Characterization of Xanthomonas axonopodis pv. phaseoli isolates

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli Campylobacters are a common cause of bacterial gastroenteritis worldwide, with Campylobacter jejuni and C. coli being the most common species isolated in human infections. If antimicrobial treatment is required, the drugs of choice at the moment are the macrolides and fluoroquinolones. In this thesis, the in vitro resistance profiles of the C. jejuni and C. coli strains were evaluated with emphasis on multidrug resistance. The aim was also to evaluate the different resistance mechanisms against the macrolides. Further, the disk diffusion method was compared to agar dilution method and its repeatability was evaluated, since it has been widely used for the susceptibility testing of campylobacters. The results of the present study showed that resistance to the fluoroquinolones is common in strains isolated from Finnish patients, but resistance to the macrolides is still rare. Multidrug resistance was associated with resistance to both ciprofloxacin and erythromycin. Among the available per oral drugs, least resistance was observed to coamoxiclav There was no resistance to the carbapenems. Sitafloxacin and tigecycline were in vitro highly effective towards Campylobacter species. A point mutation A2059G of the 23S rRNA gene was the main mechanism behind the macrolide resistance, whereas the efflux pumps did not seem to play an important role when a strain had A2059G mutation. A five amino acids insertion, which has not been described previously, in the ribosomal protein L22 of one highly-resistant C. jejuni strain without mutation in the 23S rRNA gene was also detected. Concerning the disk diffusion method, there was variation in the repeatability In conclusion, macrolides still appear to be the first-choice alternative for suspected Campylobacter enteritis. The in vitro susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials on campylobacteriosis, but in life-threatening situations, a carbapenem may be the drug of choice. More studies are needed on whether the disk diffusion test method could be improved or whether all susceptibilities of campylobacters should be done using a MIC based method.

Effects of low intensity laser in in vitro bacterial culture and in vivo infected wounds

OBJECTIVE: to compare the effects of low intensity laser therapy on in vitro bacterial growth and in vivo in infected wounds, and to analyze the effectiveness of the AsGa Laser technology in in vivo wound infections. METHODS: in vitro: Staphylococcus aureus were incubated on blood agar plates, half of them being irradiated with 904 nm wavelength laser and dose of 3J/cm2 daily for seven days. In vivo: 32 male Wistar rats were divided into control group (uninfected) and Experimental Group (Infected). Half of the animals had their wounds irradiated. RESULTS: in vitro: there was no statistically significant variation between the experimental groups as for the source plates and the derived ones (p>0.05). In vivo: there was a significant increase in the deposition of type I and III collagen in the wounds of the infected and irradiated animals when assessed on the fourth day of the experiment (p=0.034). CONCLUSION: low-intensity Laser Therapy applied with a wavelength of 904nm and dose 3J/cm2 did not alter the in vitro growth of S. aureus in experimental groups; in vivo, however, it showed significant increase in the deposition of type I and III collagen in the wound of infected and irradiated animals on the fourth day of the experiment.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Two-Photon Excited Fluorescence Detection Technology in Screening for Methicillin-Resistant Staphylococcus aureus

Control of the world-wide spread of methicillin-resistant Staphylococcus aureus (MRSA) has been unsuccessful in most developed countries. A few countries have been able to maintain a low MRSA prevalence, plausibly due to their strict MRSA control policies. Such policies require wide-scale screening of patients with suspected MRSA colonization, in order to nurse the MRSA-positive patients in contact isolation. The aim of this study was to develop and introduce a 2-photon excited fluorescence detection (TPX) technique for screening of MRSA directly from clinical samples. The assay principle involves specific online immunometric monitoring of S. aureus growth under selective antibiotic pressure. After the novel TPX approach had been set up, its applicability for the detection of MRSA was evaluated using a large MRSA collection including practically all epidemic MRSA strains identified in Finland between 1991 and 2009. The TPX assay was found both sensitive (97.9%) and specific (94.1%) in this epidemiological setting, illustrating that the method is tolerant to wide biological variation as well as to environments with rapidly emerging MRSA strains. When MRSA was screened directly from colonization samples, all patients positive for MRSA by conventional methods were positive also by the TPX assay. The assay capacity was 48 samples per a test run, and the median time required for confirmation of a true-positive screening test result was 3 h 26 min. Collectively, the findings presented in this thesis suggest that the TPX MRSA screening assay could be applicable for direct screening of MRSA colonization samples without any prior steps of isolation. This can potentially mean that contact isolation of suspected carriers testing negative could be discontinued earlier, thereby reducing the costs and burden associated with the containment of MRSA. In case of infection, a positive test result would ensure an early onset of effective therapy.

