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Brazilian Journal of Microbiology, Volume: 43, Número: 3, Publicado: 2012
  • Bioprospecting thermophiles for cellulase production: a review Review

    Acharya, Somen; Chaudhary, Anita

    Resumo em Inglês:

    Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.
  • Challenges in shrimp aquaculture due to viral diseases: distribution and biology of the five major penaeid viruses and interventions to avoid viral incidence and dispersion Review

    Seibert, Caroline H.; Pinto, Aguinaldo R.

    Resumo em Inglês:

    Shrimp aquaculture has been dramatically affected by many pathogenic diseases, mainly caused by five viruses: IHHNV, YHV, TSV, WSSV, and IMNV. Here we provide a state-of-the-art overview of these shrimp viruses, with emphasis on distribution, pathology, morphology, and genomic organization, in addition to current diagnostic methods and intervention practices.
  • Biodegradation of petroleum hydrocarbons in hypersaline environments Review

    Martins, Luiz Fernando; Peixoto, Raquel Silva

    Resumo em Inglês:

    Literature on hydrocarbon degradation in extreme hypersaline media presents studies that point to a negative effect of salinity increase on hydrocarbonoclastic activity, while several others report an opposite tendency. Based on information available in the literature, we present a discussion on the reasons that justify these contrary results. Despite the fact that microbial ability to metabolize hydrocarbons is found in extreme hypersaline media, indeed some factors are critical for the occurrence of hydrocarbon degradation in such environments. How these factors affect hydrocarbon degradation and their implications for the assessment of hydrocarbon biodegradation in hypersaline environments are presented in this review.
  • A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida dubliniensis Medical Microbiology

    Ge, Yi Ping; Wang, Le; Lu, Gui Xia; Shen, Yong Nian; Liu, Wei Da

    Resumo em Inglês:

    Candida dubliniensis is an emerging pathogen capable of causing superficial as well as systemic infections. Due to its close similarity to C. albcians, conventional methods based on phenotypic traits are not always reliable in identification of C. dubliniensis. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay to identify and discriminate between the two closely related species. The D1/D2 region of 28S rDNA was amplified by PCR and enzymatically digested by ApaI and BsiEI respectively. PCR products of both species were digested into two fragments by ApaI, but those of other yeast species were undigested. BsiEI cut the PCR products of C. albicans into two fragments but not those of C. dubliniensis. Thus two species were differentiated. We evaluated 10 reference strains representing 10 yeast species, among which C. albicans and C. dubliniensis were successfully identified. A total of 56 phenotypically characterized clinical isolates (42 C. albicans isolates and 14 C. dubliniensis isolates) were also investigated for intra-species variability. All tested isolates produced identical RFLP patterns to their respective reference strains except one initially misidentified isolate. Our method offers a simple, rapid and reliable molecular method for the identification of C. albicans and C. dubliniensis.
  • Occurrence of killer Candida glabrata clinical isolates Medical Microbiology

    O, Arroyo-Helguera; Alejandro, De Las Penas; Irene, Castaño

    Resumo em Inglês:

    In this work we characterized the occurrence of killer activity in 64 Candida glabrata clinical isolates under different conditions. We found that only 6.25 % of the clinical isolates tested were positive for killer activity against a Saccharomyces cerevisiae W303 sensitive strain. Sensitivity of killer activity to different values of pH and temperatures was analyzed. We found that the killer activity presented by all isolates was resistant to every pH and temperature tested, although optimal activity was found at a range of pH values from 4 to 7 and at 37ºC. We did not observe extrachromosomal genetic elements associated with killer activity in any of the positive C. glabrata isolates. The killer effect was due to a decrease in viability and DNA fragmentation in sensitive yeast.
  • Subtyping of Chilean Methicillin-Resistant Staphylococcus aureus strains carrying the staphylococcal cassette chromosome mec type I Medical Microbiology

    Medina, Gustavo; Otth, Carola; Otth, Laura; Fernández, Heriberto; Muñoz, Celeste; Cruz, María; Zaror, Ángela; Henriquez, Ruby; Arce, Maria; Wilson, Myra

    Resumo em Inglês:

    The cassette chromosome mec (SCCmec) present in methicillin-resistant Staphylococcus aureus (MRSA) has two essential components, the ccr gene complex and the mec gene complex. Additionally, SCCmec has non-essential components called J regions which are used for MRSA subtyping. This study was performed to determine subtypes MRSA strains carrying SCCmec type I based on polymorphism of regions located downstream of the mecA gene. A total of 98 MRSA strains carrying SCCmec type I isolated from patients hospitalized at the County Hospital of Valdivia (Chile) between May 2007 and May 2008, were analyzed by multiplex PCR designed to amplify the mecA gene and 7 DNA hypervariable regions located around the mecA gene. MRSA strains were classified into seventeen genotypes accordingly to amplification patterns of DNA hypervariable regions. Five genotypes showed amplification patterns previously described. The remaining twelve genotypes showed new amplification patterns. Genotypes 18 and Genotype 19 were the most frequently detected. Regions HVR, Ins117 and pI258 stand out as being present in more than 60% of tested isolates. The acquisition of hypervariable regions by MRSA is a continuous horizontal transfer process through which the SCCmec have been preserved intact, or even may give rise to new types and subtypes of SCCmec. Therefore it is possible to infer that most MRSA strains isolated at the County Hospital of Valdivia (Chile) were originated from two local clones which correspond to Genotype 18 and Genotype 19.
  • Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains Medical Microbiology

    Giudice, Mauro Cintra; Reis-Menezes, Adriana Araújo; Rittner, Glauce Mary Gomes; Mota, Adolfo José; Gambale, Walderez

    Resumo em Inglês:

    A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.
  • Evaluation of diagnostic methods for the detection of Helicobacter pylori in gastric biopsy specimens of dyspeptic patients Medical Microbiology

    Ramis, Ivy Bastos; Moraes, Ernani Pinho de; Fernandes, Márcia Silveira; Mendoza-Sassi, Raul; Rodrigues, Obirajara; Juliano, Carlos Renan Varela; Scaini, Carlos James; Silva, Pedro Eduardo Almeida da

    Resumo em Inglês:

    Helicobacter pylori infects nearly 50% of the world's population. This microorganism is accepted as the most important agent of gastritis and as a risk factor for peptic ulcer disease and gastric adenocarcinoma. Currently many diagnostic methods exist for detecting H. pylori, however they all have limitations, thus it is recommend a combination of at least two methods. The aim of this study was to evaluate diagnostic methods, such as in-house urease test, culture and Polymerase Chain Reaction (PCR), for the detection of the H. pylori in gastric biopsy specimens of 144 dyspeptic patients, using as gold standard the association between histology and rapid urease test. According to the gold standard used in this study, 48 (33.3%) patients were infected with H. pylori, while 96 (66.7%) were classified as not infected. The in-house urease test and the PCR were the most sensitive methods (100%), followed by culture (85.4%). However, the in-house urease test and the culture were the most specific (100%), followed by PCR (75%). In conclusion, this study showed that, in comparison with the combination of histology and rapid urease test, the in-house urease test and the PCR presented 100% of sensitivity in the diagnosis of gastric infection by H. pylori, while the in-house urease test and the culture reached 100% of specificity. These finding suggest that the combination of two or more methods may improve the accuracy of the H. pylori detection.
  • Occurrence of periodontal pathogens among patients with chronic periodontitis Medical Microbiology

    Farias, B.C.; Souza, P.R.E.; Ferreira, B.; Melo, R.S.A.; Machado, F.B.; Gusmão, E.S.; Cimões, R.

    Resumo em Inglês:

    The aim of the present study was to evaluate the presence of the periodontal pathogens that form the red complex (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) and Aggregatibacter actinomycetemcomitans in patients with chronic periodontitis. The sample consisted of 29 patients with a clinical and radiographic diagnosis of chronic periodontitis based on the criteria of the American Academy of Periodontology (3). Samples for microbiological analysis were collected from the four sites of greatest probing depth in each patient, totaling 116 samples. These samples were processed using conventional polymerase chain reaction, which achieved the following positive results: 46.6% for P. gingivalis, 41.4% for T. forsythia, 33.6% for T. denticola and 27.6% for A. actinomycetemcomitans. P. gingivalis and T. forsythia were more prevalent (p < 0.05) in periodontal pockets ≥ 8 mm. The combinations T. forsythia + P. gingivalis (23.2%) and T. forsythia + P. gingivalis + T. denticola (20.0%) were more frequent in sites with a probing depth ≥ 8 mm. Associations with the simultaneous presence of A. actinomycetemcomitans + P. gingivalis, A. actinomycetemcomitans + T. forsythia, P. gingivalis + T. forsythia and T. forsythia + T. denticola were statistically significant (p < 0.05). It was concluded that the red complex pathogens are related to chronic periodontitis, presenting a higher occurrence in deep periodontal pockets. Moreover, the simultaneous presence of these bacteria in deep sites suggests a symbiotic relationship between these virulent species, favoring, in this way, a further progression of periodontal disease.
  • Isolation of the first IMP-4 metallo-β-lactamase producing Klebsiella pneumoniae in Tianjin, China Medical Microbiology

    Li, Jing; Hu, Zhidong; Hu, Qiaojuan

    Resumo em Inglês:

    This study shows for the first time the mechanism of carbapenem resistance of a Klebsiella pneumoniae clinical isolate TJ8 recovered from Tianjin medical university general hospital ,China. The modified Hodge test and EDTA synergy test were performed for the screening of carbapenemases and metallo-β-lactamases (MBLs), respectively. Polymerase chain reactions and DNA sequencing confirmed that the strain carried IMP-4 metallo-β-lactamase (MBL) , SHV-11 and TEM-1 β-lactamase. ClassⅠintegron was positive and gave a 3.0-kb PCR amplicon .IMP-4 was located in ClassⅠintegron 5'CS. The gene determinants were organized in the order of blaIMP-4-orfII-orfIII.In all, the results show that IMP-4 MBL production caused the TJ8 resistance to carbapenems.
  • Carriage frequency, phenotypic and genotypic characteristics of Staphylococcus aureus isolated from dialysis and kidney tranplant patients at a hosptial in northern Paraná Medical Microbiology

    Giarola, Luciana Borges; Santos, Rosiane Ribeiro dos; Tognim, Maria Cristina Bronharo; Borelli, Sueli Donizete; Bedendo, João

    Resumo em Inglês:

    The objective of the present study was to determine the frequency of Staphylococcus aureus nasal carriage among dialysis and kidney transplant patients, to identify the antimicrobial resistance profile of these strains and to verify their genetic profiles with the RW3A primer. The study included 159 individuals, comprising 111 dialysis and 48 kidney transplant patients. Of the 48 transplant patients, 75% were positive for S. aureus, whereas 49% of the 111 dialysis patients were carriers. Two samples yielded conflicting results for oxacillin sensitivity between the disk diffusion and minimum inhibitory concentration (MIC) assays: both were sensitive by the disk diffusion assay and resistant by MIC (4 μg/ml). In the antibiogram by disk diffusion, ten samples were resistant to cefoxitin, among which eight were also resistant to oxacillin. The resistance of the ten samples to cefoxitin by the disk diffusion assay was confirmed by MIC. Of the ten oxacillin-resistant samples, eight harbored the mecA gene. All samples were sensitive to vancomycin, and most were resistant to penicillin and demonstrated high rates of resistance to the other antimicrobials tested. The samples from dialysis patients exhibited a more homogenous genetic profile. Among the samples with a high percent similarity, no correlation with sensitivity or resistance to oxacillin was observed. According to the results of this study, the implementation of prevention and control measures, such as increased restrictions on prescriptions for antimicrobial drugs and nasal decontamination prior to high-risk procedures, is recommended.
  • Detection of Mogibacterium timidum in subgingival biofilm of aggressive and non-diabetic and diabetic chronic periodontitis patients Medical Microbiology

    Casarin, Renato Corrêa Viana; Saito, Daniel; Santos, Vanessa Renata; Pimentel, Suzana Peres; Duarte, Poliana Mendes; Casati, Márcio Zaffalon; Gonçalves, Reginaldo Bruno

    Resumo em Inglês:

    The aim of the present study was to evaluate the frequency of detection of Mogibacterium timidum in subgingival samples of subjects with generalized aggressive periodontitis (GAgP) and uncontrolled diabetic and non-diabetic subjects with generalized chronic periodontitis (GChP). 48 patients with GAgP, 50 non-diabetic and 39 uncontrolled (glycated hemoglobin >7%) type 2 diabetic subjects with GChP were enrolled in this study. Subgingival biofilm were collected from deep pockets (probing depth > 7 mm). After DNA extraction, M. timidum was detected by Nested Polymerase Chain Reaction and chi-square test was used to data analysis (p>0.05). There were no differences in the frequency of detection of M. timidum between subjects with GAgP (35%) and non-diabetic subjects with GChP (40%) (p>0.05). The frequency of detection of M. timidum was significantly higher in deep pockets of diabetic subjects with GChP (56%) when compared to GAgP (p<0.05), but similar to non-diabetic subjects with GChP (p>0.05). The frequency of detection of M. timidum was higher in subjects GChP presenting uncontrolled type 2 diabetes mellitus, when compared to GAgP subjects.
  • Bactericidal antibiotic-phytochemical combinations against methicillin resistant Staphylococcus aureus Medical Microbiology

    Kyaw, Bhone Myint; arora, Shuchi; Lim, Chu Sing

    Resumo em Inglês:

    Methicillin resistant Staphylococcus aureus (MRSA) infection is a global concern nowadays. Due to its multi-drug resistant nature, treatment with conventional antibiotics does not assure desired clinical outcomes. Therefore, there is a need to find new compounds and/or alternative methods to get arsenal against the pathogen. Combination therapies using conventional antibiotics and phytochemicals fulfill both requirements. In this study, the efficacy of different phytochemicals in combination with selected antibiotics was tested against 12 strains of S. aureus (ATCC MRSA 43300, ATCC methicillin sensitive S. aureus or MSSA 29213 and 10 MRSA clinical strains collected from National University Hospital, Singapore). Out of the six phytochemicals used, tannic acid was synergistic with fusidic acid, minocycline, cefotaxime and rifampicin against most of strains tested and additive with ofloxacin and vancomycin. Quercetin showed synergism with minocycline, fusidic acid and rifampicin against most of the strains. Gallic acid ethyl ester showed additivity against all strains in combination with all antibiotics under investigation except with vancomycin where it showed indifference effect. Eugenol, menthone and caffeic acid showed indifference results against all strains in combination with all antibiotics. Interestingly, no antagonism was observed within these interactions. Based on the fractional inhibitory concentration indices, synergistic pairs were further examined by time-kill assays to confirm the accuracy and killing rate of the combinations over time. The two methods concurred with each other with 92% accuracy and the combinatory pairs were effective throughout the 24 hours of assay. The study suggests a possible incorporation of effective phytochemicals in combination therapies for MRSA infections.
  • Diphtheria Antibodies and T lymphocyte Counts in Patients Infected with HIV-1 Medical Microbiology

    Speranza, Francisco A. B.; Ishii, Solange K.; Thuler, Luiz C. S.; Damasco, Paulo V.; Hirata Jr, Raphael; Mattos-Guaraldi, Ana L.; Milagres, Lucimar G.

    Resumo em Inglês:

    We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.
  • Comparison of primers for RAPD-PCR from environmental isolates of Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii complex Medical Microbiology

    Pedroso, Reginaldo dos Santos; Ferreira, Joseane Cristina; Costa, Karen Regina Carim da; Candido, Regina Celia

    Resumo em Inglês:

    Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e.g., CAV2 for C. albidus) may not to be applied to some species.
  • Antimicrobial activity of meta-alkoxyphenylcarbamates containing substituted N-phenylpiperazine fragment Medical Microbiology

    Malík, Ivan; Bukovský, Marián; Andriamainty, Fils; Gališinová, Jana

    Resumo em Inglês:

    In the present investigation, the basic esters of meta-alkoxyphenylcarbamic acid bearing variously substituted N-phenylpiperazine fragment were screened for their in vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans, respectively. The most effective against Escherichia coli was found the compound 6d (MIC=195,3 μg/mL) bearing simultaneously para-fluoro substituent at the 4‑phenylpiperazin-1-yl core and meta-methoxy side chain in the lipophilic part of the molecule. From whole analyzed set of the molecules the substance 8e with propoxy side chain forming meta-alkoxyphenylcarbamoyl fragment and lipophilic, sterically bulky meta-trifluoromethyl group attached at N-phenylpiperazine moiety was evaluated as the most active against Candida albicans (MIC=97,7 μg/mL). On the contrary, all investigated structures were practically inactive against Staphylococcus aureus (MIC>1000 μg/mL)
  • A prospective study on Aeromonas in outpatients with diarrhea in the central region of Rio Grande do Sul State Medical Microbiology

    Prediger, Karoline de Campos; Pereira, Renata da Silva; Winckler Neto, Carlos Hugo Del Priore; Santos, Roberto Christ Vianna; Fadel-Picheth, Cyntia Maria Telles; Vizzotto, Bruno Stefanello

    Resumo em Inglês:

    Aeromonas spp. were identified in five (2,7%) of 182 diarrheal stool cultures, A. caviae was predominant, resistant mainly to ampicillin and cephalotin. This is the first study showing the presence of Aeromonas spp. in diarrheal stools of outpatients in the central region of Rio Grande do Sul State, Brazil.
  • Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of Enterotoxigenic Escherichia coli and CfaE-R181A mutant Medical Microbiology

    Liaqat, Iram

    Resumo em Inglês:

    Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/ CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacIq/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacIq/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.
  • Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis Medical Microbiology

    Macedo, Maíra Bidart; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; Silva, Pedro Eduardo Almeida da; Martin, Anandi

    Resumo em Inglês:

    We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.
  • Preparation, characterization and in vitro antimicrobial activity of liposomal ceftazidime and cefepime against Pseudomonas aeruginosa strains Medical Microbiology

    Torres, Ieda Maria Sapateiro; Bento, Etiene Barbosa; Almeida, Larissa da Cunha; Sá, Luisa Zaiden Carvalho Martins de; Lima, Eliana Martins

    Resumo em Inglês:

    Pseudomonas aeruginosa is an opportunistic microorganism with the ability to respond to a wide variety of environmental changes, exhibiting a high intrinsic resistance to a number of antimicrobial agents. This low susceptibility to antimicrobial substances is primarily due to the low permeability of its outer membrane, efflux mechanisms and the synthesis of enzymes that promote the degradation of these drugs. Cephalosporins, particularty ceftazidime and cefepime are effective against P. aeruginosa, however, its increasing resistance has limited the usage of these antibiotics. Encapsulating antimicrobial drugs into unilamellar liposomes is an approach that has been investigated in order to overcome microorganism resistance. In this study, antimicrobial activity of liposomal ceftazidime and cefepime against P. aeruginosa ATCC 27853 and P. aeruginosa SPM-1 was compared to that of the free drugs. Liposomal characterization included diameter, encapsulation efficiency and stability. Minimum Inhibitory Concentration (MIC) was determined for free and liposomal forms of both drugs. Minimum Bactericidal Concentration (MBC) was determined at concentrations 1, 2 and 4 times MIC. Average diameter of liposomes was 131.88 nm and encapsulation efficiency for cefepime and ceftazidime were 2.29% end 5.77%, respectively. Improved stability was obtained when liposome formulations were prepared with a 50% molar ratio for cholesterol in relation to the phospholipid. MIC for liposomal antibiotics for both drugs were 50% lower than that of the free drug, demonstrating that liposomal drug delivery systems may contribute to increase the antibacterial activity of these drugs.
  • In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China Veterinary Microbiology

    Dawei, Guo; Liping, Wang; Chengping, Lu

    Resumo em Inglês:

    Streptococcus suis is a swine pathogen and also a zoonotic agent. The formation of biofilms allows S. suis to become persistent colonizers and resist clearance by the host immune system and antibiotics. In this study, biofilm forming potentials of various S. suis strains were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and tissue culture plates stained with crystal violet. In addition, the effects of five antimicrobial agents on biofilm formation were assayed in this study. S. suis produced biofilms on smooth and rough surface. The nutritional contents including glucose and NaCl in the growth medium modulated biofilm formation. There was a significant difference in their biofilm-forming ability among all 46 S. suis strains. The biofilm-forming potential of S. suis serotype 9 was stronger than type 2 and all other types. However, biofilm formation was inhibited by five commonly used antimicrobial agents, penicillin, erythromycin, azithromycin, ciprofloxacin, and ofloxacin at subinhibitory concentrations, among which inhibition of ciprofloxacin and ofloxacin was stronger than that of other three antimicrobial agents.Our study provides a detailed analysis of biofilm formation potential in S. suis, which is a step towards understanding its role in pathogenesis, and eventually lead to a better understanding of how to eradicate S. suis growing as biofilms with antibiotic therapy.
  • Isolation and characterization of canine parvovirus type 2c (CPV-2c) from symptomatic puppies Veterinary Microbiology

    Puentes, R; Eliopulos, N; Pérez, R; Franco, G; Sosa, K; Bianchi, P; Furtado, A; Hübner, S.O.; Esteves, P.A.

    Resumo em Inglês:

    Canine parvovirus type 2 (CPV-2) is a leading cause of diarrhea in puppies in several parts of the world. In this study CPV-2 was detected and recovered from puppies showing clinical disease from Montevideo, Uruguay. Samples were processed and used to infect CRFK and MDCK cells in order to isolate the virus. Out of twelve, two samples were positive for CPV-2. A genomic region of 583 bp was amplified and the molecular characterization was performed by sequencing, phylogenetic analysis and Restriction Fragment Length Polymorphism (RFLP). Two isolated viruses (UY1 and UY2) were CPV-2c-like viruses. The comparison between the cytophatic effect (CPE) of CPV-2 (vaccinal virus) and CPV-2c (isolated virus) on primary canine cells cultures and on CRFK line cells, demonstrated that CPV-2c is less citopathogenic in CRFK than in primary cultures. Our study represents the first report on isolation and characterization of canine parvovirus type 2c (CPV-2c) in cell cultures from South American dogs.
  • Genotypic and phenotypic detection of capsular polysaccharides in Staphylococcus aureus isolated from bovine intramammary infections in Argentina Veterinary Microbiology

    Camussone, C.; Rejf, P.; Pujato, N.; Schwab, A.; Marcipar, I.; Calvinho, L.F.

    Resumo em Inglês:

    Staphylococcus aureus (n=157) isolated from intramammary infections in Argentine dairy areas were evaluated for presence of cap5 and cap8 loci. Isolates carrying cap5 and cap8 were serotyped using specific antisera. Sixty four percent of the isolates were genotyped as cap5 or cap8 and 50% of them expressed CP5 or 8.
  • Partial VP1 sequencing of Brazilian infectious bursal disease virus strains Veterinary Microbiology

    Fernandes, Maria Judite Bittencourt; Simoni, Isabela Cristina; Harakava, Ricardo; Rivas, Eliana Borges; Arns, Clarice Weis

    Resumo em Inglês:

    Infectious bursal disease virus (IBDV) is classified according to the antigenicity and virulence into classical virulent (cv), very virulent (vv), and antigenic variant strains. The molecular basis for the IBDV antigenic variation is well established and is associated to the capsid protein, VP2 (gene VP2 of segment A), whereas both VP2 and the RNA-dependent RNA polymerase, VP1 (gene VP1 of segment B), have been correlated with the virulence. In this study, seventeen Brazilian IBDV samples previously characterized by the VP2 gene as cv (three) and vv (fourteen) strains were genetically and molecularly analyzed for their VP1 gene. All of the strains kept with the same cv or vv classification except one sample, Br/03/DR. This sample was classified as vv by its VP2 gene, but it was most closely related to the cv strains by its VP1 partial sequence and phylogeny. Studies on the phylogeny of VP1 have suggested a possible reassortment event that originated the vvVP1. In this case, the sample carrying vvVP2 and cvVP1 could be a descendant of IBDV ancestors prior to the reassortment of vvVP1; alternatively, it could be the result of a genetic exchange between the segments of different strains or with a live attenuated vaccine. Nevertheless, this is the first report of natural genetic reassortment of IBDV in Brazil.
  • Detection of Genetic characterization of Porcine circovirus 2 (PCV2) in Brazilian wildlife boars Veterinary Microbiology

    Castro, A.M.M.G.; Castro Jr., F.G.; Budiño, F.E.L.; Baldin, C.M.; Silva, S.O.S.; Brandão, P.E.; Richtzenhain, L.J.

