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A direct transfer of plasmid pBP517 from C600 to J53 Escherichia coli K12 strains by electroporation, either by the standard method, or in the presence of 20 μg/mL of DNAse was carried out. In a standard experiment, donor and recipient bacteria were mixed and subjected to the electroporation procedure, about 2700 (range 1600-3700) recombinants were found, while no colonies were detected from non treated bacteria. When the same tests were performed at the presence of DNAse the number of recombinants fell to about 200 (range 183-218). This difference between the number of colonies found in the presence or in absence of DNAse was observed every time the tests were repeated. According to these observations, plasmid DNA has been transferred from donor to recipient cells via electroporation also when DNAse was added. Since the free genetic material is destroyed by the enzyme and recombination takes place it has been hypothesized that there must be a direct contact between the partner cells.

Integration of F’lac plasmid into chromosome of both gyrA(Ts) and gyrB(Ts) cells phenotypically suppress the thermosensitive mutations of the DNA gyrase enzymes. As the comparative strains isolated from dnaA(Ts), these high frequency of recombination (Hfr) derivatives were able to transfer chromosomal markers to recipient strains, showed a growth rate of about 60 min, and developed filamentous forms when incubated at the temperature of 43°C. Conversely to dnaA(Ts) Hfr selected isolates, the great majority of Hfr derivative of gyrase mutants resulted resistant to acridine orange and rifampin. Time-kill experiments carried out at the non-permissive temperature also revealed that nalidixic acid has no antibacterial activity on these Hfr strains while derivatives of dnaA(Ts) mutant, as well as the control strain HfrH, were strongly inhibited by this drug. Therefore F plasmid induced duplication of chromosome in the mutants even if the DNA gyrase enzymes are not working. Of a certain interest is that these bacteria exhibit physiological perturbations that affect the main cellular functions, however, they do not appear essential for the survival of the strains.

We report the characterization of an Acinetobacter baumannii resistant to colistin isolated from a patient treated with colistin for 22 days. The identification and initial susceptibility testing of the strain was performed at ASL3 Imperiese with the Vitek-2 automated system and than the strain was re-identified at the Sezione di Microbiologia with APINE. In vitro activity of antimicrobial agents was determined by the microdiluition methods. The detection of the beta-lactamase gene was performed by PCR and for the analysis of LPS were sequenced the genes of the PmrABC system. We compared the PFGE profile of the colistin-resistant A. baumannii with 5 A. baumannii colistin-susceptible strains isolated in the same Hospital during 2010. The strain was resistant to all antimicrobial agents tested, except to tygecyclin. The PFGE shown that the A. baumannii colistin-resistant was closery related to the colistin-susceptible strains. The sequencing of the PmrABC system showed that the strain had a single mutation in the PmrB component. Colistin-resistant strain have recently been found in several Gram-negative bacteria such A. baumannii, K. pneumoniae and P. aeruginosa. This clinical case confirms that resistance to colistin in A. baumannii can be selected in vivo following the use of colistin in therapy.

Introduction. Sysmex UF-1000 is a new flow cytometry for the analysis of urine based on a laser diode technology using specific compounds for the staining nucleic acids, characterized by a threshold of detection of bacteria equal to 1000 CFU/ml.The purpose of this study was to compare the positivity or negativity of the urine samples using standard procedures and with the response obtained from the instrument UF-1000i. Methods. During the period May and July 2011, 1024 urine samples obtained from the laboratory of the hospital in Genoa-Voltri were analyzed with the instrument Sysmex UF-1000i.The samples were stored at a temperature of 5°C during transport.The instrument after loading of the sample is able to assess the bacterial load in about 1 minute per sample. Results. 1024 samples were analyzed with Sysmex UF-1000 analyzed in parallel by the hospital laboratory in Genoa Voltri. 228 are positive results for Sysmex UF-1000 (bacterial loads in excess of 1000 CFU/ml).The data obtained were consistent with those recorded with traditional analysis. 18 samples were positive only for our instrument, but not with the traditional system used in the laboratory for comparison. Conclusion. The advantage of Sysmex UF-1000 is certainly due to obtain immediate results after the reading of each individual sample that is higher than any other instrument or method of analysis used. The slight discrepancy of the results obtained may be due to the extremely low calibration of UF-1000. This allows in a very short time to discard all negative samples with enormous saving of time and material. For positive samples and applies the criterion of the routine ie, the bacterial load has a meaning according to the type of patient considered, as in the case of pediatric or catheterized patients

