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Culture-dependent and culture-independent microbiological methods are two approaches used to study microbial community composition. Culture-dependent methods have been the standard method used for many years but have limited utility with unculturable microorganisms. Culture-independent methods, including molecular techniques, enable direct analysis of microbial DNA without requiring cultivation. Both culture-dependent and -independent methods have roles in advancing our understanding of microbiology, and a combination of these approaches often yields a comprehensive depiction of the microbial diversity within a dynamic system. Bacterial activity reaction tests (BARTs) are a common culture-dependent test used to identify bacteria growing in industrial water samples. In this study, next-generation sequencing (NGS) was used to identify the taxa growing in BARTs and compared with the BART reaction patterns. Additionally, several water samples were analyzed by both BART and NGS analysis to determine whether the bacteria found in the water were also present in the BARTs. The results showed overall agreement between NGS and BARTs, though, in some cases, the most abundant taxa found in the water samples differed from those in the BARTs. This highlights the need for further study into the microbial community dynamics of culture-dependent tests to determine whether they are representative of the original sample.

ABSTRACT Intestinal epithelial cells represent the first line of defense from invading enteric pathogens. During the course of infection, pro-inflammatory programmed cell death is an effective way to eliminate invading microbes and to create a localized inflammatory environment. On the other hand, pathogens evolved countless strategies to overcome cell death and to keep the host alive ensuring their spread. It was previously shown that Shigella flexneri apyrase interacts with OmpA to contribute to a proper polar exposition of IcsA, which mediates actin-based motility. However, apyrase is also an ATP-diphosphohydrolase whose catalytic activity function has not been elucidated yet. Herein, we demonstrated that apyrase contributes to the manipulation of host cell fate by S. flexneri since it is released within the host cell cytoplasm during infection to degrade intracellular ATP. Thus, apyrase contributes to prevent caspase-1 activation, thereby downregulating the activation of pyroptosis in infected cells. Overall, apyrase is involved in the modulation of host cell survival and dampens the inflammatory response. IMPORTANCE In this paper, we demonstrated that apyrase is released within the host cell cytoplasm during infection to target the intracellular ATP pool. By degrading intracellular ATP, apyrase contributes to prevent caspases activation, thereby inhibiting the activation of pyroptosis in infected cells. Our results show, for the first time, that apyrase is involved in the modulation of host cell survival, thereby aiding this pathogen to dampen the inflammatory response. This work adds a further piece to the puzzle of Shigella pathogenesis. Due to its increased spread worldwide, prevention and controlling strategies are urgently needed. Overall, this study highlighted apyrase as a suitable target for an anti-virulence therapy to tackle this pathogen.

Vancomycin is commonly used as a first line therapy for gram positive organisms such as methicillin resistant Staphylococcusaureus. Vancomycin-induced acute kidney injury (V-AKI) has been reported in up to 43% of patients, especially in those with higher targeted trough concentrations. The precise mechanism of injury in humans remains elusive, with recent evidence directed towards proximal tubule cell apoptosis. In this study, we investigated the protein contents of urinary exosomes in patients with V-AKI to further elucidate biomarkers of mechanisms of injury and potential responses. Urine samples from patients with V-AKI who were enrolled in the DIRECT study and matched healthy controls from the UAB-UCSD O'Brien Center Biorepository were included in the analysis. Exosomes were extracted using solvent exclusion principle and polyethylene glycol induced precipitation. Protein identity and quantification was determined by label-free liquid chromatography mass spectrometry (LC/MS). The mean peak serum creatinine was 3.7 ± 1.4 mg/dL and time to kidney injury was 4.0 ± 3.0 days. At discharge, 90% of patients demonstrated partial recovery; 33% experienced full recovery by day 28. Proteomic analyses on five V-AKI and 7 control samples revealed 2009 proteins in all samples and 251 proteins significantly associated with V-AKI (Pi-score > 1). The top discriminatory proteins were complement C3, complement C4, galectin-3-binding protein, fibrinogen, alpha-2 macroglobulin, immunoglobulin heavy constant mu and serotransferrin. Urinary exosomes reveal up-regulation of inflammatory proteins after nephrotoxic injury in V-AKI. Further studies are necessary in a large patient sample to confirm these findings for elucidation of pathophysiologic mechanisms and validation of potential injury biomarkers.

