In Vivo Assessment of Growth and Virulence Gene Expression during Commensal and Pathogenic Lifestyles of luxABCDE-Tagged Enterococcus faecalis Strains in Murine Gastrointestinal and Intravenous Infection Models

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P_16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R² > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10⁹ CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.     

Genes important for catalase activity in Enterococcus faecalis

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly.     

In vitro activity of sodium benzoate against clinically relevant Enterococcus faecalis and Enterococcus faecium isolates

The antimicrobial effects of sodium benzoate against Enterococcus faecalis and Enterococcus faecium were investigated. The MIC(90) of sodium benzoate were 64 mg/L for E. faecalis and 32 mg/L for E. faecium, while the MBC(90) were 128 mg/L and 64 mg/L, respectively. Although further studies are required for clinical evidence, sodium benzoate seems to be effective against Enterococcus spp.     

粪肠球菌和屎肠球菌耐药性分析

目的 监测我院肠球菌中粪肠球菌株和屎肠球菌株的耐药性,为临床合理应用抗菌药物提供依据。方法 采用法国生物梅里埃公司的GPI板进行细菌鉴定及药敏试验,应用whonet5软件统计粪肠球菌和屎肠球菌的耐药率。结果 粪肠球菌和屎肠球菌对氯霉素、呋喃妥因、万古霉素有较好体外抗菌活性,耐药率都在50%以下,对万古霉素的耐药率在1%以下。粪肠球菌对青霉素、高水平庆大霉素、环丙沙星、利福平、红霉素等大部分抗菌素的耐药率有逐年下降趋势,而屎肠球菌对环丙沙星、利福平、呋喃妥因等抗菌素的耐药率则有上升趋势,屎肠球菌对大多数抗菌素耐药率都高于粪肠球菌。结论 粪肠球菌和屎肠球菌呈多重耐药,临床用药应结合药敏试验结果合理选择抗菌药物。     

Identification of Aerobically and Anaerobically Induced Genes in Enterococcus faecalis by Random Arbitrarily Primed PCR

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.     

一株替代粪肠球菌的生产用芽胞杆菌筛选及其性质

目的 筛选1株能够产业化、替代粪肠球菌的芽胞杆菌。方法 从健康鸡、鸭、仔猪的粪便与肠道内容物中筛选,采用选择性培养基和耐酸耐胆盐发酵,通过耐人工肠液和人工胃液试验与粪肠球菌比较得到1株产酸能力较好的替代粪肠球菌的芽胞杆菌,并对其性质进行研究。结果 所筛选的芽胞杆菌（GY0520）对人工胃液、人工肠液有很好耐受性,90 ℃水浴15 min存活率为97%,能够产生大量有机酸,有利于提高动物机体的抗病能力及改善其生理性能,但对抗生素有一定的敏感性,不能配伍使用。结论 所筛选的芽胞杆菌能够替代粪肠球菌用于生产,为养殖业的微生态产品提高稳定性提供参考。     

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren’t. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ² test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ² test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.     

Artificial Infections of Pneumocystis carinii in the SCID Mouse and their Use in the In Vivo Evaluation of Antipneumocystis Drugs

A model for the in vivo evaluation of antipneumocystis drugs has been developed in SCID mice infected intratracheally with cryopreserved mouse-derived Pneumocystis carinii. The development of a highly reproducible fatal P. carinii pneumonia occured within 10 weeks (mean survival time ± SEM = 72.2 ± 1.2 days). Continuous administration of dexamethasone (2 mg/liter in the drinking water) exacerbated the rate of onset of severe P. carinii pneumonia (mean survival time ± SEM = 63 ± 1.3 days) in SCID mice. The number of cysts per g of lung homogenate (homogenate counts) were maximal with an inoculum of 20,000 cysts at 6 weeks post infection. Homogenate counts correlated with infection scores (graded assessments of immunofluorescent cysts on lung impression smears) suggesting that infection scoring accurately and rapidly reflects the severity of P. carinii pneumonia in SCID mice. These studies led to the development of a drug screening protocol in which Pneumocystis-free female SCID mice (20–25 g) were started on dexamethasone 7 days prior to IT inoculation with a single dose of 20,000 cysts. Drugs were evaluated either for: a) prophylaxis (continuously from day 1 post infection) or b) treatment (from day 21 post infection) until day 42 post infection, when all mice were killed and infection scores determined. Co-trimoxazole (at 250 mg sulfamethoxazole + 50 mg trimethoprim/kg/day) given in the drinking water was found to be highly effective in both the prophylaxis and treatment of mouse P. carinii pneumonia. Co-trimoxazole remained very effective in the prophylaxis P. carinii pneumonia in the SCID mouse at 125 mg sulfamethoxazole + 25 mg trimethoprim/kg/day p.o. and showed some enhancement of efficacy over sulfamethoxazole alone at 125 mg/kg/day p.o., suggesting limited synergy between sulfamethoxazole and trimethoprim. The results presented provide confirmation of the usefulness and predictability of the model.     