This study was conduced during March-May 2011 with the collaboration of 4 clinical microbiology laboratories evenly distibuited across the Ligurian area to identify the most frequent pahogens isolates from skin and soft tissue infections and to evaluate their antibiotic susceptibility patterns. Overall, 213 consecutive, non duplicate strains were collected and sent to the coordinating laboratory.The most rappresented pathogens were: S. aureus (35.7%), P. aeruginosa (14%), E. coli (12.7%), Staphylococcus coaugulase negative (6.6%) and Enterococcus spp. (4.7%). The data indicate an increase of Gram negative compared to previous years, S. aureus remains the most common pathogen.The methicillin resistance in S. aureus was 43.4% and no one Enterococcus spp. resistant to vancomicin was found.

Objectives. An epidemiological study addressed to identify the most represented pathogens isolated from blood and to evaluate their antibiotic susceptibility patterns, was conducted. Methods. Five clinical microbiology laboratories, homogenously distributed in Liguria, were required to collected all consecutive non-duplicates strains isolated from blood cultures during March 2011 to May 2011. the strains were sent to the reference laboratory (Section of Microbiology, DISC, University of Genoa, Italy). Results. A total of 159 microorganisms were enrolled, including 81 Gram positive, 69 Gram negative and 9 fungi.The most represented pathogens were: Escherichia coli (35), Staphylococcus aureus (26), S. epidermidis (20), S. hominis (10). Samples were collected mainly from medicine (59 isolates).Among the staphylococci, the most active molecules were: vancomycin (100% of susceptible strains), teicoplanin (93.4%), trimethoprim-sulfamethoxazole (83.8%) and tobramycin (61.6%). Enterococci showed rates of resistance to vancomycin of 25%. Enterobacteriaceae exhibited resistance to ampicillin (76.9%), ceftriaxone (44.4%), ciprofloxacin (43.3%), trimethoprim-sulfamethoxazole (36.6%) and ceftazidime (32.2%). Conclusions. The data show a higher incidence of Gram positive (51%) in comparison to Gram negative (43.4%). Gram-positive strains showed a high resistance level to fluoroquinolones (92.3%) while Gram-negative resulted resistant to ceftriaxone (44.4%) and fluoroquinolone (43.3%).

Objectives. An epidemiological study to identify the most represented pathogens isolated from blood and to evaluate their antibiotic susceptibility patterns, was conducted. Methods. Seven clinical microbiology laboratories, homogeneously distributed in the Ligurian area,were required to collected all consecutive non-duplicates strains isolated froom blood cultures during January 2010 to April 2010. The strains were sent to the reference laboratory (Sezione di Microbiologia del DISC, University of Genoa, Italy). Results. A total of 277 microorganisms were enrolled, including 155 Gram positive and 122 Gram negative.The most represented pathogens were: Escherichia coli (68), Staphylococcus aureus (57), Staphylococcus epidermidis (32), Staphylococcus hominis (17), Pseudomonas aeruginosa (15), Klebsiella pneumoniae (15), Enterococcus faecalis (11). Samples were collected mainly from medicine (66, 33.3%, of this number was determined by E. coli), intensive care units (33, 18.2% of this number consisted of S. epidermidis), surgery (24, 33.3% consisted of E. coli) and infectious diseases (20, of which S. aureus, E. coli and S. epidermidis equally represented 20.0%).Among the Staphylococci the most active molecules were: vancomycin and teicoplanin (100% of susceptible strains), chloramphenicol (92.3%) and trimethoprim-sulfamethoxazole (89.8%). Among the OXA-R Staphylococci (81/123, 65.9%) the most active molecules were: vancomycin and teicoplanin (100% of susceptible strains), chloramphenicol (93.8%) and trimethoprim-sulfamethoxazole (84.8%). Enterococci showed rates of resistance to vancomycin of 5.9%. Enterobacteriaceae exhibited resistance to ampicillin (77.5%), trimethoprim-sulfamethoxazole (42.6%), ciprofloxacin (41.2%), ceftriaxone (37.5%), ceftazidime (28.2%), cefepime (26.7%), cefoxitin (22.1%), piperacillintazobactam (20.4%), imipenem (4.7%) and amikacin (2.9%). The Gram negative non-Enterobacteriaceae showed rates of resistance of 100% to ceftriaxone, 81.3% to trimethoprim-sulfamethoxazole, 42.1% to ciprofloxacin and piperacillin-tazobactam, 33.3% to ceftazidime, 31.6% to cefepime, 27.8% to imipenem, 26.3 % to amikacin. Conclusions. The data show a higher incidence of Gram positive (56%) in comparison to Gram negative (44%).This confirms the high incidence of oxacillino-resistance in Staphylococci in our geographic area.Against Enterobacteriaceae rates of resistance were observed in excess of 20% for all drugs tested except imipenem (4.7%) and amikacin (2.9%). The proportion of imipenem-resistant isolates was constituted of strains of K. pneumoniae carbapenemase producers.

