Phenotypic and genotypic characterization of clinically relevant bacteria isolated from dental waste and waste workers' hands, mucosas and coats.

Infectious wastes are potential sources of pathogenic micro-organisms, which may represent a risk to the professionals who manage them. In this study, we aimed to characterize the infectious bacteria present in dental waste and waste workers. The dental waste produced over 24 h was collected and waste workers were sampled by swabbing. Isolate resistance profiles were characterized by Vitek® and PCR and biofilm formation by Congo Red agar, string test and microtitre assay. To assess similarity between the waste and the workers' samples, a random amplified polymorphic DNA test was used. Twenty-eight bacteria were identified as clinically relevant. The most frequent gene was blaTEM present in five Gram-negative micro-organisms, and one blaSHV in Klebsiella pneumoniae. All Pseudomonas aeruginosa were positive to extracellular polymeric substances formation, except one isolated from a worker. Klebsiella pneumoniae had negative results for the string test. Pseudomonas aeruginosa showed better adherence at 25°C after 48 h of incubation and K. pneumonia had the best biofilm formation at the same temperature, after 24 h. The similarity between P. aeruginosa recovered from dental waste and from workers was low, however, it is important to note that a pathogen was found on a worker's hands and that improvements in biosafety are required. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious dental waste can contain clinically relevant bacteria with important resistance and biofilm profiles. These micro-organisms could be transmitted to waste workers, other professionals and patients if the principles of biosafety measures are neglected. To our knowledge, no study has ever evaluated the microbial characterization and the potential contamination risk of dental infectious waste and waste handlers. The presence of clinically relevant bacteria in the hands and nasal mucosa of waste workers highlights the need for studies in this field to clarify the risk of these pathogens in dental healthcare services, and to stress the need for an efficient waste management.

Enterotoxigenic Escherichia coli in children with acute diarrhoea and controls in Teresina/PI, Brazil: distribution of enterotoxin and colonization factor genes.

AIMS: To investigate the distribution of the genes that encode enterotoxins and the colonization factors (CF) types as well as the antibiotic susceptibility profile of enterotoxigenic Escherichia coli (ETEC) isolated from children from the Brazilian Northeast. METHODS AND RESULTS: We conducted a 3·5-year prospective study that involved 250 children with and 150 without diarrhoea, aged 1-60 months, from low-income families in Teresina/Brazilian Northeast. All samples were assayed for E. coli, enterotoxin and CF genes and antimicrobial susceptibility by microbiological methods and PCR. ETEC strains were isolated from 9·2% children with and 4·0% without diarrhoea. Infection was more common in children aged 6-24 months in rainy months. elt⁺ /CFA/IV⁺ and elt⁺ /CS14⁺ were the most frequent genotypes. Susceptibility to nalidixic acid, ciprofloxacin and gentamicin and resistance to ampicillin, cephalothin and sulfamethoxazole-trimethoprim were common. CONCLUSIONS: elt ⁺isolates and ETEC strains harbouring genes encoding CFA/IV and CS/14 were the most common ETEC found in Brazilian Northeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data, the first generated for north-eastern Brazilian children, may be important for the development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveller's diarrhoea in the area studied.

Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate.

AIMS: To purify and partially characterize a bacteriocin produced by a Fusobacterium nucleatum strain. METHODS AND RESULTS: Following protein precipitation the effect of different treatments on a bacteriocin produced by a F. nucleatum strain named P12.2 isolated from a patient with periodontitis was evaluated. The antagonistic activity of the intracellular fraction obtained at 80% ammonium sulphate was preserved at pH values from 6.0 to 9.0 and showed to be sensitive to high temperatures and to treatment with proteases. The fraction was submitted to sequential steps of gel filtration, ion exchange, and reverse phase chromatography, and SDS-PAGE. Data obtained by mass spectrometry revealed that the molecular mass of the protein was 27,296 Da. CONCLUSIONS: For the first time a bacteriocin produced by a F. nucleatum strain was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description on characterization of a bacteriocin produced by F. nucleatum. It is possible that the bacteriocin plays a role in the regulation of population levels of periodontopathic organisms.

Physiological alterations of a Fusobacterium nucleatum strain exposed to oxidative stress.

