Scielo RSS <![CDATA[Brazilian Journal of Microbiology]]> vol. 42 num. 2 lang. pt <![CDATA[SciELO Logo]]> <![CDATA[<b>Multiresistance and endemic status of <i>acinetobacter baumannii</i> associated with nosocomial infections in a tunisian hospital</b>: <b>a critical situation in the intensive care units</b>]]> Acinetobacter baumannii is often implicated in hospital outbreaks in Tunisia. It's a significant opportunistic pathogen associated with serious underlying diseases such as pneumoniae, meningitis and urinary tract infections. The aim of our study was to evaluate its degree of endemicity and its antibiotic resistance evolution essentially in the unit care where its isolation was predominant (57%). This study used 3 methods: antibiotyping, RAPD using 2 primers VIL 1, VIL5 and PFGE with ApaI restriction enzyme. The presence of integron1 and 2 was also studied. Antibiotyping showed that 92% of patients were resistant of all ß- lactams (except Imipenem) and that the resistance to Imipenem occurred in 47% of cases. RAPD profiles obtained with the 2 arbitrarily primers VIL1 and VIL5 gave respectively 5 and 4groups and PFGE fingerprinting patterns revealed 22 different pulsotypes. Integron 1 was present in 25% of unrelated strains and type 2 integron was not detected in any of the studied strains. Among 204 strains, multiple and heterogeneous groups were detected with the genomic studies. In addition, any correlation was obtained with the antibiotyping results. These findings demonstrate the endemic status of A. baumannii in our hospital and the persistence of a large number of multiresistant strains in the unit's care. When outbreaks of A. baumannii occur, it's essential to develop restricted hygiene procedures and a serious surveillance of critical units such as ICU for very ill patients. <![CDATA[<b>A quantitative analysis of <i>Propionibacterium acnes</i> in lesional and non-lesional skin of patients with progressive macular hypomelanosis by real-time polymerase chain reaction</b>]]> Little is known about the etiology of progressive macular hypomelanosis, although it has been suggested that Propionibacterium acnes plays an important role. While microbiological culture is commonly employed to identify Propionibacterium acnes, new identification methods have been under investigation, amongst them polymerase chain reaction. To determine the cut-off point for the number of genome copies of Propionibacterium acnes in the lesional skin of patients with progressive macular hypomelanosis as a positive marker, employing quantitative real-time polymerase chain reaction and anaerobic culture, considered gold standard. An observational study with a comparison group, included 35 patients with dermatosis, attended at the Oswaldo Cruz University Hospital, Pernambuco, Brazil, between March and May 2008. Lesional skin was compared to non-lesional skin through positive testing with real-time polymerase chain reaction and culture. The Statistical Package for Social Sciences, version 12.0, was employed for the association analysis with the McNemar test, and the cut-off point with the ROC curve for maximum values. Propionibacterium acnes was most frequently encountered in lesional areas (p<0,025). The cut-off point of Propionibacterium acnes in lesional skin was 1,333 genome copies, with a sensitivity of 87,9% and a specificity of 100,0%. Since Propionibacterium acnes is a saprophyte, identifying the cut-off point may assist in determining its positivity in lesional skin in patients suffering with this dermatosis. <![CDATA[<b>Detection of <i>Bartonella henselae</i> in defibrinated sheep blood used for culture media supplementation</b>]]> Bartonella henselae was detected in defibrinated sheep blood employed in supplementing a selective bacteria culture medium by nested PCR. We recommended that highly sensitive technical tests be run to ensure a sterile culture medium for Bartonella spp. isolation, since infected blood samples used in preparation could lead to false-positive results. <![CDATA[<b>Antifungal activity of <i>Cymbopogon winterianus</i> jowitt ex bor against <i>Candida albicans</i></b>]]> Candida albicans is an opportunistic yeast and a member of the normal human flora that commonly causes infections in patients with any type of deficiency of the immune system. The essential oils have been tested for antimycotic activity and pose much potential as antifungal agents. This work investigated the activity of the essential oil of Cymbopogon winterianus against C. albicans by MIC, MFC and time-kill methods. The essential oil (EO) was obtained by hydrodistillation using a Clevenger-type apparatus. It was tested fifteen strains of C. albicans. The MIC was determined by the microdilution method and the MFC was determined when an aliquot of the broth microdilution was cultivated in SDA medium. The phytochemical analysis of EO showed presence of citronellal (23,59%), geraniol (18,81%) and citronellol (11,74%). The EO showed antifungal activity, and the concentrations 625 µg/mL and 1250 µg/mL inhibited the growth of all strains tested and it was fungicidal, respectively. The antimicrobial activity of various concentrations of EO was analyzed over time, it was found concentration-dependent antifungal activity, whose behavior was similar to amphotericin B and nystatin. <![CDATA[<b>Isolation of <i>Helicobacter pylori</i> in gastric mucosa and susceptibility to five antimicrobial drugs in Southern Chile</b>]]> Helicobacter pylori colonizes more than 50% of the world population thus, it is considered an important cause of gastric cancer. The aim of this study was to determine the isolation frequency of H. pylori in Southern Chile from patients with symptomatology compatible with gastritis or gastric ulcer and to correlate these findings with demographic parameters of infected patients and the susceptibility profiles of the isolated strains to the antimicrobial drugs used in the eradication treatments. A total of 240 patients were enrolled in the study. Each gastric biopsy was homogenized and seeded onto blood agar plates containing a selective antibiotics mixture (DENT supplement). Plates were incubated at 37° C in a microaerophilic environment for five days. The susceptibility profiles to amoxicillin, ciprofloxacin, clarithromycin, tetracycline and metronidazole were determined using the E-test method. H. pylori was isolated from 99 patients (41.3%) with slightly higher frequency in female (42% positive cultures) than male (40.2% positive cultures). With regard to age and educational level, the highest isolation frequencies were obtained in patients between 