Preparation and anti-Vibrio vulnificus hemolysin A of macrophage membrane hybrid liposome / 药学实践杂志

Objective To prepare a biomimetic nano carrier macrophage membrane hybrid liposome by heterozygous macrophage membrane and liposome, which could be used for the clearance and toxicity inhibition of Vibrio vulnificus hemolysin A (VvhA). Methods Macrophage membrane was extracted and hybridized with liposome by thin-film evaporation combined extrusion method. The hybridized liposome of macrophage membrane was constructed and characterized. The in vitro detoxification ability of the hybridized vector was evaluated by hemolysis test and cytotoxicity test. The detoxification ability of the vector was evaluated by mouse skin infection model. Results Anti toxoid studies in vivo and in vitro showed that the anti-hemolysis rate of macrophage membrane heterozygous liposomes in vitro reached 97.03%, which could effectively inhibit the skin ulceration in subcutaneous infected mice and make the survival rate of abdominal infected mice reach 80%. Conclusion The constructed macrophage membrane hybrid liposome had high detoxification ability, which could provide a potential solution and research basis for the prevention and treatment of Vibrio vulnificus infection.

Hemolysin Sph2 from Leptospira interrogans amplified inflammatory response in murine macrophages J774A.1 through HMGB1 / 中华微生物学和免疫学杂志

Objective:To investigate the possible mechanism of high mobility group box-1 (HMGB1) in amplifing inflammatory responses in Leptospira interrogans hemolysin Sph2-treated J774A.1 macrophages. Methods:Recombinant Sph2 was incubated with J774A.1 macrophages. The damage of cell membrane was detected by lactate dehydrogenase(LDH) determination; the changes of cell structure were observed by cryo-electron microscope; ELISA was used to determine the expression of HMGB1. After the commercial recombinant HMGB1 was incubated with mouse J774A.1 macrophages, the phosphorylation of NF-κB, p38-MAPK and JNK signaling pathway wsa detected by Western blot, and the expression of IL-1β, IL-6, and KC (IL-8) was detected by ELISA.Results:Recombinant hemolysin rSph2 induced significant changes in the structures of J774A.1 cells, including nucleus disappearance, cell membrane structure damage, cell lysis and membrane swelling. The yields of LDH and HMGB1 also increased significantly. Phosphorylated-NF-κB, -p38-MAPK and -JNK were increased by HMGB1. The expression of IL-1β, IL-6 and KC in J774A.1 cells was up-regulated by HMGB1 and inhibited via inhibitors of NF-κB, p38-MAPK and JNK signal pathways.Conclusions:Hemolysin rSph2 damaged the membrane of J774A.1 cells, and induced the secretion of HMGB1. Secreted-HMGB1 might induce the expression of IL-1β, IL-6 and KC in J774A.1 cells via NF-κB, p38-MAPK and JNK signal pathways, thus amplifying the inflammatory responses caused by Sph2.

Study on Sub-minimal Inhibitory Concentration of Tanreqing Injection in Inhibiting Hemolytic Activity of Staphylococcus aureus α-hemolysin / 中国实验方剂学杂志

Objective:To study whether Tanreqing injection （TRQ） can alleviate the body injury in the process of infection by inhibiting the production and release of <italic>α</italic>-hemolysin of <italic>Staphylococcus aureus</italic> under sub-minimal inhibitory concentration， and to provide experimental basis for better guidance of clinical medication. Method:The effects of TRQ on the minimum inhibitory concentration （MIC） and bacterial growth of <italic>S.aureus</italic> were determined firstly by microplate method and time-growth curve. The different sub-minimal inhibitory concentrations of TRQ were co-cultured with bacteria or bacterial supernatants， and then co-incubated with defibrillated rabbit blood to detect the inhibitory and neutralizing effects of TRQ on <italic>S.aureus</italic> <italic>α</italic>-hemolysin. Cell counting kit-8 （CCK-8） cell viability assay was used to detect the protective effect of TRQ on <italic>S. aureus</italic>-mediated damage to human alveolar epithelial cells （A549）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of sub-minimal inhibitory concentration of TRQ on the mRNA expression of <italic>S.aureus</italic> <italic>α</italic>-hemolysin regulatory genes hla and agrA. Result:The MIC of TRQ to <italic>S.aureus </italic>was 1/8 of the stock solution， and the sub-minimal inhibitory concentration （1/64MIC-1/16MIC） TRQ used in this study did not affect the growth of bacteria. 1/64MIC-1/16 MIC TRQ had the effect of inhibiting and neutralizing the hemolytic activity of <italic>α</italic>-hemolysin， with a protective effect on <italic>S.aureus</italic> supernatant-mediated A549 cell damage， and its inhibitory effect on <italic>α</italic>-hemolysin was closely related to the inhibition of hla and agrA mRNA expression. Conclusion:The sub-minimal inhibitory concentration TRQ can inhibit and neutralize the hemolytic activity of <italic>α</italic>-hemolysin of <italic>S.aureus</italic>， with a protective effect on A549 cell damage mediated by <italic>S.aureus</italic> infection， and its mechanism of inhibiting <italic>α</italic>-hemolysin is closely related to the interference with agr regulatory system.

