Epidemiological analysis of Group A Streptococcus infections in a hospital in Beijing, China.

Our study aimed to investigate the epidemiological and molecular characteristics of isolates collected from Group A Streptococcus (GAS) infections in children in Beijing China during the year 2019. Emm typing, superantigens, and erythromycin resistance genotypes were determined by PCR. Antimicrobial susceptibility testing was performed as recommended by Clinical Laboratory Standards Institute (CLSI). A total of 271 GAS isolates were collected. Thirteen different emm types, including 31 subtypes, were identified. The most prevalent emm types were emm12 (52.77%), emm1 (36.9%), emm3.1 (2.95%), and emm75.0 (2.95%). Two variant subtypes, STC36.0 and STG840.2, were identified. There was no difference in the portion of emm12 and emm1 isolates in scarlet fever, impetigo, and psoriasis. The majority of superantigens detected were smeZ (94.46%), speC (91.14%), and ssa (74.91%), followed by speH (56.46%), speI (45.76%), speJ (36.9%), and speA (34.32%). More scarlet fever isolates harbored speA (35.6%) and speJ (38.4%), more psoriasis isolates harbored speI (57.9%), and more impetigo isolates harbored ssa (89.7%). Isolates were universally susceptible to penicillin and resistant to erythromycin (94.83%). Moreover, 89.67% erythromycin resistance isolates harbored the ermB gene. The erythromycin resistance rate of the isolates from the three diseases was different. Scarlet fever is the common streptococcal infectious disease in dermatology. Emm12 and emm1 were the most prevalent emm types. The most prevalent superantigens detected were smeZ, spec, and ssa. There is association between diversity of superantigens and disease manifestation. Hence, continuous surveillance of GAS molecular epidemiological characterizations in different diseases is needed.

Panton-Valentine leukocidin and staphylococcal cassette chromosome mec characterization of community acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus (SA) represents one of the most important microorganism that is part of the normal microflora of humans, but in certain conditions can cause very serious infections. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a wide spectrum of nosocomial and community associated infections worldwide. The aim of this study was to determine community acquired MRSA (CA-MRSA), as well as the frequency of Panton-Valentine leukocidin (PVL) genes and staphylococcal cassette chromosome mec (SCCmec) types in isolates obtained from outpatients in the region of 700,000 people (Canton Sarajevo, Bosnia and Herzegovina) Methods: Our investigation included phenotypic and genotypic markers such as antimicrobial resistance, pulsed-field gel electrophoresis (PFGE), SCC typing, and PVL detection. RESULTS: Antimicrobial susceptibility: all MRSA isolates were resistant to the ß-lactam antibiotics tested, and all isolates were susceptible to trimethoprim sulphamethoxazole, rifampicin, fusidic acid, linezolid, and vancomycin. After the PFGE analysis, the isolates were grouped into five similarity groups: A-E. The largest number of isolates belonged to one of two groups: C - 60% and D - 27%. In both groups C and D, SCCmec type IV was predominant (60% and 88.8%, respectively). A total of 24% of the isolates had positive expression of PVL genes, while 76% showed a statistically significantly greater negative expression of PVL genes. CONCLUSIONS: Using combination techniques, we were able to investigate the origin and genetic background of the strains. PFGE analysis revealed two large, genetically related groups of strains consisting of 87 isolates. Our results suggest failure to apply the screening policy, and a lack of knowledge about multiresistant MRSA strains. This study showed the local epidemiological situation which should be the basis of antimicrobial empiric therapy for non-hospitalized patients.

Inhibition effect of Zedoary turmeric oil on Listeria monocytogenes and Staphylococcus aureus growth and exotoxin proteins production.

