OXA-48 and NDM-1 Klebsiella pneumoniae of Sequence Type 101 from blood in a patient with travel history abroad, Italy.

Klebsiella pneumoniae (KP) is an important pathogen involved in serious nosocomial infections all over the world. Here, we describe the first report on a blood-stream infection caused by an OXA-48/NDM-1 ST101-KP, in Italy. The patient was an Italian woman, transferred from Cairo Hospital to a Neurosurgery ward in Cuneo (IT). The detection described here enhances the need for an effective National infection control strategy in Italy.

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.

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.

Epidemic diffusion of OXA-23-producing Acinetobacter baumannii isolates in Italy: results of the first cross-sectional countrywide survey.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is emerging worldwide as a public health problem in various settings. The aim of this study was to investigate the prevalence of CRAb isolates in Italy and to characterize their resistance mechanisms and genetic relatedness. A countrywide cross-sectional survey was carried out at 25 centers in mid-2011. CRAb isolates were reported from all participating centers, with overall proportions of 45.7% and 22.2% among consecutive nonreplicate clinical isolates of A. baumannii from inpatients (n = 508) and outpatients (n = 63), respectively. Most of them were resistant to multiple antibiotics, whereas all remained susceptible to colistin, with MIC50 and MIC90 values of ≤ 0.5 mg/liter. The genes coding for carbapenemase production were identified by PCR and sequencing. OXA-23 enzymes (found in all centers) were by far the most common carbapenemases (81.7%), followed by OXA-58 oxacillinases (4.5%), which were found in 7 of the 25 centers. In 6 cases, CRAb isolates carried both bla(OXA-23-like) and bla(OXA-58-like) genes. A repetitive extragenic palindromic (REP)-PCR technique, multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine the genetic relationships among representative isolates (n = 55). Two different clonal lineages were identified, including a dominant clone of sequence type 2 (ST2) related to the international clone II (sequence group 1 [SG1], SG4, and SG5) and a clone of ST78 (SG6) previously described in Italy. Overall, our results demonstrate that OXA-23 enzymes have become the most prevalent carbapenemases and are now endemic in Italy. In addition, molecular typing profiles showed the presence of international and national clonal lineages in Italy.

Prevalence of urinary colonization by extended spectrum-beta-lactamase Enterobacteriaceae among catheterised inpatients in Italian long term care facilities.

BACKGROUND: Long Term Care Facilities (LTCFs) play a key role in guaranteeing care to patients in developed countries. Many patients, mostly elderly, access LTCFs at some time in their lives, and their healthcare pathways often require them to move back and forth between hospital and outpatient settings. These patterns bring about new challenges regarding infection control, especially healthcare associated infections. METHODS: A point prevalence study was conducted in 23 Italian LTCFs, to identify colonization in patients with urinary catheter (>24 hours). Species identification, susceptibility tests and extended spectrum beta lactamase (ESBL) production screenings were performed using Vitek 2 System. Enterobacteria identified by Vitek 2 System as ESBL-producers or suspected AmpC hyperproducers on the basis of cephamycin resistance, were sent to a research laboratory where they underwent a double-disk synergy test. Finally, ESBL-producers were screened for bla resistance genes by PCR assay. RESULTS: 211 patients with catheter were screened, 185 out of 211 patients showed positive samples for the presence of Enterobacteriaceae, 114 of these 185 patients were colonized by extended spectrum cephalosporins resistant microorganisms. We identified a total of 257 Gram negative pathogens, of which 51.8% (133/257) were extended spectrum cephalosporins resistant. 7 out of 133 cephamycin not susceptible strains proved to be AmpC-type beta-lactamases and 125/133 ESBL-producers; 1 was not further characterized. 43 out of 257 (16.7%) E. coli, 37/257 (14.4%) P. mirabilis, 20/257 (7.8%), P. stuartii, 14/257 (5.4%) M. morganii, 7/257 (2.7%), K. pneumoniae, 4/257 (1.6%) C. koseri proved to be overall ESBL-producers by double-disk synergy test. Third and fourth generation cephalosporin resistant P. mirabilis, P. stuartii and M. morganii strains mainly harboured a blaTEM gene (95.9%), while 89.1% of E. coli were positive for the blaCTX-M determinant by PCR and sequencing. Patients with decubitus had a higher risk of colonization by at least one resistant isolate (p < 0.01). Samples of patients undergoing antibiotic therapy and patients with decubitus showed a higher risk (p < 0.05) of colonization by beta-lactam resistant microorganisms. CONCLUSIONS: These data confirm the presence of high percentages of ESBL-positive Enterobacteria in Italian LTCFs and the predominance of CTX-M type ESBL in E. coli. The alarming presence of ESBL-producing Enterobacteriaceae in Italian LTCFs can seriously compromise the effectiveness of antibiotic therapy.acilities (LTCFs), Antimicrobial resistance.

