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PubMed1992

A hemolysin-encoding plasmid contributes to bacterial virulence in experimental Enterococcus faecalis endophthalmitis.

Beta-hemolysin production is a variable trait of the Lancefield group D streptococcus, Enterococcus faecalis. The E. faecalis hemolysin is encoded by large transmissible plasmids. The variable nature of this putative virulence factor provided an ideal system for testing its contribution in experimental endophthalmitis. In this study, isogenic E. faecalis strains were compared to determine whether the presence of the hemolysin-encoding plasmid affected the severity of disease in a rabbit endophthalmitis model. Experimental infections (n = 6) with 10(1)-10(4) E. faecalis organisms harboring the hemolysin-encoding plasmid resulted in a 98% loss of retinal function (by electroretinography [ERG]) and white reflex by postoperative day 3. By contrast, infections of similar numbers of plasmid-free E. faecalis organisms (n = 5) resulted in retention of some retinal function (23% per ERG) with a red reflex demonstrated on postoperative day 3. Results of light microscopy, slit-lamp examination, ERG, and indirect ophthalmoscopy indicated that infections with hemolysin-encoding plasmid-containing E. faecalis resulted in a more aggressive endophthalmitis compared with the endophthalmitis caused by plasmid-free E. faecalis. This is the first endophthalmitis model to the authors' knowledge that specifically evaluates bacterial virulence using isogenic strains.

Animals↗

PubMed1995

[Pathogenic bacteria isolated from the sputum of the patients with pulmonary emphysema].

Isolated pathogenic bacteria from sputum of the patients with pulmonary emphysema who were admitted in our hospital from 1984 to 1994 were examined to elucidate the relationship between isolated bacteria from sputum and pulmonary functions including vital capacity (VC), forced expiratory volume (FEV1.0), PaO2 and PaCO2. VC of the patients from whom MSSA (methicillin-sensitive Staphylococcus aureus) or Enterococcus faecalis (E. faecalis) were isolated was significantly lower than that of the patients from whom Streptococcus pneumoniae (S. pneumoniae), Branhamella catarrhalis (B. catarrhalis) or Haemophilus influenza (H. influenza) were isolated. FEV1.0 had a similar tendency as VC in terms of isolated organisms from the patients with emphysema. Similarly, PO2 of the patients from whom MSSA or E. cloacae were isolated was significantly lower than that of the patients from whom S. pneumoniae, B. catarrhalis or H. influenzae were isolated, and PCO2 of the patients from whom S. pneumoniae, B. catarrhalis or H. influenza were isolated. There was also impaired respiratory function in the patients from whom MSSA, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Xanthomonas maltophilia (X. maltophilia) or Enterobacter cloacae (E. cloacae) were isolated, compared with those in the patients from whom S. pneumonia, B. catarrhalis or H. influenzae were isolated. These results suggest that isolated pathogenic bacteria are shifted from S. pneumoniae, B. catarrhalis or H. influenza to MSSA, E. coli, P. aeruginosa, X. maltophilia or E. cloacae in the course of impairment of respiratory function in pulmonary emphysema. The treatment and prophylaxis for acute exacerbation in pulmonary emphysema should be based on these results.

Bacteria↗

PubMed1996

[Clinically isolated bacterii seen in complicated infections].

The spectrum of causative bacterial agents responsible for post-operative infections in the surgical field have dramatically changed with time. This could be due to recent technical progress in bacterial extraction or the result of more effective antibiotics used post-operatively. Nevertheless nosocomial infections including those caused by MRSA still demand our close attention. Commonly extracted bacterii include Staphylococcus spp., E. faecalis, E. coli, Klebsiella spp., E. cloacae, P. aeruginosa and Bacteroides spp., but with regards to pathogenicity, extraction frequency and drug resistance, agents requiring the highest note of caution may be MRSA, P. aeruginosa and Bacteroides spp. MRSA is still the bacterium which makes the most frequent appearance in the clinical field, and the only effective antibiotic to MRSA is vancomycin. In addition, P. aeruginosa is the most common bacterial agent seen in post-operative infections and also the agent to which many of the cephem drugs do not express a high degree of antibacterial effect. Furthermore, about half of the Bacteroides spp. are beta-lactamase-producing strains which deactive the effect of penicillin and cephem drugs within the infection site. A drug which proves effective against E. coli may nevertheless become somewhat ineffective against this agent when found in a mixed infection and if the drug lacks stability against beta-lactamase. Clinically, mixed infections including some or even all the above agents are known to occur, continuously drawing our close attention.

Bacteroides↗

PubMed1998

Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates.

The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.

