Toxic Shock Syndrome Toxin Research Articles

Characterisation of antibiotic resistant Staphylococcus species and molecular identification of mecA and toxic shock syndrome toxin-1 (tsst-1) genes of Staphylococcus aureus isolates from cows' milk

Staphylococcus aureus (S. aureus) is one of the most important causes of foodborne illness. Due to the interest of people to use traditional dairy products, especially milk, in the current descriptive study the frequency of S. aureus, antibiotic resistance pattern and presence of toxic shock syndrome toxin-1 gene (tsst-1 gene) in these isolated strains was investigated. Thirty-nine strains (26%) of S. aureus were identified from the samples studied, of which 6 (15.38%) were resistant to methicillin, and 9 (23.07%) were positive for mecA gene. Also, 31 (20.7%) coagulase-negative staphylococci isolates were identified. Out of the 39 strains of S. aureus, the presence of tsst gene was detected in 1 strain (2.56%). Therefore, S. aureus contamination indicates a risk to public health and can be significantly controlled using a high level of hygiene and professional training in the handling of raw milk.

Occurrence, Antibiotic Susceptibility, Biofilm Formation and Molecular Characterization of Staphylococcus aureus Isolated from Raw Shrimp in China.

The aim of this study was to determine the prevalence and characterization of Staphylococcus aureus isolated from 145 shrimp samples from 39 cities in China. The results show that 41 samples (28%) from 24 cities were positive, and most of the positive samples (39/41, 95.1%) were less than 110 MPN/g. Antimicrobial susceptibility testing showed that only seven isolates were susceptible to all 24 antibiotics, whereas 65.1% were multidrug-resistant. Antibiotic resistance genes that confer resistance to β-lactams, aminoglycosides, tetracycline, macrolides, lincosamides and streptogramin B (MLSB), trimethoprim, fosfomycin and streptothricin antibiotics were detected. All S. aureus isolates had the ability to produce biofilm and harbored most of the biofilm-related genes. Genes encoding one or more of the important virulence factors staphylococcal enterotoxins (sea, seb and sec), toxic shock syndrome toxin 1 (tsst-1) and Panton-Valentine leukocidin (PVL) were detected in 47.6% (30/63) of the S. aureus isolates. Molecular typing showed that ST15-t085 (27.0%, 17/63), ST1-t127 (14.3%, 9/63) and ST188-t189 (11.1%, 7/63) were the dominant genetic types. The finding of this study provides the first comprehensive surveillance on the incidence of S. aureus in raw shrimp in China. Some retained genotypes found in this food have been linked to human infections around the world.

Inhibition of Toxic Shock Syndrome-Associated Staphylococcus aureus by Probiotic Lactobacilli.

Staphylococcus aureus is a human pathogen with many infections originating on mucosal surfaces. One common group of S. aureus is the USA200 (CC30) clonal group, which produces toxic shock syndrome toxin-1 (TSST-1). Many USA200 infections occur on mucosal surfaces, particularly in the vagina and gastrointestinal tract. This allows these organisms to cause cases of menstrual TSS and enterocolitis. The current study examined the ability of two lactobacilli, Lactobacillus acidophilus strain LA-14 and Lacticaseibacillus rhamnosus strain HN001, for their ability to inhibit the growth of TSST-1 positive S. aureus, the production of TSST-1, and the ability of TSST-1 to induce pro-inflammatory chemokines from human vaginal epithelial cells (HVECs). In competition growth experiments, L. rhamnosus did not affect the growth of TSS S. aureus but did inhibit the production of TSST-1; this effect was partially due to acidification of the growth medium. L. acidophilus was both bactericidal and prevented the production of TSST-1 by S. aureus. This effect appeared to be partially due to acidification of the growth medium, production of H2O2, and production of other antibacterial molecules. When both organisms were incubated with S. aureus, the effect of L. acidophilus LA-14 dominated. In in vitro experiments with HVECs, neither lactobacillus induced significant production of the chemokine interleukin-8, whereas TSST-1 did induce production of the chemokine. When the lactobacilli were incubated with HVECs in the presence of TSST-1, the lactobacilli reduced chemokine production. These data suggest that these two bacteria in probiotics could reduce the incidence of menstrual and enterocolitis-associated TSS. IMPORTANCE Toxic shock syndrome (TSS) Staphylococcus aureus commonly colonize mucosal surfaces, giving them the ability to cause TSS through the action of TSS toxin-1 (TSST-1). This study examined the ability of two probiotic lactobacilli to inhibit S. aureus growth and TSST-1 production, and the reduction of pro-inflammatory chemokine production by TSST-1. Lacticaseibacillus rhamnosus strain HN001 inhibited TSST-1 production due to acid production but did not affect S. aureus growth. Lactobacillus acidophilus strain LA-14 was bactericidal against S. aureus, partially due to acid and H2O2 production, and consequently also inhibited TSST-1 production. Neither lactobacillus induced the production of pro-inflammatory chemokines by human vaginal epithelial cells, and both inhibited chemokine production by TSST-1. These data suggest that the two probiotics could reduce the incidence of mucosa-associated TSS, including menstrual TSS and cases originating as enterocolitis.

Antimicrobial Resistance and Virulence Gene Profile of Clinical Staphylococcus aureus: A Multi-Center Study from Ethiopia.

