Abscesses and wound infections due to Staphylococcus lugdunensis: report of 16 cases.

PURPOSE: Staphylococcus lugdunensis has emerged as a major human pathogen, capable of causing significant infections at many sites. It should never be dismissed as a contaminant without careful review. We report 16 cases of wound infections and skin and soft tissue abscesses caused by S. lugdunensis during a period of 3.5 years (January 2008-June 2011). These cases were isolated from clinical specimens in a tertiary hospital (250 beds) in Athens, Greece. METHODS: The identification of S. lugdunensis was based on Gram staining, catalase and coagulase test results, and 26 biochemical reactions that were included in the database of the MicroScan Walkaway 96 commercial system. The susceptibility pattern was performed with the same commercial system according to CLSI recommendations. RESULTS: Twenty-five isolates were classified as S. lugdunensis, of which 16 were considered to be clinically significant. The age distribution of the patients ranged from 29 to 65 years. Patient outcome after treatment was good with no long-term sequel. All isolated S. lugdunensis were methicillin sensitive (cefoxitin screen negative), while five isolates were ß-lactamase producers. The isolates were susceptible to most of the antibiotics tested except for a few cases that were resistant to erythromycin, tetracycline, and clindamycin. CONCLUSIONS: Coagulase-negative staphylococci isolated from traumatic and surgical wound infections should be identified by microbiological laboratories to the species level, and susceptibility testing should be performed on these isolates so as not to underrate the virulence of staphylococci resembling S. aureus.

Occurrence of microorganisms of public health and spoilage significance in fruit juices sold in retail markets in Greece.

Fruit juices are an important part of the modern diet in many countries. However, few data are available concerning the microbiological quality of the fruit juices sold in Greece. Using standard microbiological procedures, we conducted a bacteriological survey of commercially sold, pasteurized, shelf-stable fruit juices from retail markets. A total of 120 samples of fruit juices sold in various retail markets were examined for their bacteriological quality. The pH of the tested juices was 2.4-4.8. Bacteria were isolated from 51 samples (42.5%) and fungi from 78 samples (65%). Escherichia coli O157:H7 was detected in four of the analyzed samples (3.34%), and Staphylococcus aureus was detected in four different samples (3.34%). In 11 samples (9.1%), the total number of microorganisms detected was as high as 125 colony-forming units (CFU). Acidophilic microorganisms were isolated from 26 samples (21.7%) and Blastomyces was detected in 46 samples (38.3%). All samples were negative for Lactobacillus, Clostridium perfrigens, Salmonella spp., Bacillus cereus, total coliforms, E. coli, and Listeria monocytogenes. Many of the microorganisms detected may cause disease in humans; thus, a number of the tested samples did not meet the Greek guidelines for the microbiological quality of juices. Use of a Hazard Analysis Critical Control Point (HACCP) system should be generally introduced into the juice industry sector to improve the quality of fruit juices, as well as other manufactured foods.

Hybridisation of F+ RNA coliphages detected in shellfish samples with oligonucleotide probes to assess the origin of microbiological pollution of shellfish.

Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator, male-specific B+ coliphages, have been investigated as viral indicators of faecal contamination that may provide source-specific information for impacted environmental waters. This study compared the distribution of E. coli and F+ RNA bacteriophages in shellfish grown in harvesting areas of Greece and also examined the presence and proportions of the different subgroups of F+ RNA coliphages in shellfish. F+ RNA bacteriophages were present in shellfish at higher concentrations than E. coli. Elevated numbers of F+ RNA bacteriophages observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in Greece. The majority of F+ RNA coliphages detected in shellfish samples belonged to group IV which indicated the possible presence of animal faecal material in sample harvesting areas. Phages of groups II and III (human waste and human faecal material, respectively) were present at low levels. Finally, 8% of the phages hybridised were found to belong to group I. The presence of group IV showed seasonal distribution (more in winter, less in summer) whereas the other groups did not show any difference. Monitoring of F+ coliphage subgroups may indicate the presence and major sources of microbial inputs to surface waters; however, environmental effects on the relative occurrence of different groups need to be 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. METHODS AND RESULTS: Disk diffusion method was applied with a selection of antibiotics. Statistical approach was performed with hierarchical cluster analysis (CA), discriminant analysis (DA) and principal component analysis. Comparing human and animal isolates there was significant difference in levels of resistance to all antibiotics tested (P<0.05) with 46 and 24 distinct resistance patterns for human and animal isolates respectively. CA and DA separated human and animal isolates with a high average rate of correct classification (99.2%), when all animal isolates were pooled together. CONCLUSIONS: MAR analysis compared with appropriate statistical evaluation may provide a useful tool for differentiating the human or animal origin of E. coli isolates derived from environmental samples. Subsequently, determination of the source of faecal pollution becomes possible. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the source of faecal pollution enables the prediction of possible risk for public health and the application of appropriate management plans for prevention of further contamination.