21-30 (55%) and 41-50 (52.6%) years old, and patients with secondary (43.9%) and university (46.2%) educational levels. Nineteen (21.6%) strains showed resistance to at least one antimicrobial drug. Tetracycline was the most active antimicrobial in vitro, whereas metronidazole was the less active. One strain (5.3%) showed resistance to amoxicillin, clarithomycin and metronidazole, simultaneously. <![CDATA[<b>Development of conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens</b>]]> Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration . <![CDATA[<b>Nasal rhinosporiodiosis from uttar pradesh (India)</b>: <b>a non-endemic zone: first case report</b>]]> Rhinosporiodiosis is a cosmopolitan disease of man and animals, endemic in India and Sri Lanka with main focus of infection in Southern Tamil Nadu. Uttar Pradesh (UP) is not known to be an endemic zone for this disease .We present here the first case of nasal Rhinosporiodiosis from this non-endemic zone. <![CDATA[<b>Prevalence of antimicrobial resistance and integrons in <i>Escherichia Coli</i> from Punjab, Pakistan</b>]]> Antimicrobial resistance was studied in Escherichia coli strains isolated from urine samples of 457 patients suffering from urinary tract infection. High prevalence of class 1 integrons (43.56%), sulfamethoxazole resistance genes sul1 (45.54%) and sul2 (51.48%) along with occurrence of quinolone resistance genes was detected in multi drug resistance isolates. <![CDATA[<b>Resistance of <i>Klebsiella Pneumoniae</i> clinical isolates</b>: <b>linkage of outer membrane proteins (omps) with production esbls</b>]]> Three isolates of Klebsiella pneumoniae, collected from the University Hospital in Fortaleza, Brazil, were analyzed to determine their resistance to multiple antibiotics. The results of this study showed that the resistance of the clinically isolated bacteria is associated with the production of extended-spectrum beta-lactamases (ESLBs) and loss of outer membrane proteins. <![CDATA[<b>New antimicrobial combinations</b>: <b>substituted chalcones- oxacillin against methicillin resistant <i>Staphylococcus aureus</i></b>]]> Staphylococcus aureus, the most virulent Staphylococcus species, is also the prevalent pathogen isolated from hospitalized patients and the second most common from patients in outpatient settings. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents. Related studies of antimicrobial activity indicate that crude extracts containing flavonoids, triterpenes and steroids have showed significative activity against several Staphylococcus aureus strains. Combination effects between flavonoids and antibiotics also have been reported. The aim of the present work was to investigate in vitro synergism between several chalcones substituted in combination with oxacillin, an antibiotic used conventionally against S. aureus ATCC 43 300 that is resistant to meticillin, using the kinetic turbidimetric method developed earlier. The results were satisfactory for all assayed combinations and in accordance with the mechanism of bacteriostatic inhibition previously proposed, except for 2´,4´-dihydroxy-3´-methoxychalcone - oxacillin. The best combination was 2´,3´-dihydroxychalcone - oxacillin (MIC: 11.2 μg/mL). Further investigations are needed to characterize the interaction mechanism with antibiotics. Thus, chalcones - oxacillin combination could lead to the development of new antibiotics against methicillin resistant S. aureus infection. <![CDATA[<b>Evaluation of biofilm production by <i>Pseudomonas Aeruginosa</i> isolates recovered from cystic fibrosis and non-cystic fibrosis patients</b>]]> Cystic fibrosis (CF) patients typically suffer of persistent and recurrent lung infections caused by Pseudomonas aeruginosa that many times possess ability for the biofilm production. Here, biofilm production among P. aeruginosa isolates recovered from sputum of CF and non-CF patients was evaluated. Most isolates were biofilm-producing independently of the patient's condition. <![CDATA[<b>Species distribution and antimicrobial susceptibility of enterococci isolated from</b> <b>broilers infected experimentally with <i>Eimeria</i> spp and fed with diets containing different supplements</b>]]> Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40%), followed by E. casseliflavus/E. gallinarum (10.8%), E. mundtii (10.8%), E. faecium (10.8%), E. columbae (5.8%) and E. gallinarum (4.2%). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci. <![CDATA[<b>The hydrophobicity and roughness of a nasoenteral tube surface influences the adhesion of a multi-drug resistant strain of <i>Staphylococcus Aureus</i></b>]]> In this study, we examined the physiochemical properties of nasoenteral feeding tubes made from two different types of polymer: silicone materials and polyurethane. The internal surfaces of the nasoenteral feeding tubes were analyzed for their hydrophobicity, roughness, microtopography, rupture-tension and ability to stretch. We also studied the adhesion of an isolated, multi-drug resistant strain of S. aureus to these polymers. The polyurethane nasoenteral tube, which was classified as hydrophilic, was more resistant to rupture-tension and stretching tests than the silicone tube, which was classified as hydrophobic. Additionally, the polyurethane tube had a rougher surface than the silicone tube. Approximately 1.0 log of S. aureus cells adhered to the tubes and this number was not statistically different between the two types of surfaces (p > 0.05). In future studies, new polymers for nasoenteral feeding tubes should be tested for their ability to support bacterial growth. Bacterial adhesion to these polymers can easily be reduced through modification of the polymer's physicochemical surface characteristics. <![CDATA[<b>Effect of culture medium on biocalcification by <i>Pseudomonas Putida</i>, <i>Lysinibacillus Sphaericus</i> and <i>Bacillus Subtilis</i></b>]]> The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials. <![CDATA[<b>Inoculation of tomato seedlings with <i>Trichoderma Harzianum</i> and Arbuscular Mycorrhizal Fungi and their effect on growth and control of wilt in tomato seedlings</b>]]> A green house study was conducted to investigate the ability of an isolate of Trichoderma harzianum (P52) and arbuscular mycorrhizal fungi (AMF) in enhancing growth and control of a wilt pathogen caused by Fusarium oxysporum f. sp. lycopersici in tomato seedlings. The plants were grown in plastic pots filled with sterilized soils. There were four treatments applied as follows; P52, AMF, AMF + P52 and a control. A completely randomized design was used and growth