AIM: The purpose of this study was to investigate the effect of oxidative stress on physiological and genetic characteristics of Fusobacterium nucleatum and its interference on this microbial identification methods. METHODS AND RESULTS: Fus. nucleatum ssp. nucleatum ATCC 25586 (wt-strain) and an oxidative-stress-adapted strain derived from the wt-strain (aero-strain) were employed in the study. Cell-free crude protein extracts were obtained from both strains and differentially expressed proteins were identified by two-dimensional electrophoresis. Bacterium identification was performed by conventional biochemical tests, automated Rapid ID 32A system and specific PCR analysis. Genetic diversity between wt- and aero-strain was assessed by arbitrarily-primed (AP)-PCR. There were significant changes in the protein profile of aero-strain. The identification of the wt-strain was confirmed by all methods employed. Similar results were obtained for aero-strain when conventional biochemical tests and PCR were used. However, aero-strain was identified as Fusobacterium varium when submitted to Rapid ID 32A system. According to AP-PCR analysis, no significant genetic alteration was detected in aero-strain. CONCLUSIONS: The adaptive response of Fus. nucleatum to oxidative stress is associated with changes on its biology, which may lead to misidentification of the organism, according to the conventional identification methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxidative stress may act as a cause of adaptive response in Fus. nucleatum with consequences to its biology, such as alterations on biochemical and physiological profile.

iceA genotypes of Helicobacter pylori strains isolated from Brazilian children and adults.

Data concerning the geographic distribution of iceA alleles are scarce, and information on the association of the gene with the disease is rare and still controversial. Furthermore, no such study has been developed in Brazil, where duodenal ulcer and gastric adenocarcinoma are very common. We investigated, by PCR, the frequency of iceA alleles and cagA status in Helicobacter pylori strains isolated from 142 patients (62 children and 80 adults; 66 female; mean age, 30.0 years; age range, 3 to 78 years) with gastritis, duodenal ulcer, or gastric adenocarcinoma. iceA was identified in bacterium samples obtained from all patients. Eleven (7.7%) of them were infected with multiple strains. Among the patients with nonmixed infection, iceA2 allele was detected in 118 (90.1%). iceA2 allele was associated with ulcer (P = 0.02) and with carcinoma (P = 0.001). iceA2 amplicons of 229, 334, or 549 bp were detected, but none of them was associated with the patient's disorder. iceA2 strains were more frequent in patients older than 7 years (P = 0.001). The gene was also more frequent in strains obtained from males (P = 0.02). cagA was more common in strains obtained from carcinoma (P = 0.0008) and ulcer patients (P < 0.006). cagA-positive strains were more frequent in children older than 7 years (P < 0.003). No association between cagA status and sex was found (P = 0.28). In conclusion, we think iceA should not be used as a reliable marker for predicting the clinical outcome of H. pylori infection.

Microbiological and histological study of the gastrointestinal tract of germ-free mice infected with Helicobacter trogontum.

Helicobacter spp. have been the focus of considerable research because of the role of this genus in gastrointestinal diseases. We infected NIH germ-free mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the distribution of this bacterium in the gastrointestinal tract and the histopathological changes it can induce in this host. Sixteen mice were challenged with a single dose of H. trogontum (test group) and killed one and six weeks after inoculation (eight animals at each point). Eight animals were challenged with 0.85% saline alone (control group) and killed at the same time points (four at each point). Fragments from the gastric and intestinal mucosa were obtained for microbiological and histological examination. H. trogontum was isolated from the cecum and colon of all test mice and also from the gastric mucosa of several of them. All infected animals presented histological changes in at least one region of the bowel. Alterations in the gastric mucosa were also observed mainly in the six-week-infected group. The predominant histological change observed was a moderate diffuse inflammatory infiltrate of mononuclear cells in the lamina propria, often accompanied by a mild infiltration of polymorphonuclear cells. Two animals presented focal infiltration of inflammatory cells in the liver, although no bacteria were found in the liver of any animal. H. trogontum is an intestinal species that is able to elicit inflammatory responses in other regions of the gastrointestinal tract such as the gastric mucosa and the liver of gnotobiotic mice.

cagA-positive Helicobacter pylori and risk for developing gastric carcinoma in Brazil.

To evaluate a possible association between infection with cag A-positive strains and gastric carcinoma increased risk we studied 119 Helicobacter pylori-positive patients with gastric carcinoma and 119 matched controls. The presence of cag A gene was investigated by PCR in H. pylori isolates and in gastric biopsy specimens. A significant association was found between cag A-positive status and distal gastric carcinoma for both the intestinal and diffuse types of tumor for both males and females. On the other hand, no association was observed between cag A-positive status and proximal gastric carcinoma.

Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group.

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originally susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were beta-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstrated. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains.