spa Types and Staphylococcal Enterotoxin Production of Staphylococcus aureus Isolated from Wild Boar.

Little is known about the structure of S. aureus population and the enterotoxin gene content in wild boar. In 1025 nasal swabs from wild boars, 121 S. aureus isolates were identified. Staphylococcal enterotoxin (SE) genes were identified in 18 isolates (14.9%). The seb gene was found in 2 S. aureus isolates, sec in 2 isolates, the see and seh genes were found in 4 and 11 isolates, respectively. The production of SEs was evaluated in bacteria grown in microbial broth. Concentration of SEB reached 2.70 µg/ml after 24 h and 4.46 µg/ml at 48 h. SEC was produced at 952.6 ng/ml after 24 h and 7.2 µg/ml at 48 h. SEE reached 124.1 ng/ml after 24 h and 191.6 ng/ml at 48 h of culture. SEH production reached 4.36 µg/ml at 24 h and 5.42 µg/ml at 48 h of culture. Thirty-nine spa types were identified among S. aureus isolates. The most prevalent spa types were t091 and t1181, followed by t4735 and t742, t3380 and t127. Twelve new spa types, i.e., t20572‒t20583 were identified. The wild boar S. aureus population was shown to contain previously identified animal/human-associated spa types and spa types not identified in humans or animals. We also indicate that wildlife animals can be a significant reservoir of see-positive S. aureus.

Effect of Radiant Catalytic Ionization and Ozonation on Salmonella spp. on Eggshells.

Three Salmonella enterica strains were used in the study (serovars: S. enteritidis, S. typhimurim and S. virchow). This study evaluated the efficacy of radiant catalytic ionization (RCI) and ozonation against Salmonella spp. on eggshell (expressed as log CFU/egg). The egg surface was contaminated three different bacterial suspension (103 CFU/mL, 105 CFU/mL and 108 CFU/mL) with or without poultry manure. Experiments were conducted at 4 °C and 20 °C in three different time period: 30 min, 60 min and 120 min. Treatment with RCI reduced Salmonella numbers from 0.26 log CFU/egg in bacterial suspension 108 CFU/mL, 4 °C and 20 °C, with manure for 30 min to level decrease in bacteria number below the detection limit (BDL) in bacterial suspension 105 CFU/mL, 20 °C, with or without manure for 120 min. The populations of Salmonella spp. on eggs treated by ozonizer ranged from 0.20 log CFU/egg in bacteria suspension 108 CFU/mL, 20 °C, with manure for 30 min to 2.73 log CFU/egg in bacterial suspension 105 CFU/mL, 20 °C, with manure for 120 min. In all treatment conditions contamination with poultry manure decrease effectiveness the RCI and ozonation. In summary, RCI technology shows similar effectiveness to the ozonation, but it is safer for poultry plant workers and consumers.

The Impact of Graphite Oxide Nanocomposites on the Antibacterial Activity of Serum.

Nanoparticles can interact with the complement system and modulate the inflammatory response. The effect of these interactions on the complement activity strongly depends on physicochemical properties of nanoparticles. The interactions of silver nanoparticles with serum proteins (particularly with the complement system components) have the potential to significantly affect the antibacterial activity of serum, with serious implications for human health. The aim of the study was to assess the influence of graphite oxide (GO) nanocomposites (GO, GO-PcZr(Lys)2-Ag, GO-Ag, GO-PcZr(Lys)2) on the antibacterial activity of normal human serum (NHS), serum activity against bacteria isolated from alveoli treated with nanocomposites, and nanocomposite sensitivity of bacteria exposed to serum in vitro (using normal human serum). Additionally, the in vivo cytotoxic effect of the GO compounds was determined with application of a Galleria mellonella larvae model. GO-PcZr(Lys)2, without IR irradiation enhance the antimicrobial efficacy of the human serum. IR irradiation enhances bactericidal activity of serum in the case of the GO-PcZr(Lys)2-Ag sample. Bacteria exposed to nanocomposites become more sensitive to the action of serum. Bacteria exposed to serum become more sensitive to the GO-Ag sample. None of the tested GO nanocomposites displayed a cytotoxicity towards larvae.

