Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital.

BACKGROUND: Outbreaks of vancomycin-resistant Enterococcus faecium (VRE) strains is an emerging problem worldwide. Even if still relatively uncommon in European hospitals, infections caused by VRE have also been increasing recently in this continent. METHODS: In this study, we characterized 50 consecutive VRE and 23 vancomycin-sensitive E. faecium (VSE) isolates collected in an Italian hospital. The presence of the esp gene and that of genes encoding resistance to glycopeptides was investigated by polymerase chain reaction (PCR). All of the isolates were typed by multi-locus sequence typing (MLST), and a selection of them also by pulsed-field gel electrophoresis (PFGE). RESULTS: We found that all of the VRE and 18 (78%) of the VSE strains belonged to the single clonal complex-17 (CC17). The most represented sequence type (ST) was ST78 (34% of the isolates). When further analyzed by PFGE, ST78 isolates were subdivided into five pulsotypes, four of them closely related. The strong association between the esp gene and CC17 was confirmed. Interestingly, such an association was higher among vancomycin-resistant isolates. Most of the esp-positive isolates (34/46, 74%) encoded Esp4, a rare variant of this protein characterized by the absence of A repeats. CONCLUSIONS: Our findings underscore the role of the CC17 lineage in the nosocomial spread of VRE and VSE, and its rapid local evolution, underscoring the need for programs designed to provide early detection in order to prevent its spreading among the nosocomial population.

Clustering and risk factors of methicillin-resistant Staphylococcus aureus carriage in two Italian long-term care facilities.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a well-recognized agent of health care-associated infections in long-term care facilities, but few data about the circulation of MRSA in this setting in Italy are available. The aim of the study is to determine the prevalence and risk factors for MRSA carriage in nursing home residents in Vicenza (northeastern Italy). PATIENTS AND METHODS: A point prevalence survey was conducted in two long-term care facilities (subdivided into 15 wards) from 12 June 2006 to 6 July 2006. Anterior nasal swabs were obtained from residents and laboratory screening for MRSA was performed; full antibiotic susceptibility was assessed in MRSA isolates. Macrorestriction analysis of chromosomal DNA was carried out by pulsed field gel electrophoresis (PFGE). For each subject, demographic data, length of stay, dependency, cognitive function, presence of medical devices, comorbidities, current and previous antibiotic treatment, previous hospital admission and presence of infection were assessed on the day of sample collection. Factors that were found to be significantly associated with MRSA carriage at univariate analysis were introduced into multilevel logistic regression models in order to estimate the odds ratios (OR) with 95% confidence intervals (CI) for the risk of MRSA colonization, taking into account the clustering of patients within wards. RESULTS: Nasal swabs were obtained in 551 subjects; overall 43 MRSA carriers were detected (7.8%; CI = 5.7-10.4%). The rate of nasal carriers was very similar in the two institutions, and varied from 0% (0/36) to 18% (7/39) between wards. Only two out of 15 wards were found to have no MRSA carriers; overall, three pairs of colonized roommates were detected. Upon multilevel logistic regression, the risk of MRSA carriage was increased in patients with cancer (OR = 6.4; CI = 2.5-16.4), in those that had undergone recent hospitalization (OR = 2.2; CI = 1.0-4.4), and it reached OR = 4.0 (CI = 1.7-9.9) in those with three or more antibiotic treatments in the previous year; about 10% of the variability in MRSA carriage could be attributed to differences between wards. Pulsed field gel electrophoresis analysis permitted the definition of six clusters; two of these comprised 78.6% of the studied isolates and were quite similar, with one being more strongly represented among subjects hospitalized in the previous 12 months. All of the MRSA strains were resistant to ciprofloxacine; nevertheless, the majority were susceptible to most other non-betalactam antibiotics. CONCLUSION: The study suggests that nursing homes are a significant reservoir for MRSA. Statistical and PFGE analyses indicate a scenario where MRSA seems to be endemic and individual risk factors, namely recent hospitalizations and repeated antibiotic treatments, play a major role in the selection of drug-resistant organisms. Infection control measures should be coordinated among different health care settings, and the appropriate use of antibiotics has emerged as an important issue for improving the quality of care.

