Assessment of Zn and Cu in piglets' liver and kidney: impact in fecal Enterococcus spp.?

Zinc and copper have been used as growth promotors in alternative to antibiotics in pig's diet. The aim was the ascertainment of the Zn and Cu concentrations in piglets' liver and kidney and their impact in the reduced susceptibility to Zn, Cu, and antibiotics in enterococci, used as microbiota biomarker. Zn and Cu were determined in the livers and kidneys of 43 piglets slaughtered in Portugal, by flame atomic absorption spectrometry. Enterococci were isolated from feces for determining the identification of species (E. faecalis, E. faecium, and Enterococcus spp.); susceptibility to vancomycin, ciprofloxacin, linezolid, tigecycline, ampicillin, imipenem, and metals; and Cu tolerance genes. In piglets with Zn and Cu high or toxic levels, enterococci had reduced susceptibility to ions, reinforced by the presence of Cu tolerance genes and by resistance to antibiotics. The study relevance is to show the relationship between these metals' levels and decreased susceptibility to Cu, Zn, and antibiotics by enterococci. From the results, it could be supposed that the piglets were being fed with high doses of Zn and Cu which could select more resistant bacteria to both antibiotics and metals that could spread to environment and humans.

Genome and fermentation analyses of Enterococcus faecalis DB-5 isolated from Japanese Mandarin orange: An assessment of potential application in lactic acid production.

Enterococcus faecalis strain DB-5 is a lactic acid bacterium newly isolated from the Japanese mandarin orange (mikan). The DB-5 strain produces organic acid from various carbohydrate sources including glycerol and starch. To gain deeper insights into its potential application in lactic acid fermentation (LAF), the genome and fermentation analyses of E. faecalis DB-5 were performed. Whole genome sequencing was carried out using the DNBSEQ platform. After trimming and assembly, the total size of the assembled genome was revealed to be 3,048,630 bp, distributed into 63 contigs with an N50 value of 203,673. The genome has 37.2% GC content, 2928 coding DNA sequences, and 54 putative RNA genes. The DB-5 strain harbored two l-lactate dehydrogenases (L-LDHs), both of which conserved the catalytic domain sequences. The optical purity measurement showed that strain DB-5 is homofermentative and produced only l-lactic acid (LA), which correlated with genome-based pathway analysis. To confirm its LA productivity at high temperatures, open repeated batch fermentation was performed at 45 °C using sucrose as a carbon source. The volumetric LA productivity of DB-5 was averaged at 3.66 g L-1 h-1 for 24 h during the 3rd to 11th fermentation cycles. E. faecalis DB-5 could efficiently convert around 94% of sucrose to LA throughout the fermentation cycles at 45 °C. These genomic characteristics and fermentation properties of E. faecalis DB-5 provide beneficial information for a deeper understanding of the functional properties of future high-temperature LAFs from biomass resources.

Assessment of Tedizolid In Vitro Activity and Resistance Mechanisms against a Collection of Enterococcus spp. Causing Invasive Infections, Including Isolates Requiring an Optimized Dosing Strategy for Daptomycin from U.S. and European Medical Centers, 2016 to 2018.

High-level aminoglycoside resistance was noted in 30.0% of Enterococcus faecalis and 25.2% of Enterococcus faecium isolates. Only 3.3% and 2.1% of E. faecalis isolates had elevated daptomycin MIC (≥2 mg/liter) and vancomycin resistance, respectively. In contrast, 37.4% to 40.3% of E. faecium isolates exhibited these phenotypes. Tedizolid inhibited 98.9% to 100.0% of enterococci causing serious invasive infections, including resistant subsets. Oxazolidinone resistance was mainly driven by G2576T; however, optrA and poxtA genes were also detected, including poxtA in the United States and Turkey.

Assessment of vancomycin resistance transfer among enterococci of clinical importance in milk matrix.

