Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Dispersão de bioaerossóis por aeradores em uma estação de tratamento de esgoto sanitário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identificação de Enterobacter spp. através de técnicas convencionais e do sistema semi-automatizado Microscan (Autoscan-4)

A hundred and ninety-five (195) strains of Enterobacter spp., isolated from diverse clinical specimens - urine, feces, cateter, blood, wound, tracheal aspirate, vaginal fluid - were submitted to the conventional identification by biochemical tests, and were also submitted to the identification by panels NegCombo 20 of the system automated MicroScan - AutoScan- 4 (Dade Behring Inc., West Sacramento, CA, USA). The samples were from patients of the Clinical Laboratory from the School of Pharmacy and Biochemistry of UNOESTE, Presidente Prudente, SP, and from patients hospitalized at the University Hospital Domingos Leonardo Cerávolo, UNOESTE. Of the total of strains tested, 191 (97.9%) presented agreement between the two approaches utilized and 4 strains (2.1%) presented identification disagreement, that is, the genus identified was different in each approach. By this study, the conclusion is that both the approaches utilized for the identification presented advantages and disadvantages related to the cost, facility of execution, quickness, reliability and some other characteristics. Even so, our results showed that conventional methods represent a reliable tool for Enterobacter identification.

Aderência e formação do biofilme por Staphylococcus aureus ao polietilenotereftalato (PET) usado como embalagem para acondionamento de medicamentos e cosméticos

The polyethylene terephthalate (PET) is used for package drugs and cosmetics. The aim of this research was examine by scanning electron microscopy (SEM) a Staphylococcus aureus attachment and biofilm formation on a polyethylene terephthalate (PET) surface and hydrophobicity of S. aureus by adherence to hydrocarbons. A suspension of S. aureus was prepared in Mueller-Hinton broth and, coupons of polyethylene terephthalate were incubated for 30 minutes, two, 24 and 48 hours, 15 and 30 days. Afteron the coupons were removed and prepared for scanning electron microscope analysis. The attachment and biofilm formation was observed on the surfaces of PET. The SEM revealed adhesion and biofilm formation on PET surfaces. The hydrophobicity test classified S. aureus as hydrophobic.

Arbitrarily primed polymerase chain reaction for fingerprinting the genotype identification of mutans streptococci in children with Down syndrome

This study investigated the possible intrafamilial similarity of mutans streptococcal strains in some families with a child with Down syndrome using chromosomal DNA fingerprinting. The isolates were genotyped using arbitrarily primed polymerase chain reaction with the OPA 02 and OPA 03 primers. The results showed that five children with Down syndrome harbored mutans streptococci genotypes different from those of their mothers. A matching of genotypes was observed within the control pair (mother/child without Down syndrome). After six months, new samples were collected from all participants. Analysis showed that samples from children with Down syndrome were colonized by a new strain of Streptococcus mutans that did not match the previously collected one. The results suggest the S. mutans indigenous bacteria change more than once in children with Down syndrome.

Effect of 0.4% stannous fluoride gel on Streptococci mutans in relation to elastomeric rings and steel ligatures in orthodontic patients

Background: Patients with fixed orthodontic appliances often experience an absolute increase in the number of Streptococci mutans colony-forming units (cfu). The aim of this investigation was to study the development of biofilm and S. mutans cfu in connection with stainless steel ligatures and elastomeric rings in orthodontic patients treated with and without 0.4% stannous fluoride gel (SFG). Material: Forty-seven patients were divided into 2 groups: those treated with 0.4% SFG for 4 minutes (experimental) and those without 0.4% SFG (control). In each patient, elastomeric rings were used for ligation on 1 side of the dental arch midline, and stainless steel ligatures were used on the opposite side. Saliva samples were collected before and after appliance placement. At 15 and 30 days after appliance placement, biofilm samples from the stainless steel ligatures and the elastomeric rings were collected and subjected to microbiologic procedures and scanning electron microscopy (SEM) analysis. Results: The numbers of S. mutans cfu in the saliva and biofilm were not statistically different between the teeth fitted with elastomeric rings and stainless steel ligatures, or between the experimental and control groups. SEM analysis showed biofilm formation on both ligature ties. Conclusions: Topical application of 0.4% SFG in orthodontic patients with elastomeric rings or stainless steel ligatures does not cause a significant decrease in S. mutans cfu in the saliva and biofilm. Copyright © 2005 by the American Association of Orthodontists.