Molecular analysis of vanA enterococci isolated from humans and animals in northeastern Italy.

A total of 53 vancomycin-resistant vanA-positive enterococci isolates from poultry farms (17 Enterococcus faecium; 8 Enterococcus durans) and from different hospitals (23 E. faecium; 5 Enterococcus faecalis) in northeastern Italy were compared on the basis of their antibiotic susceptibilities, their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and the organization of their Tn1546-related elements. Ampicillin resistance was similar in both groups of isolates (52 and 60.7%, respectively), whereas human strains were more resistant to high-level gentamicin and streptomycin. A total of 52% of animal strains and 60% of human strains were resistant to tetracycline, and 56% and 46.4% to quinupristin/dalfopristin, respectively. In E. faecium and E. durans animal isolates, nine and six distinct PFGE patterns, respectively, were found: in two instances indistinguishable isolates were found from different farms. In E. faecium and E. faecalis human isolates, nine and six distinct PFGE patterns, respectively, were found; among E. faecium strains, 12 were identical or closely related and were isolates from the same hospital. Elements mediating vanA-glycopeptide resistance were characterized by PCR with primers that amplified 10 overlapping fragments of Tn1546. A total of 84.6% of animal strains and 64.2% of human strains contained elements indistinguishable from the prototype Tn1546. In addition, nine different types were identified, but none was common to animal and human strains.

Synergy and mechanism of interaction between pefloxacin and penicillin G against enterococci.

In vitro synergy between penicillin and pefloxacin against Enterococcus hirae and Enterococcus faecium strains with different penicillin susceptibility was studied. The combination was synergistic against penicillin-resistant strains. E. hirae R40 and E. faecium 28R, but not against the penicillin-susceptible ones (E. hirae ATCC 9790 and E. faecium 28S). Analysis of PBPs of cells, grown in the presence of pefloxacin, showed that PBP5 of penicillin-resistant strains, the PBP responsible for the resistance of enterococci to beta-lactam antibiotics, is consistently reduced while it is almost unaffected in the penicillin-susceptible strains even at the highest concentrations of pefloxacin. These results indicate that pefloxacin interferes with the mechanism of synthesis of PBPs and particularly of PBP5, a protein whose production has already been modified in resistant strains, in some way rectifying the previous alteration.

Mechanism of action of BAY v 3522, a new cephalosporin with unusually good activity against enterococci.

The in vitro activity of BAY v 3522, a new cephalosporin with unusually good activity against enterococci, was tested on 100 clinical isolates of Enterococcus faecalis. The MIC for 86.3% of the strains was 4 micrograms/ml, whereas the MIC for 13.7% ranged from 8 to 16 micrograms/ml. No differences were found between MICs determined with low- or high-density inocula. The bactericidal activity of BAY v 3522 was tested on eight clinical strains; most strains showed a ca. 3-log decrease of the original inoculum at two to eight times the MIC. The interaction of BAY v 3522 and of other beta-lactams with penicillin-binding proteins (PBPs) was studied with a laboratory strain, E. hirae ATCC 9790, producing a discernible amount of PBP 5, a protein belonging to the family of low-affinity PBPs, responsible for the low susceptibility of enterococci to beta-lactams. PBPs 3 and 5 of ATCC 9790 showed the highest affinity for the new cephalosporin. Bay V 3522 at the MIC (8 micrograms/ml) saturated these two PBPs without any significant binding to the other PBPs. This result may explain the good antienterococcal activity of BAY v 3522.

Effect of non-beta-lactam antibiotics on penicillin-binding protein synthesis of Enterococcus hirae ATCC 9790.

Fosfomycin, bacitracin and vancomycin in combination with penicillin exhibit a synergic effect against Enterococcus hirae ATCC 9790. This strain, when incubated in presence of the MIC of non-beta-lactam antibiotics, showed an alternated pattern of PBPs. Bacitracin and vancomycin caused a decrease in the density of all PBPs while fosfomycin only reduced that of PBP 6. It is suggested that the observed synergy is a consequence of the inhibition of PBP synthesis by antibiotics which act on the early stages of peptidoglycan synthesis prior to the formation of cross-links.