Dissemination of vancomycin resistance in enterococci has been associated with horizontal transfer of mobile genetic elements. Aim of the study was to evaluate if milk matrix is a suitable environment to support transferability of vancomycin resistance (vanA) gene from clinical vancomycin-resistant Enterococcus faecium to vancomycin-sensitive Enterococcus faecalis. Enterococci strains were firstly screened for the presence of cpd (inducible sex pheromone determinant) gene, vanA and tetL genes (vancomycin and tetracycline resistance markers, respectively) and the gelE (extracellular metalloendopeptidase) gene to define the mating pairs. Based on these selection markers, we investigated the transferability of eight plasmid-borne vanA harbored by E. faecium (vanA+, cpd-, tetL- and gelE-) into two E. faecalis (vanA-, cpd+, tetL + and gelE+) recipient strains in milk matrix. The strains were mated in a 1:1 ratio in 7% reconstituted milk and incubated at 37 °C. Transconjugants emerged from all 16 matings within 2 h of incubation and were evidenced by dual antibiotic resistance (vancomycin and tetracycline). The vancomycin-resistance of trasconjugants was maintained even after ten subsequent passages on nonselective medium. Transconjugants were positive for vanA, tetL and gelE genes. This study indicates milk matrix as suitable environment to support gene exchange between Enterococcus species.

Assessment of selenium bioaccumulation in lactic acid bacteria.

Selenium is an essential micronutrient for living beings, as it helps to maintain the normal physiological functions of the organism. The numerous discoveries involving the importance of this element to the health of human beings have fostered interest in research to develop enriched and functional foods. The present study evaluated the potential for bacterial strains of Enterococcus faecalis (CH121 and CH124), Lactobacillus parabuchneri (ML4), Lactobacillus paracasei (ML13, ML33, CH135, and CH139), and Lactobacillus plantarum (CH131) to bioaccumulate Se in their biomass by adding different concentrations of sodium selenite (30 to 200 mg/L) to the culture medium. Quantification of Se with UV and visible molecular absorption spectroscopy showed that the investigated bacteria were able to bioaccumulate this micromineral into their biomass. Two of the L. paracasei strains (ML13 and CH135) bioaccumulated the highest Se concentrations (38.1 ± 1.7 mg/g and 40.7 ± 1.1 mg/g, respectively) after culture in the presence of 150 mg/L of Se. This bioaccumulation potential has applications in the development of dairy products and may be an alternative Se source in the diets of humans and other animals.

A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are resistant to most classes of antibiotics. Diagnosis of VRE using standard methods takes 2 to 5 days. Development of a rapid PCR-assay that detects and identifies resistant genes in bacteria would provide time-critical information on the presence of VRE in clinical samples allowing early treatment and management of infected patients. OBJECTIVES: Investigate the use of high resolution melting analysis (HRMA) and 16S-rRNA-PCR approach for rapid and cost-effective identification of VRE. DESIGN: Descriptive antibiotic susceptibility studies. SETTING: Manchester Academic Health Sciences Centre and School of Translational Medicine, University of Manchester, UK, and Department of Clinical Laboratory Sciences, Taibah University, Saudi Arabia. MATERIALS AND METHODS: PCR-HRMA using 16S-rRNA V1-primers was used to detect and identify VRE. DNA from different strains of vancomycin-resistant and -sensitive Enterococcus faecalis (VSE) and Enterococcus faecium were amplified using V1-primer followed by HRMA in a single run. Differentiation of VRE from VSE was based on curve shapes generated against reference organisms (Bacteroides fragilis). MAIN OUTCOMES MEASURES: Amplification curves and difference plots for VRE and VSE. RESULTS: Difference plots were generated for all vancomycin-resistant and -sensitive E faecalis and E faecium strains by subtracting their fluo.rescence melting profile from that of a reference-species B fragilis. A characteristic curve shape was produced by vancomycin-sensitive E faecalis and E faecium. However, vancomycin-resistant strains of these bacteria were associated with a markedly different curve shape facilitating a clear differentiation. CONCLUSION: The 16S-PCR-HRMA approach has the potential for detecting vancomycin-resistant E faecium and E faecalis. Data with VRE provide the basis for combining VRE identification with pathogens speciation in a rapid, cheap assay able to identify a pathogen as an Enterococcus and whether it is vancomycin-sensitive or -resistant E faecium or E faecalis in a single PCR and HRMA run. LIMITATIONS: Tested on specific, but not all, reference Enterococcus species and clinical isolates. CONFLICT OF INTEREST: None.

Ex vivo assessment of synergic effect of chlorhexidine for enhancing antimicrobial photodynamic therapy efficiency on expression patterns of biofilm-associated genes of Enterococcus faecalis.

