Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

Synthesis, structure-activity relationships, and in vitro antibacterial and antifungal activity evaluations of novel pyrazole carboxylic and dicarboxylic acid derivatives.

A series of pyrazole-3-carboxylic acid and pyrazole-3,4-dicarboxylic acid derivatives were synthesized, the structures were confirmed by their NMR ((1)H and (13)C) and FT-IR spectra, and elemental analyses. The antibacterial and antifungal activities of the compounds against five bacterial and five fungal pathogens were screened using modified agar well diffusion assay. Most of the molecules have inhibitory effects on both standard and clinical Candida albicans strains. However, only the molecules 8, 10, 21, and 22 demonstrate some inhibitory effects on Candida parapsilosis, Candida tropicalis, and Candida glabrata strains. The structure-antifungal activity relationships of the compounds on the C. albicans strains were investigated by electron-conformational method. The pharmacophores and antipharmacophores responsible for the inhibition and non-inhibition of the C. albicans strains were obtained by electronic and geometrical characteristics of the reactive fragments of the molecules. These fragments along with the associated parameters can be used in designing the future more potent antifungal agents. It has been shown that both the positions of electronegative atoms like F and O in the pyrazole substituents and the amount of the associated charges on such atoms are crucial in regulating the strength of antifungal activity for the C. albicans strain.

Fe(III) reduction and U(VI) immobilization by Paenibacillus sp. strain 300A, isolated from Hanford 300A subsurface sediments.

A facultative iron-reducing [Fe(III)-reducing] Paenibacillus sp. strain was isolated from Hanford 300A subsurface sediment biofilms that was capable of reducing soluble Fe(III) complexes [Fe(III)-nitrilotriacetic acid and Fe(III)-citrate] but unable to reduce poorly crystalline ferrihydrite (Fh). However, Paenibacillus sp. 300A was capable of reducing Fh in the presence of low concentrations (2 µM) of either of the electron transfer mediators (ETMs) flavin mononucleotide (FMN) or anthraquinone-2,6-disulfonate (AQDS). Maximum initial Fh reduction rates were observed at catalytic concentrations (<10 µM) of either FMN or AQDS. Higher FMN concentrations inhibited Fh reduction, while increased AQDS concentrations did not. We also found that Paenibacillus sp. 300A could reduce Fh in the presence of natural ETMs from Hanford 300A subsurface sediments. In the absence of ETMs, Paenibacillus sp. 300A was capable of immobilizing U(VI) through both reduction and adsorption. The relative contributions of adsorption and microbial reduction to U(VI) removal from the aqueous phase were ∼7:3 in PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] and ∼1:4 in bicarbonate buffer. Our study demonstrated that Paenibacillus sp. 300A catalyzes Fe(III) reduction and U(VI) immobilization and that these reactions benefit from externally added or naturally existing ETMs in 300A subsurface sediments.

Characterization of mono- and mixed-culture Campylobacter jejuni biofilms.

Campylobacter jejuni, one of the most common causes of human gastroenteritis, is a thermophilic and microaerophilic bacterium. These characteristics make it a fastidious organism, which limits its ability to survive outside animal hosts. Nevertheless, C. jejuni can be transmitted to both humans and animals via environmental pathways, especially through contaminated water. Biofilms may play a crucial role in the survival of the bacterium under unfavorable environmental conditions. The goal of this study was to investigate survival strategies of C. jejuni in mono- and mixed-culture biofilms. We grew monoculture biofilms of C. jejuni and mixed-culture biofilms of C. jejuni with Pseudomonas aeruginosa. We found that mono- and mixed-culture biofilms had significantly different structures and activities. Monoculture C. jejuni biofilms did not consume a measurable quantity of oxygen. Using a confocal laser scanning microscope (CLSM), we found that cells from monoculture biofilms were alive according to live/dead staining but that these cells were not culturable. In contrast, in mixed-culture biofilms, C. jejuni remained in a culturable physiological state. Monoculture C. jejuni biofilms could persist under lower flow rates (0.75 ml/min) but were unable to persist at higher flow rates (1 to 2.5 ml/min). In sharp contrast, mixed-culture biofilms were more robust and were unaffected by higher flow rates (2.5 ml/min). Our results indicate that biofilms provide an environmental refuge that is conducive to the survival of C. jejuni.

