Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Bacteriocins as alternative agents for control of multiresistant staphylococcal strains

AIMS: To investigate the activity of seven staphylococcins, bacteriocins produced by staphylococci, against multiresistant Staphylococcus aureus and coagulase-negative staphylococci (CNS) involved in human infections. METHODS AND RESULTS: Four bacteriocins produced by Staph. epidermidis (Pep5, epidermin, epilancin K7 and epicidin 280) and three produced by Staph. aureus (aureocins A70, A53 and 215FN) were tested. Sixteen Staph. aureus strains, including a representative strain of the endemic Brazilian methicillin-resistant clone (MRSA), and 57 CNS strains were used as indicators. Among the staphylococcins used, Pep5 was able to inhibit 77.2% of the CNS strains and 87.5% of the Staph. aureus strains tested, including the Brazilian MRSA endemic clone, responsible for a large number of hospital-acquired infections in Brazil. On the other hand, aureocin A53 and epidermin presented a high antagonistic activity only against the Staph. aureus strains, being able to inhibit, respectively, 87.5% and 81.3% of them, including also the Brazilian MRSA endemic clone. The remaining bacteriocins inhibited only a low percentage of the nosocomial staphylococcal strains tested. CONCLUSIONS: Aureocin A53 and epidermin have potential applications against MRSA, whereas Pep5 seems to be an attractive agent against both MRSA and CNS, including mupirocin-resistant strains and the Brazilian endemic clone of MRSA, which is also found disseminated in other countries. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocins may represent alternative agents to control important nosocomial pathogens.     

Mupirocin resistance in staphylococci: development and transfer of isoleucyl-tRNA synthetase-mediated resistance in vitro

Mupirocin resistance could be transferred from highly resistant clinical isolates of Staphylococcus aureus to highly sensitive recipients of Staph. aureus, Staph. epidermidis and Staph. haemolyticus. Transconjugants of the latter two organisms could transfer this resistance into mupirocin-sensitive Staph. aureus. Moderately resistant strains did not transfer this resistance to sensitive recipients, nor did strains with high-level mupirocin resistance developed by serial transfer or habituation. The inhibitory effects of mupirocin on crude isoleucyl-tRNA synthetases (IRS) isolated from mupirocin-sensitive and -resistant strains of Staph. aureus have been determined. Drug concentrations needed to produce 50% inhibition, I50 values, were very low against IRS from a highly sensitive strain, somewhat higher against IRS from moderately resistant strains, much higher against enzyme from strains trained in vitro to high-level resistance, and considerably higher still against IRS extracted from clinical isolates possessing high-level mupirocin resistance and from the transconjugates of such strains resulting from crosses with mupirocin-sensitive strains. It is concluded that high-level resistance in clinical isolates is plasmid-mediated involving a second, mupirocin-resistant IRS whereas in moderately resistant strains, and in strains trained in vitro to high-level resistance, chromosomal mutations are likely to be responsible for decreasing IRS sensitivity.     

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.     

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.     

Serological characteristics of coagulase-positive staphylococci isolated from man and animals

A simplified method allowed Staphylococcus aureus, Staph. intermedius and coagulase-positive Staph. hyicus subsp. hyicus isolated from humans, dogs, monkey, sheep, poultry, rabbits, giant rats (Cricetomys gambianus) and other animals to be serotyped. The nine coagulase-positive staphylococcal strains of human origin possessed thermolabile and thermostable agglutinogens. Two strains of Staph. intermedius of human and canine origins examined had agglutinogen K1K2. The three Staph. aureus strains isolated from African giant rats (Cricetomys gambianus) had agglutinogens a5 and P common to them. The Staph. aureus strain isolated from a monkey belonged to serotype b1, c1, o and the caprine strain of Staph. hyicus subsp. hyicus was serotype a5, c1.     

Screening of staphylococci for the toxic shock syndrome toxin-1 (TSST-1) gene

Dot blot hybridization was used to screen 820 staphylococci for the presence of the gene coding for TSST-1. The DNA of 33 strains among 70 Staph. aureus strains isolated from suspected toxic shock syndrome (TSS) cases hybridized with the probe. These results agreed perfectly with those obtained with a phenotypic method (immunodiffusion). Among 608 Staph. aureus strains isolated over a period of one month from hospitalized patients, 66 (11%) hybridized with the probe; of these strains, 64 (97%) were found to produce TSST-1 in vitro. None of 145 coagulase-negative staphylococcal strains harboured DNA hybridizing with the probe. The data indicate that this genotypic assay is suitable for epidemiological studies.     

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°C there was no marked variation in coagulase production by the surviving strains but at 55 and 62–5dE 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 62mD5dE C resulted in loss of enterotoxin production by all the survivors except S₆ and 234, the surviving cells of which still prodused enterotoxin B after heat treatment at 55dE 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.     

Recipient capacity of clinical strains of staphylococci belonging to different phage groups]

The recipient capacity of the strains of Staph. epidermidis and Staph. areus belonging to different phage groups, as well as the possibility of epidemic distribution of the erythromycin resistance marker among the clinical staphyloccal strains on using the defective phage obtained from strain 8325 P IIde was studied. The defective phage P IIde may be the source of epidemic distribution of the drug resistance among the competent strains of Staph. aureus. All erythromycin sensitive strains of Staph. aureus lysed by the phages of groups I and III proved to be competent recipients of the erythromycin resistance marker. The strains of Staph. aureus of phage group II and phage type 80/81, as well as the strains of Staph. epidermidis were not competent recipients under our experimental conditions. It was not possible to transfer the high level of erythromycin resistance (1000 gamma/ml) on transduction to the strains of phage group I with a relatively low level of resistance to this antibiotic (20-50 gamma/ml.     

