F Biavasco

Publications of F Biavasco

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

  Authors: G Zandri, S Pasquaroli, C Vignaroli, S Talevi, E Manso, G Donelli, F Biavasco

  Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 04/2012;

  Clin Microbiol Infect ABSTRACT: Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%)Clin Microbiol Infect ABSTRACT: 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.

• New sequence types and multidrug resistance among pathogenic Escherichia coli isolates from coastal marine sediments.

  Authors: C Vignaroli, G M Luna, C Rinaldi, A Di Cesare, R Danovaro, F Biavasco

  Applied and environmental microbiology. 03/2012;

  The spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence and significance in marine environments are still limited. We examined 109 EscherichiaThe spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence and significance in marine environments are still limited. We examined 109 Escherichia coli strains from coastal marine sediments carrying virulence genes for antibiotic susceptibility, specific resistance genes, prevalence of class 1 and 2 integrons, and sequence type.Antibiotic resistance was found in 35% of strains and multiple resistances in 14%; the resistances detected most frequently were against tetracycline (28%), ampicillin (16.5%), trimethoprim/sulfamethoxazole (13%), and streptomycin (7%). The highest prevalence of resistant strains was in phylogenetic group A, whereas phylogroup B2 exhibited a significantly lower frequency compared to all the other groups. Sixty percent of multiresistant strains harbored class 1 or 2 integrase genes and about 50% carried resistance genes (particularly dfrA and aadA ) linked to class 1 integron. Multilocus sequence typing of 14 selected strains identified eight different types characteristic of extra-intestinal pathogens and three new allelic combinations.Our data suggest that coastal marine sediment may be a suitable environment for the survival of pathogenic and antimicrobial-resistant E. coli strains capable of contributing to resistance spread via integrons among benthic bacteria, and highlight a role for them in the emergence of new virulent genotypes.

• Extraintestinal Escherichia coli carrying virulence genes in coastal marine sediments.

  Authors: G M Luna, C Vignaroli, C Rinaldi, A Pusceddu, L Nicoletti, M Gabellini, R Danovaro, F Biavasco

  Applied and environmental microbiology. 09/2010; 76(17):5659-68.

  Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. WeDespite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx(1), stx(2), aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.

• Bacteria associated with the rapid tissue necrosis of stony corals.

  Authors: G M Luna, F Biavasco, R Danovaro

  Environmental microbiology. 07/2007; 9(7):1851-7.

  The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of anThe rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio-specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for > 15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology.

• VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants.

  Authors: F Biavasco, G Foglia, C Paoletti, G Zandri, G Magi, E Guaglianone, A Sundsfjord, C Pruzzo, G Donelli, B Facinelli

  Applied and environmental microbiology. 05/2007; 73(10):3307-19.

  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 transmissionVanA-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.

• Effetto del siero sull’adesività di streptococchi orali

  Authors: R. Tarsi, F. Biavasco, M. Zampini, A. Crippa, C. Pruzzo

  L’Igiene Moderna. 01/2004; 121:96.

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

  Authors: F Biavasco, C Paladini, C Vignaroli, G Foglia, E Manso, P E Varaldo

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 06/2001; 20(5):309-14.

  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 oneTwo 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.

• Glycopeptide susceptibility profiles of Staphylococcus haemolyticus bloodstream isolates.

  Authors: F Biavasco, C Vignaroli, R Lazzarini, P E Varaldo

  Antimicrobial agents and chemotherapy. 11/2000; 44(11):3122-6.

