Detection of human papillomavirus DNA and p53 gene mutations in esophageal cancer samples and adjacent normal mucosa.

BACKGROUND/AIM: There is evidence of a possible etiological role of human papillomaviruses (HPVs) in the development of esophageal tumors. Loss of function of the wild-type p53 tumor suppressor gene product by binding to E6 oncoproteins of high-risk HPVs is considered an important event in tumor development. The aim of this study was to verify the prevalence of HPV infection and p53 mutation in esophageal tumor tissue samples and in the adjacent normal mucosa in patients from a high-risk area in Italy. METHODS: DNA from 33 biopsy specimens (17 tumor sample biopsies and 16 samples of adjacent normal mucosa) was screened for HPV DNA using two polymerase chain reaction based procedures. Restriction fragment length polymorphism analysis was used for typing. Screening of p53 mutations was performed with polymerase chain reaction-single strand conformation polymorphism analysis and DNA sequencing. RESULTS: Overall, 8 of 17 patients presented HPV DNA; HPV 16 was detected in 4 of 8 samples. Samples from tumors and adjacent mucosa were positive for mucosal HPVs in 7 of 17 and 4 of 16 cases, respectively. In 1 case, HPV DNA was detected in the normal mucosa only. None of the samples contained HPVs of the epidermodysplasia verruciformis or cutaneous groups. Mutations of p53 were detected in two HPV DNA negative samples. In both cases, the mutation was present in the tumor only. CONCLUSIONS: Our results are in favor of the involvement of both aberrant p53 expression and HPV infection in the development of esophageal tumors. The high HPV infection rate in patients from a high-risk region suggests that subjects harboring HPVs (in particular HPV 16) in the esophagus should be considered at risk of esophageal malignancies.

Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans.

Gram-negative anaerobes belonging to the genera Fusobacterium, Prevotella and Porphyromonas were investigated for the presence of tetQ and ermF, which have been shown to be spread by conjugal elements. One hundred isolates from either sites of infection or various body sites in healthy subjects were studied. PCR was used to detect tetQ, and DNA-DNA hybridization studies on EcoRI chromosomal digests were undertaken to detect the presence of tetQ and ermF. Antibiotic sensitivity assays were performed on selected isolates to detect tetracycline, erythromycin and penicillin resistance. Twenty Fusobacterium isolates lacked tetQ, and were tetracycline sensitive. Twenty per cent of Prevotella spp. isolates both from clinical specimens and from healthy subjects were found to possess tetQ. Of 20 Porphyromonas isolates tested, one (Porphyromonas levii) from a case of bacterial vaginosis was shown to possess tetQ in the chromosome. The presence of tetQ was always associated with tetracycline resistance. Four isolates of Prevotella melaninogenica and one isolate of Prevotella were ermF-positive, although expression of erythromycin resistance was not consistently associated with detection of this gene. Antibiotic resistance phenotypes of Prevotella isolates were shown to be related to specific chromosomal restriction patterns by hybridization studies: tetracycline resistance and tetracycline/erythromycin resistance are conferred by Bacteroides tetracycline-resistant ERL elements, whereas the tetracycline/penicillin resistance phenotype could be due to spread of elements identified in Prevotella only. Tetracycline/erythromycin-resistant and tetracycline/erythromycin/penicillin-resistant P. melaninogenica isolates were found in this study. It appeared that the presence of tetQ and ermF in Bacteroides and Prevotella contributed to the persistence of antibiotic resistance isolates within the host and to potential spread to other organisms through conjugal elements.

Diagnostic anaerobic bacteriology in Italy: state of the art. Italian Anaerobic Study Group.

A total of 220 microbiology departments in 205 general hospitals, 12 university hospitals, and 3 military hospitals were surveyed by questionnaire during the period March-May 1996 in order to evaluate the status of diagnostic anaerobic bacteriology in Italy. Responses were received from 47 laboratories (21.3%). The number of beds in the responding facilities varied widely (129-2,200), as did the number of specimens tested for anaerobic bacteriology (50-29,900). In most cases (94%), the microbiologist made the decision to culture and to proceed to further identification of isolates (mainly by commercial systems), depending on the adequacy of samples. Antibiotic susceptibility testing was performed in 52.5% of laboratories surveyed. Although it is difficult to draw relevant conclusions owing to the poor response rate, it seems that the condition of anaerobic bacteriology in Italy is far from satisfactory. Among major limitations are low interest in the field, lack of standardization of procedures, poor utilization of reference centers, and limited technological resources.

Recovery of Bilophila wadsworthiafrom Clinical Specimens in Italy.

