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Observations on the tolerance and the paradoxical effect in enterococci.

Enterococci, already know to be relatively unaffected by several antibiotics due to their inheritant characteristics, are increasingly resistant to some very important groups of drugs, by means of acquisition or exchange of new genetic traits of resistance. Resistance or moderate susceptibility towards penicillin is an interesting characteristic of enterococci, whose low degree of susceptibility to this drug is due to a low affinity for penicillin-binding proteins (PBP). Some strains of enterococci are not killed by the action of this drug but are "tolerant" (MIC/MBC > 32 mg/l). This kind of "resistance", in which the probability of surviving under selective pressure of the drug is increased, is probably linked to the deficiency of the cell's autolytic system. Only rarely does another form of resistance called the "paradoxical effect" appear, in which higher numbers of cells survive at high concentrations than at lower concentrations. In our study the degree of bactericidal activity of some beta-lactams was considered. Our results demonstrate that: i) the paradoxical effect appears more in cultures in exponential phase compared to aged cultures; ii) mutated strains show an increased number of cells that respond paradoxically (the behavior is genetically determined); iii) different beta-lactams induced different degrees of autolysis.

Anti-Bacterial Agents↗

Molecular alterations of VanA element in vancomycin-resistant enterococci isolated during a survey of colonized patients in an Italian intensive care unit.

To screen for vancomycin-resistant enterococci (VRE) colonization in hospitalized patients and to study molecular evolution and alterations of Tn1546-like elements in VRE among potentially at-risk patients, a 3-year surveillance protocol in an Intensive Care Unit was performed. A total of 397 patients were screened in the period June, 1997-June, 2000, and VRE were isolated from rectal swabs taken at admission, weekly, and when clinically indicated. The susceptibility of the enterococci was determined by the disk diffusion and broth dilution methods. The presence of vancomycin-resistance genes (vanA, vanB, and vanC) was assessed by polymerase chain reaction (PCR); genetic clonality of isolates was assessed by pulsed-field gel electrophoresis (PFGE); Tn1546 types were obtained by restriction fragment length polymorphism (RFLP) analysis of Tn1546 PCR fragments. Thirty-four strains, 31 identified as Enterococcus faecium and 3 strains as E. faecalis, were isolated from 12 of the 397 patients (3.0%); all strains were VanA as assessed by PCR and were resistant to the other antibiotics tested and showed high-level resistance to aminoglycosides. Enterococci isolated during the study period showed that different genetic backgrounds of strains, determined by PFGE combined with RFLP of Tn1546, are present in all the strains isolated in the study. PFGE type B was predominant in 1998 and 1999, and insertion sequence movements were found to have a role in the evolution of VanA resistance elements found in all strains. This study demonstrates that single patients may be colonized by closely related VRE with several PFGE types containing a wide variety of VanA elements. Moreover, isolates with identical PFGE types may contain different VanA elements reflecting rearrangements mediated by insertion sequences in VRE strains during their stay in the gastrointestinal tract.

Adolescent↗

Characterization of high-level aminoglycoside-resistant enterococci in Kuwait hospitals.

This study investigated the distribution of genes for aminoglycoside-modifying enzymes (AME) and the genetic relatedness of high-level aminoglycoside-resistant enterococci isolated in Kuwait hospitals. A total of 117 enterococci, consisting of 109 Enterococcus faecalis, seven Enterococcus faecium, and one Enterococcus casseliflavus were studied. The MICs of gentamicin, kanamycin, amikacin, tobramycin, and streptomycin were determined by agar dilution and the genes encoding the AAC(6')- APH(2"), ANT(4'), APH(3'), APH (2")-Ib, APH (2")-Ic, APH (2")-Id, and ANT(6) enzymes were amplified by PCR. They were typed by pulsed-field gel electrophoresis (PFGE). Filter mating was used to transfer gentamicin resistance determinants. They were all resistant to kanamycin (MIC 2000 mg/L). Fifty-five isolates were resistant to gentamicin (MIC 500 mg/L), 72 were resistant to tobramycin (MIC 64 mg/L), 115 were resistant to amikacin (MIC 64 mg/L), and 97 were resistant to streptomycin (MIC 1000 mg/L). The aac(6')-Ie-aph(2")-Ia was detected in all isolates with gentamicin MIC 500 mg/L and in 15 isolates with gentamicin MIC 256 mg/L. The aph(3')-IIIa gene was detected in 101 isolates, whereas the ant(6')-Ia gene was detected in 85 of the 97 streptomycin-resistant isolates with MIC 1000 mg/L. The aac(6')-Ii gene was detected only in the seven E. faecium isolates. None of them contained ant(4')-Ia, aph(2")-Ib, aph(2")-Ic and aph(2")-Id. PFGE revealed heterogeneous patterns with no dominant clone. The results demonstrated that AME are common in aminoglycoside-resistant enterococci isolated in Kuwait. However, the absence of a dominant clone suggests that they acquired high-level aminoglycoside independently.

