The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity.

In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF-alpha) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high-virulence hemolytic isolates induced significantly stronger TNF-alpha production by infected macrophages than the low-virulence unhemolytic bacilli.     

反刍动物产志贺毒素大肠杆菌的分离鉴定和致病潜力分析

产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）是重要的食源性病原,而STEC往往以正常菌群的形式存在于牛羊等反刍动物肠道。[目的] 本研究对牛羊粪便样品中的STEC分离和鉴定并对分离株进行致病潜力分析。从江苏、云南和河北等地共分离到羊源STEC菌株11株,牛源STEC菌株1株,另新疆农业大学佟盼盼组馈赠牛源菌株10株。[方法] 通过细菌选择培养及特异性基因stx1和stx2的检测进行分离鉴定;并通过Vero细胞毒性试验、溶血活性试验和毒力因子的检测分析STEC分离株的致病潜力。[结果] 分离到羊源分离株11株,分离率17.5%（11/63）;分离得到牛源分离株1株,分离率0.7%（1/134）;11株羊源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性;11株牛源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性。11株羊源STEC分离株eae基因携带率为63.6%（7/11）,而11株牛源STEC分离株eae基因携带率仅为9.0%（1/11）。[结论] 结果表明羊源STEC菌株的分离率和致病潜力高于牛源菌株,所以,除牛外,羊作为STEC菌株宿主也应该得到更多的重视。     

Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4⁺ and CD8⁺ T cells and an increase of activated T cells expressing IL-2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-γ plays an important role in the protection of mice from P. carinii infection.     

Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages

We reconfirmed that the LD₅₀S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Early‐shared Mycobacterium bovis bacillus Calmette–Guérin sub‐strains induce Th1 cytokine production in vivo

Interleukin‐12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL‐2 and IFN‐γ. We have previously reported greater IL‐12 production from macrophages infected with early‐shared BCG sub‐strains (ex. BCG‐Japan, ‐Sweden) than from those infected with late‐shared BCG (ex. BCG‐Pasteur and ‐Connaught) 1 . In this study, we investigated the Th1 cytokine‐inducing activity of splenocytes co‐cultured with BCG‐infected DCs. Early‐shared BCG‐infected DCs produced IL‐12 and TNF‐α? Furthermore, when they were co‐cultured with purified protein derivative‐stimulated DCs, the splenocytes of mice immunized with BCG‐Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG‐Connaught. In conclusion, early‐shared BCG sub‐strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.

     

Comparative profiles of intramacrophage behavior of Mycobacterium tuberculosis and Mycobacterium avium complex with different levels of virulence

Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) strains with different levels of virulence in mice were examined for profiles of interaction with murine peritoneal macrophages (Mphis). Their growth rates in Mphis were in these orders: H37Ra strain (attenuated) > H37Rv strain (virulent) for MTB, and N-260 strain (moderate virulence) > MAC N-444 strain (low virulence) for MAC. MTB but not MAC caused the necrotic death of host Mphis in terms of increased release of lactate dehydrogenase from infected Mphis. The MTB H37Ra strain induced a greater production of reactive nitrogen intermediates (RNI) by Mphis than the MTB H37Rv strain did. However, this phenomenon was not observed with MAC, implying less important roles of RNI in the expression of Mphi antimicrobial activity against MAC organisms.     

表达GFP/mCherry白色念珠菌的构建及其在与巨噬细胞互作中的应用

白色念珠菌(Candida albicans)被巨噬细胞吞噬的效率与被吞噬后的形态观察是研究白色念珠菌与巨噬细胞互作的重要内容。【目的】以野生型菌株SC5314为母本,构建能够表达绿色荧光蛋白(green fluorescent protein, GFP)/mCherry的白色念珠菌,应用于巨噬细胞与白色念珠菌互作的研究。【方法】通过生长与形态观察、细胞活性检测及小鼠系统性感染模型确定荧光蛋白的表达对菌株生长、形态与毒力的影响;在共培养条件下,通过流式细胞术及荧光显微镜检测巨噬细胞的吞噬率及白色念珠菌的形态变化。【结果】构建的菌株在表型上与野生型菌株一致,并可用于在共培养下测定巨噬细胞吞噬率的流式细胞术以及观察白色念珠菌的形态变化。【结论】表达荧光蛋白的菌株为研究巨噬细胞与白色念珠菌的互作提供了新方法。     

Diversity and exploration of bioactive marine actinomycetes in the Bay of Bengal of the Puducherry coast of India