Methods of experimental induction of periapical inflammation. Microbiological and radiographic evaluation

Aim: To evaluate the influence of coronal filling and apical perforation on the induction of periapical inflammation. Methodology: Fifty-eight root canals in the teeth of dogs were divided into four groups. Groups I and II: root canals were exposed for 180 days; groups III and IV: root canals were exposed for 7 days and then the access cavity filled for 53 days. The root apices of groups I and III were perforated after the coronal opening, whilst those of groups II and IV remained intact. Standard radiographs were taken before and after the experimental periods. Digital images of the radiographs were created and then analysed by three examiners. After induction of periapical inflammation, the root canal contents were collected using paper points. Microbiologic evaluation of the type of microorganism was carried out by culture in different growth media. The radiographic and microbiologic data were statistically analysed using ANOVA at a 5% significance level. Results: There were a greater total number of microorganisms in groups I and II (P < 0.05). The number of anaerobes was greater than the number of aerobes (P < 0.05). The size of the periapical radiolucencies were not significantly different between the experimental groups. Conclusions: The different methods analysed induced similar areas of periapical radiolucency in dogs with predominantly anaerobic bacteria. However, the time required for induction was less when the method with coronal filling was used. © 2005 International Endodontic Journal.

Isolation of Mycobacterium gordonae from poultry slaughterhouse water in São Paulo State, Brazil

Water samples (24 untreated water, 12 treated water and 24 served water) used in different stages of the slaughter process were examined to identify a possible source of pathogenic mycobacteria. The isolates were identified based on microscopy, morphological and biochemical features, mycolic acid analysis and molecular method - PCR-restriction-enzyme analysis. Eighteen mycobacterial strains were isolated from 60 water samples: 11 from untreated water, 5 from treated water and 2 from served water. All mycobacteria isolated were identified as Mycobacterium gordonae and showed the following PRA genotypes: III (27.8%), IV (38.9%) and V (33.3%).

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Immune Response to the Rhodococcus equi infection in high and low antibody-producing mice (selection IV-A)

Rhodococcus equi is a Gram-positive, facultative intracellular bacterium which infects macrophages and causes rhodococcal pneumonia and enteritis in foals. Recently, this agent has been recognized as an opportunistic pathogen for immunocompromised humans. Several murine experimental models have been used to study R. equi infection. High (H IV-A) and Low (L IV-A) antibody (Ab)-producers mice were obtained by bi-directional genetic selections for their ability to produce antibodies against sheep and human erythrocytes (Selection IV-A). These lines maintain their phenotypes of high and low responders also for other antigens than those of selection (multispeciflc effect). A higher macrophage activity in L IV-A mice has been described for several intracellular infectious agents, which could be responsible for their intense macrophage antigens (Ag)-handling and low Ab production. Due to these differences, L IV-A mice were found to exhibit a better performance to trigger an effective immune response towards intracellular pathogens. The objective of this work was to characterize the immune response of Selection IV-A against R. equi. H IV-A and L IV-A mice were infected with 2.0 × 10 6 CFU of ATCC 33701 +R. equi by intravenous route. With regards to bacterial clearance and survival assays, L IV-A mice were more resistant than H IV-A mice to virulent R. equi. L IV-A mice presented a higher hydrogen peroxide (H 2O 2) and nitric oxide (NO) endogenous production by splenic macrophages than H IV-A mice. L IV-A expressed the most intense cellular response, available by the Delayed-Type Hypersensitivity (DTH) reaction, which activated macrophages and produced more H 2O 2 and NO. The three times higher specific antibodies titres in H IV-A indicated that Selection IV-A maintained the multispecific effect and the polygenic control of humoral and cellular responses also to R. equi.