measurements and disease assessment taken after 3, 6 and 9 weeks. Treatments that significantly (P < 0.05) enhanced heights and root dry weights were P52, AMF and a treatment with a combination of both P52 and AMF when compared the control. The treatment with both P52 and AMF significantly (P < 0.05) enhanced all growth parameters (heights; shoot and root dry weight) investigated compared to the control. Disease severity was generally lower in tomato plants grown with isolate P52 and AMF fungi either individually or when combined together, though the effect was not statistically significant (P0.05). A treatment combination of P52 + AMF had less trend of severity as compared to each individual fungus. T. harzianum and AMF can be used to enhance growth in tomato seedlings. <![CDATA[<b>A novel acidophilic, thermophilic iron and sulfur-oxidizing archaeon isolated from a hot spring of tengchong, yunnan, China</b>]]> A novel thermoacidophilic iron and sulfur-oxidizing archaeon, strain YN25, was isolated from an in situ enriched acid hot spring sample collected in Yunnan, China. Cells were irregular cocci, about 0.9-1.02 µm×1.0-1.31 µm in the medium containing elemental sulfur and 1.5-2.22 µm×1.8-2.54 µm in ferrous sulfate medium. The ranges of growth and pH were 50-85 (optimum 65) and pH 1.0-6.0 (optimum 1.5-2.5). The acidophile was able to grow heterotrophically on several organic substrates, including various monosaccharides, alcohols and amino acids, though the growth on single substrate required yeast extract as growth factor. Growth occurred under aerobic conditions or via anaerobic respiration using elemental sulfur as terminal electron acceptor. Results of morphology, physiology, fatty acid analysis and analysis based on 16S rRNA gene sequence indicated that the strain YN25 should be grouped in the species Acidianus manzaensis. Bioleaching experiments indicated that this strain had excellent leaching capacity, with a copper yielding ratio up to 79.16% in 24 d. The type strain YN25 was deposited in China Center for Type Culture Collection (=CCTCCZNDX0050). <![CDATA[<b>Enterobacteriaceae in mouth and cloaca of <i>podocnemis expansa</i> and <i>P. Unifilis</i> (testudines: chelonia) populations of national park of araguaia plains, Brazil</b>]]> Shigella flexnerii and Escherichia coli were the most frequent Gram-negative bacteria found in the mouth cavity and cloacae of the turtles Podocnemis expansa and P. unifilis on beaches in the National Park of Araguaia, Brazil. Reptiles are known as Salmonella carriers, despite rarely isolated in these turtles. <![CDATA[<b>Conservation tillage, optimal water and organic nutrient supply enhance soil microbial activities during wheat (<i>Triticum Aestivum L.</i>) cultivation</b>]]> The field experiments were conducted on sandy loam soil at New Delhi, during 2007 and 2008 to investigate the effect of conservation tillage, irrigation regimes (sub-optimal, optimal and supra-optimal water regimes), and integrated nutrient management (INM) practices on soil biological parameters in wheat cultivation. The conservation tillage soils has shown significant (p<0.05) increase in soil respiration (81.1%), soil microbial biomass carbon (SMBC) (104%) and soil dehydrogenase (DH) (59.2%) compared to the conventional tillage soil. Optimum water supply (3-irrigations) enhanced soil respiration over sub-optimum and supra-optimum irrigations by 13.32% and 79% respectively. Soil dehydrogenase (DH) activity in optimum water regime has also increased by 23.33% and 8.18% respectively over the other two irrigation regimes. Similarly, SMBC has also increased by 12.14% and 27.17% respectively in soil with optimum water supply compared to that of sub-optimum and supra-optimum water regime fields. The maximum increase in soil microbial activities is found when sole organic source (50% Farm Yard Manure+25% biofertilizer+25% Green Manure) has been used in combination with the conservation tillage and the optimum water supply. Study demonstrated that microbial activity could be regulated by tillage, water and nitrogen management in the soil in a sustainable manner. <![CDATA[<b>The osmoprotective effect of some organic solutes on <i>Streptomyces</i> sp. mado2 and <i>nocardiopsis</i> sp. mado3 growth</b>]]> The response of two marine actinomycetes such as Streptomyces sp. MADO2 and Nocardiopsis sp. MADO3 to osmotic stress in minimal medium M63 and in glycerol-asparagine medium (ISP5) was studied. The two strains were moderately halophilic and the behavior of the strain Streptomyces sp. MADO2 and Nocardiopsis sp. MADO3 towards the salt stress was varied depends on the media composition and the salinity concentration. The strain Streptomyces sp. was more sensitive to salt stress than Nocardiopsis sp. The growth of both Streptomyces sp. and Nocardiopsis sp. were inhibited at 1 M NaCl irrespective of the medium used. The Nocardiopsis sp. acquired osmoadaptation on ISP5 medium whereas the Streptomyces sp. showed poor growth on M63 medium. Glycine betaine (GB), proline and trehalose played a critical role in osmotic adaptation at high osmolarity whereas at low osmolarity they showed an inhibitory effect on the bacterial growth. The present findings confirmed that GB was the powerful osmoprotectant for Streptomyces sp. and Nocardiopsis sp. grown at 1 M NaCl both in M63 and ISP5 media. <![CDATA[<b>Microbiological monitoring mf mineral water commercialized in Brazil</b>]]> The quality of mineral water commercialized in Brazil regarding the microbial content was analyzed and the results were compared with the standards established by the current legislation. Results demonstrated there was no bacterial contamination, but several types of fungi were found. Therefore, bottled mineral water could be considered a possible route for the transmission of filamentous fungi and yeasts. <![CDATA[<b>Quality of water sources used as drinking water in a Brazilian peri-urban area</b>]]> The objective of this paper was to assess bacteriological quality of drinking water in a peri-urban area located in the Metropolitan Region of São Paulo, Brazil. A total of 89 water samples were collected from community plastic tanks and 177 water samples from wells were collected bimonthly, from September 2007 to November 2008, for evaluating bacteriological parameters including: Escherichia coli, Enterococcus and heterotrophic plate count (HPC). Clostridium perfringens was investigated in a subsample (40 samples from community plastic tank and 40 from wells). E. coli was present in 5 (5.6%) samples from community plastic tanks (2.0 - 5.1x10(4) MPN/100mL) and in 70 (39.5%) well