Microbiological evaluation of bottled non-carbonated ("still") water from domestic brands in Greece.

The microbiological quality of 1,527 samples of bottled non-carbonated ('still') mineral water, purchased from retail outlets and derived from 10 manufacturing companies in Greece, was investigated during the period 1995-2003. Applying the membrane filter technique, the aliquots of water samples (250 ml) were analyzed for the presence and enumeration of total coliforms, Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa. Also, aerobic bacteria were counted as Heterotrophic Plate Count (HPC) ml(-1) at 22 and 37 degrees C. Positive samples for the parameters tested varied significantly among brands with an overall percentage of 13.95% bottled water samples noncompliant with the Greek water regulation. Microorganisms isolated from the samples tested were identified as species of Pseudomonas, Aeromonas, Pasteurella, Citrobacter, Flavobacterium, Providencia and Enterococcus. The most frequent isolated microorganism during the period of the study was P. aeruginosa. Generally, bacterial load of the samples tested ranged in low levels. The purpose of the current study was to evaluate the microbiological quality of the bottled water provided by domestic brands in the Greek market during the period 1995-2003.

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. METHODS AND RESULTS: Pseudomonas strains (160) isolated from the aquatic environment in Greece and identified by a rapid identification commercially available system (API20NE), were subjected to whole-cell protein electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Randomly Amplified Polymorphic DNAs (RAPD) using two 10-mer primers. In general, the obtained results were in agreement. Twenty isolates that could not be identified by the API20NE system were classified by the other methods. CONCLUSIONS: Rapid identification systems may serve only for a first rough identification of environmental Pseudomonads. In order to acquire further information, so that conclusions about their role in the ecosystem and human health could be drawn, other phenotypic or genotypic methods have to be applied. SIGNIFICANCE AND IMPACT OF STUDY: It is important, from a public health point of view, to monitor the identities of environmental Pseudomonas isolates using specific methods due to their ubiquity, heterogeneity and their pathogenicity, either established or potential.

Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria.

A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. QC ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35 degrees C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96-well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35 degrees C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization.

Evaluation of microbiological quality of coastal waters in Greece.

To evaluate the microbiological water quality of bathing sites along the Achaia coastline (south western Greece), a survey was conducted to determine the concentration of faecal bacterial and phage indicators as well as the presence of human viruses. Seawater samples (234) were collected from nine bathing sites on the Achaia coastline and were analysed for the presence of: total coliforms, faecal coliforms, faecal streptococci, Escherichia coli, somatic coliphages, F-RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, enteroviruses, adenoviruses and hepatitis A viruses. Most of the bacteriological analysis results were in accordance with the European Union standards. In all sites, bacteriophages were detected occasionally. Enteroviruses and adenoviruses were detected in 24 samples (10.26%) and 37 samples (15.81%) respectively. No samples were positive for the presence of hepatitis A virus. The overall data indicates that bathing sites are impacted by human faecal material. Both bacterial indicators and phages have low predictive capability for the presence of human viruses in coastal waters. None of the environmental parameters analysed was strongly related to the presence of the indicator organisms and viruses. Appropriate and effective administrative measures that should be taken into account may be considered in order to improve water quality and reduce public health risk.