Genetic Diversity and Distribution of Virulence-Associated Genes in Y. enterocolitica and Y. enterocolitica-Like Isolates from Humans and Animals in Poland.

Yersinia enterocolitica, widespread within domestic and wild-living animals, is a foodborne pathogen causing yersiniosis. The goal of this study was to assess a genetic similarity of Y. enterocolitica and Y. enterocolitica-like strains isolated from different hosts using Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and Pulsed-Field Gel Electrophoresis (PFGE) methods, and analyze the prevalence of virulence genes using multiplex-Polymerase Chain Reaction (PCR) assays. Among 51 Yersinia sp. strains 20 virulotypes were determined. The most common virulence genes were ymoA, ureC, inv, myfA, and yst. Yersinia sp. strains had genes which may contribute to the bacterial invasion and colonization of the intestines as well as survival in serum. One wild boar Y. enterocolitica 1A strain possessed ail gene implying the possible pathogenicity of 1A biotype. Wild boar strains, represented mainly by 1A biotype, were not classified into the predominant Variable-Number Tandem Repeats (VNTR)/PFGE profile and virulotype. There was a clustering tendency among VNTR/PFGE profiles of pig origin, 4/O:3, and virulence profile. Pig and human strains formed the most related group, characterized by ~80% of genetic similarity what suggest the role of pigs as a potential source of infection for the pork consumers.

Identification of Yersinia enterocolitica isolates from humans, pigs and wild boars by MALDI TOF MS.

BACKGROUND: Yersinia enterocolitica is widespread within the humans, pigs and wild boars. The low isolation rate of Y. enterocolitica from food or environmental and clinical samples may be caused by limited sensitivity of culture methods. The main goal of present study was identification of presumptive Y. enterocolitica isolates using MALDI TOF MS. The identification of isolates may be difficult due to variability of bacterial strains in terms of biochemical characteristics. This work emphasizes the necessity of use of multiple methods for zoonotic Y. enterocolitica identification. RESULTS: Identification of Y. enterocolitica isolates was based on MALDI TOF MS, and verified by VITEK® 2 Compact and PCR. There were no discrepancies in identification of all human' and pig' isolates using MALDI TOF MS and VITEK® 2 Compact. However three isolates from wild boars were not decisively confirmed as Y. enterocolitica. MALDI TOF MS has identified the wild boar' isolates designated as 3dz, 4dz, 8dz as Y. enterocolitica with a high score of matching with the reference spectra of MALDI Biotyper. In turn, VITEK® 2 Compact identified 3dz and 8dz as Y. kristensenii, and isolate 4dz as Y. enterocolitica. The PCR for Y. enterocolitica 16S rDNA for these three isolates was negative, but the 16S rDNA sequence analysis identified these isolates as Y. kristensenii (3dz, 4dz) and Y. pekkanenii (8dz). The wild boar' isolates 3dz, 4dz and 8dz could not be classified using biotyping. The main bioserotype present within pigs and human faeces was 4/O:3. It has been shown that Y. enterocolitica 1B/O:8 can be isolated from human faeces using ITC/CIN culturing. CONCLUSION: The results of our study indicate wild boars as a reservoir of new and atypical strains of Yersinia, for which protein and biochemical profiles are not included in the MALDI Biotyper or VITEK® 2 Compact databases. Pigs in the south-west Poland are the reservoir for pathogenic Y. enterocolitica strains. Four biochemical features included in VITEK® 2 Compact known to be common with Wauters scheme were shown to produce incompatible results, thus VITEK® 2 Compact cannot be applied in biotyping of Y. enterocolitica.

Production of Staphylococcal Enterotoxins D and R in Milk and Meat Juice by Staphylococcus aureus Strains.

