Kadir Serdar Diker

Emerging pathogen Arcobacter spp. in acute gastroenteritis: molecular identification, antibiotic susceptibilities and genotyping of the isolated arcobacters

The aims of this study were as follows: (i) to isolate Arcobacter spp. from the stool samples of patients with gastroenteritis; (ii) to identify them with molecular methods; (iii) to genotype them using enterobacterial repetitive intergenic consensus (ERIC)-PCR; and (iv) to determine their antibiotic susceptibilities. For the study, a total of 3287 diarrhoeal stool samples submitted to the Microbiology Laboratory of the Kayseri Training and Research Hospital, Kayseri, Turkey, between 2010 and 2011 were analysed. Campylobacter blood-free selective medium supplemented with cefoperazone, amphotericin B and teicoplanin was used for isolation. Medium inoculated with stool samples was incubated microaerobically at 37 °C for 72-96 h. Phenotypic tests, a genus-specific PCR and a multiplex PCR were used to identify the arcobacters, whilst ERIC-PCR was used for genotyping and the antibiotic susceptibilities of the isolates were detected by E-test. Arcobacter spp. were isolated from nine of the 3287 samples. These nine isolates were identified as Arcobacter butzleri and all showed different ERIC-PCR profiles. All nine isolates were resistant to ampicillin and susceptible to gentamicin, tetracycline, erythromycin and ciprofloxacin. As far as is known, this is the first study in which A. butzleri has been isolated from human acute gastrointestinal infections in Turkey. According to these results, it is recommended that, when investigating the aetiology of infections of the digestive system in humans, Arcobacter spp. be considered for inclusion. The results of this study should contribute to our knowledge related to A. butzleri infections in humans.

In vitro infection of human fetal membranes with candida species

To assess whether Candida species can penetrate intact fetal membranes under in vitro conditions, Candida albicans, Candida tropicalis, Candida guilliermondii, Candida pseudotropicalis and Candida glabrata were inoculated onto the surface of the maternal side of the membranes obtained from 4 pregnant women undergoing repeat cesarean section. After incubation under culture conditions, membranes were evaluated by histological examination. C. albicans inoculated onto the maternal side penetrated and passed to the fetal side and caused some degeneration of the structure of the membrane epithelium. The other four Candida species grew heavily on the maternal surface but did not penetrate and invade the membranes. This effect of C. albicans on fetal membranes may explain the potential mechanism in the development of Candida infections of the amniotic fluid, fetal membranes and possibly the fetus.

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.

In vitro permeation of human chorioamniotic membranes by Campylobacter jejuni

In vitro penetration of human chorioamniotic membranes by Campylobacter jejuni was investigated by an organ culture model. Membrane permeation was detected by an immunoperoxidase technique and viable bacterial counts of membrane homogenates. Human clinical isolates of C. jejuni inoculated on the maternal side of the membranes penetrated to the fetal side suggesting that chorioamniotic membranes constituted a weak barrier against Campylobacter infection. Chicken fecal isolates did not penetrate chorioamniotic membranes. In vitro culture conditions did not affect the viability of membranes. Human placental extracts and amniotic fluids enhanced the in vitro growth of C. jejuni. These results suggest that certain strains of C. jejuni may penetrate intact fetal membranes and this event may play a role in the pathogenesis of infection.

Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR

Background: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs. Objectives: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method. Results: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates. Conclusions: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.

Pathogenicity, genotyping and antibacterial susceptibility of the Listeria spp. Recovered from stray dogs

The present study aimed to determine the prevalence of Listeria spp. in stray dogs in the Kayseri province of Turkey. In addition, serotyping, genotyping and virulence gene analysis of the isolated Listeria spp. were performed and their pathogenicity and antibacterial susceptibility were investigated. The study included 80 rectal swaps taken from 80 stray dogs of different ages and gender that were sheltered in the Kayseri Municipal Dog Shelter. Listeria selective broth and Listeria selective agar were used for the isolation of Listeria spp. and the isolates were identified using a Microbact 12L (Oxoid, England) identification test kit. 16S rDNA sequencing and species-specific polymerase chain reaction (PCR) were performed for molecular identification of the isolates, multiplex PCR and a serological test were performed for serotyping, and PCR was used for virulence gene analysis. For determining the pathogenicity of L. monocytogenes and L. innocua isolates, a total of 100 mice (50 pregnant and 50 non-pregnant) were used. The mice were infected intraperitoneally; the inoculation dose was 1 × 109 CFU/mL and 0.2 mL was used for each animal. Tissue samples obtained from infected mice were processed for the re-isolation of the Listeria spp. and then stained with hematoxylin eosin and Brown-Brenn Gram stain. The antibiotic susceptibilities of the isolates were determined by the disk diffusion method. Listeria spp. were isolated from 5 (6.25%) of the 80 fecal samples. While 1 of the isolates was identified as L. monocytogenes, 4 of them were identified as L. innocua. Serotyping by serological and molecular methods revealed the isolate of L. monocytogenes to be serotype 1/2a. According to the phylogenetic trees, L. innocua and L. monocytogenes strains were clustered in different groups. The L. monocytogenes isolate was positive for all virulence genes tested. All L. innocua isolates were positive for the plcB gene. While all L. innocua isolates were negative for the lin1068 gene, 3 L. innocua isolates were found to be positive for the lin0558 gene. In mice infected with L. monocytogenes, pathological findings were observed in the uterus, intestines, pancreas, and heart. In mice infected with L. innocua, pathological findings were observed in the stomach, intestines and spleen. L. monocytogenes- or L. innocua-related infections or other inflammatory reactions were not observed in the brains of infected animals. On histopathological examination with Gram stain, an abundance of Listeria spp. was observed in the lesions of the liver, spleen, uterus, and kidney. Moreover, while abortion was observed in all animals infected with L. monocytogenes, it was not observed in any of the animals infected with L. innocua. Antibiotic susceptibility testing revealed that all 5 isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, erythromycin, gentamicin, penicillin G, and trimethoprim-sulfamethoxazole and were resistant to nalidixic acid, streptomycin, and cefuroxime sodium. Considering also the pathogenicity of the isolated microorganisms, it can be suggested that stray dogs as carriers of Listeria spp. are a significant risk to public health. As L. innocua isolates, which are considered apathogenic, led to the occurrence of lesions similar to those caused by L. monocytogenes, detailed studies on the pathogenesis of L. innocua infections caused by L. innocua isolates recovered from various sources are required.

Neonatal calf meningitis associated with Streptococcus gallolyticus subsp. gallolyticus

Here, we report a case of neonatal calf meningitis due to Streptococcus gallolyticus subsp. gallolyticus (SGG). Clinical, pathological and microbiological findings were evaluated. API Strep, 16S rRNA gene sequencing, rpoB gene sequencing and sodA gene sequencing were used for the complete identification of SGG. This is the first documented report of neonatal calf meningitis due to SGG in veterinary medicine.