Methicillin-resistant Staphylococcus aureus in human milk

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37 degrees C, population sizes were already higher than 9.4 x 10(5) UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.     

Effects of sample handling temperatures on bovine skim milk progesterone concentrations

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph. aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50 degrees C there was no marked variation in coagulase production by the surviving strains but at 55 and 62.5 degrees C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62.5 degrees C resulted in loss of enterotoxin production by all the survivors except S6 and 234, the surviving cells of which still produced enterotoxin B after heat treatment at 55 degrees C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

C-390 as sole selective agent for isolation of Pseudomonas aeruginosa from hospital waste water

Pseudomonas aeruginosa was recovered (in numbers ranging from 10(2) to 10(5) colony-forming units per millilitre) from heavily contaminated hospital waste water when grown at 41.5 degrees C on a differential medium agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) at a final concentration of 30 micrograms/mL. The medium appeared to be highly selective for P. aeruginosa with 95-100% of all colonies isolated from four different hospital waste waters being identified as P. aeruginosa. Many strains of P. aeruginosa isolated from hospital waste waters failed to hydrolyse casein when grown on skim milk agar and this medium appeared to restrict pigment production to only pyoverdin (detectable only under ultraviolet light). However, most strains were capable of casein hydrolysis when grown on a modified skim milk medium.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Minimal water activity for enterotoxin A production and growth of Staphylococcus aureus.

The minimal water activity (alphaw) for growth was correlated with enterotoxin A formation by two strains of Staphylococcus aureus in a salt mixture broth. Within 7 days at 30 degrees C both strains grew and formed enterotoxin A minimally between alphaw 0.864 and 0.867, but at 25 degrees C, the minimal alphaw for both activities was increased to between 0.870 and 0.887 after a 2-week incubation.     

A 6 h microslide immunodiffusion assay for confirmed detection of staphylococcal enterotoxins

Use of appropriately balanced immunological reagents and incubation of microslides at 45 degrees C resulted in confirmed assay results in 6 h for staphylococcal enterotoxins. The sensitivity of the 45 degrees C-6 h assay was comparable to the 37 degrees C-24 h assay: 0.1 micrograms of staphylococcal enterotoxin A/ml.     

Effect of incubation media on the recovery of Escherichia coli K12 heated at 52 degrees C.

The exposure of exponentially grown Escherichia coli K12 to 52 degrees C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 degrees C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not. As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 degrees C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.     

Effect of the lactoperoxidase system on Listeria monocytogenes behavior in raw milk at refrigeration temperatures.

Activity of raw milk lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system on four Listeria monocytogenes strains at refrigeration temperatures after addition of 0.25 mM sodium thiocyanate and 0.25 mM hydrogen peroxide was studied. The LP system exhibited a bactericidal activity against L. monocytogenes at 4 and 8 degrees C; the activity was dependent on temperature, length of incubation, and strain of L. monocytogenes tested. D values in activated-LP system milk for the four strains tested ranged from 4.1 to 11.2 days at 4 degrees C and from 4.4 to 9.7 days at 8 degrees C. The lactoperoxidase level in raw milk declined during a 7-day incubation, the decrease being more pronounced at 8 degrees C than at 4 degrees C and in control milk than in activated-LP system milk. The thiocyanate concentration decreased considerably in activated-LP system milk at both temperatures during the first 8 h of incubation. LP system activation was shown to be a feasible procedure for controlling development of L. monocytogenes in raw milk at refrigeration temperatures.     

Simple method to observe the adaptive response of Listeria monocytogenes in food

A simple, novel method for determining stress-adaptive response of Listeria monocytogenes in food systems is presented. The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5.25 with incubation at 36 degrees C for 60 h to detect acid adaptation and plating on LSA with 70 gl-1 NaCl and incubation at 7 degrees C for 7 d to detect cold-osmotic adaptation. Adapted cells produced larger colonies (> 1 mm) under these conditions than unadapted cells. Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population. After a 5-d adaptation period at 4 degrees C in milk with 80 gl-1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with < 1% of the unstressed control population. Stress-adapted L. monocytogenes were isolated from soft cheese held for 42 d at 10 C.     

Effect of the lactoperoxidase system on Listeria monocytogenes behavior in raw milk at refrigeration temperatures.

Activity of raw milk lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system on four Listeria monocytogenes strains at refrigeration temperatures after addition of 0.25 mM sodium thiocyanate and 0.25 mM hydrogen peroxide was studied. The LP system exhibited a bactericidal activity against L. monocytogenes at 4 and 8 degrees C; the activity was dependent on temperature, length of incubation, and strain of L. monocytogenes tested. D values in activated-LP system milk for the four strains tested ranged from 4.1 to 11.2 days at 4 degrees C and from 4.4 to 9.7 days at 8 degrees C. The lactoperoxidase level in raw milk declined during a 7-day incubation, the decrease being more pronounced at 8 degrees C than at 4 degrees C and in control milk than in activated-LP system milk. The thiocyanate concentration decreased considerably in activated-LP system milk at both temperatures during the first 8 h of incubation. LP system activation was shown to be a feasible procedure for controlling development of L. monocytogenes in raw milk at refrigeration temperatures.     

Stability of plasmids in lactococci during extended incubation in growth media

The stability of plasmids in Lactococcus lactis ssp. lactis strains C2 and ML3, and L. lactis ssp. cremoris strains ML1 and SC607, was investigated by extended incubation of bacterial cells in low nutrient media under acidic conditions. Strains were grown overnight (16-18 h) in skim milk and unbuffered medium (M17-) at 32 degrees C and subsequently held at that temperature for extended periods (greater than or equal to 96 h). Lac- variants were obtained from each strain in milk and (M17-) broth. The plasmid profiles of Lac- variants when compared with their parental Lac+ strains showed loss of one or more plasmid bands. None of the Lac- mutants showed loss of smaller plasmids (less than 5 MDa) indicating that smaller plasmids in lactococci are more stable under these conditions than larger plasmids (greater than 10 MDa). Concomitant loss of the Lac+ phenotype and plasmids by the method used in the present investigation may have application for isolating mutants devoid of one or more plasmids.     

Staphylococcal enterotoxin A: a chromosomal gene product.

Physical and genetic analysis of the plasmid make-up of Staphylococcus aureus strains S6 and FRI-100 has shown that staphylococcal enterotoxin A is a chromosomal gene product.     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis.

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.