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In 70 enterococcal strains of diverse origin belonging to 16 species a hydrooxamate class siderophore was detected with chemical and biological tests. A correlation between hydrooxamate siderophore production, species affilIation and pathogenicity of enterococci was not found.
Combination of carbenicillin and cephalothin with various aminoglycosides (gentamicin, sisomicin, tobramycin, kanamycin and amikacin) were studied in vitro using 20 strains of enterococci isolated from clinical material. The addition of the aminoglycosides to carbenicillin increased the rate of bactericidal action of carbenicillin on most strains. Little benefit was obtained by adding the aminoglycosides to cephalothin and antagonistic action has been observed on some strains.
Vancomycin-resistant enterococci is a worldwide threat not only in hospitals, but also in communities. Community-acquired infections play an important role in spreading VRE because the use of avoparcin, an antimicrobial growth promoter in food animals, is strongly suggested to cause the emergence of VRE. In particular, chickens imported into Japan have been reported to be highly contaminated with VRE, and this may be a key issue in the prevalence of VRE. Nosocomial VRE infections have become a serious problem in immuno-compromised patients throughout the world, and many VREs have been isolated in Japan in the last few years; however, fortunately, nosocomial outbreaks are still rare. VRE can survive for weeks in hospital environments, which makes infection control difficult and complicated, and strains of VRE resistant to almost all antimicrobial agents are a real threat. In outbreaks of VRE, prudent antimicrobial use and prompt effective infection control are important.
In order to describe the susceptibility patterns and determine the identities of Enterococcus spp. isolated at a regional teaching hospital in Taichung during the period from June through November 1998, 96 clinical isolates of enterococci were collected for further analysis. Major sources of these isolates included urine, wound, and pus. Species identification was performed using the API 20 Strep system and supplemental tests. The minimum inhibitory concentrations (MICs) of six antimicrobial agents were determined by E-test for each isolate. Disk diffusion tests were also performed and the results were compared with those reported by the clinical laboratory. Because gentamicin susceptibility tests showed inconsistent results in many isolates, MIC determinations by the microbroth dilution method were also performed for these isolates. All isolates were tested for beta-lactamase production using the chromogenic method. The results showed that Enterococcus faecalis was the most frequently encountered species (86.5%), followed by Enterococcus faecium (7.3%), Enterococcus avium (5.2%) and Enterococcus casseliflavus (1.0%). The MIC90 of ampicillin, penicillin, vancomycin, teicoplanin, ciprofloxacin and gentamicin for total isolates were 1, 3, 2, 0.25, 1, and more than 1024 microg/mL, respectively. All isolates were susceptible to vancomycin and teicoplanin. The same rate of resistance (3.1%) was found to penicillin, ampicillin and ciprofloxacin in all isolates. There were 50 (52%) and 48 (50%) isolates with high level streptomycin and gentamicin resistance, respectively. The MIC90 of ampicillin and penicillin for E. faecium were significantly higher than those for other species (96 and > 256 vs. < or = 1 and < or = 3 microg/mL, respectively); in fact, ampicillin or penicillin-resistance was only found in E. faecium. No organism was found to produce beta-lactamase. There were 29 isolates showing discrepant results between the study findings and clinical laboratory report for gentamicin susceptibility. Most isolates (27/ 29) reported as susceptible to gentamicin by the clinical laboratory showed a high level gentamicin resistance by MIC determinations. The inconsistent results may be due to discrepancy in interpretations for heterogeneous resistance, bias in selecting colonies for the disk diffusion test, or variation in the quality of Mueller-Hinton agar used. The results suggest that any pure isolates growing some colonies within the inhibition zone should be considered as gentamicin resistant even if the zone diameter is equal or greater than the susceptible breakpoint. In order to obtain accurate results, the gentamicin MIC should be determined by the dilution method for enterococcal isolates that yield intermediate inhibition zones or zones just above the susceptible limit.
The incidence of Enterococcal nosocomial infection has increased since 1980s. Most Enterococci are intrinsically resistant to many kinds of antibiotics, therefore, therapeutic drugs for enterococal infectious disease are limited to a few antibiotics such as ampicillin, gentamicin and vancomycin. However, since the first report of penicillinase producing E. faecalis in 1983, ampicillin-resistant strains have been increasing, furthermore, these strains often associated with high-level resistance to aminoglycosides. The yearly usage of vancomycin increased because vancomycin was only effective drug against such resistant strains and worldwide spread of multidrug-resistant MRSA which vancomycin is only effective. However, High-level vancomycin-resistance strains of E. faecium were first isolated in England in 1986. In 1990s, VRE have been spreading all over the world, especially, the emergency of multidrug-resistant strains, which are resistant to high concentrations of ampicillin or gentamicin or to vancomycin in USA, brought serious therapeutic dilemmas. This review focuses on the background in emergence of VRE, the relation with use of avoparcin, is used for growth promotion in farm animals, the mechanism of resistance, and the prevention of the spread of vancomycin resistance.