Seventeen Staphylococcus aureus strains were tested for production of staphylococcal enterotoxin D (SED) and staphylococcal enterotoxin R (SER) in milk and meat juice. SED was secreted in milk by 12 S. aureus strains at 6-54 ng/mL at 24 h and 9-98 ng/mL at 48 h. Another five strains secreted SED at 0.9-1.9 µg/mL at 24 h and at 1.2-2.4 µg/mL at 48 h. Strains producing high levels of SED in milk secreted 77-666 µg/mL of SED in meat juice at 24 h and 132-1225 µg/mL at 48 h. Strains producing lower amounts of SED in milk secreted 228-1109 ng/mL of SED at 24 h and 377-1782 ng/mL at 48 h in meat juice. Tested S. aureus strains produced SER in milk at 33-183 ng/mL at 24 h and 41-832 ng/mL at 48 h. Fourteen strains produced more SER in meat juice than in milk (17- to 232-fold and 15- to 269-fold more at 24 and 48 h, respectively). Three S. aureus strains secreted less than 74 ng/mL of SER in meat juice. Expression pattern of known enterotoxin regulators, that is, agrA, sarA, hld, rot, and sigB, was similar in selected strong and weak SED producers grown in both food matrices and could not explain differences in enterotoxin protein level. This suggests that enterotoxin regulation is more complex than previously thought. We demonstrated that in a number of tested S. aureus strains, production of SED and SER was significantly decreased in milk when compared with meat juice, supporting previous reports. However, certain strains secreted high amounts of SED and SER, irrespective of environment, likely contributing to higher food safety risk.

Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene.

Twenty Staphylococcus aureus strains harboring seh gene, including one carrying also sec gene and 11 sea gene, were grown in BHI+YE broth and milk and were tested for SEA, SEC and SEH production. All strains decreased pH of BHI+YE broth at 24h and increased them at 48h. Seventeen S. aureus strains grown in milk changed pH for no >0.3 unit until 48h. Three other S. aureus strains significantly decreased pH during growth in milk. All S. aureus produced SEH in BHI+YE broth in amounts ranging from 95 to 1292ng/ml, and from 170 to 4158ng/ml at 24 and 48h, respectively. SEH production in milk by 17 strains did not exceed 23ng/ml at 24h and 36ng/ml at 48h. Three S. aureus strains able to decrease milk pH produced 107-3029ng/ml and 320-4246ng/ml of SEH in milk at 24 and 48h, respectively. These strains were grown in milk and BHI+YE broth with pH stabilized at values near neutral leading to a significant decrease of SEH production. Representative weak SEH producers were grown in milk at reduced pH resulting in moderate increase in SEH production. SEA was produced in milk by 10S. aureus strains at 24-151ng/ml at 24h, and 31-303ng/ml at 48h. SEA production in milk was higher or comparable as in BHI+YE broth in 3 strains and lower for remaining strains. Production of SEC by sec-positive S. aureus strains was lower in milk than in BHI+YE broth, ranging from 131 to 2319ng/ml at 24 and 48h in milk and 296-30,087ng/ml in BHI+YE at 24 and 48h. Both lacE and lacG transcripts involved in lactose metabolism were significantly up-regulated in milk in strong SEH producers. In these strains hld, rot and sarA transcripts were up-regulated in milk as compared to weak SEH producers. Stabilization of milk pH at a value of raw milk significantly down-regulated hld, rot and sarA RNA in strong SEH producers. Milk was generally found unfavorable for enterotoxin production. However, certain S. aureus strains were not restricted in SEH and SEA expression in milk, unlike SEC which remained down-regulated in this environment. Therefore, low safety risk related to S. aureus producing SEC in milk, as suggested previously, may not pertain to certain SEA and SEH-producing strains.

Genotype and enterotoxigenicity of Staphylococcus epidermidis isolate from ready to eat meat products.

We have previously shown that potentially pathogenic isolates of Staphylococcus epidermidis occur at high incidence in ready-to-eat food. Now, within 164 samples of ready-to-eat meat products we identified 32 S. epidermidis isolates. In 8 isolates we detected the genes encoding for staphylococcal enterotoxins, but in 7 S. epidermidis isolates these genes were not stable over passages. One isolate designated 4S was shown to stably harbour sec and sel genes. In the genome sequence of S. epidermidis 4S we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis FRI909. Alignment of S. epidermidis 4S and S. epidermidis FRI909 SePIs revealed 6 nucleotide mismatches located in 5 of the total of 29 ORFs. Genomic location of S. epidermidis 4S SePI was the same as in FRI909. S. epidermidis 4S is a single locus variant of ST561, being genetically different from FRI909. SECepi was secreted by S. epidermidis 4S to BHI broth ranging from 14 to almost 36µg/mL, to milk ranging from 6 to 9ng/mL, to beef meat juice from 2 to 3µg/mL and to pork meat juice from 1 to 2µg/mL after 24 and 48h of cultivation, respectively. We provide the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to Staphylococcus aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety.

