Colonization and infection due to carbapenemase-producing Enterobacteriaceae in liver and lung transplant recipients and donor-derived transmission: a prospective cohort study conducted in Italy.

OBJECTIVES: A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. METHODS: Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. RESULTS: Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor-recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacter aerogenes. CONCLUSIONS: This study showed a low risk of donor-recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

Colonization by multidrug-resistant organisms in long-term care facilities in Italy: a point-prevalence study.

OBJECTIVES: To determine prevalence and risk factors for colonization by multidrug-resistant organisms (MDROs) in long-term care facility (LTCF) residents in Italy. Genotypes of MDRO isolates were investigated. METHODS: A point-prevalence study was conducted at 12 LTCFs located in four Italian cities (2 February to 14 March 2015). Rectal swabs, faeces and nasal/auxiliary swabs were cultured for extended-spectrum ß-lactamase (ESBL)- and/or carbapenemase-producing Enterobacteriaceae, Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA) respectively. Antimicrobial susceptibility testing, detection of ESBL and/or carbapenemase genes and molecular typing of MDROs were performed. Risk factors for colonization were determined by univariate and multivariate analysis. RESULTS: A total of 489 LTCF residents aged ≥65 years were enrolled. The prevalence of colonization by ESBL-producing Enterobacteriaceae, MRSA and C. difficile was 57.3% (279/487), 17.2% (84/487) and 5.1% (21/409) respectively. Carriage rate of carbapenemase-producing Enterobacteriaceae was 1% (5/487). Being bedridden was a common independent risk factor for colonization by all MDROs, although risk factors specific for each MDRO were identified. ESBL-producing Escherichia coli carriage was associated with the sequence type (ST) 131-H30 subclone, but other minor STs predominated in individual LTCF or in LTCFs located in the same city, suggesting a role for intrafacility or local transmission. Similarly, MRSA from LTCF residents belonged to the same spa types/ST clones (t008/ST8 and t032/ST22) commonly found in Italian acute-care hospitals, but infrequent spa types were recovered in individual LTCFs. The prevalent C. difficile PCR ribotypes were 356/607 and 018, both common in Italian acute-care hospitals. CONCLUSIONS: MDRO colonization is common among residents in Italian LTCFs.

Application of capsular sequence typing (CST) to serotype non-viable Streptococcus pneumoniae isolates from an old collection.

Serotyping of Streptococcus pneumoniae is essential for monitoring changes in the pneumococcal population and the impact of vaccines. Recently, various DNA-based methods have become available and are increasingly used because they are cheaper and easier to perform than the Quellung reaction. Our aim was to apply a DNA-based method, capsular sequence typing (CST), to a collection of non-viable lyophilized pneumococcal isolates dating from the 1980s to elucidate the serotypes circulating in Italy 30 years ago. As a preliminary evaluation of the method, CST was applied to 68 recent pneumococcal isolates representative of the most common serotypes circulating in Italy in invasive pneumococcal disease (IPD) previously serotyped by the Quellung reaction. CST was then applied to 132 lyophilized non-viable isolates. A serotype-specific polymerase chain reaction (PCR), using primers suggested by the Centers for Disease Control and Prevention (CDC), was performed when CST did not yield a univocal serotype. Considering the control isolates, CST concordance with the Quellung reaction was 95.6 %. For the non-viable lyophilized isolates, CST identified a univocal serotype for 59.4 % of the isolates. This percentage increased to 78.1 % if CST was combined with serotype-specific PCR. The most frequent serotypes in the collection of non-viable strains were: 3 (15.6 %), 14 (11.7 %), 35B (5.5 %), 19A (5.5 %), and 8 (4.7 %). CST proved to be a valid method for serotyping pneumococcal strains and provided information about pneumococcal serotypes present in Italy 30 years ago. The combination of CST with serotype-specific PCR was an effective strategy to identify pneumococcal serotypes that can be suggested also for routine laboratories.

Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014.

Consecutive non-replicate clinical isolates (n=191) of carbapenem non-susceptible Enterobacteriaceae were collected from 21 hospital laboratories across Italy from November 2013 to April 2014 as part of the European Survey on Carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Klebsiella pneumonia carbapenemase-producing K. pneumoniae (KPC-KP) represented 178 (93%) isolates with 76 (43%) respectively resistant to colistin, a key drug for treating carbapenamase-producing Enterobacteriaceae. KPC-KP colistin-resistant isolates were detected in all participating laboratories. This underscores a concerning evolution of colistin resistance in a setting of high KPC-KP endemicity.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy.

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA.

Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: results of the first countrywide survey, 15 May to 30 June 2011.

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging as a public health problem in various settings. In Italy, a rapid and remarkable increase of carbapenem-non-susceptible Klebsiella pneumoniae has been reported since 2010. Here we report on the results of a countrywide cross-sectional survey, carried out from 15 May to 30 June 2011 to investigate the diffusion of CRE in Italy and to characterise the most prevalent resistance mechanisms and their dissemination patterns. CRE were reported from most (23 of 25) participating laboratories, with an overall proportion of 3.5% and 0.3% among consecutive non-duplicate clinical isolates of Enterobacteriaceae from inpatients (n=7,154) and outpatients (n=6,595), respectively. K. pneumoniae was the most frequent species (proportion of carbapenem-non-susceptible isolates: 11.9%), while a minority of CRE of other species were detected. Carbapenemase production was detected in the majority (85%) of CRE. KPC-type enzymes were by far the most common (89.5% of carbapenemase producers), followed by VIM-1 (9.2%) and OXA-48 (1.3%). KPC-producing K. pneumoniae (KPC-KP) were detected in most centres and contributed majorly to the epidemic dissemination of CRE recently observed in our country. Dissemination of KPC-KP was mostly sustained by strains of clonal complex 258 (ST-258 producing KPC-2 or KPC-3, and ST-512 producing KPC-3), while a minority belonged to ST-101.

Outbreak of skin and soft tissue infections in a hospital newborn nursery in Italy due to community-acquired meticillin-resistant Staphylococcus aureus USA300 clone.

BACKGROUND: Community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) is responsible for severe infections in previously healthy people acquired in the community in different areas of the world. AIM: To report an outbreak of CA-MRSA in a hospital newborn nursery in northern Italy in September-October 2010, its investigation and control measures. METHODS: The epidemiology of the outbreak is reported. The investigation included screening neonates, parents and staff for MRSA carriage. Molecular strain typing was performed on MRSA isolates. FINDINGS: The outbreak affected nine neonates with three severe infections. In addition, four mothers had postpartum mastitis, and three mothers and one father had skin infection. The outbreak strain belonged to the USA300 CA-MRSA clone. Asymptomatic carriage of the outbreak strain was found among neonates, parents and hospital staff. The implementation of appropriate infection control measures in the hospital terminated the outbreak. CONCLUSIONS: To our knowledge, this is the first report of a hospital outbreak caused by the USA300 CA-MRSA clone in Europe. It is important to reinforce infection control measures, particularly in high-risk groups, such as neonates, to prevent USA300 from becoming endemic in European hospitals.

Carbapenem non-susceptible Klebsiella pneumoniae from Micronet network hospitals, Italy, 2009 to 2012.

Carbapenem-resistant Klebsiella pneumoniae has recently been reported as a new, multidrug-resistant nosocomial pathogen in several hospitals from various Italian regions. Through Micronet, a new Italian sentinel laboratory-based surveillance network, we studied the trend of non-susceptibility of K. pneumoniae to selected carbapenems (imipenem and/or meropenem) in 14 of the 15 hospitals participating in the network. Analysis of data from 1 January 2009 to 30 April 2012 revealed a statistically significant increasing trend (p<0.01) in the proportion of carbapenem non-susceptible K. pneumoniae isolates from clinical specimens (from 2.2 % in 2009 to 19.4% in 2012). The increase in the proportion of non-susceptibility was very large for isolates from the respiratory tract (from 5.3% in 2009 to 38.5% in 2012) and blood (from 5.4% in 2009 to 29.2% in 2012). The results demonstrate the urgent need in Italy for infection control, guidelines, antibiotic stewardship programmes and utilisation of surveillance systems, such as Micronet, which are capable of receiving data from hospitals in real time for many pathogens and types of clinical specimens.

DNA microarray-based characterisation of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from Italy.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to collect and characterise the CA-MRSA isolates circulating in Italy, since only some case reports have been published. Eighteen Panton-Valentine-positive CA-MRSA isolates were collected from different Italian hospitals during the period 2005-2009 from severe infections (skin and soft tissue infections, n = 10; necrotising pneumonia, n = 7; and sepsis, n = 1). Accessory gene regulator (agr) typing, staphylococcal cassette chromosome (SCC) mec typing, spa typing, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and DNA microarray were applied to categorise isolates into clones and to compare the relevant genetic features of each clone. Six different clones were identified, the most common (7 out of 18 isolates, 38.8%) being agrI/ST8/SCCmecIV, corresponding to the USA300 clone. Six out of the seven USA300 isolates did not harbour the arginine catabolic mobile element (ACME). Four strains (22.2%) were agrIII/ST80/SCCmecIV, corresponding to the European clone. Two of the other clones, namely, agrIII/ST88/SCCmecV and agrIII/ST772/SCCmecV, corresponded to CA-MRSA clones rarely found in other countries and probably originating from Africa or the Indian subcontinent. The results of microarray hybridisations showed that the distribution of resistance genes and other virulence factors was specific to each clone. Some characteristics could be exploited as specific markers for a clone or a group of isolates, e.g. the mer operon, recovered only in ACME-negative USA300 strains. DNA microarray contributed to a more complete description of the variety of different CA-MRSA clones circulating in Italy.

Contribution of serotype and genetic background to biofilm formation by Streptococcus pneumoniae.

Streptococcus pneumoniae is the main cause of a variety of infections in children and the elderly ranging from otitis media to pneumonia. In recent years, biofilm formed by S. pneumoniae has begun to attract attention for a possible role in strains fitness and/or virulence. We evaluated the ability to form biofilm in a collection of clinical isolates, including antibiotic-resistant isolates whose genetic background had been previously ascertained. It appears that biofilm formation is a rather common feature among pneumococci, an observation which would fit with some types of infections caused by this microorganism (i.e. otitis, meningitis), which have often been associated with the ability to form biofilm. Antibiotic-susceptible isolates were able to form thicker biofilms compared to resistant strains, although no specific association could be observed with either serotypes or clones. This lack of association between the ability to form biofilm and any of the characters examined, while being a very common feature of pneumococci, may be suggestive of an important role for biofilm in pneumococcal ecology.

Vancomycin-heteroresistant phenotype in invasive methicillin-resistant Staphylococcus aureus isolates belonging to spa type 041.

The aim of this study was to characterise invasive methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains from Italy and to investigate the presence of heteroresistant vancomycin-intermediate S. aureus (h-VISA). Eighty-two MSSA and 66 MRSA strains obtained from 19 laboratories were submitted to in vitro susceptibility testing; MRSA strains were also analysed by the macro Etest (MET) and vancomycin population analysis profiles (PAP) to detect the presence of h-VISA. Genotyping included the detection of agr locus, SCCmec typing, spa typing and multilocus sequence typing (MLST). By Etest, 66% of all isolates showed a minimum inhibitory concentration (MIC) >or=1.5 microg/ml and two MRSA strains were categorised as VISA (MIC = 3 microg/ml). Twelve MRSA strains were positive by MET; of these, 9 (14% of all MRSA) were confirmed as h-VISA by PAP. MRSA strains were assigned to 14 spa types, with t001, t008 and t041 including 77% of the isolates. The most common spa type, t041, characterised as ST228/273-MRSA-I (CC5) and comprising 24 isolates, included one VISA and eight h-VISA. This is the first description of a close association between h-VISA and t041, a spa type common in Italy and in other European countries, that highlights the importance of molecular typing to identify clones of special clinical relevance.

No evidence of colonization with community-acquired methicillin-resistant Staphylococcus aureus in HIV-1-infected men who have sex with men.

To assess the prevalence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) colonization in HIV-1-infected men who have sex with men (MSM), a cross-sectional study was conducted on 104 persons attending a large STI/HIV unit in Rome, Italy in the period June 2007-June 2008. Swabs obtained from both anterior nares and S. aureus isolates were characterized by phenotypic and genotypic methods. A total of 24 individuals (23.1%) were colonized with S. aureus but none carried MRSA. No statistically significant association between colonization with S. aureus and behavioural, clinical, virological or immunological characteristics was identified. This study indicates a lack of circulation of CA-MRSA in HIV-1-infected MSM in Italy and underscores large epidemiological differences between the USA and a European country, so that only locally conducted epidemiological studies can provide insight into the local circulation of CA-MRSA in general and selected populations.

What is MRSA?

For decades methicillin-resistant Staphylococcus aureus (MRSA) has been considered the prototype of multi-resistant nosocomial pathogens, causing infections in high-risk patients. Changes in the healthcare system, coupled with the evolution of this versatile microorganism, have transformed MRSA into a cause of community-onset infections, in both patients who have contact with the healthcare system and patients without such a risk factor. New lineages of MRSA, defined as community acquired (CA)-MRSA, have emerged that have a propensity to cause infections in young individuals without risk factors. CA-MRSA primarily causes skin infections and, rarely, necrotising pneumonia. In the USA, these strains belong to a single widespread clone, designated USA300, while in Europe they belong to a variety of clones. Most strains carry genes for the Panton-Valentine leukocidin, whose role in diseases is under debate. In subjects living in the community who have contact with the healthcare system, MRSA strains of the nosocomial type are a frequent cause of infection and of pneumonia in particular. The detection of a large MRSA reservoir in pigs and the finding that professionally exposed individuals are colonised, has further shown that it is necessary to closely follow the epidemiology of MRSA if we want to combat it effectively.

Characterization of macrolide efflux pump mef subclasses detected in clinical isolates of Streptococcus pyogenes isolated between 1999 and 2005.

The macrolide efflux mechanism of resistance, mef, was characterized in community-acquired respiratory tract infections with Streptococcus pyogenes. Fifty-four (4.6%) M phenotype isolates were screen tested as negative for mef(A). Of these 54 isolates, 5 (0.4%), 27 (2.3%), and 1 (0.1%) were considered to be mef(I) positive, a novel mosaic variant of mef, or a novel subclass of mef, respectively. This study shows (i) the definitive presence of mef(E) in S. pyogenes and its global distribution, (ii) the presence of a mosaic variant of mef composed of mef(A) and mef(E), (iii) the previously undescribed presence of mef(I) in S. pyogenes, and (iv) the presence of a novel subclass of mef in S. pyogenes.

Population structure of invasive Streptococcus pneumoniae isolates in Italy prior to the implementation of the 7-valent conjugate vaccine (1999-2003).

A total of 773 pneumococcal isolates were collected from a nationwide surveillance of invasive pneumococcal diseases during 1999-2003 prior to the implementation of the 7-valent conjugate vaccine (PCV7) in Italy. The isolates included vaccine serotypes (VS, 393 isolates), vaccine-related serotypes (VRS, 93), and nonvaccine serotypes (NVS, 279). The ten most prevalent serotypes were: 14 (16.4%), 3 (8.4%), 23F (8%), 19F (7.4%), 4 (5.9%), 7F (5.8%), 9V (5.3%), 6B (4.9%), 19A (4.7%), and 1 (3.7%). VRS or NVS isolates showed a lower rate of penicillin or drug resistance than VS. Representative isolates of the major VS, VRS, and NVS were genotyped by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates examined were found to belong to 18 international clones and to eight newly described clones. VS isolates sharing clonal groups with VRS or NVS were also detected. Evidence of a past history of capsular switching events was observed in five clones.