Molecular Epidemiology of Multi-Drug Resistant Pseudomonas aeruginosa Isolates from Hospitalized Patients in Greece.

Resistant Pseudomonas aeruginosa isolates are one of the major causes of both hospital-acquired infections (HAIs) and community-acquired infections (CAIs). However, management of P. aeruginosa infections is difficult as the bacterium is inherently resistant to many antibiotics. In this study, a collection of 75 P. aeruginosa clinical isolates from two tertiary hospitals from Athens and Alexnadroupolis in Greece was studied to assess antimicrobial sensitivity and molecular epidemiology. All P. aeruginosa isolates were tested for susceptibility to 11 commonly used antibiotics, and the newly introduced Double Locus Sequence Typing (DLST) scheme was implemented to elucidate the predominant clones. The tested P. aeruginosa isolates presented various resistant phenotypes, with Verona Integron-Mediated Metallo-ß-lactamase (VIM-2) mechanisms being the majority, and a new phenotype, FEPR-CAZS, being reported for the first time in Greek isolates. DLST revealed two predominant types, 32-39 and 8-37, and provided evidence for intra-hospital transmission of the 32-39 clone in one of the hospitals. The results indicate that DLST can be a valuable tool when local outbreaks demand immediate tracking investigation with limited time and financial resources.

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.

Exotic Tourist Destinations and Transmission of Infections by Swimming Pools and Hot Springs-A Literature Review.

A growing number of people undertake international travel, and yet faster growth of such travel is expected in the tropics. Information on the hazards presented by pool and hot spring waters in tropical countries is very limited. This review aims to collate available information on pool water quality, alongside data on cases and outbreaks associated with swimming in pools in tropical regions affecting both local populations and travellers. Bacteria species commonly causing cases and outbreaks in the tropics as well as elsewhere in the world were excluded, and the review focuses on studies related to pathogens that, with the exception of Cryptosporidium, are unusual in more temperate climates. Studies concerning subtropical countries were included in the light of climate change. Diseases transmitted by vectors breeding in poorly maintained, neglected or abandoned pools were also included. 83 studies dealing with Microsporidia, Leptospira spp., Schistosomas spp., Cryptosporidium spp., Acanthamoeba spp., Naegleria spp., Clostridium trachomatis, viruses, and vectors breeding in swimming pool and hot tub waters, and fulfilling predefined criteria, have been included in our survey of the literature. In conclusion, prevention strategies for pool safety in the tropics are imperative. Public health authorities need to provide guidance to westerners travelling to exotic destinations on how to protect their health in swimming pools.

Molecular Characterization and Phylogenetic Analysis of Pseudomonas aeruginosa Isolates Recovered from Greek Aquatic Habitats Implementing the Double-Locus Sequence Typing Scheme.

The recently described double-locus sequence typing (DLST) scheme implemented to deeply characterize the genetic profiles of 52 resistant environmental Pseudomonas aeruginosa isolates deriving from aquatic habitats of Greece. DLST scheme was able not only to assign an already known allelic profile to the majority of the isolates but also to recognize two new ones (ms217-190, ms217-191) with high discriminatory power. A third locus (oprD) was also used for the molecular typing, which has been found to be fundamental for the phylogenetic analysis of environmental isolates given the resulted increased discrimination between the isolates. Additionally, the circulation of acquired resistant mechanisms in the aquatic habitats according to their genetic profiles was proved to be more extent. Hereby, we suggest that the combination of the DLST to oprD typing can discriminate phenotypically and genetically related environmental P. aeruginosa isolates providing reliable phylogenetic analysis at a local level.

Occurrence of Cryptosporidium and Giardia in recycled waters used for irrigation and first description of Cryptosporidium parvum and C. muris in Greece.

Here, we present the first time findings regarding the occurrence of Cryptosporidium and Giardia in sewage waters and the first molecular characterization of Cryptosporidium species in Greece. Biological treatment plants from three regions in Greece have been investigated. The detection of Cryptosporidium oocysts was by modified Ziehl-Neelsen acid fast (MZN-AF) and by immunofluorescence microscopy (IFT) for Cryptosporidium and Giardia (oo)cysts, whereas nested PCR based on the SSU rDNA assay was used for molecular detection of Cryptosporidium followed by sequencing for the genetic characterization of the species. In total, 73 samples (37 raw sewage samples and 38 of treated water samples) were collected and analyzed. Of the 73 water samples, 4 samples were Cryptosporidium-positive by IFT and staining, 12 samples were Cryptosporidium-positive by nested PCR; 9 samples were Giardia-positive by IFT. We showed that Cryptosporidium cysts are found both in the input and the discharge of the biological treatment plants. Molecular characterization of Cryptosporidium based on the small subunit ribosomal DNA gene resulted in the determination of Cryptosporidium parvum and Cryptosporidium muris Greek isolates. This is the first report of Cryptosporidium and Giardia occurrence in wastewaters and the first molecular identification of Cryptosporidium species in Greek environments. As the treated water is used for irrigation, or it is discharged into the sea, our findings indicate that biological treatment facilities constitute a possible risk for public health because the related species are prevalent in humans; the results invite for further epidemiological investigations to evaluate the real public health risk in Greece.

