A survey of microorganisms for thermonuclease production

A total of 1204 cultures comprising 16 genera were surveyed for production of thermonuclease (TNase) in milk. Cultures other than Staphylococcus capable of TNase production were restricted to two genera, Streptococcus and Bacillus. Nineteen percent of 338 group D streptococci comprising four species (85% of which were Streptococcus faecalis) and 17% of 60 streptococci belonging to other groups produced TNase. Nine percent of 130 Bacillus cultures comprising six species produced the enzyme. On the other hand, 99% of coagulase-positive staphylococci produced TNase and only 18% of the coagulase-negative staphylococci produced the enzyme. The amount of TNase produced by streptococci and bacilli was significantly lower than that produced by coagulase-positive staphylococci. The pH profile of the streptococci and Bacillus TNases was similar to that of the staphylococcal TNase; each enzyme exhibited a minor peak at pH 7.0 and a broad major peak ranging from pH 8.5 to 10. The nuclease produced by coagulase-positive Staphylococcus was more heat stable than the nucleases produced by Streptococcus and Bacillus; there was little loss in activity of the staphylococcal enzyme after 60 min at 100 degrees C, whereas 50% of the activity of the streptococcal and Bacillus nucleases was destroyed in 40-60 min and 60-80 min, respectively.     

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. mitior produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1-12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher (P less than 0.001, chi 2 test and P less than 0.05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus (P less than 0.001 and P less than 0.01) and of oral Staph. epidermidis over oral Staph. aureus (P less than 0.01 and P less than 0.05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological viewpoints are discussed.     

Molecular dissection of phage lysin PlySs2: integrity of the catalytic and cell wall binding domains is essential for its broad lytic activity

Virologica Sinica - The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms...     

Some Microbiological Aspects of Staphylococcal Mastitis

(1) Mannitol fermentation is a reasonably reliable method for the detection of coagulase positive staphylococci in milk. This reliability can be improved if mannitol fermentation is carried out under anaerobic conditions.(2) Among hemolytic strains of staphylococci isolated from milk, beta hemolytic staphylococci predominate. Bovine and sheep blood agar plates give similar hemolytic patterns, but the hemolysis is more pronounced on sheep blood agar.(3) Gelatin liquefaction cannot be relied upon for the selection of coagulase positive staphylococci in milk.(4) Urease production is a feature of the majority of coagulase positive staphylococci isolated from milk.(5) Tellurite Glycine agar medium is not satisfactory for the selection of coagulase positive staphylococci in milk.     

Sensitivity to antibiotics of bacterial strains isolated from clinical specimens during the years 1985-1986

Sensitivity of 312 strains of staphylococci, 386 strains of streptococci and 1193 strains of aerobic gram-negative bacilli to the selected antibiotics was tested. These strains were isolated from the clinical material at the Clinical Hospital No. 1 in Warsaw within 1985-1986. Staphylococci were sensitive to pristinamycin, cefazolin, fusidic acid, oxacillin, and clindamycin. In 1986, a decrease in the number of strains sensitive to these antibiotics, except cefazolin, was seen. In case of streptococci the most active proved chloramphenicol and gentamicin but a significant decrease in the percentage of sensitive strains was also noted in 1986. The highest number of gram-negative bacilli was sensitive to amikacin, colistin, nalidixic acid, pipemidic acid, and gentamicin. In 1986, a decrease in the percentage of sensitive strains was noted. Amikacin and colistin were the most active against Pseudomonas spp. while amikacin and nalidixic and pipemidic acids--against Proteus spp. Comparison of the results with those obtained in 1981-1984 has shown that the sensitivity of staphylococci changed the most significantly and this change was unfavourable. Gentamicin and amikacin remained the most active against gram-negative bacilli while amikacin and colimycin against Pseudomonas spp. In case of anaerobes the majority of strains was sensitive to chloramphenicol, tetracycline and clindamycin. Metronidazole was active against high percentage of Clostridium spp. and all gram-negative bacilli while the percentage of gram-positive bacilli and cocci was sensitive to metronidazole.     

A lysozyme isolated from rainbow trout acts on mastitis pathogens

The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.     

Bacteriology of Dehydrated Space Foods

The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count ( 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beef soup and gravy base were positive.     

A lysozyme isolated from rainbow trout acts on mastitis pathogens

Abstract The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.     

