A small-scale, low-cost isolation system for the incubation and rearing of low bacterial load chicks as a model to study microbial-intestinal interactions

A small-scale, economical isolator system was adapted to hatch and raise chicks in a bacteria-free environment as a means to observe bacterial interactions with the intestinal mucosa during early development. The design and construction of flexible plastic isolators for incubation and brooding are described along with methodologies for preparation of eggs for entry into the isolators, incubation and hatching. Two trials were conducted, the first in August 2005 and the second in March 2006. Results from both trials showed no differences in body weights of chicks raised in isolation when compared with those raised conventionally. Growth of bacteria was detected from rectal swabs at day 2 post-hatch, with both trials, showing a light growth of Bacillus sp., coagulase-negative staphylococci and haemolytic streptococcus in trial 1, and a light growth of Bacillus cereus only in trial 2. Although not germfree, the growth of bacteria in chicks raised in isolation was decreased or absent when compared with chicks raised conventionally. Feed was negative for contamination and surface swabs of equipment were also negative until day 3 post-hatch, suggesting possible contamination within the eggs themselves. Despite the presence of bacterial species, the isolator system was successful in producing low bacterial load chicks for comparison studies with conventionally raised chicks.     

Stimulating effect ofStreptococcus faecalis on phagocytosis in gnotobiotic piglets

The concentration of active phagocytes in peripheral blood remained in germfree pigs up to the age of one year approximately at the same level as found at the age of 7 d and did not exceed 0.3×10⁹/L of blood, whereas a steady increase was established in conventional pigs. Monoassociation of gnotobiotic piglets withStreptococcus faecalis increased during 24 h the concentration of circulating granulocytes and the concentration of active phagocytes. An even more pronounced effect was obtained when formolizedS. faecalis cells were applied intraperitoneally to germfree piglets. This treatment elevated the phagocytosis index also in conventional piglets, as well as in germfree piglets previously given cyclophosphamide.Escherichia coli O 83 or a mixture of anaerobic bacteria did not cause any serious changes in the activity of phagocytosis in gnotobiotic piglets.S. faecalis seems to be a natural factor stimulating both the release of granulocytes from their depots as well as their phagocytic activity.     

无菌小鼠的人工哺乳培育

采用无菌子宫摘取术,在隔离器中剥离子宫取仔１６只,生存１５只,在我国首次采用每２４小时插胃管灌胃５～６次的人工哺乳方法,哺乳１５只,离乳６只。粪便、垫料、棉拭子经无菌检查,均无细菌生长。同时对人工乳成分进行了分析比较,比较了人工乳灭菌温度与时间对各营养成分破坏情况,比较了人工乳、豚鼠母乳与小鼠母乳之间的差异     

Entamoeba histolytica: concurrent irreversible loss of infectivity-pathogenicity and encystment potential after prolonged maintenance in axenic culture in vitro

The NIH-200 strain of Entamoeba histolytica became avirulent after more than 2 yr maintenance in axenic culture in vitro. In an attempt to restore virulence to the amoeba, it was transferred to Locke's egg rice-flour medium with various combinations of the following bacteria: Bacteroides sp., Clostridium perfringens, Escherichia coli, and Streptococcus faecalis. Similar cultures were established with a mixed bacterial flora (comprising many unknown species), with and without rice flour, and an attempt was made to induce encystation. Subsequent inoculation of amoebae from the various amoeba-bacteria cultures into the cecum of germfree and exgermfree guinea pigs harboring the same bacteria, as the culture-produced inoculum did not in any instance produce amoebic lesions or prolonged amoebic infections of the enteric lumen. All attempts to induce encystation were unsuccessful; the amoeba had lost its encystment potential, and this was believed to be intimately related to the irreversible loss of virulence.     