samples (2.0 - 8.6x10(4) MPN/100mL). Thus, these samples were not in accordance with the Brazilian Regulation. Enterococcus was detected in 20 (22.5%) samples of the community plastic tanks (1 to 79 NC/100mL) and in 142 (80.2%) well samples (1 to &gt;200 NC/100mL). C. perfringens was detected in 5 (12.5%) community plastic tanks samples and in 35 (87.5%) wells samples (2.2 to &gt;16 MPN/100mL). HPC were above 500 CFU/mL in 5 (5.6%) waters from community plastic tanks. In wells samples, the HPC ranged from <1 to 1.6x10(4) CFU/mL. The residual chlorine did not attend the standard established in the drinking water legislation (0.2 mg/L), except in 20 (22.5%) samples. These results confirm the vulnerability of the water supply systems in this peri-urban area what is clearly a public health concern. <![CDATA[<b>Identification and characterization of the endophytic plant growth prompter <i>Bacillus Cereus</i> strain mq23 isolated from <i>Sophora Alopecuroides</i> root nodules</b>]]> Endophytes MQ23 and MQ23R isolated from Sophora alopecuroides root nodules were characterized by observing their ability to promote plant growth and employing molecular analysis techniques. Results showed that MQ23 and MQ23R are potential N2-fixing endophytes and belong to the same species as Bacillus cereus. MQ23 was shown to be able to produce siderophores, IAA, and demonstrate certain antifungal activity to plant pathogenic fungi. Co-inoculation with MQ23+MQ23II showed a more significant effect than inoculation alone in vitro for most of positive actions suggesting they have a cooperative interaction. Results of plant inoculation with endophytes indicated that the growth indexes of co-inoculated MQ23+MQ23II were higher than those of inoculated alone (p<0.05) (the exception being for root fresh weight) when compared to negative control. There have been little of any studies of nonrhizobial putative endophytes with growth-promotion attributes in S. alopecuroides root nodules. This could be exploited as potential bio-inoculants and biocontrol agents in agriculture. <![CDATA[<b>Quick adaptation of <i>Ralstonia Solanacearum</i> to copper stress to recover culturability and growth in water and soil</b>]]> Cells of Ralstonia solanacearum were exposed to Cu in distilled water, and the resulting Cu-stressed non-culturable cells were inoculated to natural (non-pasteurized) and pasteurized soils in order to examine their culturability and recovery. Exposing the cells to 20 µM CuSO4 produced transitory non-culturable cells, which exhibited a remarkable recovery in culturability after incubation in the solution for 36 h, reaching a density near the initial level by 108 h. To determine whether such non-culturable cells actually "resuscitated" or multiplied after adapting to Cu toxicity, growth curves were constructed in order to contrast the rates of increase in culturable cell numbers between Cu-stressed or non-stressed inocula. Additionally, fresh non-stressed cells were exposed to CuSO4 in the presence of nalidixic acid by adding the antibiotic at different times after the onset of Cu stress to verify any cell multiplication during the population increase. The results revealed that the non-culturable cells surviving Cu toxicity adapted very quickly to Cu and began multiplying within 12 h, because only the Cu-stressed cells that were increasing in the exponential growth phase, but not those in the stationary phase, were killed by the antibiotic. Such cells exhibited an apparent tolerance to this metal when inoculated to a freshly prepared solution of CuSO4, and also detoxified the ion in the solution in which they grew. The presence of nutrients greatly counteracted the effect of Cu in water microcosms, since culturable cells were detected and increased in number even when exposed to 40 µM CuSO4. In contrast, when inoculated to non-pasteurized soil, Cu-stressed cells showed no such recoveries. However, when the soil was pasteurized before inoculation or added with nutrients, culturable cells were recovered and increased in number. This indicates that increased nutrient availability in soil allows Cu-stressed cells to quickly overcome the stress and increase in culturable populations. <![CDATA[<b>Identification, isolation and optimization of antifungal metabolites from the <i>Streptomyces Malachitofuscus</i> ctf9</b>]]> An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8. <![CDATA[<b>"A comparison between sugar consumption and ethanol production in wort by immobilized <i>Saccharomyces Cerevisiae, Saccharomyces Ludwigii</i> and <i>Saccharomyces Rouxii</i> on Brewer's Spent Grain"</b>]]> The immobilization of Saccharomyces cerevisiae DSM 70424, Saccharomyces ludwigii DSM 3447 and Saccharomyces rouxii DSM 2531 on brewer's spent grain and then ethanol production and sugar consumption of these immobilized yeasts were investigated. The aim of this study was to investigate the abilities of these three immobilized yeasts for producing alcohol for brewing at two temperatures (7 and 12 °C) using two different sugar levels (one at original level supplied in the brewery and one with 2.5% (w/v), added glucose to the wort). Increasing both parameters resulted in higher alcohol production by all the yeasts studied. At 7 °C and with original wort density the ethanol content at the end of fermentation was 2.7% (v/v) for S. cerevisiae, 1.7% for S. ludwigii and 2.0% for S. rouxii. After the addition of 2.5% (w/v) glucose at the same temperature (7 °C), the alcohol production was increased to 4.1, 2.8 and 4.1%, respectively. Similar improvements were observed when the fermentation was carried out at 12 °C with/without the addition of glucose to the wort. However, temperature indicated greater influence on S. ludwigii than did on S. rouxii and S. cerevisiae. The immobilization as carried out in this study impacted both S. ludwigii and S. rouxii in a way that they could consume maltose under certain conditions. <![CDATA[<b>Production flush of <i>Agaricus blazei</i> on Brazilian casing layers</b>]]> This study aimed to verify the biological efficiency and production flushes of Agaricus blazei strains on different casing layers during 90 cultivation days. Four casing layers were used: mixture of subsoil and charcoal (VCS), lime schist (LSC), São Paulo peat (SPP) and Santa Catarina peat (SCP); and two genetically distant A. blazei strains. The fungus was grown in composted substratum and, after total colonization, a pasteurized casing layer was added over the substratum, and fructification was induced. Mushrooms were picked up daily when the basidiocarp veil was stretched, but before