This study was conducted during January-April 2010 with the collaboration of 7 clinical microbiology laboratories evenly distributed across the Ligurian area to identify the most frequent Gram negative species and to evaluate their antibiotic susceptibility patterns Overall, 110 consecutive multi-resistant non duplicate Gram negative isolates,were collected and sent to the coordinating laboratory (Sezione di Microbiologia del DISC, University of Genoa, Italy) together with susceptibility data obtained by routine methods. In addition, strains resistant to carbapenems were characterized by PCR. A total of 110 Gram negative multi-resistance strains were found, including 74 and 36 isolated from healthcare or nosocomial settings and community acquired infections, respectively. The most represented pathogens were: A. baumannii (38, 34.5%), E. coli (30, 27.2%), P. aeruginosa (29, 26.3%), K. pneumoniae (9, 8.2%) and P. mirabilis (4, 3.6%). A. baumannii were more frequently collected from healthcare settings or nosocomial samples, while the other strains were generally equally isolated from in- and out-patients. Amikacin was the most active molecule against E. coli and P. mirabilis (96,7% and 100% of susceptible stains respectively). Colistin was the only active molecule agains A. baumanii and P. aeruginosa (100% of susceptible strains). Against K. pneumoniae tigecycline and colistin were the most active molecules (100% of susceptible strains). Imipenem was the most active compound against E. coli and P. mirabilis (100% of susceptible strains). A large number (97.4%) of A. baumannii was resistant to imipenem. K. pneumoniae and P. aeruginosa showed rates of resistance of 88% and 34.4% respectively. A. baumannii, K. pneumoniae and P. aeruginosa isolates resistant to Imipenem, carried OXA-23, KPC and VIM carbapenemases.These data shown a significant spread of multidrug-resistant Gram negative bacteria in hospitals and in communities.The production of carbapenemase in A. baumannii, K. pneumoniae and P. aeruginosa is now an important phenomenon in our region.

Background. In this study (part of the global T.E.S.T. program) was evaluated the in vitro activity of tigecycline, member of a new class of antimicrobial agents, the glycylcyclines, against clinical isolates collected in Italy. Methods. A total of 194 clinical isolates were collected and identified in our Institution during 2010. Minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by the CLSI (2010) recommended broth microdilution method. Results. Globally 129 Gram negative and 65 Gram positive pathogens were evaluated.Tigecycline demonstrated excellent inhibitory activity against Escherichia coli, Haemophylus influenzae, Enterococcus spp., Staphylococcus aureus MetS, Streptococcus pneumoniae and Streptococcus agalactiae with MIC90 1mg/l. Conclusion. Tigecycline exhibited potent in vitro antibacterial activity (comparable to or greater than most commonly employed antimicrobials) against both Gram positive and negative clinical pathogens. These data suggest that tigecycline, with an expanded broad-spectrum antimicrobial activity, may be an effective empiric therapeutic option for the treatment of serious infections caused by clinically relevant pathogens.

Objectives. In this study the in vitro activity of tigecycline, member of a new class of antimicrobial agents, the glycylcyclines, was evaluated against clinical isolates collected in Italy. Study Design. A total of 313 clinical pathogens were collected and identified in our Institution during 2007-2008. Minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by the CLSI (2007) recommended broth microdilution method. Results. Globally 205 Gram-negative and 108 Gram-positive pathogens were evaluated.Tigecycline demonstrated excellent inhibitory activity against Acinetobacter spp., H. influenzae, E. coli, Enterococcus spp., S. aureus, S. agalactiae and S. pneumoniae with MIC90 ≤1mg/l. Conclusion. Tigecycline exhibited potent in vitro antibacterial activity (comparable to or greater than most commonly employed antimicrobials) against both Gram-positive and negative clinical pathogens.These data suggest that tigecycline, with an expanded broad-spectrum antimicrobial activity, may be an effective empiric therapeutic option for the treatment of serious infections caused by clinically relevant pathogens.