BACKGROUND: It has clearly been demonstrated that Enterococcus faecalis, as a persistent microorganism, is the major agent in the etiopatogeny of endodontic infections. Recently, the limitations of conventional endodontic therapy have given rise to many attempts to introduce antimicrobial photodynamic therapy (aPDT) as an alternative treatment. The aim of this study was to analyze the ex vivo effect of aPDT in combination with 2.0% chlorhexidine (CHX) as a conventional therapy on colony count and expression patterns of genes associated with biofilm formation of E. faecalis. MATERIALS AND METHODS: A total of 125 extracted human single-rooted teeth were divide into six groups (A-F; n = 20) and were incubated with E. faecalis. Group A- photosensitizer (indocyanine green [ICG]); B- diode laser; C- aPDT; D- 2.0% CHX; E- aPDT with photosensitizer modified by 2.0% CHX; and F- control group (no procedure was performed). Five remaining teeth were used to confirm the presence of E. faecalis biofilm via scanning electron microscope. Counts of colony forming units (CFUs) in each group were evaluated separately and quantitative real-time PCR (qRT-PCR) was then applied to monitor genes expression of fsrC, efa, and gelE involved in E. faecalis biofilm. RESULTS: The results showed that none of the tested groups achieved eradication or inhibition of biofilm. On the other hand, aPDT + 2.0% CHX, 2.0% CHX, and ICG- mediated aPDT groups showed significantly less CFU/mL than ICG and diode laser groups. The group with the lowest CFU/mL count was the aPDT + 2.0% CHX, being statistically different from all other groups that could decrease the expression levels of efa, gelE, and fsrC genes 6.8-, 8.3-, and 12.1-fold, respectively. CONCLUSION: Based on the results, the synergism effect of ICG-aPDT with 2.0% CHX leads to modulation of the virulence of E. faecalis strains biofilm model by suppressing the expression of the genes associated with biofilm formation.

Molecular assessment of virulence determinants, hospital associated marker (IS16gene) and prevalence of antibiotic resistance in soil borne Enterococcus species.

Enterococci, no more regarded as GRAS (Generally Recognized As Safe) organism, are emerging as an important source of nosocomial infections worldwide. The main contributors in pathogenesis of enterococci are the presence of various virulent factors and antibiotic resistance genes. We aimed to examine the prevalence, dissemination, antibiotic resistance and virulent factors associated with enterococci from bulk soil (BS). A total of 372 enterococci were isolated from 500 soil samples. PCR was used to identify the isolates up to species level and for carriage of 16 virulence genes including hospital associated marker (i.e. IS16). E. faecium (77%), E. faecalis (10%), E. hirae (4%) and E. casseliflavus (1%) were the major species isolated. The efaAfs was the most dominant gene (100%), followed by gelE (78.9%), sprE (76.3%) and esp (13%) in E. faecalis isolates. The E. faecium carried largely efaAfm (86.8%) and acm (50.3%) genes. Presence of entP (10%), entA (8.3%) and entB (6.9%) genes was detected mostly in E. faecium, while enlA (18%) and ef1097 (2.6%) was only detected in E. faecalis isolates. 50% E. faecalis and 2% E. faecium isolates harbored IS16, while five E. faecalis harbored both IS16 and espTIM genes providing strong evidence about the presence of espTIM gene on 64 Kb pathogenicity island. BOX and RAPD PCR analysis revealed high degree of genetic variation within the species. Degree of resistance against 12 major antibiotics showed chloramphenicol as the most effective and meropenom as the least effective antibiotic. Presence of multiple antibiotic resistant, virulent and hospital associated enterococci in bulk soil represents a potential source for further dissemination to humans and animals and poses potential impact on public health.

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

BACKGROUND/PURPOSE: Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. METHODS: The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. RESULTS: A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. CONCLUSION: The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening.

Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

Assessment of free fatty acids and cholesteryl esters delivered in liposomes as novel class of antibiotic.

