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Antimicrobial growth promoter ban and resistance to macrolides and vancomycin in enterococci from pigs.

Ninety-six enterococcus isolates from fecal samples of pigs receiving tylosin as an antimicrobial growth promoter and 59 isolates obtained in the same farms 5 to 6 months after the ban of antimicrobial growth promoters in Switzerland were tested for susceptibility to nine antimicrobial agents. A clear decrease in resistance to macrolides, lincosamides, and tetracycline was visible after the ban. Vancomycin-resistant Enterococcus faecium belonged to the same clonal lineage as vancomycin-resistant isolates previously isolated from Danish pigs.

Animals↗

The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates.

The in vitro activity of daptomycin was assessed in comparison with that of vancomycin and penicillin against a wide range of Gram-positive aerobic clinical isolates. MICs were determined by an agar dilution method on Mueller-Hinton agar (NCCLS/EUCAST) and on Isotonic agar adjusted to contain 50 mg/L free calcium (BSAC). Both media were enriched with 5% horse blood for fastidious organisms. Daptomycin MICs for all 172 staphylococci, including methicillin-susceptible and methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus, were 0.03-0.5 mg/L. For 99 of the 100 enterococci (Enterococcus faecalis, n = 50; Enterococcus faecium, n = 50), including 37 vancomycin-resistant isolates, they were 0.25-2 mg/L. For all 108 beta-haemolytic streptococci, including Streptococcus pyogenes and Streptococcus agalactiae, daptomycin MICs were 0.016- 0.25 mg/L; for 101 alpha-haemolytic streptococci, including Streptococcus pneumoniae and 'viridans' streptococci, they were 0.016-2 mg/L. For miscellaneous vancomycin-resistant isolates including Lactobacillus spp., Lactococcus spp., Leuconostoc spp., Pediococcus spp. and isolates of Enterococcus casseliflavus and Enterococcus gallinarum, daptomycin MICs were 0.03-2 mg/L; MICs for the seven isolates of Listeria monocytogenes were 0.25-4 mg/L. There was little difference between the results on Mueller-Hinton agar and on supplemented Isotonic agar The discrepant results occasionally obtained tended to be one dilution higher on supplemented Isotonic agar. Daptomycin was active (MICs < or = 2 mg/L) against all the isolates tested with the exception of one isolate each of E. faecium and L. monocytogenes (MICs = 4 mg/L). Our results indicate that daptomycin MICs are independent of methicillin and vancomycin MICs.

Anti-Bacterial Agents↗

Identification of Enterococcus species isolated from foods of animal origin.

Enterococci isolated from a large variety of fresh and prepared foods of animal origin during routine microbiologic control tests in a distribution firm, were identified to species level using API 20 STREP galleries supplemented with conventional tests, rapid ID32 STREP galleries and SDS-PAGE analysis. API 20 STREP tests correctly identified 77% of the strains, mainly Enterococcus faecium and E. faecalis. A simple presumptive identification scheme based on pigmentation, tetrazolium reduction and acid production from mannitol and raffinose identified 90% of the strains. More complex procedures were necessary to identify the remaining strains. Nearly all strains isolated from hard cheeses and prepared cheese-meat combinations were identified as E. faecium while E. faecalis was the most frequent species in crustaceans. In meat and in prepared meat products E. faecium, E. faecalis and less frequently E. hirae/E. durans were found. Three of four E. gallinarum strains were isolated from products containing turkey meat.

Animals↗

Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers.

The prevalence of resistance in enterococci to antibiotics, commonly used for therapy in poultry or as antimicrobial growth promoters (AMGPs), was determined in faecal samples of two chicken populations: broilers in which antibiotic and AMGP use is common and laying-hens with a low antibiotic usage. In addition faecal samples were examined from three human populations: broiler farmers, laying-hen farmers and poultry slaughterers. MICs of an extended panel of antibiotics for a randomly chosen gentamicin- or vancomycin-resistant enterococcal isolate from each faecal specimen were also determined. The prevalence of resistance for all antibiotics tested was higher in broilers than in laying-hens. Resistance in faecal enterococci of broiler farmers was for nearly all antibiotics higher than those observed in laying-hen farmers and poultry slaughterers. The overall resistance in broilers was correlated with the resistance in broiler farmers and in poultry slaughterers. No correlation between the results obtained in the laying-hens with any of the other populations was found. The 27 gentamicin-resistant isolates all showed high-level resistance to gentamicin and two of these isolates, both Enterococcus faecium, were resistant to all antibiotics tested, except vancomycin. The 73 vancomycin-resistant enterococci (VRE) isolated from the five populations belonged to four different species and in all isolates the vanA gene cluster was detected by blot hybridization. The pulsed-field gel electrophoresis (PFGE) patterns of these vancomycin-resistant enterococci were quite heterogeneous, but Enterococcus hirae isolates with the same or a closely related PFGE pattern were isolated at two farms from the broiler farmer and from broilers. Molecular characterization of vanA-containing transposons of these isolates showed that similar transposon types, predominantly found in poultry, were present. Moreover, similar vanA elements were not only found in isolates with the same PFGE pattern but also in other VRE isolated from both humans and chickens. The results of this study suggest transmission of resistance in enterococci from animals to man. For VRE this might be clonal transmission of animal strains, but transposon transfer seems to occur more commonly.