Introdução: O aumento de casos de malária, mesmo com o desenvolvimento de tratamentos e testes diagnósticos rápidos, apresenta-se como um grande desafio para a saúde global. A malária é uma parasitose transmitida por mosquitos do gênero Anopheles, geralmente silvestres e usualmente encontrados em água parada e límpida. Alterações no ambiente podem forçar o mosquito a conviver com o ser humano, aumentando a incidência de malária. Estas alterações incluem o desmatamento, que sofreu aumento expressivo no Brasil entre o ano de 2011 e 2021. Como visto, a malária é uma doença com forte influência na saúde humana, ambiental e animal, tornando a abordagem de One Health (Saúde Única) ideal para este estudo. Portanto, nossos objetivos são acompanhar e comparar a evolução da incidência anual de casos de malária e a variação do desmatamento em municípios da Amazônia Legal entre 2011 e 2021. Metodologia: Os dados sobre casos de malária no Brasil foram obtidos nos boletins epidemiológicos da Secretaria de Vigilância em Saúde, para cálculo da incidência de malária em cada local e período, a população de cada município foi obtida através das estimativas do Censo demográfico do Brasil de 2010 do Instituto Brasileiro de Geografia e Estatística (IBGE). Dados sobre o desmatamento foram coletados do Instituto Nacional de Pesquisas Espaciais (INPE), na plataforma TerraBrasilis. A partir da incidência da malária anual por município e dos números do desmatamento nos locais estudados, foi ajustado um modelo linear misto generalizado (GLMM) com efeitos fixos para o ano e a área desmatada e interceptos aleatórios para os municípios. O modelo foi ajustado com o pacote “GLMMadaptive” na versão 4.3.0 do software R. A variável dependente foi a incidência de malária e a distribuição usada foi a binomial negativa inflacionada de zeros. Foi realizado um teste de Kolmogorov-Smirnov para verificar a normalidade dos resíduos. Resultados: Observou-se que quanto maior a área desmatada no município maior é a incidência de malária. O coeficiente indica que, em média, um aumento de um desvio padrão na área desmatada está associado a um aumento de 0,1595 na incidência de malária (em escala logarítmica). Essa associação é estatisticamente significativa (p = 0,0164). Conclusões: Apesar da relação positiva, os resíduos não são gaussianos, podendo indicar outras variáveis que influenciam na incidência de malária dentro deste modelo, o próximo passo é investigar estas variáveis e refinar o modelo usado.

Introdução/objetivo: A expansão do tratamento preventivo da tuberculose (TPT) é estratégica na meta de eliminação da doença como problema de saúde pública até 2035. Este estudo avaliou a cascata de cuidado da infecção tuberculosa latente (ILTB) em pacientes transplantados de órgãos sólidos e transplante de células pluripotentes hematopoiéticas (TCPH) em um hospital de referência. Métodos: Estudo descritivo, transversal, epidemiológico operacional; foram incluídos pacientes candidatos/transplantados notificados para ILTB no período de janeiro de 2009 a julho de 2022. Resultados: Considerando os pacientes transplantados no período de estudo foram notificados 5,18% casos de ILTB; dentre os transplantados hepáticos (TxH), 7,03%, nos transplantados renais (TxR), 6,3%, nos TCPH, 0,95% e nenhum no transplante cardíaco. Foram avaliados 194 pacientes com ILTB: 74 candidatos à transplante hepático (TxH), 114 candidatos a transplante renal (TxR) e seis candidatos a TCPH. O diagnóstico de ILTB foi realizado por meio do teste tuberculínico em 84,02% dos pacientes, nos demais foi embasado na história clínica e exame radiológico. O regime terapêutico consistiu em isoniazida (6-9H) em 97,42%, rifampicina (4R) em 1,55% e rifapentina associada a isoniazida (3HP) em um caso. O tratamento foi concluído em 82,47% deles; 3,1% dos pacientes tiveram o tratamento suspenso por toxicidade. Após o tratamento para ILTB, apenas um paciente desenvolveu TB ativa. Conclusão: No grupo avaliado foram detectadas lacunas na cascata de cuidado associadas ao diagnóstico da ILTB, suprimento irregular do PPD, ausência de método alternativo e pactuação das diretrizes para a garantia da TPT de forma precoce em grupos com alto risco de adoecimento. O regime com 6-9H foi efetivo e seguro, poucos pacientes utilizaram os regimes 4R e 3HP.