BACKGROUND: Healthcare associated infections (HAI) with multidrug-resistant (MDR) bacteria continue to be a global threat, highlighting an urgent need for novel antibiotics. In this study, we assessed the potential of free fatty acids and cholesteryl esters that form part of the innate host defense as novel antibacterial agents for use against MDR bacteria. METHODS: Liposomes of six different phospholipid mixtures were employed as carrier for six different fatty acids and four different cholesteryl esters. Using a modified MIC assay based on DNA quantification with the fluoroprobe Syto9, formulations were tested against Gram-positive and Gram-negative bacteria implicated in HAI. Formulations with MIC values in the low µg/mL range were further subjected to determination of minimal bactericidal activity, hemolysis assay with sheep erythrocytes, and cytotoxicity testing with the human liver cell line HepG2. The potential for synergistic activity with a standard antibiotic was also probed. RESULTS: Palmitic acid and stearic acid prepared in carrier 4 (PA4 and SA4, respectively) were identified as most active lipids (MIC against MDR Staphylococcus epidermidis was 0.5 and 0.25 µg/mL, respectively; MIC against vancomycin resistant Enterococcus faecalis (VRE) was 2 and 0.5 µg/mL, respectively). Cholesteryl linoleate formulated with carrier 3 (CL3) exhibited activity against the S. epidermidis strain (MIC 1 µg/mL) and a Pseudomonas aeruginosa strain (MIC 8 µg/mL) and lowered the vancomycin MIC for VRE from 32-64 µg/mL to as low as 4 µg/mL. At 90 µg/mL PA4, SA4, and CL3 effected less than 5 % hemolysis over 3 h and PA4 and CL3 did not exhibit significant cytotoxic activity against HepG2 cells when applied at 100 µg/mL over 48 h. CONCLUSIONS: Our results showed that selected fatty acids and cholesteryl esters packaged with phospholipids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria and may augment the activity of antibiotics. Bactericidal activity could be unlinked from hemolytic and cytotoxic activity and the type of phospholipid carrier greatly influenced the activity. Thus, fatty acids and cholesteryl esters packaged in liposomes may have potential as novel lipophilic antimicrobial agents.

Performance of the EUCAST disk diffusion method, the CLSI agar screen method, and the Vitek 2 automated antimicrobial susceptibility testing system for detection of clinical isolates of Enterococci with low- and medium-level VanB-type vancomycin resistance: a multicenter study.

Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n=28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n=12) and Enterococcus faecium (n=18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n=5), Norwegian (n=13), and Swedish (n=10) laboratories using the EUCAST disk diffusion method (n=28) and the CLSI agar screen (n=18) or the Vitek 2 system (bioMérieux) (n=5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P=0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P<0.0001) or Merck Mueller-Hinton (MH) agar (P=0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P=0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges.

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis.

OBJECTIVES: To determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory. METHODS: Different MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80-200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays. RESULTS: The biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%-27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection. CONCLUSIONS: Newly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid-host association can rapidly emerge.

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods.

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMß1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.

Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut.

A total of seventy lactic acid bacteria (LAB) were isolated from the faeces of healthy humans and their identities were confirmed by sequencing of their 16S rDNA genes. Of these only 5 isolates were found to resist bile salts and indicated survival in the simulated in vitro digestion assay which reproduces the stomach and intestinal digestion indicating their tolerance to gastric enzymes and the low pH conditions. Species that showed the best resistance to these conditions were: Lactobacillus casei, Lactobacillus sp., uncultured bifidobacteria, Enterococcus faecalis and Streptococcus anginosus. These strains were investigated further to study their capacity to adhere to human intestinal Caco-2 cells. E. faecalis was the most adherent strain. Examination of the virulence determinants for this strain indicated that it was positive for efaAfs, gelE, agg, cpd, cob, ccf and cad, a profile that is similar to that of many E. faecalis isolates from food sources. The cytolysin biosynthetic genes cylA, cylB and cylM that are more associated with the clinical isolates of E. faecium were not detected in this strain. The antibiotic susceptibility tests indicated that the strain was sensitive to vancomycin, tetracycline, rifampicin and erythromycin but resistant only to kanamycin and chloramphenicol. These data suggest that the strain E. faecalis CP58 may be tested further for beneficial properties and developed as a new probiotic.

Inducible expression eliminates the fitness cost of vancomycin resistance in enterococci.

Inducible vancomycin resistance in enterococci is due to a sophisticated mechanism that combines synthesis of cell wall peptidoglycan precursors with low affinity for glycopeptides and elimination of the normal target precursors. Although this dual mechanism, which involves seven genes organized in two operons, is predicted to have a high fitness cost, resistant enterococci have disseminated worldwide. We have evaluated the biological cost of VanB-type resistance due to acquisition of conjugative transposon Tn1549 in Enterococcus faecium and Enterococcus faecalis. Because fitness was dependent on the integration site of Tn1549, an isogenic set of E. faecalis was constructed to determine the cost of inducible or constitutive expression of resistance or of carriage of Tn1549. A luciferase gene was inserted in the integrase gene of the transposon to allow differential quantification of the strains in cocultures and in the digestive tract of gnotobiotic mice. Both in vitro and in vivo, carriage of inactivated or inducible Tn1549 had no cost for the host in the absence of induction by vancomycin. In contrast, induced or constitutively resistant strains not only had reduced fitness but were severely impaired in colonization ability and dissemination among mice. These data indicate that tight regulation of resistance expression drastically reduces the biological cost associated with vancomycin resistance in Enterococcus spp. and accounts for the widespread dissemination of these strains. Our findings are in agreement with the observation that regulation of expression is common in horizontally acquired resistance and represents an efficient evolutionary pathway for resistance determinants to become selectively neutral.