Aminoglycosides↗

Concomitant high-level vancomycin and penicillin resistance in clinical isolates of enterococci.

Enterococci are important nosocomial pathogens among which resistance to multiple antibiotics is being recognized with increasing frequency. We characterized three clinical isolates from three New York City hospitals that demonstrated concomitant resistance to vancomycin (one VanA, two VanB phenotypes) and high-level resistance to penicillin. Two Enterococcus faecium strains were intrinsically highly resistant to penicillin and showed very low affinity for penicillin of penicillin-binding protein 5. Unlike previously described glycopeptide-resistant enterococci, these strains were not hypersusceptible to beta-lactam agents after vancomycin induction, and combinations of penicillin and vancomycin were not synergistic against them. A third isolate, Enterococcus faecalis, produced beta-lactamase. Two of the three strains were also highly resistant to all aminoglycosides. Emergence of concomitant high-level resistance to multiple antibiotic classes among enterococci considerably narrows the therapeutic options for treatment of infections due to these opportunistic pathogens.

Aged↗

Influence of oral glycopeptides on the fecal flora of human volunteers: selection of highly glycopeptide-resistant enterococci.

Changes in fecal flora were evaluated in 22 healthy volunteers administered oral vancomycin or teicoplanin in 1989-1991 in Belgium. Evaluation of 5 colonies per subject revealed no glycopeptide-resistant enterococci in the predominant flora before glycopeptide administration; however, large numbers (mostly Enterococcus faecium) emerged by the end of the study in 14 (64%) of the subjects. Pediococci and lactobacilli also increased in number. In 1992, 40 healthy volunteers and 33 cancer patients were evaluated by plating stool samples directly onto selective media containing vancomycin; low numbers of vancomycin-resistant enterococci (< 50 cfu/g) were found in 11 (28%) of the 40 and 4 (12%) of the 33 samples, respectively. DNA restriction fragment length polymorphism analysis showed that most isolates were different, but all contained vanA in Tn1546-like elements. These results indicate that vanA and Tn1546-like elements were common in Belgium as early as 1989 and that community-based individuals in that location likely form a major reservoir for glycopeptide-resistant enterococci.

Adult↗

In-vitro activity of LY 146032, a novel cyclic lipopeptide, alone and in combination with gentamicin or tobramycin against enterococci.

The in-vitro activity of LY 146032, a novel cyclic lipopeptide and the effect of the combination of LY 146032 and gentamicin or tobramycin against 25 strains of enterococci isolated from blood cultures were studied. All strains of enterococci were inhibited by less than or equal to 2 mg/l LY 146032. The minimal inhibitory concentrations and minimal bactericidal concentrations were within one dilution. In the time-kill study, there was slow bactericidal activity. Complete killing of 10(7) cfu/ml of enterococci at 48 h by LY 146032 alone occurred with two strains. There was synergism between LY 146032 and gentamicin or tobramycin; complete killing at 24 h and 48 h occurred with many strains. No antagonism was demonstrated.

Anti-Bacterial Agents↗

The prevalence of high-level aminoglycoside resistance among enterococci isolated from blood cultures during 1980-1988.

Two hundred and eighty-one strains of enterococci isolated from blood cultures of 281 consecutive patients during 1980-1988 and 108 strains of enterococci isolated from miscellaneous clinical materials during 1988 were studied for high-level resistance (MIC greater than 2000 mg/l) to amikacin, kanamycin, streptomycin, gentamicin, tobramycin and netilmicin. Before 1985 there was no enterococcal strain with high-level resistance to amikacin, gentamicin, tobramycin or netilmicin, but 14% were resistant to kanamycin and 21% to streptomycin. For strains isolated from blood cultures during 1985-1988, the prevalence of high-level resistance was as follows: amikacin, less than 1%; kanamycin, 35%; streptomycin, 26%; gentamicin, 9%; tobramycin, 9%, netilmicin, 4%. Prevalence of high-level aminoglycoside resistance of enterococci isolated from miscellaneous sources during 1988 was higher (amikacin, 5%; kanamycin, 37%; streptomycin, 31%; gentamicin, 24%, tobramycin, 27%; netilmicin, 10%). Among strains isolated from blood cultures between 1980-1984, 24% (30 of 126 strains) and among 1985-1988 strains, 40% (62 of 155 strains) were resistant to at least one of the aminoglycosides tested. Similarly, among the miscellaneous strains, 40% (43 of 108 strains) had high-level resistance to at least one of the aminoglycosides tested.