Summary: In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1–C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

Background and Aims: Antimicrobial susceptibility testing of anaerobic clinical isolates is of paramount importance for patient therapy and resistance monitoring. In our laboratory the MIC gradient Etest method and broth microdilution with Sensititre trays are used for susceptibility testing of anaerobes and the aim of this study was to compare the two methods on a panel of anaerobes routinely isolated from patients in the province of Bolzano, Italy. Materials and Methods: Totally, 74 non-repetitive Gram-positive and Gram-negative patient isolates were tested with Etest strips on Fastidious Anaerobe Agar (F.A.A.) and with Sensititre trays, according to manufacturer’s instructions. Interpretation of MICs was by EUCAST or CLSI criteria, resistance percentages were calculated and Categorical Agreement (CA) and Essential Agreement (EA) between the two methods were determined. Results: Of the 74 isolates, 68 (91.9%) grew on both systems and agreement for these was compared in the study. CA for all isolates was ≥90% for all tested antibiotics except moxifloxacin, whereas EA was generally lower. Resistance was generally low, except for clindamycin in all isolates and tigecycline in Gram-negatives. In our study Etest was a superior and more handy method. Conclusions: To conclude, we believe the Etest method is more suitable for routine diagnostic laboratory usage. Nevertheless, multicenter studies are required to evaluate the two methods for anaerobic susceptibility testing.

The existing literature supports the anti-inflammatory, antioxidant, and antiviral capacities of the polyphenol extracts derived from Geranium sanguineum L. These extracts exhibit potential in hindering viral replication by inhibiting enzymes like DNA polymerase and reverse transcriptase. The antiviral properties of G. sanguineum L. seem to complement its immunomodulatory effects, contributing to infection resolution. While preclinical studies on G. sanguineum L. suggest its potential effectiveness against COVID-19, there is still a lack of clinical evidence. Therefore, the polyphenols extracted from this herb warrant further investigation as a potential alternative for preventing and treating COVID-19 infections.

Besides representing one of the most relevant threats of fungal origin to human and animal health, the genus Aspergillus includes opportunistic pathogens which may infect bees (Hymenoptera, Apoidea) in all developmental stages. At least 30 different species of Aspergillus have been isolated from managed and wild bees. Some efficient behavioral responses (e.g., diseased brood removal) exerted by bees negatively affect the chance to diagnose the pathology, and may contribute to the underestimation of aspergillosis importance in beekeeping. On the other hand, bee immune responses may be affected by biotic and abiotic stresses and suffer from the loose co-evolutionary relationships with Aspergillus pathogenic strains. However, if not pathogenic, these hive mycobiota components can prove to be beneficial to bees, by affecting the interaction with other pathogens and parasites and by detoxifying xenobiotics. The pathogenic aptitude of Aspergillus spp. likely derives from the combined action of toxins and hydrolytic enzymes, whose effects on bees have been largely overlooked until recently. Variation in the production of these virulence factors has been observed among strains, even belonging to the same species. Toxigenic and non-toxigenic strains/species may co-exist in a homeostatic equilibrium which is susceptible to be perturbed by several external factors, leading to mutualistic/antagonistic switch in the relationships between Aspergillus and bees.