Vancomycin-resistant enterococci (VRE) is a world-wide emerging pathogen. The first confirmed VRE isolate in Taiwan was detected in 1995. After that, more and more isolates of VRE were found in western part of Taiwan. However, before this report, there was no any isolate of VRE found in eastern part of Taiwan. In December 1997 and January 1998, two cases of VRE urinary tract infection were found in two regional hospitals in north-eastern Taiwan. They had received prolonged treatment with multiple antibiotics for other infections before they got VRE infection. The isolates showed susceptibility to other antimicrobial agents by routine disk diffusion susceptibility test and these two patients were successfully treated. The VRE did not spread in those two hospitals. After further study, the 3 isolates from these two patients all had minimum inhibitory concentration of vancomycin >256 microg/mL. They all carried van A gene and the pulsed-field gel electrophoresis demonstrated they belonged to different DNA types. It was concluded that VRE appeared in north-eastern Taiwan but the strains were not from a common source.
Among 20 strains of genus Enterococcus isolated from different sources 12 strains multiplied in iron unsupplemented normal human serum. The excess of iron did not increase the rate of multiplication. Three strains survived in serum and growth of five strains was inhibited. The siderophores production had no effect on the multiplication of enterococci in serum unsupplemented with iron.
The study was undertaken to investigate the ability of enterococci to assimilate iron via siderophores of bacteria living in the same habitats in the human organism. The potential recipients of exogenous siderophores were six Enterococcus faecalis and six Enterococcus faecium strains, isolated from clinical materials of human origin. The donors of siderophores were Gram-negative rods (various species of the Enterobacteriaceae, Pseudomonas and Acinetobacter) and Gram-positive cocci (various species of Staphylococcus and Streptococcus). All of the investigated E. faecium and only two E. faecalis strains demonstrated the ability to utilize the siderophores of the aforementioned bacterial groups, predominantly the chelators of Gram-negative rods, those of Gram-positive cocci were utilized to a smaller extent. Four recipient strains from E. faecalis species did not demonstrate the ability to utilize siderophores synthesized by all of 40 investigated donor strains.
Very important bacterial pathogens found in hematological patients at present are vancomycin-resistant enterococci (VRE). The main goal of this retrospective study was to assess their occurrence in relation to antibiotic use. We isolated 1918 Enterococcus strains, in toto, 138 (7.2%) of which proved to be VRE. The VRE most frequently identified were Enterococcus faecium VanA (77%) and Enterococcusfaecalis VanB (12%), mostly isolated from stools (57%). Comparing the development of the selection pressure of antibiotics and percentage of VRE in each period of observation, an effect of the administration of each antibiotic group on the occurrence of VRE can be presumed. A reduction in the administration of third generation cephalosporins, glycopeptides and fluoroquinolones and its replacement by penicillin antibiotics combined with inhibitors of bacterial beta-lactamases, contributed to the cessation of VRE incidence and succeeding reduced occurrence from 15.1% in the second half of 1998 to 6.1% in the first half of 2000.
The resistance determinants were transferred from clinical strains of enterococci to Staphylococcus aureus strains. As recipients methicillin-resistant and methicillin-susceptible strains were used and the filter-mating procedure was performed. The transconjugants resistant to erythromycin were obtained in the case of all recipients, in one case the vanA determinant conferring the resistance to vancomycin was transferred together with erythromycin resistance. However the resistance was very unstable and the level was not as high as in the case of Enterococcus faecalis donor strain. The vanA determinant was easily transferred between enterococcal strains by the conjugation and a transfer occurred of vanA alone or together with erythromycin resistance.
Methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci are major hospital pathogens in hospital settings. These organisms are mainly transmitted by direct skin contact with infected/colonized patients. Air borne transmission may rarely occur in MRSA positive cases with upper respiratory infections. Contamination in immediate environment requires proper disinfection after contact. Screening patients for MRSA and VRE can be restricted against those in/from the high risk areas. The clinical pictures, methods for screening and the precautions were also discussed based on the nature of communicability in both pathogens.
One hundred strains of Enterococci obtained from various clinical specimens over a period of six months were speciated of which 56 were E. faecalis and 44 were E. faecium. Antimicrobial susceptibility pattern showed 90% of them were resistant to penicillin and 20% of them were resistant to vancomycin. Comparative evaluation of Beta-lactamase production by iodometric method and cloverleaf method showed that the latter was cost effective and easy to perform for routine use.