Differentiation of faecal Escherichia coli from human and animal sources by random amplified polymorphic DNA-PCR (RAPD-PCR).

In this study the assessment of randomly amplified polymorphic DNA (RAPD) analysis was established as a molecular epidemiological tool. RAPD analysis was performed to differentiate faecal Escherichia coli isolates from human and animal sources. E. coli strains (128) were isolated from human and animal faeces (from cattle and sheep). Genomic DNA was extracted and randomly amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting was performed. Seven arbitrary primers were tested with a view to discriminating between E. coli isolates from humans and E. coli isolates from animals. RAPD profiles were analysed with hierarchical cluster analysis using an unweighted pair group method. RAPD profiles obtained with three of the tested primers (1247, 1290 and 1254) established a distinct differentiation between E. coli isolates from humans and E. coli from animals. Low levels of misclassification and high levels of specificity make RAPD a sensitive, efficient and reliable means of distinguishing closely related strains.

Evaluation of virological quality of sewage from four biological treatment plants by a nested-PCR technique.

In order to determine the virological quality of sewage from four biological treatment plants in Greece (two in the city of Athens and two in the city of Patras), 92 raw sewage samples were analysed for the presence of enteroviruses and adenoviruses during the period from October 2000 to February 2003. A nested-PCR method was used in order to increase the sensitivity of virus detection. Enteroviruses were detected in 43 samples (47%) and adenoviruses in 75 samples (81.5%) of raw sewage by nested PCR. The more frequent isolation of adenoviruses in raw sewage indicated their stability as virological indicators of the pollution of the environment and their increased persistence in sewage.

Antimicrobial susceptibility testing of aquatic bacteria: quality control disk diffusion ranges for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 at 22 and 28 degrees C.

Quality control (QC) ranges for disk diffusion susceptibility testing of aquatic bacterial isolates were proposed as a result of a multilaboratory study conducted according to procedures established by the National Committee for Clinical Laboratory Standards (NCCLS). Ranges were proposed for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 at 22 and 28 degrees C for nine different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, gentamicin, oxolinic acid, oxytetracycline, ormetoprim-sulfadimethoxine, and trimethoprim-sulfamethoxazole). All tests were conducted on standard Mueller-Hinton agar. With >/=95% of all data points fitting within the proposed QC ranges, the results from this study comply with NCCLS guidelines and have been accepted by the NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing. These QC guidelines will permit greater accuracy in interpreting results and, for the first time, the ability to reliably compare susceptibility test data between aquatic animal disease diagnostic laboratories.

Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas.

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.

Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom.

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

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.

Multiantibiotic resistance of gram-negative bacteria isolated from drinking water samples in southwest Greece.

In this study we monitored the sensitivity of 239 gram-negative bacteria (of fecal and non-fecal origin), isolated from the old drinking water distribution network of Patras in southwestern Greece, to 20 antibiotic agents. Two methods were used to find the multiresistant bacteria (bacteria resistant to two or more antibiotics): the diffusion disk method and a serial dilution method. The gram-negative bacteria tested were: Enterobacteriaceae (62), Pseudomonas (145), Vibrionaceae (24), Chromobacter (3), Acinetobacter (2) and others (4). The highest levels of antibiotic resistance were obtained for cephalothin (86.7%), ampicillin (77.5%) and carbenicillin (71%) followed by cefoxitin (55.4%) and cefuroxime (51.2%). Intermediate resistance levels were found for ticarcillin (31.3%), ceftizoxime (31.2%), chloramphenicol (30.3%), and cefotetan (25.2%). Low resistance levels were obtained for cefotaxime (17.9%), sulfisoxazole (15.2%), ceftriaxone (12.5%), tetracycline (11.9%), trimethoprim/sulfamethoxazole (7.4%) and piperacillin (2.4%). Overall 91.3% of the gram-negative bacteria isolated from drinking water were multiresistant. No resistant strains were found to quinolones, aminoglycosides, imipenem, aztreonam, ceftazidime or cefoperazone. The high antibiotic resistance rate of the isolated microorganisms from the Patras drinking water supply is discussed.

