Identification and Characterization of Vancomycin-resistant Enterococcus species Frequently Isolated from Laboratory Mice

To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics.     

Absence of VanA- and VanB-Containing Enterococci in Poultry Raised on Nonintensive Production Farms in Brazil

We examined cloacal samples from poultry raised on nonintensive production farms in Brazil for the presence of vancomycin-resistant enterococci. No VanA- or VanB-containing enterococci were identified in a total of 200 cloacal swabs. The most prevalent species were Enterococcus gallinarum (vanC1; 13.0%) and E. casseliflavus (vanC2/3; 5.5%).     

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.     

Phenotypic and Genotypic Characterization of Bacteriocins in Enterococcal Isolates of Different Sources

A collection of 57 enterococcal isolates from different origin (including river, treatment plant, spring and garbage water, soil, animal, and vegetables from Aydın) was screened for the production of bacteriocins. Enterococci were identified at species levels as Enterococcus faecium (34), E. hirae (6), E. casseliflavus (4), E. durans (4), E. faecalis (4), E. mundtii (3) and E. avium (2). Of the 57 isolates 40 of them inhibited the growth of at least one indicator bacterium. Based on our PCR results 54 strains possesed enterocin genes. The genes of entA and entB were the most frequently detected structural genes among the PCR positive strains (54 and 53 strains, respectively) and the entB gene was always associated with entA gene. The highest combination of enterocin genes (24 of 54 strains) detected was entA, entB, entP and entL50A/B. The enterocins AS-48 and CylL_LS genes were not found. Three enterococcal isolates, 2 E. faecium and 1 E. hirae were not harbour any of tested enterocin genes. No correlation between the presence of enterocin structural genes and the origin of the strain was detected, also no relationship seemed to exist between the tested enterocin genes and the activity spectra of isolates. Genes encoding bacteriocins are widely disseminated among enterocci from different origin and more studies should be done for evaluate industrial potential of bacteriocins.     

Bacteriophage-mediated transduction of antibiotic resistance in enterococci

Aims: Temperate bacteriophages are bacterial viruses that transfer genetic information between bacteria. This phenomenon is known as transduction, and it is important in acquisition of bacterial virulence genes and antimicrobial resistance determinants. The aim of this study was to demonstrate the role of bacteriophages in gene transfer (antibiotic resistance) in enterococci. Methods and Results: Three bacteriophages from environmental samples isolated on pig host strains of Enterococcus gallinarum and Enterococcus faecalis were evaluated in transduction experiments. Antibiotic resistance was transferred from Ent. gallinarum to Ent. faecalis (tetracycline resistance) and from Ent. faecalis to Enterococcus faecium, Enterococcus hirae/durans and Enterococcus casseliflavus (gentamicin resistance). Conclusions: Bacteriophages play a role in transfer of antibiotic resistance determinants in enterococci. Significance and Impact of the Study: This study confirms previous suggestions on transduction in enterococci, in particular on interspecies transduction. Interspecies transduction is significant because it widens the range of recipients involved in antimicrobial resistance transfer.     

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

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

Prevalence of antimicrobial resistance and molecular characterization of tetracycline resistance mediated by tet(M) and tet(L) genes in Enterococcus spp. isolated from food in Southern Brazil

Enterococcus spp. are opportunistic pathogens that are widely distributed in the natural environment. Two remarkable characteristics of enterococci is their intrinsic resistance against several of the antimicrobial agents routinely prescribed in the treatment of Gram-positive cocci, and their enormous capacity to acquire different genetic markers by conjugation. The aim of this study was to evaluate the prevalence of antimicrobial resistance and the frequency of tet(M) and tet(L) genes in 112 Enterococcus spp. strains isolated from food. Fifty-two strains (64%) of Enterococcus faecalis, 10 (55%) of Enterococcus faecium, 2 (66%) of Enterococcus casseliflavus and 3 (42%) of Enterococcus gallinarum showed multidrug resistance. Tet(M) gene associated with or without the tet(L) gene was the most prevalent genotype found in food. Nine erythromycin-resistant and tetracycline-susceptible enterococci strains harbor silencing tet(M) or tet(L) genes were present in our investigation. In conclusion, antibiotic-resistant enterococci current in food may act as a reservoir of resistant strains creating a potential route of genes transference by horizontal gene transfer.     

Copper Resistance in Enterococcus faecium, Mediated by the tcrB Gene, Is Selected by Supplementation of Pig Feed with Copper Sulfate

The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes in the enterococcal species E. mundtii, E. casseliflavus, and E. gallinarum for the first time.     