Fungi in the genus Talaromyces occur in every environment in both terrestrial and marine contexts, where they have been quite frequently found in association with plants and animals. The relationships of symbiotic fungi with their hosts are often mediated by bioactive secondary metabolites, and Talaromyces species represent a prolific source of these compounds. This review highlights the biosynthetic potential of marine-derived Talaromyces strains, using accounts from the literature published since 2016. Over 500 secondary metabolites were extracted from axenic cultures of these isolates and about 45% of them were identified as new products, representing a various assortment of chemical classes such as alkaloids, meroterpenoids, isocoumarins, anthraquinones, xanthones, phenalenones, benzofurans, azaphilones, and other polyketides. This impressive chemodiversity and the broad range of biological properties that have been disclosed in preliminary assays qualify these fungi as a valuable source of products to be exploited for manifold biotechnological applications.

Mature neurotrophic factors and their propeptides play key roles ranging from the regulation of neuronal growth and differentiation to prominent participation in neuronal survival and recovery after injury. Their signaling pathways sculpture neuronal circuits during brain development and regulate adaptive neuroplasticity. In addition, neurotrophic factors provide trophic support for damaged neurons, giving them a greater capacity to survive and maintain their potential to regenerate their axons. Therefore, the modulation of these factors can be a valuable target for treating or preventing neurologic disorders and age-dependent cognitive decline. Neuroregenerative medicine can take great advantage by the deepening of our knowledge on the molecular mechanisms underlying the properties of neurotrophic factors. It is indeed an intriguing topic that a significant interplay between neurotrophic factors and various metals can modulate the outcome of neuronal recovery. This review is particularly focused on the roles of GDNF, BDNF and NGF in motoneuron survival and recovery from injuries and evaluates the therapeutic potential of various neurotrophic factors in neuronal regeneration. The key role of metal homeostasis/dyshomeostasis and metal interaction with neurotrophic factors on neuronal pathophysiology is also highlighted as a novel mechanism and potential target for neuronal recovery. The progress in mechanistic studies in the field of neurotrophic factor-mediated neuroprotection and neural regeneration, aiming at a complete understanding of integrated pathways, offers possibilities for the development of novel neuroregenerative therapeutic approaches.

Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-β bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

Precision medicine refers to the tailoring of therapeutic strategies to the individual characteristics of each patient; thus, it could be a new approach for the management of severe asthma that considers individual variability in genes, environmental exposure, and lifestyle. Precision medicine would also assist physicians in choosing the right treatment, the best timing of administration, consequently trying to maximize drug efficacy, and, possibly, reducing adverse events. Metabolomics is the systematic study of low molecular weight (bio)chemicals in a given biological system and offers a powerful approach to biomarker discovery and elucidating disease mechanisms. In this point of view, metabolomics could play a key role in targeting precision medicine.

Background: Progressive multifocal leukoencephalopathy (PML) caused by the JC virus is the main limitation to the use of disease modifying therapies for treatment of multiple sclerosis (MS). Methods: To assess the PML risk in course of ocrelizumab, urine and blood samples were collected from 42 MS patients at baseline (T0), at 6 (T2) and 12 months (T4) from the beginning of therapy. After JCPyV-DNA extraction, a quantitative-PCR (Q-PCR) was performed. Moreover, assessment of JCV-serostatus was obtained and arrangements’ analysis of non-coding control region (NCCR) and of viral capsid protein 1 (VP1) was carried out. Results: Q-PCR revealed JCPyV-DNA in urine at all selected time points, while JCPyV-DNA was detected in plasma at T4. From T0 to T4, JC viral load in urine was detected, increased in two logarithms and, significantly higher, compared to viremia. NCCR from urine was archetypal. Plasmatic NCCR displayed deletion, duplication, and point mutations. VP1 showed the S269F substitution involving the receptor-binding region. Anti-JCV index and IgM titer were found to statistically decrease during ocrelizumab treatment. Conclusions: Ocrelizumab in JCPyV-DNA positive patients is safe and did not determine PML cases. Combined monitoring of ocrelizumab’s effects on JCPyV pathogenicity and on host immunity might offer a complete insight towards predicting PML risk.