Role of House Flies in the Ecology of Enterococcus faecalis from Wastewater Treatment Facilities

Enterococci are important nosocomial pathogens, with Enterococcus faecalis most commonly responsible for human infections. In this study, we used several measures to test the hypothesis that house flies, Musca domestica (L.), acquire and disseminate antibiotic-resistant and potentially virulent E. faecalis from wastewater treatment facilities (WWTF) to the surrounding urban environment. House flies and sludge from four WWTF (1–4) as well as house flies from three urban sites close to WWTF-1 were collected and cultured for enterococci. Enterococci were identified, quantified, screened for antibiotic resistance and virulence traits, and assessed for clonality. Of the 11 antibiotics tested, E. faecalis was most commonly resistant to tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin, and these traits were intra-species horizontally transferrable by in vitro conjugation. Profiles of E. faecalis (prevalence, antibiotic resistance, and virulence traits) from each of WWTF sludge and associated house flies were similar, indicating that flies successfully acquired these bacteria from this substrate. The greatest number of E. faecalis with antibiotic resistance and virulence factors (i.e., gelatinase, cytolysin, enterococcus surface protein, and aggregation substance) originated from WWTF-1 that processed meat waste from a nearby commercial meat-processing plant, suggesting an agricultural rather than human clinical source of these isolates. E. faecalis from house flies collected from three sites 0.7–1.5 km away from WWTF-1 were also similar in their antibiotic resistance profiles; however, antibiotic resistance was significantly less frequent. Clonal diversity assessment using pulsed-field gel electrophoresis revealed the same clones of E. faecalis from sludge and house flies from WWTF-1 but not from the three urban sites close to WWTF-1. This study demonstrates that house flies acquire antibiotic-resistant enterococci from WWTF and potentially disseminate them to the surrounding environment.     

Recognition of Group D Streptococcal Species of Human Origin by Biochemical and Physiological Tests

The speciation of 262 strains of group D streptococci isolated from human sources is described. One hundred forty-two isolates from blood cultures were included; 96 of these were submitted as isolates from clinical cases of subacute bacterial endocarditis. The results show that 98 Streptococcus faecalis, 29 S. faecalis var. zymogenes, 44 S. faecalis var. liquefaciens, 27 S. faecium, 13 S. durans, 44 S. bovis, and 7 unspeciated S. bovis-like group D isolates were identified. No S. faecium var. casseliflavus, S. equinus, or S. avium (group Q streptococci) were identified among the human isolates. The speciation procedures and techniques are detailed. The procedures and limitations of the tests used are discussed. Ninety-eight percent of the 262 strains were speciated by a spectrum of tests that allowed us to recognize atypical as well as typical strains within species.     

Detection and Enumeration of Fecal Indicator Organisms in Frozen Sea Foods: II. Enterococci

Consistently high recoveries of enterococci as compared to the low numbers of coliforms obtained from the same samples of frozen sea foods are indirect evidence that enterococci are better indicators of contamination in such foods.

The use of azide dextrose broth, modified by the incorporation of bromthymol blue, and of ethyl violet azide broth as presumptive and confirmation tests, respectively, were found to be highly specific for the detection and enumeration of enterococci in these samples. Tetrazolium agar medium, when used as a third step after the confirmation test, provides a reliable differentiation of Streplococcus faecalis types from other group D streptococci. A simple procedure is described for further identification of S. faecalis varieties and other enterococcal species.

Incidence of biotypes within certain species is noted and relationships of these subgroups to the organisms described by other workers is discussed.

The striking resistance of all group D streptococci to dihydrostreptomycin and polymyxin B seems to offer promise for evolving a new selective medium for these organisms.

     

Enterococcus faecalis alters endo-lysosomal trafficking to replicate and persist within mammalian cells

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using in vitro keratinocyte and macrophage infection models, we show that E. faecalis becomes internalized and a subpopulation of bacteria can survive and replicate intracellularly. E. faecalis are internalized into keratinocytes primarily via macropinocytosis into single membrane-bound compartments and can persist in late endosomes up to 24 h after infection in the absence of colocalization with the lysosomal protease Cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in heterotypic intracellular trafficking with partial or absent labelling of E. faecalis-containing compartments with Rab5 and Rab7, small GTPases required for the endosome-lysosome trafficking. In addition, E. faecalis infection results in marked reduction of Rab5 and Rab7 protein levels which may also contribute to attenuated Rab incorporation into E. faecalis-containing compartments. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.     

A technique for rearing germfree piglets obtained without surgery

A relatively simple procedure is described for obtaining germfree piglets which does not involve hysterectomy or hysterotomy. Newborn pigs were delivered into an isolator and their freedom from microbial contamination was ensured by applying bactericidal solutions externally and a combination of antibiotics in solution per os. 40 piglets so derived have been maintained free from detectable micro-organisms, some for up to 140 days. Equipment is described which allowed the long-term holding of these animals so that nutritional balance studies could be completed.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

Complete genome sequence and comparative genomic analysis of Enterococcus faecalis EF-2001, a probiotic bacterium

Enterococcus faecalis is a common human gut commensal bacterium. While some E. faecalis strains are probiotic, others are known to cause opportunistic infections, and clear distinction between these strains is difficult using traditional taxonomic approaches. In this study, we completed the genome sequencing of EF-2001, a probiotic strain, using our in-house hybrid assembly approach. Comparative analysis showed that EF-2001 was devoid of cytolysins, major factors associated with pathogenesis, and was phylogenetically distant from pathogenic E. faecalis V583. Genomic analysis of strains with a publicly available complete genome sequence predicted that drug-resistance genes- dfrE, efrA, efrB, emeA, and lsaA were present in all strains, and EF-2001 lacked additional drug-resistance genes. Core- and pan-genome analyses revealed a higher degree of genomic fluidity. We found 49 genes specific to EF-2001, further characterization of which may provide insights into its diverse biological activities. Our comparative genomic analysis approach could help predict the pathogenic or probiotic potential of E. faecalis leading to an early distinction based on genome sequences.     

Differential Rates of Digestion of Bacteria by Freshwater and Marine Phagotrophic Protozoa

Differential decreases over time of two bacterial species, Escherichia coli and Enterococcus faecalis, in a freshwater and a marine ecosystem were observed and explained by a differential rate of digestion of these bacteria by phagotrophic flagellates and ciliates. For this purpose, fluorescence-labeled bacteria (FLB) were used and prepared from the two species cited above. The number of FLB was observed for 5 days in fresh and marine waters in the presence or absence (0.2-μm-pore-size-filtered water) of natural microbiota. These experiments showed a longer persistence of Enterococcus faecalis FLB as opposed to Escherichia coli FLB in the presence of natural microbiota. Removal of FLB was due to protozoan grazing because no decrease of FLB number was observed in the absence of natural microbiota. In short-term (about 40 min) ingestion experiments, we found similar clearance rates of Escherichia coli and Enterococcus faecalis FLB by assemblages of flagellates from the freshwater and the marine ecosystem and by cultured assemblages of ciliates from the marine ecosystem. Clearance rates of Enterococcus faecalis FLB were greater than those of Escherichia coli FLB for assemblages of ciliates from the freshwater ecosystem. Comparison of rates of ingestion and digestion of FLB by protozoa showed that Escherichia coli FLB were digested and ingested at similar rates. However, Enterococcus faecalis FLB were digested slower than they were ingested. These results suggest that a longer persistence of Enterococcus faecalis as opposed to Escherichia coli can be explained by a differential digestion by flagellates and ciliates in aquatic ecosystems. Moreover, rates of ingestion and digestion were strongly correlated for both FLB types.     

Studies on the phagocytic activity of the reticuloendothelial system (RES) to rough and smoothEscherichia coli in young conventional and germfree guinea pigs

The functional (phagocytic) capacity of the reticuloendothelial system (RES) of young conventional and germfree guinea pigs was studied using thein vivo blood clearance test of living bacteria (rough and smoothEscherichia coli). It was found that as previously shown in newborn germfree piglets, the smooth strain was taken up from the blood stream of germfree guinea pigs very slowly whereas roughEscherichia coli was phagocytosed effectively. The inability of the RES of germfree guinea pigs to phagocytose the smooth strain is not due to a functional incapability of phagocytic cells, but it reflects rather the lack of serum opsonins to this strain. This was demonstrated in experiments in which smooth bacteria, sensitized prior to injection into the blood circulation with specific antiserum, were phagocytosed as effectively as the rough strain. It is assumed that effective phagocytosis of rough strain is due to the presence of non-specific opsonins (e.g. components of the complement system). In young conventional guinea pigs both strains,i.e. smooth and rough, were taken up from the blood stream very effectively thus indicating that sufficient levels of serum opsonins for both strains were present. This fact could be correlated with the finding that in sera of conventional guinea pigs haemagglutinating antibodies to both strains ofEscherichia coli could be detected, whereas in sera of young germfree guinea pigs, no antibodies to usedEscherichia coli strain were found. The importance of serum opsonins for effective phagocytosis of bacteria by RE cellsin vivo is discussed.     

