Multi-criterion analysis of the effect of physico-chemical microbiological agents on Legionella detection in hotel water distribution systems in Crete.

Introduction: Water distribution systems in hotels have been related to outbreaks caused by Legionella spp. Certain measures, including disinfection by chlorination, maintaining increased temperatures are usually undertaken to prevent Legionella outbreaks. However, these preventive strategies are not always effective, since there are several factors (e.g., synergistic interactions with other microbes, physico-chemical factors, biofilm formation, availability of nutrients) that promote survival and proliferation of the pathogen in water pipes., Accordingly, there is a need of a holistic approach in development of preventive models for Legionella outbreaks associated with water distribution systems. Methods: Water samples were collected from hotel water systems and were tested for the presence of Legionella, E. coli, total coliforms, total mesophilic count and Pseudomonas. In each sample, temperature and chlorine were also tested. Other epidemiological factors were additionally recorded including number of rooms, stars, proximity of sampling point to the boiler, etc. Data were processed by generalized linear analysis, and modeling based on logistic regression analysis to identify independent predictive factors associated with the presence of Legionella in hotel water systems. Results: According to the generalized linear model, temperature affected (p<0.05) the presence of Legionella regardless of the species or the water supply (hot or cold). Additionally, opportunistic (P. aeruginosa) or non-opportunistic (E. coli, coliforms) pathogens were significantly associated (p<0.05) with the presence of all Legionella species. Temperature also exhibited a positive effect to all pathogens tested except for Pseudomonas according to the linear model. Multivariate analysis showed that Pseudomonas, total coliforms, HPC and temperature had a statistically significant effect on the presence of Legionella. Based on a binomial model, cold water had a positive effect on Legionella. Type of sampling and proximity of the sample to the boiler seemed to pose different effect on Legionella depending on the cfu/L. The number of hotel stars and rooms did not appear to have any effect in all tested models. Discussion: Collectively, these results indicate the need for development of individualized water safety plans tailored by the presence of other microbiological agents, and unique physico-chemical factors, which could facilitate the survival of Legionella.in hotel water systems.

Giant cell arteritis associated with acute Q fever - A case report.

Q fever is a worldwide spread zoonotic disease, caused by the gram-negative intracellular bacillus Coxiella burnetii. Apart from its most common manifestations, Q fever has been reported to occasionally mimic autoimmune diseases. We herein present a case of acute Q fever in a 69-year-old man, manifesting as prolonged fever with pneumonitis, in whom biopsy of the temporal artery revealed giant cell arteritis. Moreover, PCR testing of the biopsy specimen was positive for Coxiella burnetii, thus further supporting the possibly infectious etiology of some cases of biopsy proven giant cell arteritis, with implications for treatment.

Exploring immunogenicity of tick salivary AV422 protein in persons exposed to ticks: prospects for utilization.

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

Dynamic modelling of Legionella pneumophila thermal inactivation in water.

A predictive mathematical model describing the effect of temperature on the inactivation of Legionella pneumophila in water was developed. Thermal inactivation of L. pneumophila was monitored under isothermal conditions (51 - 61°C). A primary log-linear model was fitted to the inactivation data and the estimated D values ranged from 0.23 to 25.31 min for water temperatures from 61 to 51°C, respectively. The effect of temperature on L. pneumophila inactivation was described using a secondary model, and the model parameters z value and Dref (D-value at 55°C) were estimated at 5.54°C and 3.47 min, respectively. The developed model was further validated under dynamic temperature conditions mimicking various conditions of water thermal disinfection in plumbing systems. The results indicated that the model can satisfactorily predict thermal inactivation of the pathogen at dynamic temperature environments and effectively translate water temperature profiles to cell number reduction. The application of the model in combination with effective temperature monitoring could provide the basis of an integrated preventive approach for the effective control of L. pneumophila in plumbing systems.

In the Search of Potential Serodiagnostic Proteins to Discriminate Between Acute and Chronic Q Fever in Humans. Some Promising Outcomes.