This report is the first survey in Italy to evaluate the incidence of recovery of Bilophila wadsworthia in clinical situations. The survey was carried out at the departments of Microbiology in two Northern Italian hospitals over a one-year period. Tests for B. wadsworthia were carried out on a range of specimens from different body sites, when etiology by anaerobes was suspected. Out of a total of 350 samples examined, 67% were positive in bacteriological tests. Mixed anaerobic infections were detected in 53 specimens, corresponding to 23% of all cases. Strains of B. wadsworthia were isolated from 12 samples, equivalent to 5% and 22% of total and mixed/anaerobic infections, respectively. Bilophila wadsworthia was always isolated in mixed infections, mainly from the large intestine (67% of cases). The infectious process of B. wadsworthia was often complicated by abscess formation, regardless of body site. Interestingly, a strain was isolated from one case of bacteremia. The microorganisms most frequently isolated with B. wadsworthia were Escherichia coli for facultative species (38%), and Bacteroides fragilis, from anaerobic isolates (25%). Production of beta-lactamases by B. wadsworthia isolates was found in ten strains (83%), which appeared to be penicillin G resistant at concentration equal to or greater than the break-point (4 microg/mL). Epidemiological and clinical data from this and previous studies point to the involvement of B. wadsworthia in mixed infections. To assess the specific contribution of the species to the disease, studies of pathogenetic factors are to be considered in parallel. Nonetheless, production of beta-lactamases by most B. wadsworthia isolates could easily interfere with the therapeutical approach to infections involving the new species. The addition of a selective medium to culture specimens from the abdominal cavity should be considered in order to detect the presence of B. wadsworthia.

Characterization of a putative new HPV genomic sequence from a cervical lesion using L1 consensus primers and restriction fragment length polymorphism.

Various methods have been proposed for HPV detection and typing. Prevalence and distribution among types have varied depending upon the methods used and the populations studied. We have applied the polymerase chain reaction (PCR) followed by a Restriction Fragment Length Polymorphism (RFLP) analysis using the MY09/MY11 primers for detection of HPV in cervicovaginal lavages obtained from 323 patients who were referred to our Clinical Department either for genital complaints or an abnormal PAP smear. We assessed (i) the prevalence of HPV and (ii) the reliability of RFLP-typing. For the latter, 35 PCR-HPV products were sequenced. HPV-DNA was detected in 40/197 (20.3%) patients with normal cytology 86/111 (77.5%) with LSIL and 11/15 (73.3%) with HSIL. HPV-16 was the most common type detected in normal cervical cytology samples (10/40, 25%), whereas HPV 16 and 18 were detected in 36/97 (37.1%) of the LSIL and HSIL patients, evidencing the presence of these high-risk HPV types not only in malignant conditions. Results obtained after partial nucleotide sequencing confirmed the results obtained by RFLP analysis. In this study, a putative new HPV fragment (GA6053) was identified. Its closest homology to other known HPV types is 73.8% to HPV-62, 73.0% to HPV-61 and 67.7% to HPV-18. The use of degenerate primers, in conjunction with RFLP, proved to be a reliable method for HPV detection and typing.

Rapid polymerase chain reaction method for specific detection of toxigenic Clostridium difficile.

A rapid polymerase chain reaction (PCR) method for detection of toxigenic Clostridium difficile directly from fecal samples by amplification of toxin A gene fragments was investigated. The technique was applied to monitor the spread of the microorganism in a long-term care ward with a relatively high incidence of overt episodes of diarrhea. The PCR approach has several advantages over traditional methods, rapidly allowing the specific detection of toxigenic Clostridium difficile strains from stool samples in both symptomatic and asymptomatic subjects with toxigenic strains. This PCR method allows early detection of toxigenic Clostridium difficile and could thus represent a powerful tool for the surveillance of epidemics.

Role of structural and extracellular virulence factors in gram-negative anaerobic bacteria.

Gram-negative anaerobic bacteria belonging to the genera Bacteroides, Prevotella, and Porphyromonas represent the most common cause of endogenous, usually mixed, infections occurring after abdominal or gynecologic surgery. Anaerobes are important pathogens in oral-cavity infections as well as in systemic infections that originate from the mouth. Clinical interest in these organisms is linked to the therapeutic problems usually encountered in treating mixed infections. Despite their clinical relevance, very little is known about the pathogenetic mechanism of anaerobic infections. In Bacteroides species, the capsule has been thought to be important, and initially it was considered unique to Bacteroides fragilis, the most common pathogen. It has been claimed that the capsule is involved in adhesion, abscess formation, and impaired phagocytosis. However, other structures such as pili and extracellular substances, including metabolic by-products (e.g., short-chain fatty acids), have to be considered as potentially relevant pathogenetic mechanisms in anaerobic infections. Several extracellular enzymes have been investigated, but no clear evidence is available for establishing their relevance in disease mechanisms. Special attention should be devoted to enzymes able to digest IgA (IgA proteases), a first-line defense mechanism that is active in the mucosal membranes.

Evaluation of an automated system for identification of anaerobic bacteria.