Agriculture↗

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.

Animals↗

Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers.

An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.

Base Sequence↗

Antimicrobial susceptibility of enterococci recovered from commercial swine carcasses: effect of feed additives.

Enterococcus faecium is an important nosocomial pathogen often displaying multiple antibiotic resistance. The increase in clinical isolates can be attributed in part to hospital practices in antibiotic usage, but there is concern that antibiotic-resistant strains might also originate in animals fed rations containing antibiotic growth promoters. Ingestion of meat from carcasses contaminated with faecal enterococci might then result in human colonization or resistance gene transfer to human enterococci. Because there are few comparisons of bacteria isolated from matched animals that have, or have not, been fed a diet containing antibiotic, two such groups of pig carcasses were sampled at a commercial abattoir. Forty isolates from each group of pigs were tested for their resistance to avilamycin and tylosin. Although a modest number of pigs was examined, and the number of strains of E. faecium tested was small, there was no evidence that the feeding of a growth promoter caused selection of enterococci resistant to tylosin or avilamycin.

Abattoirs↗

Selection of enterococci for potential canine probiotic additives.

Enterococci are important inhabitants of animal intestine and are widely used in probiotic products. A potentially successful probiotic strain is expected to have several desirable properties in order to be able to exert its beneficial effects. Forty enterococcal isolates from dog faeces were tested for characters believed to be important for probiotic strains; bacteriocin production, resistance or tolerance to antibiotics, low pH, bile tolerance and adhesive activity. The total count of enterococci was found to be 3.3-7.3log(10)CFU/g of faeces. Most identified strains were Enterococcus faecium. All strains were sensitive to vancomycin, ampicillin, penicillin and chloramphenicol. Thirty-three percentage of strains were resistant to erythromycin and 28% to tetracycline. Among 40 isolates, 75% showed a broad inhibitory spectrum only against Gram-positive indicator bacteria. Seven strains with broad bacteriocin activity were selected for further assays. In the presence of 1% bile, the survival rate of selected strains ranged between 72 and 98%. Survival of strains at pH 3.0 was found in the range between 76 and 87% after 3h. The adhesion of the tested strains to intestinal mucus ranged from 4 to 11% for canine mucus and from 5 to 8% for human mucus. E. faecalis EE4 and E. faecium EF01 showed the best probiotic properties. It indicates that they could be used as new candidate probiotic strains after in vivo testing.

Animals↗

In vitro activity of LY333328 (oritavancin) against Gram-positive aerobic cocci and synergy with ciprofloxacin against enterococci.

Gram-positive cocci are a major cause of nosocomial bacteraemias and are often resistant to most antibiotics. The emergence of enterococci with reduced susceptibility to vancomycin has created an urgent need for novel antibiotics to combat infections associated with these bacteria. In this study the in vitro activity of LY333328 (oritavancin), a semi-synthetic glycopeptide, was evaluated. LY333328 was effective against all strains of staphylococci, streptococci and enterococci tested. A combination of LY333328 and ciprofloxacin was additive for 50% of Enterococcus faecium strains resistant to both vancomycin and ciprofloxacin, and for 100% of vancomycin-susceptible and either ciprofloxacin-susceptible or -resistant strains.

Anti-Bacterial Agents↗

Emergence of 4',4"-aminoglycoside nucleotidyltransferase in enterococci.