  Twelve clinical strains of Staphylococcus haemolyticus (eight methicillin resistant and three methicillin susceptible), isolated from blood cultures between 1982 and 1997, were investigated forTwelve clinical strains of Staphylococcus haemolyticus (eight methicillin resistant and three methicillin susceptible), isolated from blood cultures between 1982 and 1997, were investigated for teicoplanin and vancomycin susceptibility profiles. On the basis of conventional MIC tests and breakpoints, four isolates were susceptible (MICs, 1 to 8 microgram/ml) and eight were resistant (MICs, 32 to 64 microgram/ml) to teicoplanin while all were susceptible to vancomycin (MICs, 1 to 2 microgram/ml). All four strains for which the conventional teicoplanin MICs were within the range of susceptibility expressed heterogeneous resistance to teicoplanin and homogeneous vancomycin susceptibility. Of the eight strains for which the conventional teicoplanin MICs were within the range of resistance, six expressed heterogeneous and two expressed homogeneous teicoplanin resistance while seven showed heterogeneous vancomycin resistance profiles (with subpopulations growing on 8 microgram of the drug per ml at frequencies of >/=10(-6) for six strains and 10(-7) for one) and one demonstrated homogeneous vancomycin susceptibility. Of six bloodstream isolates of other staphylococcal species (S. aureus, S. epidermidis, and S. simulans), for all of which the conventional teicoplanin MICs were >/=4 microgram/ml and the vancomycin MICs were </=2 microgram/ml, none exhibited heterogeneous susceptibility profiles for teicoplanin while three showed homogeneous and three showed heterogeneous susceptibility profiles for vancomycin (with subpopulations growing on 8 microgram of the drug per ml found for only one strain). The results of this study indicate that a heterogeneous response to glycopeptides is a common feature of S. haemolyticus isolates and suggest that susceptibility to glycopeptides as determined by conventional MIC tests may not be predictive of the outcome of glycopeptide therapy.

• Glycopeptide resistance in coagulase-negative staphylococci.

  Authors: F Biavasco, C Vignaroli, P E Varaldo

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 07/2000; 19(6):403-17.

  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 toCoagulase-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.

• Antistaphylococcal and antistreptococcal killing kinetics of ciprofloxacin, ofloxacin, sparfloxacin, and trovafloxacin.

  Authors: M Prenna, M P Montanari, M Mingoia, F Biavasco, S Ripa, P E Varaldo

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 12/1998; 17(11):816-9.

• Lectin reactivity and virulence among strains of Listeria monocytogenes determined in vitro using the enterocyte-like cell line Caco-2.

  Authors: B Facinelli, E Giovanetti, G Magi, F Biavasco, P E Varaldo

  Microbiology (Reading, England). 02/1998; 144 ( Pt 1):109-18.

  Forty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested forForty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested for lectin agglutination using a four-lectin set. Lectin reactivity (i.e. agglutination by one or more of the four lectins) was observed in all 12 clinical isolates, 16 of the 23 food isolates and eight of the 11 collection strains. Virulence was evaluated in vitro based on strains' ability to invade the human enterocyte-like cell line Caco-2. In gentamicin survival experiments, recovery of viable intracellular bacteria among lectin-unreactive strains was usually 100-1000-fold lower than among lectin-reactive haemolytic strains, and lower than among nonhaemolytic strains. Considerable cytopathogenic effects were produced by lectin-reactive haemolytic strains in trypan-blue-stained cell monolayers, whereas lectin-unreactive and nonhaemolytic strains produced no detectable cytopathogenic effect. Among lectin-reactive strains, the number of listerial cells associated with Caco-2 monolayers was more than tenfold greater than among lectin-unreactive strains. Cell invasion was inhibited by pretreatment of Caco-2 cells with sugars recognized by the lectins or of listeriae with enzymes which removed the same sugars from the bacterial surface. The results suggest that the study of lectin interactions could be helpful in understanding the pathogenicity potential of isolates of food and environmental origin.

• Coagulase-negative Staphylococcus species as unusual causes of infection.

  Authors: P E Varaldo, F Biavasco

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 01/1998; 25(6):1491-3.

• In vitro antibacterial activity of LY333328, a new semisynthetic glycopeptide.

  Authors: F Biavasco, C Vignaroli, R Lupidi, E Manso, B Facinelli, P E Varaldo

  Antimicrobial agents and chemotherapy. 11/1997; 41(10):2165-72.