In vitro response to bactericidal activity of cell wall-active antibiotics does not support the general opinion that enterococci are naturally tolerant to these antibiotics.

The incidence of tolerance and paradoxical response to bactericidal activity of penicillin was investigated in 50 clinical isolates of Enterococcus faecalis. Of the isolates tested, 86% exhibited the paradoxical phenomenon whereby there were more survivors at high than at low concentrations above the MIC. Low penicillin concentrations caused decreases equal to or higher than 99.9% in 11 strains, from 99.9 to 99.5% in 23 strains, and lower than 99.5% in 9 strains. Of the total strains, 14% were killed to the same extent by all concentrations above the MIC. The bactericidal activities of other beta-lactams (ampicillin and piperacillin) and other cell wall inhibitors (vancomycin and daptomycin) were also tested against some of these strains. In general, beta-lactams exhibited the best bactericidal activity at 2 x MIC. Piperacillin was the most active, as at 2 x MIC it reduced the original inoculum by 99.9% or more in most of the strains. No concentration of vancomycin above the MIC caused 99.9% killing of the strains, whereas daptomycin was bactericidal at 8 x MIC in most cases. Paradoxical response to bactericidal activity of beta-lactams was abolished by incubation of the inoculum with 2 x MIC before exposure to higher antibiotic concentrations. These findings suggest that enterococci are not always tolerant to cell wall-active antibiotics and that accurate in vitro bactericidal tests may be useful for the choice of appropriate therapy for infections caused by these microorganisms.

Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin.

Ten clinical isolates of Enterococcus faecalis were examined for susceptibility to the bactericidal activity of penicillin. Four of these had MBCs of penicillin equal to 2 to 4 x the MIC, and six exhibited a paradoxical response to penicillin, i.e., the bactericidal activity of the antibiotic had a concentration optimum at 2 to 4 x the MIC and decreased significantly at concentrations above this. We found that the paradoxical response to penicillin was an intrinsic and stable property of a strain, but that its phenotypic expression was not homogeneous; only a fraction of the cell population that died at low concentrations was able to survive at high penicillin concentrations. The size of this fraction increased with increasing antibiotic concentration and reached a maximum in the late-log phase of growth. All 10 strains produced a lytic enzyme that was active on Micrococcus luteus heat-killed cells, whereas only some strains lysed E. faecalis heat-killed cells. Strains producing large amounts of the latter enzyme did not show the paradoxical response to penicillin, whereas mutants of these strains that lacked this enzymatic activity paradoxically responded to the antibiotic activity. In addition, from strains that showed paradoxical response to penicillin and produced only the enzyme that was active on M. luteus, it was possible to isolate mutants that were also capable of lysing E. faecalis cells and that were killed with similar efficiency by all concentrations above the MBC. On the basis of these findings, the paradoxical response to penicillin is explained as a property of certain strains of E. faecalis; this property is genetically characterized by alterations in synthesis or activity of one autolysin but phenotypically expressed only by a few cells that are in a particular physiological condition when exposed to high concentrations of antibiotics.

Purification of Streptococcus faecium penicillin binding protein 5, a multifunctional penicillin-binding protein.

Penicillin-binding protein 5 of Streptococcus faecium has been solubilized and partially separated from other membrane proteins by covalent affinity chromatography. PBP 5 was successively purified to homogeneity by resolution on SDS-polyacrylamide gel, elution and renaturation of penicillin-binding activity. The purification procedure does not alter the properties that the protein exhibits in the membranous environment.