Characterization of Staphylococcus aureus Small Colony Variant (SCV) Clinical Isolates in Zaria, Nigeria

Staphylococcal isolates from specimen submitted to the Medical Microbiology laboratory of Ahmadu Bello University Teaching Hospital, Zaria were collected over a period of 6 months (February-July 2012), characterized by microbiological standard procedures and the S. aureus small colony variant (SCV) isolates were isolated. The antibiotic susceptibility pattern of the isolates was determined by the Kirby-Bauer-CLSI modified disc agar diffusion (DAD) technique. The SCV isolates were assessed for the carriage of four virulence genes; sdrE (putative adhesin) icaA (intracellular adhesin) hlg (hemolysin), Cna (collagen adhesin). A total of 258 non-duplicate staphylococcal isolates made up of 219 (84%) S. aureus and 39 (15%) coagulase-negative staphylococci (coNS) where obtained. A total of 48 (22%) isolates were determined to be S. aureus SCV mainly from wound/abscess (31%). S. aureus SCV isolates were generally resistant to all the nine antibiotics tested with only minimal sensitivity to tigecyclin (10.4%) and ciprofloxacin (18.8%). Statistically, there was no significant difference between the microbial load and the different antibiotics that were used, (P ≥0.05). None of the S. aureus SCV isolates carried the four virulence genes which were tested in this study. The results have therefore proved that S. aureus small colony variant exist in our environment and they are more resistant to most antimicrobial agent than their wild type.

Identification of staphylococcus aureus lineage ST59 by the combined detection of delta hemolysin allelic variant G10S and beta hemolysin / 中华检验医学杂志

Objective To investigate the identification of staphylococcus aureus lineage ST59 using the combined detection of delta hemolysin allelic variant G10S(HldG10S) and beta hemolysin(β-toxin). Methods Perspective study.A total of 82 non-duplicate clinical staphylococcus aureus were collected from November 2017 to April 2018 in the department of Clinical laboratory, the Third Xiangya Hospital of Central South University, China.The strains were routinely identified by MALDI-TOF MS and the mass spectra were obtained. According to the m/z expression intensity of delta hemolysin(Hld), all strains were divided into three groups:HldG10S (3036±2.0)m/z, Hld (3006±2.0)m/z and ND [no (3036±2.0)m/z and no (3006±2.0)m/z]. The distribution of ST59 in the three groups was detected by MLST. Reverse synergic hemolysis test was used to determine theβ-toxin phenotype. And the sensitivity, specificity and accuracy of HldG10S,β-toxin and the combined detection of HldG10S and Hld to identify ST59 were compared. Results Among the 82 strains, 21 strains expressed HldG10S toxin, accounting for 25.6％. 39 strains expressed Hld toxin, accounting for 47.6％.22 strains did not express HldG10S and Hld toxin, accounting for 26.8％. In HldG10S group,16 strains were ST59, accounting for 76.19％(16/21).ST59 was not found in both Hld and ND groups. All 16 strains of ST59 in HldG10S group producedβ-toxin, while none of the 5 strains of non-ST59 producedβ-toxin. The specificity(100％) and accuracy(100％) of the combined detection was significantly higher than that of HldG10S andβ-toxin single detection of specificity(92.4％, 77.3％) and accuracy(80.5％, 81.7％) (χ2=19.472, P<0.001;χ2=17.792, P<0.001). Conclusion The combined detection of HldG10S andβ-toxin can preliminarily and rapidly identify ST59, which can assist the routine monitoring of the change trend of staphylococcus aureus epidemic.