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 blaKPC2 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.

Emergence of a VIM-1 MBL and CTX-M-15 ESbL-producing Klebsiella pneumoniae clone from acute and rehabilitation hospitals in Italy.

We report the emergence of VIM-1 MBL and CTX-M-15-producing K. pneumoniae isolates collected from patients at two acute care hospitals (I.R.C.C.S. "S. Matteo" and "Casa Sollievo della Sofferenza" Hospital) and a long-term rehabilitation facility in Northern Italy (I.R.C.C.S. "S. Maugeri"). Between February 2007 and October 2008, 30 K. pneumoniae strains showing decreased susceptibility to carbapenems were collected. PCR and sequencing experiments revealed the presence of blaVIM-1 gene in 14/30 isolates. All the above isolates carried the blaSHV-5 determinant as well; interestingly, 8/14 VIM positive isolates were also CTX-M-1- like producers. VIM-1 positive strains were present in all hospitals. PFGE genomic profiles of the 14/30 isolates showed that 2 different clones, A and B, were responsible for outbreaks. The coexistence in the same bacterial cell of compatible plasmids carrying epidemiologically important emerging resistance genes, such as MBLs and CTX-Ms, is worrisome since it could predict the generation and spread of pan-resistant bacteria and the consequent treatment option limitations that can lead to significant morbidity and mortality. Control measures should be applied to detect MBL-producing strains and to contrast the vertical and plasmidic diffusion of carbapenem-resistant K. pneumoniae in acute care and rehabilitation facilities.

Evaluation of brilliance CRE agar for the detection of carbapenem-resistant gram-negative bacteria.

The aim of this work was to evaluate the performance of the new chromogenic medium BrillianceTM CREAgar (Thermo Fisher Scientific) for determining the limit of detection of carbapenem-resistant enterobacteria (CRE). A total of 70 clinical isolates were studied. Of these, 30 were well-characterized CRE, including Klebsiella pneumoniae strains producing KPC-, VIM-, and OXA-type enzymes, VIM-positive Enterobacter cloacae and Escherichia coli, NDM-positive E. coli, and enterobacterial isolates characterized by porin loss associated with ESBL production or AmpC hyperproduction. Ten carbapenem-resistant non-fermentative isolates were also included as well as 30 carbapenem-susceptible isolates. Carbapenem-resistant strains were inoculated at three different concentrations onto Brilliance CRE Agar (from 1.5x101 CFU/ml up to 1.5x104 CFU/ml) whereas carbapenem-susceptible isolates were inoculated at a concentration of 1.5x102 CFU/ml. The medium sustained the growth of carbapenem-resistant isolates, showing detection limits from 1.5x101 CFU/ml (in 31/40 cases) to 1.5x104 CFU/ml. No growth was observed with carbapenem-sensitive control strains. Our results indicate that the Brilliance CRE Agar allows the growth of carbapenem-resistant isolates with low detection limits and could represent a useful screening medium for both enterobacteria and non-fermentative Gram-negative strains resistant to carbapenems.

Molecular epidemiology of KI and WU polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co-infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients.

Differences in biofilm formation and aggregative adherence between beta-lactam susceptible and beta-lactamases producing P. mirabilis clinical isolates.

Biofilm formation of multidrug resistant (MDR) extended-spectrum beta-lactamase (ESbetaL) producing Proteus mirabilis isolates from a long-term care and rehabilitation facility (LTCRF) in Northern Italy was evaluated. A total of 10 strains, 4/10 producing the acquired AmpC beta-lactamase CMY-16, 3/10 producing the ESbetaL TEM-92 and the remaining negative for the presence of beta-lactamase genes, were studied using standard adherence assays on titer plates. Tests were performed in three different media, including Luria Bertani (LB), LB diluted and urine. Three representative strains were also tested for biofilm production in microtiter in presence of beta-lactam sub-MIC concentrations. The same isolates were screened for aggregative adherence onto monkey kidney cells (LLC-MK2). All strains studied were capable of biofilm formation, though at different levels. The beta-lactamase positive strains were statistically better significant in biofilm formation than negative ones regardless of growth medium. Cellular adherence assays showed a preferential ability of all isolates, regardless of beta-lactamase production, to adhere to inert surfaces rather than to cells. Although the results did not fully support a direct correlation between beta-lactamase production and biofilm formation, both mechanisms can greatly contribute to bacterial persistence in the urinary tract.

Complete Genome and Plasmids Sequences of a Clinical Proteus mirabilis Isolate Producing Plasmid Mediated NDM-1 from Italy.

Background: The spread of carbapenemase genes, such as blaNDM-1, in Proteus mirabilis poses a public health threat. The aim of the study was to characterize the genome and plasmids sequences of an NDM-1-positive strain (IBCRE14), which was isolated in 2019 from a catheterized patient hospitalized in Italy. Methods: Whole genome sequencing (WGS) of IBCRE14 was performed on extracted genomic DNA using Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases from the Center for Genomic Epidemiology. Results: IBCRE14 had a genome size of 4,018,329 bp and harboured genes coding for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ), and trimethoprim (dfrA1). A large plasmid (pIB_NDM_1) harboured antibiotic resistance genes against sulphonamide (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr-2), aminoglycosides (aadA1, aph3-VI), and beta-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) harboured a qnrD1 gene coding for quinolone resistance. Conclusion: The ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could both acquire more resistance genes and easily disseminate. To our knowledge, this is the first report on an untypable plasmid harbouring blaNDM-1 in P. mirabilis, in Italy.

Deadly Puppy Infection Caused by an MDR Escherichia coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6)-Ib-cr - Positive in a Breeding Kennel in Central Italy.

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired ß-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring bla TEM-1b, bla CTX-M-15, bla OXA-1, aph(6)-Ib, aac(6')Ib-cr, aac(3)-Ila, aph(6)-Id, aadA1, qnrB1, sul2, catA1, catB3, tetA, and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, bla DHA-1, qnrB4, mph(A), sul1, and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A bla CMY-2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA, iss, astA, and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6')-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir.

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

BACKGROUND: Acinetobacter baumannii is emerging as an important nosocomial pathogen. Multidrug resistance, as well as ability to withstand environmental stresses, makes eradication of A. baumannii difficult, particularly from hospital settings. RESULTS: Over a six-year period, 73 isolates of A. baumannii 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 A. baumannii 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 A. baumannii 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 A. baumannii SMAL. CONCLUSIONS: Our results indicate that exposure to subinhibitory concentrations of imipenem can stimulate biofilm formation and induce iron uptake in a pathogenic strain of A. baumannii, with potential implications on antibiotic susceptibility and ability to persist in the human host.

Trends in frequency and in vitro antifungal susceptibility patterns of Candida isolates from women attending the STD outpatients clinic of a tertiary care hospital in Northern Italy during the years 2002-2007.

Vulvovaginal candidiasis is a common mucosal infection caused by saprophytic and opportunistic yeasts belonging to the Candida genus. 518 vaginal swabs, with positive fungal culture were collected from unselected women attending the Sexually Transmitted Disease clinic of an Italian tertiary care hospital over a six year period to determine the pathogen prevalence in vulvovaginal candidiasis and to evaluate in vitro the antifungal susceptibilities of yeast recovered by Sensititre YeastOne antifungal panel plates according to CLSI document M27-A2. The isolates belonging to the genus Candida were 495 (95.5%) with Candida albicans percentage equal to 61.2%. Voriconazole was highly active (MIC50 0.008; MIC90 0.5 microg/ml), regardless of the species tested. On the contrary, fluconazole susceptibility was based upon the species. The intrinsic resistance to fluconazole of C. krusei was confirmed.

Complete Nucleotide Sequence of Plasmids of Two Escherichia coli Strains Carrying bla NDM- 5 and bla NDM - 5 and bla OXA - 181 From the Same Patient.

Aim of this study was to genetically characterize two carbapenemase-producing Escherichia coli strains obtained from a pediatric patient affected by diarrhea, expressing OXA-181 and/or NDM-5 type enzymes. The above microorganisms were collected in the same Desenzano hospital (Northern Italy) where the bla NDM- 5 gene was detected for the first time in Italy 3 years ago. One strain (5P), belonged to sequence type ST405/ST477 (according to Pasture/Oxford schemes) and serotype O102:H6. It was characterized by a 130562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) enclosing two main antibiotic resistance islands: (i) ARI-I, 10030 bp in size, carried genes coding for ß-lactam- (bla OXA- 1, bla CTX-M- 15), fluoroquinolone/aminoglycoside- (aac(6')-lb-cr) and phenicol- resistance (catB3), (ii) ARI-II, 15326 bp in size, carried genes coding for sulfonamide- (sul1), ß-lactam- (bla NDM- 5, bla TEM- 1 B), phenicol- (catB3), trimethoprim- (dfrA17), antiseptic- (qacEΔ1), and aminoglycoside- (aadA5, rmtB) resistance. The other isolate (5M), belonged to sequence type ST2659/ST759 and serotype O50/02:H18, and carried four plasmids: a 153866 bp multi-replicon IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), an 89866 bp IncI1 plasmid, a 51480 bp IncX3 plasmid (pISV_IncX3_OXA181), and a 41143 bp IncI plasmid (pISV_IncI_CMY-42). pISV_IncFII_NDM-5 carried two main antibiotic resistance islands: (i) ARI-III, 12220 bp in size, carried genes coding for ß-lactam- (bla OXA- 1), fluoroquinolone/aminoglycoside- (aac(6')-lb-cr), tetracycline- (tet(B)) and phenicol- resistance (catB3, catA1), and ii) ARI-IV, 26527 bp in size, carried determinants coding for macrolide- (erm(B), mph(A)), sulfonamide- (sul1), beta-lactam- (bla NDM- 5, bla TEM- 1 B), trimethoprim- (dfrA14, dfrA12), antiseptic- (qacEΔ1), and aminoglycoside- resistance (aadA5). pISV_IncI_CMY-42 harbored the bla CMY- 42 gene coding for beta-lactam resistance, pISV_IncX3_OXA181 harbored genes encoding fluoroquinolone- (qnrS1) and beta-lactams- resistance (bla OXA- 181). In conclusion, the detection of two different NDM-5 E. coli strains from a pediatric patient with a history of travel to the Far East countries strongly highlight an increasing trend and risk of importation from such areas.

Can Insertion Sequences Proliferation Influence Genomic Plasticity? Comparative Analysis of Acinetobacter baumannii Sequence Type 78, a Persistent Clone in Italian Hospitals.

Acinetobacter baumannii is a known opportunistic pathogen, dangerous for public health mostly due to its ability to rapidly acquire antibiotic-resistance traits. Its genome was described as characterized by remarkable plasticity, with a high frequency of homologous recombinations and proliferation of Insertion Sequences (IS). The SMAL pulsotype is an A. baumannii strain currently isolated only in Italy, characterized by a low incidence and a high persistence over the years. In this present work, we have conducted a comparative genomic analysis on this clone. The genome of 15 SMAL isolates was obtained and characterized in comparison with 24 other assemblies of evolutionary related isolates. The phylogeny highlighted the presence of a monophyletic clade (named ST78A), which includes the SMAL isolates. ST78A isolates have a low rate of homologous recombination and low gene content variability when compared to two related clades (ST78B and ST49) and to the most common A. baumannii variants worldwide (International Clones I and II). Remarkably, genomes in the ST78A clade present a high number of IS, including classes mostly absent in the other related genomes. Among these IS, one copy of IS66 was found to interrupt the gene comEC/rec2, involved in the acquisition of exogenous DNA. The genomic characterization of SMAL isolates shed light on the surprisingly low genomic plasticity and the high IS proliferation present in this strain. The interruption of the gene comEC/rec2 by an IS in the SMAL genomes brought us to formulate an evolutionary hypothesis according to which the proliferation of IS is slowing the acquisition of exogenous DNA, thus limiting genome plasticity. Such genomic architecture could explain the epidemiological behavior of high persistence and low incidence of the clone and provides an interesting framework to compare ST78 with the highly epidemic International Clones, characterized by high genomic plasticity.

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.

Prevalence, antimicrobial resistance, and extended-spectrum beta-lactamases characterization of Salmonella isolates in Apulia, southern Italy (2001-2005).

The prevalence, antimicrobial susceptibility, and production of extended-spectrum beta-lactamases (ESBLs) of Salmonella collected from several hospitals in Apulia (southern Italy) were evaluated. The most common Salmonella isolates were Salmonella enterica serovar Typhimurium (44.6%), S. enterica serovar Enteritidis (33.4 %), S. enterica serovar Infantis (3.2 %), S. enterica serovar Typhi (1.5%), and S. enterica serovar Bovismorbificans (1.5%). The other serovars accounted for less than 1% each. Our data show a high resistance to ampicillin, tetracycline, and chloramphenicol. The isolates were pansensitive (53.5%), resistant to one antimicrobial agent (10.5%), resistant to two antimicrobial agents (22.1%), resistant to three antimicrobial agents (10.8%), and to four antimicrobial agents (2.7%). Resistance to more than four antibiotics was observed in 0.5% of strains. The presence of ESBL was found in only one strain of S. enterica serovar Bovismorbificans. The CTX-M-1 type-producing strain was identified by isoelectric focusing and molecular analysis. Results were consistent with the presence of a pI 8.6 ESBL active on cefotaxime, ceftazidime, cefepime, and aztreonam. Mating experiments showed that the CTX-M-1 determinant was transferable. To the best of our knowledge, this is the first report of CTX-M-1 type ESBL in Salmonella serovar Bovismorbificans.