M. Papapetropoulou

Evaluation of Potential Indicators of Viral Contamination in Shellfish and Their Applicability to Diverse Geographical Areas

ABSTRACT The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10–100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre‐enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.

DETECTION OF ENTEROVIRUSES, ADENOVIRUSES AND HEPATITIS A VIRUSES IN RAW SEWAGE AND TREATED EFFLUENTS BY NESTED-PCR

To determine the good working condition of the biological treatment plant of the University Hospital of Patras, Greece, twenty-four raw sewage samples and twenty-four effluent samples were analyzed for the presence of enteroviruses, adenoviruses and Hepatitis A virus (HAV), during the period of March 1995 to March 1996. We used a nested-PRC approach, to increase the sensitivity of the detection. Enteroviruses and adenoviruses were detected in twelve samples (50%) and fourteen samples (58.3%) of raw sewage, respectively. HAV was not detected in any of the raw sewage samples. The more frequent isolation of adenoviruses in raw sewage, indicates their stability as virological indicators of the pollution of the environment. In addition, a seasonal distribution of the detection of enteroviruses and adenoviruses was observed. The absence of HAV is in agreement with the lack of HAV infections in the hospital during that period of time. In contrast with the raw sewage, we were unable to detect the presence of enteroviruses, adenoviruses in samples collected after the biological treatment plant. This fact indicates the effective treatment of sewage by the local biological purification plant.

Environmental Mycobacteria in Drinking Water Before and After Replacement of the Water Distribution Network

Environmental mycobacteria (M. chelonae (8), M.flavescens (6), M. gordonae (6), M. fortuitum (5), M. kansasii (4), M. phlei (2) and M. terrae (2)) were isolated from21.3% of drinking water samples before replacement of thedistribution network of Patras. After replacement of the networkonly 1.8% of the samples contained environmental mycobacteria(M. chelonae (2)). The identification of environmentalmycobacteria was performed by Restriction EnzymeAnalysis-Polymerase Chain Reaction. Our results showed no statistically significant correlationbetween the presence of mycobacteria and the bacteriologicalfecal indicators (p >0.05). Moreover, we determined thatconcentrations of free residual chlorine equal to or greaterthan 0.5 mg L-1 in the water supply network were needed toeliminate environmental mycobacteria.We conclude that the presence of a biofilm in the old Patrasdrinking water network significantly increased the number ofenvironmental mycobacteria in the drinking water. This problemwas resorted after the replacement of the network pipes.

Microbiological quality of bottled water in Greece

microbiological quality of the water produced by 38 bottling plants in Greece between 1987 and 1992 is presented and discussed. The presence of Pseudomonas spp. is also considered.

Differentiation of faecal Escherichia coli from humans and animals by multiple antibiotic resistance analysis

Aims:  Multiple antibiotic resistance (MAR) was performed on 128 Escherichia coli isolates, recovered from faecal samples of humans and animals (cattle, goat, sheep) to determine and compare their antibiotic resistance patterns and to evaluate them statistically in order to specify the source of the faecal material.

Non-Tuberculosis Mycobacteria in Hospital Water Supplies

Non-tuberculosis mycobacteria (NTM) were found in 10 (15.6%) out of 64 examined samples of tap water collected from 64 different points from the water supplies of the five main hospitals of the city of Patras (Peloponnese, Greece), during a six month period. The range of mycobacteria isolated from all the hospital water supplies was from 5-205 CFU L−1. The identification of non-tuberculosis mycobacteria was performed with PCR-Restriction Enzyme Analysis (PCR-REA). The dominant isolates were Mycobacterium chelonae (3), Mycobacterium gordonae (2), Mycobacterium flavescens (1) and Mycobacterium terrae (1). One strain was unidentifiable and another two were lost during subculturing. All examined samples were negative for faecal indicators. The periodical examination of potable water of certain hospital units (such as the ones taking care of immunocompromised hosts, haemodialysis units, endoscopy units, Intensive Care Units etc.) for non-tuberculosis mycobacteria is considered worthwile.

Comparative typing of Pseudomonas species isolated from the aquatic environment in greece by SDS-PAGE and RAPD analysis

Aims:  Three broadly used typing methods were employed in order to assess and compare the identification and classification of environmental Pseudomonas strains. The reproducibility, typeability and discriminatory power of the methods were also compared to evaluate their application. Finally, the potential impact on public health of the isolates is to be discussed.

Regulation of translation initiation in mussels (Mytilus galloprovincialis, lam.) following contamination stress.

The use of mussels (Mytilus sp.) as bioindicators of the aquatic environment is a valuable approach to monitor environmental contamination. Applying batteries of biomarkers is an essential prerequisite, since the complexity of environmental contaminants can induce in mussels a variety of structural and functional responses, which are not necessarily correlated. In an attempt to correlate translation responses to contamination stress, the sedimentation profiles of runoff ribosomes isolated from digestive gland cells of control or contaminated Mediterranean mussels (Mytilus galloprovincialis, Lam.) were examined and the efficiency of these ribosomes to accomplish protein synthesis was determined. While the major species of ribosomal material was 80S monomers, native 60S and 40S ribosomal subunits were also detected independently of the contamination level in the surrounding waters. However, concomitant with the increase of contamination stress, the level of 80S ribosomes was reduced in favor of free ribosomal subunits. In addition, ribosomes isolated from contaminated mussels and programmed with poly(U) were less efficient to enzymatically bind AcPhe-tRNA, compared with ribosomes from control samples. These results suggest that the contamination stress causes stoichiometric aberrations in the ribosomal particle pool and reduction of translation machinery capability to initiate protein synthesis. Data support the notion that downregulation of translation is an important component of the cellular stress response and may be exploited as a biomarker of environmental contamination.

Microbiological Quality of Drinking Water in South-Western Greece

A study was carried out in order to estimate the presence of enteric and non-enteric indicators in the water distribution systems of Western Greece and to evaluate different methods for culture and isolation of coliforms in that region, under several incubation conditions and using different media. According to the different media and techniques used, the numbers of water samples found unsafe for consumption represented 21, 17 and 10% of the total, when mT7 agar, M-Endo agar LES (Membrane Filtration technique) and Most Probable Number (MPN) techniques were used, respectively. In one third of the samples oxidase positive microorganisms were present, which were almost eliminated by using anaerobic incubation of the media. Faecal streptococci were found only in 5% of the samples tested. The MPN technique proved to be significantly less efficient in recovering the coliform colonies than the Membrane Filtration (MF) technique (x2 = 125.758 < 182.405). The use of m-Endo agar LES and mT7 agar showed no statistically significant difference in detecting total coliforms (x2 = 162.55 > 162.422). However, a larger mean number of colonies per sample developed on mT7 agar, indicating that the latter medium should be used in our region for the detection of total coliforms.