Real-time recombinase polymerase amplification for detection of Vibrio parahaemolyticus / 预防医学

Objective@# To establish real-time recombinase polymerase amplification（RPA）for the rapid detection of Vibrio parahaemolyticus（VP）.@*Methods@#An exo probe and primers were designed according to the conserved sequence of thermolabile hemolysin（tlh）gene of VP and then RPA for detection of VP was established. The sensitivity of the assay was evaluated by detecting different concentration of VP;the specificity was evaluated by detecting different bacteria;the stability was evaluated by repeat trials;the application effect was evaluated by detecting food samples which were simultaneously tested with traditional culture method according to GB 4798.7-2013 Detection of VP.@*Results@#A real-time RPA was established to complete VP amplification within 20 min at a constant temperature of 39 ℃. The analytical sensitivity of the assay was five pg per reaction and no cross-reactivity with other pathogenic bacteria observed. The RPA detection results with different concentration of VP and E. coli DNA templates at three time points were consistent. The detection results of 51 food samples by RPA were the same as those by traditional culture method.@*Conclusion@#The established real-time RPA can qualitatively detect VP,with simple operation and interpretation of results,which is suitable for rapid detection of VP in public health emergencies and food safety supervision.

Relationships between Virulence Factors and Antimicrobial Resistance among Escherichia coli Isolated from Urinary Tract Infections and Commensal Isolates in Tehran, Iran

OBJECTIVES: Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs). Here, we determined whether sensitivity to antibiotics was related to the prevalence of iron scavenging genes, or to biofilm and hemolysis formation. METHODS: A total of 110 UPEC and 30 E coli isolates were collected from the urine of UTI patients and feces of healthy individuals without UTI, respectively. The presence of iron receptor genes and phenotypic properties were evaluated by polymerase chain reaction and phenotypic methods, respectively. Susceptibility to routine antibiotics was evaluated using the disc diffusion method. RESULTS: The prevalence of iron scavenging genes ranged from 21.8% (ireA) to 84.5% (chuA) in the UPEC. Resistance to ceftazidime and cefotaxime was significantly correlated with the presence of fyuA and iutA iron genes. Biofilm production was significantly associated with the prevalence of fyuA and hma iron genes. A higher degree of antibiotic resistance was exhibited by isolates that produced biofilms than by their non-biofilm producing counterparts. CONCLUSION: Our study clearly indicates that biofilm production is associated with antibiotic resistance, and that iron receptors and hemolysin production also contribute to reduced antibiotic sensitivity. These results further our understanding of the role that these virulence factors play during UPEC pathogenesis, which in turn may be valuable for the development of novel treatment strategies against UTIs.

Investigation of Single-stranded Deoxyribonucleic Acid Translocation Through α-Hemolysin Nanopore with Ionic Liquid Supporting Electrolytes / 分析化学

Due to the difference in spatial configuration and charge of the bases in a DNA molecule, characteristic translocation current pulses through a single nanopore could be obtained. This could become the basis of DNA sequencing method. However, due to the fast translocation speed (sub-micro seconds) and the small current change (about pA), it is still a challenge to obtain the accurate molecular substructure with present electronic techniques. In this work, in order to control the translocation behavior of ssDNA, two kinds of ionic liquids with high viscosity and conductivity were introduced to establish a viscosity gradient with the α-hemolysin single nanopore interface and the acidity of the solution was optimized. The trans chamber contained pure BmimPF6 and the cis chamber contained 1 mol/ L BmimCl and 10 mmol/ L Tris-HCl ( pH 5. 5 ). Preliminary experiment results under this electrolyte configuration showed that poly ( dC) 15 , poly ( dC) 15 , poly(dC) 30 and poly(dC) 50 exhibited obvious long duration pulses with high current suppression ratio. The blocking depth reached more than 95％ of long blocking events. The duration time of long blocking events prolonged to tens or hundreds of milliseconds. Meanwhile, the peak-peak of baseline noise was reduced by about 30％ .