the lamella were exposed. The biological efficiency (BE) was determined by the fresh basidiocarp mass divided by the substratum dry mass, expressed in percentage. The production flushes were also determined over time production. The BE and production flushes during 90 days were affected by the strains as well as by the casing layers. The ABL26 and LSC produced the best BE of 60.4%. Although VCS is the most used casing layer in Brazil, it is inferior to other casing layers, for all strains, throughout cultivation time. The strain, not the casing layer, is responsible for eventual variations of the average mushroom mass. In average, circa 50% of the mushroom production occurs around the first month, 30% in the second month, and 20% in third month. The casing layer water management depends on the casing layer type and the strain. Production flush responds better to water reposition, mainly with ABL26, and better porosity to LSC and SCP casing layers. <![CDATA[<b>Growth of the Ectomycorrhizal Fungus <i>Pisolithus Microcarpus</i> in different nutritional conditions</b>]]> The most important plant species employed in reforestation programs depend on ectomycorrhizal fungi for their establishment and growth. The exploitation of this symbiosis to improve forest productivity requires fungal inoculants in a large scale level. To develop such a technology it is necessary to define the optimal composition of the culture medium for each fungus. With these objectives in mind, the effect of the composition of the culture medium on biomass production of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) was studied. The original composition of two culture media, already employed for cultivation of ectomycorrhizal fungi, was submitted to several variations with the C/N ratio as the main variable. A variation of the Pridham-Gottlieb medium was the most efficient for the production of biomass. Therefore, it was submitted to a factorial assay where glucose, peptone and yeast extract components were the factors analyzed. Results showed that the glucose concentration may be increased up to 40 % in order to promote higher biomass production. Peptone had a positive effect on this variable, whereas yeast extract promoted a deleterious effect. These results indicate that it is advisable to eliminate yeast extract from the medium and replace it with peptone prior to use. <![CDATA[<b>Comparative study of two purified inulinases from thermophile <i>Thielavia Terrestris</i> NRRL 8126 and mesophile <i>Aspergillus Foetidus</i></b> <B>NRRL</B> <b>337 grown on <i>Cichorium Intybus</i> l</b>]]> Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml) and higher maximum initial velocity (115 µmol/min/mg of protein) for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. <![CDATA[<b>Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis</b>]]> We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaça and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaça samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent. <![CDATA[<b>Optimization of clavulanic acid production by <i>Streptomyces</i> daufpe 3060 by response surface methodology</b>]]> Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study. <![CDATA[<b>Isolation and characterization of a <i>Pichia anomala</i> strain</b>: <b>a promising candidate for bioethanol production</b>]]> A yeast strain designated as Y-1 was isolated and characterized from wine yeast ("Jiuqu"). Based on the morphological and biochemical results, along with the rDNA internal transcribed spacer region (ITS), Y-1 was identified to be a Pichia anomala strain. Y-1 is an ethanol-tolerant strain, enduring ethanol concentrations of up to 14 %. Y-1 growth medium conditions were optimized, results showing good growth in medium with pH ranges from 3.5-6.5, temperature ranges from 25-30 °C, and inoculums range of 8 %-12 %, while optimum growth conditions were reached at a temperature of 30 °C, pH 5.0, and inoculums of 10 %. Furthermore, when the alkaline hydrolyzed Shatian pummelo peel solutions were inoculated with 10 % Y-1 and fermented at 30 °C for 6 d, 4.7 % pure ethanol (w/w) was produced, as evidenced by gas chromatography analysis. Our present study shows potential for the Y-1 strain to be a promising candidate for bioethanol production. <![CDATA[<b>Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers</b>]]> A culture of Saccharomyces cerevisiae M30 entrapped in loofa-reinforced alginate was used for continuous ethanol fermentation in a packed-bed reactor with initial sugar concentrations of 200-248 g/L. Maximum ethanol productivity of 11.5 g/(L·h) was obtained at an ethanol concentration of 57.4 g/L, an initial sugar concentration of 220 g/L and a dilution rate (D) of 0.2 h-1. However, a maximum ethanol concentration of 82.1 g/L (productivity of 9.0 g/(L·h)) was obtained at a D of 0.11 h-1. Ethanol productivity in the continuous culture was 6-8-fold higher than that in the batch culture. Due to the developed carrier's high biocompatibility, high porosity, and good mechanical strength, advantages such as cell regeneration, reusability, altered mechanical strength, and high capacity to trap active cells in the reactor were achieved in this study. The immobilized cell reactor was successfully operated for 30 days without any loss in ethanol productivity. The average conversion yield was 0.43-0.45 throughout the entire operation, with an immobilization yield of 47.5%. The final total cell concentration in the reactor was 37.3 g/L (17.7 g/L immobilized cells and 19.6 g/L suspended cells). The concentration of suspended cells in the effluent was 0.8 g/L. <![CDATA[<b>Characterization of class 1 integrons and antibiotic resistance genes in multidrug-resistant <i>Salmonella enterica</i> isolates from foodstuff and related sources</b>]]> In recent years, an increase in the occurrence of antimicrobial resistance among Salmonella enterica has been observed in several countries, which is worrisome because S. enterica is one of the most common causes of human gastroenteritis worldwide. The aim of this study was to characterize class 1 integrons and antibiotic resistance genotypes in Salmonella enterica isolates recovered from foodstuff and related sources. Nineteen multidrug-resistant (MDR) Salmonella enterica isolates were recovered. Higher resistance rates to tetracycline (90%), streptomycin (80%), sulfamethoxazole-trimethoprim (80%), ampicillin (60%) and nalidixic acid (70%) were related to the presence of the tetA, aadA, sul1/sul2, blaTEM-1 genes, and a codon mutation at position 83 of the gyrA gene, respectively. Class 1 integrons