Low prevalence of vancomycin-resistant enterococci in clinical samples from hospitalized patients of the Canary Islands, Spain

Over the last decade vancomycin-resistant enterococci (VRE) have emerged as nosocomial pathogens. The aim of this study was to determine the prevalence of VRE in clinical samples from hospitalized patients in the Canary Islands. From April to November 2000, 437 enterococci were isolated from patients hospitalized at the four main health care centers in those islands. Identification to the species level was performed with the GPS-TA (Vitek 1) or the Wider I system. A PCR assay was used to determine the genotype of glycopeptide resistance (vanA, vanB, vanC1, and vanC2/C3 genes). Only three (0.7%) VRE were detected: one vanA Enterococcus faecalis, and two vanC1 Enterococcus gallinarum. To our knowledge, this is the first VRE study carried out in the Canary Islands hospitals, and the results showed a low prevalence of VRE. Electronic Publication     

Persistence of Vancomycin-Resistant Enterococci in New Zealand Broilers after Discontinuation of Avoparcin Use

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was ≥256 μg/ml for 98.7% of isolates, and the avilamycin MIC was ≥8 μg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.     

Antimicrobial activity of isoprenoid-substituted xanthones from Cudrania cochinchinensis against vancomycin-resistant enterococci.

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against vancomycin-resistant enterococci (VRE). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against five VRE strains (VanA, VanB and VanC) (MICs = 1.56 microg/ml). Four xanthones, 1,3,7-trihydroxy-2-prenylxanthone, gerontoxanthone I, alvaxanthone and isoalvaxanthone, showed weaker antibacterial activity against these VREs (MICs = 3.13-6.25 microg/ml). .     

Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources

Aim: Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin‐resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production. Methods and Results: Among the isolates, 27 (8·9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against Gram‐positive and some Gram‐negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS‐48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL_L gene in eight. Among the strains showing the cylL_L gene, only two E. faecalis expressed a β‐haemolysis. Conclusions: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. Significance and Impact of the Study: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits.     

Phenotypic and genotypic characterization of bacteriocins in clinical <Emphasis Type="Italic">enterococcal</Emphasis> isolates of Tunisia

The presence of bacteriocin structural genes (entA, entB, entP, entQ, entAS-48, entL50A/B, bac31, and cylL) encoding different bacteriocins (enterocin A, enterocin B, enterocin P, enterocin Q, enterocin AS-48, enterocin L50A/B, bacteriocin 31 and cytolysin L, respectively), and the production of bacteriocin activity were analysed in 139 E. faecalis and 41 E. faecium clinical isolates of Tunisia. Forty-eight of 139 E. faecalis isolates (34%) and 7 of 41 of E. faecium isolates (17%) were bacteriocin producers. Sixty-two per cent of the bacteriocin-producing enterococci showed inhibitory activity against L. monocytogenes. Different combinations of entA, entB, entP, and entL50A/B genes were detected among the seven bacteriocin-producer E. faecium isolates, and more that one gene were identified in all the isolates. The entA gene was associated in most of the cases with entB gene in E. faecium isolates. Cyl _LS were the unique genes detected among E. faecalis (in 24 of 48 bacteriocin-producer isolates, 50%). A β-hemolytic activity was demonstrated in 19 of the 24 cyl _LS -positive E. faecalis isolates (79%), this activity being negative in the remaining five isolates. The presence of different bacteriocin structural genes and the production of antimicrobial activities seems to be a common trait of clinical enterococci.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.     

Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers

Wild flowers in the South of Spain were screened for Enterococcus faecalis and Enterococcus faecium. Enterococci were frequently associated with prickypear and fieldpoppy flowers. Forty-six isolates, from 8 different flower species, were identified as E. faecalis (28 isolates) or E. faecium (18 isolates) and clustered in well-defined groups by ERIC-PCR fingerprinting. A high incidence of antibiotic resistance was detected among the E. faecalis isolates, especially to quinupristin/dalfopristin (75%), rifampicin (68%) and ciprofloxacin (57%), and to a lesser extent to levofloxacin (35.7%), erythromycin (28.5%), tetracycline (3.5%), chloramphenicol (3.5%) and streptomycin (3.5%). Similar results were observed for E. faecium isolates, except for a higher incidence of resistance to tetracycline (17%) and lower to erythromycin (11%) or quinupristin/dalfopristin (22%). Vancomycin or teicoplanin resistances were not detected. Most isolates (especially E. faecalis) were proteolytic and carried the gelatinase gene gelE. Genes encoding other potential virulence factors (ace, efaA _fs, ccf and cpd) were frequently detected. Cytolysin genes were mainly detected in a few haemolytic E. faecium isolates, three of which also carried the collagen adhesin acm gene. Hyaluronidase gene (hyl _Efm ) was detected in two isolates. Many isolates produced bacteriocins and carried genes for enterocins A, B, and L50 mainly. The similarities found between enterococci from wild flowers and those from animal and food sources raise new questions about the puzzling lifestyle of these commensals and opportunistic pathogens.     