Enterococcus faecium BM4102 was resistant to macrolide-lincosamide-streptogramin B-type (MLS) antibiotics; tetracycline-minocycline; and high levels of kanamycin, neomycin, tobramycin, and dibekacin but not gentamicin. This aminoglycoside resistance phenotype is new in enterococci. The genes conferring resistance to aminoglycosides and MLS antibiotics in this strain were carried on a plasmid, pIP810, that was self-transferable to to other Enterococcus strains. Resistance to tobramycin and structurally related aminoglycosides, kanamycin, neomycin, and dibekacin, was due to synthesis of a 4',4"-aminoglycoside nucleotidyltransferase. Homology was detected by hybridization between pIP810 DNA and a probe specific for a gene encoding an enzyme with identical site specificity in staphylococci. The bacteriostatic activity of amikacin apparently was not affected by the presence of the enzyme, although it was modified in vitro. However, the bactericidal activity of amikacin and the synergism of this aminoglycoside with penicillin were abolished.

Anti-Bacterial Agents↗

Recovery from a single blood culture of two enterococcus gallinarum isolates carrying both vanC-1 and vanA cluster genes and differing in glycopeptide susceptibility.

Two Enterococcus gallinarum isolates distinguished by different colony sizes were recovered from the same blood culture from a woman with acute myeloid leukemia. They were designated E31 (the one with larger colonies) and E32 (the one with smaller colonies). Both isolates were glycopeptide resistant, but the MICs of vancomycin and teicoplanin for E31 (32 and 2 microg/ml, respectively, consistent with the VanC phenotype) and E32 (128 and 16 microg/ml, respectively, consistent with the VanA phenotype) were different. E31 and E32 had the same plasmid profile and showed identical pulsed-field gel electrophoresis patterns after digestion of total DNA with NotI and a two-band variation after digestion with SmaI. Polymerase chain reaction experiments showed that both isolates had both the vanC-1 and vanA genes and carried a Tn1546-related transposon lacking orf1, vanY, and vanZ. The absence of these three genes was confirmed by Southern analysis with appropriate probes. Southern hybridization experiments using a vanA probe showed that this atypical Tn1546-related element appeared to be located on the chromosome. In both E31 and E32, the vanA probe hybridized to EcoRV and HindIII fragments larger in size than the hybridizing fragments observed in the VanA prototype strain Enterococcus faecium BM4147, suggesting the lack of the relevant EcoRV and HindIII restriction sites.

Anti-Bacterial Agents↗

Nosocomial bloodstream infections in United States hospitals: a three-year analysis.

Nosocomial bloodstream infections are important causes of morbidity and mortality. In this study, concurrent surveillance for nosocomial bloodstream infections at 49 hospitals over a 3-year period detected >10,000 infections. Gram-positive organisms accounted for 64% of cases, gram-negative organisms accounted for 27%, and 8% were caused by fungi. The most common organisms were coagulase-negative staphylococci (32%), Staphylococcus aureus (16%), and enterococci (11%). Enterobacter, Serratia, coagulase-negative staphylococci, and Candida were more likely to cause infections in patients in critical care units. In patients with neutropenia, viridans streptococci were significantly more common. Coagulase-negative staphylococci were the most common pathogens on all clinical services except obstetrics, where Escherichia coli was most common. Methicillin resistance was detected in 29% of S. aureus isolates and 80% of coagulase-negative staphylococci. Vancomycin resistance in enterococci was species-dependent--3% of Enterococcus faecalis strains and 50% of Enterococcus faecium isolates displayed resistance. These data may allow clinicians to better target empirical therapy for hospital-acquired cases of bacteremia.

Bacteremia↗

Mechanisms of resistance to imipenem and ampicillin in Enterococcus faecalis.

We found ampicillin- and imipenem-resistant isolates of vanA-possessing Enterococcus faecalis with MICs of 8 to 16 microg/ml and 4 to 32 microg/ml, respectively. There have been few reports about penicillin- and imipenem-resistant E. faecalis. Two mechanisms of beta-lactam resistance in E. faecalis, the production of beta-lactamase and the overproduction of penicillin-binding proteins (PBPs), have been reported. The resistant isolates in the current study did not produce any beta-lactamases and analysis of the PBPs showed no overproduction. However, the affinities of PBP4 for beta-lactams in the resistant strains were lower than those of susceptible strains but the affinities of other PBPs for beta-lactams did not change. Accordingly, whole pbp4 fragments from these resistant isolates were sequenced. Two amino acid substitutions at positions 520 and 605 were observed in the highly resistant strains compared to the susceptible ones, Pro520Ser and Tyr605His, and a single Tyr605His amino acid substitution was found in the low-resistance strains. These two point mutations exist in the region between the active-site-defining motifs SDN and KTG of the penicillin-binding domain, the main target of beta-lactams. A strong correlation was seen between these substitutions and decreasing affinities of PBP4 to beta-lactams. In E. faecalis, resistance due to mutations in PBPs has not been reported, though it has in Enterococcus faecium. Our results suggest that development of high-level resistance to penicillins and imipenem depends on point mutations of PBP4 at positions 520 and 605.