harboring aadA, blaTEM-1, sul1 or dhfr1 genes were detected in nine (45%) Salmonella enterica strains belonging to serotypes Brandenburg, Panama, Agona, Mbandaka and Alachua. Finally, clonal dissemination of S. Panama, S. Derby and S. Mbandaka was confirmed by PFGE. Detection of clonally related MDR Salmonella enterica suggests that endemic serotypes can be supported by class 1 integron-borne gene cassettes and/or mutations in drug targets. Emergence and dissemination of multidrug-resistant Salmonella enterica can have a major public health impact in an environment where large-scale suppliers ship their products. <![CDATA[<b>Brazilian kefir</b>: <b>structure, microbial communities and chemical composition</b>]]> Microbial ecology and chemical composition of Brazilian kefir beverage was performed. The microorganisms associated with Brazilian kefir were investigated using a combination of phenotypic and genotypic methods. A total of 359 microbial isolates were identified. Lactic acid bacteria (60.5%) were the major isolated group identified, followed by yeasts (30.6%) and acetic acid bacteria (8.9%). Lactobacillus paracasei (89 isolates), Lactobacillus parabuchneri (41 isolates), Lactobacillus casei (32 isolates), Lactobacillus kefiri (31 isolates), Lactococcus lactis (24 isolates), Acetobacter lovaniensis (32 isolates), Kluyveromyces lactis (31 isolates), Kazachstania aerobia (23 isolates), Saccharomyces cerevisiae (41 isolates) and Lachancea meyersii (15 isolates) were the microbial species isolated. Scanning electron microscopy showed that the microbiota was dominated by bacilli (short and curved long) cells growing in close association with lemon-shaped yeasts cells. During the 24 h of fermentation, the protein content increased, while lactose and fat content decreased. The concentration of lactic acid ranged from 1.4 to 17.4 mg/ml, and that of acetic acid increased from 2.1 to 2.73 mg/ml. The production of ethanol was limited, reaching a final mean value of 0.5 mg/ml. <![CDATA[<b><i>Arcobacter butzleri</i></b>: <b>first isolation report from chicken carcasses in costa rica</b>]]> Arcobacter butzleri isolation from chicken carcasses in Costa Rica is reported for the first time. The isolated strains (P and R) were presumptively identified by their phenotypic characteristics. Definitive identification was made using a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. These first isolations indicate the necessity of further investigation about the prevalence, distribution, ecology and interactions with human beings of this and other Arcobacter species. <![CDATA[<b>The neem [<i>Azadirachta indica</i> a</b>: <b>juss (<i>meliaceae</i>)] oil reduction in the <i>in vitro</i> production of zearalenone by <i>Fusarium graminearum</i></b>]]> Zearalenone, a mycotoxin produced by fungi of the genus Fusarium, including F. graminearum, triggers reproduction disorders in certain animals and hyperestrogen syndromes in humans. Current research investigates three concentrations of neem oil extract (0.1, 0.25 and 0.5%) in reducing the production of zearalenone. Neem oil extract decreased zearalenone amount in the three concentrations but highest inhibition (59.05%) occurred at 0.1%. <![CDATA[<b>Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic <i>Escherichia coli</i> isolated from food and cattle in Argentina</b>]]> We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes. <![CDATA[<b>Optimization of media components for enhanced production of <i>streptococcus phocae</i> pi80 and its bacteriocin using response surface methodology</b>]]> The standard MRS components were optimized using response surface methodology for increasing yield of Streptococcus phocae PI80 viable cells and its bacteriocin. The highest amounts of bacteriocin activity and viable cells were recorded from prediction point of optimized MRS medium and achieved two fold higher (33049.8 AU.mL-1 and 14.05 LogCFU.mL-1) than un-optimized counterpart. <![CDATA[<b><i>Mycoplasmas hyorhinis</i> in different regions of cuba</b>: <b>diagnosis</b>]]> M. hyorhinis is considered one of the etiological agents of arthritis in sucking pigs, but recently as seen, some strains can produce pneumonia that could not be distinguished from the mycoplasmosis caused by M. hyopneumoniae. The study was conducted to research the presence of Mycoplasma hyorhinis (M. hyorhinis ) in different regions of the country from exudates of pig lungs with typical EP lesions. Exudates from 280 pig lungs with typical EP lesions were studied using molecular techniques such as PCR, real time PCR and amplification of the 16S-23S rRNA. It was detected that the 66% of the samples studied resulted positive to M. hyorhinis, and the presence of this species was detected in all the provinces. Amplification and studies on the intergenic region 16S-23S of M. hyorhinis rRNA demonstrated the existing variability among strains of a same species. This study is the first report on M. hyorhinis detection in Cuba. <![CDATA[<b>Tuberculosis determined by <i>Mycobacterium bovis</i> in captive waterbucks (<i>Kobus ellipsiprymnus</i>) in São Paulo, Brazil</b>]]> Two waterbucks from São Paulo Zoo Foundation exhibited respiratory symptoms in July 2004. After euthanasia, granulommas in lungs and mediastinic lymph nodes were observed. Acid-fast bacilli isolated were identified as Mycobacterium bovis spoligotype SB0121 by PRA and spoligotyping. They were born and kept in the same enclosure with the same group, without any contact to other species housed in the zoo. This is the first detailed description of M. bovis infection in Kobus ellipsiprymnus. <![CDATA[<b>Production of reactive oxygen</b> <b>(H<sub>2O2</sub>) and nitrogen (NO) intermediates and TNF-</b>α <b>in mice genetically selected for high (H) and low (L) antibody response and experimentally infected with <i>Leptospira</i> serovar pomona</b>]]> The aim of the present study was to evaluate the activity of macrophages, and the production of TNF-α and antibodies against experimental infection by Leptospira serovar Pomona in mice genetically selected for High (H) or Low (L) humoral immune response. To evaluate macrophagic activity, peritoneal and splenic lavages were performed for determination of oxygen (H2O2) and nitrogen (NO) intermediates. The production of the tumor necrosis factor (TNF-α) was investigated through bioassays in serum and homogenates of splenic and hepatic cells of control and infected animals, as was as specific antibodies production. The immune response against serovar Pomona in those lines, was characterized by high antibody production, especially in later periods of the infectious process, whereas values of bacterial recovery in culture medium were lower. The production of reactives oxygen and nitrogen intermediate, also helped to eliminate Leptospira Pomona in both lines; H2O2 production an important factor in H IV-A, as well as NO production in L IV-A, especially in later post-inoculation periods. The same was detected for TNF-α. Results suggest that such lines could be an important model to investigate the pathogenesis and the immune response of animals against the several Leptospira serovars. <![CDATA[<b>Identification and antimicrobial resistance of microflora colonizing feral pig (<i>Sus scrofa</i>) of Brazilian Pantanal</b>]]> Antimicrobial resistance of bacteria is a worldwide problem affecting wild life by living with resistant bacteria in the environment. This study presents a discussion of outside factors environment on microflora of feral pigs (Sus scrofa) from Brazilian Pantanal. Animals had samples collected from six different body sites coming from two separated geographic areas, Nhecolandia and Rio Negro regions. With routine biochemical tests and commercial kits 516 bacteria were identified, with 240 Gram-positive, predominantly staphylococci (36) and enterococci (186) strains. Among Gram-negative (GN) bacteria the predominant specimens of Enterobacteriaceae (247) mainly represented by Serratia spp. (105), Escherichia coli (50), and Enterobacter spp. (40) and specimens not identified (7). Antimicrobial susceptibility was tested against 17 drugs by agar diffusion method. Staphylococci were negative to production of enterotoxins and TSST-1, with all strains sensitive towards four drugs and highest resistance toward ampicillin (17%). Enterococci presented the highest sensitivity against vancomycin (98%), ampicillin (94%) and tetracycline (90%), and highest resistance pattern toward oxacillin (99%), clindamycin (83%), and cotrimoxazole (54%). In GN the highest resistance was observed with Serratia marcescens against CFL (98%), AMC (66%) and AMP (60%) and all drugs was most effective against E. coli SUT, TET (100%), AMP, TOB (98%), GEN, CLO (95%), CFO, CIP (93%). The results show a new profile of oxacillin-resistant enterococci from Brazilian feral pigs and suggest a limited residue and spreading of antimicrobials in the environment, possibly because of low anthropogenic impact reflected by the drug susceptibility profile of bacteria isolated. <![CDATA[<b>Immune response to dna vaccine expressing transferrin binding protein a gene of<i> Pasteurella multocida</i></b>]]> Haemorrhagic Septicaemia (HS), an acute and fatal disease of cattle and buffalo is primarily caused by serotype B:2 or E:2 of Pasteurella multocida. The transferrin binding protein A (TbpA) has been found to act as immunogen and potent vaccine candidate in various Gram negative bacteria including P. multocida. The present study was carried out to evaluate the potential of this antigen as a DNA vaccine against HS in mice model. The tbpA gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and along with murine IL2 gene as immunological adjuvant to produce monocistronic and bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines was evaluated based on serum antibody titres and lymphocyte proliferation assay. A significant increase in humoral and cell mediated immune responses was observed in mice vaccinated with DNA vaccines as compared to non immunized group. Additionally, the bicistronic DNA vaccine provided superior immune response and protection level following challenge as compared to monocistronic construct. The study revealed that DNA vaccine presents a promising approach for the prevention of HS. <![CDATA[<b>Identification of fungi of the genus <i>Aspergillus</i> section <i>nigri</i> using polyphasic taxonomy</b>]]> In spite of the taxonomy of the Aspergillus species of the Nigri Section being regarded as troublesome, a number of methods have been proposed to aid in the classification of this Section. This work aimed to distinguish Aspergillus species of the Nigri Section from foods, grains and caves on the basis in Polyphasic Taxonomy by utilizing morphologic and physiologic characters, and sequencing of ß-tubulin and calmodulin genes. The morphologic identification proved useful for some species, such as A. carbonarius and Aspergillus sp UFLA DCA 01, despite not having been totally effective in elucidating species related to A. niger. The isolation of the species of the Nigri Section on Creatine Sucrose Agar (CREA) enabled to distinguish the Aspergillus sp species, which was characterized by the lack of sporulation and by the production of sclerotia. Scanning Electron microscopy (SEM) allowed distinguishing the species into two distinct groups. The production of Ochratoxin A (OTA) was only found in the A. carbonarius and A. niger species. The sequencing of β-tubulin gene was efficient in differing most of the Aspergillus species from the Nigri Section with the exception of Aspergillus UFLA DCA 01, which could not be distinguished from A. costaricaensis. This species is morphologically similar to A. costaricaencis for its low sporulation capacity and high sclerotia production, but it differs morphologically from A. costaricaensis for its conidial ornamentation and size of vesicles. Equally, based on partial calmodulin gene sequence data Aspergillus UFLA DCA 01 differs from A. costaricaensis. <![CDATA[<b>Assessment of the quality of dna extracted by two techniques from <i>Mycobacterium tuberculosis</i> for fast molecular identification and genotyping</b>]]> We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping. <![CDATA[<b>Variations in the sensitivity of different primers for detecting <i>Wolbachia</i> in <i>Anastrepha</i> (diptera: tephritidae)</b>]]> Wolbachia are endosymbiont bacteria of the family Rickettsiacea that are widespread in invertebrates and occur between 20% and 60% of Neotropical insects. These bacteria are responsible for reproductive phenomena such as cytoplasmic incompatibility, male killing, feminization and parthenogenesis. Supergroups A and B of Wolbachia are common in insects and can be identified using primers for 16S rDNA, ftsZ and wsp; these primers vary in their ability to detect Wolbachia. The ftsZ primer was the first primer used to detect Wolbachia in Anastrepha fruit flies. The primers for 16S rDNA, ftsZ and wsp and the corresponding PCR conditions have been optimized to study the distribution of Wolbachia and their effect on the biology of Anastrepha in Brazil. In this work, we examined the ability of these primers to detect Wolbachia in Anastrepha populations from