Staphylococcus aureus causes a wide range of infections from mild skin and soft tissue to severe life-threatening bacteremia. The pathogenicity of S. aureus infections is related to various bacterial surface components and extracellular proteins such as toxic-shock syndrome (TSS) toxin and Panton-Valentine leukocidin (PVL). In this study we determine the antimicrobial resistance of isolated strains and their virulence genes in Ethiopia. A total of 190 archived S. aureus isolates from four Ethiopia Antimicrobial Resistance (AMR) Surveillance sites were analyzed. The identification of S. aureus was done by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF Biotyper) and antimicrobial susceptibility test (AST) was done using VITEK® 2. Multiplex PCR was used to detect mecA, mecC, pvl and spa genes and super-antigens (sea, seb, sec, seh and sej staphylococcal enterotoxins). A total of 172 isolates were confirmed as S. aureus, 9 (5.23%) were methicillin-resistant S. aureus (MRSA) and 163 (94.76%) were methicillin-susceptible S. aureus (MSSA). AST showed that 152 (88.4%) isolates were resistant to penicillin; 90 (52.32%) resistant to trimethoprim-sulfamethoxazole; and 45 (26.16%) resistant to tetracycline. A total of 66 (38.37%) isolates harbored at least one staphylococcal enterotoxin gene and 31 (46.96%) isolates had more than one. The most frequent enterotoxin gene encountered was seb 28 (16.28%). The TSST-1 gene was detected in 23 (13.37%). Presence of staphylococcal enterotoxin gene showed significant association with antibiotic resistance to cefoxitin, benzylpenicillin, oxacillin, erythromycin, clindamycin, tetracycline and SXT. The pvl gene was detected in 102 (59.3%) of isolates. Isolates from patients below 15 years of age showed significantly high numbers of pvl gene (P = 0.02). Presence of sej (P = 0.011) and TSST-1 (P <0.001) genes were associated with the presence of pvl gene. In this study, isolates were highly resistant to oral antibiotics and the pvl, seb, sea and TSST-1 genes were prevalent.

Persistence of the Staphylococcus aureus epidemic European fusidic acid-resistant impetigo clone (EEFIC) in Belgium.

In August 2018, a public health alert was issued in Belgium regarding clusters of impetigo cases caused by the epidemic European fusidic acid-resistant impetigo clone (EEFIC) of Staphylococcus aureus. As a result, the Belgian national reference centre (NRC) was commissioned to update the epidemiology of S. aureus causing community-onset skin and soft tissues infection (CO-SSTI) to assess the proportion of EEFIC among them. For 1 year, Belgian clinical laboratories were asked to send their first three S. aureus isolated from CO-SSTI each month. Isolates were tested for antimicrobial susceptibility to oxacillin, mupirocin and fusidic acid. Resistant isolates were also spa typed and tested for the presence of the genes encoding the Panton-Valentine leucocidin, the toxic shock syndrome toxin and the exfoliatins A and B. MLST clonal complexes were deduced from the spa types. Among the 518 S. aureus strains analysed, 487 (94.0%) were susceptible to oxacillin. Of these, 79 (16.2%) were resistant to fusidic acid, of which 38 (48.1%) belonged to the EEFIC. EEFIC isolates were mostly isolated from young patients with impetigo and showed a seasonal late summer peak. These results suggest the persistence of EEFIC in Belgium. Furthermore, its prevalence may lead to reconsideration of the treatment guidelines for impetigo.

Methicillin-Resistant Staphylococcusaureus Strains Isolated from Burned Patients in a Tunisian Hospital: Molecular Typing, Virulence Genes, and Antimicrobial Resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. Their spread poses a serious public health problem worldwide. Nevertheless, in Tunisia and other African countries, very little molecular typing data on MRSA strains is currently available. In our study, a total of 64 MRSA isolates were isolated from clinical samples collected from burned patients hospitalized in the Traumatology and Burns Center of Ben Arous in Tunisia. The identification of the collection was based on conventional methods (phenotypic and molecular characterization). The characterization of the genetic support for methicillin resistance was performed by amplification of the mecA gene by polymerase chain reaction (PCR), which revealed that 78.12% of S. aureus harbors the gene. The resistance of all the collection to different antibiotic families was studied. Indeed, the analysis of strain antibiotic susceptibility confirmed their multi-resistant phenotype, with high resistance to ciprofloxacin, gentamicin, penicillin, erythromycin, and tetracycline. The resistance to the last three antibiotics was conferred by the blaZ gene (73.43%), the erm(C) gene (1.56%), the msr(A) gene (6.25%), and tet(M) gene (7.81%), respectively. The clonal diversity of these strains was studied by molecular typing of the accessory gene regulator (agr) system, characterization of the SCCmec type, and spa-typing. The results revealed the prevalence of agr types II and III groups, the SCCmec type III and II cassettes, and the dominance of spa type t233. The characterization of the eight enterotoxins genes, the Panton-Valentine leukocidin and the toxic shock syndrome toxin, was determined by PCR. The percentage of virulence genes detected was for enterotoxins (55%), tst (71.88%), leukocidin E/D (79.69%), and pvl (1.56%) factors. Furthermore, our results revealed that the majority of the strains harbor IEC complex genes (94%) with different types. Our findings highlighted the emergence of MRSA strains with a wide variety of toxins, leukocidin associated with resistance genes, and specific genetic determinants, which could constitute a risk of their spread in hospitals and the environment and complicate infection treatment.