Ação de pesticidas em Metarhizium anisopliae no solo

The present study aimed to analyze the action of some acaricides, fungicides, insecticides and herbicides containing different active ingredients on Metarhizium anisopliae Metsch. (Sorokin) inoculated into autoclaved soil. The action of the pesticides was evaluated based on the fungal respiratory activity. The first assessment was done at 48h after inoculation. The pesticides were then added and respiratory activity was determined nine times every 48h and an additional five times every 4 days. Except for the fungicides, no significant effect (P > 0.05) of the pesticides on M. anisopliae was observed. A reduction in CO2 production was observed for the mancozeb treatment from day 4 to day 6 of incubation, and for tebuconazol between days 4 and 6, 8 and 10, and 32 and 36. The same was observed for copper oxychloride between days 10 and 12 and 32 and 36, and for chlorothalonyl between 8 and 10, 10 and 12, and 32 and 36 days of incubation. Identical effect occurred for the acaricides abamectin and fenbutatin oxide, with a reduction in CO2 production between 20 and 24 days of incubation. The herbicides glyphosate, trifluralin and ametrin reduced the respiratory activity of the fungus between days 10 and 12, while the insecticide trichlorfon reduced respiratory activity only from 32 to 40 days of incubation. The results indicate that the toxic action of pesticides on the fungus in soil is small, suggesting that this pest control bioagent can be used in combination with pesticides without compromising its activity.

Bioaerossóis bacterianos em um hospital

Microbial contamination of an enclosed area may come from outside or be generated within the area itself. In the study described here, bioaerosol levels in hospital air were quantified and related to those found in the air outside, taking into account some of the environmental variables that affect air quality, such as the number of occupants of a room and the type of ventilation. Airborne bacteria were collected for five minute by blowing air at 500 L min -1 in high speed jets on to the surface on plates culture in a single-stage bioaerosol impactor. The mean viable count of bacteria in the air outside in the hospital, São Paulo State, was 77 ± 4 CFU m -3, while in the air inside the hospital it was 302 ± 260 CFU m-3. The operating theatre was the only space sampled with a controlled environment and, after orthopedic surgery, it had the highest bioaerosol count recorded (867 ± 482 CFU m-3). In the enclosed environments, nine bacterial species were identified. Despite the difficulty in establishing precise numbers of bacteria in the bioaerosols in hospital environments, the values obtained demonstrate a need to instigate suitable programs to keep the microbial density low in these environments, and eliminate microorganisms presenting a significant risk to their occupants. It is also recommended that such programs include the monitoring of hospital air, with the aim of defining standards for acceptable numbers of bacteria in the bioaerosol.

Caracterização da população portadora de tuberculose do município de Américo Brasiliense/SP, no período de 1992 a 2002

Tuberculosis (TB) is a serious public health problem all over the world, which was recognized by the WHO in 1993 as a global emergency because of its very high incidence in the 22 countries that concentrate 80% of TB cases. One of these countries is Brazil, where TB occurrences are well-documented in major cities, but little is known of its spread in rural areas and small towns. Therefore, an epidemiological study was done on medical records of TB sufferers in the rural district and small town of Américo Brasiliense, São Paulo state, from 1992 to 2002, with the aim of improving TB prevention and treatment. The results showed that the incidence of TB peaked in the years of intense migration of rural workers, largely cane-cutters. Among these, the disease attacks mainly the men, in their productive years (20 to 40 years old). The predominant clinical form observed was pulmonary tuberculosis. The treatment abandonment rate was less than 1.8%, while the cure rate was around 90%. The disease detection rate by examination of sputum for acid-fast bacilli was around 60%.