Manuela Tamburro

Prevalence of virulence-associated genes and cytolethal distending toxin production in Campylobacter spp. isolated in Italy.

The prevalence of virulence and cytolethal distending toxin (CDT) genes and the cytotoxic activity in Vero and HEp-2 cells was estimated in 29 Campylobacter jejuni and 36 Campylobacter coli from foods, animals and humans isolates. All C. jejuni showed flaA, cadF, cdtA, cdtB, cdtC and cdt cluster genes fragments, except for ceuE (86.2%) and cdt genes (93.1%). Amongst C. coli strains, a lower prevalence of ceuE gene (83.3%) was detected than that for cdtA, cdtB, cdtC genes (97.2%), cdt gene cluster (94.4%) and cdt genes (86.1%); whereas flaA and cadF genes were amplified in all isolates. Despite the high prevalence of CDT genes only 8 (27.6%) C. jejuni and 1 (2.8%) C. coli showed evidence for cytotoxin production in HEp-2 cells. However, how CDT positive and CDT negative strains differ in their biological properties remains unknown, but the relative higher prevalence of cytotoxicity in C. jejuni could be consistent with its predominant epidemiological role in human infections.

Prevalence and biomolecular characterization of Campylobacter spp. isolated from retail meat.

We estimated the prevalence of Campylobacter spp. in retail meat (n = 352 samples; 104 chicken, 106 pork, and 142 beef) collected in Campobasso, Italy, comparing two microbiological methods. All the isolates were characterized by biomolecular techniques for epidemiological purposes. Campylobacter isolation was performed by selective culture and membrane filtration methods. Phenotypic and genotypic methods for genus and species identification were evaluated together with antimicrobial resistance and plasmid profiling. Sixty-nine (86.2%) samples were positive by selective culture, 49 (61.2%) by membrane filtration, and 38 (47.5%) by both methods. Only 74 of 80 strains were confirmed as Campylobacter spp. by PCR, and two Campylobacter coli were identified as Campylobacter jejuni. Chicken meat was more frequently contaminated than other meats. Selective culture was more sensitive than membrane filtration (85 versus 66%), and specificity of the methods was 98 and 100%, respectively. Among Campylobacter isolates from chicken meat, 86.5% were multidrug resistant. Resistance to ciprofloxacin (51.3%) and enrofloxacin (52.7%) was lower than to nalidixic acid (71.6%). C. coli strains showed the highest cross-resistance for quinolones (82.6%) and fluoroquinolones (60.9%) as well as a high resistance to tetracycline. Plasmids were isolated from six C. coli and two C. jejuni isolates, but no association was detected between antimicrobial resistance and plasmid DNA carriage. Selective culture is considered as the optimal method for Campylobacter isolation, although it was unable to detect all contaminated samples. Membrane filtration provided more specific results but with low sensitivity. A combination of both techniques may offer better results.

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.

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.

Prevalence and genotype identification of human papillomavirus in women undergoing voluntary cervical cancer screening in Molise, Central Italy

We examined the prevalence of HR- and LR-HPV by Linear Array genotyping test in 299 women aged 18-63 years who consecutively visited Molise Region main hospitals for routine Pap smear between February and August 2008. Ninety women were positive for any HPV (30.1%), and 66 for any HR-HPV (22.1%). The most prevalent HR-HPV types were HPV 16 (22.2% of all women with HPV infection), HPV 53 (14.4%), and HPV 66 (14.4%). HPV infections increased from 15.8% in the 18-20 years group to 50.0% in the 21-23 years group and then decreased to 9.1% in those aged 50 years or more (p=0.008). Multiple HPV infections were observed in 15.7% of the study sample (52.2% of all HPV positive). There is a significantly higher prevalence of multiple infections in 18-32 years group women (24.5%) compared with females aged 33 years or more (6.8%) (p<0.005). Current smokers were at increased risk of HPV infection (44.2% of HPV infections compared with 23.5% in never smokers, and 25.3% of multiple HPV infections compared with 11.3%; p=0.001). HR-HPV infections were higher in women never been pregnant (27.1% compared with 7.7%; p=0.001). Oral contraceptive use was completely unrelated to infection. Among the 122 women who had both cytological examination and HPV results, multiple HR-HPV types were found in 36.8% of those with abnormal cervical findings, and in 13.6% of those with normal cervical findings (p=0.05). The results of the present investigation provide further evidence for the notion that cervical HPV infection is more widespread than previously suggested.