Experimental study of combined staphylococcal, streptococcal and adenovirus infections in tissue culture]

Associated adenoviral, staphylococcus, and streptococcus infection was studied in the cultures of cells HEp-2 and PAO. Under conditions of monoinfection the cell culture largely inhibited the reproduction of staphylococci, and failed to influence the streptococci. In double and triple associated infections staphylococci overcame the inhibitory action of the cell culture. The pathogenic properties of cocci (plasma coagulation, hemotoxic properties) grown in the cell culture both under conditions of monoinfection, and in associations, failed to change. In double and triple associated infections adenoviruses did reproduce, but in lower titre than in monoinfection. Under conditions of mixed infection cocci penetrated and reproduced in the cell cytoplasm more intensively than in monoinfection. The cytopathic action was determined by viral associate, and was identical by its character to adenoviral monoinfection. A statistically significant increase in the activity of aldolase and transaminase enzymes was noted in mixed infection. The changes in the enzyme activity proved to depend on the character of the associations studied.     

A study of the microbiological status of Irish farmhouse cheeses with emphasis on selected pathogenic and spoilage micro-organisms

H.M. COVENEY, G.F. FITZGERALD AND C. DALY. 1994. Ninety-six 25 g samples from 25 Irish farmhouse cheeses, two Irish non-farmhouse cheeses and four foreign cheeses were evaluated for the presence of a variety of micro-organisms, namely, coliforms, faecal streptococci, Staphylococcus aureus , yeasts, moulds, salmonellas and shigellas. Seventeen cheeses, i.e. the soft and semi-soft types, were examined for Listeria monocytogenes. Most of the farmhouse cheeses are currently manufactured from raw milk, but some producers now use heat-treated milk. The incidence of coliforms and faecal coliforms was higher in soft, semi-soft and semi-hard cheeses than in hard types. High levels of contamination by faecal streptococci and non-pathogenic (coagulase-negative) Staph. aureus prevailed in a high proportion of the cheeses. Pathogenic (coagulase-positive) staphylococci, however, were also isolated from 50% of the cheeses, some of which were manufactured from pasteurized milk. Yeasts were found mainly in unpasteurized varieties, especially in the category of soft cheeses. Moulds were isolated from five non-mould-ripened cheeses, as well as from mould-ripened varieties. Salmonellas, shigellas and Listeria monocytogenes were not detected after direct enrichment.     

Antagonism of Lactic Streptococci Toward Staphylococcus aureus in Associative Milk Cultures

The inhibition of growth of Staphylococcus aureus by lactic streptococci in associative cultures in milk was not due to hydrogen peroxide produced by the streptococci. Dialyzed whey from the milk culture of lactic streptococci was more inhibitory than dialyzed whey from milk acidified with lactic acid, indicating that material other than lactate was also involved. Analyses of cation and anion exchange fractions from the dialyzed whey showed that only the neutral fraction was inhibitory.     

Effect of probiotic-fermented milk administration on gastrointestinal survival of Lactobacillus casei ATCC 393 and modulation of intestinal microbial flora

The aim of the present study was to assess the survival of free and immobilized Lactobacillus casei ATCC 393 on apple pieces, contained in probiotic-fermented milk, after gastrointestinal (GI) transit and to investigate the potential regulation of intestinal microbial flora in a rat model. In in vitro GI stress tolerance tests, immobilized L. casei ATCC 393 exhibited significantly higher survival rates compared to free cells. At a second stage, probiotic-fermented milk produced by either free or immobilized cells was administered orally at a single dose or daily for 9 days in Wistar rats. By 12 h after single-dose administration, both free and immobilized cells were detected by microbiological and molecular analysis at levels ≥6 logCFU/g of feces. Moreover, daily administration led to significant reduction of staphylococci, enterobacteria, coliforms and streptococci counts. In conclusion, L. casei ATCC 393 contained in fermented milk survived GI transit and modulated intestinal microbiota.     

Sensitivity of the wound microflora of oncological patients to some new antibiotics]

Sensitivity of the microflora of the oncological patients' wounds to the new antibiotics, such as gentamicin, kanamycin, oxacillin, ampicillin and lincomycin was studied with the help of the disc method. The discs with the above antibiotics were prepared under laboratory conditions in accordance with the respective instructions in the WHO. Sensitivity of 429 bacterial cultures, including 98 cultures of pathogenic staphylocci, 45 cultures of Enterococci, 43 hemolytic streptococci, 143 cultures of Escherichia, 50 cultures of Ps. aeruginosa and 50 cultures of Proteus was determined. The studies showed that gentamicin was the most active antibiotic aganist all the microbial species isolated from the surgical and other wounds of oncological patients. It may be used in treatment of the infections caused by association of the microbes belonging to different species, as well as in treatment of purulent processes before elucidating their etiology, 16.7 per cent of the Enterococcal isolates were resistant to gentamicin. Monomycin, kanamycin, oxacillin, lincomycin and macrolide antibiotics at present are sufficiently active against pathogenic staphylococci and hemolytic streptococci.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. minor produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1–12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher ( P < 0·001, χ² test and P < 0·05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus ( P < 0·001 and P < 0·01) and of oral Staph. epidermidis over oral Staph. aureus ( P < 0·01 and P < 0·05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological veiwpoints are discussed.     

Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR

Aims:  Breast milk has been described as a source of bacteria influencing the development of the infant gut microbiota. Up to the present, few studies have been focused on the application of culture-independent techniques to study bacterial diversity in breast milk. In this context, the aim of this study was to characterize the breast milk microbiota of healthy women by applying the quantitative real-time PCR technique (qRTi-PCR).
Methods and Results:  A total of 50 breast milk samples were analysed by qPCR to assess the presence of different bacterial genera or clusters, including the Bifidobacterium , Lactobacillus , Staphylococcus , Bacteroides , Enterococcus , Streptococcus , Clostridium cluster IV and Clostridium cluster XIVa–XIVb groups. Staphylococcus , Streptococcus , Bifidobacterium and Lactobacillus were the predominant groups and were detected in all the samples. Clostridium XIVa–XIVb and Enterococcus were detected in most of the samples in contrast to the Bacteroides and Clostridium cluster IV groups.
Conclusions:  Our results confirm the abundance of bacterial DNA in breast milk samples and suggest that the qRTi-PCR technique has a huge potential in the microbiological analysis of human milk.
Significance and Impact of the study:  qRTi-PCR allowed the detection of bacterial DNA of streptococci, staphylococci, lactic acid bacteria and bifidobacteria in the samples of human milk, which confirms that breast milk can be an important source of bacteria and bacterial DNA to the infant gut.     

Evaluation of media for monitoring fecal streptococci in seawater

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Evaluation of media for monitoring fecal streptococci in seawater.

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Comparison of bacterial flora in the oral cavity of children and adults

The aim of the study was to assess levels of occurrence and number of aerobic bacteria hemolysing and non-hemolysing, anaerobic bacteria, streptococci hemolysing and non-hemolysing, staphylococci, and bacteria responsible for tooth decay (Streptococcus mutans and Lactobacillus spp.) on oral cavity in children and adults. The results obtained indicate the difference of the level of occurrence of, aerobic bacteria hemolysing and non-hemolysing, anaerobic bacteria, streptococci hemolysing and non-hemolysing, staphylococci, and bacteria responsible for tooth decay (Streptococcus mutans and Lactobacillus spp.) was not statistically significant in either group. The counts and average values of the counts for aerobic bacteria non-hemolysing, anaerobic bacteria, streptococci hemolysing and non-hemolysing and Streptococcus mutans turned out to be statistically significantly larger in adults than in children. However for aerobic bacteria hemolysing, staphylococci and Lactobacillus spp. the difference of the counts was not statistically significant in either group.     

Detection and characterization of erythromycin-resistant methylase genes in Gram-positive bacteria isolated from poultry litter

The epidemiology of four erythromycin-resistant methylase ( erm) genes, ermA, ermB, ermC and msrA, was determined in erythromycin-resistant staphylococci, enterococci and streptococci isolated from poultry litter. All isolates were resistant to multiple antibiotics. Southern hybridization indicated that 4 of the 20 staphylococci contained the ermC gene on plasmids: on a 2.2 kb plasmid in Staphylococcus hominis and S. sciuri, on a 6.0 kb plasmid in S. xylosus, and on a 7.0 kb plasmid in S. lentus. In 16 of the 20 staphylococci, the ermA gene was harbored exclusively on the chromosome, as a double chromosomal insert on 8.0 and 6.2 kb EcoRI fragments. None of the staphylococci harbored the msrA gene. Dot-blot analysis indicated that all enterococci and streptococci hybridized with a biotinylated ermB gene probe. Southern hybridization indicated that only 2 of the 19 erythromycin-resistant enterococci contained the ermB gene on plasmids. The gene was localized on 4.0 kb and 5.9 kb plasmids, respectively, in two Enterococcus faecium isolates. Results from our studies indicate that the patterns of occurrence of erm genes, the sizes of the plasmids and the copy numbers of the inserts were different from the existing information on the presence of erm genes in clinical strains of Staphylococcus spp.