Study on Interaction of Protamine and Single-stranded DNA by Current Pulses Through Single Nanopore / 分析化学

Single nanopore current pulse method is a new, rapid and simple detection method, which is promising for single-molecule DNA sequencing and bio-sensing. Due to the short duration and the low current amplitude of the pulses caused by molecular translocation under normal conditions, pulse detection system with fast response and high sensitivity is required. In this work, based on a lab-established pulse detection system, the effect of protamine in the regulation of single-stranded (ssDNA) current pulses with α-hemolysin (α-HL) single nanopore interface was investigated. Experimental results showed that the pulses positive charged protamine and negative ssDNA probes were both well observed with the established system, and both the pulse amplitude and duration of ssDNA were increased as a result of interaction with protamine. This study provides a way to improve the resolving power of current pulses based on molecular interactions.

Inhibitory effects of aloin on growth activity and hemolytic toxin expression of Staphylococcus aureus / 临床检验杂志

Objective To investigate the inhibitory effects of aloin on the growth of Staphylococcus aureus and its virulence factors α-hemolysin in vitro.Methods Broth dilution was used to measure the minimum inhibitory concentration (MIC) of water-soluble aloin on S.aureus.Agar drilling method was used to observe the size of inhibition zone of aloin for S.aureus.Plasma coagulase test was used to detect the changes of S.aureus coagulase and absorbance was measured to detect the changes of hemolytic activity when S.aureus was exposed to aloin.Real time PCR was used to detect the effects of aloin on the expressions of hla and agrA mRNA.Results The soluble aloin inhibited the growth of S.aureus in a dose-dependent manner.The inhibition zone diameter of a standard strain of S.aureus (ATCC 25923) was 21.5 mm with MIC of 12.5 mg/mL and 17 mm for the clinical isolate SA1.5 with MIC of 15 mg/mL.After treated with soluble aloin,the coagulase titers of ATCC 25923 were 16,4 and 2 for 1/2 MIC,1 MIC and 2 MIC respectively compared with titer 32 of the control group without soluble aloin.The expression of α-hemolysin of S.aureus ATCC 25923 was down-regulated by soluble aloin and the hemolytic activity of S.aureus ATCC 25923 with 1/2 MIC,1 MIC and 2 MIC groups were (77.4 ±3.41) ％,(42.2 ± 2.4) ％ and (38.7 ± 2.4) ％ respectively.The expression levels of hla were 0.020 3 (0.019 6,0.028 8),0.011 6(0.010 6,0.013 1) and 0.033 7(0.020 2,0.042 9) respectively in the 1/2 MIC,1 MIC and 2 MIC group respectively,and there were significant differences among the three groups (H =16.807,P ＜ 0.05).The expression levels of agrA was 0.074 6 (0.066 2,0.098 2),0.020 8 (0.012 2,0.032 6) and 0.021 3 (0.010 2,0.029 6) in the 1/2 MIC,1 MIC and 2 MIC group respectively,and there were significant differences among the three groups (H =16.320,P ＜ 0.05).Conclusion Aloin may inhibit the growth of S.aureus and could effectively inhibit the expression of α-hemolysin.

Effects of Periplaneta americana extract Ento-A on immune function in immunosuppressed mice / 中国药理学通报

Aim To investigate the effects of Periplane-ta americana extract Ento-A on the immune function in immunosuppressed mice . Methods Immunosup-pressed mouse model was induced by intraperitoneal injection of cyclophosphamide in KM mice .To evalu-ate the effects of Ento-A on the immune function in im-munosuppressed mice , neutral red method and MTT assay were used respectively to detect the effects of En-to-A on the phagocytosis of peritoneal macrophages and T cell proliferation rate in mice; with sheep red blood cell as immunogen , the effects of Ento-A on the pro-duction of serum hemolysin were evaluated;peripheral blood was tested and immune organ index calculated . Results Compared with model control group , the high, medium and low doses of Ento-A could improve the expression of serum hemolysin in immunosup-pressed mice ( P<0.01 ) , and increase the spleen in-dex(P<0.01) and thymus index (P>0.05), signifi-cantly increased the content of WBC ( P<0.01 ) , PLT ( P<0.01 ) , HGB ( P<0.01 ) , while the contents of RBC was on the rise , with no significant difference ( P>0.05 ) in peripheral blood , significantly enhanced phagocytic function and T lymphocyte proliferative abil-ity in a dose-dependent manner ( P<0.01 ) .Conclu-sion Ento-A can enhance the immune function of im-munosuppressed mice .

Pertussis: enfermedad reemergente / Pertussis: reemerging disease / Pertussis: doença reemergente

RESUMEN Introducción: Actualmente a pesar de las estrategias de vacunación a nivel mundial, B. pertussis se ha convertido en un problema de salud pública, sigue siendo una de las enfermedades menos prevenibles por vacunación en todo el mundo, aún en países desarrollados con amplia cobertura de vacunación. Objetivo: Describir los principales mecanismos de virulencia asociados a la infección mediante los cuales la bacteria logra evadir la respuesta inmune, además de dar a conocer un panorama actual del estado de inmunización contra la tos ferina y la problemática de su reemergencia a nivel mundial. Métodos: Se realizó una revisión mediante búsqueda electrónica de literatura; entre las estrategias de búsqueda se destaca el empleo de las bases de datos como PubMed, ScienceDirect, SciELO, Redalyc; y búsqueda en portales de salud; sobre aspectos generales de la enfermedad y su agente causal, además de últimas actualizaciones sobre el tema. Resultados: Como lo confirman recientes estudios, el incremento del riesgo de infección por B. pertussis sigue presentándose en adolescentes y adultos debido a la disminución en la respuesta inmune inducida por la vacunación y la infección natural, por ende, la información actual indica que está reemergiendo la tos ferina en todo el mundo, situación que es necesario conocer para un oportuno diagnóstico y tratamiento. Conclusiones: Análisis de la literatura demuestra la necesidad de ampliar el uso de técnicas moleculares; llevar a cabo la modificación de los programas de vacunación, con la administración de dosis de refuerzo entre adolescentes y adultos; además programas de monitoreo epidemiológico eficaces de recolección de casos con tos ferina y sistemas adecuados de notificación para lograr la reducción equitativa y sustentable de la morbilidad y mortalidad de ésta enfermedad.

ABSTRACT Introduction: Currently despite vaccination strategies worldwide, B. pertussis has become a public health problem, it remains one of the least vaccine preventable diseases in the world, even in developed countries with extensive coverage of vaccination. Objective: To describe the main mechanisms of virulence associated with the infection through which the bacterium manages to evade the immune response, as well as to present a current picture of the state of immunization against pertussis and the problem of its reemergence worldwide. Methods: Electronic literature search was performed; among the search strategies we highlight the use of databases such as PubMed, ScienceDirect, SciELO, Redalyc; and search in health portals; on general aspects of the disease and its causal agent, in addition to the latest updates on the subject. Results: As confirmed by recent studies, the increased risk of B. pertussis infection continues to occur in adolescents and adult's due to the decrease in the immune response induced by vaccination and natural infection, therefore, current information indicates that reemerging whooping cough around the world, a situation that is necessary to know for a timely diagnosis and treatment. Conclusions: Analysis of the literature demonstrates the need to expand the use of molecular techniques; to carry out the modification of the vaccination programs, with the administration of doses of reinforcement between adolescents and adults; In addition to effective epidemiological monitoring programs for the collection of pertussis cases and adequate notification systems to achieve an equitable and sustainable reduction in the morbidity and mortality of this disease.

RESUMO Introdução: Atualmente, apesar das estratégias de vacinação em todo o mundo, B. pertussis tornou-se um problema de saúde pública, continua a ser uma das doenças menos evitáveis pela vacina no mundo, mesmo em países desenvolvidos com ampla cobertura de vacinação. Objetivo: Descrever os principais mecanismos de virulência associados à infecção através dos quais a bactéria consegue evadir a resposta imune, bem como apresentar uma imagem atual do estado da imunização contra a tosse convulsa e o problema da ressurgência em todo o mundo. Métodos: Pesquisa eletrônica de literatura foi realizadantre as estratégias de Investigação, destacamos o uso de bancos de dados como PubMed, ScienceDirect, SciELO, Redalyc; e investigação em portais de saúde; sobre aspectos gerais da doença e seu agente causal, além das atualizações mais recentes sobre o assunto. Resultados: Conforme confirmado por estudos recentes, o aumento do risco de infecção por B. pertussis continua a ocorrer em adolescentes e adultos devido à diminuição da resposta imune induzida por vacinação e infecção natural, portanto, informações atuais indicam que a ressurreição de tosse convulsa ao redor do mundo, uma situação que é necessário conhecer para um diagnóstico e tratamento oportunos. Conclusões: A análise da literatura demonstra a necessidade de ampliar o uso de técnicas moleculares; para realizar a modificação dos programas de vacinação, com a administração de doses de reforço entre adolescentes e adultos; além de programas efetivos de monitoramento epidemiológico para a coleta de casos de tosse convulsa e sistemas de notificação adequados para alcançar uma redução sustentável e equitativa na morbidade e mortalidade desta doença.

To explore the preparation method of different animal erythrocytehemolysin in experimental teaching of medical immunology / 国际检验医学杂志

Objective To detect different animal erythrocyte hemolysins titer,and compare the application of these hemolysins in immunological experimental teaching,for selecting the better method of preparing high titer hemolysin for experimental teaching of medical immunology.Methods A total of 40 experiment rabbits were divided into 4 groups in this study,and immunized by sheep red blood cell(SRBC) and porcine red blood cell(PRBC) through different immunization procedures to prepare the hemolysin,detect and compare these 4 groups hemolysins titer by the complement hemolysis test.Results Rabbit Anti-SRBC in the group A was 1∶4 800,rabbit Anti-PRBC in the group B was 1∶1 200,rabbit Anti-SRBC in the group C was 1∶1 000,rabbit Anti-PRBC in the group D was 1∶200.Conclusion The hemolysin titer of the rabbit Anti-PRBC was lower than that of the rabbit Anti-SRBC by the same immunization procedures,and the immunization procedure by intradermal multi-point and auricular vein injection is the better method of preparing high titer hemolysin,so PRBC could replace SRBC as antigen,and immunize the rabbits for preparing hemolysin,which could be used in experimental teaching of medical immunology.

Recent progress in Staphylococcus aureus α-hemolysin / 中华微生物学和免疫学杂志

Staphylococcus aureus(S.aureus) secretes a variety of pathogenic toxins and one of the most prominent toxins is α-hemolysin,which is considered as the main virulence factor in skin necrosis and severe infections caused by S.aureus infection. α-hemolysin is so named because of its ability to dissolve red blood cells. However,it has a wide range of effects on various cells and may cause different pathogenic re-sponses in different host cells. This review summarizes recent studies on the structure of α-hemolysin and its interaction with different cells in order to better understand the role of α-hemolysin in the process of S.aureus infection,which may provide a reference for developing new approaches to the prophylaxis and treatment of S.aureus infection.

Prevalence and virulence factors of Candida spp.associated with blow flies

Objective:To investigate the prevalence of Candida spp.and the virulence factors of Candida albicans (C.albicans) isolated from external surfaces of blow flies collected from Mae Sot,Tak Province,Thailand,Methods:The blow flies were collected by sterile sweep nets from three areas in Mae Sot.Yeast isolation was first performed on Sabouraud dextrose agar (SDA) supplemented with chloramphenicol and cycloheximide.The yeast isolates were then identified by using chromogenic agar,a yeast identification test kit,a germ tube formation test and a carbohydrate utilization test.The β-hemolysis was determined on 7％ sheep blood agar,while phospholipase activity was measured on SDA agar supplemented with 10％ egg yolk suspension.Antifungal susceptibility testing was determined by broth micro-dilution testing against ketoconazole and amphotericin B.Results:The prevalence rate of Candida spp.on the external surfaces of the blow flies was 78.1％.All C.albicans isolated from the blow fly demonstrated β-hemolysin and potent phospholipase activities and 47.1％ of C.albicans were resistant to ketoconazole with MIC values 128 μg/mL.Conclusions:The result s indicate that blow flies could play an essential role in the transmission of potentially pathogenic and antifungal resistant C.albicans into the environment.Further investigation on other virulence factors and genetic relatedness among isolates from the blow flies,the environment and clinical specimens is required to confirm this role.