Amino Acid Substitution↗

The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis.

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.

Bacterial Proteins↗

Prevalence of vancomycin-resistant enterococci in Europe.

The aim of the present study was to determine the prevalence of vancomycin-resistant enterococci (VRE) in Europe. Overall, 49 laboratories in 27 countries collected 4,208 clinical isolates of enterococci. Species identification, susceptibility testing, and van gene determination by polymerase chain reaction were performed in a central laboratory. Overall, 18 vanA and 5 vanB isolates of VRE were found. The prevalence of vanA VRE was highest in the UK (2.7%), while the prevalence of vanB VRE was highest in Slovenia (2%). Most vanA and vanB VRE were identified as Enterococcus faecium. Most VRE isolates originated from the patient's urogenital tract, skin, or digestive tract. VRE were equally distributed among clinical departments, with no clear preponderance in any single patient group. A total of 71 isolates containing the vanC gene were identified. The prevalence of vanC VRE was highest in Latvia and Turkey, where rates were 14.3 and 11.7%, respectively. Two-thirds of these isolates were identified as Enterococcus gallinarum and one-third as Enterococcus casseliflavus; the majority of these isolates were cultured from feces. Almost all isolates were obtained from hospitalized patients, mostly children. The highest prevalence of high-level gentamicin-resistant enterococci was seen in Turkey and Greece. In general, the distribution of this resistance type seemed unrelated to the occurrence of VRE. The prevalence of vanA/ vanB VRE in Europe is still low; the majority of the VRE isolates exhibit the vanC genotype and colonize the gastrointestinal tract of hospitalized children.

Adolescent↗

Low in vitro selection frequencies of enterococcal and staphylococcal mutants resistant to the oxazolidinone AZD2563.

Mutants with oxazolidinone MICs, 2- to 16-fold higher than those of parents, were selected from two of five clinical isolates of Enterococcus faecalis during exposure to AZD2563, but only at frequencies of ca. 10(-8). Resistance was not selected in Enterococcus faecium, Staphylococcus aureus or coagulase-negative staphylococci (CoNS). Mutants of one E. faecalis isolate had a G2576-->U 23S rRNA mutation; mutants derived from the second E. faecalis isolate lacked this mutation.

Enterococcus↗

Linezolid for treatment of vancomycin-resistant enterococcal peritonitis.

A 42-year-old woman on continuous ambulatory peritoneal dialysis (CAPD) developed peritonitis secondary to vancomycin-resistant Enterococcus faecium and Candida albicans while hospitalized for pneumonia. She was treated successfully with intravenous linezolid, fluconazole given by nasogastric tube, and removal of the peritoneal catheter. A concentration of linezolid above the minimum inhibitory concentration for most gram-positive pathogens, including vancomycin-resistant E faecium, was achieved in the dialysate fluid after an oral loading dose of 1200 mg. Additional data are needed to establish the role of linezolid in treating CAPD-associated peritonitis.

Acetamides↗

Distribution and characterization of Enterococcus species in municipal sewages.

The occurrence, distribution and characterization of enterococci in municipal sewages from two geographically different regional sewage treatment plants in East Slovakia was studied. The total counts of enterococci in samples varied from 10(4) to 10(6) CFU/ml. The vast majority of the strains identified were Enterococcus faecium (50%), with E. gallinarum (25.5%) and E. casseliflavus (10.1%). The species which accounted for 14.4% of the cultures were not specified. Lactic acid production from twenty isolates ranged from 0.085 mol l-1 to 0.914 mol l-1. Urease activity ranged from 1.21 nkat ml-1 to 10.49 nkat ml-1. The strains were resistant to at least one (except for vancomycin) and at most to six antibiotics tested. The majority of isolates were biresistant. Although nine strains were without bacteriocin production, the other strains inhibited the growth of enterococcal as well as staphylococcal isolates. E. faecium AL 40 produced bacteriocin which inhibited the growth of all indicators reaching 12 mm zones of inhibition. E. faecium was the most prevalent species in all samples, but the source position of isolates as well as the sewage composition did not affect enterococcal distribution. The ability of isolates to produce bacteriocin indicates their possible use in biotechnological environmental processes.

Bacteriocins↗