Gene expression in Listeria monocytogenes exposed to sublethal concentration of benzalkonium chloride

In this study, tolerance at sublethal concentration of benzalkonium chloride and transcription levels of mdrL, ladR, lde, sigB and bcrABC genes in Listeria monocytogenes strains were evaluated. Viable cells reduction occurred in 45% of strains and clinical isolates showed lower sensitivity than isolates from foods. An increased transcription of an efflux system encoding gene was found in 60% of strains, and simultaneous mdrL overexpression and ladR underexpression occurred in 30% of isolates. A significant association between reduced benzalkonium chloride activity and both mdrL and sigB overexpression was observed; sigB expression also correlated with both mdrL and ladR genes. The bcrABC gene was only found in six strains, all isolated from foods and sensitive to benzalkonium chloride, and in four strains an underexpression was observed. Disinfection at sublethal concentration was less effective in clinical isolates, and mdrL and sigB expression was significantly affected by disinfection. Further insights are needed to understand the adaptation to benzalkonium chloride and to evaluate whether changes in gene expression could affect the L. monocytogenes virulence traits and persistence in the environment.

Type-specific persistence and associated risk factors of human papillomavirus infections in women living in central Italy

OBJECTIVE We examined persistence and clearance of human papillomavirus (HPV) infections and risk factors associated with persistence in 79 women based on the results of two sequential tests performed over 12-24 months. STUDY DESIGN Between February 2008 and August 2009, 398 women aged 18-63 years were examined for presence of cervical HPV infection by cervical scrape specimen and PCR. Detection was performed using Linear Array (LA) HPV Genotyping Test. All women were interviewed, and a short questionnaire was administered to collect information on socio-demographic characteristics, sexual and reproductive history, smoking habits, oral contraceptive use, history of sexually transmitted diseases, and Chlamydia trachomatis or Mycoplasma spp. infections. Pearsons χ² test was used to verify the association between all independent variables with the response variable. RESULTS Initially, high risk-HPV (HR-HPV) and low risk-HPV (LR-HPV) infection was detected in 69.6% and 30.4% of the women, respectively, whilst multiple infections occurred in 53.2%. HPV 16 was the most common (20.2%) high-risk type, followed by 52, 31 and 53. At follow-up, HR-HPV infection was detected in 50.6% of the women; among these, 67.5% had persistent infection, while 12.5% acquired other high-risk types, and 20.0% of those positive for LR-HPV at entry had a new HR-HPV infection. Multiple infections were detected in 38.0% of the women. HPV 16 and 31 were the most frequent types, followed by HPV 73. Type-specific HR-HPV persistence was found in 49.1% of women. HPV 31, 39 and 73 were the most frequently persistent types, whilst HPV 16 was the least persistent. No significant age difference between women with persistence or clearance was found. The highest HR-HPV persistence occurred in the 22-27 years old group, whereas clearance increased in women aged 28-33 years. No significant association between persistent HR-HPV infection and oral contraceptive use, smoking habits and history of sexually transmitted disease was detected both at entry and follow-up study. The association between C. trachomatis or Mycoplasma spp. and HPV persistence could not be investigated because of the low detection rate of these microorganisms. CONCLUSIONS The persistence of HR-HPV infection level was similar to that reported elsewhere, and HPV 31, 39 and 73 showed the highest likelihood of persistence, partially in agreement with other studies. The clinical relevance of the low persistence of HPV 16 and other HR-HPV is unknown. Persistent HR-HPV infection in women aged 22-27 years was in agreement with other authors. To the best of our knowledge, this is the first report on persistence of HR-HPV infections in Italy in a general population, although we examined a small sample in a short follow-up time.