ANTIMICROBIAL, PHYTOCHEMICAL AND TOXICOLOGICAL PROFILE OF ESSENTIAL OIL EXTRACTED FROM PANDANUS ODORIFER WITH AN AVAILABLE SIMULATION STUDY

Plant-derived essential oils are natural sources of antimicrobial and pharmaceutical agents besides their broad spectrum of use in the cosmetic, food, and perfume industries. The essential oil from the flowers of P. odorifer was extracted using the hydro distillation method. The major compound from the essential oil was identified by high-resolution mass spectroscopy (HRMS) analysis the identified phytochemicals include p-benzoquinone, 2-phenethyl alcohol, p-cymene, 2-phenethyl methyl either alpha-Terpineol, Psoralen, Isoplumbagin, Genipin, Artemidiol, Pinocembrin, (-)-Glycinol and, Iprobenfos. Two of its major phytochemicals,2-phenethyl methyl ether (PEME) and alpha-terpineol identified through HRMS were procured from Sigma-Aldrich. The antimicrobial properties of the PEME, essential oil, and terpineol was determined using agar well diffusion and micro broth dilution method against four clinically isolated multidrug-resistant (MDR) pathogenic bacteria viz. Staphylococcus aureus, Streptococcus pyogenes, Acinetobacter baumanni, and Pseudomonas aeruginosa. PEME and alpha-terpineol exhibited significant antibacterial activity against all the isolated pathogenic MDR bacteria. No pathological abnormalities were noticed even if it was provided at the highest treatment dose of 800 mg/kg body weight. Further In silico study of molecular interaction analyses have been performed for the proteins Extracellular Adhesion Protein (EAP), Protein A, and Toxic Shock Syndrome Toxin (TSST) of Staphylococcus aureus with the ligand 2-methoxyethyl benzene which shows better interaction with the target. This study validated the robust antibacterial activity of the essential oil of P. odorifer against both gram-positive and negative bacteria. Further, it is validated that the antibacterial activity was mainly because of the presence of two major phytochemicals, PEME and terpineol identified by HRMS analysis.

Staphylococcus aureus in the Processing Environment of Cured Meat Products.

The presence of Staphylococcus aureus in six dry-cured meat-processing facilities was investigated. S. aureus was detected in 3.8% of surfaces from five facilities. The occurrence was clearly higher during processing (4.8%) than after cleaning and disinfection (1.4%). Thirty-eight isolates were typified by PFGE and MLST. Eleven sequence types (STs) were defined by MLST. ST30 (32%) and ST12 (24%) were the most abundant. Enterotoxin genes were detected in 53% of isolates. The enterotoxin A gene (sea) was present in all ST30 isolates, seb in one ST1 isolate, and sec in two ST45 isolates. Sixteen isolates harbored the enterotoxin gene cluster (egc) with four variations in the sequence. The toxic shock syndrome toxin gene (tst) was detected in 82% of isolates. Regarding antimicrobial resistance, twelve strains were susceptible to all the antibiotics tested (31.6%). However, 15.8% were resistant to three or more antimicrobials and, therefore, multidrug-resistant. Our results showed that in general, efficient cleaning and disinfection procedures were applied. Nonetheless, the presence of S. aureus with virulence determinants and resistance to antimicrobials, particularly multidrug-resistant MRSA ST398 strains, might represent a potential health hazard for consumers.

Molecular identification, antimicrobial resistance and virulence gene profiling of Staphylococcus spp. associated with bovine sub-clinical mastitis in Bangladesh

This study was conducted to investigate the diversity and antimicrobial resistance profiling of Staphylococcus species causing sub-clinical mastitis (SCM) in dairy herds in Bangladesh as well as putative risk factors associated with the infections. Individual quarter milk samples were collected from a total of 284 lactating cows from 30 dairy farms were screened by means of California mastitis test; 178 (62.7%) of them had at least of quarter affected by SCM. After conventional microbiological isolation procedures, PCR tests were used for Staphylococcus species identification and detection of antimicrobial resistance and virulence genes. S. chromogenes (65.7%) was the most predominant species followed by, S. epidermidis (20.2%), S. haemolyticus (19.1%), S. aureus (15.7%), and S. sciuri (5.6%). High levels of antimicrobial resistance to ampicillin and amoxicillin/clavulanic acid were observed in S. aureus (82.1% and 75%) and S. sciuri (80% and 70%), while resistance to cefepime was markedly higher in S. chromogenes (95.7%), S. haemolyticus (94.1%), and S. epidermidis (97.2%). Multidrug resistance isolates were identified in all five species. The mecA gene was detected in S. aureus (32.1%) and S. chromogenes (5.98%). In addition, 20% S. sciuri and 17.7% S. haemolyticus carried the cytotoxin (pvl) gene, while 14.3% S. aureus harbored the toxic shock syndrome toxin (tst) gene. Multivariable logistic regression analysis identified “Old aged” (OR [CI]: 3.5 [1–12.4]); “Early stage of lactation” (OR [CI]: 3.4 [1.2–9.7]) and, “Firm udder condition” (OR [CI]: 4.2 [1.2–14.6]) as risk factors associated with SCM caused by S. aureus, S. chromogenes, and S. haemolyticus, respectively. Moreover, “Use of antimicrobials” (OR [CI]: 10.4 [3.4–32.1] and “History of previous clinical mastitis” (OR [CI]: 4.9 [1.2–19.7] for the carriage of methicillin-resistant Staphylococcus spp.

Methicillin-Resistant Staphylococcus aureus Isolated from Nasal Swab: Profiling of Some Virulence Genes

Background: Healthcare workers (HCWs) carrying Staphylococcus aureus may act as a source of infection. This study was carried out to investigate the prevalence of MRSA and the genetic make-up of S. aureus nasal colonizers among HCWs. Methodology: Nasal swabs were obtained from 202 HCWs and 205 NHCWs and molecular characterization of identified methicillin resistant S. aureus isolates was achieved using polymerase chain reaction technique for leukocidin toxin, pyrogenic toxins and toxic shock syndrome toxin. Results: seventy-four HCWs (36.6%) were colonized with S. aureus (MSSA = 51; MRSA = 23). The overall MRSA colonization rate was 11.3 % (n/N=23/202) and 31.1% (n/N=23/74) of those colonized with S. aureus harbored MRSA. There was statistically significant difference in S. aureus colonization rate between HCWs and NHCWs (P = 0.003). Most of the 74 isolates contained at least one of the 13 enterotoxin genes. The most prevalent enterotoxin was seg (63.5%), followed by sei (55.4%), sea (45.9%), seb (36.4%), see (9.4%) and sej (5.4%). Considerable genetic diversity was detected. The present study demonstrated a relatively high rate of tst (48.6%), eta (44.5%) and luk (31%) as well as the wide occurence of enterotoxin genes among colonizing S. aureus. The highest combination between positive mecA isolates and other virulence genes was found in mecA-seg (41.8%), mecA-sei (40.5%), and mecA-sea-seb-seg-sei (24.3%). Conclusions: These findings provide insight into the virulence genes of S. aureus nasal carriage among the HCWs and NHCWs in Gaza Strip.

アトピー性皮膚炎を基礎疾患とし肺動脈弁感染性心内膜炎を発症した1例(A case of isolated pulmonary valve infective endocarditis with atopic dermatitis)

