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PubMed2006

Detection of human-derived fecal pollution in environmental waters by use of a PCR-based human polyomavirus assay.

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 microl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking "toolbox."

Animals↗

PubMed2007

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates.

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.

Anti-Bacterial Agents↗

PubMed1997

Are clinical laboratories in California accurately reporting vancomycin-resistant enterococci?

In order to determine whether hospital-based clinical laboratories conducting active surveillance for vancomycin-resistant enterococci in three San Francisco Bay area counties (San Francisco, Alameda, and Contra Costa counties) were accurately reporting vancomycin resistance, five vancomycin-resistant enterococcal strains and one vancomycin-susceptible beta-lactamase-producing enterococcus were sent to 31 of 32 (97%) laboratories conducting surveillance. Each strain was tested by the laboratory's routine antimicrobial susceptibility testing method. An Enterococcus faecium strain with high-level resistance to vancomycin (MIC, 512 microg/ml) was correctly reported as resistant by 100% of laboratories; an E. faecium strain with moderate-level resistance (MIC, 64 microg/ml) was correctly reported as resistant by 91% of laboratories; two Enterococcus faecalis strains with low-level resistance (MICs, 32 microg/ml) were correctly reported as resistant by 97 and 56% of laboratories, respectively. An Enterococcus gallinarum strain with intrinsic low-level resistance (MIC, 8 microg/ml) was correctly reported as intermediate by 50% of laboratories. A beta-lactamase-producing E. faecalis isolate was correctly identified as susceptible to vancomycin by 100% of laboratories and as resistant to penicillin and ampicillin by 68 and 44% of laboratories, respectively; all 23 (74%) laboratories that tested for beta-lactamase recognized that it was a beta-lactamase producer. This survey indicated that for clinically significant enterococcal isolates, laboratories in the San Francisco Bay area have problems in detecting low- to moderate-level but not high-level vancomycin resistance. Increasing accuracy of detection and prompt reporting of these isolates and investigation of cases are the next steps in the battle for control of the spread of vancomycin resistance.

Ampicillin↗

PubMed1992

[The effect of culture media on bacteriocin production in various strains of bacteria].

Some staphylococcus and enterococcus strains were used to investigate the effect of culture medium on bacteriocin production. Staphylococcus cohnii SC7, Staphylococcus sp. ZTJ 151, S. saprophyticus SS 877, Enterococcus faecium EF1 and E. faecalis EFG2 were isolated from the rumen wall and contents of lambs, calves and fallow deer, Enterococcus gallinarum EG10 and E. avium EA12 were isolated from the caecum of Japanese quail. The tested bacteria belong to producers with a wide antimicrobial effectiveness spectrum, they have low to medium adherence and urease activity (Tab. III). These culture media were used to test the effect of culture medium on bacteriocin production: nutrient agar no. 2 and VL agar enriched with 2% of glucose and lactose (ZAG, ZAL, VLG, VLL), agar for isolation of faecal streptococci (SA) and the base for blood agar no. 4 and no. 2 (KA4, KA2). The strains Streptococcus bovis AO 24/85 and Staphylococcus aureus Oxford 209 P were used as indicator bacteria. Tables I and II show the results of these tests. The tested strains produced the widest inhibition zones (6 mm) with both indicators on SA medium, and this indicates massive bacteriocin production. On ZAG medium, the zones of enterococci with the AO 24/85 strain were larger size than those of staphylococci, but the zones were dim. All strains with the 209P indicator produced dim zones of the 2mm size. The larger inhibition zones (2-5mm) in comparison with staphylococci were observed in enterococci on the ZAL medium with the AO24/85 strain. The production of tested strains was balanced on VLG agar with respect to the use of both indicators.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriocins↗

PubMed2002

Antimicrobial activity of imipenem against isolates from complicated urinary tract infections.

Antimicrobial activity of imipenem was measured using 4725 strains isolated from patients with complicated urinary tract infections (CUTIs) between 1988 and 2000. Imipenem was inactive against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, Enterococcus faecium and some non-fermenting Gram-negative rods. Resistant strains (MIC>16 mg/l) were observed in Staphylococcus haemolyticus (22%), Enterococcus faecalis (4%), Enterococcus avium (8%), Serratia marcescens (5%) and Pseudomonas aeruginosa (7%). Although the prevalence of imipenem-resistant strains of S. aureus, S. epidermidis and P. aeruginosa was sporadically high in some years, no steady increase was seen over the period. Resistant strains were rare in other major uropathogenic species. These results suggest that imipenem is still one of the most reliable antimicrobial drugs.

Anti-Bacterial Agents↗

PubMed2002

Clinical and bacteriological results from the administration of Cefepime (Maxipime) in patients with severe sepsis.