Paratuberculosis in a dairy Gyr herd in the State of Paraíba, Brazil

This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (Johne's disease) in a dairy Gyr herd in the State of Paraíba, northeastern Brazil. An eight years old cow with chronic unresponsive diarrhea was clinically examined and euthanized for pathological evaluation. Fecal samples from all 160 animals over 12 months of age from the herd were collected for isolation of Mycobacterium avium subsp. paratuberculosis. Clinically, the index case cow was severely dehydrated, cachectic, with profuse mucous diarrhea. The main post-mortem findings were emaciation and thickened intestinal wall. Microscopically, the intestinal lamina propria and submucosa were infiltrated by macrophages, epithelioid cells, and Langhans giant cells with numerous alcohol-acid resistant bacilli in the cytoplasm. Two fecal samples displayed growth in slants of Herrold's egg-yolk agar supplemented with mycobactin J, 150 days after incubation. No growth was noticed in slants without mycobactin J. Microscopic examination of the isolated microorganisms stained by Ziehl-Neelsen revealed considerable amounts of alcohol-acid resistant bacilli, morphologically compatible with Mycobacterium spp. Based on the clinical signs, gross and histological lesions, growth time, bacterial morphology in Ziehl-Neelsen staining, and dependence of mycobactin J, the first diagnosis of paratuberculosis in Zebu cattle was made.

Absence of intestinal colonization by vancomycin-resistant enterococci in nonhuman primates

The animal reservoirs of vancomycin-resistant enterococci (VRE) have important role in the epidemiology of the bacteria and resistant genes. The present work searched fecal samples taken off nonhuman primates for the presence of VRE. Resistance profiles, virulence traits, and genetic variability among enterococci isolates were also analyzed. The samples included Capuchin monkeys (Cebus apella, n=28) and Common marmoset (Callithrix penicillata, n=37) housed in the Primate Center of the University of Brasília, Brazil. Most individuals were captive monkeys from the Central-West and South-East regions of Brazil (n=48). We collected rectal swabs and carried out selective isolation followed by multiplex Polymerase Chain Reaction (PCR) to identify species and resistance genes. No vanA or vanB-containing enterococci were found. The carriage rates ranged from 1.5% for the VanC-type E. casseliflavus and E. gallinarum until 12.3% (n=8) for Enterococcus faecalis. All E. faecalis isolates showed susceptibility to vancomycin, teicoplanin, ampicillin, gentamicin, and streptomycin. The virulence genes ace and esp were prevalent (100.0%, 87.5%). Multilocus variable number of tandem repeats (MLVA) revealed diversity in the number of repeats among E. faecalis isolates and targets, which was higher for espC, efa5, and efa6. We identified six different MLVA genotypes that were divergent from those described in human beings. Also, they were clustered into two genogroups that showed host-specificity for the species Cebus apella or Callithrix penicillata. In conclusion, no vanA- or vanB-containing enterococci were found colonizing those primate individuals. This finding suggested that the primate individuals investigated in our study are not directly involved in the epidemiological chain of high-level vancomycin-resistant genes vanA or vanB in Brazil. Our study also showed that E. faecalis isolated from nonhuman primates carry virulence traits and have ability to spread their lineages among different individuals.

Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg

The serological response of beef calves was evaluated with different vaccination regimens against blackleg, using an official strain (MT) and a field-collected strain of Clostridium chauvoei as antigens. Sixty calves were randomly allocated to four different groups and were submitted to distinct vaccination protocols with a commercial polyvalent vaccine. Group G1 was first vaccinated at four months of age and a booster shot was given after weaning, at eight months. Group G2 was given the first dose at eight months and a booster shot 30 days later. Group G3 was vaccinated only once at eight months and the control group was not vaccinated. These alternative vaccination regimens were proposed in an effort to adequately protect cattle under open-field farming conditions. Serological evaluations were made by Elisa at 4, 8, 9 and 10 months of age. Both groups receiving booster shots had a significantly increased serological response 30 days later. However, the serum IgG levels against C. chauvoei were significantly higher in the calves that were first vaccinated at four months. At 10 months, the two booster shot groups (G1 and G2) had similar serological responses, while the calves that were treated with a single dose of vaccine at weaning (G3) had a response that was similar to that of the control group. The serological response of the calves was significantly inferior at several of the evaluation times when the field strain of the bacteria was used as a challenge antigen instead of the official MT strain. The serological response of calves that are vaccinated twice was found to be satisfactory, independent of the first injection being made at four or eight months of age. It was also concluded that it would be useful to include local bacterial strains in commercial vaccine production.

