Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea

AIMS: To investigate the molecular epidemiological study of Staphylococcus aureus from staphylococcal food poisoning (SFP) incidents in South Korea. METHODS AND RESULTS: Three hundred and thirty-two strains isolated from ten provinces between June 1999 and January 2002 were characterized by staphylococcal enterotoxin genes, toxic shock syndrome toxin 1 (tst) gene, and exfoliative toxin genes. Toxin genotypes were sea-seh (n=197), sea (n=51), sea-seg-sei (n=14), seg-sei (n=10), seb (n=10), seb-sed-seg-sei-sej (n=3), sea-seg-seh-sei (n=1), sea-seb (n=1), sea-sec (n=1), seg-sei plus eta (n=4), and sea-seg-sei plus tst (n=40). Most of the strains could be classified into three clusters of pulsed-field gel electrophoresis (PFGE) types A and B with coagulase type VII and type E with coagulase type IV. Of the ten sequence types (ST), ST1, ST59, and ST30 were frequently showed by multilocus sequence typing. CONCLUSIONS: The strain belonging to PFGE pattern A with sea-seh gene, coagulase VII, and ST1 was the most epidemic clone of SFP incidents in Korea.     

雾化吸入金葡素诱导主动性免疫防护降低兔金黄色葡萄球菌的感染效应

目的探索雾化吸入金葡素(staphylococcin)是否诱导主动性免疫防护以降低兔金黄色葡萄球菌(Staphylococcus aureus)的感染效应。方法将金黄色葡萄球菌感染的新西兰大白兔模型30只(雌雄各半,平均体质量2.45 kg)随机分为对照组、雾化组,每组15只。对照组采用生理盐水进行雾化吸入,雾化组采用生理盐水+金葡素雾化吸入,每天1次,共20 d。ELISA法检测各组兔模型采用干预因素处理前后血清IgG抗体滴度,碱性磷酸酶-抗碱性磷酸酶桥联酶染色法(APAAP法)检测T细胞亚群CD4^+、CD8^+T淋巴细胞比例和CD4^+/CD8^+比值的变化。结果雾化组处理后兔血清IgG抗体滴度显著高于对照组,差异有统计学意义(χ^2=18.9451,P<0.01)。同时,雾化组CD4^+T淋巴细胞比例显著高于对照组(t=2.1340,P<0.01),CD4^+/CD8^+比值也明显高于对照组(t=5.2983,P<0.05)。结论雾化吸入金葡素能有效诱导金黄色葡萄球菌感染的兔产生主动性免疫防护,降低金黄色葡萄球菌感染的效应。     

The profiles of enterotoxin genes in Staphylococcus aureus from nasal carriers

AIMS: To evaluate the occurrence of enterotoxin genes in Staphylococcus aureus recovered from nasal carriers. METHODS AND RESULTS: Eighty S. aureus strains were tested for the presence of 17 new enterotoxin genes using multiplex-PCR. Sixty-one isolates were found to carry enterotoxin genes. The majority of the enterotoxigenic isolates carried enterotoxin gene cluster (egc) genes, namely seg, sei, sem, sen and seo. The egc type containing the seu gene was found in 19 of the 47 isolates with egc-like genes. Interestingly, no seu-containing egc coexisted with sec and sel, as was the case for a considerable portion of the isolates carrying a seu-negative egc. The tst gene was detected in two isolates carrying sec and sel only and in eight isolates carrying seu, but not in the isolates containing the seu-negative egc type. CONCLUSIONS: The genes forming an egc were found to be predominant in S. aureus from nasal carriers. The coexistence of a seu-positive egc with tst in contrast to an egc lacking the seu gene apparently is not associated with the presence of tst and can reflect a difference between these gene groupings. SIGNIFICANCE AND IMPACT OF THE STUDY: The egc types carried by the analysed isolates seem to have an influence on the distribution of other genes located on staphylococcal pathogenicity islands, which may modulate the repertoire of virulence factors carried by a single S. aureus strain.     

Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity

Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 μg ml^?1) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms

A novel benzimidazole molecule that was identified in a small‐molecule screen and is known as antibiofilm compound 1 (ABC‐1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC‐1 was tested in various biofilm‐forming strains of S. aureus. It was demonstrated that ABC‐1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall‐associated protein dependent or cell wall‐ associated extracellular DNA (eDNA). Of note, ABC‐1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC‐1 treated strains, implying that ABC‐1 affects not only SpA but also other factors. Indeed, ABC‐1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC‐1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.     