Screening of In Vivo Activated Genes in Enterococcus faecalis during Insect and Mouse Infections and Growth in Urine

Enterococcus faecalis is part of the commensal microbiota of humans and its main habitat is the gastrointestinal tract. Although harmless in healthy individuals, E. faecalis has emerged as a major cause of nosocomial infections. In order to better understand the transformation of a harmless commensal into a life-threatening pathogen, we developed a Recombination-based In Vivo Expression Technology for E. faecalis. Two R-IVET systems with different levels of sensitivity have been constructed in a E. faecalis V583 derivative strain and tested in the insect model Galleria mellonella, during growth in urine, in a mouse bacteremia and in a mouse peritonitis model. Our combined results led to the identification of 81 in vivo activated genes. Among them, the ef_3196/7 operon was shown to be strongly induced in the insect host model. Deletion of this operonic structure demonstrated that this two-component system was essential to the E. faecalis pathogenic potential in Galleria. Gene ef_0377, induced in insect and mammalian models, has also been further analyzed and it has been demonstrated that this ankyrin-encoding gene was also involved in E. faecalis virulence. Thus these R-IVET screenings led to the identification of new E. faecalis factors implied in in vivo persistence and pathogenic potential of this opportunistic pathogen.     

Enterococcus faecalis Subverts and Invades the Host Urothelium in Patients with Chronic Urinary Tract Infection

Bacterial urinary tract infections (UTI) are a major growing concern worldwide. Uropathogenic Escherichia coli has been shown to invade the urothelium during acute UTI in mice and humans, forming intracellular reservoirs that can evade antibiotics and the immune response, allowing recurrence at a later date. Other bacterial species, such as Staphylococcus saprophyticus, Klebsiella pneumonia and Salmonella enterica have also been shown to be invasive in acute UTI. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the elderly population, is poorly understood. Moreover, the species of bacteria involved remains largely unknown. A previous study of a large cohort of non-acute LUTS patients found that Enterococcus faecalis was frequently found in urine specimens. E. faecalis accounts for a significant proportion of chronic bladder infections worldwide, although the invasive lifestyle of this uropathogen has yet to be reported. Here, we wanted to explore this question in more detail. We harvested urothelial cells shed in response to inflammation and, using advanced imaging techniques, inspected them for signs of bacterial pathology and invasion. We found strong evidence of intracellular E. faecalis harboured within urothelial cells shed from the bladder of LUTS patients. Furthermore, using a culture model system, these patient-isolated strains of E. faecalis were able to invade a transitional carcinoma cell line. In contrast, we found no evidence of cellular invasion by E. coli in the patient cells or the culture model system. Our data show that E. faecalis is highly competent to invade in this context; therefore, these results have implications for both the diagnosis and treatment of chronic LUTS.     

Molecular Screening of Enterococcus Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10⁻³ per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3′), aph(6′), and aac(6′)/aph(2′), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.     

Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F

The ability of the vancomycin-resistant Enterococcus faecalis (V583) to restore redox homeostasis via antioxidant defense mechanism is of importance, and knowledge into this defense is essential to understand its antibiotic-resistance and survival in hosts. The flavoprotein disulfide reductase AhpR, composed of the subunits AhpC and AhpF, represents one such vital part. Circular permutation was found to be a feature of the AhpF protein family. E. faecalis (V583) AhpF (EfAhpF) appears to be a representative of a minor subclass of this family, the typically N-terminal two-fold thioredoxin-like domain (NTD_N/C) is located at the C-terminus, whereas the pyridine nucleotide-disulfide oxidoreductase domain is encoded in the N-terminal part of its sequence. In EfAhpF, these two domains are connected via an unusually long linker region providing optimal communication between both domains. EfAhpF forms a dimer in solution similar to Escherichia coli AhpF. The crystallographic 2.3 Å resolution structure of the NTD_N/C domain reveals a unique loop-helix stretch (₄₀₉ILKDTEPAKELLYGIEKM₄₂₆) not present in homologue domains of other prokaryotic AhpFs. Deletion of the unique ₄₁₅PAKELLY₄₂₁-helix or of ₄₁₅PAKELL₄₂₀ affects protein stability or attenuates peroxidase activity. Furthermore, mutation of Y421 is described to be essential for E. faecalis AhpF's optimal NADH-oxidative activity.     

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.