Phospholipid Metabolism in Mouse Sciatic Nerve In Vivo

To probe the activities of various pathways of lipid metabolism in peripheral nerve, six phospholipid-directed precursors were individually injected into the exposed sciatic nerves of adult mice, and their incorporation into phospholipids and proteins was studied over a 2-week period. Tritiated choline, inositol, ethanolamine, serine, and glycerol were mainly used in phospholipid synthesis; in contrast, methyl-labeled methionine was primarily incorporated into protein. Phosphatidylcholine was the main lipid formed from tritiated choline, glycerol, and methionine precursors. Phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol were the main lipids formed from serine, ethanolamine, and inositol, respectively. With time there was a shift in label among phospholipids, with higher proportions of choline appearing in sphingomyelin, glycerol in phosphatidylserine, ethanolamine in phosphatidylethanolamine (plasmalogen), and inositol in polyphosphoinositides, especially phosphatidylinositol 4,5-bisphosphate. We suggest that the delay in formation of these phospholipids, which are concentrated in peripheral nerve myelin, may, at least in part, be due to their formation at a site(s) distant from the sites where the bulk of Schwann cell lipids are made. We propose that separating the synthesis of these myelin-destined lipids to near the Schwann cell's plasma membrane would facilitate their concentration in peripheral nerve myelin sheaths. At earlier labeling times, ethanolamine and glycerol were more actively incorporated into phosphatidylcholine and phosphatidylinositol, respectively, than later. The transient labeling of these phospholipids may reflect some unique role in peripheral nerve function.     

New World Simian Foamy Virus Infections In Vivo and In Vitro

Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a β-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence.     

Infections with Van B phenotype Enterococcus faecium and E. faecalis in patients with immune deficiency during the course of hematologic neoplasms

In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. The aim of this study was to characteristics of the types of resistance to glycopeptide antibiotic and to check the resistance patterns of these pathogens. A trial of monitoring the risk factors for colonization or infection with VRE as well as epidemiological investigation were undertaken. Genus identification of the isolates was performed according to the method of Facklam and Collins, and species were identified using the API 20 Strep test. MICs of different antimicrobial agents were determined by the E-test method. The isolates collected during the investigation demonstrated resistance to multiple antimicrobials, which is a common characteristic of VRE. Isolates were found to be uniformly resistant to penicillin, fluoroquinolones, tetracycline and to high concentrations of aminoglycosides. The only drugs with in vitro activity against the isolates were ampicillin (VRES), linezolid (VRES, VREM) and quinupristin-dalfopristin (VREM). Except for a single VREM isolate, they all revealed the typical VanB phenotype with resistance to vancomycin and susceptibility to teicoplanin. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene claster, including the regulatory genes vanRB and -SB, and the vancomycin--inducible promoter PYB. Our studies have found an association between colonization or infection with VRE and the mean duration of hospital stay, previously administration of glycopeptide, cephalosporins and imipenem. These organisms were a common cause of monoethiological bloodstream infections.     

Effect of various growth media upon survival during storage of freeze-dried Enterococcus faecalis and Enterococcus durans

AIMS: The effects of three different growth media (MRS, M17 and Lee's) on survival during freeze-drying and subsequent storage of six strains of Enterococcus faecalis and two strains of E. durans were investigated. METHODS AND RESULTS: Distinct Enterococcus spp. strains were grown on M17, MRS and Lee's broth, freeze-dried and stored at 20 degrees C in air under darkness. At regular intervals throughout storage, freeze-dried samples were rehydrated and then plated on M17 agar. CONCLUSIONS: A higher survival rate during storage of dried E. durans was obtained when growth occurred in MRS. The same effect was not observed, however, for the majority of E. faecalis strains, which clearly survived better in the dried state when this organism had been grown in M17 or Lee's medium. SIGNIFICANCE AND IMPACT OF STUDY: The survival of the dried Enterococcus spp. tested during storage was shown to be strain-specific and dependent on the growth medium.