Assessment of high-level gentamicin and glycopeptide-resistant Enterococcus faecalis and E. faecium clonal structure in a Portuguese hospital over a 3-year period.

This study focussed on the clonal structure and temporal distribution of E. faecalis and E. faecium with high-level resistance to gentamicin (HLGR) and glycopeptides (GR) collected from clinical samples during 2004 to 2006 at a Portuguese Hospital. The findings were an E. faecalis-dominant and epidemic clone (PFGE-AO), the maintenance of a major epidemic E. faecium clone (PFGE-c) and a high prevalence of putative virulence genes--asa1 (aggregation substances), gelE (gelatinase), cylA (cytolysin), esp (enterococcal surface protein), and hyl (hyaluronidase)--most of them significantly associated with the major clones of both species. The E. faecalis GR isolates ST6 and the E. faecium GR isolates ST17, ST18 and ST280 belong to the clonal complexes E. faecalis-CC2 and E. faecium-CC17, which are well adapted to the nosocomial setting and are disseminated worldwide. This study highlights the need for continuous and active surveillance in this Portuguese hospital in order to follow the evolution of these epidemic and persistent clones.

Assessment of the bacterial diversity of human colostrum and screening of staphylococcal and enterococcal populations for potential virulence factors.

In contrast to breast milk, little is known about the bacterial composition of human colostrum. The objective of this work was to analyze the bacterial diversity of colostrum obtained from healthy women and to characterize the dominant bacterial species for the presence of possible virulence factors. Samples of colostrum obtained from 36 healthy women were inoculated into different culture media. Several isolates from each medium were selected and identified. Staphylococcal and enterococcal isolates were submitted to genetic profiling. One representative of each profile was included in a genetic and phenotypic characterization scheme, including detection of potential virulence traits/genes and sensitivity to antibiotics. Staphylococcus epidermidis and Enterococcus faecalis were the dominant species, followed by Streptococcus mitis, Propionibacterium acnes and Staphylococcus lugdunensis. Among the 48 S. epidermidis isolates selected on the basis of their genetic profiles, the biofilm-related icaD gene and the mecA gene were detected in only 11 and six isolates, respectively. In parallel, 10 enterococcal isolates were also characterized and none of them contained the cylA, vanA, vanB, vanD, vanE and vanG genes. All of them were sensitive to vancomycin. There were no indications that the colostrum samples contained harmful bacteria.

A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species.

BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene. RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases. CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.

Survivability of vancomycin resistant enterococci and fitness cost of vancomycin resistance acquisition.

AIMS: To investigate the survivability of vancomycin resistant enterococci (VRE) under dry starvation conditions and the fitness cost of vancomycin resistance. METHODS: VRE colonies on cotton swabs were incubated at room temperature in a sterile box and cultured weekly until cultures no longer showed growth. Negative swabs inoculated into brain heart infusion (BHI) broth were subcultured to blood agar after 24, 48, and 72 hours of incubation to resuscitate viable but non-culturable cells. Stability of the vancomycin resistance determinant and of the DNA fingerprint pattern was determined by multiplex polymerase chain reaction (PCR) and repetitive PCR, respectively. Tests for fitness cost were carried out on the same VRE isolates and 28 hospital vancomycin sensitive enterococci (VSE) isolates by incubation and measurement of optical density using a microplate reader and comparing maximum growth rate and lag phase duration between VRE and VSE, using independent samples t tests. RESULTS: Mean maximum time of recovery by primary culture was 8.5 weeks for Enterococcus faecalis VRE and 21.8 weeks for E. faecium VRE. Two of two E. faecalis isolates were resuscitated after 24 hours in BHI broth, and two of five E. faecium isolates after 72 hours. No fitness cost of vancomycin resistance was demonstrated. CONCLUSIONS: VRE can survive for prolonged periods in a dry starvation state, retaining their genetic complement, including vancomycin resistance determinants, and show little or no fitness cost of vancomycin resistance. Thus, the rate of entry required for VRE to become, and remain, endemic in the community is relatively small.