Anti-Bacterial Agents↗

PubMed1986

Use of penicillin-binding proteins for the identification of enterococci.

The results of 20 physiological and fermentation tests and examination of the penicillin-binding proteins (PBPs) of 85 enterococcal strains demonstrated that the genus Enterococcus could be divided into at least nine distinct species: E. faecalis, E. faecium, E. durans, E. hirae, E. avium, E. gallinarum, E. casseliflavus, E. malodoratus and E. mundtii. Each species had a specific pattern of at least five PBPs, with molecular masses in the range of about 40-130 kDa. The pattern of PBPs may be useful for identification purposes, since some strains with unusual fermentation characteristics were assigned to species by this technique.

Bacterial Proteins↗

PubMed2002

Subgingival microbiota of Brazilian subjects with untreated chronic periodontitis.

BACKGROUND: Different periodontopathogenic microbiota have been associated with periodontal diseases in several populations. The present investigation determined the subgingival microbiota of untreated chronic periodontitis Brazilians using the checkerboard DNA-DNA hybridization technique. METHODS: Twenty-five periodontitis patients (mean age, 41 +/- 2; mean probing depth [PD], 3.3 +/- 0.2; mean attachment level [AL], 3.6 +/- 0.2) with no history of previous periodontal therapy and a control group of 14 healthy subjects (mean age, 34 +/- 0.6; mean PD, 1.8 +/- 0.2; mean AL, 1.7 +/- 0.1) were selected. Measurements of PD, AL, bleeding on probing, plaque accumulation, and suppuration were recorded at 6 sites/tooth. Subgingival plaque samples were obtained from 4 sites in each tooth/subject in both groups. The presence and levels of 41 subgingival species were determined in 4,032 plaque samples using whole genomic DNA probes and the checkerboard method. RESULTS: Periodontal pathogens, as well as some unusual species (E. faecalis, E. coli and Bartonella sp.), were detected significantly more often and/or in higher levels in the periodontitis group (P < 0.05). Most species were more frequently detected in interproximal sites. B. forsythus, P. gingivalis, E. nodatum, and F. nucleatum ss vincentii showed a significant positive correlation with mean PD and AL (P < 0.05). CONCLUSIONS: The subgingival microbiota of Brazilians with untreated chronic periodontitis were complex, including high proportions of periodontopathogens commonly found in other populations, as well as some unusual species.

Adult↗

PubMed2003

Species identification and detection of vancomycin resistance genes in enterococci of animal origin by multiplex PCR.

A multiplex PCR assay for the differentiation of species and detection of vancomycin (van) resistance genes in enterococci was established. Three hundred sixty-seven enterococcal strains isolated from food were selected from a sample collection and were examined with regards to the existence of four vancomycin resistance genes (vanA, vanB, vanC1 and vanC2) and to species differentiation (E. faecalis, E. faecium, E. casseliflavus and E. gallinarum) by multiplex PCR. Apart from unambiguous results for the majority of strains, the E. faecium specific primers used here, showed weak points in the specificity and sensitivity, respectively. Despite these rare instances, we succeeded in optimising the multiplex PCR assay by variation of annealing temperature and primer combination. The assay presented here, with its optimised parameters, is therefore suitable for routine use and because of time and material saving, it is an alternative and rapid method in comparison to conventional tests.

Animals↗

PubMed2005

Bacteria induce expression of apoptosis in human spermatozoa.

An increased number of sperm undergoing apoptosis has been observed during inflammatory processes in the male genital tract, which might be associated with elevated reactive oxygen species (ROS) levels. However, another factor to stimulate apoptosis could be the direct contact with bacteria or its products, even in the absence of ROS. The aim of this study was to investigate whether bacteria can directly initiate apoptosis in human spermatozoa. Human spermatozoa selected by density gradient centrifugation were incubated with polymorphonuclear granulocytes (PMN) isolated from blood and/or E. faecalis, E. coli or S. aureus. As ROS inductor in PMN, phorbol-12-myristate-13-acetate was used. After incubating the cells for 60 min at 37 degrees C, ROS were determined by chemiluminescence and phosphatidyl serine (PS) externalization was analyzed by flow cytometry with Annexin V-FITC and propidium iodide (PI). The increase in the percentage of spermatozoa Annexin V-FITC-positive/ PI-negative (early event of late apoptosis) was significant after the incubation with PMN plus PMA, PMN plus E. coli and E. coli alone. The percentage of spermatozoa Annexin V-FITC-positive/ PI-positive (apoptosis/necrosis) increased significantly in sperm incubated with E. coli and S. aureus (20.3% +/- 3 and 13.6% +/- 3.2 compared to sperm alone, 6% +/- 0.5). Sperm incubated with PMN-PMA activated showed only a relative increase in apoptosis/necrosis (8.4% +/- 1). Our results show that bacteria directly increase the PS externalisation in ejaculated human sperm. This way of inducing apoptosis does not require external ROS and may result from anyone of the molecular mechanisms that account for changes in motility, vitality and DNA integrity, that are characteristics of spermatozoa in male genital tract infection.