Objectives. An epidemiological study, addressed to identify the pathogens isolated from blood, and their antibiotic susceptibility patterns, was conducted. Methods. 12 laboratories, homogeneously distributed in a Northern area of Italy, were required to collected all consecutive non-duplicated strains isolated from blood during February 2008 to February 2009 and sent them to the reference laboratory. Results. A total of 1092 microorganisms were collected, including 653 gram-positive, 385 gram-negative and 54 fungi. Escherichia coli 234, Staphylococcus epidermidis 205, S. aureus 142, S. hominis 87, Enterococcus faecalis 47, S. haemolyticus 33, Klebsiella pneumoniae 33, Pseudomonas aeruginosa 32, Candida albicans 28, Enterobacter cloacae 21 were the prevalent microrganisms found. Samples were collected mainly from medicine (255 strains), intensive care units (154), surgery (99), infectious diseases (93), paediatrics (62) and nephrology (62). Antibiotic resistance (in %) in staphylococci was 65.7 (methicillin), 33.5 (gentamicin), 61.8 (azithromycin), 59.6 (erythromycin), 45.2 (ciprofloxacin) 14.8 (chloramphenicol), 2.0 (teicoplanin), and 24.1 (trimethoprim-sulfamethoxazole) no vancomycin-resistant strain was found. Enterococci showed resistance to vancomycin (10.8), ampicillin (34.4), gentamycin (42.9), ciprofloxacin (42.2) teicoplanin (7.6), erythromycin (54.7) and chloramphenicol (17.5). Enterobacteriaceae exhibited resistance to ciprofloxacin(27.0), ampicillin (74.1), ceftazidime (15.8), cefoxitin (14.7), cefepime (13.3), ceftriaxone (15.0), both imipenem and amikacin (0.95), piperacillin-tazobactam (5.1) and trimethoprim-sulfamethoxazole (32.7). Non fermenting gram negative strains were found resistant to ciprofloxacin (27.3), ceftazidime (9.5), cefepime (14.6), ceftriaxone (81.6), both imipenem and amikacin (18.6), trimethoprim-sulfamethoxazole (65.2), and piperacillin-tazobactam (7.5). Conclusions.These data show a prevalent incidence of gram-positive (59.7 %) in comparison to gram-negative (35.3%) bacteria isolated from blood.A high percentage of methicillin-resistant staphylococci as well as ceftazidime-resistance among Enterobacteriaceae is also observed suggesting that this phenomenon requires periodically surveillance.

In order to evaluate whether ophthalmic preparations can be safely used within 12 hours after first opening, four different sterile ocular medications,were opened and tested for spontaneous bacterial contamination after exposure to air. Samples (10ml) were collected from 5 containers of each ophthalmic preparation after 0, 2, 4, 8 and 24 hours. No viable microorganisms were found during and at the end of the evaluation time. In order to assess if the ocular bacterial population might contaminate the medications, about 105 microbial cells of different species (Staphylococcus aureus, S. epidermidis, S. haemolyticus, Streptococcus pneumoniae, S. pyogenes, Corynebacterium spp., Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii, and Candida albicans) were added to the containers and incubated at 35°C or at room temperature. Samples were collected and the number of viable microrganisms was estimated.The medicaments showed a bacteriostatic or bactericidal activity (

Introduction. An epidemiological study addressed to identify gram-negative bacteria, isolated from laboratories in a Northern area of Italy, and their antibiotic resistance patterns was conducted. Methods. Twelve laboratories distributed on Ligurian territory or neighbouring areacollected all consecutive gram-negative isolates belonging to the Enterobacteriaceae family and non-fermenter group for 2 months and sent them to a reference laboratory. Results. A total of 1880 pathogens were collected, including 899 and 981 strains isolated from nosocomial- and community-acquired infections, respectively. Escherichia coli (63.3% of total) was the most frequently isolated pathogen followed by Pseudomonas aeruginosa (9.6%), Proteus mirabilis (8.9%) and Klebsiella pneumoniae (5.4%). Nosocomial samples were collected mainly from patients in general medicine wards (19.9%) and healthcare settings (14.1%). Urine was the most common clinical sample (79.9% of the total). Other samples were sputum and bronchoaspirates (8%), skin wounds including those from decubitus (5.3%) and blood (4.1%). E. coli and P. mirabilis were collected mainly from urinary tract infection while P. aeruginosa appears more involved in respiratory or other infections. Considering the resistance to representative classes of antibiotics, it was higher (%) for piperacillin-tazobactam in P. mirabilis (30.3), for ceftazidime in Enterobacter aerogenes (40.8) and in Providencia stuartii (40), for imipenem and amikacin in P. aeruginosa (16.2 and 13.7 respectively), for ciprofloxacin in P. stuartii (66.6) and in P. mirabilis (44.7) than in others bacteria. Conclusions. The increasing age of the population in general medical wards and healthcare settings is associated with urinary tract and bedsore infections. E. coli confirms its epidemiologic and pathogenic role, but P. mirabilis and P. aeruginosa are emerging as alternativechallenges.