BACKGROUND: The aim of the present study was to investigate the clinical and bacteriological efficacy of Cefepime administered to critically ill sepsis patients, as well as possible adverse reactions and interactions. MATERIAL/METHODS: 30 patients with severe sepsis of different origin were treated with Cefepime a dosage of 2.0 g bid. Of these patients, 26 survived (86.67%). The clinical and bacteriological effects of antibiotic administration were recorded at the end of treatment. RESULTS: The treatment outcomes in terms of clinical results were as follows: 17 patients cured (56.67%), 7 improved (23.33%), 2 with unsatisfactory outcome (6.67%), and 4 failures (13.33%). Cefepime treatment resulted in a reduction of the APACHE II score from ca. 20 at the beginning to 9.44 at the end, while the SOFA score fell from 7 to 2.62. This indicates improved clinical status and reversal of multiple organ failure. The bacteriological results were as follows: pathogen eradication in 17 cases (56.67%), pathogen persistence in 8 cases (26.67%), and superinfection in 5 cases (16.66%). Out of 80 total hemocultures, 63 proved to be sterile. In two cases in vitro Enterococcus faecium was isolated with antibiotic resistance. Of 81 total cultures of infectious foci, 10 showed resistance: 4 Pseudomonas aeruginosa, 4 Acinetobacter baumannii, and 2 Enterococcus faecalis. No side effects of Cefepime administration were recorded. CONCLUSIONS: Cefepime administration resulted in low mortality, clinical improvement, minimal resistance, and lack of side effects. Cefepime is an effective drug of first choice for critically ill sepsis patients

Adult↗

PubMed2003

Purification and characterization of a novel bacteriocin produced by Enterococcus faecalis strain RJ-11.

Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.

Amino Acid Sequence↗

PubMed2005

Pleiotrophic effects of 2 Enterococcus faecalis sagA-like genes, salA and salB, which encode proteins that are antigenic during human infection, on biofilm formation and binding to collagen type i and fibronectin.

BACKGROUND: We have shown previously that Enterococcus faecium SagA has broad-spectrum binding to extracellular matrix (ECM) proteins. In the present study, 2 sagA-like genes, salA and salB, were identified in Enterococcus faecalis. METHODS: We compared the salA and salB mutants; their parental strain, OG1RF; and the salB-complemented strain for binding to ECM proteins and biofilm formation. RESULTS: The salB mutant (TX5123) grew more slowly but showed greater binding (approximately 10%-20% of cells bound) to fibronectin (FN) and collagen type I (CI) than did OG1RF (approximately 1% of cells bound) (P<.001). Although TX5123 showed decreased biofilm formation in tryptic soy broth plus 0.25% glucose (TSBG) (P<.001 vs. OG1RF), a marked increase in biofilm formation was shown by TX5123 but not by OG1RF when they were grown in TSBG plus horse serum (HS) or TSBG plus FN, and the increase was coincident with increased attachment and hydrophobicity of TX5123. Complementation of the salB mutant restored the wild-type phenotypes. CONCLUSIONS: Whether SalB expression is ever sufficiently low in vivo to enhance adherence to ECM proteins or the serum-elicited increase in biofilm formation seen with the salB mutant in vitro is not currently known, but it is a potential way in which this organism could increase its adherence and biofilm formation during infection.

Bacterial Adhesion↗

PubMed2004

Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin.

The quinolone resistance determining regions of gyrA and parC in four species of enterococci from environmental samples with reduced susceptibility to ciprofloxacin were sequenced. The nucleotide sequence variations of parC could be related to the different enterococcal species. Mutations in Enterococcus faecalis and Enterococcus faecium related to reduced susceptibility were identical to mutations detected in E. faecalis and E. faecium of clinical origin. A minimal inhibitory concentration of 8 microg ml(-1) to ciprofloxacin was not associated with any mutations in the gyrA and parC gene of Enterococcus casseliflavus and Enterococcus gallinarum. These two species may be intrinsically less susceptible to ciprofloxacin.

Amino Acid Sequence↗

PubMed2001

Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci.

Five genotypes of acquired glycopeptide resistance have been documented in enterococci, with vanA and vanB being the most globally widespread and prevalent. Resistance results from the production of peptidoglycan precursors with reduced binding affinity for glycopeptides and is encoded by complex clusters of van genes. The prototype VanA element is Tn1546, a 10.8-kb transposon that carries the vanRSHAXYZ genes. Diverse VanA elements exist, but all share the vanRSHAX cluster and are believed to be derived from a Tn1546 progenitor. The sequences of these genes are remarkably conserved, with only a few point mutations identified. VanA elements do however vary by the presence of deletions and insertion sequences (IS) in nonessential genes (orf1, orf2, vanY, and vanZ) and intergenic regions. IS transposition probably plays a key role in VanA element evolution. By contrast, vanB gene clusters show greater sequence divergence. Three vanB alleles have been reported, of which vanB2 appears to be the most widespread, generally as part of Tn5382 and related elements. To date, only four Enterococcus faecium strains with VanD resistance have been reported, and each contained a distinct vanD allele. The VanE and VanG types have each been identified in single strains of Enterococcus faecalis. The existence of distinct genotypes, together with the allelic nature of vanB and vanD, suggests that van clusters have transferred to enterococci on multiple occasions from undefined donor species, with subsequent horizontal dissemination, particularly of VanA and VanB elements, among enterococci. Characterization of glycopeptide resistance elements yields information on their evolution and broadens our insights into the epidemiology of resistant enterococci.