The present study was designed to investigate the Puducherry coast of the Bay of Bengal, India for the diversity of bioactive actinomycetes. A total of 50 actinomycete strains were isolated from the marine sediments and most of the strains were belongs to Streptomyces. These strains were identified by means of morphological physiological, biochemical and cultural characteristics. The isolates were subjected to shake flask fermentation and the secondary metabolites were extracted with ethyl acetate and screened for cytotoxicity, hemolytic activity and antimicrobial activity against selected bacterial and fungal pathogens. The cytotoxic activity was evaluated using HeLa cell lines by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay, hemolytic activity on mouse erythrocytes and the antifungal activity was evaluated by MTT cytotoxic assay against Aspergillus niger, Aspergillus fumigatus and Candida albicans. The antibacterial activity was studied against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae. The cytotoxicity and antimicrobial activity of secondary metabolite was found to be concentration dependent and nearly 24% of isolates showed significant antimicrobial, hemolytic and cytotoxic activity. The results of our study indicate the diversity and bioactive potential of marine actinomycetes isolated in the Puducherry coast.     

In Vitro Proliferative Activity of Spleen Cells in Mice Infected with Attenuated Salmonella typhimurium

Proliferative activity of cultured spleen cells obtained from mice 1 to 5 weeks after infection with attenuated strains of Salmonella typhimurium was examined in the presence or absence of lipopolysaccharide (LPS) or concanavalin A (Con A). Spontaneous uptake of ³H-thymidine (TdR) by cells taken from infected mice at the 2nd and 3rd weeks was obviously lower than that by cells from uninfected, control mice. Cells from infected mice at the 4th and 5th weeks also showed a lower proliferative response to LPS than that of the controls. However, the responses of the cells to Con A remained virtually unchanged during the entire period. Furthermore, the reduction of spontaneous ³H-TdR uptake by the cells could be achieved also by the injection of heat-killed instead of living organisms. The T- and B-lymphocyte populations of these spleen cells were examined by the dye exclusion cytotoxic test using rabbit anti-mouse T- and anti-mouse B-lymphocyte sera, respectively. There was some alteration of the populations in the cells, but it did not correlate with the reduction in ³H-TdR uptake. Results of expriments with cultured cells reconstituted with lymphocytes and macrophages isolated from spleen cells suggested that the spontaneous reduction of proliferative activity observed in cells taken from the infected mice could be attributed to the dysfunction of macrophages.     

A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674^T and S. equorum subsp. linens DSM 15097^T, respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA–DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.     

Biotreatment of restaurant wastewater with an oily high concentration by newly isolated bacteria from oily sludge

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P?<?0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.

     

Biomaterial-Associated Infection with Candida albicans in Mice

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial-associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end-point attached heparinized polyethylene (H-PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H-PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non-modified H-PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).     

Characterization of Clinical and Environmental Mycobacterium avium Spp. Isolates and Their Interaction with Human Macrophages

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.     

Immunosuppressive Activities of Peritoneal and Splenic Macrophages in Murine Leprosy: Effect on Lymphocyte Transformation and Tumor Growth

The ability of peritoneal macrophages (PM) and splenic macrophages (SM) to suppress tumor growth and lymphocyte transformation in vitro was studied in infected mice with Mycobacterium lepraemurium (MLM). Both PM and SM of leprous mice showed cytostatic activity against tumor cells in vitro. However, such cells showed significantly less cytostatic activity on a per cell basis than highly activated macrophages obtained from Corynebaclerium parvum-immunized mice. Furthermore, this cytostatic activity declined as the infection progressed. Mitogen-induced transformation of splenic lymphocytes was also suppressed in the presence of adherent PM and SM from leprous mice. PM from leprous mice showed significantly less activity than PM from C. parvum-immunized mice in terms of suppression of lymphocyte transformation. Moreover, PM from leprous mice treated with C. parvum or sodium thioglycollate broth demonstrated significantly less ability to suppress lymphocyte transformation than did PM from similarly treated normal mice or untreated leprous mice. These findings demonstrated that MLM infection stimulates the mononuclear phagocyte system but does not activated it to the extent that it confers enhanced resistance to MLM on the host.     