Prevalence and virulence factors of Candida spp. associated with blow flies

Objective To investigate the prevalence of Candida spp. and the virulence factors of Candida albicans (C. albicans) isolated from external surfaces of blow flies collected from Mae Sot, Tak Province, Thailand. Methods The blow flies were collected by sterile sweep nets from three areas in Mae Sot. Yeast isolation was first performed on Sabouraud dextrose agar (SDA) supplemented with chloramphenicol and cycloheximide. The yeast isolates were then identified by using chromogenic agar, a yeast identification test kit, a germ tube formation test and a carbohydrate utilization test. The β-hemolysis was determined on 7% sheep blood agar, while phospholipase activity was measured on SDA agar supplemented with 10% egg yolk suspension. Antifungal susceptibility testing was determined by broth micro-dilution testing against ketoconazole and amphotericin B. Results The prevalence rate of Candida spp. on the external surfaces of the blow flies was 78.1%. All C. albicans isolated from the blow fly demonstrated β-hemolysin and potent phospholipase activities and 47.1% of C. albicans were resistant to ketoconazole with MIC values 128 μg/mL. Conclusions The results indicate that blow flies could play an essential role in the transmission of potentially pathogenic and antifungal resistant C. albicans into the environment. Further investigation on other virulence factors and genetic relatedness among isolates from the blow flies, the environment and clinical specimens is required to confirm this role.

Expression, purification and antibody preparation of beta-hemolysin from Staphylo-coccus aureus / 军事医学

Objective To express the beta hemolysin ( Hlb), an important toxin secreted by Staphylococcus aureus ( S.aureus) and the mutant protein Hlb H-149-N , to detect the hemolytic activity of Hlb and Hlb H-149-N on sheep erythrocytes , and to prepare the specific antibodies against Hlb which can inhibit the hemolytic activity of Hlb .Methods Hlb gene was amplified by PCR using S.aureus NCTC-8325 genome DNA as template.The expression vector pET-28a-hlb was constructed and transformed into E.coli BL21(DE3).The expression vector pET28a-hlbH-149-Nwas constructed through point mutation.The recombinant Hlb and Hlb H-149-N protein were expressed and purified by Ni 2+affinity chromatography .The hemolytic activity of Hlb and Hlb H-149-N was measured by sheep erythrocyte lysis assay .Results Recombinant Hlb protein and the mutant were obtained .Further investigations showed that Hlb could significantly induce the lysis of SRBC while HlbH-149-N could not.The specific polyclonal antibodies against Hlb (anti-Hlb) were prepared.It was found that anti-Hlb recognized Hlb and Hlb H-149-N .Moreover , it was found that anti-Hlb blocked the hemolytic activity of Hlb .Conclusion The recombinant Hlb protein with high hemolytic activity and Hlb H-149-N without hemolytic activity are obtained while its neutralized antibody is pepared .Hlb from S.aureus has different hemolytic effects on erythrocytes from various species .Our findings will facilitate the investigation on the role of Hlb in the pathogenesis of S.aureus.

Analysis of the hemolysin and virulence-related genes of incomplete hemolysis Staphylococcus aureus / 中华临床感染病杂志

Objective To analyze hemolysin and virulence -related genes in incomplete hemolytic Staphylococcus aureus.Methods Fifty strains of incomplete hemolytic Staphylococcus aureus were isolated from patients admitted in the Second Affiliated Hospital of Soochow University during 2013 and 2014, and the isolates with complete hemolytic phenotype were also collected at the same period as the control strains . All the strains were inoculated and subcultured on four kinds of sheep blood agar plates supplied by different manufacturers to compare their hemolytic phenotype .The relative mRNA expressions of hemolysin genes (hla, hlb, hlc, hld) in standard strain, complete and incomplete hemolytic phenotype strains were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and valued by 2 -△△Ct method.t test was used to compare mRNA expressions of hemolysin genes .Western blot was performed to analyze the expression of α-hemolysin.Antibiotic susceptibility test of incomplete hemolytic strains was performed using broth microdilution method.Resistant gene mecA and virulence genes pvl, tst were detected by PCR.Results The steady and hereditary incomplete hemolysis was observed in 50 strains of incomplete hemolytic Staphylococcus aureus on the sheep blood agar plates from different suppliers .Taking mRNA expression of hla, hlb, hlc, hld in standard strain as 1, the relative mRNA expressions of hemolysin genes in incomplete hemolytic strains were 0.02, 7.51, 0.06 and 0.12 respectively, there were statistical differences between standard strain and incomplete hemolytic strains (t =8.46, -56.40, 8.12 and 7.61, all P <0.05).And the expression of α-hemolysin was decreased in incomplete hemolytic strains .All the strains were identified as methicillin resistant Staphylococcus aureus (MRSA).Three strains exhibited different minimum inhibitory concentrations of teicoplanin and linezolid after subcultured , but the differences had no impact on the final results of antibiotic susceptibility test .mecA, pvl and tst genes were positive in incomplete hemolytic strains . Conclusion Staphylococcus aureus with incomplete hemolytic phenotype is methicillin resistant with higher expression of β-hemolysin and lower expressions of α-hemolysin, γ-hemolysin and δ-hemolysin.It carries plv and tst virulence genes and is of high virulence .