An overview of pool and spa regulations in Mediterranean countries with a focus on the tourist industry.

The Mediterranean area is densely populated and a very popular tourist destination. This study aimed at gathering up-to-date information on current national pool and spa regulations. We formulated and duly distributed to 20 Mediterranean countries a questionnaire divided into two sections: for pool and spa facilities, respectively. The questionnaire was formulated in such a way that a positive answer would imply that the specific topic was addressed effectively. While all 20 countries responded to the swimming-pool-related questionnaire, only 11 of these countries reported the existence of spa-related regulations. When combining the response overall of all countries for pool and spa facilities together, of a grand total of 606 answers, 183 (30.2%) were positive. The positive answers in the pool section of the questionnaire were 29% of the total of 420 answers while positive answers in the spa section of the questionnaire were fewer (27.8% of the total of 176 answers). The countries were grouped by geographical area of the Mediterranean basin where they are situated. Also, the questions were grouped into broad thematic categories. The paper presents conclusions drawn on the basis of the data received according to these geographical and thematic groupings.

Cupriavidus metallidurans biomineralization ability and its application as a bioconsolidation enhancer for ornamental marble stone.

Bacterially induced calcium carbonate precipitation of a Cupriavidus metallidurans isolate was investigated to develop an environmentally friendly method for restoration and preservation of ornamental stones. Biomineralization performance was carried out in a growth medium via a Design of Experiments (DoE) approach using, as design factors, the temperature, growth medium concentration, and inoculum concentration. The optimum conditions were determined with the aid of consecutive experiments based on response surface methodology (RSM) and were successfully validated thereafter. Statistical analysis can be utilized as a tool for screening bacterial bioprecipitation as it considerably reduced the experimental time and effort needed for bacterial evaluation. Analytical methods provided an insight to the biomineral characteristics, and sonication tests proved that our isolate could create a solid new layer of vaterite on marble substrate withstanding sonication forces. C. metallidurans ACA-DC 4073 provided a compact vaterite layer on the marble substrate with morphological characteristics that assisted in its differentiation. The latter proved valuable during spraying minimum amount of inoculated media on marble substrate under conditions close to an in situ application. A sufficient and clearly distinguishable layer was identified.

Prediction of Salmonella in seawater by total and faecal coliforms and Enterococci.

The power of total coliforms (TC), faecal coliforms (FC) and Enterococci to predict the presence of Salmonella in seawater was investigated. Indicator cut-off values with the most satisfactory combination of sensitivity and specificity in predicting Salmonella presence were 1,000 CFU 100ml(-1)TC, 200 CFU 100ml(-1)FC, 500 CFU 100ml(-1) Enterococci. When TC or FC were used for Salmonella prediction in logistic regression, then the addition of another indicator did not have a statistically significant effect. When Enterococci were used for prediction, then the addition of either of the two other indicators led to a statistically significant improvement (P=0.001 for TC, P=0.003 for FC). These results suggest that using either TC or FC alone provided an adequate indicator of Salmonella presence, but a statistically significant improvement is possible over using Enterococci alone. Concerning Enterococci, European Union limits for excellent coastal water quality (100 CFU 100 ml(-1)) and United States Environmental Protection Agency criteria for marine bathing waters (35 CFU 100 ml(-1)) have the same value in predicting Salmonella absence (92.5%).

Prevalence study of Legionella spp. contamination in Greek hospitals.

Water and swab samples were collected from 13 hospitals and analyzed for Legionella counts. Legionella was detected in eight out of 13 hospitals and in 22 of 130 water and swab-collected samples. A total of 72.7% of the strains were L. pneumophila ser. 1, 22.7% were L. pneumophila ser. 2-14, and 4.5% did not belong to any of these groups. AFLP typing of the L. pneumophila ser. 1 strains generated two distinguishable AFLP types. There was no significant correlation to the sample type with Legionella recovery. Legionella isolation was more likely to occur in the cooling towers than the water system. Water temperatures of 30-40 degrees C seem to favor Legionella growth. Of the 265 serum samples taken from the medical and technical staff for the control of IgG titre, 89.4% were negative, 7.2% were positive, and for 3.4% the result was doubtful. No association between IgG titre and maximum observed level of Legionella occurrence was detected.

Correlation between bacterial indicators and bacteriophages in sewage and sludge.

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.