Transition from resistance to hypersusceptibility to beta-lactam antibiotics associated with loss of a low-affinity penicillin-binding protein in a Streptococcus faecium mutant highly resistant to penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium has been shown to have a very low affinity for penicillin, and this PBP was suggested to be responsible for both the natural low susceptibility and high resistance to the antibiotic in this species (R. Fontana, R. Cerini, P. Longoni, A. Grossato, and P. Canepari, J. Bacteriol. 155:1343-1350, 1983). In this study, an S. faecium mutant (Rev 14) hypersusceptible to penicillin was derived from the highly resistant S. faecium R40 treated with novobiocin, and its properties were compared with those of the parent and S. faecium PS, a relatively susceptible strain from which R40 was isolated. The hypersusceptible strain did not synthesize PBP 5, but it did resemble the parent in cell morphology, growth rate, and autolytic activity. In addition, it was highly susceptible to other beta-lactams but remained as susceptible as R40 and PS to antibiotics of a different mechanisms of action. The affinity of individual PBPs for the beta-lactams tested was the same in all the strains. This finding suggested that Rev 14 hypersusceptibility was due to the lack of PBP 5 and strongly supported the role of this protein in the mechanism of both natural low susceptibility and high-level resistance to beta-lactams in S. faecium.

The photosensitizing activity of haematoporphyrin on mollicutes.

The photosensitizing activity of haematoporphyrin (HP) on Mycoplasma hominis and Acholeplasma laidlawii was studied as a function of the phase of growth and the amount of sterols in the cell membrane. Less HP was bound to cells when the membrane had a high sterol content. Both strains in the exponential but not in the stationary phase of growth were sensitive to HP treatment (above 1 microgram ml-1) in the dark. Visible light irradiation of HP-loaded cells caused in all cases a decrease of cell survival, with concomitant changes in the pattern of membrane proteins that suggested protein-protein cross-linking, and the appearance of ultrastructural alterations (rounded and lysed cells); the photosensitivity was indirectly related to the sterol content of the cell membrane. On the whole, our findings suggest that the cell membrane is a major target for HP photosensitization of mycoplasma cells.

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.

Survival of genital mycoplasma on various bacteriological swabs and transport media.

An approach to diagnostic microbiology necessitates measures which maintain the flora unchanged during the transportation of the specimens to the laboratory, preserving the viability of labile and fastidious organisms such as mycoplasma. Some of these problems have been studied in the present investigation. Five different swabs (untreated, albu, charcoal, alginate and phosphate-buffered albumin) and three transport media (Stuart, Amies and MTB) were examined using genital mycoplasma. Absorbent effect of swabs treated with albumin is lower than all the others while the antimycoplasma activity, due to the toxic substances present in the fiber of tip swab, is always lower for the charcoal swab. Concerning transport media, the most favourable results were obtained with the MTB medium. In this medium M. hominis and U. urealyticum counts decreased about 0.5 log and M. fermentans decreased 1.25 log after seven days at 4 degrees C. Furthermore, after inoculation of mycoplasma, MTB can be frozen at -20 degrees C without greatly modifying the titer. The reasons of the different performance of transport media on mycoplasma survival were discussed.

[Research on enzymes of Mycoplasma: enolase of Mycoplasma hominis]. / Ricerche sugli enzimi dei micoplasmi: enolasi del Mycoplasma hominis

A screening of enzymes on cell-free extracts of various species of mycoplasmas revealed the presence of enolase (EC 4.2.1.11) in significative amount in M. pneumoniae and M. fermentans, in lower amounts in M. hominis, A. laidlawii and in trace only in U. urealyticum. The value of activity of the various mycoplasmas could be correlated with their metabolism. From 40 g of cell paste of M. hominis, 2.5 mg of enolase purified over 70 folds, was obtained with successive steps of salt fractionation and column chromatography. Kinetic studies gave the following constants: Km for 2-phospho-D-glicerate, 0.7 x 10(-4)M; optimum of Mg++ concentration, 1 x 10(-3)M; optimum of pH, 7.7 inhibition by fluoride and phosphate, I = 0.77 X 10(-12)M4. Molecular weight estimation indicated for the native enzyme a value of about 100,000 daltons. These data suggest closer similarities with the enolase of microorganisms like E. coli and yeast than with the genus Thermus or B. stearothermophilus.