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Antibiotic resistance monitoring: the Spanish programme. The VAV Network. Red de Vigilancia de Resistencias Antibióticas en Bacterias de Origen Veterinario.

Antimicrobial resistance is a problem in modern public health and antimicrobial use and especially misuse, the most important selecting force for bacterial antibiotic resistance. As this resistance must be monitored we have designed the Spanish network 'Red de Vigilancia de Resistencias Antibióticas en Bacterias de Origen Veterinario'. This network covers the three critical points of veterinary responsibility, bacteria from sick animals, bacteria from healthy animals and bacteria from food animals. Key bacteria, antimicrobials and animal species have been defined for each of these groups along with laboratory methods for testing antimicrobial susceptibility and for data analysis and reporting. Surveillance of sick animals was first implemented using Escherichia coli as the sentinel bacterium. Surveillance of E. coli and Enterococcus faecium from healthy pigs was implemented in 1998. In July 1999, data collection on Salmonella spp. was initiated in poultry slaughterhouses. Additionally, the prevalence of vancomycin resistant E. faecium was also monitored. This network has specific topics of interest related to methods of determining resistance, analysis and reporting of data, methods of use for veterinary practitioners and collaboration with public health authorities.

Animals↗

Epidemiology of glycopeptide-resistant enterococci colonizing high-risk patients in hospitals in Johannesburg, Republic of South Africa.

Recent cases of infections caused by glycopeptide-resistant enterococci (GRE) have highlighted the emergence of these organisms in the Republic of South Africa. During May 1998 we conducted a prevalence study in four hospitals in Johannesburg and obtained 184 rectal swabs from patients identified as being at high risk for GRE colonization. Twenty enterococcal isolates showing various glycopeptide resistance genotypes were recovered: 3 Enterococcus faecium vanA isolates, 10 E. faecium vanB isolates, 6 E. gallinarum vanC1 isolates, and 1 E. avium vanA isolate. Macrorestriction analysis was used to demonstrate the clonal spread of GRE strains within hospitals. Evidence also demonstrated the likely persistence of the original E. faecium vanA isolate associated with the first confirmed death contributed to by GRE infection in South Africa in March 1997.

Adolescent↗

Phylogeny and identification of Enterococci by atpA gene sequence analysis.

The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.

Animals↗

Successful treatment of ventriculostomy-related meningitis caused by vancomycin-resistant Enterococcus with intravenous and intraventricular quinupristin/dalfopristin.

We report a case of ventriculostomy-related meningitis caused by vancomycin-resistant Enterococcus faecium (VRE). The patient was successfully treated with administration of quinupristin/dalfopristin by both intravenous and intraventricular routes. A brief review of the literature is provided, which indicates that optimal management with quinupristin/dalfopristin should include daily intraventricular doses of at least 2 mg.

Drug Therapy, Combination↗

Comparative in vitro activity of moxifloxacin against Gram-positive clinical isolates.

The in vitro activity of moxifloxacin was compared with that of 15 antibacterial agents against 513 Gram-positive microorganisms. The MIC(90) (mg/L) of moxifloxacin was 0.06 for quinolone-susceptible Staphylococcus aureus and Staphylococcus epidermidis, 0.12 for Streptococcus pyogenes and Streptococcus agalactiae; 0.25 for Streptococcus pneumoniae, Streptococcus mitis, Streptococcus bovis, Streptococcus anginosus and Actinomyces pyogenes; 0.5 for Streptococcus sanguis and Listeria monocytogenes, 2 for Corynebacterium jekeium and Bifidobacterium bivius. Over 50% of Enterococcus faecalis, Enterococcus faecium, quinolone-resistant staphylococci, Nocardia steroides and Clostridium difficile were susceptible to 2 mg/L moxifloxacin. Moxifloxacin and trovafloxacin demonstrated comparably high activity towards Gram-positive cocci; moxifloxacin and clinafloxacin were most active against Gram-positive bacilli.

