Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Submandibular lymphadenitis due to Mycobacterium malmoense: first reported case from Greece.

An 8-year-old girl with subacute submandibular lymphadenitis and no other complaints is described. After failure of parenteral antistaphylococcal therapy, she underwent incision and drainage of the involved lymph node. The responsible pathogen was identified as Mycobacterium malmoense by GenoType CM assay and sequencing of the 16S ribosomalRNA (rRNA) gene. The patient remains healthy, 11 months after surgery, even though it took approximately 4 months for the surgical incision to heal completely. While M. malmoense is a relatively common cause of non-tuberculous mycobacteria (NTM) lymphadenitis in Northern Europe, this is the first reported case from Greece. We conclude that in a young child with lymphadenitis without systemic symptoms, the microbiology laboratory should be notified in advance in order to extend the duration of mycobacterial cultures. Application of molecular methods will increase the number of reported cases of rare NTM in the future.

Cavitary tuberculosis re-emerging in immigrant children.

Cavitary pulmonary tuberculosis in children is uncommon in areas with a low tuberculosis burden. We present two cases in an 11-year old immunocompetent girl and an 8-year old immunocompromised boy. Both children were immigrants. No other cavitary tuberculosis cases have been observed in a population of 103,781 children in Crete, Greece for the past 25 years. CONCLUSION: Severe forms of tuberculosis may re-emerge and BCG vaccination should remain part of the immunisation programme in immigrant populations.

The epidemiology of Brucellosis in Greece, 2007-2012: a 'One Health' approach.

Background: Brucellosis remains a disease that is very difficult to control and eradicate in Greece. Information exchange between the responsible authorities is crucial in order to support public health infrastructure in the sense of the 'One-Health' strategy model. Methods: The data for 2007-2012 were retrieved from the notifiable diseases system and analysed statistically for correlations between human brucellosis cases and the disease in small ruminants. Disease-related risk factors were also estimated with parallel exploitation mapping software. Results: In Greece the dominant strain for brucellosis is Brucella melitensis. The average incidence in Greece was estimated to be 1.43/100,000. The majority of human cases were males (67.60%). The age distribution of brucellosis patients differs significantly between men and women. Brucellosis in male patients was related to high risk jobs and animal contact, while brucellosis in females was related to recent consumption of dairy products. Seasonality of the disease was different in relation to the European countries an observation attributed to the traditional customs. There was a statistically significant difference in human brucellosis incidence between the eradication and vaccination zones. Conclusion: The updated information on brucellosis in Greece revealed differences in seasonality and transmission patterns. A more active cooperation between the involved public health-related sectors should be followed in order to effectively fight brucellosis as there are still foci of brucellosis in Greece.

Major pathogen microorganisms except yeasts can be detected from blood cultures within the first three days of incubation: A two-year study from a University Hospital.

The knowledge of the expected time-to-positivity (TTP) of blood cultures by major pathogens is essential both clinically and economically. To this end, we conducted the present two-year study in our Institution, aiming to assess the TTP of all the major microorganisms including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetoacter baumannii, Enterococcii spp, Staphylococcus aureus and yeasts, to determine whether a 3-day interval is sufficient for their detection. The TTP for each case of strain isolation per patient was determined as the TTP of the first bottle among a set of bottles collected within the same period of time to be flagged as positive per patient. Based on our results, almost all major Gram-negative (99.30%), Gram-positive microbia (99.01%) and yeasts (98.85%) were detected within the first 5-days of incubation, leading to the solid conclusion that a 5-day period of incubation is adequate to detect almost all the major routine pathogens. By contrast, when a 3-day period was examined acceptable results were only found for Gram-negative (98.33%) and Gram-positive (98.51%) microbia. A significant proportion of yeasts (8.05%) could not be detected within this time frame. Therefore, regarding the yeasts, a 3-day incubation period cannot be considered as adequate and is not advocated.

Factors Influencing Non-albicans Candidemia: A Case-Case-Control Study.