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Marine-derived fungi are an important source of many valuable compounds with original structures and diverse physico-chemical properties. In this work, the metabolomic profile of a strain of Penicillium brevicompactum, recovered from a snakelocks sea anemone (Anemonia sulcata), was investigated through the parallel application of LC-ESI-HRMS, GC-MS, and NMR. Our strategy allowed the identification of mycophenolic acid, brevianamide A, and several compounds belonging to the thiosilvatins. Among the latter, five products are reported for the first time in this species. The main product of this series, cis-bis(methylthio)silvatin, was also tested for antiproliferative activity on both cancer and non-tumoral colon cell lines.

Background and aim. Despite the availability of a vaccine,measles continues to be endemic in Italy, where an increase of cases was reported during 2016. This study describes the measles outbreaks in Emilia-Romagna Region (ERR), one of the Italian regions mostly affected. Materials and Methods. A total of 101 suspected cases were reported in ERR during 2016. Laboratory diagnosis by serological and/or molecular methods was performed on 142 specimens (78 urine, 19 oral fluid and 45 sera) related to 97 suspected cases. For positive cases, measles virus (MV) strains involved were identified. Results. Among 101 suspected cases, 72 (71.3%) were confirmed. Vaccination status was known for 61 (84.7%) cases, of which 56 (91.8%) were unvaccinated. The highest incidence was found in the age group 15-39 years. In addition, for the 34.7% (25/72) of confirmed cases, the transmission occurred in nosocomial settings, where healthcare workers were involved (60% of cases). Roma/Sinti population were also involved in 12.5% (9/72)or confirmed cases. Both groups are considered hard-to-reach for immunization. The phylogenetic analysis showed circulation of MV strains belonging to genotype B3 and D8 in 45 (80.4%) and 11 cases (19.6%), respectively. In 94.7% of cases, the measles endemic transmission was demonstrated. Conclusions. This data obtained through active surveillance showed the endemic transmission of MV within a population with immunity gaps including healthcare workers (20.8% of confirmed cases), among which the spread of two endemic MV strains was observed.

Human cases of West Nile virus (WNV) disease have been reported in Italy since 2008. So far, most cases have been identified in north-eastern Italy, where, in 2012, the largest outbreak of WNV infection ever recorded in Italy occurred. Most cases of the 2012 outbreak were identified in the Veneto region, where a special surveillance plan for West Nile fever was in place. In this outbreak, 25 cases of West Nile neuroinvasive disease and 17 cases of fever were confirmed. In addition, 14 WNV RNA-positive blood donors were identified by screening of blood and organ donations and two cases of asymptomatic infection were diagnosed by active surveillance of subjects at risk of WNV exposure. Two cases of death due to WNND were reported. Molecular testing demonstrated the presence of WNV lineage 1 in all WNV RNA-positive patients and, in 15 cases, infection by the novel Livenza strain was ascertained. Surveillance in other Italian regions notified one case of neuroinvasive disease in the south of Italy and two cases in Sardinia. Integrated surveillance for WNV infection remains a public health priority in Italy and vector control activities have been strengthened in areas of WNV circulation.

