Antimicrobial resistance global emergence: healthcare facilities or environmental microbiota as the most important reservoir of antibiotic resistant microorganisms?

The ecosystems contamination caused by drugs and by their biologically active transformation products has become an emerging environmental issue: recently developed analytical and sensitive methods have allowed the detection of these pollutants in different matrices (like water, soil, sediment). [...

Extended-spectrum TEM- and SHV- Type beta-lactamase-producing Klebsiella pneumoniae strains causing Outbreaks in Intensive Care Units in Italy.

The aim of the present study was to investigate the production of extended-spectrum beta-lactamases (ESbetaLs) and the epidemiological correlations in a total of 107 Klebsiella pneumoniae strains resistant to third- and fourth-generation cephalosporins. The strains were collected from patients in four intensive care units (3 neonatal and 1 general) in three hospitals in Italy between March 1996 and July 1997. All strains were found to produce ESbetaLs. Phenotypic (antibiotyping and ESbetaL patterns) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analyses showed that a single strain had been responsible for each outbreak in each of the four intensive care units. Isoelectric focusing, activity on substrates and gene sequencing showed that the strains produced SHV-5, SHV-2a, SHV-12 and TEM-52 beta-lactamases. This is not only the first time that ESbetaL-producing Klebsiella pneumoniae strains have been reported as causing epidemics in Italian hospitals, it is also, to the best of our knowledge, the first time that an outbreak caused by a TEM-52 ESbetaL-producing Klebsiella pneumoniae strain has been reported. The data presented here illustrate the complexity of determining the epidemiological pattern of ESbetaL producers in large hospitals that do not have an ESbetaL-monitoring program

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

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

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

Hypoxia inducible factor-1β regulates a pro-invasive phenotype in acute monocytic leukemia

Hypoxia inducible transcription factors (HIFs) are the main regulators of adaptive responses to hypoxia and are often activated in solid tumors, but their role in leukemia is less clear. In acute myeloid leukemia (AML), in particular, controversial new findings indicate that HIF-1β can act either as an oncogene or a tumor suppressor gene, and this may depend on the stage of leukemia development and/or the AML sub-type. In this study, we find that HIF-1β promotes leukemia progression in the acute monocytic leukemia sub-type of AML through activation of an invasive phenotype. By applying a list of validated HIF-1β-target genes to different AML sub-types, we identified a HIF-1β signature that typifies acute monocytic leukemia when compared with all other AML sub-types. We validated expression of this signature in cell lines and primary cells from AML patients. Interestingly, this signature is enriched for genes that control cell motility at different levels. As a consequence, inhibiting HIF- 1β impaired leukemia cell migration, chemotaxis, invasion and transendothelial migration in vitro, and this resulted in impaired bone marrow homing and leukemia progression in vivo. Our data suggest that in acute monocytic leukemia an active HIF-1β-dependent pro-invasive pathway mediates the ability of leukemic cells to migrate and invade extramedullary sites and may be targeted to reduce leukemia dissemination

Rapid and sensitive detection of bla KPC gene in clinical isolates of Klebsiella pneumoniae by a molecular real-time assay.

The aim of this study was the rapid identification of bla KPC gene in 38 Klebsiella pneumoniae clinical isolates with reduced susceptibility to carbapenems. The modified Hodge Test (MHT) was carried out to phenotypically determine whether resistance to carbapenems was mediated by a carbapenemase. The detection of the bla KPC gene was performed by real-time acid nucleic sequence-based amplification (NASBA™™), specifically designed for the detection of KPC RNA target.Thirty-two/38 isolates evaluated by MHT showed the production of carbapenemases, while all the strains exhibited the production of KPC by inhibition test with phenylboronic acid (the combined disk test with IPM/IPM plus phenylboronic acid). The detection of bla KPC gene by Nuclisens EasyQ KPC yielded positive results in 38/38 (100\%) strains. The presence of bla KPC gene was confirmed in all K. pneumoniae isolates when tested by the gold standard PCR assay.In consideration of the serious challenge represented by infections due to K. pneumoniae it appears necessary the rapid identification of carbapenemases in clinical settings as it is made possible by the use of NASBA™ assay

OXA-244-Producing ST131 Escherichia coli From Surface and Groundwaters of Pavia Urban Area (Po Plain, Northern Italy)

The study aimed to investigate (i) the occurrence of third-generation cephalosporins and/or carbapenems non-sensitive Enterobacterales in Pavia surface and groundwaters, (ii) their resistance determinants, and (iii) the clonal features of the most relevant strains. During May 13 and 14, 2019, n = 18 water samples from n = 12 sampling sites in the urban/peri-urban area of Pavia (Po Plain, Northern Italy) have been evaluated. At first, hydrochemical analysis and bacterial plate counts were carried out on all the water samples. One milliliter of each water sample was then screened on both MacConkey agar (MC) added with cefotaxime (1 mg/L; 2 mg/L) and MC plus meropenem (0.25 mg/L; 4 mg/L). Species identification and antimicrobial susceptibilities were assessed by MicroScan autoSCAN-4. Double Disk Synergy (DD) test, CT103XL microarray, acc(6')-Ib-cr, qnrS, blaCTX-M-/MOX-/VEB-/OXA-type genes targeted PCR and sequencing, Pulsed-Field Gel Electrophoresis (PFGE), MultiLocus Sequence Typing (MLST), and Whole-Genome Sequencing on selected strains were performed. A total of n = 30 isolates grown on β-lactams enriched MC: Escherichia coli (n = 21; 70%), Klebsiella spp. (n = 5; 16.6%), Citrobacter freundii (n = 2; 6.7%), and Kluyvera intermedia (n = 2; 6.7%). All E. coli and K. pneumoniae were ESβL-producers by DD. The 66.6, 38.0, and 19.0% of E. coli were ciprofloxacin/levofloxacin, trimethoprim-sulfamethoxazole, and gentamicin resistant (EUCAST 2019 breakpoints), respectively. A blaCTX-M-type determinant was identified in E. coli (n = 20/21; 95.2%) and K. pneumoniae (n = 2/3; 66.7%). The remaining E. coli was blaVEB-1 and blaMOX-2 genes positive. The aac(6')-Ib-cr determinant was found in n = 7 E. coli and n = 1 K. pneumoniae, while qnrS was found in n = 1 E. coli and n = 2 K. pneumoniae. PFGE showed clonal heterogeneity among ESβL-E. coli. Two out of four E. coli detected as blaOXA-244-positive, belonged to the pandemic ST131. One XDR K. pneumoniae from a stream sample, detected as blaKPC-2 positive, resulted of ST258. The epidemiological impact of blaOXA-244 ST131 E. coli and blaKPC-2 ST258 K. pneumoniae presence in surface waters of an urban area in Northern Italy must not be underestimated

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