The study identified factors predisposing to non-albicans candidemia with special interest to prior antimicrobial treatment. A retrospective, case-case-control study was performed at the University Hospital of Heraklion, Greece, from November 2007 through September 2011 including adult patients. The study had three groups. The first included 58 patients with non-albicans candidemia, the second 48 with C. albicans candidemia, while the third (control) 104 without candidemia. Each of the two candidemia groups was compared with the control using multivariate logistic regression model. The mean (SD) age of the non-albicans, the albicans and the control patients was 67 (12), 67 (18) and 59 (19) years, respectively. The most common non-albicans Candida spp. isolated were C. parapsilosis in 19 patients (33%), C. glabrata in 17 (29%) and C. tropicalis in 15 (26%). Independent risk factors for non-albicans candidemia were prior treatment with quinolones (p < 0.001), b-lactam-b-lactamase inhibitors (p = 0.011) and presence of central venous catheter (p = 0.05), while for C. albicans candidemia were prior treatment with quinolones (p < 0.001), carbapenems (p = 0.003) along with cardiac disease (p < 0.001). Neither duration of hospitalization nor in-hospital mortality [41% for the non-albicans vs 29% for C. albicans group (p = 0.192)] was significantly different between the two candidemia groups. The study reveals the role of antimicrobial exposure as a risk factor for candidemia caused by different species. Prior treatment with b-lactam-b-lactamase inhibitors was associated with non-albicans, while with carbapenems with C. albicans candidemia. Prior use of quinolones was associated with candidemia in general.

Non-O1, non-O139 Vibrio cholerae bacteremic skin and soft tissue infections.

BACKGROUND: Non-O1, non-O139 Vibrio cholerae can cause sporadic cases of gastroenteritis and extra-intestinal invasive infections, following exposure to contaminated seawater or freshwater or after consumption of raw seafood. Bacteremic infections with skin and soft tissue manifestations are uncommon and in most cases are associated with liver cirrhosis, haematologic malignancies, diabetes mellitus and other immunosuppressed conditions. METHODS: The medical literature was reviewed and we found 47 published cases of non-O1, non-O139 Vibrio cholerae bacteremic skin and soft tissue infections. A fatal case of bacteremia with bullous cellulitis in a 43-year-old patient with liver cirrhosis is described, which is the first reported in Greece. RESULTS: From January 1974 to May 2015, a total of 48 patients with non-O1, non-O139 Vibrio cholerae bacteremia with skin and soft tissue infections were reported. Males predominated. Liver cirrhosis, chronic liver disease and alcohol abuse were common comorbidities. The soft tissue lesions most commonly described were localised cellulitis, with or without bullous and haemorrhagic lesions (66.7%), while necrotising fasciitis was more rare (29.2%). Of the 48 patients with non-O1, non-O139 V. cholerae bacteremic skin and soft tissue infections, 20 (41.7%) died despite treatment. CONCLUSION: Although rarely encountered, non-O1, non-O139 Vibrio cholerae should be included in the differential diagnosis of bacteremic skin and soft tissue infections in patients with underlying illnesses and epidemiologic risk factors. Timely and appropriate antibiotic and surgical treatments are important in the management of the infection.

Risk factors for carbapenem-resistant Klebsiella pneumoniae infection/colonization: a case-case-control study.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is increasingly reported worldwide. The aim of the present study was to identify risk factors associated with the development of CRKP infections. A retrospective, case-case-control study was performed at the University Hospital of Heraklion, Greece. The study population included 83 patients from whom CRKP was isolated, 79 from whom carbapenem-sensitive K. pneumoniae (CSKP) was isolated and 161 (control group) from whom K. pneumoniae was not isolated. The median age of CRKP and CSKP patients was 79 (28-101) and 80 (39-97) years, respectively, while that of the controls was 75 (18-100) years. K. pneumoniae was isolated predominantly from urine in both case groups, followed by blood. Independent risk factors for CRKP infection/colonization were admission to ICU (p = 0.004), prior surgical procedure (p = 0.036) and presence of renal disease (p = 0.037), while for CSKP were neurological disease (p = 0.007), and older age (p = 0.011). No association between CRKP and prior antimicrobial exposure was found. Of the entire cohort 40 patients (12%) died; 22 (27%) in the CRKP, 12 (15%) in the CSKP and 6 (4%) in the control group. Isolation of any K. pneumoniae strain was associated with higher mortality compared to the control group (21% vs. 4%; p < 0.005). Mortality was not statistically different between those infected/colonized/with a CRKP or a CSKP strain (p = 0.084). According to these results prior ICU stay, prior surgical procedure and renal disease were independent risk factors for the development of a CRKP infection/colonization.