Anti-Infective Agents↗

Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.

BACKGROUND/AIMS: Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. METHODS: Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. RESULTS: Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n=23), and response to pheromones in E. faecalis culture filtrate (n=16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n=31) but not in Enterococcus faecium (n=2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. CONCLUSIONS: Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.

Adult↗

Evaluation of 15 motility media and a direct microscopic method for detection of motility in enterococci.

Isolation of motile, vanC enterococci has yet to be a major infection control concern; however, rapid detection still is important. We evaluated 15 motility media from three manufacturers and a 2-h direct microscopic method for accurate detection of 89 enterococcal strains, including 72 vanC enterococcal strains. Resistance genes were confirmed by a multiplex PCR method with the vanC gene detected in all motile enterococci. Motility in the 72 vanC enterococci was detected at 30 degrees C within 72 h in BD Biosciences motility nitrate medium and in Remel motility B medium, motility B medium supplemented with methyl-alpha-D-glucopyranoside (investigational), motility S medium, motility test medium, and motility test medium with tetrazolium indicator. Motility was also observed for all vanC enterococci with the 2-h direct (30 degrees C incubation) microscopic detection method. All Enterococcus faecalis and Enterococcus faecium isolates were observed to be nonmotile in all media and by the direct microscopic method. Since differences between the various motility media tested were observed, the medium used for detection of enterococcal motility must be selected carefully.

Bacterial Proteins↗

Daptomycin activity and spectrum: a worldwide sample of 6737 clinical Gram-positive organisms.

BACKGROUND: Increasing antimicrobial resistance among bacterial pathogens has prompted attempts to develop new antimicrobial agents active against multidrug-resistant Gram-positive pathogens. OBJECTIVES: To evaluate the in vitro activity of daptomycin against a worldwide collection of clinical bacterial isolates. METHODS: Daptomycin is a novel cyclic lipopeptide recently approved by the United States Food and Drug Administration. Daptomycin and selected comparators were tested against 6737 clinical Gram-positive strains from more than 70 centres located in Europe, North America and South America. RESULTS: The overall distribution of daptomycin MIC values were in the range < or = 0.12-8 mg/L and 99.4% of all strains were inhibited at < or = 2 mg/L. Despite resistances to other antimicrobial agents, >99.9% of staphylococcal isolates were inhibited at < or = 1 mg/L of daptomycin (MIC90 0.5 mg/L for staphylococci). Streptococcal isolates were very susceptible to daptomycin independent of their susceptibility to penicillin. MIC50/90 values were < or = 0.12 and 0.25 mg/L, respectively. Enterococci showed the highest daptomycin MIC values, but all isolates tested were inhibited at < or = 4 mg/L (except for one Enterococcus faecium isolate which showed a daptomycin MIC of 8 mg/L). CONCLUSIONS: Daptomycin exhibited excellent in vitro activity against a wide spectrum of Gram-positive organisms and may represent a therapeutic option for infections caused by multidrug-resistant pathogens worldwide.

Bacillus↗

In vitro activity of ampicillin/sulbactam against enterococci determined by the time-kill method.

Time-kill studies demonstrated that at clinically achievable serum concentrations, ampicillin/sulbactam was equivalent in activity to ampicillin alone against non-beta-lactamase producing isolates of Enterococcus faecalis and Enterococcus faecium. Sulbactam possessed no antibacterial activity against these organisms. It is not yet known if the activity of ampicillin will be increased with the addition of sulbactam when tested against beta-lactamase-producing enterococci.

Ampicillin↗

In vitro activity of RP 59500 (quinupristin/dalfopristin) against antibiotic-resistant strains of Streptococcus pneumoniae and enterococci.

The activity of RP 59500 (quinupristin/dalfopristin) was evaluated in vitro against antibiotic-resistant strains of Streptococcus pneumoniae (N = 15) and Enterococcus spp. (N = 43). By broth dilution MIC tests RP 59500 was highly active against penicillin-resistant S. pneumoniae and vancomycin-resistant Enterococcus faecium, but showed poor activity against E. faecalis. In time-kill studies the drug was rapidly bactericidal against S. pneumoniae but failed to kill most enterococci, even in the presence of gentamicin or human serum.