Coxiella burnetii is the agent that causes acute and chronic Q fever infections in humans. Although the isolates studied so far have shown that the two forms of the disease differ in virulence potential thus, implying a variance in their proteomic profile, the methods used do not deliver enough discriminatory capability and often, human infections may be mis-diagnosed. The current study adds further knowledge to the results that we have already published on the Coxiella outer membrane protein 1 (Com1). Herein we identified the proteins GroEL, Ybgf, OmpH, and UPF0422 as candidates for serodiagnostics of Q fever; following cloning, expression and purification they were further used as antigens in ELISA for the screening of patients' sera associated with chronic Q fever endocarditis, sera negative for phase I IgG, sera with at least one sample positive for phase I IgG and sera from patients who suffered from various rheumatic diseases. Blood donors were used as the controls. Sensitivity, specificity, positive predictive value, negative predictive value, and Cohen's kappa coefficient (κ) were calculated and we also performed binary logistic regression analysis to identify combinations of proteins with increased diagnostic yield. We found that proteins GroEL and Ybgf, together with Com1, play the most significant role in the correct diagnosis of chronic Q fever. Of these three proteins, it was shown that Com1 and GroEL present the highest sensitivity and specificity altogether. The results add to the existing knowledge that an antigen-based serodiagnostic test that will be able to correctly diagnose chronic Q fever may not be far from reality.

Antibiotic Resistance of Legionella pneumophila in Clinical and Water Isolates-A Systematic Review.

The current systematic review investigates the antibiotic susceptibility pattern of Legionella pneumophila isolates from the 1980s to the present day, deriving data from clinical and/or water samples from studies carried out all over the world. Eighty-nine papers meeting the inclusion criteria, i.e., "Legionella pneumophila" and "resistance to antibiotics", were evaluated according to pre-defined validity criteria. Sixty articles referred to clinical isolates, and 18 articles reported water-related L. pneumophila isolates, while 11 articles included both clinical and water isolates. Several methods have been proposed as suitable for the determination of MICs, such as the E-test, broth and agar dilution, and disk diffusion methods, in vivo and in vitro, using various media. The E-test method proposed by the European Society of Clinical Microbiology and Infectious Diseases (EUCAST) seems to be the second most frequently used method overall, but it is the preferred method in the most recent publications (2000-2019) for the interpretation criteria. Erythromycin has been proved to be the preference for resistance testing over the years. However, in the last 19 years, the antibiotics ciprofloxacin (CIP), erythromycin (ERM), levofloxacin (LEV) and azithromycin (AZM) were the ones that saw an increase in their use. A decrease in the sensitivity to antibiotics was identified in approximately half of the reviewed articles.

Q fever in Greece: Findings of a 13 years surveillance study.

Q fever is an endemic disease in different parts of Greece. The current study aimed to investigate the prevalence of acute Q fever disease in Greece through the operation of the national reference centre for Q fever. A total of 5397 sera were received from febrile patients under the suspicion of Q fever infection during a 13 years period (2001-20013). A questionnaire was filled in by the clinicians containing certain clinical/epidemiological/demographic information. The diagnosis was based both on IFA (IgG and IgM phase II antibodies against Coxiella burnetii) and on molecular means. A total of 685 (12.7 %) samples were initially tested positive for acute Q fever. The mean (±SD) age of patients was 55.3 years (±18.7). Out of the 489 convalescent samples, 134 (27.4 %) samples indicated a minimum of a four-fold seroconversion and were considered as laboratory confirmed cases of acute Q fever. Pneumonia was the most frequently encountered clinical symptom with presence in 6.8 % of all positive samples. Forty six (46) patients were laboratory confirmed as chronic Q fever cases. Climate seemed to influence the distribution of Q fever cases throughout the years. The findings of the current study comply with past studies carried out elsewhere that had demonstrated a clear relation of the disease with temperature, south winds, etc. This study represents the first large scale attempt to gather a long period information on Q fever infection in Greece. The findings of the current study support the fact that Q fever is an important endemic zoonotic disease in Greece and needs increased awareness by clinical physicians and health care system.