冬凌草甲素对金黄色葡萄球菌生物膜的抑制机制

【背景】金黄色葡萄球菌是一种常见的食源性致病菌,易在食品及加工器具表面形成生物膜,引起食品腐败和疾病的传播,威胁食品安全。【目的】研究冬凌草甲素抑制金黄色葡萄球菌生物膜形成的作用机制。【方法】使用结晶紫染色法和扫描电镜观察冬凌草甲素对金黄色葡萄球菌生物膜形成的抑制作用,刚果红平板法定性检测冬凌草甲素对细胞间多糖黏附素(polysaccharideintercellular adhesion,PIA)合成的影响,分光光度法测定冬凌草甲素对供试菌株胞外DNA (eDNA)释放量的影响,RT-PCR技术检测冬凌草甲素对供试菌株ica A、cid A、agr A和sar A基因表达量的影响。【结果】冬凌草甲素对金黄色葡萄球菌生物膜形成有较强的抑制作用;冬凌草甲素能显著抑制PIA的合成,且呈浓度剂量依赖;冬凌草甲素能抑制供试菌株e DNA的释放量,其中1/4最小抑菌浓度(minimum inhibitory concentration,MIC)的冬凌草甲素作用金黄色葡萄球菌16 h后,与对照组相比,e DNA的释放量降低了48.62%;冬凌草甲素可显著抑制金黄色葡萄球菌生物膜形成相关基因的表达,其中1/2MIC的冬凌草甲素作用金黄色葡萄球菌16 h后,ica A、cid A、agr A和sar A基因的表达量分别比对照降低了91.6%、94.7%、77.6%和70.4%。【结论】冬凌草甲素通过抑制ica A和cid A基因的表达,影响PIA的合成和eDNA的释放,进而干预生物膜的形成。     

Interlaboratory proficiency tests for the detection of staphylococcal enterotoxin type A in food

Staphylococcal enterotoxins (SEs) are among the leading causes of food intoxications, affecting consumer health even in nanogram (ng) amounts. In the European Union, certain food safety criteria are specified, including the absence of SEs in cheeses, milk powder and whey powder. Until 2019, the analytical reference method used was the European Screening Method, which was replaced by EN ISO 19020. For the official laboratories involved in food control, the German Reference Laboratory for coagulase-positive staphylococci including Staphylococcus aureus organized three interlaboratory proficiency tests (ILPTs) to detect SE type A in food during the years 2013–2018. The selected food products (cream cheese and vanilla pudding) were successfully tested beforehand with regard to easy handling, homogeneity and stability of the added toxin. In 2013, ILPT participants overall were not competent in detecting SE type A in food. The following factors were identified to improve the performance: (i) concentration of sample extract using dialysis; (ii) selection of a sensitive detection kit; and (iii) proper sample handling. By taking these factors into account and instructing and training the laboratories, their competence greatly improved. In 2018, all performance criteria (specificity, sensitivity and accuracy) were >90%, even at very low concentrations of SE type A of approximately 0·01 ng g^–1 food.     

Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus

AIMS: This paper reports on a new putative enterotoxin SEU encoded by the enterotoxin gene cluster egc from Staphylococcus aureus and on a real-time polymerase chain reaction (PCR) assay for detecting the seu gene. METHODS AND RESULTS: PCR and sequencing revealed a new putative enterotoxin SEU encoded by some egc clusters. The seu gene resulted from sequence divergence in the psient1 and psient2 pseudogenes previously described in the egc cluster (Jarraud et al. [2001] Journal of Immunology166, 669). The presence of the seu gene was investigated in a collection of S. aureus strains by conventional PCR and by a specific 5'-nuclease PCR assay. Among the 24 strains harbouring the egc cluster, four tested positive for the seu gene. CONCLUSIONS: The existence of the seu gene adds to the number of newly described se genes and underlines the need for a better understanding of their role in the pathogenesis of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: A thorough study of the seu gene should provide further insight into the phylogenetics of the staphylococcal enterotoxins.     

Diversity of Staphylococcus aureus enterotoxin types within single samples of raw milk and raw milk products

AIM: To find out if testing of up to 10 Staphylococcus aureus isolates from each sample from raw milk and raw milk products for staphylococcal enterotoxin (SE) might increase the chances of identifying potential sources of food intoxication. METHODS AND RESULTS: Altogether 386 S. aureus isolates were tested for the presence of SE by reversed passive latex agglutination (SET-RPLA), and SE genes (se) by a multiplex polymerase chain reaction (PCR). In 18 of 34 (53%) S. aureus positive samples a mixture of SE and/or se positive and negative isolates were identified. Multiplex PCR increased the number of potential SE producing strains, i.e. isolates that harboured se, with 51% among the product and 48% among the raw bovine milk isolates. Examination by pulsed-field gel electrophoresis mostly confirmed clonal similarity among isolates sharing SE/se profile, but did not further differentiate between them. CONCLUSIONS: Isolates of S. aureus collected from one sample may show great diversity in SE production and different plating media seem to suppress or favour different strains of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Several isolates of S. aureus from each sample should be tested for enterotoxin production in cases with typical SE intoxication symptoms with methods that are able to reveal new SE/se.     

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802

AIMS: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products. METHODS AND RESULTS: Specificity and reliability of the PCR detection methods used were ascertained by using nine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seA to seE; seG, seH, seI, seM, seJ, seN and seO) and egc (containing the following sequence of genes: seO, seM, seI, phient1, phient2, seN and seG). Of 109 wild Staphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11 strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference and wild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicate that the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv). CONCLUSIONS: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare. SIGNIFICANCE AND IMPACT OF THE STUDY: During this study it was shown that at least five different egc may exist in S. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.     