    Resumo em Inglês:

    A semi-intensive wildlife boars farm presented a clinical history of high mortality in 70 - 90 days-old pigs (> 50 %). Two 90 days-old animals with weight loss and wasting were necropsied and the samples tested for PCV2 by polymerase chain reaction (PCR). The genetic material of PCV2 was sequenced and classified into the PCV2a genotype together with PCV2 sequences obtained from samples of Poland, Brazil, Slovenia and Greece wild boars.
  • Molecular characterization of vanA-containing Enterococcus from migratory birds: song thrush (Turdus philomelos) Veterinary Microbiology

    Silva, Nuno; Igrejas, Gilberto; Felgar, Ana; Gonçalves, Alexandre; Pacheco, Rui; Poeta, Patrícia

    Resumo em Inglês:

    Vancomycin-resistant enterococci (VRE) were detected in two faecal samples (1.3%) of song thrush in Portugal. vanA isolates showed high level vancomycin/teicoplanin resistance, as well as resistance to ciprofloxacin, quinupristin-dalfopristin and cloranfenicol. Thrush can be a reservoir of VRE and transmit these resistant bacteria to other animals including humans.
  • Antimicrobial susceptibility of Clostridium perfringens isolated from piglets with or without diarrhea in Brazil Veterinary Microbiology

    Salvarani, Felipe Masiero; Silva, Rodrigo Otávio Silveira; Pires, Prhiscylla Sadanã; Cruz Júnior, Eduardo Coulaud da Costa; Albefaro, Isabella Silva; Guedes, Roberto Maurício de Carvalho; Lobato, Francisco Carlos Faria

    Resumo em Inglês:

    The minimum inhibitory concentration (MIC) was determined for 13 antibiotics against Clostridium perfringens isolated from Brazilian piglets. The collection of isolates was performed in June to October 2010. All isolates were susceptible to amoxicillin and ceftiofur, whereas most were resistant to tetracycline and lincomycin. Avilamycin and narasin were more effective against isolates from non-diarrheic than from diarrheic piglets. The other antimicrobials were less active in need of high concentrations to inhibit the growth of the C. perfringens type A. These results suggest the need for further studies evaluating molecular factors related to the antimicrobial resistance of C. perfringens.
  • First isolation of Mycobacterium avium subsp paratuberculosis from commercial pasteurized milk in Argentina Veterinary Microbiology

    Paolicchi, Fernando; Cirone, Karina; Morsella, Claudia; Gioffré, Andrea

    Resumo em Inglês:

    Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina.
  • Molecular identification and thermoresistance to boiling of Nocardia farcinica and Nocardia cyriacigeorgica from bovine bulk tank milk Veterinary Microbiology

    Condas, L.A.Z.; Ribeiro, M.G.; Gonoi, T.; Matsuzawa, T.; Yazawa, K.; Motta, R.G.; Franco, M.M.J.; Listoni, F.J.P.

    Resumo em Inglês:

    Two strains of Nocardia spp. were isolated from bovine milk of two individual bulk tank. Molecular identification classified the strains as Nocardia farcinica and Nocardia cyriacigeorgica. The thermorresistance to boiling of the isolates was carried out and was observed bacterial growth after boiling. Our findings indicate the potential risk of pathogen transmission to humans through contaminated milk with Nocardia spp.
  • Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process Industrial Microbiology

    Singh, Ranjan; Gaur, Rajeeva; Tiwari, Soni; Gaur, Manogya Kumar

    Resumo em Inglês:

    Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.
  • Medium optimization of protease production by Brevibacterium linens DSM 20158, using statistical approach Industrial Microbiology

    Shabbiri, Khadija; Adnan, Ahmad; Jamil, Sania; Ahmad, Waqar; Noor, Bushra; Rafique, H.M.

    Resumo em Inglês:

    Various cultivation parameters were optimized for the production of extra cellular protease by Brevibacterium linens DSM 20158 grown in solid state fermentation conditions using statistical approach. The cultivation variables were screened by the Plackett-Burman design and four significant variables (soybean meal, wheat bran, (NH4)2SO4 and inoculum size were further optimized via central composite design (CCD) using a response surface methodological approach. Using the optimal factors (soybean meal 12.0g, wheat bran 8.50g, (NH4)2SO4) 0.45g and inoculum size 3.50%), the rate of protease production was found to be twofold higher in the optimized medium as compared to the unoptimized reference medium.
  • Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation Industrial Microbiology

    Moretti, Marcia M. S.; Bocchini-Martins, Daniela A.; Silva, Roberto Da; Rodrigues, André; Sette, Lara D.; Gomes, Eleni

    Resumo em Inglês:

    Twenty-seven thermophilic and thermotolerant fungal strains were isolated from soil, decaying organic matter and sugarcane piles based on their ability to grow at 45ºC on medium containing corn straw and cardboard as carbon sources. These fungi were identified in the genera Aspergillus, Thermomyces, Myceliophthora, Thermomucor and Candida. The majority of the isolated strains produced xylanase and cellulases under solid state fermentation (SSF). The highest cellulase and xylanase productions were obtained by the cultivation of the strains identified as Aspergillus fumigatus M.7.1 and Myceliophthora thermophila M.7.7. The enzymes from these strains exhibited maximum activity at pH 5.0 and at 60 and 70ºC. The endo-glucanase from A. fumigatus was stable from 40ºC to 65ºC and both endo-glucanase and xylanase from M. thermophila were stable in this temperature range when in absence of substrate. The enzymes were stable from pH 4.0 to 9.0.
  • Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor Industrial Microbiology

    Mukhtar, Hamid; Ikram-ul-Haq,

    Resumo em Inglês:

    We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37ºC while that of alpha amylase was observed at 40ºC. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.
  • Rapid screening of tannase producing microbes by using natural tannin Industrial Microbiology