Molecular evidence of Anaplasma phagocytophilumin aborted goat fetuses and placenta.

Anaplasma phagocytophilum, transmitted by Ixodes ticks, is an intracellular pathogen of zoonotic interest. Regarding animals of veterinary importance, infection by this agent has been linked mainly to high fever, neutropenia, reduced milk production, but hemorrhagic diathesis, abortion and impaired spermatogenesis have also sporadically been reported. In Greece, A. phagocytophilum has been detected in dogs, ticks and humans, while so far only A. ovis had been detected in farm animals. Following the occurrence of multiple abortions in two goat farms in Northern Greece, samples were collected from aborted animals. Stomach contents and placental tissue from aborted animals tested positive for A. phagocytophilum by molecular assays and negative for other infectious and parasitic agents. Treatment with oxytetracycline LA stopped the abortions. In tick risk areas clinicians should consider A. phagocytophilum as a cause of abortion in goats.

Genotyping of Coxiella burnetii in sheep and goat abortion samples.

BACKGROUND: Q fever, caused by Coxiella burnetii, is a zoonosis that presents a worldwide distribution and affects both humans and animals. The route of dispersal of the pathogen by ruminants into the environment usually involves stages of abortion and parturition, nevertheless the agent can, also, be detected in other animal samples. Therefore it is considered as important in terms of proper diagnosis, as well as, for epidemiology and surveillance purposes, to genotype the pathogen. The aim of the current study was to investigate the presence of different genotypes of the agent in animals that had suffered from abortion during a two-year survey in Greece. RESULTS: Sixty nine tissue samples (37 stomach contents, 11 liver samples, 21 cotyledons) were collected from 59 abortion cases in sheep (N = 45) and goats (N = 14) from 65 farms at eight different areas of Greece. Samples were screened by qPCR and positive ones were further genotyped using a 10-locus multiple loci (ms 1, 3, 7, 12, 20, 21, 22, 26, 30 and 36) variable number of tandem repeat analysis (MLVA) method. Three genotypes were identified in sheep (A, B, C). Samples representing each of the obtained MLVA profile were further used for MST genotyping. Ten spacers (Cox 2, 5, 6, 18, 20, 22, 37, 51, 56 and 57) were amplified. A close relatedness among the identified MLVA genotypes was confirmed since they all belonged to MST group 32. CONCLUSIONS: The current study introduces into the aspect of genotyping of C. burnetii in Greece. Further studies are needed to explore the presence of more genotypes, to associate the genotypes circulating in the animal and tick population with those causing human disease in order to further expand on the epidemiological aspects of the pathogen.

Golden jackals (Canis aureus) as hosts for ticks and tick-borne pathogens in Serbia.

The golden jackal (Canis aureus) is a medium-sized canid species native to Europe. This species is characterized by rapid large-scale expansion. A similar trend is also observed in Serbia, where the species is now distributed in more than a half of the territory. Although jackals prefer habitats in human-dominated landscapes, these animals have not been studied well enough from an eco-epidemiological point of view, and little is known about their potential for carrying zoonotic pathogens. In a study conducted during a three-year period (01/2010-02/2013), a total of 216 hunted or road-killed golden jackals were collected from 10 localities in Serbia. Ticks, when present, were removed, and after necropsy, spleen samples were collected from each animal. All tick and spleen samples were tested for the DNA of bacterial and protozoan tick-borne pathogens (Borrelia species, Bartonella species, Rickettsia species, Anaplasma species, Coxiella burnetii, Francisella species and Babesia species) by multiplex real-time PCR, conventional PCR and sequencing analyses. The DNA of Babesia canis was detected in nine out of 216 (4.2%) spleen samples, and two samples (0.9%) tested positive for Anaplasma phagocytophilum. In 118 ticks collected from jackals, the DNA of two Babesia species (Ba. canis and Ba. microti), three Borrelia species (Bo. garinii, Bo. valaisiana, and Bo. lusitaniae) and A. marginale was detected. From the aspect of public health surveillance, the potential role of the golden jackal in the maintenance of vector-borne zoonotic pathogens in Serbia must be considered, and further eco-epidemiological studies should be performed to determine the precise role of this animal species in zoonotic disease transmission cycles.