MicroRNAs expression patterns in the response of poplar woody root to bending stress

AbstractMain conclusionThe paper reports for the first time, in poplar woody root, the expression of five mechanically-responsive miRNAs. The observed highly complex expression pattern of these miRNAs in the bent root suggest that their expression is not only regulated by tension and compression forces highlighting their role in several important processes, i.e., lateral root formation, lignin deposition, and response to bending stress. Mechanical stress is one of the major abiotic stresses significantly affecting plant stability, growth, survival, and reproduction. Plants have developed complex machineries to detect mechanical perturbations and to improve their anchorage. MicroRNAs (miRNAs), small non-coding RNAs (18–24 nucleotides long), have been shown to regulate various stress-responsive genes, proteins and transcription factors, and play a crucial role in counteracting adverse conditions. Several mechanical stress-responsive miRNAs have been identified in the stem of Populus trichocarpa plants subjected to bending stress. However, despite the pivotal role of woody roots in plant anchorage, molecular mechanisms regulating poplar woody root responses to mechanical stress have still been little investigated. In the present paper, we investigate the spatial and temporal expression pattern of five mechanically-responsive miRNAs in three regions of bent poplar woody taproot and unstressed controls by quantitative RT-PCR analysis. Alignment of the cloned and sequenced amplified fragments confirmed that their nucleotide sequences are homologous to the mechanically-responsive miRNAs identified in bent poplar stem. Computational analysis identified putative target genes for each miRNA in the poplar genome. Additional miRNA target sites were found in several mechanical stress-related factors previously identified in poplar root and a subset of these was further analyzed for expression at the mRNA or protein level. Integrating the results of miRNAs expression patterns and target gene functions with our previous morphological and proteomic data, we concluded that the five miRNAs play crucial regulatory roles in reaction woody formation and lateral root development in mechanically-stressed poplar taproot.

Evaluation of transcription levels of inlA, inlB, hly, bsh and prfA genes in Listeria monocytogenes strains using quantitative reverse-transcription PCR and ability of invasion into human CaCo-2 cells.

Listeria monocytogenes virulence depends on the activity of well-characterized virulence factors. In this study, transcription levels of inlA, inlB, hly, bsh and prfA genes in L. monocytogenes strains, and the ability of invasion into CaCo-2 cells were investigated. Serotyping, multiplex-PCR for serovar identification and restriction fragment analysis of inlA were performed. Transcription levels and invasiveness were evaluated by quantitative reverse-transcription PCR and by in vitro assays, respectively. The isolates were of serovars 1/2a, 4b, 1/2c, 1/2b and 3a. Full-length inlA profiles were found for nine of ten clinical isolates, while five of seven cultures from foods showed truncated profile. The analysis of transcription levels of virulence factors encoding genes demonstrated a substantial inter-strain heterogeneity, with clinical strains showing higher levels for almost all genes than isolates from food. A correlation between transcription levels of inlA and inlB, as well as between bsh and prfA, was observed. Significant differences between clinical strains and food isolates in the invasion of CaCo-2 cells were found. Analysis of gene transcription and invasiveness of human cells suggests different virulence phenotypes among L. monocytogenes populations, and this characterization could be a useful tool for risk assessment purposes and for the development of public health strategies.

High Prevalence of Human Papillomavirus Type 58 in HIV infected men who have sex with men: A preliminary report in Central Italy

Human papillomavirus (HPV) infection and type‐specific prevalence at anal, oral, coronal sulcus, and urethral mucosa in fifty HIV positive men having sex with men (MSM) were evaluated; patients were enrolled in a non‐metropolitan area of Central Italy. Clinical and socio‐demographic information, drug, and sexual behaviors were obtained for each participant. HPV was detected by PCR from an overall of 200 specimens, and genotyping was performed by both Restriction Fragment Length Polymorphism analysis and sequencing.