Epidemiologia locale di MRSA, con riferimento alla circolazione di cloni ospedalieri e comunitari e al possibile ruolo della catena alimentare

ABSTRACT Local epidemiology of MRSA,with reference to the circulation of hospital and community strains and the possible role of the food chain Introduction The aim of this PhD project is to evaluate the local epidemiology of MRSA strains and the related risk to the healthcare environment, the community and the food production chain. According to literature, MRSA is a rising problem in different settings and new strains are spreading all over the world. In addition to HA-MRSA, CA-MRSA e LA-MRSA are new important problems to face. In this contest, this study is focused to estimate the frequency of isolation of MRSA in different kind of samples and to analyze the main features of the collected strains. Comparing the isolates each other, it is possible to underline similarities and correlations and finally understand their specify identity. Materials and methods MRSA strains were isolated using Brain Hearth Infusion (BHI) broth and mannitol salt agar plates. After a further incubation at 35°C for 48 h, suspected colonies were confirmed to be S. aureus and tested for resistance in Müller-Hinton agar with oxacillin (6 μg/ml). MRSA strains are analyzed with antibiograms (Kirby-Bauer), E-test and D-test. Molecular typing techniques as MLVA, PCR multiplex for SCCmec determination, MLST, PCR for tst-1were used for the isolated MRSA. A selection of strains is analyzed with microarray, whole genome sequencing and spa typing. Results Between January 2010 and December 2013 samples of different origin were collected and MRSA was detected. In details, we analyzed: 145 food samples, 71 animal samples, 2452 nasal swabs from the children hospitalized in the NICU (Neonatal Intensive Care Unit) in the University Hospital of Palermo and 500 nasal swabs from healthy children up to 3 years old attending public nursery schools in Palermo. From food and animal samples none MRSA was isolated. Within the food samples 12 MSSA strains were collected (8.3%). 7 The study developed in collaboration with the NICU takes is based upon a four-year surveillance program started in June 2009 and now still ongoing. The frequencies of colonization of MRSA fell from 27% in the first year of study 2009/10 to 10% in the fourth years 2012/2013. In contrast, the rate of colonization of MSSA rose from 5% to 16% in the same four-year period. Most of the isolated MRSA do not have important antibiotic resistances and were recognized with a particular MLVA profile. The dominant strain is identified with MLST as ST22 and SSCmec IVa with PCR multiplex for SCCmec determination. This strain was identified with microarray as CC22-MRSA IV tst1+/ UK-EMRSA-15 “Middle Eastern Variant” and confirmed by tst-1 PCR. The whole genome sequencing was used to estimate the presence of about 350-400 SNPs between a selection of strain collected from 2009 to 2013. To have a whole sight of the situation in the NICU it is useful to specify that different MRSA strains were identified during the study and we want to underline especially the spreading of CA-MRSA ST1 for almost five months with 14 cases of colonization. The study in healthy children attending the nursery schools detected 10 MRSA (2%) and 68 MSSA (13.6%). These ten children positive for MRSA were attending nine different day care centers. No association with specific risk factors was found. Eight MRSA isolates were tst1+ as CC22-MRSA IV tst1+/ UK-EMRSA-15 “Middle Eastern Variant” and identified by spa typing as spa t223. The other two isolates were identified ST1-IVa and ST398-V. They tested negative for PVL and tst1 genes and were resistant to clindamycin and erythromycin. ST398-V was also vancomycin resistant with MIC of 3 mg/L. Two isolates, ST22-IVa and ST1-IVa respectively, were recovered from two children attending the same day care center. None of the family members of the child carrying ST398-MRSA-V reported any exposure to livestock. Conclusions The exposure to the hospital environment is the main risk factor for MRSA. The colonization rates are considerably higher in the NICU than in the nursery schools (10% vs 2% in the last year of study). On the other hand, the emergent spreading of MRSA in the community should be monitored locally. In this study it is demonstrated the circulation of the same MRSA strain CC22-MRSA IV tst1+/ UK-EMRSA-15 “Middle Eastern Variant” spa t223 in the NICU and in the nursery schools. A single MRSA strain is able to spread in hospital and community setting as an evidence of the great ability to adaptation. This same strain was before identified in Abu 8 Dhabi and USA from foreign people. In this contest, our evaluation of the local epidemiology of MRSA is very important to understand what is happening globally and what we may expect for the future. In addition, the surveillance program done in the NICU was a very helpful tool to control the circulation of MRSA and reduce the rate of colonization and the study in the nursery schools is important to evaluate the situation in the community

Preliminary study on trace metal content in manna extracted from Fraxinus angustifolia cultivars in Madonie (Sicily, Italy)

The manna is a product obtained from the elaborate sap solidification that comes out from the incisions made on the stem of some species of the genus Fraxinus L. In this study, we have analyzed 12 samples of manna to determine the chemical composition. Twenty elements (Al, As, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Rb, Sb, Se, Sr, U, V, Zn) were determined using an Inductively Coupled Plasma Mass Spectrometer. The results show that the most abundant elements are Fe > Rb > Al > Cu > Ba > Zn > Sr with median concentrations in the range of 0.9 – 10mg kg-1. The remaining elements exhibit median concentrations in the range of 0.0003 -0.5 mg kg-1. The manna also can be considered as good sources of essential elements for subjects with diseases related to diabetes. Moreover, the manna could be recommended for a daily diet

tst1-positive ST22-MRSA-IVa in healthy Italian preschool children

A survey was performed in May 2013 to assess methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization in healthy children attending 26 municipal daycare centres in Palermo, Italy. Of the 500 children, ten (2 %) tested positive. Eight MRSA isolates were tst1-positive ST22-MRSA-IVa, spa t223; the other two isolates were identified as ST1-IVa and ST398-V, respectively. tst1-positive ST22-MRSA, spa t223 has been previously identified only in the Middle Eastern area

Healthcare associated pathogens in a changing world

In developed countries about 10% of the hospitalizations are complicated by a healthcare-associated infection [1]. Up to 75% of these infections are due to multidrug-resistant organisms (MDROs) [1]. Antimicrobial resistant bacterial infections are associated to higher morbidity, mortality and healthcare costs than those caused by susceptible organisms [1]. The findings of the point prevalence survey in European acute care hospitals published in 2013 by the European Centre for Disease Control and Prevention (ECDC) show large variations between countries and between different regions of the same country, with Italy being allocated within the high-endemic areas for both MRSA and MDROs [2]. Despite antimicrobial resistance affects most bacterial species, MDR Gram negatives represent the most serious threat. In a few years Enterobacteriaceae, mainly Escherichia coli and Klebsiella pneumoniae, have evolved from extended spectrum β-lactamase (ESBL) producing to carbapanem-resistant organisms [3]. Simultaneously, Acinetobacter baumannii has quickly become extremely or pan-drug resistant [4]. Carbapenem resistant Gram negatives heavily impact on clinical outcomes with mortality rates significantly higher than the susceptible strains of the same species [1]. Of further concern, very few antimicrobial agents are available for an effective treatment of these infections and new agents active against these organisms are not currently in development. Many intertwining factors are driving these epidemiological changes, involving patients, healthcare delivery systems, infection control practices and, most important, misuse and inappropriate use of antibiotics in all healthcare facilities, in community and in animal husbandry. In particular, the transition of the healthcare delivery systems from a hospital-centered model to a healthcare facility network has gradually blurred the borders between hospital and community and the patients’ travel within this network has critically contributed to disseminate MDROs [5]. As a consequence, antimicrobial resistance is now as common, if not more so, in post-acute clinical facilities, such as long term care settings and nursing homes [5]. The “revolving door” is the very efficacious image used as the paradygm of the spreading routes of organisms with hospital and community reservoirs, as E. coli or MRSA. The revolving door, indeed, enlightens how the colonized patients entering back and forth several healthcare settings drive the amplification of the antibiotic resistance [6]. Stringent infection control and prevention practices and wise use of antibiotics are unanimously agreed as the key actions to fight MDROs. Of course, we need new antibiotics, but first we have to learn how to protect them from a precipitous erosion of their effectiveness

Methicillin-resistant Staphylococcus aureus nasal colonization in a level III neonatal intensive care unit: Incidence and risk factors

Objective: To describe epidemiologic features and identify risk factors for methicillin-resistant Staphylococcus aureus (MRSA) acquisition in a level III neonatal intensive care unit (NICU). Setting: A prospective, cohort study in a university-affiliated NICU with an infection control program including weekly nasal cultures of all neonates. Methods: Demographic, clinical, and microbiologic data were prospectively collected between June 2009 and June 2013. Molecular characterization of MRSA isolates was done by multilocus variable number tandem repeat fingerprinting, staphylococcal cassette chromosome mec typing, and on representative isolates by multilocus sequence typing and s. pa typing. Results: Of 949 neonates, 217 (22.87%) had a culture growing MRSA, including 117 neonates testing positive at their first sampling. Of these latter infants, 96 (82.05%) were born with the infection and 59 (50.43%) had been transferred from the nursery. Length of stay and colonization pressure were strong independent predictors of MRSA acquisition. Among MRSA isolates, 7 sequence types were identified, with ST22-IVa, spa type t223, being the predominant strain. Conclusions: In an endemic area, early MRSA acquisition and high colonization pressure, likely related to an influx of colonized infants from a well-infant nursery, can support persistence of MRSA in NICUs. Surveillance, molecular tracking of strains, and reinforcement of infection control practices, involving well-infant nurseries in a comprehensive infection control program, could be helpful in containing MRSA transmission

Outbreak of ST395 KPC-Producing Klebsiella pneumoniae in a Neonatal Intensive Care Unit in Palermo, Italy

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Methicillin-resistant Staphylococcus aureus colonization: a three-year prospective study in a neonatal intensive care unit in Italy.

Background: Methicillin resistant Staphylococcus aureus (MRSA) is a major etiological agent of infection in neonatal intensive care units (NICUs). Routes of entry of this organism can be different and the transmission pathway complex. Colonized neonates are the main endogenous reservoir. Methods and Results: We conducted a prospective three-year study on MRSA colonization recruiting 722 neonates admitted between 2009 and 2012. Nasal swabs were cultured weekly and MRSA isolates were submitted to molecular typing. The annual incidence density of acquisition of MRSA ranged from a maximum of 20.2 cases for 1000 patient-days during the first year to a minimum of 8.8 cases in the second one to raise again up to 13.1 cases during the third year. The mean weekly colonization pressure fluctuated from 19.1% in the first year to 13.4% in the second year and 16.8% in the third year. It significantly correlated with the number of MRSA acquisitions in the following week. Overall, 187 (25.9%) subjects tested positive for MRSA. A non multiresistant, tst positive, ST22-MRSA-IVa spa t223 strain proved to be endemic in the NICU, being identified in 166 (88.8%) out of 187 colonized neonates. Sporadic or epidemic occurrence of other strains was detected. Conclusions: An MRSA strain belonging to the tst1 positive, UK-EMRSA-15/ ‘‘Middle Eastern Variant’’ appeared to be endemic in the NICU under investigation. During the three-year period, substantial changes occurred in case-mix of patients moving towards a higher susceptibility to MRSA colonization. The infection control procedures were able to decrease the colonization rate from more than 40% to approximately 10%, except for an outbreak due to a CA-MRSA strain, ST1-MRSAIVa, and a transient increase in the colonization prevalence rate coincident with a period of substantial overcrowding of the ward. Active surveillance and molecular typing contributed to obtain a reliable picture of the MRSA dissemination in NICU

The Increasing Challenge of Multidrug-Resistant Gram-Negative Bacilli: Results of a 5-Year Active Surveillance Program in a Neonatal Intensive Care Unit

Colonization and infection by multidrug-resistant gram-negative bacilli (MDR GNB) in neonatal intensive care units (NICUs) are increasingly reported.We conducted a 5-year prospective cohort surveillance study in a tertiary NICU of the hospital "Paolo Giaccone," Palermo, Italy. Our objectives were to describe incidence and trends of MDR GNB colonization and the characteristics of the most prevalent organisms and to identify the risk factors for colonization. Demographic, clinical, and microbiological data were prospectively collected. Active surveillance cultures (ASCs) were obtained weekly. Clusters of colonization by extended spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae were analyzed by conventional and molecular epidemiological tools.During the study period, 1152 infants were enrolled in the study. Prevalences of colonization by MDR GNB, ESBL-producing GNB and multiple species/genera averaged, respectively, 28.8%, 11.7%, and 3.7%. Prevalence and incidence density of colonization by MDR GNB and ESBL-producing GNB showed an upward trend through the surveillance period. Rates of ESBL-producing E coli and K pneumoniae colonization showed wide fluctuations peaking over the last 2 years. The only independent variables associated with colonization by MDR GNB and ESBL-producing organisms and multiple colonization were, respectively, the days of NICU stay (odds ratio [OR] 1.041), the days of exposure to ampicillin-sulbactam (OR 1.040), and the days of formula feeding (OR 1.031). Most clusters of E coli and K pneumoniae colonization were associated with different lineages. Ten out of 12 clusters had an outborn infant as their index case.Our study confirms that MDR GNB are an increasing challenge to NICUs. The universal once-a-week approach allowed us to understand the epidemiology of MDR GNB, to timely detect new clones and institute contact precautions, and to assess risk factors. Collection of these data can be an important tool to optimize antimicrobials use and control the emergence and dissemination of resistances in NICU

The burden of Candida species colonization in NICU patients: a colonization surveillance study

Fungal infections are an important cause of morbidity and mortality in neonatal intensive care units (NICUs). The identification of specific risk factors supports prevention of candidemia in neonates. Effective prophylactic strategies have recently become available, but the identification and adequate management of high-risk infants is still a priority. Prior colonization is a key risk factor for candidemia. For this reason, surveillance studies to monitor incidence, species distribution, and antifungal susceptibility profiles, are mandatory. Among 520 infants admitted to our NICU between January 2013 and December 2014, 472 (90.77%) were included in the study. Forty-eight out of 472 (10.17%) patients tested positive for Candida spp. (C.), at least on one occasion. All the colonized patients tested positive for the rectal swab, whereas 7 patients also tested positive for the nasal swab. Fifteen out of 472 patients (3.18%) had more than one positive rectal or nasal swab during their NICU stay. Moreover, 9 out of 15 patients tested negative at the first sampling, suggesting they acquired Candida spp. during their stay. Twenty-five of forty-eight (52.1%) colonized patients carried C.albicans and 15/48 (31.25%) C.parapsilosis. We identified as risk factors for Candida spp. colonization: antibiotic therapy, parenteral nutrition, the use of a central venous catheter, and nasogastric tube. Our experience suggests that effective microbiological surveillance can allow for implementing proper, effective and timely control measures in a highrisk setting

Outbreak of colonizations by extended-spectrum β-lactamase-producing Escherichia coli sequence type 131 in a neonatal intensive care unit, Italy.

BACKGROUND: Extended spectrum β-lactamases (ESBLs) often associated with resistance to aminoglycosides and fluoroquinolones have recently emerged in community-associated Escherichia coli. The worldwide clonal dissemination of E. coli sequence type (ST)131 is playing a prominent role. We describe an outbreak of colonizations by ESBL-producing E. coli (ESBL-E. coli) in the neonatal intensive care unit (NICU) of the University Hospital, Palermo, Italy. METHODS: An epidemiological investigation was conducted with the support of molecular typing. All children admitted to the NICU and colonized by ESBL-E. coli between January and June 2012, were included in the study. Cases were defined as infants colonized by E. coli resistant to third generation cephalosporins and fluoroquinolones. A case–control study was also performed to identify possible risk factors. RESULTS: During the outbreak period, 15 infants were found to be colonized by ESBL-E. coli. The epidemic strain demonstrated continuous transmission throughout the outbreak period. Case–control study identified a lower birth weight as the only risk factor for colonization. The strain belonged to the sequence-type 131 community-associated clone. Transmission control interventions, including contact precautions and cohorting, restriction of the new admissions, sanitization of surfaces and equipment and targeted training sessions of the NICU staff, were successful in interrupting the outbreak. CONCLUSIONS: Although invasive infections did not develop in any of the 15 colonized neonates, our report highlights the need to strictly monitor the spill in the NICU setting of multidrug resistant community-associated organisms. Our findings confirm also the role of active surveillance in detecting the silent spread of ESBL-producing Gram negatives in a critical healthcare setting and trigging the implementation of infection control measures. As β-lactam and fluoroquinolone resistant E. coli strains are increasingly spreading in the community, this event could become a more serious challenge