Anti-Bacterial Agents↗

PubMed1998

Occurrence of bacteriocin production among environmental enterococci.

The occurrence of enteroccoci in cattle dung water from the basins of 25 cattle farms of 15 northeastern Slovakia districts was screened as well as bacteriocin production among selective enterococcal isolates. The average total count of enterococci detected reached 5.0 x 10(3) cfu ml-1. Enterococcus faecium was the predominant species (25%) followed by Ent. casseliflavus (19.2%), Ent. faecalis (9.6%), Ent. avium and Ent. durans (1.9%). The antagonistic activity of isolates showed a mainly antilisterial effect. Enterococcus faecalis V24 strain produced a heat stable, largely hydrophobic antimicrobial substance with best production in the range pH 4 to 7 and with a strong inhibitory effect even against Gram-negative bacteria.

Animals↗

PubMed1999

Practical approach to the identification of clinically relevant Enterococcus species.

Enterococci have become important nosocomial pathogens, with Enterococcus faecalis and then Enterococcus faecium predominating. Because of the emergence of glycopeptide (vancomycin and teicoplanin) resistance in enterococci, laboratories have been required to screen for resistant strains and to identify them to the species level. This has resulted in the need for accurate identification of species less commonly associated with clinical infections, such as Enterococcus casseliflavus and Enterococcus gallinarum, which are inherently resistant to the glycopeptides. Studies evaluating commonly used commercial identification systems, have found error rates for enterococcal species identification of 2-21% for E. faecalis, 5-9% for E. faecium, and 14-79% for other species. Reporting errors may have adverse effects on the management of clinical infections, as well as in the control of multidrug-resistant strain outbreaks. The purpose of this document is to present a simplified approach to the identification of Enterococcus species that uses a combination of rapid, readily available, and inexpensive tests.

Anti-Bacterial Agents↗

PubMed1996

Selective isolation of vancomycin-resistant enterococci.

Broth formulations of two media selective for enterococci, Enterococcel, M-Enterococcosel broths were supplemented with 6 micrograms of vancomycin per ml and evaluated for isolation of vancomycin-resistant enterococci (VRE). Each broth was challenged with various concentrations of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and vancomycin-susceptible and vancomycin-resistant enterococci and with 193 perianal specimens obtained from patients at risk in our institution for VRE colonization. Both the Enterococcosel and M-Enterococcus broths with vancomycin detected as few as 1 to 9 CFU of VRE while inhibiting growth of the other organisms tested. Enterococcus faecium organisms (MIC, > 256 micrograms/ml) were recovered from 66 perianal swab cultures in the enterococcosel-vancomycin broth, and VRE were recovered from 62 perianal swab cultures in the M-Enterococcus-vancomycin broth. Enterococcosel-vancomycin broth detected VRE in perianal specimens 48 h earlier than did M-Enterococcus-vancomycin broth. Enterococcosel broth with 6 micrograms of vancomycin per ml can be used for the rapid and selective isolation of VRE from surveillance specimens.

Anti-Bacterial Agents↗

PubMed2004

High antibiotic susceptibility among bacterial pathogens in Swedish ICUs. Report from a nation-wide surveillance program using TA90 as a novel index of susceptibility.

Local infection control measures, antibiotic consumption and patient demographics from 1999-2000 together with bacteriological analyses were investigated in 29 ICUs participating in the ICU-STRAMA programme. The median antibiotic consumption per ICU was 1147 (range 605-2143) daily doses per 1000 occupied bed d (DDD1000). Antibiotics to which > 90% of isolates of an organism were susceptible were defined as treatment alternatives (TA90). The mean number of TA90 was low (1-2 per organism) for Enterococcus faecium (vancomycin:VAN), coagulase negative staphylococci (VAN), Pseudomonas aeruginosa (ceftazidime:CTZ, netilmicin: NET) and Stenotrophomonas maltophilia (CTZ, trimethoprim-sulfamethoxazole: TSU), but higher (3-7) for Acinetobacter spp. (imipenem:IMI, NET, TSU), Enterococcus faecalis (ampicillin:AMP, IMI, VAN), Serratia spp. (ciprofloxacin:CIP, IMI, NET), Enterobacter spp. (CIP, IMI, NET, TSU), E. coli (cefuroxime:CXM, cefotaxime/eftazidime:CTX/CTZ, CIP, IMI, NET, piperacillin-tazobactam:PTZ, TSU), Klebsiella spp. (CTX/CTZ CIP, IMI, NET, PTZ, TSU) and Staphylococcus aureus (clindamycin, fusidic acid, NET, oxacillin, rifampicin, VAN). Of S. aureus isolates 2% were MRSA. Facilities for alcohol hand disinfection at each bed were available in 96% of the ICUs. The numbers of TA90 available were apparently higher than in ICUs in southern Europe and the US, despite a relatively high antibiotic consumption. This may be due to a moderate ecological impact of the used agents and the infection control routines in Swedish ICUs.