Molecular typing and cdt genes prevalence of Campylobacter jejuni isolates from various sources.

The genetic diversity of 168 Campylobacter jejuni isolates originating from human (n=30), cattle (n=36), sheep (n=44), dog (n=35), and poultry (n=21) and cdt genes prevalence of the isolates were investigated. To determine the genetic diversity of these strains, random amplified polymorphic DNA-polymerase chain reaction (PCR) using a random primer (M13) was performed. The numbers of genotypes determined in human, cattle, sheep, dog, and poultry isolates were 19, 18, 17, 18, and 6, respectively. To find out the prevalence of cdt genes in C. jejuni isolates simultaneously, a multiplex PCR was performed. The prevalence of the separate cdt genes was found to vary from 69% to 100% for cdtA, 92% to 100% for cdtB, and 39% to 98% for cdtC. These rates without host discriminating were 95%, 98%, and 93% for cdtA, cdtB, and cdtC, respectively. The prevalence of all three cdt genes in strains originating from human, cattle, sheep, dog, and poultry were 87%, 67%, 84%, 89%, and 39%, respectively. These results showed the relatively high genetic heterogeneity and variation of cdt genes among C. jejuni isolates from various sources except for poultry isolates. This study gives baseline data on molecular characterization of C. jejuni strains from different sources.

Mouse intestine colonization ability of Campylobacter coli strains.

Campylobacter coli is an etiological agent of gastrointestinal and extraintestinal infections in man and animals, and can be found as a commensal in gastrointestinal tract of animals. In this study, we aimed to determine differences among C coli strains in colonization of the intestinal tract of mice. Seven C coli strains isolated from diarrheic patients, asymptomatic hosts and chicken carcasses were used for this study. Each strain was inoculated with 0.1 ml of a bacterial suspension (3 x 10(8) CFU/ml) to 5 weanling mice, intragastrically. For the isolation of C coli, faecal pellets collected before inoculation and after inoculation at particular intervals were cultured on Campylobacter Selective Agar. Seven C. coli strains were divided into 3 colonization groups, based on faecal shedding. Group I showed immediate colonization, with prolonged excretion of organism in all mice. Group II showed delayed and short time colonization of C. coli. Group III could not colonize mice. Division of isolates into colonization groups was as follows: Group I included 3 strains from gastrointestinal disease; Group II included 2 strains from asymptomatic hosts and Group III included 2 strains from chicken carcasses. The study showed that there were marked differences among C coli strains with respect to their colonization potential and it may depend upon the origin of the strain. For understanding the complete pathogenesis of Campylobacter spp., a greater number of strains from different sources and geographical locations require to be tested in further investigations in the light of our findings.

Slime production and antibiotic resistance of Enterococcus faecalis isolated from arthritis in chickens.

Slime factor production and antibiotic resistance of 67 Enterococcus faecalis strains isolated from chicken arthritis were investigated in this study. Slime factor productions of enterococci were found as 59.7%. The antibiotic resistances were investigated by testing gentamycin, penicillin, streptomycin, vancomycin, danofloxacin, and enrofloxacin. The resistance rates were found as 62.68%, 76.11%, 67.16%, 13.43%, 47.76%, 43.28%, respectively. For slime factor positive enterococci, the antibiotic resistance rates were found as follows respectively; 82.50%, 87.50%, 92.50%, 17.50%, 72.50%, and 60.00%. In conclusion; the slime factor might play a role as a colonization factor for chicken arthritis and slime factor positive enterococci were found to be more resistant to these antibiotics. The resistance rates between slime factor positive and negative enterococci against the tested antibiotics except for vancomycin were found statistically significant (p<0.05).