Aminoglycosides↗

Rapid increase in the prevalence of high-level aminoglycoside resistance among enterococci isolated from blood cultures during 1989-1991.

At St Elizabeth Hospital Medical Center in Youngstown, Ohio, USA, the first blood culture isolate of Enterococcus faecalis with high-level gentamicin resistance (MIC > 2000 mg/L) was seen in 1985, and the prevalence of high-level gentamicin resistance among enterococci isolated from blood cultures during 1985-8 was 9%. During the period 1989-91, the prevalence of high-level gentamicin resistance among enterococci isolated from blood cultures increased to 35% (44 of 126 strains). Increases in the prevalence of high-level resistance to amikacin, tobramycin, netilmicin, kanamycin and streptomycin were also demonstrated. Ten of 44 strains (23%) with high-level gentamicin resistance did not exhibit high-level resistance to streptomycin. Of the 126 strains of enterococci, 52% had high-level resistance to at least one aminoglycoside.

Aminoglycosides↗

High-level aminoglycoside resistance among enterococci and group A streptococci.

The occurrence of high-level aminoglycoside resistance (HLAmR) was determined for 73 enterococci and 54 group A streptococci by the high-load disc method, tube macrodilution and the polymerase chain reaction (PCR). The PCR method revealed the presence of genes coding for aminoglycoside-3'-O-phosphoryltransferase-III (APH(3')-III), aminoglycoside-6'-N-acetyltransferase/2''-O-phosphoryltransferase (AAC(6')/APH(2'')), or both, in 20.6%, 9.6% and 4.1% of the enterococci, respectively. The prevalence of HLAmR to at least one aminoglycoside among local enterococci was 37% (27/73). Only one of 54 Streptococcus pyogenes isolates produced APH(3')-III and exhibited high-level resistance to kanamycin and streptomycin. In general, the three methods yielded comparable results, with only three discrepancies among the 127 isolates examined. High-load disc screening and tube macrodilution proved to be practical, reliable and reproducible, and thus suitable for routine screening. Of 20 Enterococcus faecalis strains tested, all were penicillin-tolerant. Only one of seven penicillin-tolerant S. pyogenes strains was HLAmR. No association between the two forms of resistance was found.

Aminoglycosides↗

Unorthodox antibiotic combinations including ciprofloxacin against high-level gentamicin resistant enterococci.

Development of high-level gentamicin resistance among enterococci represents a serious therapeutic problem as it precludes synergy between aminoglycosides and cell-wall active agents. As part of a search for active antibiotic combinations against enterococci with high-level gentamicin resistance, we tested by the time kill curve method the efficacy of ciprofloxacin combined with ampicillin, trimethoprim-sulphamethoxazole, vancomycin or teicoplanin against ten isolates of Enterococcus faecium, three of Enterococcus casseliflavus and 13 of Enterococcus faecalis that exhibited a MIC of gentamicin > or = 2000 mg/L. Most of the E. faecium were also resistant to ampicillin and to ciprofloxacin. The combination of ciprofloxacin with ampicillin was bactericidal against five of seven E. faecium strains that exhibited a ciprofloxacin MIC < or = 4 mg/L, but was inactive against the three E. faecium that were highly resistant to ciprofloxacin. This combination was also bactericidal against the E. casseliflavus and all the E. faecalis strains. The combination of ciprofloxacin with trimethoprim-sulphamethoxazole was bactericidal against five of the seven E. faecium and seven of the nine E. faecalis strains with a ciprofloxacin MIC < or = 4 mg/L. No bactericidal activity of this combination was seen against the enterococci that were highly resistant to either ciprofloxacin or to trimethoprim-sulphamethoxazole. The combination of ciprofloxacin with glycopeptides was inactive against E. faecium and E. casseliflavus and against E. faecalis, it was either ineffective or antagonistic; in only one case it was bactericidal. Five strains of E. faecium were resistant to all antibiotic combinations tested.

Ampicillin↗

Molecular analysis of diverse elements mediating VanA glycopeptide resistance in enterococci.