Adult↗

PubMed2003

Enterococci in foods--a conundrum for food safety.

Enterococci form part of the lactic acid bacteria (LAB) of importance in foods. They can spoil processed meats but they are on the other hand important for ripening and aroma development of certain traditional cheeses and sausages, especially those produced in the Mediterranean area. Enterococci are also used as human probiotics. However, they are important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics, but antibiotic resistance alone cannot explain the virulence of some of these bacteria. Virulence factors such as adhesins, invasins and haemolysin have been described. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some harbour virulence traits and generally, Enterococcus faecalis harbours more of them than Enterococcus faecium. Regulations in Europe stipulate that safety of probiotic or starter strains is the responsibility of the producer; therefore, each strain intended for such use should be carefully evaluated. For numerous questions, immediate answers are not fully available. It is therefore suggested that when considering an Enterococcus strain for use as a starter or probiotic culture, it is imperative that each particular strain should be carefully evaluated for the presence of all known virulence factors. Ideally, such strains should harbour no virulence determinants and should be sensitive to clinically relevant antibiotics. In general, E. faecium appears to pose a lower risk for use in foods, because these strains generally harbour fewer recognised virulence determinants than E. faecalis. Generally, the incidence of such virulence determinants among E. faecium strains is low, as compared to E. faecalis strains, probably as a result of the presence of pheromone-responsive plasmids.

Animals↗

PubMed2001

Evaluation of an automated system for the identification and antimicrobial susceptibility testing of enterococci.

The performance of a new version of an automated system panel, the Positive Combo Panel Type 11 of MicroScan WalkAway 96 (WA96; Dade Behring) was evaluated and compared to that of reference methods for the identification and for antimicrobial susceptibility testing of the different enterococcal species. A total of 376 enterococcal isolates were tested. The MicroScan WA96 correctly identified 99.6% (266/267), 78.3% (18/23) and 68.6% (59/86) of Enterococcus faecalis, Enterococcus faecium and species other than E. faecalis and E. faecium, respectively. Although low probability of accurate identification was obtained for 37 (9.8%) strains, the system indicated that supplementary tests were necessary for precise identification of 8 (9.3%) among the 86 strains included in the non-faecalis/non-faecium group and of 3 (13.0%) among the E. faecium isolates. In comparison to the agar screening method, the percentage of agreement for detection of resistance markers by the automated system was 90.2% (37/41) for ampicillin, 90.6% (48/53) for high-level resistance to streptomycin (HLRS), 96.4% (80/83) for high-level resistance to gentamicin (HLRG), and 100% (14/14) for vancomycin. The results indicate that the MicroScan WA96 performed well for the identification of E. faecalis and typical E. faecium isolates, and for the detection of resistance to vancomycin and HLRG. However, the system still needs further improvement in order to provide reliable results for the characterization of the other enterococcal species, including atypical variants of E. faecium.

Autoanalysis↗

PubMed2005

Homofermentative lactic acid bacteria of a traditional cheese, Comlek peyniri from Cappadocia region.

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5.3 after 6 h incubation at 30 degrees C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.

Animals↗

PubMed1997

Enterococci acquire new kinds of resistance.

In recent years, enterococci have become increasingly resistant to a broad range of antimicrobial agents. The development of high-level resistance to aminoglycosides, penicillins, and glycopeptides singly and in combination has important clinical implications. Strains of Enterococcus faecium that are resistant to every useful available antibiotic have been described. Resistance to penicillin can be due to overproduction of penicillin-binding protein (which has low affinity for penicillins) or to production of beta-lactamase. High-level resistance of enterococci to gentamicin is due to the synthesis of a modifying enzyme. In this case, the synergistic activity of the combination of penicillin with any aminoglycoside (except for streptomycin) is totally abolished. Acquired resistance to glycopeptides is often plasmid-mediated and is associated with a major epidemic potential since certain plasmids are self-transferable from E. faecium to a variety of gram-positive organisms, including Staphylococcus aureus.

Cross Infection↗