Genetic Diversity and Incidence of Virulence-Associated Genes of Arcobacter butzleri and Arcobacter cryaerophilus Isolates from Pork, Beef, and Chicken Meat in Poland.

Incidence of 9 virulence-associated genes and genetic diversity was determined in 79 A. butzleri and 6 A. cryaerophilus isolates from pork, beef, and chicken meat. All A. butzleri isolates harboured the tlyA gene, and most of them carried ciaB, mviN, pldA, cadF, and cj1349 genes. ciaB was found to occur with higher frequency in poultry if compared with pork (p = 0.0007), while irgA was more frequent in poultry than in beef (p = 0.007). All 6 A. cryaerophilus isolates harboured the ciaB gene, while mviN and tlyA were detected in 3 out of these isolates. Only one isolate carried the cadF gene. All beef-derived A. cryaerophilus isolates carried ciaB, mviN, and tlyA genes. A. cryaerophilus isolates from chicken meat harboured ciaB gene only. The pork-derived isolate harboured ciaB and cadF genes. Seventy-four genotypes were distinguished within 79 A. butzleri isolates. Nineteen from 21 isolates derived from beef and pork were found to be closely related to A. butzleri from chicken meat. Each of the 6 A. cryaerophilus isolates was found to have unique genotype. We demonstrated that closely related genotypes can spread within pork, beef, and chicken meat populations of A. butzleri but not A. cryaerophilus.

Population Structure and Oxacillin Resistance of Staphylococcus aureus from Pigs and Pork Meat in South-West of Poland.

The genotypes and oxacillin resistance of 420 S. aureus isolates from pigs (n = 203) and pork (n = 217) were analyzed. Among 18 spa types detected in S. aureus from pig t011, t021, t034, t091, t318, t337, and t1334 were the most frequent. Among 30 spa types found in S. aureus isolates from pork t084, t091, t499, t4309, t12954, and t13074 were dominant. The animal S. aureus isolates were clustered into MLST clonal complexes CC7, CC9, CC15, CC30, and CC398 and meat-derived isolates to CC1, CC7, and CC15. Thirty-six MRSA were isolated exclusively from pigs. All MRSA were classified to spa t011 SCCmecV. BORSA phenotype was found in 14% S. aureus isolates from pigs and 10% isolates from pork meat. spa t034 dominated among BORSA from pigs and t091 among meat-derived BORSA. This is the first report on spa types and oxacillin resistance of S. aureus strains from pigs and pork meat in Poland. Besides S. aureus CC9, CC30, and CC398 known to be distributed in pigs, the occurrence of genotype belonging to CC7 in this species has been reported for the first time. To our knowledge it is also the first report concerning CC398 BORSA isolates from pigs and pork meat.

Production of staphylococcal enterotoxin R by Staphylococcus aureus strains.

Staphylococcal enterotoxin D and R (SED, SER) production was determined in 24 S. aureus strains harboring sed gene. Seven of them were not able to produce SED as evidenced by enzyme-linked immunosorbent assay and Western blotting. Sequencing revealed that all these strains harbor a variant of sed gene. Expression of SER was detectable in 22 out of 24 isolates, with variance in productivity ranging from ∼40 to 450 ng/mL. Out of the seven isolates not able to produce SED, three produced high amounts of SER (249-396 ng/mL), two produced less than 200 ng/mL of SER, and two were found to express no detectable amount of SER. Three of those were assigned to spa type t1677 with two being of agr type III and one of agr type I. One strain was t084, agr type II, one t603, agr type II, one 2920, agr type III, one t2920, agr type III, and one t5160, agr type I. Because conventional screening procedures involve only the detection of classical enterotoxins in food, the isolates not able to produce SED presented in this study could pose a threat to human health due to SER production.