Differences were examined among 24 distinct elements mediating VanA-type glycopeptide resistance in enterococci isolated from hospital patients and non-human sources in the UK. The methods used included long-PCR restriction fragment length polymorphism (L-PCR RFLP) analysis and DNA hybridization. All elements had conserved vanRSHAX genes, but variation occurred upstream of vanR and downstream of vanX. Twenty-one VanA elements had significant alterations upstream of vanR in the transposition genes orf1 and orf2: either parts of these genes were absent or they were disrupted by IS1216V or IS3-like insertion sequences. Among VanA elements with alterations downstream of vanX, seven lacked vanY, one lacked both vanY and vanZ, and ten had copies of insertion sequence IS1216V between vanX and vanY. All VanA elements of group D (from geographically and temporally diverse enterococci) were characterized by the presence of an IS1216V/IS3-like/orf1 complex and a point mutation in vanX, both of which were absent from the other 23 groups of VanA elements. This finding is consistent with the dissemination of a stable resistance element. We conclude that L-PCR RFLP analysis, combined with DNA hybridization, merits further development for studying the evolution and epidemiology of VanA resistance elements in enterococci.

Animals↗

Evaluation of selective and enrichment media for isolation of glycopeptide-resistant enterococci from faecal specimens.

OBJECTIVES: Vancomycin-resistant enterococcus enrichment broth (VEB) and vancomycin-resistant enterococcus selective agar with vancomycin 6 mg/L (VSA) are novel azide-aesculin agar-based media that contain meropenem as an additional selective agent. The media were compared with enterococcosel broth (EB) and enterococcosel agar with vancomycin 6 mg/L (EA) for the isolation of glycopeptide-resistant enterococci (GRE) from routine faecal screening specimens. METHODS: Two hundred and eighteen routine faecal screening specimens from patients at Addenbrooke's Hospital were examined. The majority were from patients on haematology wards (155) or the intensive therapy unit (ITU) (21). Specimens were inoculated on to VSA and EA directly, and after enrichment in VEB and EB, respectively. RESULTS: One hundred and twenty-eight GRE isolates were recovered from 93 (43%) specimens with enterococci carrying vanA or vanB genes. There were no statistically significant differences between media (specimens positive; numbers of GRE isolates) on direct plating on VSA (87; 104) or EA (86; 97) or following 24 h enrichment in VEB (89; 103) or EB (86; 98). There was no significant advantage to enrichment compared with direct plating. Incubation of enrichment broth cultures for only 6 h appeared detrimental. Enterococci with vanC were isolated significantly less frequently from VEB and VSA than from EB and EA. Growth of organisms other than GRE was more common on VSA than on EA. CONCLUSIONS: VEB and VSA were at least as effective as EB and EA for the recovery of GRE from faecal screening specimens, but substantially more non-GRE grew on VSA than on EA. Enrichment culture offered no significant advantages over direct plating.

Agar↗

Epidemiology of antimicrobial resistance in enterococci of animal origin.

OBJECTIVE: We evaluated the epidemiology of antimicrobial resistance in enterococci from animal farms and the potential relation of resistance to antimicrobial use. METHODS: Enterococci from faecal samples from 18 beef cattle, 18 dairy cattle, 18 swine, 13 chicken, and eight turkey farms were prospectively evaluated over a 6 year period from 1998 to 2003. RESULTS: We evaluated 1256 isolates of Enterococcus faecium and 656 isolates of Enterococcus faecalis. None was vancomycin resistant. Quinupristin/dalfopristin, gentamicin and ciprofloxacin resistance rates in E. faecium were 2%, 0% and 55% in beef cattle, 8%, 7% and 47% in dairy cattle, 21%, 1% and 47% in swine, 85%, 12% and 23% in chicken, and 52%, 13% and 24% in turkey isolates, respectively. For E. faecalis, gentamicin resistance rates were 0% in beef cattle, 24% in dairy cattle, 37% in swine, 32% in chicken, and 29% in turkey isolates, whereas 12%, 9%, 21%, 64% and none of isolates from beef, dairy, swine, chicken, and turkey farms, respectively, were resistant to ciprofloxacin. Quinupristin/dalfopristin resistance in E. faecium was more common on chicken and turkey farms using virginiamycin (P<0.0001 for both) compared with farms not using a streptogramin, gentamicin resistance was more common on dairy farms using gentamicin (P<0.0001) compared with farms not using this antibiotic, and ciprofloxacin resistance was more common on turkey and dairy farms using enrofloxacin compared with those with no enrofloxacin use (P=0.02 and P=0.04, respectively). For E. faecalis, gentamicin resistance was more frequently detected on dairy and swine farms using gentamicin (P<0.0001 and P=0.0052, respectively) and ciprofloxacin resistance was more common on beef farms using enrofloxacin (P<0.0001) compared with farms not using these antimicrobials. PFGE showed multiple strain types with some clones common between animals of the same animal species. CONCLUSIONS: This study shows the presence of a significant reservoir of antibiotic-resistant enterococci among farm animals. Resistance was more common on farms using antimicrobial agents.

Agriculture↗

High occurrence and persistence of antibiotic-resistant enterococci in poultry food samples in Portugal.

OBJECTIVES: We determined the presence of antibiotic-resistant enterococci (ARE) in commercialized poultry samples from Portugal and analysed their clonal diversity and the resistance genes harboured by these strains. METHODS: Ninety-nine retail poultry samples of 10 widely commercialized brands were studied (1999-2001). Samples were enriched and plated on selective media with and without vancomycin, gentamicin, streptomycin or kanamycin. Antibiotic susceptibility was established following standard criteria. Identification and detection of genes coding for resistance were determined by PCR. Clonal relatedness was established by PFGE. RESULTS: A high percentage of samples contained vancomycin-resistant enterococci (VRE) (48%), or enterococci highly resistant (HLR) to gentamicin (34%), streptomycin (32%) or kanamycin (30%). Co-resistance to tetracycline, erythromycin, ciprofloxacin and quinupristin/dalfopristin was observed in most of these isolates. VRE were classified as VanA phenotype-vanA genotype (38% of samples), VanB phenotype-vanA (13%) or VanC phenotype-vanC1 (23%). All HLR to gentamicin isolates contained aac(6')-Ie-aph(2'')-Ia. We detected erm(B) in both erythromycin-resistant and -susceptible isolates. Some VRE and HLR to gentamicin strains were recovered from different samples and brands. Long-term persistence of particular VRE strains (>2 years), exhibiting different Van phenotypes, was observed. CONCLUSIONS: High occurrence of ARE suggests maintenance of selective pressure by the use of antibiotics/other substances in the Portuguese poultry environment. Persistence of a number of widespread PFGE types containing different resistance genes might reflect environmental/host-adapted enterococcal strains that might contribute to the maintenance of antibiotic resistance, thus constituting a resistance reservoir that is non-sensitive to banning interventions.

Acetyltransferases↗

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry.

OBJECTIVES: To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. METHODS: A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. RESULTS: In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. CONCLUSIONS: This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.

Animals↗

Interactions of vancomycin resistant enterococci with biomaterial surfaces.

Enterococci are a frequent cause of nosocomial infections and are often found adherent to indwelling catheters. Concern about such device associated infections has increased with the appearance of vancomycin resistant (VR) enterococci. However, the possible influence of vancomycin resistance in the pathogenesis of biomaterial centered infection has not yet been assessed. Using polyethylene terephthalate (PET) disks as model surfaces, the authors evaluated possible differences in the adherence and persistence of vancomycin sensitive (VS) and VR strains of Enterococcus faecium and Enterococcus faecalis on biomaterial surfaces in vitro and in vivo. The results indicate that: 1) as expected, the clearance of free VR and VS organisms after intraperitoneal injection into normal mice is equally efficient. 2) In vitro, VR bacteria are roughly twice as adherent to plasma coated PET surfaces as are VS organisms. 3) However, in vivo persistence of VS organisms preadherent to biomaterial implants is 5- to 10-fold better than that of preadherent VR organisms. The authors now believe that a discrete change in bacterial cell wall composition between VR and VS enterococci may contribute to the substantial differences in bacterial adhesion and survival of adherent organisms.

Animals↗

Vancomycin-resistant staphylococci and enterococci: epidemiology and control.

PURPOSE OF REVIEW: This review updates information on the development and spread of vancomycin resistance in staphylococci and enterococci. RECENT FINDINGS: New information on the genetic characterization of vancomycin-resistant Staphylococcus aureus isolates from the US indicates that each of the four was the result of an independent genetic event. New data suggest that vancomycin-intermediate S. aureus isolates, particularly those showing heteroresistance, are clinically significant. Finally, vancomycin-resistant enterococci continue to be reported from around the world. Novel infection control measures, however, may aid in reducing the spread of these organisms in healthcare settings. SUMMARY: The exchange of genetic information, particularly the vanA gene, between and among staphylococci and enterococci will continue to challenge physicians, microbiologists, and infection control practitioners in efforts to identify, treat, and prevent infections with these pathogens.

Enterococcus↗

Motility of enterococci isolated from the urinary tract and from other sources.

The identity and motility of enterococci isolated as urinary pathogens were compared with those of strains from other sources. The majority of isolates from all sources proved to be Streptococcus faecalis of S. faecium but eight of 117 urinary strains and seven of 127 non-urinary strains belonged to a group intermediate between S. faecium. Of the seven "intermediate" strains from non-urinary sources, four were motile. None of the urinary enterococci was motile. The results do not indicate that urinary enterococci are more likely to be motile than are non-urinary strains.

Humans↗