Francesca Biavasco

Glycopeptide Resistance in Coagulase-Negative Staphylococci

Abstract Coagulase-negative staphylococci (CNS) were the first organisms in which acquired glycopeptide resistance was recognized. Ever since the early reports, it has been apparent that resistance to teicoplanin is more common than that to vancomycin and that resistance occurs mostly in species such as Staphylococcus haemolyticus and Staphylococcus epidermidis. The minimum inhibitory concentrations (MICs) of teicoplanin for CNS usually fall over a wide range, and, especially in some methicillin-resistant isolates of the two above-mentioned species, they can reach and even exceed the resistance breakpoint, whereas vancomycin MICs tend to remain more stable over a narrower range within the limits of susceptibility. CNS strains intermediately susceptible and even resistant not only to teicoplanin but also to vancomycin have, however, been isolated, most frequently from patients subjected to prolonged glycopeptide treatment. Laboratory detection of glycopeptide-resistant CNS may be problematic, mainly because susceptibility tests, particularly those for teicoplanin, are influenced by various technical factors, and agar diffusion tests may yield false susceptibility data. In studies with experimental glycopeptides, some molecules have exhibited improved in vitro activity compared with teicoplanin and vancomycin, but these encouraging microbiological findings have not usually been followed by in vivo trials. Stepwise and single-step exposure to teicoplanin and vancomycin has allowed stable clones for which glycopeptide MICs are increased to be obtained from susceptible CNS strains, particularly strains of Staphylococcus haemolyticus and Staphylococcus epidermidis. In these studies, resistance to teicoplanin was generally easier to obtain than resistance to vancomycin, and the levels of teicoplanin resistance were higher. Population studies have demonstrated the usually heterogeneous nature of glycopeptide resistance in CNS. Although glycopeptide-resistant CNS have been shown to differ in several features from their glycopeptide-susceptible counterparts, the exact mechanism of staphylococcal glycopeptide resistance remains unknown.

VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants

ABSTRACT VanA-type human (n = 69), animal (n = 49), and food (n = 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.

Staphylococcus delphini sp. nov. a Coagulase-Positive Species Isolated from Dolphins

A new coagulase-positive species of the genus Staphylococcus, Staphylococcus delphini, is described on the basis of a study of two strains isolated from purulent skin lesions of dolphins. The new species is established and differentiated from the other coagulase-positive Staphylococcus species primarily on the basis of its deoxyribonucleic acid-deoxyribonucleic acid hybridization relationships, its cell wall composition, its bacteriolytic activity pattern, its penicillin-binding protein profile, its biochemical reactions, and the relatively high guanine-plus-cytosine content of its deoxyribonucleic acid. The type strain is strain Heidy (= DSM 20771).

In vitro conjugative transfer of VanA vancomycin resistance betweenEnterococci andListeriae of different species

In a study designed to gain data on the in vitro transferability of vancomycin resistance from enterococci of the VanA phenotype to listeriae of different species, three clinicalEnterococcus isolates —Enterococcus faecium LS10,Enterococcus faecalis LS4, andEnterococcus faecalis A3208, all harboring a plasmid that strongly hybridized with avanA probe — were used as donors in transfer experiments. Strains of fiveListeria species were used as recipients. FromEnterococcus faecium LS10, glycopeptide resistance was transferred toListeria monocytogenes, Listeria ivanovii, andListeria welshimeri recipients, whereas no transfer occurred toListeria seeligeri orListeria innocua strains. From the twoEnterococcus faecalis isolates, no transfer occurred to anyListeria recipient. MICs of both vancomycin and teicoplanin were ≥256 mg/l for all transconjugants tested. Furthermore, all transconjugants harbored a plasmid that strongly hybridized with thevanA probe, withvanA consistently located in anEcoRI fragment of about 4 kb. Exposure ofListeria transconjugants to vancomycin resulted in synthesis of a membrane protein similar in size (39 kDa) to a vancomycin-induced membrane protein ofEnterococcus faecium LS10. In retransfer experiments withListeria transconjugants used as donors, glycopeptide resistance was transferred to allListeria recipients tested, including strains ofListeria innocua andListeria seeligeri, which were unable to receive the resistance fromEnterococcus faecium LS10. The frequency ofvanA transfer to listerial recipients was greater in retransfer experiments than in the primary matings. These findings suggest that thevanA resistance determinant might spread to the established pathogenListeria monocytogenes, both directly from a resistant enterococcus and through strains of nonpathogenicListeria species acting as intermediate resistance vehicles.

Further characterization of borderline methicillin-resistant Staphylococcus aureus and analysis of penicillin-binding proteins.

Eighty-nine Staphylococcus aureus strains were grouped according to their susceptibility or resistance to methicillin and oxacillin. The role of beta-lactamase in borderline methicillin resistance was confirmed by tests with beta-lactamase inhibitors, particularly when salt-supplemented medium was used. A penicillin-binding protein assay indicated that borderline methicillin-resistant S. aureus strains do not produce PBP 2a. Images

Aquaculture can promote the presence and spread of antibiotic-resistant Enterococci in marine sediments.

Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic environment through release of large amounts of chemicals, including antibiotics. Moreover, the presence of organic matter and bacteria of different origin can favor gene transfer and recombination. Whereas the consequences of such activities on environmental microbiota are well explored, little is known of their effects on allochthonous and potentially pathogenic bacteria, such as enterococci. Sediments from three sampling stations (two inside and one outside) collected in a fish farm in the Adriatic Sea were examined for enterococcal abundance and antibiotic resistance traits using the membrane filter technique and an improved quantitative PCR. Strains were tested for susceptibility to tetracycline, erythromycin, ampicillin and gentamicin; samples were directly screened for selected tetracycline [tet(M), tet(L), tet(O)] and macrolide [erm(A), erm(B) and mef] resistance genes by newly-developed multiplex PCRs. The abundance of benthic enterococci was higher inside than outside the farm. All isolates were susceptible to the four antimicrobials tested, although direct PCR evidenced tet(M) and tet(L) in sediment samples from all stations. Direct multiplex PCR of sediment samples cultured in rich broth supplemented with antibiotic (tetracycline, erythromycin, ampicillin or gentamicin) highlighted changes in resistance gene profiles, with amplification of previously undetected tet(O), erm(B) and mef genes and an increase in benthic enterococcal abundance after incubation in the presence of ampicillin and gentamicin. Despite being limited to a single farm, these data indicate that aquaculture may influence the abundance and spread of benthic enterococci and that farm sediments can be reservoirs of dormant antibiotic-resistant bacteria, including enterococci, which can rapidly revive in presence of new inputs of organic matter. This reservoir may constitute an underestimated health risk and deserves further investigation.

Multidrug-Resistant Enterococci in Animal Meat and Faeces and Co-Transfer of Resistance from an Enterococcus durans to a Human Enterococcus faecium

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6′)-Ie aph (2′′)-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.

Antibiotic pressure can induce the viable but non-culturable state in Staphylococcus aureus growing in biofilms

OBJECTIVES Staphylococcal biofilms are among the main causes of chronic implant-associated infections. We have recently suggested that their transformation into viable but non-culturable (VBNC) forms (i.e. forms capable of resuscitation) could be responsible for the recurrent symptoms. This work aims to establish whether Staphylococcus aureus biofilms can give rise to VBNC forms capable of being resuscitated in suitable environmental conditions, the role of different stressors in inducing the VBNC state and the conditions favouring resuscitation. METHODS S. aureus 10850 biofilms were exposed to different concentrations of antibiotic (vancomycin or quinupristin/dalfopristin) and/or to nutrient depletion until loss of culturability. The presence of viable cells and their number were examined by epifluorescence microscopy and flow cytometry. Gene expression was measured by real-time PCR. Resuscitation ability was tested by growth in rich medium containing antioxidant factors. RESULTS Viable subpopulations were detected in all non-culturable biofilms. However, viable cell numbers and gene expression remained constant for 150 days from loss of culturability in cells from antibiotic-exposed biofilms, but not in those that had only been starved. Resuscitation was obtained in rich medium supplemented with 0.3% sodium pyruvate or with 50% filtrate of a late-log culture. CONCLUSIONS Our findings demonstrate that S. aureus can enter the VBNC state in infectious biofilms. The presence of vancomycin or quinupristin/dalfopristin can inadvertently induce a true VBNC state or its persistence in S. aureus cells embedded in biofilms, supporting previous findings on the role of staphylococcal biofilms in recurrent infections.

Nosocomial outbreak of systemic candidosis associated with parenteral nutrition.

Eight patients in two surgical units developed systemic candidosis during a 40-day period from June 5 to July 13, 1987 (in five cases Candida albicans was identified). Three of them died. All cases belonged to a group of 27 patients receiving parenteral nutrition (PN), while among the 108 patients who did not receive PN, no cases were observed (p = .000001). Candida was cultured from two PN bags administered to the cases. A specialized nutrition nurse was responsible for the PN compounding and for maintaining administration sets in the two wards involved. An epidemiological investigation, in which 19 uninfected patients who had had PN were used as controls, showed no significant difference between cases and controls except that lipids were more frequently added to bags administered to cases (p = .0005). Furthermore, the bags administered to cases contained a higher average number of multidose constituents (p = .0008) when the comparison was focused on the two days before the onset of symptoms. Given the favorable medium provided by lipids, even a low level contamination of PN solutions during compounding and/or administration could have been responsible for the exposure of cases to multidose vials suggests, although not conclusively, that an extrinsic contamination occurred during compounding. Six isolates of C albicans were available from four cases. C albicans was cultured from the pharyngeal swabs of two physicians and three nurses, including the specialized nutrition nurse.

Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays

Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%) samples contained viable but non-culturable (VBNC) cells; the majority were positive on real-time PCR specific for Staphylococcus epidermidis (one also for Staphylococcus aureus). Viable cells were significantly (p<0.01) associated with CVCs from febrile patients, three of whom showed S. epidermidis-positive blood cultures, suggesting that CVC-associated biofilms can be reservoirs for staphylococci in the VBNC state. The possible role of VBNC staphylococci in persistent infections related to medical devices requires further investigation.