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BACKGROUND: Campylobacter spp. are together with Salmonella spp. the leading causes of human bacterial gastroenteritis worldwide. The most commonly isolated species in humans are Campylobacter jejuni and C. coli. The isolation, identification, and antimicrobial resistance of Campylobacter spp. from poultry and raw meat from slaughterhouses, has been investigated for the first time in Greece. During the period from August 2005 to November 2008 a total of 1080 samples were collected: (a) 830 fecal samples from five poultry farms, (b) 150 cecal samples from chicken carcasses in a slaughterhouse, and (c) 100 fecal samples from one pig farm near the region of Attica. The identification of the isolates was performed with conventional (sodium hippurate hydrolysis and commercial identification system (Api CAMPY system, bioMerieux, France), as well as with and molecular methods based on 16S rRNA species specific gene amplification by PCR and subsequent sequence analysis of the PCR products. RESULTS: Sixteen Campylobacter strains were isolated, all collected from the poultry farms. None of the strains was identified as C. jejuni. Antimicrobial susceptibility to six antimicrobials was performed and all the strains were susceptible to ciprofloxacin, amoxicillin-clavulanic acid, and gentamicin. Thirteen out of 14 C. coli were resistant to erythromycin and all C. coli strains were resistant to ampicillin. CONCLUSION: Our results emphasize the need for a surveillance and monitoring system with respect to the prevalence and antimicrobial resistance of Campylobacter in poultry, as well as for the use of antimicrobials in veterinary medicine in Greece.

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BACKGROUND AND OBJECTIVE: Campylobacter jejuni is one of the most common enteric pathogens worldwide. The bacterium is transmitted to humans via contaminated food and water. In the majority of cases the disease is self-limiting, but treatment is indicated in immunocompromised patents, in severe cases with septicemia, and in children. The subtyping of clinical, animal, and food C. jejuni isolates is very important for epidemiological studies. In the present study, 192 Campylobacter jejuni isolates characterized by pulsed-field gel electrophoresis (PFGE) of SmaI digested genomic DNA were further examined with respect to their antimicrobial resistance and their flagellin A (flaA) genotypes in order to disclose any correlation between a certain flaA type and a specific antimicrobial susceptibility pattern. METHODS: C. jejuni clinical isolates were collected from infected children up to 14 years of age from five general hospitals in the area of Attica, Greece, during the period 2004-7. C. jejuni strain isolation and identification from stool samples were performed by conventional bacteriological methods. SmaI restriction fragments were prepared as described previously for the PFGE analysis. Antimicrobial susceptibility was tested and interpreted by determination of the minimal inhibitory concentration (MIC) by use of the agar dilution method as described by the Clinical Laboratory Standards Institute. FlaA typing was performed by PCR amplification of the corresponding gene, and the product was digested with DdeI and visualized by agarose gel electrophoresis. Data were analyzed using the software Gene Profiler 1-D Gel Analysis and Data Basing for Windows((R)). RESULTS: A statistically significant correlation between certain flaA genotypes (flaA 17 Greece [GR], flaA 19 GR and flaA 39 GR) and resistance to some antimicrobial agents (ampicillin, amoxicillin/clavulinic acid [co-amoxiclav], erythromycin, nalidixic acid, and ciprofloxacin) was detected in C. jejuni strains isolated from infected children. CONCLUSIONS: Further investigations on a molecular basis are warranted in order to clarify whether certain C. jejuni flaA types are associated with specific antimicrobial resistance attributes.

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The distribution of Yersinia strains in animal reservoirs was examined in 835 food animals (pigs, chickens, sheep, cows) from different Greek departments (Attica, Fthiotida, Viotia and Evia) over a one year period. The isolated strains were characterized with respect to the presence of chromosomal (yst) and plasmid-encoded virulence determinants (virF, yadA) and their antimicrobial susceptibility was tested. In total, Yersiniaspp. were obtained from 9.94% of the 835 food animals at slaughter that were sampled in this study. There was no statistically significant seasonal distribution, nor was any significant departmental distribution observed. From the 83 isolated Yersinia strains, 76 (91,57%) belonged to Y. enterocolitica (58 were of serotype O:3/biotype 4 and 18 strains were non O:3, non O:9), 3 belonged to Y. pseudotuberculosis, 2 to Y. kristensenii and 2 to Y. intermedia. Y. enterocolitica O:3/4 was mainly isolated from the pigs, while Y. enterocolitica non O:3, non O:9 was from the chickens. The strains were grouped into 5 genotypes, with respect to the presence or absence of the virulence genes. A significant predominance of genotype V, the one carrying all the three virulence genes, was observed in the strains isolated from the pigs. Complete susceptibility to most of the 3rd and to the 4th generation cephalosporins and to ciprofloxacin, was observed among the isolates. Remarkable was the association between the presence of each virulence gene separately and resistance to some antimicrobials, a matter of further investigation.