Occurrence, Structure, and Mobility of Tn1546-Like Elements in Environmental Isolates of Vancomycin-Resistant Enterococci

The occurrence, structure, and mobility of Tn1546-like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored vanA. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as Enterococcus faecium (n = 13), E. casseliflavus (n = 6), E. mundtii (n = 3), E. faecalis (n = 3), and E. durans (n = 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn1546-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn1546 in the prototype strain E. faecium BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for E. casseliflavus strains. Indistinguishable Tn1546-like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to E. faecium BM4105 was demonstrated for E. faecalis, E. mundtii, and E. durans. This study indicates that VRE, including species other than E. faecium and E. faecalis, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn1546-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.     

Nosocomial infection caused by vancomycin‐susceptible multidrug‐resistant Enterococcus faecalis over a long period in a university hospital in Japan

Compared with other developed countries, vancomycin‐resistant enterococci (VRE) are not widespread in clinical environments in Japan. There have been no VRE outbreaks and only a few VRE strains have sporadically been isolated in our university hospital in Gunma, Japan. To examine the drug susceptibility of Enterococcus faecalis and nosocomial infection caused by non‐VRE strains, a retrospective surveillance was conducted in our university hospital. Molecular epidemiological analyses were performed on 1711 E. faecalis clinical isolates collected in our hospital over a 6‐year period [1998–2003]. Of these isolates, 1241 (72.5%) were antibiotic resistant and 881 (51.5%) were resistant to two or more drugs. The incidence of multidrug resistant E. faecalis (MDR‐Ef) isolates in the intensive care unit increased after enlargement and restructuring of the hospital. The major group of MDR‐Ef strains consisted of 209 isolates (12.2%) resistant to the five drug combination tetracycline/erythromycin/kanamycin/streptomycin/gentamicin. Pulsed‐field gel electrophoresis analysis of the major MDR‐Ef isolates showed that nosocomial infections have been caused by MDR‐Ef over a long period (more than 3 years). Multilocus sequence typing showed that these strains were mainly grouped into ST16 (CC58) or ST64 (CC8). Mating experiments suggested that the drug resistances were encoded on two conjugative transposons (integrative conjugative elements), one encoded tetracycline‐resistance and the other erythromycin/kanamycin/streptomycin/gentamicin‐resistance. To our knowledge, this is the first report of nosocomial infection caused by vancomycin‐susceptible MDR‐Ef strains over a long period in Japan.     

The opportunistic pathogen Enterococcus faecalis resists phagosome acidification and autophagy to promote intracellular survival in macrophages

While many strains of Enterococcus faecalis have been reported to be capable of surviving within macrophages for extended periods, the exact mechanisms involved are largely unknown. In this study, we found that after phagocytosis by macrophages, enterococci‐containing vacuoles resist acidification, and E. faecalis is resistant to low pH. Ultrastructural examination of the enterococci‐containing vacuole by transmission electron microscopy revealed a single membrane envelope, with no evidence of the classical double‐membraned autophagosomes. Western blot analysis further confirmed that E. faecalis could trigger inhibition of the production of LC3‐II during infection. By employing cells transfected with RFP‐LC3 plasmid and infected with GFP‐labelled E. faecalis, we also observed that E. faecalis was not delivered into autophagosomes during macrophage infection. While these observations indicated no role for autophagy in elimination of intracellular E. faecalis, enhanced production of reactive oxygen species and nitric oxide were keys to this process. Stimulation of autophagy suppressed the intracellular survival of E. faecalis in macrophages in vitro and decreased the burden of E. faecalis in vivo. In summary, the results from this study offer new insights into the interaction of E. faecalis with host cells and may provide a new approach to treatment of enterococcal infections.     