阿萨希毛孢子菌的溶血活性及生物膜形成能力与其基因型关系研究

目的 分析临床分离自尿路感染患者的阿萨希毛孢子菌的体外溶血活性及生物膜形成能力与其基因型的关系,为临床诊治提供依据.方法 玻璃珠法提取总DNA,并采用PCR技术利用IGS1区特异性引物确定其基因型别;同时,平板法和XTT还原比色法分别检测阿萨希毛孢子菌的溶血活性及生物膜形成能力,并分析其与基因型的关系.结果 10株分离自尿路感染的阿萨希毛孢子菌基因型分别属于Ⅰ、Ⅲ、Ⅳ,其中以Ⅳ型为主;所分离菌株均具有不同程度的溶血活性;除1例分离株外,其余各分离株均具有在聚苯乙烯表面形成生物膜的能力;基因型Ⅲ型菌株具有较强的溶血活性和生物膜形成能力.结论 分离自尿路感染患者的10株阿萨希毛孢子菌以Ⅳ型为主,而其中Ⅲ型菌株表现出较强的溶血活性和生物膜形成能力.     

Virulence ofAeromonas species as assessed through mouse lethality studies

The relative virulence of 32Aeromonas isolates, primarily of clinical origin, were evaluated for mouse lethality by intraperitoneal inoculation of 10⁷ CFU into albino mice. Three categories could be distinguished on the basis of this assay, including a highly virulent group (80%–100% mortality), a low to moderate virulence category (20%–60% mortality), and strains that were completely avirulent. Of theA. sobria isolates tested, 82% fell into the highly virulent category (P<0.005), whereasA. hydrophila strains were intermediate in virulence potential, andA. caviae strains studied were avirulent. There was no apparent correlation between highly virulentAeromonas isolates and phenotypes associated with enterotoxigenicity, hemolytic activity, cytotoxin production, or serum resistance; this suggests that a cell surface property may be important in mouse pathogenicity. The results of these studies indicate that mouse lethality assays may be an appropriate model for the study of invasive disease clinically produced byA. sobria andA. hydrophila.     

Virulence and antibiotic susceptibility of Aeromonas spp. isolated from drinking water

Aeromonas isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, cytotoxins, phospholipase, DNase, hydrophobicity and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics of Aeromonas isolates was also examined. Majority of the isolates displayed hemolytic activity against sheep erythrocytes, while only 7 of the 23 Aeromonas strains displayed DNase activity and 4 of the 23 Aeromonas strains tested were regarded as positive for phospholipase production. Most of the isolates showed cytotoxic activities in culture filtrate dilutions at titer of 1/8 or lower. No general relation between the strain isolated and the ability to interact with epithelial cells could be established. Using the bacterial adherence to hydrocarbons method, most of the strains were classified as highly hydrophilic. All five Aeromonas jandaei strains isolates, 9 of the 12 Aeromonas sp strains and four of the five Aeromonas hydrophila were multidrug resistant. The most active antimicrobial was ciprofloxacin (susceptible in 100% of the isolates), and the least active antibiotic was ampicillin (resistance in 92% of the isolates). The majority of the isolates tested were not killed by chlorine at 1.2 mg/l. Whether the high tolerance to chlorine of Aeromonas isolates can be linked to greater virulence is not know.     

Liposome-mediated therapy of human immunodeficiency virus type-1 and mycobacterium infections

Abstract

We review our recent work on the use of liposomes for the delivery of antiviral agents to human immunodeficiency virus type-1 (HIV-1) infected cells, and antimycobactcrial drugs to cells harboring Mycobacterium avium complex or Mycobacterium tuberculosis. Soluble CD4 has been used to target liposomes to HIV-1-infected cells. Antisense oligodeoxynucleotides have been effectively delivered into HIV-1-infected macrophages using pH-sensitive liposomes. pH-sensitive liposomes with serum stability are being developed as in vivo delivery vehicles. Liposomes encapsulating an HIV-1 protease inhibitor were more effective in inhibiting virus production in infected macrophages than the free drug. Anionic liposomes were found to inhibit HIV-1 infectivity, while cationic liposomes had a differential toxicity for HIV-1-infected macrophages. Lipophilic sulfated cyclodextrins have been synthesized as novel antiviral agents. Liposome-encapsulated ciprofloxacin treatment reduced the number of viable M. avium in macrophages more than the free antibiotic. Liposome-encapsulated paromomycin and sparfloxacin were effective against M. tuberculosis inside macrophages, including multi-drug-resistant strains. Streptomycin encapsulated in liposomes and delivered intravenously or subcutaneously reduced the number of viable M. tuberculosis in infected mice and prevented mortality.