Fate of Staphylococci and Enteric Microorganisms Introduced into Slurry of Frozen Pot Pies

A slurry was prepared from six frozen pot pies diluted 1:5 with distilled water, two chicken, two turkey, and two beef pies of different brands. This slurry formed a reference sample and was placed in sterilized jars, frozen, and used as needed throughout the experiments. A second slurry was prepared in a similar manner from a frozen beef pot pie and a chicken pot pie, and was used as a control in only one experiment. The total count of microorganisms and the number of coliforms, Escherichia coli, salmonellae, and coagulase-positive staphylococci per gram were determined. Samples of slurry were inoculated in decimal dilutions with one or more of the following: Salmonella typhimurium, E. coli, and a strain of staphylococcus that causes food poisoning. The natural flora was found to exert an inhibitory effect upon the growth of the added microorganisms after incubation at 35 C for 18 hr. The inhibitory effect on growth was in part due to pH. The predominating organism isolated from the natural flora after incubation was a lactobacillus, which, when added in mixture with the test organisms in sterilized slurry, did not exert the profound inhibitory effect observed in the case of the natural flora. Some factors which may be concerned with the inhibition were investigated.     

Staphylococci in Competition: I. Growth of Naturally Occurring Mixed Populations in Precooked Frozen Foods during Defrost

In chicken pot pies, it was not possible to promote the growth of appreciable numbers of staphylococci under any condition of defrost. The pies spoiled under the same conditions, however. In macaroni and cheese dinners, tremendous numbers of saprophytic bacteria developed, but only after extended incubation at room temperature in which even spoilage was carried to an extreme. Under these conditions, staphylococci also multiplied vigorously. At the extreme temperature of 37 C, rank spoilage of such an advanced state as to render the product completely inedible was reached in less than 24 hr. Staphylococci grew very well under these extreme conditions.     

Comparative Evaluation of Five Selective and Differential Media for the Detection and Enumeration of Coagulase-Positive Staphylococci in Foods

Five selective media for the detection and enumeration of coagulase-positive staphylococci were evaluated for their efficiency in the recovery of 17 strains of coagulase-positive staphylococci from foods. They were Staphylococcus Medium 110 (SM-110), tellurite-glycine-agar (TGA), egg-tellurite-glycine-pyruvate-agar (ETGPA), tellurite-egg-agar (TEA), and tellurite-polymyxin-egg yolk-agar (TPEY). Statistical analysis by the rank correlation method of the efficiency with which these media recovered staphylococci from pure 24-hr Brain Heart Infusion cultures revealed the following efficiencies in descending order: (i) TPEY, (ii) ETGPA, (iii) TGA, (iv) TEA, (v) SM-110. Growth of 17 strains of coagulase-negative cocci on these media showed the following approximate descending order of inhibition to these organisms: (i) ETGPA, (ii) TEA, (iii) SM-110, (iv) TGA, (v) TPEY. The appearance of colonies of the various coagulase-negative strains on each medium was studied for the degree to which they could be confused with colonies of coagulase-positive strains. Nineteen food contaminants, including Proteus vulgaris, Bacillus sp., Escherichia coli, Erwinia sp., fecal streptococci, and others, were also studied for similarities in appearance to staphylococci and for ability to grow on the selective media. The influence of five sterile food homogenates (frozen chicken and tuna pies, custard, smoked ham, and raw whole egg) on recovery of 1,500 enterotoxigenic staphylococci (three strains) per milliliter was determined by statistical analysis. Three main effects (culture, media, and food) and three interactions (media with food, food with cultures, and media with culture) were found to be significant. Recovery on TPEY was influenced less by food than the other selective media and showed optimal recovery ability from sterile custard, eggs, and ham. TGA recovered well from sterile chicken pie and custard, SM-110 from sterile custard, and TEA from sterile ham. None of the media was outstanding in recovering staphylococci from tuna pie. The ability of the five selective media to recover 1,500 enterotoxigenic staphylococci (three strains) per ml from three sterile foods in the presence of 10 strains of contaminating bacteria added at the 0, 10⁵, and 10⁶ levels per milliliter was also studied and analyzed statistically. Only three factors were significant under these conditions—cultures, foods, and the interaction of media with the level of added contamination. Efficiency of recovery of TGA, SM-110, and ETGPA was found not to be dependent upon the level of contamination. Recovery on TPEY decreased with increases in the number of contaminants. TEA increased in efficiency at the 10⁵ level, but decreased at the 10⁶ level. When recovery on Trypticase Soy Agar was considered to be 100%, the average percentage of recovery by each of the selective media under all experimental conditions was determined.     