アトピー性皮膚炎（atopic dermatitis: AD）は慢性皮膚炎であり，感染性心内膜炎（infective endocarditis: IE）など重症感染症の合併報告はコントロール不良なAD例であり，さらに肺動脈弁の孤立性IE報告例はない。症例は50歳代の男性，既往はコントロール良好な小児期からのADあり。手指消毒剤により指尖部乾燥が増悪していた。入院5日前に発熱し近医から抗菌薬を処方されたが，入院前日に体動困難となり他院へ搬送され敗血症の診断で入院した。翌日精査加療目的に当院へ転院した。来院時意識清明，呼吸循環動態は保たれていたが，40℃の発熱と右肘の発赤腫脹，前腕の有痛性浮腫性紅斑を認めた。血液検査で血小板が3.6万に低下しており敗血症と診断し広域抗菌薬を投与し救命センターに入室とした。入院後右肘穿刺液，左前腕筋間の膿瘍，血液培養からメチシリン感受性ブドウ球菌が検出されセファゾリンにて治療を継続した。入院15日目まで複数回の経胸壁・食道心臓超音波検査とCT検査では疣贅を認めず，ガリウムシンチグラフィーでも有意な所見を認めなかったが，入院33日目に施行した造影CT検査にて肺動脈弁の疣贅を確認した。血液培養陰性化後6週間抗菌薬を投与し，入院51日目に退院した。ADの既往のある菌血症症例は肺動脈弁領域のIEの可能性も念頭に繰り返し評価し考慮する必要がある。 Atopic dermatitis (AD) is a common chronic dermatitis, and severe infectious diseases triggered by AD have been reported. We describe a 58–year–old Japanese male with AD who developed pulmonary–valve infective endocarditis. The dryness of his fingertips had been exacerbated by hand sanitizer. Five days before his admission, he had a fever and was prescribed antibiotics. Two days before admission, he was admitted to a general hospital but was then transferred to our hospital for treatment. On admission, he had a fever of 40℃ and painful edematous erythema of his forearm, but his respiratory and circulatory dynamics were maintained. Platelets had dropped to 36,000, and sepsis was diagnosed. Methicillin–sensitive staphylococci were detected in the right elbow’s fluid and blood culture, and we changed the antibiotics to cefazolin. Enhanced CAT scanning and cardiac ultrasound showed no abnormalities until the 15th day of hospitalization. On the 33rd day of hospitalization, vegetation of the pulmonary valve was confirmed. Antibiotics were administered for 6 weeks, and the patient recovered. Clinicians should be aware that patients with a history of AD may develop serious infections even if their AD is well controlled. アトピー性皮膚炎（atopic dermatitis: AD）は日常診療でよく遭遇する疾患である 1が，皮膚の保護機能破綻から血流感染を起こし，感染性心内膜炎（infective endocarditis: IE）などの重篤な感染症を起こすことがあるとされている 2。過去にIEを合併した報告はADのコントロールが不良なものがほとんどであり，さらに肺動脈弁に孤立性にIEを認めた報告例はない。今回ADの増悪を契機として，孤立性肺動脈弁IEを合併した1例を経験し，ADの既往がある患者の診療に関して示唆を与えるため報告する。 なお，本論文は症例報告のため倫理委員会の承諾を得る必要はなく，個人情報保護法に基づき匿名化している。また，患者より論文の出版に関する同意を得ている。 患 者：50歳代の男性 既往歴：小児期にADを発症したが保湿剤のみでコントロールは良好であった。外傷・直近の歯科治療歴はない。 現病歴：入院5日前に38℃台の発熱を認め近医を受診，肺炎を疑われクラリスロマイシンを処方された。入院前日に呼吸困難を自覚し体動困難となり他院へ救急搬送された。前医搬送時，40度の発熱，咽頭発赤，右肘に痂疲を伴う発赤および腫脹，四肢の湿疹を認めた。重症軟部組織感染の可能性が考えられ当院に加療目的に転送された。 来院時現症：意識レベル；Glasgow coma scale（GCS）E4V5M6，体温；40℃（腋窩），血圧；110/52mmHg，心拍数；106/分（整），呼吸数；24/分（整），経皮的酸素飽和度；99%（室内気），瞳孔；2.0mm/2.0mm，対光反射；迅速/迅速，眼球結膜出血斑なし，呼吸音；清，心雑音なし，腹部平坦・軟・圧痛なし。皮膚；右肘伸側に痂疲形成を伴う発赤腫脹と，両側前腕に直径2～3cm大の有痛性の浮腫性紅斑周囲には粟粒大の紅色丘疹を認めた。指尖部に一部表皮剥脱・剥離を認めた。 来院時検査所見： ＜血液検査＞ 血算；WBC 4,500/µL，RBC 278万/µL，Hb 10.7g/dL，Ht 30.5%，PLT 3.6万/µL，生化学；Na 135mmol/L，K 3.7mmol/L，Cl 103mmol/L，Ca 7.5mg/dL，IP 1.7mg/dL，Mg 1.4mg/dL，Glu 115mg/dL，ALB 1.7g/dL，BUN 14.8mg/dL，CRE 0.85，T–Bil0.4mg/dL，γ–GTP 57IU/L，AST 43IU/L，ALT 32IU/L，CK 402IU/L，CRP 29.12mg/dL，凝固；PT 79.8%，PT比1.14，PT–INR 1.16，APTT 40.5sec，FDP 4.9µg/mL ＜動脈血液ガス分析（室内気）＞ pH 7.508，pCO2 25.7mmHg，pO2 78.1mmHg，HCO3− 23.4mmol/l，BE −1.2mmol/L，Lac 1.0mmol/L ＜画像所見＞ 胸部レントゲン；肺野に異常陰影を認めない。全身CT；四肢・体幹に膿瘍を疑う所見を認めない。 経過：臨床経過から敗血症を疑いバンコマイシンとメロペネムを投与し集中治療室に入室とした。入院2日目，経食道心臓超音波を施行したが疣贅と弁膜症を疑う所見は認めなかった。血液と右肘伸側皮下の液体貯留部の培養からは黄色ブドウ球菌が検出された（Fig. 1）。 