PURPOSE: Zedoary turmeric oil (ZTO), the steam extract of Curcuma zedoaria Rosc was researched for its chemical composition, antibacterial activity, and mechanism for countering two major food-borne pathogenic species, Listeria monocytogenes and Staphylococcus aureus. METHODOLOGY: Gas chromatography-mass spectrometry (GC-MS) was used to analyse and characterize the chemical composition of ZTO. Its MICs for the two bacterial species and growth curves were measured. Western blot and real-time reverse-transcription (RT)-PCR assays were utilized to elaborate the mechanism of the antibacterial effect of ZTO by examining the expression levels of virulence-related extracellular proteins. ELISA was used to explore the biological relevance. RESULTS: GC-MS revealed high contents of curzerene, eucalyptol, germacrone and (-)-g-elemene representing 28.45, 10.94, 10.77 and 10.54  %, respectively, of the whole components. The MICs of ZTO that combatted L. monocytogenes and S. aureus were similar (1-2 mg ml-1 ). After adding ZTO at increasing concentrations, there was an evident reduction in the transcription of hly, iap, hla, sea, seb and agrA in a dose-dependent manner. Furthermore, TNF-α accumulation in RAW264.7 cells stimulated by L. monocytogenes and S. aureus supernatants was restricted by a 1/4 MIC of ZTO. CONCLUSION: Overall, L. monocytogenes and S. aureus were comparably susceptible to ZTO. These data demonstrated that ZTO's antimicrobial property was mediated by the repression of the production of virulence factors involved in L. monocytogenes and S. aureus pathogenesis, a finding that can potentially further progress in the development of new anti-virulence drugs.

Spread of seb-Positive Methicillin-Resistant Staphylococcus aureus SCCmec Type II-ST764 Among Elderly Japanese in Nonacute Care Settings.

We investigated the prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among 356 residents of nine long-term care facilities (LTCFs) in Japan during 2015 and 2017. In total, 800 specimens were tested and 39 MRSA isolates were recovered from 31 (8.71%) residents. PCR-based open reading frame typing (POT) and pulsed-field gel electrophoresis typing were performed for the 39 MRSA isolates; five of them showing identical pulsotypes, and POT scores were excluded in further analysis. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing, and toxin gene detection were performed for one representative MRSA isolate per resident. Among the 34 unrelated MRSA isolates, 15 (44.1%) and 19 (55.9%) were of SCCmec types II and IV, respectively, and belonged to seven sequence types (STs). Among the 15 SCCmec II isolates, 11 (73.3%), 3, and 1 belonged to ST764 (clonal complex [CC]5), ST5 (CC5), and ST630 (CC8), respectively. Among the 19 SCCmec IV isolates, 13 (68.4%), 3, 2, and 1 belonged to ST1 (CC1), ST474 (CC1), ST8 (CC8), and ST380 (CC8), respectively. Among the 14 CC5 lineage-SCCmec II isolates, one ST5 isolate and 7 of the 11 ST764 isolates (63.6%) carried seb gene, and 14 (87.5%) of 16 CC1 lineage-SCCmec IV isolates had sea gene (p < 0.05). The results indicate that the seb-positive SCCmec type II-ST764 clone has spread in Japanese LTCF environments. As LTCF residents have multiple comorbidities and increased susceptibility to infections, it is necessary to monitor MRSA colonization in LTCFs through periodic screening to prevent dissemination.

Methicillin-resistant Staphylococcus aureus intracranial abscess: An analytical series and review on molecular, surgical and medical aspects.