Drug Resistance, Microbial↗

Bacterial 2-component signal transduction systems: a fluorescence polarization screen for response regulator-protein binding.

Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the Van RSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements.

Anti-Bacterial Agents↗

Therapeutic value of a Lactobacillus gasseri and Bifidobacterium longum fixed bacterium combination in acute diarrhea: a randomized, double-blind, controlled clinical trial.

A multicenter, parallel-group, randomized, double-blind, active-controlled clinical trial, involving 169 outpatients at 9 centers, was conducted to assess the efficacy of a fixed bacterium combination of Lactobacillus gasseri and Bifidobacterium longum in the therapy of acute diarrhea. In particular, this clinical trial was designed to prove equivalent therapeutic efficacy of a fixed bacterium combination versus an exhaustive investigated mono-bacterium medicinal product. All patients, free to carry on usual daily activities, received 1 capsule 3 times a day of either a fixed bacterium combination of Lactobacillus gasseri and Bifidobacterium longum or Enterococcus faecium mono-bacterium. All treatments were continued for a maximum of 6 days in line with the normal course of acute diarrhea. Primary efficacy criterion was the severity and duration of diarrhea assessed by the ensemble of stool frequency as change from baseline and stool consistency at trial Day 2, 3 and 4, and time in days until normal bowel function (recovery). The median duration of diarrhea was 2.70 days versus 2.67 days (fixed bacterium combination of Lactobacillus gasseri and Bifidobacterium longum versus mono-bacterium Enterococcus feacium). The total mean difference of duration of diarrhea was 0.072 days. This result can be considered equivalent. However, the proportion of patients with complete recovery tended to be higher in the fixed bacterium combination group (92.6% versus 87.1%) resulting in a number needed to treat (NNT) of 18.1. The fixed bacterium combination reduced the number of unformed stools by 80% and the mono-bacterium by 75% during the first 2 days of treatment. Both treatments were well tolerated. Oral therapy with a fixed combination of Lactobacillus gasseri and Bifidobacterium longum shortens the duration and decreases the severity of acute self-limiting diarrhea in adults comparable to an effective mono-bacterium medicinal product. It therefore appears to be a useful and safe treatment for this disease.

Acute Disease↗

Resuscitation rate in different enterococcal species in the viable but non-culturable state.

AIMS: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. METHODS AND RESULTS: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1:10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). CONCLUSIONS: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.

Bacterial Proteins↗

Excretion of vancomycin-resistant enterococci by wild mammals.

A survey of fecal samples found enterococcal excretion in 82% of 388 bank voles (Clethrionomys glareolus), 92% of 131 woodmice (Apodemus sylvaticus), and 75% of 165 badgers (Meles meles). Vancomycin-resistant enterococci, all Enterococcus faecium of vanA genotype, were excreted by 4.6% of the woodmice and 1.2% of the badgers, but by none of the bank voles.

Animals↗

Enterococci highly resistant to penicillin: characterizing isolates from Singapore hospitals.

Enterococci are increasing in importance as nosocomial pathogens and causes of severe sepsis in immunocompromised patients. From September to November 1989, a survey of 898 enterococcal isolates showed that 52 had acquired high-level resistance to penicillin and ampicillin (MIC greater than 100 mg/l). These were all Enterococcus faecium, did not produce beta-lactamase and showed high-level resistance to gentamicin and streptomycin as well. The majority were urinary isolates, but a few caused bacteraemia in severely ill patients. The potential spread of these highly-resistant enterococci would limit the therapeutic options for systemic infections.

Adult↗

Quinupristin/dalfopristin-resistant enterococci of the satA (vatD) and satG (vatE) genotypes from different ecological origins in Germany.