Weak phenotypic reversion of ivermectin resistance in a field resistant isolate of Haemonchus contortus by verapamil

Recent advances in anthelmintic resistant phenotype reversion by Pgp modulating drugs in ruminant nematodes indicate that this can be a useful tool to helminth control. The aim of the present study was to evaluate the efficacy of ivermectin (IVM) in combination with verapamil (VRP), in oil or water-based vehicle, against an IVM-resistant field isolate of Haemonchus contortus through a larval migration assay and experimental infection trial. In the in vitro assay was observed a phenotypic reversion of H. contortus resistance to ivermectin at a high concentration of VRP, increasing IVM efficacy from 53.1% to 94.3. In the in vivo trial, IVM + VRP demonstrated 36.02% efficacy compared to the 7.75% of IVM alone. The vehicle formulation showed no influence in efficacy. These are the first results demonstrating the effect of VRP as a partial IVM-resistance phenotype reverser in a field isolate of IVM-resistant H. contortus experimentally inoculated in sheep.

Impact of ivermectin-resistant gastrointestinal nematodes in feedlot cattle in Argentina

The aim was to evaluate for 75 days the impact on production of the remaining burden of ivermectin (IVM)-resistant parasites in naturally infected feedlot calves. The herds came from tick-infested areas of cattle breeding where the systematic use of IVM to control tick increases the gastrointestinal parasites resistant to this drug. This investigation was carried out in two commercial feedlots in Buenos Aires province. In feedlot A, two groups of 35 animal each received IVM 1% and the other received ricobendazole (RBZ) 10% respectively. The same was done in feedlot B. On day 0, two groups of 35 animals were made in feedlots A and B. Fecal samples were taken on days 0, 22, 54 and 75 pos-treatment (PT), and body weight was registered, from each animal. Fecal samples were processed for individual count of eggs per gram (EPG) and pooled fecal culture was carried out for identification of the parasite genus in each sampling. Fecal egg count reduction test (FECR) was calculated on day 22 PT. The study design used was a totally randomized block, with commercial feedlot and sex as block variables. For data analysis, a mixed model of the SAS statistical program was used. The FECR average on day 22 was 28.4% in the IVM group, and 94,2 % in the RBZ group . From this date on, significant differences in EPG were kept until day 54. EPG counts were only equal near the end of the trial, on day 75 (p=0.16). In both commercial feedlots, especially in the IVM group, Cooperia spp. was the most prevalent parasite in the fecal cultures. Significant differences in weight (P<0.01) on post-treatment day 75 was found between the average weight in the RBZ and the IVM group (246 vs. 238 kg respectively), what means a difference of 8.3% in gains. The importance for production in the antiparasite failure treatment in commercial feedlots was demonstrated, and the need of pos-treatment controls to evaluate the efficacy of the antiparasitic administered is emphasized.

Occurrence and characterization of Campylobacter spp.isolates in dogs, cats and children

To improve the understanding of implications of Campylobacterspp. infections in pets and children of different environments were analysed 160 faecal samples from children and 120 from pets (103 dogs and 17 cats). Campylobacter spp. were detected in 6.87% of the children and in 18.3% of the dogs and cats. From 33 stool samples positive for Campylobacter spp., 57.6% were identified as C. jejuni, and 33.4% were identified as C. coli. More than 50% of the isolates in pets were resistant to ceftiofur, sulphazotrim, norfloxacin and tetracycline. In humans, most of the isolates were resistant to amoxicillin, cefazolin, ceftiofur, erythromycin and norfloxacin. From 19 isolates of C. jejuni, 11 isolates from children and 5 from dogs contained two to four of the virulence genes flaA, pldA, cadF or ciaB.We found an association between the presence of virulence genes and diarrhoea. Furthermore, an association was observed between the presence of Campylobacter spp. and diarrhoea in dewormed pets with blood picture suggestive of bacterial infection, and the therapeutic use of antibiotics was associated with more positive detection of Campylobacterspp. in the faeces of pets. Our data indicate that virulent strains of Campylobacter spp. can be risk factor to diarrhoea in animals, and that high resistance to antimicrobial agents is common in pets.

A field survey of crop management practices and distribution of ALS resistant Euphorbia heterophylla in two states in southern Brazil

Herbicide resistance was reported in Brazil almost ten years ago. One of the main weeds with herbicide resistance is wild poinsettia (Euphorbia heterophylla). This work evaluates the distribution of ALS-resistant E. heterophylla in two states in southern Brazil and determines the major contributing management causes for weed resistance selection in the area. E. heterophylla seeds from 148 sites located in Paraná and Rio Grande do Sul were sampled during 2001 and 2002. Farmers provided specific site data for weed control, tillage system, crop rotation and harvesting operations during previous years. ALS resistant E. heterophylla biotypes were found widely distributed in the survey area. Data analysis suggests seed dissemination is unlikely to explain the widespread distribution of resistance. The most probable factor for the selection of the resistant E. heterophylla is the persistent high use of ALS-inhibiting herbicides over time. Indirect evidence is presented demonstrating the need to educate legislators and farmers about the importance of herbicide mixtures as a strategy to prevent herbicide resistance.