PURPOSE: Intracranial abscess caused by methicillin-resistant Staphylococcus aureus (MRSA) is rare and unexplored. The aim of the present study is to examine the prevalence, clinical and molecular characteristics, treatment options and outcome of MRSA intracranial abscess over a period of 6 years. PATIENTSAND METHODS: A total of 21 patients were included in this retrospective study. The demographic and clinical details of all the patients were collected. Molecular typing including staphylococcal cassette chromosome mec typing, spa typing and polymerase chain reaction of Panton-Valentine leucocidin toxin (PVL) gene for the latter 6 isolates was performed. RESULTS: The paediatric population was the most affected group (33.3%). The primary route of infection was post-operative/trauma in 7 (33.3%) cases. All the patients were treated surgically either by aspiration or excision. Fifteen (71%) patients received anti-MRSA treatment with vancomycin or linezolid, where linezolid-treated patients showed better prognosis. Of the 11 patients who were on follow-up, unfavourable outcome was observed in 3 (27.3%) cases and 8 (72.7%) cases improved. The molecular typing of six isolates revealed four community-associated (CA) MRSA, one each of livestock-associated (LA) and healthcare-associated MRSA with PVL gene noted in all. CONCLUSION: We propose that timely diagnosis, surgical intervention and appropriate anti-MRSA treatment would contribute to better outcome. The occurrence of CA-MRSA and LA-MRSA infection in the central nervous system signifies the threat from the community and livestock reservoir, thus drawing attention towards surveillance and tracking to understand the epidemiology and implement infection control measures.

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 µg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.

Molecular and Phenotypic Characterization Revealed High Prevalence of Multidrug-Resistant Methicillin-Susceptible Staphylococcus aureus in Chongqing, Southwestern China.

Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for ∼40% of staphylococcal infections in China. However, the molecular characterization of MSSA is not well described. In this study, 124 MSSA strains collected in 2013 from a comprehensive teaching hospital in Chongqing, Southwestern China, were subjected to antibiotics susceptibility testing and molecular typing, including multilocus sequence typing, staphylococcal protein A (spa) gene typing, accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) typing, Panton-Valentine leukocidin (pvl) gene detection, and antibiotic-resistant gene detection. MSSA strains exhibited high genetic heterogeneity. A total of 10 PFGE groups, 26 sequence types, and 47 spa types were identified. Type I (62.9%) was the most frequent agr type, followed by type II (15.3%), type IV (11.3%), and type III (10.5%). The prevalence of pvl genes was 27.4% (34/124). Notably, 44.4% (55/124) of MSSA strains were multidrug resistance (MDR), and MDR isolates were mostly resistant to penicillin, erythromycin, and clindamycin. The resistance gene blaZ was present in 84.7% of strains, ermC was present in 85.5% of strains, ermA was present in 28.2% of strains, tetK was present in 16.1% of strains, tetM was present in 6.5% of strains, and aacA-aphD was present in 2.6% of strains. These data demonstrated the high prevalence of MDR MSSA in Chongqing, thereby indicating the need to control MSSA infection.

Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R2=0.9984) between the fluorescent intensity and the target DNA concentration in the samples.

Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study.

Several candidate vaccines against Shigella spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered Shigella vaccine development over the past several decades. Bioconjugation technology, exploited here for an Shigella flexneri 2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel S. flexneri 2a bioconjugate vaccine made of the polysaccharide component of the S. flexneri 2a O-antigen, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 µg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant S. flexneri 2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against S. flexneri 2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to S. flexneri 2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.).

Inhibition of streptococcal pyrogenic exotoxin B using allicin from garlic.

Streptococcal pyrogenic exotoxin B (SpeB) is an important virulence factor of group A streptococci (GAS) and inactivation of SpeB results in the significantly decreased virulence of the bacterium. The protein is secreted as an inactive zymogen of 40 KDa (SpeBz) and undergoes proteolytic truncation to result in a 28 KDa mature active protease (SpeBm). In this study the effect of allicin on the proteolytic activity of SpeBm was evaluated using azocasein assay. Allicin neutralized the SpeBm proteolytic activity in a concentration dependent manner (IC50 = 15.71 ± 0.45 µg/ml). The loss of activity was completely reversed by subsequent treatment with a reducing agent, dithiothreitol (DTT; 10 mM final concentration), suggesting that allicin likely inhibits the SpeBm by forming a disulfide linkage with an active thiol group in its active site. This mechanism of action was further confirmed with the fact that DTT did not reverse the SpeBm activity in the presence of E-64, a cysteine protease-specific inhibitor, which works specially by forming a thioether linkage with free sulfhydryl groups in enzymes active site. The MIC of allicin against GAS was found to be 32 µg/ml. Exposure of GAS culture to allicin (25 µg/ml) inhibited maturation of SpeBz to the SpeBm. In conclusion, the results of this study suggest that allicin inhibits the maturation of SpeBz and proteolytic activity of SpeBm and could be a potential therapeutic agent for the treatment of GAS infections.