A fully automated computer-assisted system (ATB system, bioMérieux, France) which uses disposable microenzymatic panels was evaluated for its ability to identify 215 strains of anaerobic bacteria (clinical isolates and reference strains). All strains were examined using conventional identification protocols and by gas chromatographic analysis of short-chain fatty acids. Automated reading of Rapid ID32A test kits (bioMérieux, France) by the ATB system gave correct identification for 195 strains (90.7%): 92.25% of gram-negative anaerobes (116 strains) and 89% of gram-positive anaerobes (99 strains) were correctly identified. Twelve strains (5.6%) were incorrectly identified and 8 strains (3.7%) were not identified by the system. For some strains in the Bacteroides fragilis group, for Clostridium difficile and for the Fusobacterium genus, additional tests suggested by the ATB software were necessary to reach a final identification at the species or genus level. On the basis of the high incidence of correct identifications and the comparison of these results with those obtained previously using other commercially available kits, the ATB system was found to be a reliable method for identification of anaerobic bacteria in clinical laboratories.

Characterization of mesophilic Aeromonas from clinical specimens by computerized analysis of SDS-PAGE protein profiles and by enzymatic activity.

Mesophilic Aeromonas (Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae) have recently been considered important aetiological agents of human diseases, mainly gastrointestinal infections. Although several findings have pointed out the significance of this group of microorganisms as enteric pathogens and suggested the presence of virulence factors, epidemiological and clinical studies are limited by the difficulty of correctly identifying mesophilic Aeromonas at the species level. SDS-PAGE of radiolabelled total protein profiles and bacterial enzymatic activities were used to type 31 clinical isolates (6 A. hydrophila, 7 A. sobria and 18 A. caviae) from patients with gastroenteritis and from healthy controls. Analysis of SDS-PAGE protein patterns, reinforced by the UPGMA-grouping system (AMBIS software) provided a good characterization of A. caviae strains as a homogeneous group of microorganisms, possessing significant differences from the other two species of mesophilic Aeromonas, in good agreement with biochemical and enzymatic tests. Data obtained in analyzing A. sobria protein profiles clearly showed two groups, with a correlation coefficient (CC) = 0.70, which in our experience is a doubtful value for assigning two strains to the same species. Strains biochemically identified as A. hydrophila showed a CC = 0.64, which is equally not acceptable for species assignment. Inter-species comparison highlighted this heterogeneity, showing two mixed subgroups, both containing strains that were assigned to A. sobria and A. hydrophila species on the basis of biochemical features.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid identification of Bacteroides species by high-performance liquid chromatography.

High-performance liquid chromatography was evaluated for the rapid identification of Bacteroides species of clinical interest. Each isolate was inoculated into a defined chemical medium containing primarily carbohydrates and was incubated aerobically at 37 degrees C for 1 h. After centrifugation, the supernatants were placed on ice to stop further enzymatic reactions. Specimens were injected into an Aminex HPX-87H column in order to determine carbohydrates and acid metabolic products. Peak areas of carbohydrates for each isolate were compared with those for uninoculated medium. As the utilization indexes of raffinose, lactose and arabinose were found to be particularly significant, the patterns of carbohydrate utilization could be used for the identification of Bacteroides species. This method can be adapted for diagnostic laboratory use and has good potential for automated microbial identification.

Evaluation of a computer-assisted method of analysing SDS-PAGE protein profiles in tracing a hospital outbreak of Serratia marcescens.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of bacterial proteins have been successfully used for taxonomical purposes. More recently this technique has been applied to epidemiological investigations in respect of various micro-organisms including Neisseria meningitidis, Staphylococcus aureus and Clostridium difficile. The main limitations of the methods so far described are lack of standardisation in extraction and separation as well as in the analysis of results. Although reproducibility in the same laboratory has been shown to be satisfactory, comparison of results among laboratories is still difficult. Moreover, assessment of differences and/or similarities among chromatograms or autoradiographs showing many bands depends upon qualitative descriptions. Interpretation of densitometric scannings is laborious and time-consuming. In this paper we present our experience of a completely standardised, fully computer-controlled procedure for SDS-PAGE (AMBIS System) in analysing 35S-methionine-labelled total proteins. The methodology proved very useful in monitoring a hospital outbreak of Serratia marcescens. It allowed us to make quantitative comparison in a shorter time as well as to handle easily a great amount of data and usefully integrate it with those obtained with other systems such as serotyping. Furthermore, when the two systems are used together, more precise information can be gained. In this epidemic, serotyping indicated the presence of two groups which would have been missed by PAGE analysis alone. Electrophoretotyping, however, focused on similarities of cellular proteins among the epidemic strains. This allowed us to distinguish them from epidemiologically unrelated strains of the same serogroup.(ABSTRACT TRUNCATED AT 250 WORDS)