Interplay among IncA and blaKPC-Carrying Plasmids in Citrobacter freundii.

We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, blaKPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying blaKPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.

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.

Colonization of long-term care facility residents in three Italian Provinces by multidrug-resistant bacteria.

Background: Rationale and aims of the study were to compare colonization frequencies with MDR bacteria isolated from LTCF residents in three different Northern Italian regions, to investigate risk factors for colonization and the genotypic characteristics of isolates. The screening included Enterobacteriaceae expressing extended-spectrum ß-lactamases (ESßLs) and high-level AmpC cephalosporinases, carbapenemase-producing Enterobacteriaceae, Pseudomonas aeruginosa or Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Methods: Urine samples and rectal, inguinal, oropharyngeal and nasal swabs were plated on selective agar; resistance genes were sought by PCR and sequencing. Demographic and clinical data were collected. Results: Among the LTCF residents, 75.0% (78/104), 69.4% (84/121) and 66.1% (76/115) were colonized with at least one of the target organisms in LTCFs located in Milan, Piacenza and Bolzano, respectively. ESßL producers (60.5, 66.1 and 53.0%) were highly predominant, mainly belonging to Escherichia coli expressing CTX-M group-1 enzymes. Carbapenemase-producing enterobacteria were found in 7.6, 0.0 and 1.6% of residents; carbapemenase-producing P. aeruginosa and A. baumannii were also detected. Colonization by MRSA (24.0, 5.7 and 14.8%) and VRE (20.2, 0.8 and 0.8%) was highly variable. Several risk factors for colonization by ESßL-producing Enterobacteriaceae and MRSA were found and compared among LTCFs in the three Provinces. Colonization differences among the enrolled LTCFs can be partially explained by variation in risk factors, resident populations and staff/resident ratios, applied hygiene measures and especially the local antibiotic resistance epidemiology. Conclusions: The widespread diffusion of MDR bacteria in LTCFs within three Italian Provinces confirms that LTCFs are an important reservoir of MDR organisms in Italy and suggests that future efforts should focus on MDR screening, improved implementation of infection control strategies and antibiotic stewardship programs targeting the complex aspects of LTCFs.

Characterization of an Outbreak of Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae in a Neonatal Intensive Care Unit in Italy.

Here we report an outbreak of extended spectrum ß-lactamase-producing Klebsiella pneumoniae that occurred in a neonatal intensive care unit in Northern Italy and involved 97 patients. Progressively tightened sets of containment measures were implemented but the epidemic event was stopped only 9 months later. The final, effective, containment strategy consisted of the application of strict geographic cohorting of colonized infants and their nursing staff, the suspension of any new admission and a rigorous daily sterilization protocol for all surfaces and fomites in the ward. A posteriori characterization of the outbreak strain was performed using both traditional microbiology and molecular biology techniques, and whole genome sequencing, allowing to compare outbreak isolates with other strains collected in the previous two years. The results allowed to determine that the outbreak strain had been circulating inside the ward since the year before. Genomic characterization revealed that the strain carried a wide array of virulence and antibiotic resistance determinants, including gene blaTEM-206, which had never been reported in a clinical isolate of K. pneumoniae before. The presence of such a high number of determinants for antibiotic resistance imposes significant therapeutic limitations on the treatment of infections, thus, further epidemiological investigations are needed to evaluate the prevalence of the newly described variant.

Occurrence of Extended Spectrum ß-Lactamases, KPC-Type, and MCR-1.2-Producing Enterobacteriaceae from Wells, River Water, and Wastewater Treatment Plants in Oltrepò Pavese Area, Northern Italy.

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-ß-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 blaCTX-M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX-M-1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV-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 blaDHA-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 IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò 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.

Evolving beta-lactamase epidemiology in Enterobacteriaceae from Italian nationwide surveillance, October 2013: KPC-carbapenemase spreading among outpatients.

Extended-spectrum beta-lactamases (ESBLs), AmpC-type beta-lactamases (ACBLs) and carbapenemases are among the most important resistance mechanisms in Enterobacteriaceae. This study investigated the presence of these resistance mechanisms in consecutive non-replicate isolates of Escherichia coli (n = 2,352), Klebsiella pneumoniae (n = 697), and Proteus mirabilis (n = 275) from an Italian nationwide cross-sectional survey carried out in October 2013. Overall, 15.3% of isolates were non-susceptible to extended-spectrum cephalosporins but susceptible to carbapenems (ESCR-carbaS), while 4.3% were also non-susceptible to carbapenems (ESCR-carbaR). ESCR-carbaS isolates were contributed by all three species, with higher proportions among isolates from inpatients (20.3%) but remarkable proportions also among those from outpatients (11.1%). Most ESCR-carbaS isolates were ESBL-positive (90.5%), and most of them were contributed by E. coli carrying blaCTX-M group 1 genes. Acquired ACBLs were less common and mostly detected in P. mirabilis. ESCR-carbaR isolates were mostly contributed by K. pneumoniae (25.1% and 7.7% among K. pneumoniae isolates from inpatients and outpatients, respectively), with blaKPC as the most common carbapenemase gene. Results showed an increasing trend for both ESBL and carbapenemase producers in comparison with previous Italian surveys, also among outpatients.

Emergence of Escherichia coli Sequence Type 131 (ST131) and ST3948 with KPC-2, KPC-3 and KPC-8 carbapenemases from a Long-Term Care and Rehabilitation Facility (LTCRF) in Northern Italy.

Aim of the study was to characterize KPC-producing Escherichia coli (KPC-Ec) clinical isolates among a Northern Italy Long-Term Care and Rehabilitation Facility (LTCRF) residents. Thirteen consecutive non repeated MDR E. coli isolates showing ertapenem Minimum Inhibitory Concentrations (MICs) >0.5 mg/L, collected during the period March 2011 - May 2013 from ASP "Redaelli" inpatients, were investigated. The bla KPC/CTX-M/SHV/TEM/OXA genes were identified by PCR and sequencing. KPC-Ec isolates underwent phylotyping, Pulsed-Field Gel Electrophoresis (PFGE), multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) profiling. Incompatibility groups analysis and conjugation were also performed. Eleven out of 13 isolates, resulted bla KPC-type positive, were consistently resistant to third generation cephalosporins, fluoroquinolones and trimethoprim-sulphametoxazole (84.6 %), retaining susceptibility to colistin (EUCAST guidelines). At least n = 4/11 of KPC-Ec patients received ≥48 h of meropenem therapy. Sequencing identified 9 bla KPC-2, 1 bla KPC-3 and 1 bla KPC-8 determinants. KPC-Ec plasmids belonged to IncF group (FIIk replicon); conjugation confirmed bla KPC/TEM-1/OXA-9 genes transferability for 10 KPC-Ec. Although three pulsotypes (A, B, C) were identified, all KPC-Ec belonged to phylogenetic group B2. Clone B (B-B5) caused an outbreak of infection involving nine inpatients at five wards. Rep-PCR showed relatedness for seven representative KPC-Ec isolates. Here we report a LTCRF outbreak caused by a ST131-B2 E. coli associated with bla KPC-2 and bla KPC-8 genes, and the emergence of the new ST3948. Elderly people with co-morbidities are at risk for ST131 colonization. KPC-Ec clones local monitoring appears essential both to avoid their spreading among healthcare settings, and to improve therapeutic choices for LTCRF residents.

Detection of an IncA/C plasmid encoding VIM-4 and CMY-4 ß-lactamases in Klebsiella oxytoca and Citrobacter koseri from an inpatient in a cardiac rehabilitation unit.

A 62-year-old patient was transferred to the cardiac rehabilitation unit of the I.R.C.C.S. Fondazione S. Maugeri after undergoing a heart transplantation at the Acute Care Hospital I.R.C.C.S. S. Matteo of Pavia. On 1 August 2013 and during hospitalization in the rehabilitation unit, Klebsiella oxytoca and Citrobacter koseri clinical isolates were simultaneously recovered from the patient's preputial swab. Both the K. oxytoca and C. koseri strains were carbapenem- resistant by MicroScan System (Beckman Coulter). Carbapenem-resistant K. pneumoniae had previously been reported in the same rehabilitation facility. The aim of the study was to identify the carbapenem resistance mechanisms among the enterobacterial species recovered. Phenotypic screening tests useful to detect the ß-lactamases/carbapenemases were performed. Carbapenem MICs were obtained by Etest. AmpC and MBL encoding genes were identified by PCR and sequencing. Conjugation assays and plasmid characterization were performed. Both of the K. oxytoca and C. koseri isolates were multi drug resistant, showing resistance to amoxicillin-clavulanic acid, three generation cephalosporins, ertapenem (K. oxytoca MIC, >32 mg/L; C. koseri MIC, 4 mg/L), imipenem (K. oxytoca MIC, 4 mg/L; C. koseri MIC, 12 mg/L), thrimethoprim sulphamethoxazole and gentamicin. Susceptibility was retained to fluoroquinolones, colistin and tigecycline. Molecular characterization confirmed the co-presence of blaCMY-4 and blaVIM-4 determinants in a 150 Kb transferable plasmid of IncA/C group. This case is the first detection in Italy of the K. oxytoca and C. koseri clinical isolates co-producing the CMY-4 and VIM-4 enzymes.