Legionella spp. Risk Assessment in Recreational and Garden Areas of Hotels.

Several Travel-associated Legionnaires' disease (TALD) cases occur annually in Europe. Except from the most obvious sites (cooling towers and hot water systems), infections can also be associated with recreational, water feature, and garden areas of hotels. This argument is of great interest to better comprehend the colonization and to calculate the risk to human health of these sites. From July 2000-November 2017, the public health authorities of the Island of Crete (Greece) inspected 119 hotels associated with TALD, as reported through the European Legionnaires' Disease Surveillance Network. Five hundred and eighteen samples were collected from decorative fountain ponds, showers near pools and spas, swimming pools, spa pools, garden sprinklers, drip irrigation systems (reclaimed water) and soil. Of those, 67 (12.93%), originating from 43 (35.83%) hotels, tested positive for Legionella (Legionella pneumophila serogroups 1, 2, 3, 6, 7, 8, 13, 14, 15 and non-pneumophila species (L. anisa, L. erythra, L. taurinensis, L. birminghamensis, L. rubrilucens). A Relative Risk (R.R.) > 1 (p < 0.0001) was calculated for chlorine concentrations of less than 0.2 mg/L (R.R.: 54.78), star classification (<4) (R.R.: 4.75) and absence of Water Safety Plan implementation (R.R.: 3.96). High risk (≥104 CFU/L) was estimated for pool showers (16.42%), garden sprinklers (7.46%) and pool water (5.97%).

Potential exposure of humans to Rickettsia felis in Greece.

Rickettsia felis is a flea-transmitted pathogen however, in Greece, much work has been done on another flea-borne pathogen, R. typhi; human cases have been described and high-risk areas have been characterized. Nevertheless, little is known about human infections caused by R. felis in the country since human cases are not routinely tested for antibodies against this pathogen. During the past seven years, we have set up a protocol at the National Reference Centre in order to improve the testing of tick-borne diseases in Greece. Based on this protocol, R. conorii, R. typhi R. slovaca, R. felis, and R. mongolotimonae have been added into the routine analysis; during these last years, eight (8) cases of potential exposure to R. felis were identified by serology. On an environmental investigation carried out at the residences of the patients, the pathogen was detected in C. felis only. The demonstration of R. felis potential presence highlights the need for better testing and surveillance of the pathogen.

Legionellosis: a Walk-through to Identification of the Source of Infection.

OBJECTIVES: Although a number of human Legionnaires' disease in tourists are recorded annually in Europe, there are few cases where a direct link can be made between the infected person and the source of infection (hotel or other accommodation). We present a scheme followed in order to track down and identify the source of infection in a tourist suffering from L. pneumophila sg 5 infection, who was accommodated in seven different hotels during his holidays in the island of Crete, and we comment on various difficulties and draw-backs of the process. METHOD: Water samples were collected from the seven hotels where the patient had resided and analyzed at the regional public health laboratory using cultivation and molecular tests. RESULTS: Of 103 water samples analyzed, 19 (18.4%) were positive for Legionella non-pneumophila and 8 (7.8%) were positive for L. pneumophila. A successful L. pneumophila sg 5 match was found between the clinical and environmental sample, which led us to the final identification of the liable hotel. CONCLUSION: Timely notification of the case, within the the European Legionnaires' Disease Surveillance Network (ELDSNet) of the partners involved, is crucial during a course of travel associated with Legionella case investigation. Moreover, the urinary antigen test alone cannot provide sufficient information for the source identification. However, acquiring clinical as well as environmental isolates for serogroup and SBT identification is highly important for the successful matching.

Canine tick-borne pathogens in Cyprus and a unique canine case of multiple co-infections.