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics.

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12 836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.

Hickman catheter-related bacteremia caused by Gordonia sputi in a patient with breast cancer.

The case of a Hickman catheter-related bacteremia caused by Gordonia sputi in a patient with breast cancer is presented. Blood cultures grew a Gram-positive rod, susceptible to several antimicrobials, subsequently identified by 16S rRNA gene sequencing as Gordonia sputi. The infection resolved after successful treatment with antibiotics and catheter removal.

Antibiotic use and the risk of carbapenem-resistant extended-spectrum-{beta}-lactamase-producing Klebsiella pneumoniae infection in hospitalized patients: results of a double case-control study.

OBJECTIVES: To identify the roles of various antibiotics as risk factors for carbapenem-resistant extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (KP) infection (ESBL-KP infection). METHODS: Data were collected over 26 months in a tertiary care university hospital with established endemicity of carbapenem-resistant ESBL-KP (ESBL-CRKP). Using a case-case-control design, patients who presented an infection caused by carbapenem-susceptible ESBL-KP (ESBL-CSKP) and patients with ESBL-CRKP infection were compared with a common control group of hospitalized patients. Effects of treatment and duration of treatment with antibiotics were examined, adjusting for major non-antibiotic risk factors and controlling for confounding effects among the antibiotics via logistic regression models. RESULTS: Ninety-six ESBL-CRKP cases, 55 ESBL-CSKP cases and 151 controls were analysed. Multivariate analysis, adjusting for major non-antibiotic risk factors, showed that the risk of ESBL-CRKP infection rose with increasing duration of prior treatment with ß-lactam/ß-lactamase inhibitor combinations [odds ratio (OR) 1.15 per day increase; P = 0.001] and revealed that increased duration of treatment with fluoroquinolones amplified the impact of exposure to carbapenems (and vice versa) on ESBL-CRKP infection risk (OR 1.02 for interaction term; P = 0.009). Duration of prior treatment with fluoroquinolones was also associated with increased risk of ESBL-CSKP infection (OR 1.07 per day increase; P = 0.028), while prior receipt of carbapenems presented a protective effect against ESBL-CSKP infection (OR 0.21; P = 0.003). CONCLUSIONS: This study highlights the major role of treatment and duration of treatment with ß-lactam/ß-lactamase inhibitor combinations and combinations of carbapenems with fluoroquinolones. Clinicians should counterweight the potential benefits of administering these antibiotics against the increased risk of ESBL-CRKP infection.

In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA.

In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91-->Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91-->Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87-->Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.

Candida albicans versus non-albicans bloodstream infection in patients in a tertiary hospital: an analysis of microbiological data.

Considerable changes in the relative frequency of systemic infections due to various Candida species as well as their in vitro susceptibility patterns have been noted in several parts of the world. We performed an analysis of microbiological data of patients with candidaemia at the University general (tertiary) hospital of Heraklion, Greece. During the study period (March 2001 to July 2006) 140 patients had candidaemia. Among them, 64/140 (46%) had candidaemia due to C. albicans and 76/140 (54%) due to non-albicans species (19/76, 25%, C. glabrata; 30/76, 40%, C. tropicalis; 20/76, 26%, C. parapsilosis; 2/76, 3%, C. lusitaniae; 3/76, 4%, C. krusei; and 2/76, 3%, C. guilliermondii). 75 isolates were tested for in vitro susceptibility to antifungal agents with E-test. No isolate was found to be resistant to amphotericin. From 34 C. albicans isolates, 5 (15%) were not susceptible to itraconazole, and 1 (3%) to fluconazole. The C. guilliermondii and the C. lusitaniae isolates were not susceptible to itraconazole. All 11 C. glabrata isolates were not susceptible to ketoconazole and itraconazole, with only 5 (45%) to fluconazole. In line with results of other relevant studies, we documented that a considerable proportion of Candida bloodstream infections were due to non-albicans Candida species.