Genotypic Characterization of Methicillin-Resistant Staphylococcus aureus Recovered at Baseline from Phase 3 Pneumonia Clinical Trials for Ceftobiprole.

Baseline methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with nosocomial and community-acquired pneumonia collected during Phase 3 trials for ceftobiprole were characterized. Eighty-four unique isolates from patients enrolled in Europe (50.0%), Asia-Western Pacific region (APAC; 20.2%), North America (19.0%), Latin America (8.3%), and South Africa (2.4%) were included. Antimicrobial susceptibility testing was performed by broth microdilution and isolates screened for Panton-Valentine leukocidin. SCCmec and agr types were determined. Strains were subjected to pulsed-field gel electrophoresis and spa typing. Clonal complexes (CCs) were assigned based on spa and/or multilocus sequence typing. Most isolates were CC5-MRSA-I/II/IV (44.0%; 37/84), followed by CC8-MRSA-IV (22.6%; 19/84) and CC239-MRSA-III (21.4%; 18/84). Other MRSA formed seven clonal clusters. Isolates from North America were associated with USA100, while those from South America belonged to the Cordobes/Chilean CC. A greater clonal diversity was observed in Europe; however, each country had CC5, CC8, or CC239 as prevalent lineages. Isolates from APAC were CC5-MRSA-II (47.1%; 8/17) or CC239-MRSA-III (47.1%; 8/17). Isolates carrying SCCmec I and III had ceftobiprole MIC50 values of 2 µg/ml, while those isolates with SCCmec II and IV had MIC50 values of 1 µg/ml. Ceftobiprole inhibited 96% and 100.0% of the isolates at ≤ 2 and ≤ 4 µg/ml, respectively. These isolates represented common circulating MRSA clones. Ceftobiprole demonstrated in vitro activity with a slight variation of minimum inhibitory concentrations (MICs) according to SCCmec or clonal type.

Protective effect of DNA vaccine encoding pseudomonas exotoxin A and PcrV against acute pulmonary P. aeruginosa Infection.

Infections with Pseudomonas aeruginosa have been a long-standing challenge for clinical therapy because of complex pathogenesis and resistance to antibiotics, thus attaching importance to explore effective vaccines for prevention and treatment. In the present study, we constructed a novel DNA vaccine by inserting mutated gene toxAm encoding Pseudomonas Exotoxin A and gene pcrV encoding tip protein of the type III secretion system into respective sites of a eukaryotic plasmid pIRES, named pIRES-toxAm-pcrV, and next evaluated the efficacy of the vaccine in murine acute Pseudomonas pneumonia models. Compared to DNA vaccines encoding single antigen, mice vaccinated with pIRES-toxAm-pcrV elicited higher levels of antigen-specific serum immunoglobulin G (IgG), enhanced splenic cell proliferation and cytokine secretion in response to Pseudomonas aeruginosa antigens, additionally PAO1 challenge in mice airway resulted in reduced bacteria burden and milder pathologic changes in lungs. Besides, it was observed that immunogenicity and protection could be promoted by the CpG ODN 1826 adjuvant. Taken together, it's revealed that recombinant DNA vaccine pIRES-toxAm-pcrV was a potential candidate for immunotherapy of Pseudomonas aeruginosa infection and the CpG ODN 1826 a potent stimulatory adjuvant for DNA vaccination.

Methicillin-resistant Staphylococcus aureus (MRSA) detected at four U.S. wastewater treatment plants.