Skin findings. (a) right elbow Day1, (b) left forearm Day1, (c) right forearm Day3, (d) right lower limbs Day3 入院3日目，十分な輸液にもかかわらず平均血圧は50mmHgに低下し，ノルアドレナリンを開始，他の病原体による感染の可能性を考慮しミノサイクリンを追加投与した。右肘部の発赤は改善したが四肢の発赤は拡大し，入院5日目に血液培養からMSSA（methicillin–susceptible Staphylococcus aureus）が検出されたため，抗菌薬をセファゾリンに変更した。さらに毒素性ショック症候群（toxic shock syndrome: TSS）の可能性も考慮しクリンダマイシンを追加投与した。 入院7日目に造影CTを熱源精査目的に施行したが血管内に有意所見は認められなかった。 入院8日目血液培養は陰性となり，体温も解熱傾向かつ紅斑は退色傾向となったが左前腕に紅斑が残存していたため，超音波で観察したところ撓側手根伸筋と腕橈骨筋の筋間に液体貯留を認め，切開・洗浄を実施した。同部位から提出した培養においてもMSSAが検出された。 入院9日目にノルアドレナリンは終了し，TSST–1（toxic shock syndrome toxin–1）陰性を確認したためクリンダマイシンも終了した（Fig. 2）。 The clinical course. Time course of the patient in therapeutic interventions. その後状態は安定していたが37.5℃前後の微熱が継続しており，IEの可能性を考え経胸壁心臓超音波検査を数日おきに繰り返し施行し，入院12日目にCT検査を施行したが有意な所見は認めなかった。入院15日目には経食道心臓超音波検査を施行したが，有意な所見は認めなかった。その後も感染の可能性を念頭に入れ，入院20日目にCT検査，21日目にガリウムシンチグラフィー検査を施行したが有意な所見は認めなかった。精査にて有意な所見は認めなかったが，血液培養が陽性であったことからIEに準じて抗菌薬を投与する方針とした。その後も経胸壁心臓超音波検査や身体所見に変化を認めなかったが，入院33日目に造影CT検査を施行したところ肺動脈弁に疣贅を疑う所見を認めた（Fig. 3）。経食道心臓超音波を実施したところ肺動脈弁に疣贅を認めたため肺動脈弁の孤立性のIEと診断した（Fig. 4）。右心系のIEであり手術は選択せず，血液培養の陰性化した入院8日目から6週間セファゾリンを経静脈的に投与した。 Enhanced CAT–scan. Day33rd. φ8mm vegetation in pulmonary valve. Transesophageal echocardiography. 8×3mm abnormal echo in pulmonary valve’s anterior semilunar cusp. 入院51日目に経胸壁心臓超音波検査を実施し疣贅に著変なく弁破壊も認めないことから，入院54日目に独歩で自宅退院した。 本症例はADの既往がある患者においてIEを肺動脈弁に合併した症例であり，ADの既往がある患者に対する診療において2つの点で示唆を与える。 1つ目はIEの合併についてである。ADを基礎疾患に持つ患者はIE，化膿性脊椎炎，腸腰筋膿瘍などの重篤な感染を合併することがあると報告され 3，慢性的な掻爬による皮膚の破綻が病原体の侵入門戸となる可能性 4に加え，免疫システムとして存在する抗菌ペプチドのcathelicidins（LL–37）とβデフェンシン2（HBD–2）の減少は，LL–37とHBD–2の組合せが黄色ブドウ球菌への相乗的な抗菌活性を持っていることから黄色ブドウ球菌に対する脆弱性 5とコロニー形成に対する感受性を高める役割を果たしている可能性 6が示唆されている。ADにIEを合併した報告例においては，ADが重症でコントロール不良である症例がほとんどである 6, 9, 10, 11, 12, 13, 14が，本症例では頸部・四肢屈側にも苔癬化を認めておらず，ADのコントロールは良好であったと考えられた。また齲歯や注射痕も認めず，当初侵入門戸は判然としなかったが，後に患者が新型コロナウイルス感染症の流行に伴いアルコールを用いて頻回に手指消毒を行うようになり，手指の皮膚に乾燥を自覚していたことと手指の乾燥と亀裂を認めたことから，指尖部の皮膚損傷が侵入門戸となり，同側の前腕，肘に膿瘍形成をし，敗血症に至った可能性が考えられた。一方で下肢にも入院時に紅斑を認めたが，皮下膿瘍や，足趾・爪辺縁含め外傷痕も認められなかった。IEの皮疹として結節性紅斑が特異的との報告もあり 7，紅斑の存在にも留意が必要と考える。 ADでは，炎症が軽快して一見正常に見える皮膚も，組織学的には炎症細胞が残存し，再び炎症を引き起こしやすい状態にあることが多いといわれており 8，さらに手指消毒によりADが増悪する可能性も示唆されている 9ことからADの既往がある患者においては，重篤な感染のリスクがあることを念頭に入れておく必要がある。 2つ目は肺動脈弁にIEを発症したことである。孤立性肺動脈弁IEはIE全体の1.5～2.0%と稀であるとされ 13，さらに過去にADを基礎疾患として肺動脈弁にIEを合併した報告例はない。右心系IEの要因として薬物常習，先天性心疾患，消化管術後などとされている 13が，本症例ではAD以外に特殊な既往歴はなくこれらの発症要因は認めなかった。本症例では，繰り返し施行していた心臓超音波検査において有意な所見は認めず，心不全も合併することなく経過しており，さらにCT検査においても右心系IEに合併するとされる肺化膿症や肺塞栓などの所見は認めず，肺動脈弁単独にIEを発症した理由は判然としなかった。本症例のように原因不明なものは過去の報告では30%あるともされており 14，稀ではあるが肺動脈弁にIEが発生することにも注意が必要であると考える。 本症例では手術を選択しなかったが，欧州心臓病学会会議のガイドラインに基づく右心系IEにおける手術考慮例は，①真菌などの治療抵抗性の病原体が原因である，②適切な抗菌薬治療を行っても7日以上血液培養陽性が続く，③三尖弁の疵贅が20mmを超え，反復性肺塞栓を認める，④内科的治療に対しての反応不良であり重度三尖弁逆流による右心不全を来した場合，とされる。本症例では持続菌血症は8日以内に陰転化しており弁破壊・逆流増悪も認められなかったことや治療に対する反応が良好であり疣贅の増大も認めなかったため手術を選択せず内科的に加療を継続した。 さらに本症例では経食道超音波検査を繰り返し施行したにもかかわらず，初期に疣贅を確認できず肺動脈弁IEと診断したのは入院33日目であった。本症例では熱源の精査目的に施行した造影CT検査により肺動脈弁に疣贅を偶発的に疑い，確定診断に至った。IEの診断においては，経食道心臓超音波検査が優れているとされている 16が，早期の病変描出は困難とされ，臨床的に疑われる場合は3～7日おきに繰り返し施行することが推奨されている 17。本症例においては，入院2日目，15日目に経食道心臓超音波検査を施行していたが有意な所見は認めなかった。後方視的に振り返ると臨床的に疑い，15日目以降も検査を繰り返し施行していればより早期に診断に至った可能性はあるが，皮下・筋膜膿瘍も形成しており全身性の多発小膿瘍形成が菌血症の長期化につながりIEを来した可能性も考慮される。 ADの既往のある菌血症症例においては当初疣贅を確認できなくても，IEの可能性を念頭に入れて評価を丹念に繰り返していく必要がある。 アトピー性皮膚炎の既往がある患者における持続菌血症は血管内病変として肺動脈弁領域も考慮し丹念に評価をする必要がある 利益相反はない。