Uptake of ciprofloxacin and ofloxacin by 2 Brucella strains and their fluoroquinolone-resistant variants under different conditions. An in vitro study.

The existence of an efflux-mediated mechanism involved in the drug accumulation process of the 2 resistant Brucella strains was investigated; 2 susceptible Brucella strains, Brucella melitensis biovar abortus 2308 (S2308) and AFbortus 3196 (S3196) strains, were the reference strains cultured on trypticase soy agar (TSA) medium and incubated to gradient concentrations of ciprofloxacin and ofloxacin antibiotics. The uptake level of 2 antibacterial agents in 2 susceptible and their fluoroquinolones-resistant variants of Brucella strains obtained in vitro at neutral (pH 7) and acidic (pH 4.5) media, in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), was analyzed spectrophotometrically. A decreased accumulation was found in all strains at the acidic medium. Accumulation of both ciprofloxacin and ofloxacin was found to be lower in resistant strains than in the susceptible ones. Preincubation with the CCCP had an obvious effect in increased intracellular fluoroquinolone fluorescence. The decrease in drug accumulation seen in 2 resistant Brucella strains could be attributed to an energy-depended efflux mechanism that mediated the resistance to fluoroquinolones, which was often associated with porin alterations (resulting to decreased outer membrane permeability).

In vitro susceptibilities of Brucella melitensis isolates to eleven antibiotics.

BACKGROUND: Brucellosis is an endemic disease present in many countries worldwide, but it is rare in Europe and North America. Nevertheless brucella is included in the bacteria potentially used for bioterrorism. The aim of this study was the investigation of the antibiotic susceptibility profile of brucella isolates from areas of the eastern Mediterranean where it has been endemic. METHODS: The susceptibilities of 74 Brucella melitensis isolates derived from clinical samples (57) and animal products (17) were tested in vitro. The strains originate from Crete (59), Cyprus (10), and Syria (5). MICs of tetracycline, rifampicin, streptomycin, gentamicin, norfloxacin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, ampicillin, amoxicillin/clavulanic acid, and erythromycin were detected by E-test method. The NCCLS criteria for slow growing bacteria were considered to interpret the results. RESULTS: All the isolates were susceptible to tetracycline, streptomycin, gentamicin, ciprofloxacin, norfloxacin, and levofloxacin. Two isolates presented reduced susceptibility to rifampicin (MIC value: 1.5 mg/l) and eight to SXT (MIC values: 0.75-1.5 mg/l). Erythromycin had the highest (4 mg/l) MIC90value and both norfloxacin and erythromycin the highest (1.5 mg/l) MIC50 value. CONCLUSION: Brucella isolates remain susceptible in vitro to most antibiotics used for treatment of brucellosis. The establishment of a standardized antibiotic susceptibility method for Brucella spp would be useful for resistance determination in these bacteria and possible evaluation of bioterorism risks.

Epidemiology and molecular analysis of intestinal colonization by vancomycin-resistant enterococci in greek hospitals.

From 1,246 specimens collected from 13 Greek hospitals, 266 vancomycin-resistant enterococci strains were isolated from 255 patients (20.5%). The VanA phenotype was present in 82 (30.8%) strains, the VanB phenotype in 17 (6.4%) strains, the VanC1 phenotype in 152 (57.1%) strains, and the VanC2/C3 phenotypes in 15 (5.6%) strains. When only VanA and VanB phenotypes were considered, the overall prevalence was 7.5%. Eighty-six isolates exhibiting the VanA or VanB phenotype were analyzed by pulsed-field gel electrophoresis (PFGE), and 46 PFGE groups were found.