BACKGROUND: The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections is increasing in the United States, and it is possible that municipal wastewater could be a reservoir of this microorganism. To date, no U.S. studies have evaluated the occurrence of MRSA in wastewater. OBJECTIVE: We examined the occurrence of MRSA and methicillin-susceptible S. aureus (MSSA) at U.S. wastewater treatment plants. METHODS: We collected wastewater samples from two Mid-Atlantic and two Midwest wastewater treatment plants between October 2009 and October 2010. Samples were analyzed for MRSA and MSSA using membrane filtration. Isolates were confirmed using biochemical tests and PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed by Sensititre® microbroth dilution. Staphylococcal cassette chromosome mec (SCCmec) typing, Panton-Valentine leucocidin (PVL) screening, and pulsed field gel electrophoresis (PFGE) were performed to further characterize the strains. Data were analyzed by two-sample proportion tests and analysis of variance. RESULTS: We detected MRSA (n = 240) and MSSA (n = 119) in 22 of 44 (50%) and 24 of 44 (55%) wastewater samples, respectively. The odds of samples being MRSA-positive decreased as treatment progressed: 10 of 12 (83%) influent samples were MRSA-positive, while only one of 12 (8%) effluent samples was MRSA-positive. Ninety-three percent and 29% of unique MRSA and MSSA isolates, respectively, were multidrug resistant. SCCmec types II and IV, the pvl gene, and USA types 100, 300, and 700 (PFGE strain types commonly found in the United States) were identified among the MRSA isolates. CONCLUSIONS: Our findings raise potential public health concerns for wastewater treatment plant workers and individuals exposed to reclaimed wastewater. Because of increasing use of reclaimed wastewater, further study is needed to evaluate the risk of exposure to antibiotic-resistant bacteria in treated wastewater.

Interactions of extracts from selected chewing stick sources with Aggregatibacter actinomycetemcomitans.

BACKGROUND: Aggregatibacter actinomycetemcomitans produces a leukotoxin that activates a pro-inflammatory death of human monocytes/macrophages. A specific clone of this bacterium (JP2) has a 530-base pair deletion in the leukotoxin promoter gene and significantly enhanced expression of leukotoxin. This specific clone of A. actinomycetemcomitans is common in some African populations and has a strong association with periodontal attachment loss in adolescents in these populations. Chewing sticks of plant origin are commonly used as oral hygiene tool in Africa, but their role as a therapeutic agent in periodontal disease is poorly investigated. RESULTS: Ethanol extracts were made from 7 common plants used as chewing sticks in West-Africa. None of the tested extracts inhibited growth of A. actinomycetemcomitans. However, extracts from Psidium guajava (Guava) completely neutralized the cell death and pro-inflammatory response of human leukocytes induced by the leukotoxin. None of the six other tested chewing stick extracts showed this effect. CONCLUSIONS: The discovery that extracts from Guava efficiently neutralizes A. actinomycetemcomitans leukotoxicity might lead to novel therapeutic agents and strategies for prevention and treatment of aggressive forms of periodontitis induced by infections with the highly leukotoxic JP2 clone of this bacterium.

An immunotoxin targeting the gH glycoprotein of KSHV for selective killing of cells in the lytic phase of infection.

Amongst the pathologies associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV), multicentric Castleman's disease is distinctive for involvement of the lytic phase of the virus replication cycle. This B cell lymphoproliferative disorder has shown clinical responsiveness not only to generalized immunotherapy and cytotoxic chemotherapy, but also to inhibitors of herpesvirus DNA replication, consistent with the involvement of lytic phase of replication. These findings suggest that selective killing of virus-producing cells might represent a novel therapeutic strategy. We designed an immunotoxin, YC15-PE38, containing a single chain variable region fragment of a monoclonal antibody against KSHV glycoprotein H (gH) linked to the effector domains of Pseudomonas aeruginosa exotoxin A. Purified YC15-PE38 displayed highly selective and potent killing of a gH-expressing transfectant cell line (subnanomolar IC(50)). The immunotoxin also strongly inhibited production of infectious KSHV virions from an induced chronically infected cell line, by virtue of selective killing of the virus-producing cells. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir. These results provide support for the development of anti-KSHV strategies based on targeted killing of infected cells expressing lytic phase genes.

Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus.

BACKGROUND: The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins. METHODOLOGY/PRINCIPAL FINDINGS: A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). CONCLUSIONS/SIGNIFICANCE: The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with ß-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.

Peppermint oil decreases the production of virulence-associated exoproteins by Staphylococcus aureus.

The present study aimed to evaluate the antimicrobial activity of peppermint oil against Staphylococcus aureus, and further investigate the influence of peppermint oil on S. aureus virulence-related exoprotein production. The data show that peppermint oil, which contained high contents of menthone, isomenthone, neomenthol, menthol, and menthyl acetate, was active against S. aureus with minimal inhibitory concentrations (MICs) ranging from 64-256 µg/mL, and the production of S. aureus exotoxins was decreased by subinhibitory concentrations of peppermint oil in a dose-dependent manner. The findings suggest that peppermint oil may potentially be used to aid in the treatment of S. aureus infections.

[Construction, expression and immunogenicity analysis of a fusion protein containing M2e of influenza A virus fused to a modified Pseudomonas aeruginosa exotoxin A].

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.

Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli.

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.

Clinical features, epidemiology, antimicrobial resistance, and exotoxin genes (including that of Panton-Valentine leukocidin) of gentamicin-susceptible methicillin-resistant Staphylococcus aureus (GS-MRSA) isolated at a paediatric teaching hospital in New South Wales, Australia.

AIMS: To describe the epidemiological, clinical, and laboratory features of gentamicin-susceptible methicillin-resistant Staphylococcus aureus (GS-MRSA) seen at a paediatric teaching hospital. METHODS: Patients from whom GS-MRSA was isolated between 1 January 2001 and 31 December 2002 were enrolled. Retrospective chart review was performed. Susceptibility testing was performed with the Vitek2 system; PCR confirmed methicillin resistance. Phage typing and pulsed field gel electrophoresis (PFGE) was performed (utilising MLST/SCCmec-defined control strains). PCR detection of tst, luk-PV, and entA-entE was performed. RESULTS: Eighty-five per cent of all Staphylococcus aureus isolates during the study period were methicillin-sensitive, and 15% were MRSA (9% GS-MRSA, 6% gentamicin resistant-MRSA). 100 GS-MRSA infections in 98 children were identified: 59 cases of skin/soft tissue, four bone and joint, four surgical site infections, three pneumonia, eight other types, and 22 represented colonisation. Ninety-nine isolates were non-multidrug resistant, but 17 strains were resistant to erythromycin, 7 to tetracyclines, 12 to ciprofloxacin, 11 to fusidic acid, 1 each to rifampicin and mupirocin. 44 isolates were Oceania strain (ST30-MRSA-IV), 20 were Queensland strain (ST93-MRSA-IV), ten were UK EMRSA-15 (ST22-MRSA-IV), eight were WA MRSA-1 (ST1-MRSA-IV), two were WA MRSA-5 (ST8-MRSA-IV), one was WA MRSA-2 (ST78slv-MRSA-IV), one was WA MRSA-15 (ST59-MRSA-IV), and the remainder were sporadics. Twenty patients were Pacific Islanders, of whom 12 had the Oceania strain; ten were Aboriginal, of whom eight had the Queensland strain. Sixty-eight isolates possessed luk-PV, including all Queensland strains and 91% of Oceania strains. Enterotoxin genes were detected in 25% of the isolates, and tst was detected in four isolates. CONCLUSIONS: GS-MRSA is a significant paediatric problem in New South Wales: two minority groups are over-represented, multiple epidemic strains were detected, most community strains possess luk-PV, and many isolates are multidrug resistant.