Fifty samples of chicken, duck and geese faeces were obtained from 13 wet markets in Kuala Lumpur to study the prevalence of vancomycin-resistant enterococci (VRE) among local market poultry. Biotyping of colonies grown on azide agar incubated at 45 degrees C yielded E. pseudoavium, E. faecalis, E. faecium and E. gallinarum from chicken faeces and E. malodoratus, E. faecalis, E. faecium, E. gallinarum, E. hirae/dispar, and E. durans from goose and duck faeces. On agar containing 6 mg/ l of vancomycin, one strain of E. flavescens was identified, giving a VRE detection rate of 2.0%. This isolate had a vancomycin M.I.C. of 8 mg/l as determined by the Etest, and the van C-3 gene that was identified by PCR followed by sequence analysis. The prevalence of VRE among poultry sold in local markets appears to be low, and may reflect the infrequent use of antimicrobials in our poultry farms. Nevertheless, the possibility of human acquisition of microbes via the food chain cautions against the use of antimicrobials in animal husbandry that may encourage the emergence and spread of multi-drug resistant organisms like the VRE among animal microbial flora.
Enterococci respond to iron deprivation in vitro by increasing the expression of a number of iron-regulated proteins. They detected in whole protoplasts lysates and corresponded to proteins with apparent molecular masses of the region 14.4-43 Kda. Their occurrence were correlated to siderophore production.
In recent years enterococci have been isolated with increasing frequency in association with serious infections. Suitable therapy for empirical treatment can only be chosen on the basis of susceptibility studies. The synergistic effects obtained by the combination of aminoglycosides with penicillin or vancomycin disappear in strains that show high level resistance (HLR) to the former. This highlights the importance of correct identification of the species of Enterococcus (especially Enterococcus faecium, a particularly resistant species), and of studies designed to examine HLR in severe infections. The preferred methods are dilution in solid or liquid medium with 2,000 micrograms of aminoglycoside per ml, or diffusion in solid medium from discs impregnated with 120 micrograms of gentamicin (which also predicts the microorganism's susceptibility to tobramycin and netilmicin) or kanamycin (which also predicts the response to amikacin), and 300 micrograms of streptomycin, for Enterococcus faecalis. The reliability of automated systems for the prediction of HLR to aminoglycosides has yet to be assessed.
The new identification system STREPTOtest (fy. Lachema, Brno) was evaluated. A total 118 well-known strains of genus Enterococcus, Streptococcus, Aerococcus, Stomatococcus and Gemella were tested. The STREPTOtest system rapidly and reliably distinguished genus Enterococcus from genus Streptococcus. This method identified correctly to the species level 65.9% of enterococci strains and 52.5% of streptococci strains. The STREPTOtest was used for identification of A. viridans and S. mucilaginosus and it was possible to separate these strains from similar ones. A new differentiation chart for identification of all 13 recently described enterococcal species was proposed.
Between April 1, 1999 and June 30, 2000, 144 isolates of enterococci (one per patient) from cultures of several anatomic sites were collected. One hundred and nineteen (82.6%) E. faecalis, 11 (7.6%) E. faecium and 14 (9.7%) of other species (5 E. raffinosus, 4 E. avium, 3 E. casseliflavus, 1 E. pseudoavium, and 1 E. dispar) were associated with clinical infections. The most common sites of isolation were: the urinary tract 54.9%, abdominal cavity 12.5%, surgical wounds 12.5%, abscesses 6.9% and diabetic foot 6.2%. High-level resistance to gentamicin or streptomycin or both was detected in 48.6% of the isolates. E. faecium and E. raffinosus were significantly more resistant than E. faecalis to ampicilin and imipenem. None of the strains exhibited beta-lactamase activity. One strain of E. faecium (0.7%) was resistant to vancomicin and teicoplanin (Van A phenotype) and two strains of E. casseliflavus (1.4%) showed low level of resistance to vancomicin (Van C phenotype). Because of these diverse antimicrobial resistance mechanisms, successful treatment and control of enterococcal infections with current antimicrobial agents are becoming increasingly difficult.
In the pool of 70 enterococcal strains of the genus Enterococcus 61.4% released citrate into the medium. This metabolite has occurred more frequently in E. faecium strains. There was no correlation between hydroxamate siderophores production and citrate releasing. Only nine (10, 3%) of 70 strains have used Fe3+-dicitrate complex as iron sources. Iron restricted condition causing moderate inhibition of growth have not increased citrate releasing. When iron deficiency has caused stronger growth inhibition, E. faecalis strains did not release citrate and E. faecium strains its smaller amounts. The resting cells grown in iron-restricted condition have incorporated 59Fe3+ complexed by citrate more active than cells grown in the medium with excess of iron. So, citrate has not been a siderophore in enterococci.