three regions in the State of São Paulo, southeastern Brazil. All of the samples were positive for Wolbachia supergroup A when screened with primers for 16S A rDNA and wsp A; the wsp B primer also gave a positive result, indicating cross-reactivity. The ftsZ primer showed a poor ability to detect Wolbachia in Anastrepha and generated false negatives in 44.9% of the samples. These findings indicate that reliable PCR detection of Wolbachia requires the use of primers for 16S rDNA and wsp to avoid cross-reactions and false negatives, and that the ftsZ primer needs to be redesigned to improve its selectivity. <![CDATA[<b>Diversity and uncommon HPV types in HIV seropositive and seronegative women attending an STI clinic</b>]]> Given the causal relationship between specific types of HPV with cervical cancer and precursor lesions, it is important to identify the viral type involved. The aim of this study is to access the prevalence of HPV types in HIV seropositive and seronegative women. Accordingly, 77 HPV positive cervical samples were obtained from 284 women (seropositive (n=112) and seronegative (n=172) for HIV) who attended a Sexually Transmitted Infection clinic, in Vitoria, Southeastern Brazil. Viral DNA was amplified by PCR using MY09/MY11 degenerated primers and the genotyping was performed by Restriction Fragment Length Polymorphism. Seventy five out of the 77 HPV samples were genotyped: 6, 11, 13, 16, 18, 26, 31, 31b, 32, 33, 34, 35, 52, 53, 55, 56, 58, 59, 61, 62, 64, 66, 71, 81, 83, 84. The most prevalent type was HPV16 followed by HPV types 6, 11 and 53. Fifty five percent and 45% belonged to high and low risk types, respectively. High risk types corresponded to 59% and 54.5% of the HPV detected in HIV seronegative and seropositive women, respectively. The uncommon HPV 13 type in cervical samples was also observed in this study. The oncogenic types were more common in the HIV seronegative samples and the number of cases with multiple infections was similar for the two groups. HPV typing is not only important clinically for the establishment of monitoring and treatment of a patient, it also provides knowledge of the viral types circulating in a population, which is of interest in the development of prevention and treatment programs for this disease. <![CDATA[<b>Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen <i>Cylindrocarpon Destructans</i></b>]]> Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi's orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters resulted in high levels of enzyme (PG and PL) production after ten days of incubation at a pH of 5.0 at 25°C in the presence of 1.5% pectin. Among different nitrogen sources, urea and peptone showed high production of PG and PL, respectively. The enzyme production and mycelial growth seems to have direct influence on the culture conditions; therefore, at stationary state high enzyme production and mycelial growth were obtained than agitation state. Along with this, optimization of enzyme activity was also determined using various physiological parameters like, temperature, incubation time and pH. Taguchi's data was also analyzed using one step ANOVA statistical method. <![CDATA[<b><i>Mycobacterium avium subsp. Paratuberculosis</i></b><b> and the expression of selected virulence and pathogenesis genes in response to 6°c, 65°c and ph 2.0</b>]]> The aim of this work was to study the expression of selected Mycobacterium avium subsp. paratuberculosis (MAP) genes connected with MAP virulence, adhesion and stress response. The temperature of 6°C and 65°C were chosen with regard to the food industry, storage conditions (refrigerator) and low-temperature pasteurization. A pH of 2.0, using lactic acid, was selected to mimic the natural environment of the stomach. Expression of selected genes was studied using real time reverse transcription PCR on three different MAP isolates. MAP isolates were chosen according to the number of their preceding cultivations. While isolates 8672 and 8819 were previously cultivated only once, MAP isolate 12146 went through four passages. Different expression profiles were observed in each of the three MAP isolates. However, particular similar patterns were observed. SigE, sigF and ahpC were up-regulated, while sigL was down-regulated under temperature stress. Mmp gene was found to be down-regulated under acidic conditions. Low passage isolates (8672 and 8819) showed certain level of acid resistance. <![CDATA[<b>Optimization of growth medium for protease production by <i>Haloferax Lucentensis</i> VKMM 007 by response surface methodology</b>]]> The production of halophilic thermostable protease by Haloferax lucentensis VKMM 007 was optimized using a statistical approach. In accordance with factorial design, soluble starch, gelatin, KCl and MgSO4 were selected among 27 variables tested. Next, a second-order quadratic model was estimated and optimal medium concentrations were determined based on quadratic regression equation generated by model. These were 5.14 g L-1 of KCl, 6.57 g L-1of MgSO4, 9.05 g L-1of gelatin and 5.27 g L-1of soluble starch in high salts media supplemented with 0.5% (w/v) of beef extract and peptone, respectively. In these optimal conditions, the obtained protease concentration of 6.80 U mL-1 was in agreement with the predicted protease concentration and was further improved to 7.02 U mL-1 by increasing the concentration of NaCl in the medium to 25% (w/v). An overall 4.0-fold increase in protease production was achieved in the optimized medium compared to activity obtained in initial medium. <![CDATA[<b>ERRATA - vol.42 no.1 São Paulo Jan./Mar. 2011</b>]]> The production of halophilic thermostable protease by Haloferax lucentensis VKMM 007 was optimized using a statistical approach. In accordance with factorial design, soluble starch, gelatin, KCl and MgSO4 were selected among 27 variables tested. Next, a second-order quadratic model was estimated and optimal medium concentrations were determined based on quadratic regression equation generated by model. These were 5.14 g L-1 of KCl, 6.57 g L-1of MgSO4, 9.05 g L-1of gelatin and 5.27 g L-1of soluble starch in high salts media supplemented with 0.5% (w/v) of beef extract and peptone, respectively. In these optimal conditions, the obtained protease concentration of 6.80 U mL-1 was in agreement with the predicted protease concentration and was further improved to 7.02 U mL-1 by increasing the concentration of NaCl in the medium to 25% (w/v). An overall 4.0-fold increase in protease production was achieved in the optimized medium compared to activity obtained in initial medium.