Multifocal diffusion of a KPC-3 producing ST512 K. pneumoniae clone in Northern Italy

Sequence Type 258 (ST258) together with its allelic single- and double-locus variants have mostly been associated with the dissemination of KPC-producing Klebsiella pneumoniae in Europe. A total of 56 nonreplicate K. pneumoniae isolates with decreased carbapenem-susceptibility, collected at 7 different hospitals located in Northern Italy were investigated for the occurrence of blaKPC-type genes. PCR and sequencing results highlighted the presence of blaKPC-2 or blaKPC-3 determinants in 10/56 and 5/56 cases respectively. Here we describe the intra- and inter-hospital spread in Northern Italy of a K. pneumoniae ST512 clone harboring the blaKPC-3 gene

Usefulness of Microscan System panels with EUCAST clinical breakpoints to evaluate the antimicrobial susceptibility of ß-lactamase producing- Gram negative isolates

The study aimed to evaluate the ability of NBC45, NBC46 and NB40 Microscan (MS) panels, updated to 2010 EUCAST breakpoints, to identify at species level and to correctly define the susceptibility to ß-lactams of 61 ß-lactamases (BLs) producing Gram-negative isolates. A collection of 73 fully identified strains was analyzed: 21 Klebsiella spp., 17 E. coli, 15 P. mirabilis, 9 A. baumannii (Ab), 7 P. aeruginosa and 4 Enterobacter spp.. 61/73 were BLs and/or carbapenemases producers: 15 were CTX-M-1/-2/-14/-15 positive, and among them two were also VIM-1 positive. Four were TEM-52/-92, 3 PER-1, 2 SHV-12/-18 and 6 CMY-16 producers, while 11 were KPC-2/-3, 9 OXA-51/-58/-23, 8 VIM-1 and 2 IMP-13 positive. One K. oxytoca K-1 iper-producer, 11 non-BL producers/ATCC control strains and a OprD2 porin lacking P. aeruginosa were also included. All isolates were identified by Api-20E and VITEK-2 System and antibiotic susceptibilities were obtained by broth microdilution method. Resistance genes were identified by PCR and sequencing. All 73 isolates were correctly identified and a complete agreement for susceptibility patterns was observed for both ATCC control strains and non-BL clinical isolates. MS failed to detect a BL/Extended-Spectrum-ß-Lactamase (ESâL) production in 5/61 cases: any ESßL alert was detected using NBC46 panel for 3/15 CTX-M positive strains and 2 VIM-1/CTX-M-15 producing K. pneumoniae isolates. Intermediate resistance to cefoxitin (MIC 16 mg/L), susceptibility to cefepime (MIC 8 mg/L for ertapenem (ETP), according to previously results. All VIM-1 producers resulted intermediate/resistant to imipenem (IP) and meropenem (MP); decreased MIC values were observed in 2/8 cases. Carbapenem MICs >8 mg/L were detected for IP-13 P. aeruginosa producers; 6/9 OXA carbapenemases- producing Ab showed IP MIC >8 mg/L and 3/6 MP MIC >8 mg/L. 3/9 Ab OXA-58/-51 producers, tested using NB40 panel, were intermediate or resistant to doripenem and meropenem. Regarding the detection of BLs overall agreement between MS and reference methods was 91.9%. Carbapenems MIC values resulted a fold lower than previously determined. Nevertheless using 2010 EUCAST breakpoints for ETP, MP and IP was possible to detect all carbapenemases- producers. MS System represents a useful tool to perform identification of BL- producing Gram negative bacteria

Growth in glucose-based medium and exposure to subinhibitory concentrations of imipenem induce biofilm formation in a multidrug-resistant clinical isolate of Acinetobacter baumannii

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>is emerging as an important nosocomial pathogen. Multidrug resistance, as well as ability to withstand environmental stresses, makes eradication of <it>A. baumannii </it>difficult, particularly from hospital settings.</p> <p>Results</p> <p>Over a six-year period, 73 isolates of <it>A. baumannii </it>were collected from infected patients in two hospitals in Italy. While 69 out of the 73 isolates displayed identical multidrug antibiotic resistance pattern, they were susceptible to carbapenems. Genetic profiles of these 69 isolates, determined by Pulsed Field Gel Electrophoresis (PFGE), indicated that they were genetically related and could be clustered in a specific clone, called SMAL. We tested the ability of the SMAL clone to form biofilm, an important determinant for bacterial colonization of the human host and for persistence in the hospital environment. Biofilm formation by <it>A. baumannii </it>SMAL, measured as surface adhesion to polystyrene, is strongly affected by growth conditions, being impaired in rich growth media such as LB, while being favoured in glucose-based medium. Surface adhesion in glucose-based media is inhibited by treatment with cellulase, suggesting that it depends on production of cellulose or of a chemically related extracellular polysaccharide. Exposure of <it>A. baumannii </it>SMAL to subinhibitory concentrations of imipenem resulted in biofilm stimulation and increased production of iron uptake proteins. Growth in iron-supplemented medium also stimulated surface adhesion, thus suggesting that increased intracellular iron concentrations might act as an environmental signal for biofilm formation in <it>A. baumannii </it>SMAL.</p> <p>Conclusions</p> <p>Our results indicate that exposure to subinhibitory concentrations of imipenem can stimulate biofilm formation and induce iron uptake in a pathogenic strain of <it>A. baumannii</it>, with potential implications on antibiotic susceptibility and ability to persist in the human host.</p

Colistin resistance in KPC-producing Klebsiella pneumoniae strains from a high specialization rehabilitation facility

The worldwide rapid spread of KPC carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) represents an increasing problem in clinical settings. Reports on KPC-Kp epidemic spread in Italian hospitals began to appear since 2010; colistin (COL) represents one of the few remaining therapeutic options available for the treatment of such multi drug-resistant (MDR) pathogens. Here we report the presence and diffusion of COL resistant KPC-Kp isolates from a High Specialization Rehabilitation Facility located in Northern Italy. Species identification and antimicrobial susceptibilities were obtained by NBC46/NM40 Microscan panels (Siemens); imipenem, meropenem and ertapenem MICs were also evaluated by Etest and broth microdilution method; blaKPC-like genes PCR were performed. PFGE (XbaI) was used to investigate clonal relatedness; epidemiological data were collected from the hospital database. Seventy-five carbapenem-resistant K. pneumoniae isolates were collected from the Fondazione S. Maugeri hospital during the period January-June 2011. Seven out of 75 MDR KPC-Kp isolates by Microscan System showed COL resistance (MIC >2 mg/L). Among them, 5/7 were collected from coma and 2/7 from cardiology and rehabilitation cardiology wards. Most of these strains were from urine (5/7); the remaining 2/7 were from blood and bronco-alveolar lavage. The 85.7% of the strains showed susceptibility to tigecycline and fosfomycin; 71.4% only to gentamicina, 28.5% to trimethoprim/sulfamethoxazole and 14.2% to amikacin. The PFGE profiles obtained analyzing 5/7 isolates from patients hospitalized from almost 10 days, showed clonal relatedness between 4/5 isolates, thus confirming the high epidemic potential of almost one KPC-Kp clinical strain collected from 4 different wards.The emergence of COL resistance in KPC-Kp, dramatically reduces the available therapeutic options. These results underline the ability of a COL resistant KPC-producing clone to rapidly spread within this Rehabilitation Facility

Evaluation of two commercially available methods used for the rapid detection of ESBL-producing strains

Detection of ESBL production by Enterobacteriaceae remains a challenge for microbiologists. Although recent changes in the breakpoints of third-generation cephalosporins decreased the likelihood of reporting ESBL producers as susceptible to these compounds, ESBL detection is of interest for prevention of dissemination of ESBL-producers by cross-transmission and for epidemiological purpose. The aim of this study was to evaluate the sensitivity and the specificity of two commercial methods suitable for rapid ESBL-detection in Gram negative bacilli: the ChromID ESBL medium (bioMérieux) and the Cica-ß-Test (Mast Group, Merseyde, UK). 121 Enterobacteriaceae collected between February 2008 and April 2009 at the Laboratory Analysis IRCCS Humanitas were tested for ESBL-production by PhoenixTM, E-test (ESBL reference test), ChromID ESBL medium and Cica-β-Test. ChromID showed 100% of sensitivity and specificity for the screening of ESBL in E. coli; lower values of specificity were found in the case of P. mirabilis (81%) and Klebsiella spp. (92%). The Cica-β-Test always showed high specificity levels, but the poor sensitivity found for both E. coli (90%), P. mirabilis (73%) and Klebsiella spp. (85%), discourages its use for screening of ESBL in Gram negative bacilli from blood-cultures. Rapid identification of ESBL producers is of interest to implement hygiene precautions. In that case, using a very sensitive primary test is of major interest

Occurrence of extended spectrum \u3b2-lactamases, KPC-Type, and MCR-1.2-producing enterobacteriaceae from wells, river water, and wastewater treatment plants in Oltrep\uf2 Pavese area, Northern Italy

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepo Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-beta-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of bla(CTX-M)-type was identified in 19/30 isolates. In further two E. coli strains, a bla(CTX-M-1) gene co-existed with a bla(SHv)-type ESBL determinant. A bla(SHv-12) gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains, A bla(DHA)-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative lncX4 plasmid (33.303 bp in size) from a stream of Oltrepo Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms

Candida albicans in a neonatal intensive care unit: antifungal susceptibility and genotypic analysis.

Invasive candidiasis in neonates has become an increasing problem over the past decade in Neonatal Intensive Care Units (NICUs). From August 2005 to January 2006, six invasive candidiasis occurred in neonates in NICU of the S. Matteo hospital of Pavia. The study focused on the species involved and their in vitro antifungal susceptibility. Genotyping was conducted to determine clonal relatedness. A total of 22 yeasts were isolated from different biological samples of neonates during six months. The infants were infected with or colonized by Candida albicans and six patients developed C. albicans deep infections. The genotyping of the transposable intron region of C. albicans strains showed that they belonged to the genotype A (17 isolates) and genotype B (5 isolates). The RAPD confirmed these results. These data suggest that nosocomial transmission of C. albicans could be take into account as a mode of acquisition by neonates in NICUs at this hospital