Canine tick-borne pathogens such as Ehrlichia canis and Hepatozoon canis are widespread in the Mediterranean basin but have never been reported or investigated in Cyprus. We describe herein the presence of canine tick-borne pathogens in three dogs with clinical signs compatible with vector-borne diseases from Paphos area of Cyprus. Molecular and phylogenetic analysis revealed the presence of E. canis, Anaplasma platys, H. canis, Babesia vogeli and Mycoplasma haemocanis in Cyprus. One dog co-infected with E. canis, H. canis, B. vogeli and M. haemocanis is, to the best of our knowledge, the first report of this multiple co-infection in dogs. The tick-borne pathogens reported in the current study should be considered in the differential diagnoses in dogs exposed to ticks in Cyprus.

Use of MALDI-TOF mass spectrometry in the battle against bacterial infectious diseases: recent achievements and future perspectives.

INTRODUCTION: Advancements in microbial identification occur increasingly faster as more laboratories explore, refine and extend the use of mass spectrometry in the field of microbiology. Areas covered: This review covers the latest knowledge found in the literature for quick identification of various classes of bacterial pathogens known to cause human infection by the use of MALDI-TOF MS technology. Except for identification of bacterial strains, more researchers try to 'battle time' in favor of the patient. These novel approaches to identify bacteria directly from clinical samples and even determine antibiotic resistance are extensively revised and discussed. Expert commentary: Mass spectrometry is the future of bacterial identification and creates a new era in modern microbiology. Its incorporation in routine practice seems to be not too far, providing a valuable alternative, especially in terms of time, to conventional techniques. If the technology further advances, quick bacterial identification and probable identification of common antibiotic resistance might guide patient decision-making regarding bacterial infectious diseases in the near future.

Environmental investigation for the presence of Vibrio species following a case of severe gastroenteritis in a touristic island.

Global changes have caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves, and corals. Following a possible human case of infection due to V. cholerae in the island of Kos (eastern Aegean, Greece), environmental samples were collected and tested for the presence of Vibrio species. Using chromogenic agar and MALDI-TOF MS, V. parahaemolyticus, V. vulnificus V. furnisii, V. alginolyticus, and V. fluvialis were isolated and/or identified. The presence of V. cholerae was established by PCR-sequencing analysis only. Following the susceptibility testing of the Vibrio isolates, only one, V. furnisii, showed intermediate resistance to ciprofloxacin. The rest of the isolates were susceptible to all antibiotics tested. The presence of Vibrio species in aquatic samples reveals potential dangers due to exposure to contaminated seawaters.

Species composition, distribution, ecological preference and host association of ticks in Cyprus.

Tick population and species depend on the effect of biotic and abiotic factors, especially vegetation, climate and host density; Cyprus, due to the mild climate, favors the appearance and spread of tick-borne infections. Our objective was to identify the tick species present in the island, to investigate their geographical distribution and their epidemiological implications. During a three-year study (2004-2006) we collected ticks from domestic and wild animals over the island of Cyprus. Data on temperature, humidity, altitude and vegetation, were also recorded. Each tick was identified by species using existing taxonomic keys. The results were mapped on a county level. During the current study 3057 ticks belonging to 11 tick species and four genera were collected from 441 (24.6%) infested animals. Rhipicephalus sanguineus was the predominant species (38.5%), followed by R. turanicus (21.3%) and R. bursa (17.8%). Most infestations occurred in May (24.0%), followed by March (13.6%) and June (12.2%). Rhipicephalus sanguineus had a positive correlation with humidity and temperature, R. bursa and Ixodes gibbosus had a positive correlation with altitude and a negative correlation with temperature. Contrary, Hyalomma excavatum had a negative correlation with altitude. Climate and the availability of hosts are among the major factors influencing ticks.

Retraction: First human case of Rickettsia sibirica mongolotimonae infection in Northern Greece in a teenager from Thrace.

This article has been retracted by the authors under the agreement between the Editor-in-Chief, Masayuki Saijo and authors.