Molecular diversity and transferability of the tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M), carried on Tn<Emphasis Type="Italic">916-1545</Emphasis> family transposons,in enterococci from a total food chain

In the present study, 20 enterococci belonging to the species Enterococcus faecalis (12 strains), Enterococcus faecium (4), Enterococcus durans (2), Enterococcus hirae (1) and Enterococcus mundtii (1) and originating from a total production chain of swine meat commodities were analysed to investigate the diversity of their tetracycline resistance gene tet(M). PCR–RFLP and sequence analysis showed that the tet(M) gene of most strains can be correlated with the Tn916 transposon. Conversely, tet(M) of six E. faecalis and the E. hirae strain, all isolated from pig faecal samples, may be associated with previously undescribed members of the Tn916-1545 transposon family. In vitro filter conjugation trials showed the ability of 50% of the enterococcal strains, including E. mundtii, to transfer the tet(M) gene (and the associated Tn916 and new transposons) to E. faecalis or Listeria innocua recipient strains. tet(M) gene transfer to L. innocua recipient was also directly observed in meat food products. Collectively, these sequence and conjugation data indicate that various transposons can be responsible of the spread of tetracycline resistance in enterococci and validate the opinion that Enterococcus species are important sources of antibiotic resistance genes for potentially pathogenic bacteria occurring in the food chain. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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     

Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.     

The SKPO-1 Peroxidase Functions in the Hypodermis to Protect Caenorhabditis elegans From Bacterial Infection

In recent years, the synergistic relationship between NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes and peroxidases has received increased attention. Peroxidases utilize NOX/DUOX-generated H₂O₂ for a myriad of functions including, but not limited to, thyroid hormone biosynthesis, cross-linking extracellular matrices (ECM), and immune defense. We postulated that one or more peroxidases produced by Caenorhabditis elegans would act in host defense, possibly in conjunction with BLI-3, the only NOX/DUOX enzyme encoded by the genome that is expressed. Animals exposed to RNA interference (RNAi) of the putative peroxidase genes were screened for susceptibility to the human pathogen Enterococcus faecalis. One of three genes identified, skpo-1 (ShkT-containing peroxidase), was studied in depth. Animals mutant for this gene were significantly more susceptible to E. faecalis, but not Pseudomonas aeruginosa. A slight decrease in longevity was also observed. The skpo-1 mutant animals had a dumpy phenotype of incomplete penetrance; half the animals displayed a dumpy phenotype ranging from slight to severe, and half were morphologically wild type. The SKPO-1 protein contains the critical catalytic residues necessary for peroxidase activity, and in a whole animal assay, more H₂O₂ was detected from the mutant compared to the wild type, consistent with the loss of an H₂O₂ sink. By using tissue-specific skpo-1 RNAi and immunohistochemical localization with an anti-SKPO-1 antibody, it was determined that the peroxidase is functionally and physically present in the hypodermis. In conclusion, these results characterize a peroxidase that functions protectively in the hypodermis during exposure to E. faecalis.     

Measuring Movement to Determine Physiological Roles of Acetylcholinesterase Classes in Caenorhabditis elegans

A difference in movement has been hypothesized to exist between Caenorhabditis elegans strains lacking one of two main genes for acetylcholinesterase (AChE), ace-1(+) and ace-2(+). We explored the precision of movement as an endpoint by measuring and comparing the movements of these strains (VC505 and GG202, respectively) and of N2 (wild-type). The order of movement of the strains is: N2 > VC505 > GG202; therefore, loss of the ace-2(+) gene is more detrimental to movement. We then compared the sensitivities of the three strains to an AChE inhibitor (propoxur) by generating movement-concentration curves, identifying effective concentrations that decreased movement by 50% (EC₅₀), and comparing them. EC₅₀ show an order of: N2 ≈ GG202 < VC505. Therefore, the enzymes encoded by ace-1(+) were more susceptible to propoxur than those of ace-2(+), suggesting that the innate difference in the AChE classes'' contributions to movement will not always determine the strain sensitivity. Measuring movement was sufficiently precise to record differences following genetic manipulation and further chemical exposure.     

Novel ROSA26 Cre-reporter Knock-in C57BL/6N Mice Exhibiting Green Emission before and Red Emission after Cre-mediated Recombination

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F₁ mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F₁ mice and R26GRR /Ins1-Cre F₁ mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).     

Bacteriocin Protein BacL1 of Enterococcus faecalis Is a Peptidoglycan d-Isoglutamyl-l-lysine Endopeptidase

Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL₁ and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL₁ and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL₁ and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL₁ and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL₁ nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL₁ alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL₁ has a peptidoglycan d-isoglutamyl-l-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL₁ serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells.     

Multidrug-Resistant Enterococci in Animal Meat and Faeces and Co-Transfer of Resistance from an <Emphasis Type="Italic">Enterococcus durans</Emphasis> to a Human <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6′)-Ie aph (2′′)-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.