Background and aims: Blood culture (BC) results are essential to guide antimicrobial chemotherapy for patients with sepsis. However, BC is a time-consuming exam, which can take several days. Reducing BCs turn around time (TAT) could impact on multiple outcome parameters and TAT monitoring is an important tool for measurement of microbiology laboratory performance. The aim of this study was to provide an overview of BC TATs among Italian microbiology laboratories. Materials and methods: Five laboratories collected and recorded, for a month period, date and time of the BC processing events. Cumulative TATs were analysed using the GraphPad software. Results: Participating laboratories reported data from 302 sepsis episodes. The median time from when the BC system produced a positive signal until Gram-stain results were reported was 7.6 hours. A rapid molecular identification and antimicrobial susceptibility testing (AST) was performed in 26.5% of BCs. Mean TAT for identification report was significantly lower when a molecular approach was adopted (12 vs. 28.7 hours, PConclusions: BC is a time-consuming exam, however, molecular identification and AST methods can drastically reduce time to results. The lack of difference between TATs observed for laboratories working 7 days/week and 6 days/week, coupled with a high rate of BCs turning positive during the night enable to conclude that the most urgent measure to reduce TATs is the expansion of laboratory regular duty hours.

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.

Sepsis is a serious clinical condition, associated with high mortality despite advanced modern medical treatment. Traditionally, the detection and identification of bacteria and fungi circulating in the blood-stream is based on blood cultures. A number of factors influence the yield of blood culture, most of them concerning the microbiologist skill and the laboratory organization. In order to collect information about the practices and procedures used for the detection of microrganisms in blood cultures in the italian laboratory (lab), an e-mail with the invitation to participate in the survey was sent to 2000 members of the Italian Association of Clinical Microbiology. Responses were received from 100 lab, located from all over the country (in 18/20 italian regions). The results presented hereby concern specimen collection, culture techniques, rapid identification and susceptibility testing, laboratory organization, relationships with physicians. In summary, most lab use automated systems (96%), the bottles are incubated immediately during public holidays in 72/96 lab (75%) and in 49/97 lab at night (50.5%), the lenght of incubation was 5 or 7 days in 93% of the lab, although it is common to extend the incubation period when brucellosis (74 lab), endocarditis (49 lab), systemic mycosis (33 lab) is suspected. A wide variety of media are employed for subcultures. All lab process the positive bottles at least once a day, while only in 42 of 81 (51.9%) lab the positive blood are processed on holiday. Communication between clinicians and microbiologist include: distribution of specimen collection guidelines (96/100 lab), availability to microbiologist of patients’ clinical situation (77/96 lab, 80.2%), and adding to report the microbiologist’ suggestion (75/98 lab, 76.5%). The results, compared with those collected with a similar questionnaire in 2001, show a greater adherence to guidelines: the number of bottles examined by lab yearly is almost doubled, the length of incubation is shortened to 5 days in 42% (vs 9.2% in 2001), direct susceptibility tests seem to be performed more frequently (in 29% of lab vs18.6% in 2001, mostly in larger hospitals), more lab process positive bottles on Sunday, cooperation with clinicians is improved.