The semisynthetic streptogramin combination quinupristin/dalfopristin (Synercid) is a promising alternative for treatment of infections due to multiply resistant gram-positive bacteria including vancomycin-resistant Enterococcus faecium. Resistance is mediated by acetyltransferases SatA (VatD) or SatG (VatE). Recent papers have indicated a possible link between the use of the streptogramin virginiamycin S/M as a feed additive in commercial animal husbandry and a selection of quinupristin/dalfopristin-resistant E. faecium (QDRE). We screened manure samples from two different turkey farms and from six different pig farms (using virginiamycin), samples from a sewage water treatment plant, 24 broiler carcasses, 10 pork samples, and 200 stool samples of nonhospitalized humans for QDRE. Our strain culture collection of hospital E. faecium isolates from the last 2 years was also reviewed for QDRE. All manure and sewage samples were positive for QDRE, as well as 11 from broiler carcasses (46%), 1 from pork (10%), and 28 from human stool specimens (14%). Thirty-six hospital isolates of E. faecium exhibited resistance to quinupristin/dalfopristin. In 141 QDRE of different origin satA (vatD) and satG (vatE) genes were detected (seven isolates from humans with an unknown resistance mechanism). Streptogramin resistance determinants were tansferable in filtermating experiments for 5 of 10 satA (vatD) and 9 of 22 satG (vatE) isolates. Different EcoRI patterns of satG (vatE) plasmids and corresponding hybridizations of the satG (vatE) gene indicated nonhomologous resistance plasmids in isolates of different origin. The results of this study indicate a common gene pool for streptogramin resistance in E. faecium of different ecological origin. A selection of QDRE using the streptogramin virginiamycin S/M as a feed additive and a spread of the resistance via the food chain to humans is probable.

Acetyltransferases↗

The lantibiotic mersacidin inhibits peptidoglycan biosynthesis at the level of transglycosylation.

The lantibiotic mersacidin has been previously reported to interfere with bacterial peptidoglycan biosynthesis, [Brötz, H., Bierbaum, G., Markus, A., Molitor, E. & Sahl, H.-G. (1995) Antimicrob. Agents Chemother. 39, 714-719]. Here, we focus on the target reaction and describe a mersacidin-induced accumulation of UDP-N-acetylmuramoyl-pentapeptide, indicating that inhibition of peptidoglycan synthesis occurs after the formation of cytoplasmic precursors. In vitro studies involving a wall-membrane particulate fraction of Bacillus megaterium KM demonstrated that mersacidin did not prevent the synthesis of lipid II [undecaprenyl-diphosphoryl-N-acetylmuramoyl-(pentapeptide)-N-ac ety lglucosamine] but specifically the subsequent conversion of this intermediate into polymeric nascent glycan strands by transglycosylation. Comparison with other inhibitors of transglycosylation shows that the effective concentration of mersacidin in vitro is in the range of that of the glycopeptide antibiotic vancomycin but 2-3 orders of magnitude higher than that of the competitive enzyme inhibitor moenomycin. The analogy to the glycopeptides may hint at an interaction of mersacidin with the peptidoglycan precursor rather than with the enzyme. Unlike vancomycin however, mersacidin inhibits peptidoglycan formation from UDP-N-acetylmuramoyl-tripeptide and is active against Enterococcus faecium expressing the vanA resistance gene cluster. This indicates that the molecular target site of mersacidin differs from that of vancomycin and that no cross-resistance exists between the two antibiotics.

Amino Acid Sequence↗

High prevalence of VanA-type vancomycin-resistant Enterococci in Austrian poultry.

Fecal samples from humans and food-producing animals were analyzed for the presence of vancomycin-resistant enterococci (VRE). The VRE carriage rate in humans was 6%, and there was a predominance of VanC-type resistance. Enterococcus faecium with vanA-mediated resistance was frequent in broiler chickens (42%) but rare in cattle and pig samples.

Adolescent↗