages was studied by application of modified Boyden chambers. Slight induction of chemotaxis of cells preincubated with enzyme was found. Phagocytic activity of rat lung macrophages was investigated by application of Varian E3 spectrometer. EPR signals emitted by paramagnetic graphite phagocytized by macrophages were measured. Increased phagocytic activity of macrophages subjected to preincubation with enzyme in concentration of 10 micrograms/ml was observed. It seems that staphylococcal proteinase is engaged in interaction of staphylococci with immunological system cells.

[Effect of Staphylococcal proteinase on adherence of Candida albicans to mucous membrane cells in vitro]. / Wplyw proteinazy gronkowcowej na przyleganie grzybów Candida albicans do komórek blony sluzowej in vitro.

The study was aimed at evaluation of influence of staphylococcal proteinase on adherence of Candida albicans to the cheek mucous membrane cells. Epithelial cells were preincubated with the enzyme which was followed by adherence tests. Significant increase of number of cells of Candida albicans adhering to epithelial cells preincubated with enzyme in concentrations of 10 micrograms/ml and 50 micrograms/ml, was detected. Staphylococcal serine protease seems to play an important role in mixed infections caused by fungi and bacteria.

Human lactoferrin binding in clinical isolates of Staphylococcus aureus.

Human lactoferrin (HLf) is an iron-binding protein and a host-defence component at the mucosal surface. Recently, a specific receptor for HLf has been identified on a strain of Staphylococcus aureus associated with toxic shock syndrome. We have looked for the occurrence of 125I-HLf binding among 489 strains of S. aureus isolated from various clinical sources. HLf binding was common among S. aureus strains associated with furunculosis (94.3%), toxic shock syndrome (94.3%), endocarditis (83.3%) and septicaemia (82.8%) and other (nasal, vaginal or ocular) infections (96.1%) with a mean binding (in fmol) of 29.1, 21.9, 16.9, 22.2 and 29.2 respectively; the differences between mean HLf binding values of 29.1-29.2, 21.9-22.2 and 16.9 were significant. Furunculosis-associated (low-invasive or localised) isolates were high-to-moderate binders of HLf; 50% gave positive results at a threshold of greater than 31 fmol of 125I-HLf bound. In contrast, endocarditis-associated (high-invasive or systemic) isolates demonstrated low binding and did not bind 125I-HLf at the above threshold level. S. aureus recognised human or bovine Lf. However, bound 125I-HLf was more effectively inhibited in a dose-dependent manner by unlabelled bovine Lf than by homologous HLf. Binding of 125I-HLf to staphylococci was optimal with organisms grown in agar compared with those from broth cultures. The binding capacity of S. aureus was abolished when strains were grown on carbohydrate- and salt-rich agar media. HLf-binding ability of S. aureus did not correlate with fibronectin, fibrinogen, immunoglobulin G or laminin binding.

Bovine lactoferrin receptors in Staphylococcus aureus isolated from bovine mastitis.

A total of 103 Staphylococcus aureus strains isolated from bovine mastitis were tested for bovine lactoferrin binding in a 125I-labeled protein binding assay. More than 85% of the strains demonstrated high to moderate and a few showed little or no binding. Bovine lactoferrin binding to S. aureus cells was high when grown on blood, nutrient, or proteose-peptone agar, but the binding capacity was low with cells grown on salt rich media, in skim milk, or in broth. The kinetics of 125I-labeled bovine lactoferrin binding required approximately 90 min for complete saturation with optimal interaction in the pH range 4.0 to 7.0. The lactoferrin-staphylococci interaction was specific with a high affinity (association constant, Ka 14 x 10(6) L/mol). Scatchard plot analysis estimated the number of binding sites per cell at 7200 on strain SA-340. Unlabeled bovine lactoferrin effectively displaced the binding of the labeled ligand to strain SA-340 in a dose-dependent manner. Bovine lactoferrin binding was inhibited or displaced by human lactoferrin. Various plasma, connective tissue, or mucosal secretory proteins tested did not inhibit lactoferrin-staphylococci interaction. Bovine lactoferrin binding components on SA-340 were resistant to glycolytic enzymes and moderately susceptible to proteolytic digestion. Two proteins with an estimated molecular weight of approximately 92 and 67 kDa were identified as bovine lactoferrin binding components of S. aureus strain SA-340.

Serine proteinase from Staphylococcus aureus enhances elastin degradation by elastases in the presence of human alpha-1-proteinase inhibitor.

During staphylococcal pneumonia massive destruction of lung tissue is often observed. Staphylococcal serine proteinase (SSP) inactivates alpha-1-proteinase inhibitor (alpha 1PI) a major factor which protects lungs from phagocyte proteases. We investigated the effect of SSP on elastin degradation by porcine pancreatic elastase (PE) and crude extract of human neutrophil elastase (NE) in solution and gel containing alpha 1PI. SSP having no elastase activity enhanced PE and NE-induced elastinolysis in solution when added to alpha 1PI before mixing with elastase and then with elastin. SSP added simultaneously with alpha 1PI to PE had no influence on elastin degradation. However, SSP added simultaneously, 30 min before or 30 min after PE significantly increased elastin digestion in elastin-agarose plate with alpha 1PI. Maximal increase in elastinolysis about 3-fold was for SSP added 30 min prior to PE. Since elastin is the major component of the alveolar walls it is possible that lung damage in the course of staphylococcal infection may partly depend on action of SSP.