Apoptosis↗

PubMed1995

Characterisation and antimicrobial susceptibility of Enterococcus species from the intestines of ducks in Assam.

The Enterococcus flora of duck intestines consisted of E faecalis and E faecium in ducklings less than eight weeks old and E faecalis, E faecium and E gallinarum in ducks more than eight weeks old. The enterococci were resistant to several antibiotics; chloramphenicol and gentamycin sulphate were the only antibiotics of those tested which were moderately effective. All the Enterococcus species isolates were resistant to the macrolide and lincosamide antibiotics.

Age Factors↗

PubMed2002

Preparation and antibacterial activity of 3-methyl-1-p-substituted phenylpyrazole-5-thiol.

3-Methyl-1-phenylpyrazole-5-thiol (3a) and its p-nitro- (5) and p-fluorophenyl (8) derivatives were prepared as potential antimicrobial agents in relatively good yields. Compounds 3a and 8 showed good antibacterial activities against MRSA, S. aureus, S. epidermidis, E. faecalis, E. faecium, and S. pyogenes. Moreover, compound 3a also showed a synergistic effect with some aminoglycosides.

Anti-Bacterial Agents↗

PubMed2002

Preparation and Antibacterial Activity of 3-Methyl-1-p-substituted Phenylpyrazole-5-thiol.

3-Methyl-1-phenylpyrazole-5-thiol (3a) and its p-nitro-(5) and p-fluorophenyl (8) derivatives were prepared as potential antimicrobial agents in relatively good yields. Compounds 3a and 8 showed good antibacterial activities against MRSA, S. aureus, S. epidermidis, E. faecalis, E. faecium, and S. pyogenes. Moreover, compound 3a also showed a synergistic effect with some aminoglycosides.

Journal Article↗

PubMed2000

Identification of enterococci at the species level by sequencing of the genes for D-alanine:D-alanine ligases.

A PCR assay based on the use of degenerate oligodeoxyribonucleotides allowed characterization of a fragment internal to the ddl genes encoding D-alanine:D-alanine ligases in Enterococcus columbae, E. durans, E. malodoratus, E. mundtii, E. raffinosus, E. seriolicida, E. solitarius, and E. sulfureus. Phylogenetic analysis of the sequence of the amplification products and of those already obtained from E. aviuni, E. casseliflavus, E. cecorum, E. dispar, E. faecalis, E. faecium, E. flavescens, E. gallinarurm, E. hirae, E. pseudoavium, and E. saccharolvticus yielded an evolutionary tree with a topology similar to that based on 16S rRNA sequences. Partial sequencing of the ddl gene can therefore be used for genotypic identification of Enterococcus spp.

Amino Acid Sequence↗

PubMed2001

Occurrence of the vanA and vanC2/C3 genes in Enterococcus species isolated from poultry sources in Malaysia.

Enterococcus species isolated from poultry sources were characterized for their resistance to antibiotics, plasmid content, presence of van genes and their diversity by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The results showed that all isolates were multi-resistance to the antibiotics tested. Ampicillin (15/70) followed by chloramphenicol (37/70) were the most active antibiotics tested against the Enterococcus spp. isolates, while the overall resistant rates against the other antibiotics were between 64.3% to 100%. All vancomycin-resistant E. faecalis, E. durans, E. hirae and E. faecium isolates tested by the disk diffusion assay were positive in PCR detection for presence of vanA gene. All E. casseliflavus isolates were positive for vanC2/C3 gene. However, none of the Enterococcus spp. isolates were positive for vanB and vanC1 genes. Plasmids ranging in sizes between 1.1 to ca. 35.8 MDa were detected in 38/70 of the Enterococcus isolates. When the genetic relationship among all isolates of the individual species were tested by RAPD-PCR, genetic differences detected suggested a high genetic polymorphisms of isolates in each individual species. Our results indicates that further epidemiological studies are necessary to elucidate the role of food animals as reservoir of VRE and the public health significance of infections caused by Enterococcus spp.

Animals↗

PubMed2001

Lactobacillus agilis is an important component of the pigeon crop flora.