Characteristics of the composition of higher fatty acids in methicillin-resistant and methicillin-sensitive Staphylococci

The composition of fatty acids of methicillin-resistant (MR) and methicillin-sensitive (MS) strains of Staph. aureus and Staph. epidermidis was determined with the method of reactive gas liquid chromatography. The MS staphylococci of the above species differed by the content of acids with branched chains of iso- and anteisostructures and straight chains. Anteisoacids in the cells of Staph. epidermidis amounted almost to 80 per cent of the total number of the acids, while in the cells of Staph. aureus, their total number amounted only to a half of the fatty acid pool. Comparison of the composition of the fatty acids of the MS and MR strains of Staph. aureus revealed differences in the proportions of the anteiso- and isoacids. The total number of the long-chain C20.0 acid in the cells of Staph. epidermidis resistant to methicillin was lower as compared to that in the sensitive cells.     

Interactive Growth of Staphylococcus aureus Strains with a Poultry Skin Microflora in a Diffusion Apparatus

The growth of different biotypes of Staphylococcus aureus strains isolated from poultry was studied using the NBS Ecologen in which the interacting mixed culture was derived from the microfiora of hen skin and separated from the Staph. aureus culture by a membrane of 0.4 μm pore size. Inhibition of growth of the Staph. aureus cultures occurred with strains from each biotype. Marked inhibition of growth was always accompanied by the production of large numbers (>10⁹/ml) of plaque forming units (phage). In the mixed culture chamber poultry phage group C strains became the predominant Staph. aureus type. The phages produced by the mixed culture showed a wide spectrum of lytic activity for the propagating strains of the human, bovine and poultry phage sets.     

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A-bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml-1 and 103 cfu ml-1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase-negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at approximately 50 cfu ml-1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.     

Comparative antibiotic sensitivity of different species of staphylococci isolated from humans]

A total of 206 strains of various staphylococcal species isolated from various sources were studied with respect to their sensitivity to 18 antibiotics. The number of strains poly-resistant to the antibiotics was almost the same among Staph. aureus and Staph. epidermidis, i. e. 54.8 and 51.3 per cent respectively. The coagulase-negative and mannitol-negative variants of Staph. aureus and Staph. epidermidis possessing high biological activity (10-14 properties) were resistant to more antibiotics as compared to the low active strains.     

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.     

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.     

Expression of glycocalyx by coagulase-negative Staphylococcus species isolated from bovine milk

Two hundred and six strains of coagulase-negative Staphylococcus species were assessed for expression of glycocalyx on serum soft agar, india ink and adherence techniques. The organisms were maintained on trypticase soy agar plates at 4 degrees C for 30 d (120 strains) or stored at -80 degrees C in skim milk for 90 d (60 strains). Additionally, 26 milk samples from cows known to have excreted coagulase-negative staphylococci were used to inoculate serum soft agar directly. Nine of 26 direct culture samples and 43 of 180 strains maintained for an extended period had diffuse-type growth on serum soft agar. The proportion that exhibited an unstained halo by india ink was similar regardless of storage time. Slime production determined by in vitro adherence revealed a higher proportion of positive strains than had been predicted by serum soft agar or india ink techniques. More strains of Staphylococcus chromogenes, Staph. epidermidis, Staph. hominis, Staph. simulans and Staph. warneri expressed glycocalyx than other coagulase-negative Staphylococcus species. These results suggest that most coagulase-negative staphylococci produce slime rather than a capsule. However, evidence for classical encapsulation was demonstrated in several strains by india ink. The finding that Staphylococcus species other than Staph. aureus isolated from bovine milk are capable of glycocalyx production may be of importance in investigations on the relationship between staphylococci and host defence mechanisms.     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

Plasmids of Staphylococcus aureus associated with live and processed poultry

A series of 23 strains of Staphylococcus aureus originally isolated from processed poultry was screened for the presence of plasmids. Plasmids were more common in strains of Staph. aureus characteristically associated with live poultry than with strains endemic in poultry plants and strains of human origin. Two plasmids with sizes of 1.65 and 18.2 kilobase pairs (kBp) were present in three strains considered typical of Staph. aureus forma specialis 'altilis' and two plasmids with sizes of 1.65 and 17 kBp were present in three of four strains of Staph. aureus var. gallinae. A 1.65 kBp plasmid was present in all seven strains of these poultry biotypes and in three of 14 'endemic' strains. All the 1.65 kBp plasmids were shown by blot hybridization to share sequence homology. There was also some sequence homology between the 18.2 kBp and 17 kBp plasmids. These results were supported by restriction enzyme digest analyses. A study of cured derivatives of strain PS221 f.sp. 'altilis' suggested that the 18.2 kBp plasmid encoded the genetic determinant(s) responsible for caseolysis. Both the 1.65 and the 18.2 kBp plasmids also exerted an effect on the production of acid from lactose. In no other characteristic did cured strains resemble the plasmid-free 'endemic' strains. This was therefore consistent with the notion that the genetic determinants associated with the cultural characteristics of endemic strains are chromosomally located.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.