    Jana, Arijit; Maity, Chiranjit; Halder, Suman Kumar; Pati, Bikas Ranjan; Mondal, Keshab Chandra; Mohapatra, Pradeep Kumar Das

    Resumo em Inglês:

    Use of natural tannin in the screening of tannase producing microbes is really promising. The present work describes about the possibility and integrity of the newly formulated method over the previously reported methods. Tannin isolated from Terminalia belerica Roxb. (Bahera) was used to differentiate between tanninolytic and nontanninolytic microbes. The method is simple, sensitive and superior for the rapid screening and isolation of tannase-producing microbes.
  • Glucose(xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues Industrial Microbiology

    Chanitnun, Kankiya; Pinphanichakarn, Pairoh

    Resumo em Inglês:

    Streptomyces sp. CH7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose) isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose) isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.
  • Agro-industrial residues and starch for growth and co-production of polyhydroxyalkanoate copolymer and α-amylase by Bacillus sp. CFR-67 Industrial Microbiology

    Shamala, T. R.; Vijayendra, S.V.N.; Joshi, G.J.

    Resumo em Inglês:

    Polyhydroxyalkanoates (PHA) and α-amylase (α-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1) were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH) or rice bran (RBH) individually or in combination (5-20 g L-1, based on weight of soluble substrates-SS). In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L-1 of SS) along with ammonium acetate (1.75 g L-1) and corn starch (30 g L-1) produced maximum quantity of biomass (10 g L-1) and PHA (5.9 g L-1). The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L-1) in the medium enhanced fermentative yield of α-amylase (2-40 U mL-1 min-1). The enzyme was active in a wide range of pH (4-9) and temperature (40-60ºC). This is the first report on simultaneous production of copolymer of bacterial PHA and α-amylase from unhydrolysed corn starch and agro-industrial residues as substrates.
  • Study on Thermodynamics and Adsorption kinetics of Purified endoglucanase (CMCase) from Penicillium notatum NCIM NO-923 produced under mixed solid-state fermentation of waste cabbage and Bagasse Industrial Microbiology

    Das, Arpan; Ghosh, Uma; Mohapatra, Pradeep Kumar Das; Pati, Bikas Ranjan; Mondal, Keshab Chandra

    Resumo em Inglês:

    In the current study, one thermostable endoglucanase was purified from Penicillium notatum NCIM NO-923 through mixed solid state fermentation of waste cabbage and bagasse. The molecular weight of the purified enzyme was 55kDa as determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had low activation energy (Ea) of 36.39KJ mol-1 for carboxymethyl cellulose hydrolysis and the enthalpy and entropy for irreversible inactivation was 87 kJ mol −1 and 59.3 J mol −1 K−1 respectively. The enzyme was quite thermostable with a Tm value of 62.2˚C. The pKa1 and pKa2 of ionizable groups of the active sites were 2.5 and 5.3 respectively. Apparent Km, Vmax and Kcat of the enzyme were found to be 5.2 mg mL-1, 80 U/gds and 322.4 sec-1 respectively. The enzyme showed about 1.4 fold increased activity in presence of 10mM MgSO4. Adsorption of endoglucanase on Avicel at wide pH range was studied at different temperatures. Langmuir type adsorption isotherm at 10˚C showed maximum adsorption strength of enzyme at pH 3.0, which was in a range of optimum pH of the enzyme.
  • Activity of disinfectants against foodborne pathogens in suspension and adhered to stainless steel surfaces Food Microbiology

    Cabeça, Tatiane Karen; Pizzolitto, Antonio Carlos; Pizzolitto, Elisabeth Loshchagin

    Resumo em Inglês:

    The purpose of this study was to investigate and compare the efficacy of various disinfectants on planktonic cells and biofilm cells of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. Numbers of viable biofilm cells decreased after treatment with all tested disinfectants (iodine, biguanide, quaternary ammonium compounds, peracetic acid and sodium hypochlorite). Sodium hypochlorite was the most effective disinfectant against biofilm cells, while biguanide was the least effective. Scanning electron microscopy observations revealed that cells adhered on stainless steel surface after treatment with the disinfectants. No viable planktonic cells were observed after treatment with the same disinfectants. Based on our findings, we concluded that biofilm cells might be more resistant to disinfectants than plancktonic cells.
  • Combination of Origanum vulgare L. essential oil and lactic acid to inhibit Staphylococcus aureus in meat broth and meat model Food Microbiology

    Barros, Jefferson C. de; Conceição, Maria Lúcia da; Gomes Neto, Nelson Justino; Costa, Ana Caroliny Vieira da; Souza, Evandro Leite de

    Resumo em Inglês:

    This study assessed the occurrence of an enhancing inhibitory effect of the combined application of Origanum vulgare L. essential oil and lactic acid against Staphylococcus aureus by the determination of Fractional Inhibitory Concentration (FIC) index and cell viability in meat broth and meat model. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the oil was 0.6 and 1.25 µL.mL-1, respectively. Lactic acid showed MIC and MBC of 2.5 and 5µL.mL-1, respectively. FIC indices of the combined application of the oil and lactic acid were 0.5 showing a synergic interaction. The essential oil and lactic acid showed similar (p>0.05) anti-S. aureus effect in meat broth over 96 h of exposure. Treatment with essential oil or lactic acid presented a smaller anti-staphylococcal effect in meat in comparison to meat broth. No significant difference (p>0.05) was found for the microbial counts in meat treated with each antimicrobial alone or in mixture. These results could arise as an interesting approach for the improvement of food preservation using more natural procedures, considering the current demand of consumer and sensory quality of foods.
  • Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment Food Microbiology

    Oliveira, I.C.; Almeida, R.C.C.; Hofer, E.; Almeida, P.F.

    Resumo em Inglês:

    A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO) were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8) pfu/mL) without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL), after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.
  • Pathogenic variation in isolates of Pseudomonas causing the brown blotch of cultivated mushroom, Agaricus bisporus Food Microbiology

    Abou-Zeid, Mohamed A.