Although the postantibiotic effect (PAE) is a well recognized phenomenon, the mechanism by which it is induced has not fully elucidated yet. It has been suggested that PAE is the time required by bacteria to synthesize proteins or mRNA characterized by a short half-life that are consumed during antibiotic treatment.This phenomenon is widely studied on Gram-positive cocci and Gram-negative rods, while information about Gram-positive rods and Gram-negative cocci are scanty.To gain new insights on the PAE, this study was addressed to evaluated the time required by Moraxella catarrhalis and Lactobacillus planctarum to resume their physiological growth rate after exposure to various antibiotics. Methods PAE was estimated in accordance with the method of Craig and Gudmundsson using the following drugs: penicillin, piperacillin-tazobactam, cefalotin, ceftazidime, imipenem, ciprofloxacin, gentamycin and azithromycin. Log-phase bacteria were exposed to drug at a concentration corresponding to 4 times the MIC value for 1h.The drug was inactivated by 1:1000 dilution. Bacterial counts were determined at time zero, immediately after drug dilution, and at each hour after removal for 6 - 7h by a pour-plate technique. The PAE was defined as the difference in time required by test and control cultures to increase by 1 log in CFU number. Results All drugs tested induced a PAE on the strains studied. M. catarrhalis registered PAE values ranging between 0,5 (gentamycin) and 2 (ceftazidime, imipenem and azithromycin).With respect to L. plantarum a PAE between 0,8 (cefalotin) and 3 hours (ciprofloxacin) were detected. Conclusion. These findings demonstrated that all the drugs tested were able to induce a PAE on the strains tested.This observation differs from that observed on Gram-negative rods characterised by negative PAE values induced by penicillins and cephalosporins.This results might reflect the different target of these compounds on these Gram-positive rods or the different affinity of the antibiotics to several PBPs that required more time to restore the physiological fitness of the bacterial cell.

Background: Synergism between cotrimoxazole (SXT) and rokitamycin (ROK) was observed carrying out antimicrobial susceptibility tests on S. pyogenes and S. pneumoniae with the disk diffusion method (CLSI, 2005). The aim of this study was to confirm this phenomenon on a large number of isolates displaying different macrolide resistance phenotypes. Methods: Synergism between SXT and ROK on 104 S. pyogenes and 102 S. pneumoniae recently isolated was detected by a double-disk screening test.Time kill experiments were also performed on representative strains adopting standard procedures (CLSI 2005). Results: The combination of SXT plus ROK was synergistic against 93% S. pyogenes strains and 51% S. pneumoniae strains. On pneumococci SXT-S this percentage arise to 64%, while, on SXT-R it was 29%. In no instances antagonism was demonstrated. Synergism was not observed against S. pyogenes strains showing cMLSB phenotype. In S. pneumoniae no relationship between different mechanisms of macrolide resistance and the results of interactions was found. Results of time-kill experiments confirmed those obtained with double-disk assay in all the strains tested. Conclusion: Synergism between SXT and ROK was more frequently encountered among S. pyogenes than S. pneumoniae strains.This drug association may be synergistic or not when acting on different strains of the same bacterial species. Different macrolide-resistance mechanisms (reduced binding due to modification of the 50S subunit or efflux pump) among the various bacteria may eplain the observed differences.

Introduction. Staphylococcus aureus is a major opportunistic pathogen especially in nosocomial settings. An increasing proportion of strains has intrinsic resistance to methicillin (MRSA) and recent reports documented the emergence of MRSA also in the community.The aim of this study was to evaluate methicillin-resistance in Liguria. Methods. 767 isolates of Staphylococcus spp have been collected from 10 laboratories, including 508 S. aureus: 292 (57.5%) hospital-acquired, 188 (37%) community-acquired, and 28 (5.5%) from healthcare settings; 259 coagulase- negative staphylococci (CNS): (193 (74.5%) hospital-acquired, 56 (21.6%) community-acquired, and 10 (3.9%) healthcare settings. Susceptibility tests were carried out by the disk diffusion method (CLSI, 2006) using cefoxitin (CFX) (Oxoid, Milan). MRSA has been also evaluated by PBP2’ latex agglutination test (Oxoid, Milan) on 52 randomly selected S. aureus. Results. The data obtained indicated an incidence of MRSA in nosocomial settings up to 45.2%, while among the community-acquired isolates the percentage was 6.4%. With respect to CNS the figures were 61,8% and 10,6% for the nosocomial-and community-acquired organisms respectively. A 99% agreement between data of the coordinating centre and the various laboratories was found. Methicillin-resistance was confirmed in 52 S. aureus by PBP2’ latex agglutination test. Conclusions. Present findings indicate that MRSA in community in the Ligurian area exists and this phenomenon requires future surveillance.