  LY333328 is a semisynthetic N-alkyl derivative of LY264826, a naturally occurring structural analog of vancomycin. LY333328 was evaluated for its in vitro inhibitory and bactericidal activities inLY333328 is a semisynthetic N-alkyl derivative of LY264826, a naturally occurring structural analog of vancomycin. LY333328 was evaluated for its in vitro inhibitory and bactericidal activities in comparison with those of the two currently available glycopeptides (vancomycin and teicoplanin). Glycopeptide-susceptible test strains included a total of 311 isolates (most of clinical origin) from the genera Staphylococcus, Enterococcus, Streptococcus, Aerococcus, Gemella, Lactococcus, Listeria, Corynebacterium, and Clostridium. Test strains resistant or intermediate to vancomycin and/or teicoplanin included 56 clinical isolates of Enterococcus (of the VanA, VanB, and VanC phenotypes) and 32 clinical isolates of Staphylococcus (S. haemolyticus, S. epidermidis, and S. aureus), 31 strains of gram-positive genera outside the spectrum of activity of vancomycin (Leuconostoc, Pediococcus, Lactobacillus, and Erysipelothrix), and laboratory-derived organisms obtained after exposure of susceptible Staphylococcus isolates to teicoplanin (6 strains) or laboratory-derived organisms with resistance determinants received from VanA enterococci (2 Enterococcus and 25 Listeria transconjugants). LY333328 was highly active against staphylococci, enterococci, and listeriae (whether they were clinical or laboratory-derived strains) resistant to the currently available glycopeptides. In particular, the MICs of LY333328 did not vary substantially between teicoplanin-susceptible and teicoplanin-resistant staphylococci and between vancomycin-susceptible and vancomycin-resistant enterococci. LY333328 demonstrated fairly good inhibitory activity even against most strains of Leuconostoc, Pediococcus, and Erysipelothrix (MIC range, 1 to 8 microg/ml), whereas it proved less active (although much more active than vancomycin or teicoplanin) against Lactobacillus strains. In minimal bactericidal concentration (MBC) and time-kill studies, LY333328 demonstrated excellent bactericidal activity; enterococci, in particular, which were largely tolerant of vancomycin and teicoplanin, were uniformly killed by LY333328, with MBC-to-MIC ratios of 4 to 8 for most vancomycin-susceptible and vancomycin-resistant strains. In attempts to select for resistant clones, no survivors stably growing in the presence of 10 microg of LY333328 per ml were obtained from the Staphylococcus and Enterococcus test strains exposed to the drug.

• News & notes: adhesiveness of Bacteroides fragilis strains isolated from feces of healthy donors, abscesses, and blood.

  Authors: C A Guzmàn, F Biavasco, C Pruzzo

  Current microbiology. 06/1997; 34(5):332-4.

  Bacteroides fragilis strains attached to oral epithelial cells (ECs) and the cell line Intestine 407 and associated with human phagocytes with different efficiencies depending on their source. TheBacteroides fragilis strains attached to oral epithelial cells (ECs) and the cell line Intestine 407 and associated with human phagocytes with different efficiencies depending on their source. The 58%, 75%, and 40% of strains isolated from feces, abscesses, and blood respectively adhered to ECs with good efficiency (11-40 bacteria/cell). Of the strains from feces and abscesses, 17% and 20% exhibited a high adherence (>40 bacteria/cell); however, none of the blood isolates presented this property. Similar results were obtained with the cell line Intestine 407 and human phagocytes. Of the isolates from feces, abscesses, and blood, 20%, 56%, and 71% respectively also exhibited hemagglutination ability, indicating that this property is a virulence trait more frequently present among pathogenic isolates than in commensal strains.

• In vitro conjugative transfer of VanA vancomycin resistance between Enterococci and Listeriae of different species.

  Authors: F Biavasco, E Giovanetti, A Miele, C Vignaroli, B Facinelli, P E Varaldo

  European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 02/1996; 15(1):50-9.

  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 clinical EnterococcusIn 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 clinical Enterococcus isolates-Enterococcus faecium LS10, Enterococcus faecalis LS4, and Enterococcus faecalis A3208, all harboring a plasmid that strongly hybridized with a vanA probe-were used as donors in transfer experiments. Strains of five Listeria species were used as recipients. From Enterococcus faecium LS10, glycopeptide resistance was transferred to Listeria monocytogenes, Listeria ivanovii, and Listeria welshimeri recipients, whereas no transfer occurred to Listeria seeligeri or Listeria innocua strains. From the two Enterococcus faecalis isolates, no transfer occurred to any Listeria recipient. MICs of both vancomycin and teicoplanin were > or = 256 mg/l for all transconjugants tested. Furthermore, all transconjugants harbored a plasmid that strongly hybridized with the vanA probe, with vanA consistently located in an EcoRI fragment of about 4 kb. Exposure of Listeria transconjugants to vancomycin resulted in synthesis of a membrane protein similar in size (39 kDa) to a vancomycin-induced membrane protein of Enterococcus faecium LS10. In retransfer experiments with Listeria transconjugants used as donors, glycopeptide resistance was transferred to all Listeria recipients tested, including strains of Listeria innocua and Listeria seeligeri, which were unable to receive the resistance from Enterococcus faecium LS10. The frequency of vanA transfer to listerial recipients was greater in retransfer experiments than in the primary matings. These findings suggest that the vanA resistance determinant might spread to the established pathogen Listeria monocytogenes, both directly from a resistant enterococcus and through strains of nonpathogenic Listeria species acting as intermediate resistance vehicles.

• Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis.

  Authors: F Biavasco, A Miele, C Vignaroli, E Manso, R Lupidi, P E Varaldo

  Microbial drug resistance (Larchmont, N.Y.). 02/1996; 2(2):231-7.

  Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience forDespite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.

• An electron microscopic study of clinical and laboratory-derived strains of teicoplanin-resistant Staphylococcus haemolyticus.

  Authors: E Giovanetti, F Biavasco, A Pugnaloni, R Lupidi, G Biagini, P E Varaldo

  Microbial drug resistance (Larchmont, N.Y.). 02/1996; 2(2):239-43.

  Staphylococcal resistance to glycopeptides (which involves more teicoplanin than vancomycin) is uncommon and largely confined to Staphylococcus haemolyticus, an emerging nosocomial pathogen with aStaphylococcal resistance to glycopeptides (which involves more teicoplanin than vancomycin) is uncommon and largely confined to Staphylococcus haemolyticus, an emerging nosocomial pathogen with a tendency to develop antibiotic resistance. In this study, six S. haemolyticus strains, including two isogenic pairs of teicoplanin-susceptible/-resistant strains and two resistant clinical isolates, were used in a morphologic and morphometric electron microscope investigation. Cells from both clinical and laboratory-derived teicoplanin-resistant strains exhibited abnormally roughened, irregular outlines when observed by transmission electron microscopy. However, no significant differences in cell wall thickness resulted from morphometric analysis when the susceptible/resistant cells of the two isogenic pairs were compared. By scanning electron microscopy, an abnormally roughened, blistered surface was associated with teicoplanin-resistant cocci. A certain variability was noted between strains, not clearly related to the resistance level. In freeze-fracture investigations, a higher number per square micrometer of intramembrane particles, more significant in the E than in the P membrane fracture face, was observed in the laboratory-derived resistant clones as compared to susceptible parent strains. Further studies are needed to understand the cause-effect relation between these ultrastructural alterations and staphylococcal resistance to teicoplanin (but not to vancomycin).

• Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli.

  Authors: C A Guzmàn, G Piatti, L H Staendner, F Biavasco, C Pruzzo

  FEMS microbiology letters. 06/1995; 128(2):189-94.

  Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coliBordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (Ion protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.

• Bilateral acanthamoeba keratitis.

  Authors: F Biavasco, E Manso, A Giovannini, G Frongia, M Scaglia, R Lupidi, E Giovanetti, R Tittarelli, A D Steele, G S Visvesvara

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 05/1994; 18(4):661-2.

• Bilateral Acanthamoeba keratitis in a gas-permeable contact lens wearer.

  Authors: A Giovannini, R Tittarelli, E Bertelli, G B Frongia, C Mariotti, E Manso, F Biavasco

  Ophthalmologica. Journal international d'ophtalmologie. International journal of ophthalmology. Zeitschrift für Augenheilkunde. 02/1994; 208(6):321-4.

  Acanthamoeba keratitis is an uncommon but potentially devastating complication of contact lens wear that has been reported with increasing frequency since its first description in 1974. We describe aAcanthamoeba keratitis is an uncommon but potentially devastating complication of contact lens wear that has been reported with increasing frequency since its first description in 1974. We describe a case of bilateral Acanthamoeba keratitis, which occurred in a 24-year-old man, wearing gas-permeable contact lenses. This is to our knowledge the first case of Acanthamoeba keratitis reported in Italy, and the fourth bilateral case reported in the literature. Medical therapy (carried out after the first week by a different institution, due to the absence of essential antiamebic drugs on the Italian market) was effective in stopping the progression of the disease.