Background:Tuberculosis (TB) represents a major health problem both in developing and both in industrialized countries.The identification of individuals latently infected with Mycobacterium tuberculosis (Mtb) play a key role for the efficacy of TB control. These individuals with a latent tuberculosis infection (LTBI), especially those with high risk of reactivation (e.g. HIV + / AIDS-infected individuals, patients undergoing immunosuppressive therapy and children younger than 5 years) could benefit from a preventive treatment with isoniazid reducing the risk of progression from LTBI to active TB. Until recently, detection of LTBI has relied on the tuberculin skin test (TST), but despite the widespread use in clinical practice,TST does not reliably diagnose LTBI because several drawbacks, e.g. lacking in specificity, particularly in who were exposed to non-tuberculous mycobacteria (NTM) or were vaccinated with Bacille Calmette-Guerin (BCG) In addition, in young subjects,TST sensitivity is hampered by impaired T cell function leading frequently to false negative results.These several drawbacks limit the use of TST for the diagnose an LTBI in patients who may benefit from preventive chemotherapy. On the other hand, an accurate diagnosis of LTBI avoid the over-treatment of those patients with a positive TST results but not latently infected with Mtb. Recently, new tests based on the detection of interferon-gamma (IFN-γ) after stimulation with Mtb-specific antigens: Early secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10) have been proposed for the diagnosis of active TB and LTBI. Methods: During the period from January 2009 to June 2009, in our laboratory 70 patients were tested with T-SPOT.TB (Oxford Immunotech, Abingdon, United Kingdom).We enrolled transplant patients and subjects ongoing transplant, patients immigrants from high prevalence TB countries, patients screened for immunosuppressive treatment, HIV / AIDS – infected individuals.We also tested 3 patients with clinical / radiological suspicion of active TB and 3 patients with positive tuberculin skin test and with a positive direct examination for mycobacteria in the urinary sediment. Results: In 2 patients with symptoms suggestive of TB in place,T-SPOT.TB showed a higher response of (IFN-g), more than 100 spots.Among individuals ongoing renal transplant, 6 patients tested T-SPOT.TB positive and 4 subjects were T.SPOT.TB -negative. Two patients with an autoimmune disease showed an high response to Mtb-specific antigens with T-SPOT.TB test tested before to start any treatment.T-SPOT.TB test tested strongly negative in 4 paediatric patients and in one HIV-infected individuals, regardless a positive response to a internal positive response (phytohaemagglutinin (PHA), suggesting a normal immune response. Conclusions:This preliminary data suggest that the T.SPOT.TB showed high sensitivity and specificity, producing a strongly negative response to Mtb-specific antigens in subjects who had a history of previous BCG-vaccination. In addition, T-SPOT.TB test provides, unlike the TST, indication about the potential immunosuppression of tested patient with an internal positive control that can highlight the production of IFN- γ by lymphocytes resulting in the application of this test in immunocompromised patients, e.g. children and transplantated patients and others.

Introduction. The bacteriology processes almost all the clinical specimens through manually performed cultures and therefore needs to evolve toward automated systems.We report our experience on the benefits of combination between liquid-based flocked swab transport systems (Copan Innovation) and platforms of plating and automatic streaking of agar plates.The main innovation is that, for the first time, the automation can be performed not only for urine and other few liquids materials, but also to most of other biological samples. Methods. Our considerations are based on more than one year of experience on the instrument InocuLAB® (Dynacon Inc.) in two Italian Hospitals, supplemented by theoretical knowledge of other marketed in Italy instruments.The three instruments currently available in Italy (A: InocuLAB® Copan, B:WASP® Copan and C: Previ-Isola bioMérieux®) can inoculate and streak liquid or eluated specimens from liquidbased flocked swab transport systems on any agar plate.The main differences are: streaking of already deposited material on plate (only A and B); choice among various protocols of streak and inoculate (only A and B); automatic opening and recapping of the specimen containers (only A and B); automatic choice of the plates (B: 9 columns and C: 5 columns); partition of the streaked plates in groups (B: up to 4 group s and C: up to 3); forced use of disposables devices (only instrument C). No instrument present problems of carryover and the quality of plated specimens is excellent. Conclusions. Although significantly different, all the platforms combined with flocked swabs represent a pre-analytical workstation for microbiology automation (inoculating, streaking, and bar-coding of liquid specimens). The main advantages can be seen in the operator’s production time, in safety and in quality of streak.The almost complete elution of the biological sample in liquid-based flocked swab transport systems is a standard base which allows to perform many diagnostic tests, including quantitative streaking for quantitative evaluation and multiple culture for additional investigation.