Antibiotic resistance in faecal bacteria (Escherichia coli,Enterococcus spp.) in feral pigeons

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli and Enterococcus spp. in feral pigeons (Columba livia forma domestica) in the Czech Republic. Methods and Results: Cloacal swabs of feral pigeons collected in the city of Brno in 2006 were cultivated for antibiotic‐resistant E. coli. Resistance genes, class 1 and 2 integrons, and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). The samples were also cultivated for enterococci. Species status of enterococci isolates was determined using repetitive extragenic palindromic‐PCR. Resistance genes were detected in resistant enterococci by PCR. E. coli isolates were found in 203 of 247 pigeon samples. Antibiotic resistance was recorded in three (1·5%, n_E. coli = 203) isolates. Using agar containing ciprofloxacin, 12 (5%, n_samples = 247) E. coli strains resistant to ciprofloxacin were isolated. No ESBL‐producing E. coli isolates were detected. A total of 143 enterococci were isolated: Ent. faecalis (36 isolates), Ent. faecium (27), Ent. durans (19), Ent. hirae (17), Ent. mundtii (17), Ent. gallinarum (12), Ent. casseliflavus (12) and Ent. columbae (3). Resistance to one to four antibiotics was detected in 45 (31%) isolates. Resistances were determined by tetK, tetL, tetM, tetO, aac(6′)aph(2′′), ant(4′)‐Ia, aph(3′)‐IIIa, ermB, pbp5, vanA and vanC1 genes. Conclusions: Antibiotic‐resistant E. coli and Enterococcus spp. occurred in feral pigeons in various prevalences. Significance and Impact of the Study: Feral pigeon should be considered a risk species for spreading in the environment antimicrobial resistant E. coli and enterococci.     

Coliforms and enterococci isolated from the intestinal tract of conventional mice

Coliforms and enterococci were isolated from the intestinal tract of infant (12-day-old) and adult (6-to 8-week-old) conventional mice. Eighty coliform isolates and eighty enterococcal strains were grouped according to their ability to ferment or hydrolyze various substrates. Sixty-one of the coliform isolates were identified asEscherichia coli. The remaining 19 strains were similar toE. coli, but did not produce-galactosidase. The enterococci belonged to two species:Streptococcus faecium andS. faecalis. Four biotypes ofS. faecium and two biotypes ofS. faecalis were detected. Xylosefermenting enterococci were isolated with a higher frequency from infant mice than from adults.     

Effect of banning vancomycin analogue avoparcin on vancomycin-resistant enterococci in chicken farms in Taiwan

Avoparcin, a vancomycin analogue, was banned as a feed additive in Taiwan in 2000. A nationwide surveillance was conducted to study the prevalence of vancomycin-resistant enterococci (VRE) on chicken farms between 2000 and 2003. Among the 1021 E. faecalis and 967 E. faecium isolates studied, resistance to tetracycline, erythromycin, high-level aminoglycosides, ciprofloxacin and chloramphenicol either increased or remained high except vancomycin. The proportion of VRE decreased, between 2000 and 2003, from 13.7% (22/161) to 3.7% (11/299) for E. faecalis, and 3.4% (4/119) to 0% (0/300) for E. faecium. Only 8.8% (7/80) of the chicken farms surveyed harboured VRE in 2003 compared with 25% (15/60) in 2000. All VRE were resistant to tetracycline and erythromycin. All VRE possess the vanA gene but nearly all (79 of 83 isolates) were susceptible to teicoplanin, indicating VanB phenotype. Some clones were detected from different farms in various regions over the years. We conclude that the frequency of VRE in chicken farms decreased in association with a ban on avoparcin; and the continued presence of VRE may be due to the ability of some strains to persist in the farms, transfer of vancomycin resistance determinants or co-selection by the continued use of other antibiotics.     

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.     

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.     

Characterization of dominant lactic acid bacteria isolated from São Jorge cheese, using biochemical and ribotyping methods

Aims: To identify, using phenotypic and genotypic methods, the dominant lactic acid bacteria (LAB) present in São Jorge cheese – one of the 11 Portuguese cheeses currently bearing an Appéllation d’Origine Protegée status. Methods and Results: A total of 225 isolates from milk, curd and cheeses throughout ripening were identified to the genus level, 108 to the species level and ten to the strain level. Phenotypic methods indicated that lactobacilli, followed by enterococci, were the dominant bacteria. The most frequently isolated species were Lactobacillus paracasei, Lactobacillus rhamnosus, Enterococcus faecalis and Enterococcus faecium. Ribotyping differentiated three L. paracasei, two E. faecalis and one Lactobacillus plantarum types. Enterococcus spp. exhibited the highest esterase and β-galactosidase activities among all isolates. Conclusions: The dominant LAB in São Jorge cheese are L. paracasei, L. rhamnosus, E. faecalis and E. faecium. Enterococcus likely plays a leading role upon acidification and aroma development in said cheese. Significance and Impact of the Study:  Our results support that a combination of conventional biochemical methods with genotypic methods allows for a thorough characterization and identification of isolates. Despite the limited number of isolates subject to molecular subtyping, a few specific Enterococcus and Lactobacillus strains were found that are promising ones for development of a starter culture. Hence, L. paracasei and E. faecalis are good candidates for a tentative starter culture, designed for manufacturing of São Jorge cheese at large – which takes advantage of actual isolates, in attempts to eventually standardize the quality of said cheese variety.     

