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

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

Multicenter prospective study on the prevalence of colistin resistance in escherichia coli: Relevance of mcr-1-positive clinical isolates in Lombardy, Northern Italy

Background: The emergence of the plasmid-mediated colistin resistance mechanism in Escherichia coli has raised concern among public health experts as colistin is a last-line antimicrobial resort. The primary aim of the study was to investigate the prevalence of this resistance trait in E. coli isolates circulating in the Lombardy region, Northern Italy. The presence of mcr-type genes and their genetic relationship were also studied. Materials and methods: A prospective study was performed during a 4-month period (May to August, 2016) in six acute care Hospitals. Consecutive nonduplicate clinical isolates of E. coli from any type of clinical specimen, with the exception of rectal swabs, were included in the study. Isolates that exhibited MIC values for colistin >2 mg/L were further investigated. Bacterial identification was obtained by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Amplification of mcr-type genes (-1 to -5 variants) and microarray analysis were accomplished. Repetitive sequence-based PCR (Rep-PCR) and multilocus sequence typing (MLST) analysis were used for genotyping. Results: Overall, 3,902 consecutive E. coli isolates (2,342 from outpatients, 1,560 from inpatients) were evaluated during the study period. Of them, 18/3,902 (0.5%), collected from 4/6 centers, showed resistance to colistin. These isolates were mostly obtained from urine of both outpatients (n=12) and inpatients (n=6). Colistin MIC values ranged from 4 to 8 mg/L. The mcr-1 gene was detected in 10/18 isolates (7 from outpatients, 3 from inpatients). Rep-PCR and MLST analysis revealed the presence of nine different clusters. Further mcr-type genes were not detected. Conclusion: Resistance to colistin in E. coli clinical isolates appears low in our geographic area. With regard to mcr-1-positive isolates, they accounted for approximately 50% of colistin-resistant E. coli isolates, thus representing a relevant resistance mechanism in this context. Although overall limited, the presence of mcr-1 determinant in our region should not be ignored and great concern should be given to the continuous surveillance

Helicobacter pylori Bacteremia: An Unusual Finding

We report a case of Helicobacter pylori transient bacteremia in a woman with ulcerated antral gastric cancer. The patient was hospitalized for laparoscopy and subtotal gastrectomy. After surgery she developed fever (39°C) and was empirically treated with levofloxacin. Blood cultures, collected and sent immediately to Laboratory, were positive for a spiral Gramnegative bacterium. This isolate was identified as H. pylori and the specific susceptibility test was performed. One day after the fever was decreased but antibiotic treatment with levofloxacin was continued and it was maintained until discharge. In summary, H. pylori transient bacteremia may occur as a rare complication after stomach surgery. Further studies are necessary to elucidate the potential role of H. pylori presence in blood

Multifocal diffusion of KPC-3-producing ST512 Klebsiella pneumoniae in Italy

Introduction. The dissemination of carbapenemase-producing Klebsiella pneumoniae strains is an increasing problem worldwide. KPC ß-lactamases are Ambler class A enzymes mostly plasmid-encoded; their global spread represents a threat to clinical patients care and public health. Multi locus sequence type (ST)258 is currently the most spread K. pneumoniae clone associated with KPC enzymes. Here we report the first identification and multifocal spread of KPC-3 producing K. pneumoniae clinical strains belonging to ST512 in Italy. Materials and Methods. Fifty six carbapenem-resistant K. pneumoniae isolates were collected from 7 Italian hospitals during the period June 2009-May 2011. Isolates were obtained from different wards (spinal unit, medicine, hematology, etc.) and biological samples (mostly rectal swabs, urine and blood). Species identification and antimicrobial susceptibilities were obtained by NBC46/NM40 Microscan panels (Siemens). MICs values were interpreted according to EUCAST 2011 breakpoints. Modified Hodge test and combined disk test with phenyl-boronic acid (BOR) and EDTA were performed.The presence of blaKPC genes were confirmed by PCR and sequencing. A complete characterization of the produced ß-lactamases (BLs) was obtained by IEF followed by PCR experiments using primers specific for the detection of blaCTX-M-, blaTEM- and blaSHV type genes. PFGE and multilocus sequence typing (MLST) were both used to investigate clonal isolates relatedness. Results. All 56 isolates resulted positive for the presence of KPC-type carbapenemases by both phenotypical and molecular analysis. Fifteen isolates, chosen as representative, were further investigated. Ten out of 15 isolates harboring the blaKPC-2 gene clustered with the known ST258, while the remaining 5/10 belonged to the newly described ST512 and harbored the blaKPC-3 gene. ST512 isolates, from 3/7 hospitals, were collected from rectal swabs (40%), blood (20%), endotracheal aspirate (20%) and urinary (20%) samples. Carbapenem MICs ranged from 2 mg/L to more than 16 mg/L; the isolates were characterized by the same multi-drug-resistant (MDR) profile. Only in 1/5 cases such antimicrobial resistance profile was extended also to colistin (>2 mg/L).Three different PFGE clones were identified (A, B and C); one resulting characteristic of each hospital. Coherently with pIs obtained by IEF, the presence of additional BLs were investigated by PCR; clones B and C resulted positive for blaCTX-M and blaSHV genes, while blaSHV and blaTEM genes were found in clone A. Conclusion.Intra- and inter-hospital spread of KPC-positive strains was demonstrated.The heterogeneity of PFGE profiles showed the spread of different K. pneumoniae ST258 clones and the emergence of the new ST512 coupled with blaKPC-3 genes in Italian clinical settings. Strategies for carbapenemase detection and appropriate hospital hygiene precautions are indicated to limit the spread of the here described ST512 clone appears essential

Prevalence of colistin resistance in Escherichia coli: mcr-1-positive clinical isolates in Lombardy, Northern Italy

Background: The recent emergence of the plasmid-mediated colistin resistance mechanism in Escherichia coli, Klebsiella pneumoniae and Salmonella spp. has raised concern among public health experts as colistin is a last-line antibiotic for the treatment of infections by carbapenem resistant Enterobacteriaceae. The aim of the study was to investigate i) the prevalence of this resistance trait in E. coli, and ii) the presence of mcr-1 and mcr-2 genes among colistin-resistant isolates in the Lombardy region, Northern Italy. Material/methods: The study was performed during a 4-month period (May-August 2016) in six acute care Hospitals. Non duplicated E. coli isolates from clinical samples of both outpatients and inpatients were included in the study (surveillance rectal swabs were excluded). Bacterial species identification was obtained by MALDI-TOF mass spectrometry. Colistin resistance (MIC > 2 mg/L) was first evaluated by routine automated systems and then confirmed by the reference broth microdilution method (EUCAST, 2016). Amplification of mcr-type genes, and microarray analysis were accomplished. Phylogroup identification, rep-PCR (DiversiLab) and PFGE (XbaI) were used for genotyping. The mcr-1-positive plasmids were characterized by PBRT kit and PCR (IncX). Results: Overall, 3902 E. coli isolates were evaluated (1560 from inpatients, 2342 from outpatients). Of them, 18/3902 (0.5%), collected from 4/6 centers, showed resistance to colistin. These isolates were obtained from both inpatients (n=6) and outpatients (n=12). In particular, hospitalized patients were from medical (3/6), rehabilitation (2/6), and surgical (1/6) wards. The great majority of colistinresistant isolates were from urine (16/18) but two of them were from blood cultures. Colistin MIC values ranged from 4 to 16 mg/L (as assessed by the reference method). The mcr-1 gene was detected in 10/18 isolates, all of which from urine cultures (7 from outpatients, 3 from inpatients). In these cases, colistin MIC values ranged from 4 to 8 mg/L. Of note, 6/10 mcr-1-positive E. coli isolates were also resistant to ciprofloxacin and trimethoprim-sulfamethoxazole, and 2 of them were also positive for the SHV-12 ESBL enzyme. All mcr-1-positive E. coli strains resulted unrelated by PFGE analysis, while rep-PCR showed eight different clusters. The identified phylogroups were A and D (in four cases each), B1 and B2 (in one case each). The incompatibility groups were IncX4 in 7/10 strains, IncHI2 in the remaining 3/10. The mcr-2 gene was never detected. Conclusions: According to most recent surveillances, our study demonstrates a low prevalence of colistin resistance among E. coli isolates also in the Lombardy region. In addition, our data show that the mcr-1 gene plays a prominent role in this context. Of particular concern, it appears the frequent detection of mcr-1 gene among isolates from urine cultures of outpatients, thus suggesting the opportunity of routine testing the antimicrobial susceptibility for colistin in these cases

Emergence of KPC-type producing Escherichia coli ST131 from three acute care hospitals and two long term and rehabilitation facilities in northern Italy

Background: Although carbapenem resistant Escherichia coli remains uncommon in Europe, Italy is one of the four countries showing the highest resistance percentages, with 2010-2013 increasing trends reported. The aim of the study was to investigate the population structure and beta-lactamases (BLs) content of nine KPC-2 and three KPC-3 E. coli strains from five hospitals in Northern Italy. Material/Methods: In the period April 2011-May 2013, 12 MDR E. coli isolates obtained from five Northern Italy Facilities were studied using PFGE, DiversiLab automated rep-PCR system, MLST and Phylogenetic group analysis. Conjugation and replicon typing were also performed. Carbapenem MIC values were evaluated by Etest. Other BLs presence was assessed by blaCTX-M/SHV/TEM/OXA genes amplification and sequencing. Results: Out of the 12 strains, six were from rehabilitation wards, four from intensive care units (ICUs), and two from nephrology and neurosurgery wards, respectively. Nine KPC-E. coli were from urine, two from sputum samples and one from blood. Two isolates retained susceptibility to ertapenem, eight to meropenem and nine to imipenem (EUCAST 2015 breakpoints). blaCTX-M-group 1 (blaCTX-M-15) and blaCTX-M-group 9 genes (blaCTX-M-27) were detected in 4/12 strains only, while blaTEM-1 and blaOXA-9 determinants were detected in 9/12 and 10/12 isolates respectively. All but one strain resulted of phylogenetic group B2. Eight different PFGE profiles (A-H) and four rep-PCR profiles were distinguishable, while five MLSTs (ST131, ST3948, ST3426, ST5839, ST3861) were detected. All KPC-2 strains, detected in each hospital, belonged to ST131 Complex (Cplx), being 8/9 of ST131 and 1/9 of ST3948, while five different pulsotypes were distinguishable. The three KPC-3 E. coli showed unique PFGE fingerprints and belonged to the here newly identified ST5839 and ST3681, in addition to the already reported ST3426, occurring in 2012 in UK. IncF, IncFII, groups were detected in all but one cases; the ST3426 KPC-3 strain showed, in addition to IncF, IncU and IncX1 plasmids. Carbapenem resistance gene transfer was obtained in three cases; for two of the above cases, the acquisition of the conjugative plasmid from a Klebsiella pneumoniae previously responsible for a patient’s infection, was ascertained. The PFGE clone F, that caused an intra-hospital outbreak during the period June 2012-May 2013 at a Long Term and Rehabilitation Facility (LTCF), showed the highest inter-hospital spreading potential, being identified also in an ICU of an Acute Care Hospital (ACH), over the same period. Conclusions: Here we report the spread of several clones of KPC-positive E: coli isolates both from ACHs and LTCFs in Northern Italy. While KPC-2 E. coli resulted linked to the ST131 Cplx, the KPC-3 strains showed a multi-clonal dissemination. The low meropenem and/or imipenem MIC values contributed to the inter-hospital dissemination and an underestimation of the real presence of such pathogen in these settings