Impact of biocides on biofilm formation by methicillin-resistant Staphylococcus aureus (ST239-SCCmecIII) isolates

Procedures of sterilization and disinfection are essential to ensure that medical and surgical instruments will not transmit infectious pathogens to patients. In the present paper, we tested the residual effect of these compounds on biofilm formation and its efficiency in disrupting preformed biofilms using methicillin-resistant Staphylococcus aureus (MRSA) isolates of the lineage ST239-SCCmecIII. All compounds examined, except 70% alcohol, caused a significant impairment in biofilm formation with concomitant inhibition of cell growth. Among the compounds examined, 10% povidone-iodine (PVP-I) was the only antiseptic that exhibited more than 90% reduction of both biofilm formation and dispersion. In the group of sterilants and disinfectants, a formulation containing 7% hydrogen peroxide and 0.2% peracetic acid (HP-PA), and sodium hypochlorite with 1% active chlorine (NaOCl) were equally effective.     

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.     

Prevention of Staphylococcus aureus biofilm formation and reduction in established biofilm density using a combination of phage K and modified derivatives

Aims: To investigate the ability of a mixture of phage K and six of its modified derivatives to prevent biofilm formation by Staphylococcus aureus and also to reduce the established biofilm density. Methods and Results: The bioluminescence‐producing Staph. aureus Xen29 strain was used in the study, and incubation of this strain in static microtitre plates at 37°C for 48 h confirmed its strong biofilm‐forming capacity. Subsequently, removal of established biofilms of Staph. aureus Xen29 with the high‐titre phage combination was investigated over time periods of 24 h, 48 h and 72 h. Results suggested that these biofilms were eliminated in a time‐dependant manner, with biofilm biomass reduction significantly greater after 72 h than after 24–48 h. In addition, initial challenge of Staph. aureus Xen29 with the phage cocktail resulted in the complete inhibition of biofilm formation over a 48‐h period with no appearance of phage resistance. Conclusions: In general, our findings demonstrate the potential use of a modified phage combination for the prevention and successful treatment of Staph. aureus biofilms, which are implicated in several antibiotic‐resistant infections. Significance and Impact of the Study: This study highlights the first use of phage K for the successful removal and prevention of biofilms of Staph. aureus.     

Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus

It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC‐737 showed strong co‐aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix‐like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin.     

Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3–10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.     

Effect of cinnamaldehyde on biofilm formation and sarA expression by methicillin‐resistant Staphylococcus aureus

Aims: To investigate the antibiofilm effect of cinnamaldehyde on methicillin‐resistant Staphylococcus aureus (MRSA) and analyse the effect of subminimum inhibitory concentrations (MICs) of cinnamaldehyde on the expression of the biofilm‐related gene sarA. Methods and Results: The MICs and minimum bactericidal concentrations (MBCs) were determined using a microtitre broth dilution method. Biofilm susceptibility was determined using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) staining and colony forming unit (CFU) counting assays. Antibiofilm effects were studied with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). SarA expression was assessed by real‐time PCR. MICs and MBCs were in the range 0·0625–0·5% (v/v). The killing effects were concentration dependent. At a concentration of 5× MIC, all strains in biofilm were decreased to lower than 20% of the control groups. SEM and CLSM images indicated that a 5× MIC concentration of cinnamaldehyde was able to detach and kill existing biofilms. Apart from strain JB‐06, real‐time PCR showed that the expression of sarA of all other strains was decreased upon exposure to sub‐MICs of cinnamaldehyde. Conclusions: These data showed the strong killing effect of cinnamaldehyde against MRSA within biofilms. Significance and Impact of the Study: This study indicated the potential of cinnamaldehyde as an inhibitory agent for use in MRSA biofilm‐related infections.     

Clonal distribution of enterotoxigenic Staphylococcus aureus on handles of handheld shopping baskets in supermarkets

Aims: Shopping carts and handheld shopping baskets in supermarkets are subject to accidental bacterial contamination through contacts with a variety of food. We investigated the prevalence of Staphylococcus aureus on the handles of handheld shopping baskets in four supermarkets distantly located in Osaka district, Japan. Methods and Results: Fifty two strains of Staph. aureus were isolated from 760 basket handles. Among these, six strains were positive for staphylococcal enterotoxin B (SEB) production, representing 12% of total. This SEB producer ratio is considerably higher than among Staph. aureus isolated from nasal swabs of the supermarket workers (2%) and from independently collected clinical specimens (4%). These SEB‐producing Staph. aureus strains from the basket handles are clonal and belong to ST12. Coagulase typing showed that they are in group VII, which is the most common cause of food poisoning in Japan. Biofilm assays indicated that SEB gene (seb)‐positive strains including this clone produced a significantly higher amount of biofilm than seb‐negative strains. Conclusions: The frequent isolation of seb‐positive Staph. aureus on shopping basket handles raises the possibility that they could be a hidden reservoir for Staph. aureus with a potential to cause food poisoning and draws attention to the importance of shopping basket sanitation.