AIMS: To examine the presence of lactic acid bacteria (LAB: enterococci, streptococci and lactobacilli) in the pigeon crop. METHODS AND RESULTS: The crops of 10 pigeons were sampled and inoculated on agar plates for isolation of streptococci, enterococci and lactobacilli. The isolates were identified using tDNA-PCR. Lactobacillus agilis, a species described in 1981 from municipal sewage, was the dominant component in eight of these pigeon crop sacs. A Lactobacillus species related to L. fermentum and L. mucosae but probably not belonging to one of these species was isolated from five birds. Three pigeons carried Enterococcus cecorum. Minor species found were E. columbae, E. faecalis, E. hirae, L. johnsonii, L. salivarius, and Streptococcus gallolyticus. A description is given of the phenotypic characteristics of the L. agilis pigeon strains. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: L. agilis is found to be the main component of the LAB flora in the pigeon crop.

Animals↗

PubMed1999

Calibration of disk diffusion antibiotic susceptibility testing: species-related trovafloxacin interpretive zone breakpoints and selection of disk potency.

International comparisons of antibiotic susceptibility require the use of common minimum inhibitory concentration (MIC) limits. Disk diffusion test results are not directly suitable for such comparisons, since different standards are often used and zone breakpoints issued might reflect different MIC limits. We have used single strain regression analysis (SRA) for the calibration of the disk test, both according to species and individual laboratory, and for quality control of trovafloxacin disk diffusion tests in 5 laboratories in Sweden. Preliminary controls using histogram analysis including subtraction histograms of reference strains revealed marked differences between different laboratories. SRA was performed on 4 reference strains, S. aureus, E. faecalis, E. coli and P. aeruginosa, using disks containing 1, 3, 10, 30 and 100 microg trovafloxacin. The results using SRA showed a difference between laboratories using Biodisk PDM medium, which produced smaller zones, and those using Oxoid IsoSensitest. Species-related regression lines for laboratories using either medium were calculated and corresponding interpretive zone breakpoints determined for MIC limits. Rational criteria for the selection of a suitable disk content of an antibiotic were also defined and applied to trovafloxacin. The 10 microg disk selected by NCCLS (National Committee for Clinical Laboratory Standards) proved optimal.

Anti-Infective Agents↗

PubMed2000

Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy.

The emergence of vancomycin-resistant enterococci (VRE) in Europe has been ascribed to the long-time use of the glycopeptide antibiotic avoparcin as feed additive in food animals, until its ban in 1997 in EU. The pres- ence of VRE in food of animal origin is believed to represent a potential risk for the consumer. We studied the fecal carriage of VRE in broiler chickens and slaughter pigs in Italy before the avoparcin ban and eval- uated the impact of avoparcin withdrawal on the presence of VRE in raw meat products. Broilers and pigs were both found to be frequently colonized by VRE, as 36% and 24.6% of the flocks or the herds, respec- tively, were positive. Molecular typing of VRE strains by PFGE showed that animals housed in different pens within the same farm were colonized by clonally related strains. After the avoparcin ban, a decrease in the rate of VRE contamination in meat products was observed. Such a decrease was statistically significant in poultry (from 18.8% to 9.6%) but not in pork products (from 9.7% to 6.9%). The majority of VRE from all sources carried the vanA resistance gene and included Enterococcus faecium, E. faecalis, E. hirae, E. durans, and E. gallinarum. None of the strains carried the vanB gene, whereas constitutively resistant vanC-positive strains were frequently found. Our results show that avoparcin withdrawal has been successful in reducing VRE contamination in meat products. However, this measure needs to be complemented by a prudent use of glycopeptide antibiotics in human medicine.

Animals↗

PubMed2001

Evaluation of ribotyping for characterization and identification of Enterococcus haemoperoxidus and Enterococcus moraviensis strains.

Seven Enterococcus moraviensis and 16 Enterococcus haemoperoxidus as well as nine reference cultures of other enterococcal species obtained from the Czech Collection of Microorganisms were characterized using ribotyping with EcoRI and HindIII in the present work. The ribopatterns obtained by both restriction enzymes clearly distinguished all E. moraviensis and E. haemoperoxidus strains from the other enterococci (E. faecalis, E. faecium, E. avium, E. raffinosus, E. pseudoavium, E. malodoratus) and they differentiated both species from each other as well. Although all strains were isolated from different sampling sites, many strains shared the same band patterns. E. moraviensis formed four ribogroups using EcoRI and two ribogroups using HindIII restriction enzyme. E. haemoperoxidus gave six different patterns with EcoRI and five using the HindIII restriction enzyme.

DNA, Bacterial↗

PubMed2006

Using DNA microarrays to identify library-independent markers for bacterial source tracking.

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.

Animals↗