Isolation of methicillin-resistant Staphylococcus pseudintermedius from breeding dogs

The overuse of antimicrobials can select resistant bacteria strains; staphylococci have the ability to become resistant to all beta-lactam antimicrobials and are a significant concern in human medicine and a growing issue for veterinary medicine. Because antimicrobials are sometimes incorrectly used in breeding kennels, the objective of the work was to assess the occurrence of methicillin-resistant coagulase-positive staphylococci in breeding dogs. The research was carried out in 13 kennels that were allotted to three categories according to the intensity of antimicrobial use. Vaginal and milk swabs were taken from 87 healthy bitches around parturition and also from multiple organs of 27 of their pups that died within the first 2 weeks. Standard bacteriological examinations were carried out and coagulase-positive staphylococci were identified. All the coagulase-positive staphylococci resulted to be Staphylococcus pseudintermedius. Susceptibility to oxacillin and the presence of the mecA gene were tested. Nine out of 89 strains (six isolated from the bitches' milk and three from dead puppies, all belonging to kennels characterized by an excessive use of antimicrobials) were multidrug-resistant, methicillin-resistant and mecA positive.Our results confirm that excessive use of antimicrobials entails the risk of selecting resistant staphylococci strains. Our data also indicate that the bacterial flora of healthy dogs belonging to specific populations may act as a reservoir of resistance genes.     

Effect of pH and Oxygen Tension on Staphylococcal Growth and Enterotoxin Formation in Fermented Sausage

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 × 10⁷ cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.     

Bacteriological Survey of the Frozen Prepared Foods Industry: I. Frozen Cream-Type Pies

During Food and Drug Administration inspections of 12 imitation cream pie producers, 453 finished product samples and 350 line samples were collected and analyzed bacteriologically. Sanitary conditions in the plants varied from good to poor and, in general, were reflected in the bacteriological results. The survey revealed that, in the great majority of cases, frozen imitation-cream pies produced in plants operating under good conditions of sanitation had the following bacteriological content: (i) a most probable number (MPN) of fewer than three Escherichia coli cells per gram (i.e., absent from all tubes in the methodology employed), (ii) an average MPN of fewer than 50 coliforms per gram (10 or more pies), (iii) the absence of coagulase-positive staphylococci in 0.1-g portions, and (iv) an aerobic plate count of fewer than 25,000 per gram (geometric mean of 10 or more pies).     

Further Studies on Staphylococci in Meats, : III. Occurrence and Characteristics of Coagulase-positive Strains from a Variety of Nonfrozen Market Cuts

From 34 retail grocery stores and meat markets, 209 samples of nonfrozen meats were obtained and analyzed for coagulase-positive Staphylococcus aureus, employing six selective media. Sixty-seven (38.7%) of 173 samples obtained from 27 stores yielded S. aureus. No coagulase-positive S. aureus was isolated from 36 samples obtained from 7 of the stores. The 67 meats yielded 272 isolates from 10 different kinds of meats. There were 162 physiological strains represented when classified by store and 36 strains classified without regard to store of origin. The larger stores yielded fewer meats with staphylococci than the smaller stores. The meats from which S. aureus was recovered in the order of frequency of percentage recovery are as follows: chicken, pork liver, fish, spiced ham, round beef steak, hamburger, beef liver, pork chops, veal steak, and lamb chops. The following seven meats did not yield staphylococci: bologna, shucked oysters, olive and pickle loaf, salami, wieners, and chopped ham. Eighty-eight per cent of the isolates produced pigment, 85% were gelatinase positive, only 1 strain failed to form a precipitate on egg yolk agar, 92% formed deoxyribonuclease, 87% produced bound coagulase, 91% produced the α-hemolysin, 70% the δ-, 22% the β-, and 6% were nil in this regard. The isolates are compared with hospital and other food strains, and their possible source in the meats is discussed.     