Detection of virulent of Klebsiella isolates from ñlinical samples / Монголын Анагаах Ухаан

IntroductionKlebsiella spp is a well-known opportunistic pathogen associated with nosocomial infections such asurinary tract, septicaemia and pneumonia number of multi-drug resistant strains and infections causedby Klebsiella has progressively increased, causing treatment limitations.GoalIdentify of phenotype of Klebseilla isolates from ñlinical samplesMaterials and MethodsA total of 112 Klebsiella strains were isolated from clinical samples in State Central First Hospital and StateCentral Third Hospital from July 2015 through December 2015. The bacterial isolates were identifi edaccording to cultural characteristics, biochemical test and API20E. The serum resistance, capsule andhypermucoviscosity, cell surface protein (curly), a-hemolysin and ability to form biofi lm were sought byphenotypic assays. Antimicrobial susceptibility was tested by diffusion method.ResultA total of 112 Klebsiella samples were collected. The bacterial isolates were identifi ed according tocultural characteristics, biochemical test and API20E, the results revealed that 16.1 percent isolateswere identifi ed as K.oxytoca all of them 83.9 percent isolates were belong to K.pneumonia. Therewere observed for ampicillin (99 percent), nitrofurantoin (53.6 percent), cepalotin (50.6 percent) and51 percent of isolates were considered as a multiple drug resistant. Serum resistance properties ofK.pneumoniae was resistance 89.4 percent, intermediately susceptible 4.3 percent, sensitive 6.4percent and for K.oxytoca resistance 88.9 percent, intermediately susceptible 5.6 percent, sensitive 5.6percent. The hemolysin àalpha was detected in 32.2 percent, and gamma, beta in 66.96 percent, 0.9percent respectively. The capsule was observed in 46.5 percent and hypermucoviscosity in 27.7 percentof isolates. The cell surface protein (curly) and biofi lm were detected in 100 percent.Conclusion:Both K.pneumoniae and K.oxytoca isolates from clinical samples have similar virulent properties, andthe a-hemolysin and hypermucoviscosity positive isolates were more resistance to antibiotics.

Amplification of tlh gene in other Vibrionaceae specie by specie-specific multiplex PCR of Vibrio parahaemolyticus

Background The surveillance of Vibrio parahaemolyticus in the Chilean coast has been mainly performed by multiplex PCR amplification of three different hemolysin genes, which are specie-specific virulence factors. These genes are also employed in the determination of V. parahaemolyticus pathogenic load in seafood and for characterization of pathogenic strains associated to diarrhea cases in human. During environmental surveillance that we performed every summer, we occasionally observed a thermolabile hemolysin (tlh) PCR product of a slightly smaller size than expected, which was coincident with low loads of V. parahaemolyticus in the environment. In order to understand this observation, we probed the specificity of tlh primers for the detection of V. parahaemolyticus at different bacterial loads and DNA concentrations. Results Primers used for the detection of V. parahaemolyticus specific tlh amplified a slightly smaller tlh gene, which is found in Vibrio alginolyticus and other related strains. These amplicons were observed when V. parahaemolyticus was absent or in undetectable loads in the environment. Conclusions Surveillance of V. parahaemolyticus using tlh primers can be imprecise because amplification of a V. parahaemolyticus specific marker in V. alginolyticus and other related strains occurs. This situation complicates potentially the estimation of bacterial load in seafood, because do not ensure the correct identification of V. parahaemolyticus when his load is low. Additionally, it could complicate the tracking of outbreaks of V. parahaemolyticus infections, considering the genetic markers used would not be specie-specific.