Objectives: Pseudomonas aeruginosa is an opportunistic human pathogen which is intrinsically resistant to many antibiotics and easily develops resistance towards many currently available agents. Intrisic resistance can be attributed to the low permeability of the P. aeruginosa outer membrane to a variety of antibiotics, including glycopeptides (GLYs). These drugs are active against Gram-positive bacteria and resistance is very rare, it appeared of some interest to evaluate the effect of combining these antimicrobial agents with antibiotics that might disorganize the structure of the outer membrane allowing the entry of glycopeptides into the Gram-negative cells. In order to verify this hypothesis, ceftazidime (CAZ) has been tested in association with vancomycin (VAN) or teicoplanin (TEI). The same experiments have been carried out also in the presence of azithromycin (AZI), which has been shown to interfere with some cellular synthesis in P. aeruginosa. Methods: A bacterial suspension of about 109CFU/ml was seeded on plates containing a fixed concentration of GLYs (500 mg/l) and increasing doses (2x,4x,8x,16x) of CAZ. Survivors were counted after 48 hs at 37°C. Results were interpreted as synergism (99%), additivity (90%), and indifference (10%) of the CFU/ml reduction found in the drugs combination in comparison to the drug alone. The same experiments have been repeated adding AZI (16 mg/l) and using GLYs at concentrations ranging from 500 to 300 mg/l. Results: CAZ in combination with GLYs reacted synergically in 20 out of 59 cases, additivity was found in 31/59 interactions and indifference was noted in 8/59 tests. Preliminary results (12 tests performed) indicated that the addition of AZI increased the incidence of synergisms and additivities even when using GLYs concentration of 300 mg/l (figure I). Conclusions: CAZ combined with GLYs gave additive or synergistic results in the geat majority of experiments, while the simultaneous combination of AZI, CAZ and a GLY increased the incidence of additive or synergistic effect against P. aeruginosa.These data, given the high concentration of GLYs employed, could be of particular interest in clinical situations where the drugs could be topically administered.

Introduction.The microbial adhesion to dental material was studied employing a acrylic resin and representative microrganisms. Method. Bacterial suspensions (about 108 CFU/ml) were mixed to dental material with different surface finishes. Bacterial counts were carried out before and after 1 hour at 37°C.A tube without dental material was used as control.The same experiments were also performed at the presence of saliva. Results. Candida albicans demonstrated to adhere to free energy material at an incidence 64% while Staphylococcus aureus and Streptococcus oralis showed more adhesive properties for roughness material (34 and 52% respectively). The presence of saliva influenced the adhesiveness to all materials with the exception of S. aureus that increased from 20 to 29% the number of cells on the surface of free energy material. Conclusion. The surface finishes appeared did not significantly influence the adhesion of microrganisms to this material.

Objectives: To evaluate the Uro-Quick system for detection of extended-spectrum beta-lactamase (ESBL) among nosocomial strains isolated from urine during a 2 months period. Methods:A total of 221 strains collected from nosocomial patients were tested for antibiotic susceptibility by the Uro-Quick system. About 106 cell/ml were used to seed 2.5 ml of broth in vials containing ceftazidime (10 μg/mL) and ceftazidime (10 μg/mL) plus clavulanic acid (10 μg/mL). After incubation the results were plotted as growth curves. All results were confirmed by Kirby-Bauer method. Control strains were included. Results: Using an inoculum of 106 cell/ml the instrument was capable to print out the result after 5 hours of incubation.Among the pathogens studied, 46 strains resulted resistant to ceftazidime (presence of growth).These results were then compared with those obtained with the Kirby-Bauer method, and it was found that among these 46 ceftazidime-resistant clones all were ESBL-producers, even if 12 showed an intermediate, and 12 a susceptible antibiotic phenotype by Kirby-Bauer method. Conclusion: Present findings suggest that Uro-Quick represents a useful technology to detect ESBL-producing strains especially those that required further confirmatory tests for their identification.The period of time needed to achieve the results, in some cases less than 5 hours, might be an advantage over the usual methodologies.

L’Uro-Quick, un sistema automatizzato ampiamente utilizzato per lo screening delle batteriurie sui campioni d’urina, è stato precedentemente impiegato per la valutazione della sensibilità agli antibiotici negli uropatogeni e per l’identificazione di resistenze ben caratterizzate veicolate da diverse specie batteriche. In questo studio sono stati esaminati utilizzando la metodica classica Kirby-Bauer per la determinazione dell’antibiotico sensibilità patogeni isolati durante il periodo settembre 2003 - marzo 2004 in reparti di terapia intensiva di un grande ospedale italiano e i risultati sono stati confrontati con quelli ottenuti con il nuovo sistema rapido Uro-Quick. L’antibiotico (in concentrazione appropriata) è stato introdotto in una cuvetta Uro-Quick contenente 2 ml di Mueller-Hinton brodo, successivamente sono stati addizionati 0.5 ml di sospensione del ceppo da saggiare (5x105 CFU/ml). Una cuvetta priva di farmaco è stata utilizzata come controllo. Dopo 3 o 5 ore di incubazione (per i ceppi Gram-negativi o Gram-positivi rispettivamente) i risultati sono stati interpretati nel seguente modo: l’assenza di sviluppo indicava sensibilità, mentre una curva di crescita analoga a quella del controllo rappresentava un ceppo resistente. I microrganismi Gram-negativi sono stati saggiati con ciprofloxacina (CIP), ampicillina (AM), piperacillina (PIP), aztreonam (ATM), amoxicillina-clavulanato (AMC), piperacillina/tazobactam (TZP), imipenem (IPM), ceftazidime (CAZ), cefotaxime (CTX), cefepime (CFP), cefuroxime (CXM), ceftriaxone (CRO), amikacina (AN), gentamicina (GM) e trimethoprim-sulfametossazolo (SXT). I Gram-positivi, invece, sono stati saggiati con ciprofloxacina (CIP), clindamicina (CM), eritromicina (E), rifampicina (RA), ampicillina (AM), penicillina (P), oxacillina (OXA), imipenem (IPM), gentamicina (GM), streptomicina (S), tetraciclina (TE), trimethoprim – sulfametazolo (SXT), vancomicina (VA) e linezolid (LZD). Sono stati esaminati 197 ceppi Gram-negativi con una concordanza media del 93.9% e 251 Gram positivi, con un accordo del 95.6% tra le due metodiche. Nei confronti di Stafilococchi, Enterococchi e membri appartenenti alla famiglia dell’Enterobacteriaceae è stato sempre riscontrato un accordo superiore al 90% tra sistema Uro- Quick e Kirby-Bauer. Sulla base di questi risultati l’impiego del sistema rapido Uro-Quick nelle infezioni nosocomiali gravi si rivela un valido aiuto consentendo di determinare rapidamente (3 o 5 ore) la sensibilità agli antibiotici ed instaurare in tempi brevi la terapia più efficace.

The Clinical Microbiology Laboratory of the University of Genoa participated, during the year 2003, in an international antimicrobial surveillance program.The collection of isolates was done according to the site of infection and/or type of patient. Four hundred twenty (420) clinical isolates were analyzed during this year and the frequencies of the different pathogens were investigated. A reference centre carried out susceptibility tests. Oxacillin-resistant Staphylococcus aureus represented 47.6% of all S. aureus isolates from blood stream infections and 33.3% of all S. aureus isolated from skin and soft tissue infections in hospitalised patients.These strains showed resistance to most of the antimicrobial agents evaluated, except vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid which registered 100% of susceptibility. Some isolates from blood stream infections such as E. coli demonstrated resistance to ciprofloxacin (23.3%), levofloxacin (20%), and gatifloxacin (16.6%), and Klebsiella pneumoniae was resistant (18%) to all fluoroquinolones tested. Pseudomonas aeruginosa manifested resistance to ciprofloxacin (16.6%), while 27.7% of these strains were resistant both to levofloxacin and gatifloxacin. All the Enterobacter cloacae isolated from blood were susceptible to ciprofloxacin, levofloxacin and gatifloxacin. Haemophilus influenzae and Moraxella catarrhalis collected from community-acquired respiratory tract infections were all inhibited by ciprofloxacin, levofloxacin and gatifloxacin. E. coli isolated from urinary tract infections in hospitalised patients were resistant to ciprofloxacin, levofloxacin and gatifloxacin (2.7%). All Salmonella spp. collected from samples of patients affected by infections of the gastro-intestinal tract were susceptible to all fluoroquinolones. Penicillin resistance in Streptococcus pneumoniae was found in 21.4% of isolates from patients with respiratory tract infections. Fluoroquinolone resistance was very rare among pneumococci except for ciprofloxacin. High rates of resistance to this drug (100%); resistance to levofloxacin and gatifloxacin was detected only in 3.6% of cases while resistance to moxifloxacina was detected in 1.8% of cases. Macrolide resistance rates were around 35% to 41%, tetracycline and trimethoprim/sulfamethoxazole resistance rates were relatively elevated (37.5% and 32.1% resistance respectively).

During the period june-october 2003 the urine samples were examined employing routine methods for strain identification and the Kirby-Bauer technique for antibiotic susceptibility tests.This usual system was compared with the new rapid Uro-Quick method employed on samples resulting positive and mono-microbial after Gram coloration. Antibiotic (in appropriate concentration) was introduced in a vial containing 2 ml of Mueller-Hinton broth, then 0.5 ml of urine were added in each vial containing the antimicrobial molecules and even in a vial without drug used as control.After 3-5 hours of incubation (for Gram negative or Gram positive strains respectively) the instrument shows the results. No growth and a growth curve like the control are representative of a susceptible and resistant strain respectively). Gram negative strain were tested against ciprofloxacin, nitrofurantoin, co-clavulanate, ceftazidime, fosfomycin, imipenem, amikacin, and trimethoprim-sulfamethoxazole, while Gram positive bacteria against ciprofloxacin, nitrofurantoin, co-clavulanate, ampicillin, fosfomycin, gentamycin, oxacillin and trimethoprim-sulfamethoxazole.The Gram negative strains isolated were 1172 and the Gram positive were 261.With the first group agreement between the two methods was always more than 90%, against Gram positive pathogens there was more than 80% of agreement. In conclusion against the mayor urinary tract pathogens (E. coli, Enterococci, Klebsiella spp. and Proteus spp.) agreement between the Uro-Quick system and the Kirby- Bauer was more than 90%.The rapid method appears useful not only for the determination of the antibiotic susceptibility of common uropathogens, but, on the basis of the present findings, it could be suggested the use of this rapid method in more severe nosocomial infections.

During the year 2002, 19 Clinical Microbiology Laboratories including 3 private centres, distributed in Ligurian and 2 in South Piemonte area have studied 26 strains carrying well-characterized antibiotic-resistant traits. Only 7 laboratories analysed the complete collection of 26 isolates and 12 sites studied at least 20 strains. Overall 85.4% of the microrganism identifications and 82.8% of the antibiotic susceptibility tests were correct. Gramnegative bacteria were more easily identified than the Gram-positive counterpart being E. faecium the most difficult pathogen to identify. Discrepancies were also observed with the antibiotic susceptibility tests. A significant number of tests, 22.6% of the identifications and 29.9% of the antibiotic susceptibility determinations were not carried out due to local economic and structural problems. Many of the pathogens are rarely encountered by Clinical Microbiology laboratories and sometime are difficult to detect, however, a large number of the centres was able to identify correctly these particular pathogens and their antibiotypes.

The Uro-Quick system, previously used to carry out antibiotic susceptibility tests directly on urine samples, has been employed for the detection of antibiotic resistance traits that might be not easily detected in well-characterized bacterial species.The period of time requested by the instrument to identify the drug resistance were: penicillin-resistant (PEN-R), constitutive and inducible oxacillin-resistant (OXA-R) Staphylococcus aureus, OXA-R S. epidermidis 480 min.Teicoplanin-resistant (TEI-R) S. haemolyticus, Vancomycin- (VanA,VanB,VanC), aminoglycoside- resistant (HLAR) Enterococcus spp, 24h. S. pyogenes exhibiting the three erythromycin-resistance phenotypes (Ery-R,C, I e M) 480 min.Ampicillin- (AMP-R), ciprofloxacin (CIP-R) and clavulanic acid-resistant (IRT) Escherichia coli 180 min. Imipenem-resistant (IMI-R) Pseudomonas aeruginosa 24h. For each strain was evaluated the optimal experimental conditions including inoculum size, time of incubation and antibiotic concentration. The results obtained indicated that Uro-Quick might be employed for the correct detection of the above antibiotic resistant markers.