Changes in protein composition and hydrolytic enzyme activity of Escherichia coli and Hafnia alvei grown in human fluids.

Growing of Escherichia coli and Hafnia alvei cells in several cell-free human fluids, such as normal serum, serum from diabetic patients, pleural, ascitic and spinal fluid, revealed that various biochemical changes occurred. Protein profile on SDS-PAGE as well as acid and alkaline phosphohydrolytic enzymes on native gels of cell extracts were affected after culturing of bacteria in the above fluids. Gelatinolytic and hyaluronolytic activity was of interest because both of them are histolytic enzymes. Although there was a potential appearance of gelatinolytic bands on gelatin-SDS-PAGE in cells starved in seawater, none of these activities were expressed in cells grown in human fluids. A hyaluronolytic activity of approximately 45 KDa was present in cells cultured in Mueller Hinton broth. This enzyme was decreased either in cells starved in seawater or in cells grown in human fluids to an almost invisible band on hyaluronan-SDS-PAGE.

Detection of adenovirus outbreak at a municipal swimming pool by nested PCR amplification.

In July 1995 an outbreak of pharyngoconjuctivitis caused by adenoviruses occurred among athletes participating in a swimming contest in a town in southern Greece (Peloponnese). At least 80 persons displayed symptoms of the illness, with the predominant ones being high fever, sore throat, conjuctivitis, headache, and abdominal pain. Poor chlorination was probably the cause of the outbreak (residual chlorine <0.2 mg/l), as after hyperchlorination the spread of adenoviruses stopped. Rapid detection of adenoviruses in the municipal swimming pool water by nested polymerase chain reaction (PCR) amplification allowed quick control of the outbreak.

Changes in ribosomal activity of Escherichia coli cells during prolonged culture in sea salts medium.

The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the kmax/Ks ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the kmax/Ks value. (kmax is the apparent catalytic rate constant of peptidyl transferase, and Ks is the dissociation constant of the encounter complex between acetyl (Ac)[3H]Phe-tRNA-poly(U)-ribosome and puromycin.) Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.

Electrophoretic analysis of hydrolytic enzymes of Escherichia coli cells starved in seawater and drinking water: comparison of gelatinolytic, caseinolytic, phosphohydrolytic and hyaluronolytic activities.

Starvation of four Escherichia coli clinical strains in seawater and drinking water for nine days revealed that various changes of hydrolytic enzymes were induced. Several gelatinolytic and caseinolytic activities differing in mol mass were detected both in seawater and drinking water starved cells by substrate gel electrophoresis. The major activities of gelatinase migrated with mol masses of approximately 170 kDa and approximately 45 kDa. On the contrary, hyaluronolytic activities were detected only in cells cultured in Mueller Hinton broth with average mol masses of 36 kDa and 45 kDa. Acid and alkaline phosphohydrolytic activities were detected by native electrophoresis. Both activities were decreased in number of bands in E. coli cells starved either in seawater or drinking water.

Environmental mycobacteria in bottled table waters in Greece.

A hundred and fifty samples of bottled table water sold by Greek factories were examined for the presence of environmental mycobacteria. Environmental mycobacteria were found in 23 (15.6%) of the 150 tested samples. Bacterial numbers of 1-100, 101-300, 301-1000, and > 10(3) CFU/L were found in 8, 2, 1, and 4% of the samples, respectively. The identification of the environmental mycobacteria was performed by both polymerase chain reaction-restriction enzyme analysis (PCR-REA) and biochemical methods. The environmental mycobacteria found were 14 Mycobacterium chelonae, 3 Mycobacterium phlei, 4 Mycobacterium gordonae, and 2 Mycobacterium flavescens. The relatively high number of environmental mycobacteria in bottled table water leads us to believe that the search of these opportunistic microorganisms in bottled water could be a useful index of their hygienic quality when this water is to be consumed by immunologically compromised patients. No statistically significant correlation was found between the presence of mycobacteria and the bacteriological faecal indicators (P < 0.005).