    Resumo em Inglês:

    Twenty seven bacterial isolates were isolated from superficial brown discolorations on the caps of cultivated Agaricus bisporus. After White Line Assay (WLA) and the assist of Biolog computer-identification system, isolates were divided into groups: (I) comprised ninteen bacterial isolates that positively responded to a Pseudomonas "reactans" reference strain (NCPPB1311) in WLA and were identified as Pseudomonas tolaasii, (II) comprised two isolates which were WLA+ towards the reference strain (JCM21583) of P. tolaasii and were proposed to be P. "reactans". The third group comprised six isolates, two of which weakly responded to the strain of P. tolaasii and were identified as P. gingeri whereas the other four were WLA- and identified as P. fluorescens (three isolates) and P. marginalis (one isolate). Isolates of P. tolaasii showed high aggressiveness compared with those of P. "reactans" in pathogenicity tests. Cubes of 1 cm³ of A. bisporus turned brown and decreased in size when were inoculated with 10 µl of P. tolaasii suspension containing 10(8) CFU ml-1, whereas a similar concentration of P. "reactans" caused only light browning. Fifty µl of the same concentration of P. tolaasii isolates gave typical brown blotch symptoms on fresh mushroom sporophores whereas the two P. "reactans" isolates caused superficial light discoloration only after inoculation with 100 µl of the same concentration. Mixture from both bacterial suspensions increased the brown areas formed on the pileus. This is the first pathogenicity report of P. tolasii and P. "reactans" isolated from cultivated A. bisporus in Egypt.
  • Inhibitory effect of essential oils against Lactobacillus rhamnosus and starter culture in fermented milk during its shelf-life period Food Microbiology

    Moritz, Cristiane Mengue Feniman; Rall, Vera Lúcia Mores; Saeki, Margarida Júri; Fernandes Júnior, Ary

    Resumo em Inglês:

    The use of essential oils in foods has attracted great interest, due to their antagonistic action against pathogenic microorganisms. However, this action is undesirable for probiotic foods, as products containing Lactobacillus rhamnosus. The aim of the present study was to measure the sensitivity profile of L. rhamnosus and a yogurt starter culture in fermented milk, upon addition of increasing concentrations of cinnamon, clove and mint essential oils. Essential oils were prepared by steam distillation, and chemically characterised by gas chromatography-mass spectrometry (GC-MS) and determination of density. Survival curves were obtained from counts of L. rhamnosus and the starter culture (alone and in combination), upon addition of 0.04% essential oils. In parallel, titratable acidity was monitored over 28 experimental days. Minimum inhibitory concentration values, obtained using the microdilution method in Brain Heart Infusion medium, were 0.025, 0.2 and 0.4% for cinnamon, clove and mint essential oils, respectively. Cinnamon essential oil had the highest antimicrobial activity, especially against the starter culture, interfering with lactic acid production. Although viable cell counts of L. rhamnosus were lower following treatment with all 3 essential oils, relative to controls, these results were not statistically significant; in addition, cell counts remained greater than the minimum count of 10(8)CFU/mL required for a product to be considered a probiotic. Thus, although use of cinnamon essential oil in yogurt makes starter culture fermentation unfeasible, it does not prevent the application of L. rhamnosus to probiotic fermented milk. Furthermore, clove and mint essential oil caused sublethal stress to L. rhamnosus.
  • Increase of stress resistance in Lactococcus lactis via a novel food-grade vector expressing a shsp gene from Streptococcus thermophilus Food Microbiology

    Tian, Hongtao; Tan, Jianxin; Zhang, Lifang; Gu, Xinxi; Xu, Wentao; Guo, Xinghua; Luo, Yunbo

    Resumo em Inglês:

    The effects of the expression of a small heat shock protein (shsp) gene from Streptococcus thermophilus on stress resistance in Lactococcus lactis under different environmental stresses were investigated in this study. pMG36e-shsp, an expression vector, was first constructed by inserting a shsp open reading frame (ORF) cloned from S. thermophilus strain St-QC into pMG36e. Then, a food-grade expression vector, pMG-shsp, was generated by deleting the erythromycin resistance gene from pMG36e-shsp. The transformation rate of pMG-shsp was comparable to that of pMG36e-shsp when each of these two vectors was introduced into L. lactis. These results demonstrated that the shsp ORF could successfully used as a food-grade selection marker in both pMG-shsp and pMG36e-shsp. Furthermore, the growth characteristics were almost the same between L. lactis ML23 transformants harboring pMG36e or pMG-shsp. The survival rate of L. lactis ML23 expressing the shsp ORF were increased to 0.032%, 0.006%, 0.0027%, 0.03%, and 0.16% under the following environmental stresses: heat, acid, ethanol, bile salt and H2O2, respectively. These results indicated that the expression of the shsp gene in the food-grade vector pMG-shsp conferred resistance to environmental stresses without affecting the growth characteristics of L. lactis ML23.
  • Diversity of leaf endophytic fungi in mangrove plants of Northeast Brazil Environmental Microbiology

    Costa, Isabella P. M. Wanderley; Maia, Leonor Costa; Cavalcanti, Maria Auxiliadora

    Resumo em Inglês:

    With the aim of increasing the knowledge about endophytic fungi, a group of microorganisms with high biotechnological potential and a valuable source of useful metabolites, a survey in leaves of mangrove plants (Avicennia schaueriana, Laguncularia racemosa, and Rhizophora mangle) was performed at the Itamaracá Island, PE, Brazil. Leaves were collected, during two seasons, dry and rainy, superficially sterilized and fragments maintained in Petri dishes with Potato dextrose agar (PDA) at 28º ± 2º C until isolation of the fungi. Fourty taxa were isolated: 25 species representing 19 genera and 15 morphotypes determined as Mycelia sterilia. Leaves of L. racemosa hosted the highest number of colony forming units (CFU) and taxa. Guignardia sp. and Colletotrichum gloeosporioides were the most frequently isolated, while Glomerella cingulata was the only species found in association with the three host plants. The proportional importance of each fungus differed among hosts. The similarity of fungi species between the two seasons reached only 4.2%, and that between the hosts was also low, with the maximum (A. schaueriana x L. racemosa) reaching 24.2%. Sphaerosporium, as well as Chloridium virescens var. virescens, Microsphaeropsis arundinis, Penicillium pinophilum, Periconia cambrensis, Phoma herbarum, P. diachenii, P. obscurans, Sordaria prolifica and Torula elisii are reported for the first time as endophytic in tropical regions.
  • Chemical characterization of bioactive compounds from the endophytic fungus Diaporthe helianthi isolated from Luehea divaricata Environmental Microbiology