Temporal expression of staphylococcal enterotoxin h in comparison with accessory gene regulator-dependent and -independent enterotoxins.

Using sandwich enzyme-linked immunosorbent assay (ELISA), the production of staphylococcal enterotoxin (SE) H was determined in 22 Staphylococcus aureus isolates bearing the seh gene. Samples of supernatants were taken at four time points corresponding to exponential phase (optical density at 600 nm [OD(600)] 0.3 to 0.6), late exponential phase (OD(600) 2 to 4), early stationary phase (OD(600) 4 to 6), and late stationary phase (OD(600) 7 to 12). In four isolates, SEH was detectable at a very low level at the first time point. In 18 isolates, the earliest SEH production was detected in the late exponential phase. For all isolates, there was an increase of SEH concentration with time. Western blot analysis revealed that SEH production, similar to SEA, started in the early exponential phase (OD(600) ∼ 0.5). Isolates with high SEH productivity, as measured by ELISA, demonstrated a higher seh transcription as well. sec transcription was induced in the stationary phase. An induction in the sea transcript was observed during mid- to late exponential phase. Expression profile of seh was similar to that of sea. We showed that the seh expression profile is similar to that of Agr-independent sea and not to that of Agr-dependent sec genes. SEH can be effectively expressed at low bacterial counts, meaning that even in an environment not favorable for S. aureus growth, seh-bearing strains can pose a risk for food safety.

Genotypes, antibiotic resistance, and virulence factors of staphylococci from ready-to-eat food.

Sixty-seven staphylococcal isolates belonging to 12 species were obtained from 70 ready-to-eat food products. Staphylococcus aureus (n=25), and Staphylococcus epidermidis (n=13) were dominant. Susceptibility to penicillin, oxacillin, tetracycline, clindamycin, gentamicin, erythromycin, ciprofloxacin, and vancomycin was determined. All investigated S. aureus isolates were resistant to at least one antibiotic, and fifteen isolates were resistant to four and more antibiotics. Thirty-eight coagulase-negative staphylococci (CNS) isolates were resistant to at least one antibiotic, and seventeen to four and more antibiotics. Fifteen CNS isolates were mecA positive, and grew in the presence of 6 µg/mL oxacillin. All S. aureus isolates were mecA-negative. Arginine catabolic mobile element (ACME) was found in seven S. epidermidis isolates. Five S. epidermidis isolates harbored ica operon, ACME and were able to form biofilm. Three of them also possessed IS256 element and were mecA-positive. The expression of icaA gene was comparable in five ica-positive S. epidermidis isolates. One of six mecA positive S. epidermidis isolates was classified as sequence type (ST)155, one as ST110, and two as ST88. Two methicillin-resistant Staphylococcus epidermis (MRSE) belonged to new STs, that is, ST362, and ST363. Enterotoxin genes were found in 92% of S. aureus isolates. No enterotoxin gene was detected in analyzed CNS population. We show that ready-to-eat products are an important source of antibiotic-resistant CNS and potentially virulent strains of S. epidermidis, including genotypes undistinguishable from hospital-adapted clones.

Characterization of borderline oxacillin-resistant Staphylococcus aureus isolated from food of animal origin.

In this study, the molecular characteristics of food-derived oxacillin-resistant Staphylococcus aureus were determined. Eight borderline oxacillin-resistant strains with MICs of 2 to 4 microg/ml were identified from 132 S. aureus isolates of food origin. One of the two isolates with a MIC of 4 microg/ml was methicillin-resistant determinant (mecA) gene positive, and the other six with MICs of 2 microg/ml were mecA negative. The mecA-positive isolate was classified as sequence type (ST)228, staphylococcal protein A (spa) type t041, and carried the staphylococcal cassette chromosome mec type I element. Two borderline oxacillin-resistant strains were classified as spa t008 and ST8, and the remaining five as spa t164 and ST20. The mecA-positive strain and four borderline oxacillin-resistant strains were found enterotoxigenic. The enterotoxin genes detected in these strains included selp, egc1, and sed-sej-selr. The borderline-resistant S. aureus isolates from a manually handled product, i.e., minced pork, were shown genetically related to strains associated with human infections. This suggests that humans can be considered as a source of contamination of this food with oxacillin-resistant S. aureus strains. The genotypes of the investigated milk borderline-resistant isolates were shown to occur not only in cows, but also in humans. Since manual handling is reduced in raw milk production, a human origin of S. aureus seems unlikely. Because knowledge of the genotypes of animal staphylococci is limited, more research is needed to address the question of the origin of antibiotic-resistant S. aureus strains in food.

Genotypes and enterotoxin gene content of S. aureus isolates from poultry.

Little is still known about the genotypes and prevalence of enterotoxin genes in animal-derived Staphylococcus aureus strains. In this study, spa type, the presence of known enterotoxin genes, and mecA gene was determined in 42 S. aureus isolates from poultry. All these isolates were classified as mecA-negative. t002, found in 19 staphylococci, was the most prevalent spa type in the studied population. t034 was found in 11 isolates. MLST performed on three t034 isolates confirmed its attachment to ST398. A strong association between the CC5 genetic background and the egc gene grouping in staphylococci of animal origin was confirmed, since all 23 isolates with t002, t214, and t010, belonging to CC5, contained egc1. No enterotoxin genes were found in 15 S. aureus isolates. In this population the most prevalent genotype was t034, found in 11 isolates. It was demonstrated that MSSA strains with the t034 ST398 genetic background also occur in poultry. This may imply, that ST398-type strains occur in a wider range of livestock species than previously believed.

Enterotoxin gene content in Staphylococcus aureus from the human intestinal tract.

Enterotoxigenic Staphylococcus aureus has been associated with staphylococcal food poisoning, which in a number of patients is accompanied by gastroenteritis. It has also been found to persist asymptomatically in the human intestinal tract, being considered one of the sources of pathogen transmission to manually handled food. However, very little is known about the incidence and enterotoxigenicity of intestinal S. aureus not associated with enteritis. There are practically no data on the frequency of some enterotoxin genes in intestinal S. aureus. Six thousand six hundred and twenty-one fecal swabs from 6-month- to 8-year-old children were analyzed for S. aureus. Growth of S. aureus was found in 347 samples. Two hundred and eight S. aureus isolates (4.2% of 4900 swabs) were from patients with sporadic enteritis and 139 isolates (8% of 1721 swabs) from patients who did not develop diarrhea during hospitalization. The genes encoding 16 members of the enterotoxin family (sea-see, seg-selp, and selu) and tst were present in 174 (83%) S. aureus isolates accompanying enteritis and in 101 (72%) isolates derived from patients with no enteritis symptoms. The genes of the classical enterotoxins (sea-see) and tst were present in 56% and 59% of the enteritis-associated and nonenteritic isolates, respectively.

Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food.

Extensive analysis of the Staphylococcus aureus genome has allowed the identification of new genes encoding enterotoxin-like superantigens (SEls). Some of these are thought to be involved in staphylococcal food poisoning, while others do not elicit any emetic effect. The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity. In this paper the distribution of the genes coding for enterotoxin-like superantigens in S. aureus isolated from food was studied. Fifty isolates of S. aureus were examined and 27 were shown to be enterotoxigenic. Only 9 of the 27 strains carried genes encoding enterotoxins SEA-SEE. In 18 SEA-SEE-negative strains the presence of newly described enterotoxin genes was detected. All SEA-SEE-positive strains simultaneously carried genes of new SEls. We show that the gene encoding SElH (staphylococcal enterotoxin-like enterotoxin H) was the most frequently detected (n=14), while genes encoding SElI together with SElG accompanied by the other genes of the egc locus were detected in three strains. We also detected the presence of three less investigated genes: sep, sel, and sek. These genes were present in eight, two, and one isolate, respectively. In one strain, sep was accompanied by genes of other SEls, while in the remaining seven it was the only enterotoxin-like gene detected. The high prevalence of newly discovered enterotoxin genes, including the genes encoding emetic toxins, was demonstrated in food-derived strains. This supports the need for additional work on its role in food poisoning and, alternatively, to monitor its presence in S. aureus isolated from food. Our results suggest that yet unknown genetic elements encoding enterotoxin genes may exist.