Bovine lactoferrin binding to six species of coagulase-negative staphylococci isolated from bovine intramammary infections.

Bovine lactoferrin (BLf), an acute-phase iron-binding secretory protein present in secretions of the bovine udder, was demonstrated to bind to the following staphylococcal species associated with bovine intramammary infections: S. epidermidis, S. warneri, S. hominis, S. xylosus, S. hyicus, and S. chromogenes. The degree of 125I-labeled BLf uptake significantly varied among the blood agar-grown cells of all six species of coagulase-negative staphylococci tested. Isolates identified as S. xylosus demonstrated the highest (mean, 35.1 x 10(6) +/- 13.3 x 10(6) nmol) and S. hyicus the lowest (mean, 10.7 x 10(6) +/- 5.9 x 10(6) nmol) binding to 125I-BLf. BLf binding was optimum at an acidic pH, with time-dependent binding saturation ranging from 70 min for S. warneri to 240 min for S. hominis. The BLf-binding mechanism was specific, with affinity constants (Ka values) ranging between 0.96 x 10(6) and 11.90 x 10(6) liters/mol. The numbers of BLf-binding sites per cell, as determined by using Scatchard analysis, were as follows: S. epidermidis, 3,600; S. warneri, 1,900; S. hominis, 4,100; S. xylosus, 4,400; S. hyicus, 6,100; and S. chromogenes, 4,700. 125I-BLf binding to all species was inhibited by unlabled BLf and unlabeled human lactoferrin, whereas none of the various plasma, connective tissue, or mucosal secretory proteins or carbohydrates tested caused significant interference. BLf-binding receptors of the six coagulase-negative staphylococcal species demonstrated marked differences in patterns of susceptibility to proteolytic or glycolytic enzyme digestion and to heat or periodate treatment. These data suggest that the BLf-binding components in S. epidermidis and S. warneri are proteins containing glycosidyl residues. In the remaining four species, the proteinaceous nature of the BLf-binding component was evident, but the involvement of glycosidyl residues was not clear. Results of this study establish the presence of specific binding components for BLf on coagulase-negative staphylococci isolated from bovine intramammary infections.

Alkaloids in the tissue culture of Holarrhena antidysenterica.

In the present study the tissue culture of a medicinal plant Holarrhena antidisenterica was established. Phytochemical analysis revealed a presence of a few alkaloids in the callus tissues. Two of them were determined by TLC as conessine and conimine. A screening of antibacterial activity of alkaloid extract was performed.

Anti-staphylococcal serine proteinase and other serum factors in phagocytosis.

The interactions between polymorphonuclear cells (PMN), Staphylococcus saprophyticus cells and rabbit antibodies against Staphylococcus aureus V8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocytosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocytosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase gamma-globulin serum fraction, anti-serine proteinase IgG, non-immunized rabbit serum or with complement.

Staphylococcal wound infection in the pig: Part I. Course.

Full-thickness excision wounds infected with Staphylococcus hyicus, a pig pathogen, or Staphylococcus aureus, a human pathogen, were produced in pigs. The inoculated wounds were kept occluded for 2 days and then exposed and biopsied at intervals for 9 to 12 days. The exposed lesions were edematous and exudative. The S. aureus model was experimentally advantageous because the infection remained localized to the wound without systemic infection or signs of discomfort. The S. hyicus infection caused a rash with skin blisters; thus, its use is discouraged. The concentration of S. hyicus in the wound on day 2 was log 8.6 +/- 0.4 CFU/g (mean +/- standard deviation). On day 4 the mean was log 9.2. For S. aureus the values were log 8.0 +/- 0.9 on day 2 and 6.9 on day 4 (p less than 0.05). Of the 50 individual values in the S. aureus series, 45 were above log 5. The inflammatory reaction was more pronounced after the infection with S. hyicus, whereas with S. aureus the fibroblast response came earlier and was more pronounced. The model parallels typical clinical courses of staphylococcal infection.

Staphylococcal wound infection in the pig: Part II. Inoculation, quantification of bacteria, and reproducibility.

Bacteria from full-thickness excision wounds with staphylococcal infection in the pig were cultured quantitatively. The bacterial concentration increased with the size of the inoculum, and after 2 days it had already reached a stable, maximal level of approximately log 8 CFU/g in the tissue. The correlation coefficient was 0.753 in comparing concentrations from superficial and deep biopsy halves and 0.145 from "surface wash" and superficial biopsies. The "within wound" sample distribution was logarithmic. The gain in precision when assessing the mean bacterial concentration from three instead of one biopsy was 45%. The coefficient of variation of between wound and within wound determinations was in the same range (i.e., 7.1-12.5%). Paired observations from a sample population with 7 sites (wounds) were needed to determine a 10% change in the bacterial concentration at a significance level of p less than 0.05, and a similar change could be determined from unpaired observations based on two populations with eleven sites.

Effect of milk on fibronectin and collagen type I binding to Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis.

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (Fn) [corrected], whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (Cn) [corrected] type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 125I-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 125I-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding.