Alphitobius diaperinus Panzer (Insecta, Coleoptera) in a single house of a broiler production facility as a potential source of pathogenic bacteria for broilers and humans.

Pest infestation in any stage can lead to a quality reduction in the finished products. This study aimed to detect Escherichia coli, Salmonella spp., Campylobacter spp., and Staphylococcus aureus in Alphitobius diaperinus adults, and in samples from broiler swabs, administered water and feed collected in a single house from a broiler production facility in central Italy. Three samplings were carried out, each collecting ninety adult beetles for microbial detection in the external, faecal and internal content; ten cloacal swab samples; and one sample of both administered feed and water. Microbiological cultures and biochemical identification were performed on suspected cultures and confirmed by species-specific PCRs. A. diaperinus was abundantly found near the windows, under the manger and in the corners of the facility. Salmonella enterica serovar Cholerasuis was found at the external surface of the beetles, while Staphylococcus xylosus and E. coli were in the faecal content. The latter micro-organism together with Staphylococcus lentus, S. xylosus and other staphylococcal species were detected in the internal microbiota. E. coli and Campylobacter spp. were observed in cloacal swabs, and S. xylosus in one feed sample. The study findings support evidence for Salmonella spp. and E. coli, and remark that adherence to sanitation rules and biosecurity procedures are required.

New Delhi metallo-ß-lactamase (NDM-1)-producing Klebsiella pneumoniae of sequence type ST11: first identification in a hospital of central Italy.

The emergence of novel resistant markers hampers the efficacy of beta-lactam antibiotics to treat infections caused by micro-organisms carrying such resistances. This study investigated the antimicrobial susceptibility pattern, the carpapenem-associated determinants and the molecular epidemiology of Klebsiella pneumoniae showing a New Delhi (NDM) metallo-ß-lactamase phenotype, isolated from a patient admitted to intensive care unit of the main hospital for acute care of Molise region, central Italy. Antimicrobial susceptibility was assessed for nineteen antibiotics by disc diffusion and agar dilution methods. Carbapenem-associated resistance determinants were detected through gene-specific amplifications, targeting blaNDM-1 , blaSHV and blaTEM , blaCTX-M , blaKPC , blaVIM , blaIMP , blaGES and blaOXA-48-lixe . Molecular characterization was carried out through multilocus sequence typing. The strain showed a multidrug resistant profile, and PCR and sequencing confirmed the presence of blaNDM-1 gene. Among the multiple resistance-associated determinants tested, the isolate, which was assigned to the sequence type ST11, only harboured blaSHV and blaTEM genes. This is the first report of NDM-1 variant in the regional healthcare setting for acute patients, raising significant concerns about the increase in the antimicrobials resistance spread through a different mechanism from the endemic KPC carbapenemase, and underlining the circulation of a virulent clone never identified before in this area.

Procedures in endoscope reprocessing and monitoring: an Italian survey.

BACKGROUND: The high diffusion of endoscopes worldwide and the need for effective reprocessing methods requested the development of guidelines and implementation of surveillance procedures at local level. STUDY DESIGN: In order to collect data on everyday's practice and adherence to available guidelines, endoscopy units from different public institutions were surveyed using a dedicated questionnaire. METHODS: Between July and November 2015 a survey was carried in 12 main hospitals from 10 different Italian regions, involving 22 endoscopy units. The state of the art of national and international guidelines was investigated to compare the protocols adopted at local level. RESULTS: In all the surveyed hospitals, the reprocessing activity is based on pre-established protocols in adherence with principal guidelines. Enzymatic detergents, which are recommended by the international guidelines, are used in 55.6% of units and peracetic acid is currently the most widely used chemical disinfectant. Discrepancies were observed in the application of periodic quality controls. CONCLUSION: Updated guidelines are generally applied in reprocessing practice. Quality controls may represent a critical issue to improve effectiveness and surveillance. The whole of acquired data can promote a positive trend towards the application of best practices.

A multicentre study on epidemiology and prevention of needle stick injuries among students of nursing schools.

INTRODUCTION: Among the health professions with a long period of training, the students of the Nursing Bachelor's Degree are the most exposed to biological risk resulting from accidents, in particular with needles and cutting edges. The aim of the study was to estimate the frequency and the circumstances for the occurrence of needle stick injuries, as a knowledge base for targeted prevention interventions. METHODS: The study was carried out between May and July 2017 in 11 Universities in Italy and 1 in Albania (associated with the "Tor Vergata" University of Rome). An anonymous semi-structured questionnaire was proposed to 1st (second semester), 2nd and 3rd year students of Nursing Bachelor's Degree. RESULTS: A total of 2742 questionnaires were collected. The average age of participants was 22.9 years (median 22, range 19-60 years), 73% of whom were females. A total of 381 injuries were reported. Three hundred and sixteen students (11.8%) underwent at least 1 injury (12.7% among females, 9.7% among males); 41 students declared two or more injuries; four students did not report the number of injuries occurred. The first injury occurred, as an average, 17 days after the start of the internship (median 15 days) and, in 25% of the cases, during the first 9 days. The highest percentage of accidents occurred during the first internship (25.3% of the total) and decreased with the progress of the training path. The injuries occurred in 38% of cases during drug preparation, 24% when disposing of sharp devices, 15% while re-capping needles, 13% during blood sampling and 10% in other circumstances. In 51.2% of cases, the needle was not sterile. Among the nursing students who suffered a needle stick injury, 58.1% declared that they had performed the post-exposure prophylaxis. 96% of students stated to be vaccinated against Hepatitis B virus. Amongst the students who had their serological status checked (74%), 18% stated the antibody titre was not protective. 49.8% of students answered to have been trained in advance on the correct procedures to avoid needle stick and cutting edges injuries in each clinical ward attended, 41.2% referred that this occurred only in some wards and 10% in no ward at all. CONCLUSION: The results of this study show a high percentage of needle stick injuries in students of the Nursing Bachelor's Degree. Therefore, there is a need for careful reflection on the most effective methods of targeted training acquisition of knowledge, skills and behavioural models useful for the exercise of the profession.

High resolution melting analysis for the characterization of lineage II Listeria monocytogenes serovars 1/2a and 1/2c based on single nucleotide polymorphisms identification within the Listeria Pathogenicity Island-1 and inlAB operon: a novel approach for epidemiological surveillance.

AIMS: A high resolution melting (HRM) assay was developed for characterizing lineage II Listeria monocytogenes based on the amplification and the melting profiles analysis of 81 fragments targeting the region from the prs to ldh loci, including the Listeria Pathogenicity Island-1 (LIPI-1) genes and the inlAB operon. METHODS AND RESULTS: Real-time PCR and HRM protocols were standardized using 10 replicate assays from L. monocytogenes EGD-e reference strain (serovar 1/2a). Twenty wild-type isolates of serovar 1/2a and two of serovar 1/2c were tested, and differences between EGD-e strain and the wild-type isolates were defined if the melting temperature (Tm ) of an amplicon was not within the lower and the upper limits calculated from replicate testing on EGD-e. The analysis revealed 17 and 19 HRM profiles with respect to prs/LIPI-1/ldh and inlAB target regions (Simpson's Index of Diversity 0·979 and 0·983) respectively. The 1/2c cultures showed 98·1% similarity to melting characteristics with EGD-e, whilst 1/2a isolates had the greatest heterogeneity that was related to inlA, inlB and actA genes. Sequencing of amplicons generating different Tm values from EGD-e confirmed the presence of point mutations. CONCLUSIONS: This method was useful for L. monocytogenes subtyping based on single nucleotide polymorphisms detection through the melting behaviour analysis of main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the effectiveness of HRM in differentiating L. monocytogenes strains with high discriminatory power, thus rendering it useful for epidemiological surveillance.

High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature.

INTRODUCTION: Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of microbial populations, and for effective epidemiological surveillance. The introduction of a novel method based on the analysis of melting temperature of amplified products, known as High Resolution Melting (HRM) since 2002, has found applications in epidemiological studies, either for identification of bacterial species or molecular typing, as well as an extensive and increasing use in many research fields. HRM method is based on the use of saturating third generation dyes, advanced real-time PCR platforms, and bioinformatics tools. OBJECTIVE: To describe, by a comphrehensive review of the literature, the use, application and usefulness of HRM for the genotyping of bacterial pathogens in the context of epidemiological surveillance and public health. MATERIAL AND METHODS: A literature search was carried out during July-August 2016, by consulting the biomedical databases PubMed/Medline, Scopus, EMBASE, and ISI Web of Science without limits. The search strategy was performed according to the following keywords: high resolution melting analysis and bacteria and genotyping or molecular typing. All the articles evaluating the application of HRM for bacterial pathogen genotyping were selected and reviewed, taking into account the objective of each study, the rationale explaining the use of this technology, and the main results obtained in comparison with gold standards and/or alternative methods, when available. RESULTS: HRM method was extensively used for molecular typing of both Gram-positive and Gram-negative bacterial pathogens, representing a versatile genetic tool: a) to evaluate genetic diversity and subtype at species/subspecies level, based also on allele discrimination/identification and mutation screening; b) to recognize phylogenetic groupings (lineage, sublineage, subgroups); c) to identify antimicrobial resistance; d) to detect and screen for mutations related to drug-resistance; e) to discriminate gene isoforms. HRM method showed, in almost all instances, excellent typeability and discriminatory power, with high concordance of typing results obtained with gold standards or comparable methods. Conversely, for the evaluation of genetic determinants associated to antibiotic-resistance or for screening of associated mutations in key gene fragments, the sensitivity and specificity was not optimal, because the targeted amplicons did not encompass all the crucial mutations. CONCLUSIONS: Despite the recent introduction of sequencing-based methods, the HRM method deserves consideration in research fields of infectious diseases, being characterized by low cost, rapidity, flexibility and versatility. However, there are some limitations to HRM assays development, which should be carefully considered. The most common application of HRM for bacterial typing is related to Single Nucleotide Polymorphism (SNP)-based genotyping with the analysis of gene fragments within the multilocus sequence typing (MLST) loci, following an approach termed mini-MLST or Minim typing. Although the resolving power is not totally correspondent to MLST, the Simpson's Index of Diversity provided by HRM method typically >0.95. Furthermore, the cost of this approach is less than MLST, enabling low cost surveillance and rapid response for outbreak control. Hence, the potential of HRM technology can strongly facilitate routine research and diagnostics in the epidemiological studies, as well as advance and streamline the genetic characterization of bacterial pathogens.

[Molecular epidemiology of infectious diseases: analytical methods and results interpretation]. / Epidemiologia molecolare delle malattie infettive:metodi di analisi ed interpretazione dei risultati .

Molecular typing and fingerprinting of microbial pathogens represent an essential tool for the epidemiological surveillance, outbreak detection and control of infectious diseases. Indeed, epidemiological investigation without genotyping data may not provide comprehensive information to allow the most appropriate interventions; despite this consideration, some barriers still hamper the routine application and interpretation of molecular typing data. In this paper, the most important methods currently used for characterization of pathogenic microorganisms for microbial source tracking and for the identification of clonal relationships among different isolates, are described according to their principles, advantages and limitations. Criteria for their evaluation and guidelines for the correct interpretation of results are also proposed. Molecular typing methods can be grouped into four categories based on different methodological principles, which include the characterization of restriction sites in genomic or plasmid DNA; the amplification of specific genetic targets; the restriction enzyme digestion and the subsequent amplification; sequence analysis. Although the development and the extensive use of molecular typing systems have greatly improved the understanding of the infectious diseases epidemiology, the rapid diversification, partial evaluation and lack of comparative data on the methods have raised significant questions about the selection of the most appropriate typing method, as well as difficulties for the lack of consensus about the interpretation of the results and nomenclature used for interpretation. Several criteria should be considered in order to evaluate the intrinsic performance and practical advantages of a typing system. However none of the available genotyping methods fully meets all these requirements. Therefore, the combined use of different approaches may lead to a more precise characterization and discrimination of isolates than a single method, especially if used in a hierarchical manner. The interpretation of the molecular results differs according to the typing system's characteristics: for example in the restriction fragments-based analysis, the divergences or the similarity percentages among the profiles are evaluated, whilst the differences in terms of number and intensity of bands are analyzed in the amplification-based approaches. Moreover, a correct interpretation of molecular results significantly depends by other critical factors, such as the comprehension of the typing system and data quality, the microbial diversity, and the epidemiological context in which the method is used. The analysis of PFGE data, considered as the "gold standard", is based on the differences of the number and position of bands patterns, although recent recommendations are now available from the Centers for Diseases Control and Prevention (CDC) for a more accurate interpretation, which also include the evaluation of the gel quality, the genetic diversity of the microorganism, the time and geographical scale of an epidemic event. Future advances in the molecular typing technologies indeed will provide rapid methodological improvements, such as a greater degree of automation, better resolution, higher throughput, and a greater availability of dedicated bioinformatics tools. These factors will all contribute to an increasing application of genotyping methods to better understand the epidemiology of infectious diseases, and to implement, along with the strengthened international and interdisciplinary partnerships, more effective control and prevention strategies for Public Health improvements.

[Vibrio spp. infections of clinical significance and implication for public health]. / Infezioni da Vibrio spp. di interesse clinico e problematiche di Sanitå Pubblica.

Vibrio spp. infections still are a Public Health concern. Vibrio spp. can be found in marine, estuarine, and freshwater environments, and can be able to cause diseases in fish, shellfish, mammals, as well as in humans. Since '80 to date, the number of species within the genus increased from 21 to more than 100. The most important is Vibrio cholerae, the etiological agent of the cholera, responsible of seven pandemics; serotypes O1 and O139 can produce cholera toxin, while serotypes non-O1/non-O139 are generally associated with sporadic cholera cases and extraintestinal infections. Vibrio parahaemolyticus is an important cause of gastroenteritis associated with contaminated seafood consumption, whereas Vibrio vulnificus and V. alginolyticus can be related to wound infections or seafoodborne primary septicemia in immunocompromised patients. Disease prevention is mainly based on the application of proper individual or collective preventive measures.

Microbiological and toxicological quality of dried herbs.

AIMS: The microbiological and toxicological quality of 51 samples of dried herbs (Melissa officinalis, Salvia officinalis, Malva sylvestris, Matricaria chamomilla, Alchemilla vulgaris and Centaurea cyanus) cultivated in family-managed farms in Molise Region (Italy) was evaluated. METHODS AND RESULTS: All the samples were analysed by using conventional methods, and for samples preparation, an alternative Washing and Shaking (WaS) protocol was developed to reduce release of antimicrobial compounds. None of the samples were of unsatisfactory quality with respect to aflatoxin B1, and only three samples from Malva sylvestris exceeded the limit of total aflatoxins according to Recommendation 2004/24/EC. The International Commission on Microbiological Specifications for Foods limits for mesophilic bacteria and total coliforms were exceeded in the 29.4 and 3.9% of samples, respectively: 7.8% of samples also exceeded the limit for Escherichia coli established by European Spice Association. When the 'WaS' method was used, higher microbial counts were obtained, especially for A. vulgaris, S. officinalis and M. officinalis. CONCLUSIONS: Herbs cultivated in family-managed small agricultural areas showed a good microbiological and toxicological quality, irrespectively of preliminary washing or selection procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: Herb matrices may contain antimicrobial activity which should be considered when applying the conventional microbiological methods for sample preparation. Alternative preparation protocols may have advantages to reduce antimicrobial effects and should be further evaluated.

Typing of Listeria monocytogenes strains isolated in Italy by inlA gene characterization and evaluation of a new cost-effective approach to antisera selection for serotyping.

AIMS: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost-effective methodological approach based on preliminary PCRs screening was proposed. METHODS AND RESULTS: The isolates were analysed by conventional serotyping, multiplex-PCRs for serogroup and lineage identification and PCR-RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58.10%), II (22.85%), III (12.38%) and IV (6.67%). Among clinical strains, lineage I was more represented (68.75%) than lineage II; whereas, lineage II was more associated with food (90.24%) and environmental (85.72%) isolates. Most of food (89.02%) and environmental (85.71%) isolates were classified into truncated InlA profiles, whereas the 93.75% of clinical strains were associated with a complete form of the protein. CONCLUSION: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs-based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost-effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.

Alloantibodies and outcomes of deceased donor kidney allografts.

Analysis of the anti-HLA antibody status of 100 recipients of kidneys from deceased donors demonstrated that presensitization and the development of alloantibodies after transplantation are associated with the development of antibody mediated as well as cellular rejection. This finding indicates that the humoral arm of the immune response is also involved in cell-mediated rejection and/or that there may be a continuum between these two forms of rejection. Most episodes of rejection were successfully reversed in our population, as shown by the overall 3-year actuarial survival of 98% in nonsensitized and 91% in sensitized recipients, emphasizing the importance of comprehensive antibody studies.

HLA antigens in alkaptonuric patients.

BACKGROUND: In alkaptonuric patients a disabling ochronotic arthropathy develops, due to the deposit of a pigmented polymer of homogentisic acid. Since in inherited diseases the clinical expressions may be multifactorial, involving genetic and environmental factors, where the HLA system may play a role, we studied HLA antigens in ochronotic patients. METHODS: The study was carried out in 21 members of three families of six ochronotic patients and in two isolated ochronotic patients. The HLA typing has been done testing for antigens from loci A, B and C, by international standard microlymphocytotoxicity method, and for loci DR and DQ, by fluorescence method on immunologically isolated cells by means of antibody-coated microspheres. The chi square test was used for statistical analysis, with Yates correction due to the low number of observations. RESULTS: Despite the limited number of subjects, due to the rarity of the disease, a significantly higher prevalence of HLA-DR7 antigen was found in the alkaptonuric patients when compared to a general population (p<0.02), suggesting a possible association, while the prevalence of HLA A, B, C and DQ showed no significant differences. CONCLUSIONS: It might play a role in the pathophysiology and in the clinical expression of the disease.

Effect of retinoids on follicular cells.

It has been demonstrated that topical application of all-trans retinoic acid and other retinoids can alter the hair-growth cycle in the C3H mouse model. The anagen phase is prolonged and the telogen phase is shortened. This effect is similar to the effect of minoxidil on the hair-cycle dynamics in this animal model. The levels of cellular retinoic acid binding protein measured by radioreceptor assay in whole skin of C3H mice were higher during anagen and lower during telogen. Topical application of certain retinoids caused elevated levels of cellular retinoic acid-binding protein (cRABP) in the whole skin homogenates during both phases of the cycle. Of the retinoids tested, those most effective in altering the levels of cRABP in the skin of the mice were also capable of significantly altering the hair-cycle dynamics. There appeared to be a relationship between the ability of retinoid to increase cRABP, increase 3H-thymidine incorporation, and alter the dynamics of the hair cycle. Only cRABP-II is detectable in human cultured dermal fibroblasts and dermal papilla cells. Dermal fibroblasts showed higher amounts of cRABP-II as compared to dermal papilla cells. The difference in cRABP-II expression might explain a distinct response to RA by these two cell populations. Whether the difference in expression of cRABP-II might be of physiologic importance remains to be determined. Treatment of human dermal papilla cells in culture with retinoic acid does not appear to affect proliferation, at least at the doses tested.