Vancomycin-resistance Transferability from VanA Enterococci to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In last decade methicillin-resistant Staphylococcus aureus with high level of vancomycin-resistance (VRSA) have been reported and generally the patients with VRSA infection were also infected with a vancomycin-resistant Enterococcus (VRE). Considering that the high level of vancomycin-resistance in VRSA isolates seems to involve the horizontal transfer of Tn1546 transposon containing vanA gene from coinfecting VRE strains, the authors have studied the “in vitro” conjugative transfer of this resistance from VanA enterococci to S. aureus. Out of 25 matings performed combining five vancomycin-resistant enterococci as donors (three Enterococcus faecalis and two Enterococcus faecium), and five S. aureus as recipients, all clinical isolates, two have been successful using E. faecalis as donor. The transfer of vancomycin-resistance was confirmed by vanA gene amplification in both transconjugants and the resistance was expressed at lower levels (MIC 32 μg/ml) in comparison with the respective VRE donors (MIC > 128 μg/ml). The vancomycin-resistance of trasconjugants was maintained even after subsequent overnight passages on MSA plates containing subinhibitory levels of vancomycin. This study shows that the vanA gene transfer can be achieved through techniques “in vitro” without the use of laboratory animals employed, in the only similar experiment previously carried out by other authors, as substrate for the trasconjugant growth. Moreover, in that previous experiment, contrary to this study, the vancomycin resistant S. aureus trasconjugants were selected on erythromycin agar and not by direct vancomycin agar selection.     

Source of Enterococci in a Farmhouse Raw-Milk Cheese

Enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. Their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. The source of enterococci in raw-milk cheese is unknown. In the present study, an epidemiological approach with pulsed-field gel electrophoresis (PFGE) was used to type 646 Enterococcus strains which were isolated from a Cheddar-type cheese, the milk it was made from, the feces of cows and humans associated with the cheese-making unit, and the environment, including the milking equipment, the water used on the farm, and the cows' teats. Nine different PFGE patterns, three of Enterococcus casseliflavus, five of Enterococcus faecalis, and one of Enterococcus durans, were found. The same three clones, one of E. faecalis and two of E. casseliflavus, dominated almost all of the milk, cheese, and human fecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination, suggesting that a niche where enterococci could grow was present and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human feces are due to consumption of the cheese. Cow feces were not considered the source of enterococci in the cheese, as Enterococcus faecium and Streptococcus bovis, which largely dominated the cows' intestinal tracts, were not found in either the milk or the cheese.     

Bacteriophage biocontrol has the potential to reduce enterococci on hospital fabrics,plastic and glass

Enterococci, particularly vancomycin resistant enterococci (VRE), are a cause of continuing concern in hospitals due to their intrinsic resistance to many antibiotics and the increasing prevalence of acquired resistance to vancomycin. Furthermore, enterococci are able to survive for prolonged periods on surfaces making disinfection more difficult. This study investigated bacteriophage biocontrol as a novel means of disinfecting hard and porous surfaces contaminated with enterococci. Four enterococcal bacteriophages, designated ΦSUT1, ΦSUT2, ΦSUT3 and ΦSUT4, were isolated from sewage and landfill leachate, characterised and used as biocontrol agents in studies investigating the efficacy of phage in the disinfection of vancomycin susceptible enterococci (VSE) and VRE on glass and fabrics commonly found in hospitals. Sterile glass slides and swatches of fabrics were inoculated with 10⁵ cfu enterococci and 10⁹ pfu/ml phage. Inoculated surfaces were incubated at ambient temperature for 2 h and bacterial populations were recovered by plating onto KF streptococcus agar. ΦSUT1 produced significant reductions in growth of Enterococcus faecalis on all surfaces tested, while ΦSUT4 produced significant reductions in growth of VRE (M168600) on all surfaces except for polyester. Phage cocktails containing ΦSUT1 and ΦSUT6 produced greater reductions in growth of VRE (M252807) on all surfaces compared to treatments using either phage alone. Although the results from this study varied considerably, the promising findings from ΦSUT1 studies indicated that phage biocontrol of enterococci should be further explored using broader host range phages.     

Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 10³ CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.