    Specian, Vânia; Sarragiotto, Maria Helena; Pamphile, João Alencar; Clemente, Edmar

    Resumo em Inglês:

    Endophytic microorganisms, defined as fungi or bacteria that colonize the interior of plants without causing any immediate negative effects or damages, have reciprocal relationships with host plants. In some cases their presence is beneficial to the host due to the synthesis of bioactive compounds, among which several alcohols, esters, ketones and others that may react with other compounds and may be lethal to pathogenic microorganisms. Diaporthe helianthi (Phomopsis helianthi in its anamorphic phase) is available worldwide, especially in Europe, Asia and America. Isolated in Europe as an agent of the sunflower stem cancer, it has also been endophytically isolated from tropical and temperate plants. A D. helianthi strain isolated from Luehea divaricata has been employed in current research. An investigation of the secondary metabolite from D. helianthi by CC and NMR of ¹H and 13C yielded the separation of 10 fractions and the identification of the phenolic compound 2(-4 hydroxyphenyl)-ethanol (Tyrosol). Its antimicrobial reaction was tested and the ensuing antagonistic effects on the human pathogenic bacteria Enterococcus hirae, Escherichia coli, Micrococcus luteus, Salmonella typhi, Staphylococcus aureus, phytopathogenic Xanthomonas asc. phaseoli and phytopathogenic fungi were demonstrated. Results show that bioactive compounds and Tyrosol produced by D. helianthi have a biotechnological potential.
  • Bacterial exopolysaccharide and biofilm formation stimulate chickpea growth and soil aggregation under salt stress Environmental Microbiology

    Qurashi, Aisha Waheed; Sabri, Anjum Nasim

    Resumo em Inglês:

    To compensate for stress imposed by salinity, biofilm formation and exopolysaccharide production are significant strategies of salt tolerant bacteria to assist metabolism. We hypothesized that two previously isolated salt-tolerant strains Halomonas variabilis (HT1) and Planococcus rifietoensis (RT4) have an ability to improve plant growth, These strains can form biofilm and accumulate exopolysacharides at increasing salt stress. These results showed that bacteria might be involved in developing microbial communities under salt stress and helpful in colonizing of bacterial strains to plant roots and soil particles. Eventually, it can add to the plant growth and soil structure. We investigated the comparative effect of exopolysacharide and biofilm formation in two bacterial strains Halomonas variabilis (HT1) and Planococcus rifietoensis (RT4) in response to varying salt stress. We found that biofilm formation and exopolysaccharide accumulation increased at higher salinity. To check the effect of bacterial inoculation on the plant (Cicer arietinum Var. CM-98) growth and soil aggregation, pot experiment was conducted by growing seedlings under salt stress. Inoculation of both strains increased plant growth at elevated salt stress. Weight of soil aggregates attached with roots and present in soil were added at higher salt concentrations compared to untreated controls. Soil aggregation was higher at plant roots under salinity. These results suggest the feasibility of using above strains in improving plant growth and soil fertility under salinity.
  • Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation Microbial Physiology

    Chaisawang, Montri; Verduyn, Cornelis; Chauvatcharin, Somchai; Suphantharika, Manop

    Resumo em Inglês:

    Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.
  • Oxygen limitation favors the production of protein with antimicrobial activity in Pseudoalteromonas sp Microbial Physiology

    López, Ruth; Monteón, Víctor; Chan, Ernesto; Montejo, Rubí; Chan, Manuel

    Resumo em Inglês:

    This study examined the effect of dissolved oxygen concentration on the production of biomass and metabolites with antimicrobial activity of Pseudoalteromonas sp cultured at 0, 150, 250, or 450 revolutions per minute (rev. min-1). Dissolved oxygen (D.O) was monitored during the fermentation process, biomass was quantified by dry weight, and antimicrobial activity was assessed using the disk diffusion method. The bacterium Pseudoalteromonas reached similar concentration of biomass under all experimental agitation conditions, whereas antimicrobial activity was detected at 0 and 150 rev. min-1 registering 0% and 12% of D.O respectively corresponding to microaerophilic conditions. Antibiotic activity was severely diminished when D.O was above 20% of saturation; this corresponded to 250 or 450 rev. min-1. SDS-PAGE electrophoresis revealed a protein with a molecular weight of approximately 80 kilodaltons (kDa) with antimicrobial activity. Pseudoalteromonas is capable of growing under oxic and microaerophilic conditions but the metabolites with antimicrobial activity are induced under microaerophilic conditions. The current opinion is that Pseudoalteromonas are aerobic organisms; we provide additional information on the amount of dissolved oxygen during the fermentation process and its effect on antimicrobial activity.
  • Amylase production by endophytic fungi Cylindrocephalum sp. isolated from medicinal plant Alpinia calcarata (Haw.) Roscoe Microbial Physiology

    Sunitha., V. H.; Ramesha., A.; Savitha., J.; Srinivas., C

    Resumo em Inglês:

    Amylases are among the most important enzymes used in modern biotechnology particularly in the process involving starch hydrolysis. Fungal amylase has large applications in food and pharmaceutical industries. Considering these facts, endophytic fungi isolated from the plant Alpinia calcarata (Haw.) Roscoe were screened for amylolytic activity on glucose yeast extract peptone agar (GYP) medium. Among thirty isolates of endophytic fungi, isolate number seven identified as Cylindrocephalum sp. (Ac-7) showed highest amylolytic activity and was taken for further study. Influence of various physical and chemical factors such as pH, temperature, carbon and nitrogen sources on amylase production in liquid media were studied. The maximal amylase production was found to be at 30ºC and at pH 7.0 of the growth medium. Among the various carbon and nitrogen sources tested, maltose at 1.5% and Sodium nitrate at 0.3% respectively gave optimum amylase production.
  • Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot Genetics And Molecular Microbiology

    Armesto, Cecilia; Maia, Fernanda Gonçalves Martins; Abreu, Mário Sobral de; Figueira, Antonia dos Reis; Silva, Bruno Marques da; Monteiro, Fernando Pereira

    Resumo em Inglês:

    Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br