Qualitative versus quantitative blood cultures in the diagnosis of catheter-related bloodstream infections in children with malignancy.

BACKGROUND: Paired quantitative and qualitative blood cultures have been introduced for the diagnosis of catheter-related bloodstream infections (CRBI) with the catheter in situ. The aim of the study was to compare the diagnostic performance and the prognostic value of the two methods in the evaluation of febrile episodes without an apparent source in children with cancer. PROCEDURE: During a 4-year period, in every febrile episode without an apparent focus, blood was drawn simultaneously from the catheter lumen and a peripheral vein in order to perform paired quantitative (Isolator) as well as qualitative (BacT/Alert) blood cultures. The diagnosis of a CRBI was defined as either a case of greater (at least 10 fold) or earlier (differential time to positivity >2 h) bacterial growth from the catheter compared to the peripheral blood sample, respectively. RESULTS: Nineteen febrile episodes manifested in 16 children (total period of observation 11,150 catheter-days) were evaluated with both methods. A concordant diagnosis of CRBI was stated with both methods in six episodes; one episode was diagnosed as CRBI only with qualitative culture criteria. Treatment failure resulted in catheter removal in five out of the seven episodes defined as CRBI with either method. Episodes where a CRBI was ruled out with both methods had a favorable outcome. CONCLUSIONS: In this study the two methods showed comparable results in the diagnosis of CRBI and both were of prognostic significance, regarding the outcome of the treatment. However, large scale studies are required in order to evaluate the clinical relevance and the cost effectiveness of performing routinely paired blood cultures with either method.

Capnocytophaga gingivalis bacteremia detected only on quantitative blood cultures in a child with leukemia.

Capnocytophaga species are inhabitants of the normal mouth flora. We describe the case of a 6-year-old-girl with leukemia and poor oral hygiene who developed bacteremia caused by Capnocytophaga gingivalis. The organism was detected only on quantitative blood cultures.

Prognostic value of quantitative blood cultures for the outcome of central venous catheters in children.

Quantitative blood cultures have been used in order to define catheter-related bloodstream infection (CRBI) in pediatric patients with malignancy and central venous catheters (CVCs). We prospectively followed 32 patients with a total of 38 CVCs for a period of 4 y (14,068 catheter-days). Of a total of 35 cases of bacteremia, 9 were considered to be CRBI (25%). The incidence of bacteremia in our study was 2.48 episodes/1,000 catheter-days and 20/38 CVCs (52%) were affected by bacteremia. The incidence of CRBI was 0.63 episodes/1,000 catheter-days and it was detected in 9/38 CVCs (23%). The catheter salvage rate in cases of bacteremia, irrespective of etiology, was 30/35 (85%). The catheter salvage rate in cases of CRBI was only 4/9 (44%), whereas all the catheters (26/26) in non-catheter-related cases of bacteremia were salvaged. We suggest that the use of quantitative blood cultures is a useful tool for the evaluation of bacteremia in patients with CVCs and is of prognostic value.

Fourteen-year seroepidemiological study of zoonoses in a Greek village.

A seroepidemiological study carried out in a high-risk village in Crete in 1985-1987 and 1998 showed that although the awareness of the people concerning zoonoses had increased during this period, the situation did not improve: there was a significant increase of the spread of seroprevalence in time and space of Coxiella burnetii, Rickettsia typhi, Brucella sp., and Entamoeba histolytica. Toxoplasma gondii, Rickettsia conorii, Borrelia burgdorferi, Echinococcus granulosus, Leishmania sp., and Fasciola hepatica stayed at the same levels. This first study of Bartonella henselae in Crete showed that 15.9% of the children tested were seropositive. The results indicate that reservoirs and vectors of the pathogens studied are widespread in the environment, and the way of life of the people favors contact with them. Seven of 30 milk samples were positive for Brucella sp. by seminested polymerase chain reaction.