Drug resistant coagulase-positive staphylococci strains in food

From the 1572 food samples, examined in Microbiology Department of Frozen Food Industry Research Laboratory in Lód?, 79 (5.0%) coagulase-positive staphylococci strains were isolated. All the strains were sensitive to vancomycin, gentamycin and chloramphenicol. Only individual staphylococci strains were resistant to erythromycin (1.3%), lincomycin (2.5%) and ciprofloxacin (2.5%). 20.3% strains, isolated mainly from raw meat, were resistant to doxycycline and 6.3% to oxacillin. 38.0% of coagulase-positive staphylococci strains had positive results of cefinase test. One strain isolated from minced meat was resistant to methicillin and at the same time it was producing beta-lactamases.     

Limitations of Fibrinogen-Polymyxin Medium in Detecting Coagulase-Positive Staphylococci in Raw Milk

A fibrinogen-polymyxin medium and Staphylococcus Medium 110 were used in the isolation of coagulase-positive staphylococci in raw milk. Results indicated that both media allow the growth of some rods and of many coagulase-negative cocci. A significantly greater number of coagulase-positive staphylococci were identified by the tube test than were revealed by halo formation on fibrinogen-polymyxin medium.     

Prevalence of Enterotoxigenic Staphylococci in Nose,Throat and Skin Lesions in Meat-Workers

The frequency of enterotoxigenic coagulase-positive and coagulase-negative staphylococci in nose and throat and in hand lesions was investigated in 86 meat cutters and dressers. Enterotoxin-producing staphylococci were demonstrated in nasal swabs from 22% of clinically well workers and from 42% of a group with mild coryza. The corresponding rates in throat swabs were 6 and 12%. Four of 16 superficial lesions of the hand harbored enterotoxigenic staphylococci. The implications for contamination of food and outbreaks of staphylococcal food poisoning are discussed.     

Pathogenic conversion of coagulase-negative staphylococci

Humans and animals are colonized by members of the genus Staphylococcus, however only some of these species evolved to cause invasive disease. The genetic basis for conversion of commensal staphylococci into pathogens is not known. We hypothesized that Staphylococcus aureus genes for coagulation and agglutination in vertebrate blood (coa, vwb and clfA) may support pathogenic conversion. Expression of coa and vwb in Staphylococcus epidermidis or Staphylococcus simulans supported a coagulase-positive phenotype but not the ability to cause disease in a mouse model of bloodstream infection. However, the simultaneous expression of coa, vwb and clfA in coagulase-negative staphylococci enabled bacterial agglutination in plasma and enhanced survival of S. simulans in human whole blood. Agglutination of S. simulans in the bloodstream of infected mice upon expression of coa, vwb and clfA provided also a mean for dissemination and replication in distal organs. Thus, the acquisition of genes for bacterial agglutination with fibrin appear sufficient for the conversion of commensal staphylococci into invasive pathogens.     

Production of Diarrheal Enterotoxins and Other Potential Virulence Factors by Veterinary Isolates of Bacillus Species Associated with Nongastrointestinal Infections

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.     

Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22

Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 10⁷ cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.     

Putative Virulence Factor Expression by Clinical and Food Isolates of Bacillus spp. after Growth in Reconstituted Infant Milk Formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.     

Effect of coliform and Proteus bacteria on growth of Staphylococcus aureus

Cultures of coliform and Proteus bacteria, mostly from foods, were tested for their effect on growth of Staphylococcus aureus in Trypticase Soy Broth. Inhibition of the staphylococcus by these competitors increased with increasing proportions of inhibiting (effector) bacteria in the inoculum and decreasing incubation temperatures (37 to 10 C). Time required for 2 × 10⁴ staphylococci to increase to 5 × 10⁶ cells per milliliter, the minimal number assumed to be necessary for food poisoning, varied with the species of effector, the original ratio of effector bacteria to staphylococci in the medium, and the incubation temperature. When the original ratio was 100:1, the staphylococci did not reach 5 × 10⁶ cells per milliliter at 10, 15, 22, or 30 C (with one exception), when growing with cultures representing six species of coliform bacteria and two of Proteus. When the ratio was 1:1, all effectors either greatly delayed the staphylococcus or prevented it from reaching hazardous numbers at 15 C, six of the eight caused a delay of 2 to 3 hr at 22 C, and only Escherichia coli delayed the coccus at 30 C. All effectors were ineffective at 22 and 30 C when original numbers of effectors and staphylococci were in the ratio 1:100. Greatest overall inhibition was by E. coli, E. freundii, and Proteus vulgaris, and these species were more effective than the others at 22 and 30 C. Aerobacter cloacae and Paracolobactrum aerogenoides were more effective at 15 C. In general, results were similar with different strains of a species. Except for Aerobacter aerogenes, Klebsiella sp., and P. aerogenoides, which apparently did not compete for nutrients, inhibition of the staphylococcus was by a combination of antibiotic substances and competition for nutrients.     

Occurrence of Coagulase-Positive Staphylococci in Cheddar Cheese

Samples (13) from several lots of cheddar cheese incriminated in staphylococcal food poisoning and 343 samples of cheddar cheese purchased over a 3-year period in retail markets were examined quantitatively for coagulase-positive staphylococci with the smear plate technique. Of the food-poisoning samples, 11 contained coagulase-positive staphylococci in numbers that ranged from 50 to several million per g. Of the 343 market cheese samples, 20% contained coagulase-positive staphylococci in concentrations ranging from less than 50 to more than 200,000 per g. The phage patterns of 64 of 89 cultures isolated from the food-poisoning samples placed them in the miscellaneous phage group (44A) or in phage group IV and the miscellaneous group (42D/44A); 14 had phage patterns that involved group III, the group with which food poisoning has usually been associated. In contrast, over 50% of 104 cultures from the market cheese, which were typed at 100 times the critical test dilution, had phage patterns that involved group III. Of nine selected cultures isolated from the food-poisoning cheese, three (all in phage group III) were positive for enterotoxin by intravenous injection test of cats.     

Staphylococci in Competition: III. Influence of pH and Salt on Staphylococcal Growth in Mixed Populations

Previous results showed definite repressive effects on the growth of staphylococci in mixed cultures due to the competitive growth of psychrophilic saprophytes. This study was continued, and the influence of other environmental factors, pH and salt, on the competition between staphylococci and saprophytes was investigated. Initial pH values varied from 5 to 9. At the extremes of the pH range, staphylococci failed to grow, while the saprophytes grew under all of the conditions tested. At pH 5, the growth curves for the saprophytes were markedly altered from those obtained at neutral pH. The lag phases were greatly lengthened at and below 20 C, but normal numbers of saprophytes were reached in the stationary phase. At pH 6 and 8, staphylococcal growth showed the same inhibition observed at pH 7, at and below 20 C; normal multiplication was observed above this temperature, but with accelerated death phases. Thus, pH did not primarily effect staphylococcal growth through its influence on saprophyte growth and competition, but rather directly affected the growth of Staphylococcus cultures. Salt concentrations from 3.5 to 9.5% were investigated for influence on staphylococcal growth in mixed populations. Above 3.5% salt, staphylococcal inhibition at and above 20 C was not as marked as in the controls, although normal numbers were never reached. The saprophytes were increasingly inhibited, and their lag phases materially lengthened as salt concentration was increased. Salt acted directly on the Staphylococcus population and also, by repressing saprophyte growth, decreased competition, which allowed the staphylococci to grow.     

Isolation and Characterization of Mercury Resistant Bacillus sp. from Soils with an Extensive History as Substrates for Mercury Extraction in Mexico

Metal phytoextraction assisted by bacteria plays an important role in bioremediation systems. In this work, mercury-resistant bacterial strains were isolated from soils with high levels of mercury (San Joaquin, Queretaro State, Mexico) and identified as Bacillus sp. based on the 16S rDNA gene sequence analysis. The bacterial strains were found to exhibit different multiple mercury-resistance and carbon source utilization characteristics. The mercury reduction ability was tested through a volatilization assay. The bacterial isolates were also evaluated for their ability to promote growth and mercury uptake in tomato plants. In a roll towel assay, the maximum vigor index of tomato plants was obtained with the inoculation of Bacillus sp. A2, A12, B11, B15 and C1, while in a pot assay, the maximum vigor index was obtained with the inoculation of Bacillus sp. A6, A7 and B20, compared with un-inoculated controls in the presence of HgCl₂. Maximum Hg accumulation in the roots and shoots of tomato plants was obtained only with Bacillus sp. A7 in the roll towel assay, whereas in the pot assay, maximum accumulation was obtained with Bacillus sp. A12 compared with un-inoculated controls. Our results show that mercury accumulation in tissue is enhanced by these plant growth promoting bacterial strains, which recommends their possible use as microbe-assisted phytoremediation systems in mercury-polluted soils.     

Genotyping and Toxigenic Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.     

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using “artificially” Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.