A multicenter retrospective survey of funguria was run at 6 Italian hospitals (Bergamo,Novara,Varese, Florence, Ancona, Pescara) from January 1, 2001 to December 31, 2002. The aim of the study was to evaluate the incidence of recovery of yeasts from urine cultures, the distribution among the hospital wards, the involved species and the number of patients with concurrent fungemia and funguria. Microorganisms (either bacteria or yeasts) were isolated from the 21% of urine cultures: overall, 2% of them were positive for yeasts, whereas 19% for bacteria.Yeasts were recovered from the 8% of the positive urine cultures. Yeasts in the urine were mostly observed in Intensive Care Units (24% of positive urine cultures), and less frequently in Medical and Surgical wards. Candida albicans was the most frequently recovered species (63%), followed by C. glabrata (18%), C. tropicalis (9%), C. parapsilosis (3%); other Candida species, Trichosporon asahii and Saccharomyces cerevisiae were occasionally isolated, whereas moulds were never recovered. Overall, 5% of patients (55/1119) with funguria had concurrent fungemia and in 41 cases the same species was recovered from both urine and blood. C. albicans was the most frequently recovered species, followed by C. glabrata, C. tropicalis and C. parapsilosis.

Sepsis is an important cause of morbidity and mortality; Blood cultures are the standard for identifying the responsible pathogen, bacteria or fungi.A number of factors influence the yield of blood culture, most of them concerning the microbiolist skill and the laboratory organization. In order to collect information about the practices and procedures used for the detection of microrganisms in blood cultures in the italian laboratory, a questionnaire was sent to all the 2000 members of the Italian Association of Clinical Microbiology. Responses were received from 110 laboratories, located from all over the country (2.028.581 hospital admission).The results presented hereby concern specimen collection, culture techniques, rapid identification and susceptibility testing. In summary, most laboratories use automated systems (83.6%), the lenght of incubation was 7 days in two out of three laboratories, although it is common to extend the incubation period when brucellosis (83 lab), endocarditis (47 lab), systemic mycosis (27 lab) is suspected.A wide variety of media are employed for subcultures. All laboratories examine the bottles at least once a day, while only 32 of 95 (33.7%) laboratories processe the positive blood cultures on holiday. Communication between clinicians and microbiologist include: distribution of specimen collection guidelines by 93 (84.5%) laboratories, availability of patients’ clinical situation in 35 (32.4%) laboratories, and adding to report the suggestion of potentialy contaminated culture (i.e.“a positive results does not necessarly indicate bacteremias”) in 31 (28.4%). Only laboratories perform direct, tests 18.6% antimicrobial susceptibility test, and 9.3% perform rapid direct identification.

Abstract Background Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains. Results All S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness. Conclusions The main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Abstract Background In a recent multi-centre Italian survey (2003–2004), conducted in 45 laboratories throughout Italy with the aim of monitoring microorganisms responsible for severe infections and their antibiotic resistance, Acinetobacter baumannii was isolated from various wards of 9 hospitals as one of the most frequent pathogens. One hundred and seven clinically significant strains of A. baumannii isolates were included in this study to determine the in vitro activity of tigecycline and comparator agents. Methods Tests for the susceptibility to antibiotics were performed by the broth microdilution method as recommended by CLSI guidelines. The following antibiotics were tested: aztreonam, piperacillin/tazobactam, ampicillin/sulbactam, ceftazidime, cefepime, imipenem, meropenem tetracycline, doxycycline, tigecycline, gentamicin, amikacin, ciprofloxacin, colistin, and trimethoprim/sulphametoxazole. The PCR assay was used to determine the presence of OXA, VIM, or IMP genes in the carbapenem resistant strains. Results A. baumannii showed widespread resistance to ceftazidime, ciprofloxacin and aztreonam in more than 90% of the strains; resistance to imipenem and meropenem was 50 and 59% respectively, amikacin and gentamicin were both active against about 30% of the strains and colistin about 99%, with only one strain resistant. By comparison with tetracyclines, tigecycline and doxycycline showed a higher activity. In particular, tigecycline showed a MIC90 value of 2 mg/L and our strains displayed a unimodal distribution of susceptibility being indistinctly active against carbapenem-susceptible and resistant strains, these latter possessed OXA-type variant enzymes. Conclusion In conclusion, tigecycline had a good activity against the MDR A. baumannii strains while maintaining the same MIC90 of 2 mg/L against the carbapenem-resistant strains.