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BACKGROUND: Pulsed-field gel electrophoresis (PFGE) typing has been recognized by several groups as a relatively simple and quick method for genotyping of Campylobacter jejuni (C. jejuni). The present study was carried out to determine the genetic variations among clinical isolates of C. jejuni from Greece and to establish a database, which could be used for future epidemiological and clinical studies. METHODS: A total of 93 C. jejuni clinical isolates of known flagellin subunit A (flaA) genotype, serotype, and antimicrobial susceptibility pattern, were collected from a general hospital in the Attica region of Greece, between the years 2000 and 2003. The PFGE profiles of SmaI DNA digests of each strain were compared using a bin analysis based on 44 molecular size intervals. RESULTS: Forty-three different PFGE types, designated as C. jejuni (C. j.) 1 Greece (GR) to C. j. 43 GR, were identified. There was no statistically significant association of PFGE type with flaA genotype, serotype, or antimicrobial susceptibility pattern. However, PFGE typing did show a remarkable discriminatory ability within the non-serotypable group. CONCLUSION: Evaluating our results, we observed that (i) there was no statistically significant clonality of a certain PFGE type among the strains examined, and (ii) the discriminatory ability of PFGE typing was much better than that of the other typing methods. This is the first report of the use of bin patterns to compare the PFGE genotypes identified.

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BACKGROUND: Flagellin subunit A gene (flaA) typing of Campylobacter has been recognized by several groups as a relatively simple and quick genotyping method. The present study aimed to create, for the first time in Greece, a database with flaA restriction patterns, which could be used for future epidemiological and clinical studies. A total of 207 C. jejuni clinical isolates of known serotype were collected from 5 general hospitals of the area of Attica, during the period 2000-2003. RESULTS: The RFLP profiles of each strain were matched in 44 bins of 0 or 1. Thirty nine different flaA types, designated as flaA 1 GR to flaA 39 GR (GR: Greece) were found. There was no significant association of certain genotypes with certain serotypes. However flaA typing showed a remarkable discriminatory ability inside the non-typable (NT) group. CONCLUSIONS: Evaluating our results we observed (i) that there was no clonality of a certain flaA type among the strains and the serotypes examined and (ii) that the discriminatory ability of flaA typing was much better than that of serotyping. Giving a simple and detailed description of the data analysis, we are the first who publish the bin patterns for the flaA genotypes found.

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INTRODUCTION: The presence of four virulence genes (racR, wlaN, cgtB, virB11) in 356 Campylobacter jejuni strains isolated from confirmed clinical cases was examined by PCR and sequence analysis. The investigated genes were chosen on the basis of their variation in prevalence. METHODS: The virulence genes were detected by PCR and the amplified products were submitted for sequence analysis. RESULTS: The gene with the highest prevalence was racR (87.08%). virB was present in only 1.69% of the C. jejuni strains, and wlaN and cgtB were detected in 16.01% and 24.44%, respectively. Five strains associated with Guillain-Barré syndrome and Miller-Fischer syndrome out of the total of 356 (1.40%) were positive for cgtB. CONCLUSION: Our findings suggest that racR may encode factors necessary for bacterial pathogenicity in humans, while the roles of the other three genes remain ambiguous.

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BACKGROUND: During the course of multiple sclerosis (MS) intrathecal oligoclonal IgGs are present in the cerebrospinal fluid (CSF). The intracellular human pathogen Chlamydia pneumoniae may play a role either as a causative pathogenetic agent in the disease, or C. pneumoniae-infected MS patients could be immunologically less able to clear the agent from the central nervous system (CNS). METHODS: CSF samples were studied in 100 individuals -- 70 MS patients and 30 age-matched controls with other neurological diseases. CSF was taken by lumbal puncture; cell cultures were performed by the cell vial technique, followed by a 4-day incubation at 37 degrees C. A nested PCR was performed. RESULTS: C. pneumoniae was detectable in the CSF of only 2.9% of the MS patients and none of control patients (with no significant difference between the MS patients and controls). IgG antibodies were positive in only 1.43% of the MS patients and 3.33% of the controls. IgA antibodies were positive in 6.66% of the control patients and none of the patients were positive for IgM antibodies. There was no statistically significant difference between the two groups of patients with respect to the three antibody classes. CONCLUSIONS: The results confirm the high leave of controversy surrounding a possible link between C. pneumoniae and MS, and the matter requires further thorough investigation.

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BACKGROUND: Whipple's disease (WD) is a rare multisystemic bacterial infection, with variable clinical manifestations occasionally involving the central nervous system. As the cultivation of the etiologic agent, Tropheryma whippelii, is difficult, a laboratory diagnosis is usually based on histological methods. In the last few years, molecular detection of the bacterial 16SrRNA genes by PCR, with 2 primer sets, has greatly contributed to the ability of clinicians to diagnose this disease. We present a cerebral case of WD in a 48-year-old male, successfully diagnosed using PCR with T. whippelii in the blood and feces. As far as we know this is the first case reported from Greece. METHODS: For the diagnosis of WD, histological examination of duodenum biopsy for diastase-resistant, non-acid fast, periodic acid Schiff (PAS)-positive inclusions in macrophages, and molecular detection of the 16SrRNA genes of T. whippelii by PCR in cerebrospinal fluid, blood, and feces, were performed. RESULTS: The histological detection was negative. PCR results were positive in the blood and feces of the patient and negative in the cerebrospinal fluid. Seven months after the onset of antimicrobial therapy, PCR was negative in all three clinical specimens. CONCLUSIONS: The application of PCR proved to be an invaluable tool for the recognition, differential diagnosis and early initiation of antimicrobial therapy for the patient diagnosed with WD, a disease which is generally fatal if it remains untreated.

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