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains’ sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

The Prevalence and Clinical Characteristics of Multidrug-resistant Hospital-acquired Staphylococcus aureus in Medina, Saudi Arabia

Hospital acquired-Staphylococcus aureus (HA-Staphylococcus aureus), particularly methicillin-resistant Staphylococcus aureus (MRSA), are an important source of nosocomial infections with high morbidity and mortality rates. Few reports showed that infections due to HA-Staphylococcus aureus in Saudi Arabia is increasing, particularly infections attributed to HA-MRSA. The study aimed to explore the prevalence and clinical characteristics of HA-Staphylococcus aureus for the first time in Medina, Saudi Arabia. A total of 1262 clinical samples of hospitalized patients were examined for the presence of Staphylococcus aureus through selective culturing on mannitol salt agar. Vitek Compact System and conventional methods were followed to confirm the isolates. Vitek Compact System tested the antimicrobial susceptibility of isolates whereas the standard PCR was employed to detect the genes encoding antimicrobial resistance (mecA and vanA) and virulence factors (tst, et, and LukS-PV). The overall HA-Staphylococcus aureus prevalence was low (6.58%, n = 1262) of which 84.34% (n = 83) were MRSA. Approximately, 57 samples of the 70 MRSA (81.5%) exhibited a multidrug-resistance (MDR) pattern. All the 83 HA-Staphylococcus aureus isolates were negative for the genes encoding toxic shock syndrome toxin, exfoliative toxin, and Panton-Valentine leukocidin. The study was conducted during the Covid-19 pandemic under partial lockdown, restricted hospitalization, and increased disinfection and infection control measures. Therefore, the low prevalence of HA-Staphylococcus aureus should be carefully interpreted and further multicenter investigations could reveal its true incidence in the city. The high prevalence of MDR HA-MRSA is alarming as it highlights inappropriate antibiotic prescriptions to counter staphylococcal infections. HA-Staphylococcus aureus investigated in this study might lack certain virulence factors. However, their MDR traits and invasive nature could worsen the situation if not properly handled.

Molecular Epidemiology of Community-Acquired Methicillin-Resistant Staphylococcus aureus and Clinical Characteristics of Different Sites of Infection.

Since community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized, the molecular epidemiology of CA-MRSA in China has been diverse. It is unclear whether different sites of CA-MRSA infection differ in antimicrobial resistance and clinical characteristics. The purpose of this study was to identify the molecular types, virulence factors and antimicrobial resistance of CA-MRSA strains and to analyze the clinical characteristics of different sites of CA-MRSA infection. 26 CA-MRSA strains were screened from Beijing Chao-Yang Hospital from 2014 to 2022. SCCmec type, MLST type, spa type, Panton-Valentine leukocidin (PVL), hemolysin α (Hla), phenolic soluble regulatory protein α (PSMα), toxic shock syndrome toxin-1 (TSST-1), and enterotoxin (SE) A to E were detected by PCR and gene sequencing. Antimicrobial susceptibility tests and the clinical features of CA-MRSA infection cases were collected for statistical analysis. The predominant type of CA-MRSA was ST59-t437-IV. New non-epidemic types, SCCmec VII, were also found. PVL was seen in 65.4% of CA-MRSA strains and TSST-1 was only be detected in 3.8% of CA-MRSA strain which caused poor prognosis. There were three types of infections: pneumonia (61.5%), infective endocarditis (7.7%), and skin and soft tissue infections (SSTIs) (30.8%). CA-MRSA pneumonia cases were secondary to influenza infection (37.5%). Patients with CA-MRSA-associated infective endocarditis were more likely to have underlying cardiac diseases. Patients with CA-MRSA-associated SSTIs were more likely to have a history of diabetes mellitus, and strains in this group were more susceptible to erythromycin and clindamycin. ST59-t437-IV was the primary CA-MRSA type in our research and in China. We proposed that TSST-1 might be one of the indicators to predict the severity and prognosis of CA-MRSA infection. Different sites of CA-MRSA infection had difference in antibiotics susceptibility testing and underlying diseases of patients. It could provide a new perspective on treating different types of CA-MRSA infection.

The emerging threat of methicillin-resistant Staphylococcus aureus (MRSA) clone ST22-PT, carrying both Panton-Valentine leucocidin and toxic shock syndrome toxin 1 genes.

Some MRSA strains produce Panton-Valentine leucocidin (PVL) and/or toxic shock syndrome toxin 1 (TSST-1), which are associated with severe infectious diseases. Although PVL- or TSST-1-positive strains have been isolated worldwide, strains carrying both PVL and TSST-1 genes are rare and sporadic. The objective of this study was to characterize these strains from Japan. A total of 6433 MRSA strains isolated in Japan between 2015 and 2021 were analysed. Molecular epidemiological and comparative genomic analyses were conducted on PVL- and TSST-1-positive MRSA strains. A total of 26 strains from 12 healthcare facilities were PVL positive and TSST-1 positive, and all were classified as clonal complex (CC) 22. These strains exhibited similar genetic features to each other and were named as ST22-PT according to a previous report. Twelve and one of the ST22-PT strains were identified in patients with deep-seated skin infections and toxic shock syndrome-like symptoms, which are typical clinical features of PVL-positive and TSST-1-positive Staphylococcus aureus, respectively. Whole-genome comparative analysis revealed that the ST22-PT strains were highly similar to PVL- and TSST-1-positive CC22 strains isolated in several countries. Evaluation of the genome structure showed that ST22-PT possessed ΦSa2 harbouring PVL genes and a unique S. aureus pathogenicity island harbouring the TSST-1 gene. ST22-PT strains have recently emerged from several healthcare facilities in Japan, and ST22-PT-like strains have been identified in several countries. Our report highlights that the risk of international spread of PVL- and TSST-1-positive MRSA clone ST22-PT needs to be further investigated.

Staphylococcal Enterotoxin B and C Mutants and Vaccine Toxoids.

Three mutants individually of both staphylococcal enterotoxins B and C were prepared by site-specific mutagenesis of enterotoxin amino acids that contact host T lymphocyte immune cell receptor sites (N23A, Q210A, and N23A/Q210A); these amino acids are shared between the two enterotoxins, and mutations reduce the interaction with the variable part of the β-chain of the T lymphocyte receptor. The mutant proteins, as expressed in Staphylococcus aureus RN4220, lacked biological toxicity as measured by the loss of (i) stimulation of rabbit splenocyte proliferation, (ii) pyrogenicity, and (iii) the ability to enhance the lethality of endotoxin shock, compared to wild-type enterotoxins. In addition, the mutants were able to vaccinate rabbits against pyrogenicity, the enhancement of endotoxin shock, and lethality in a pneumonia model when animals were challenged with methicillin-resistant S. aureus. Three vaccine injections (one primary and two boosters) protected rabbits for at least 3.5 months postvaccination when challenged with wild-type enterotoxins (last time point tested). These mutant proteins have the potential to function as toxoid vaccines against these two causes of nonmenstrual toxic shock syndrome (TSS). IMPORTANCE Toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxins B and C cause the majority of cases of staphylococcal toxic shock syndrome. Previously, vaccine toxoids of TSST-1 have been prepared. In this study, vaccine toxoids of enterotoxins B and C were prepared. The toxoids lost biological toxicity but were able to vaccinate rabbits against lethal TSS.

Antimicrobial Resistance, SCCmec, Virulence and Genotypes of MRSA in Southern China for 7 Years: Filling the Gap of Molecular Epidemiology.

As the prevalence of Staphylococcus aureus infections is of worldwide concern, phenotype and genotype in prevalent MRSA strains require longitudinal investigation. In this study, the antibiotic resistance, virulence gene acquisition, and molecular type were determined on a large scale of nosocomial S. aureus strains in Southern China during 2009-2015. Bacterial identification and antimicrobial susceptibility to 10 antibiotics were tested by Vitek-2. Virulence genes encoding staphylococcal enterotoxins (SEA, SEB, SEC, SED, and SEE), exfoliative toxins (ETA and ETB), Panton-Valentine leukocidin (PVL), and toxic shock syndrome toxin (TSST) were detected by PCR, with SCCmec typing also conducted by multiplex PCR strategy. Genotypes were discriminated by MLST and spaA typing. MLST was performed by amplification of the internal region of seven housekeeping genes. PCR amplification targeting the spa gene was performed for spa typing. No resistance to vancomycin, linezolid, or quinupristin and increase in the resistance to trimethoprim/sulfamethoxazole (55.5%) were identified. A total of nine SCCmec types and subtypes, thirteen STs clustered into thirteen spa types were identified, with ST239-SCCmec III-t037 presenting the predominant methicillin-resistant S. aureus (MRSA) clone. Typically, SCCmec type IX and ST546 were emergent types in China. Isolates positive for both pvl and tsst genes and for both eta and etb genes were also identified. Important findings in this study include: firstly, we have provided comprehensive knowledge on the molecular epidemiology of MRSA in Southern China which fills the gap since 2006 or 2010 from previous studies. Secondly, we have presented the correlation between virulence factors (four major groups) and genotypes (SCCmec, ST and spa types). Thirdly, we have shown evidence for earliest emergence of type I SCCmec from 2012, type VI from 2009 and type XI from 2012 in MRSA from Southern China.

Multiplex PCR for Detection of Exfoliative Toxins, and Toxic Shock Syndrome Toxin 1 Genes in MRSA Strains of Clinical Staphylococcus aureus Isolates

This research approaches to investigate toxin gene content of the methicillin resistant Staphylococcus aureus strains that are clinically collected from human skin infection cases by performing multiplex polymerase chain reaction (PCR) technique (irrespective of whether the strain produces the toxin or not) and for that a single PCR reaction was employed, two pairs of exfoliative toxin and one pair toxic shock syndrome toxin-1. Amplicon sizes ranged between 93, 226 and 326bp, to facilitate electrophoretic separation. Results of the current study had revealed that the major gene detected was tst found in six isolates. Multiple toxin gene combinations were also observed. This work was conducted at several institutions including Tikrit Teaching Hospital’s lab; college of science biology department labs/ Tikrit University; and the Biotechnology Research Centre/ Al-Nahrain University.

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4+ memory T cells

Severe bacterial or viral infections can induce a state of immune hyperactivation that can culminate in a potentially lethal cytokine storm. The classic example is toxic shock syndrome, a life-threatening complication of Staphylococcus aureus or Streptococcus pyogenes infection, which is driven by potent toxins known as superantigens (SAgs). SAgs are thought to promote immune evasion via the promiscuous activation of T cells, which subsequently become hyporesponsive, and act by cross-linking major histocompatibility complex class II molecules on antigen-presenting cells to particular β-chain variable (TRBV) regions of αβ T cell receptors (TCRs). Although some of these interactions have been defined previously, our knowledge of SAg-responsive TRBV regions is incomplete. In this study, we found that CD4+ and CD8+ T cells expressing TRBV12-3/12-4+ TCRs were highly responsive to streptococcal pyrogenic exotoxin C (SpeC) and toxic shock syndrome toxin-1 (TSST-1). In particular, SpeC and TSST-1 specifically induced effector cytokine production and the upregulation of multiple coinhibitory receptors among TRBV12-3/12-4+ CD4+ and CD8+ memory T cells, and importantly, these biological responses were dependent on human leukocyte antigen (HLA)-DR. Collectively, these data provided evidence of functionally determinative and therapeutically relevant interactions between SpeC and TSST-1 and CD4+ and CD8+ memory T cells expressing TRBV12-3/12-4+ TCRs, mediated via HLA-DR.