Roberto Cornax

Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

Abstract The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.

Evaluation of different bacteriophage groups as faecal indicators in contaminated natural waters in southern England

Abstract River and seawater affected by faecal discharges were analysed to evaluate the reliability of coliphages and F-specific RNA bacteriophages as indicators of the microbiological quality of waters. F-specific RNA bacteriophages showed no direct relationship with the levels of faecal pollution, and this group was never detected in samples with a low level of enteroviruses (1–10 pfu/10 l.). In contrast, coliphages were constantly detected in the same samples. The concentration of coliphages detected in the samples with 1–10 and > 10 pfu/101. of enteroviruses were similar and higher than the Economical European Community guide and imperative levels of faecal coliforms (100 and 2000 FC/100 ml, respectively). Therefore, coliphages would be considered as an optimal indicator of the microbiological quality of the natural waters.

Coliphages as an indicator of faecal pollution in water : their survival and productive infectivity in natural aquatic environments

The capability of coliphages as indicators of faecal pollution was tested, on the basis of their survival and infectivity in two natural aquatic environments (marine and river). The results obtained indicate that coliphages show a similar inactivation rate to Salmonella in all types of water studied. On the other hand, the phage productive infection of Escherichia coli cells in water and environmental conditions depends on the physiological characteristics of the host bacteria, which generally cannot grow optimally in these conditions. In conclusion, the coliphages may be considered good indicators of faecal pollution in natural waters.

Relationships between salmonella spp and indicator microorganisms in polluted natural waters

Abstract The relationships between Salmonella spp and indicator microorganisms (total coliforms, faecal coliforms, faecal streptococci, Clostridium perfringens and coliphages) were investigated in three different aquatic environments affected by wastewater discharges. The results indicated that the statistical correlations obtained depended, basically, on the source of faecal discharges. The microorganisms most closely correlated with Salmonella spp were faecal coliforms and C. perfringens , the latter also yielding the highest linear regression slope values. The detection percentages of Salmonella spp were high even at a low degree of pollution, which allowed detection of the pathogens in the absence of classical indicator microorganisms in the sample.

Significance of several bacteriophage groups as indicators of sewage pollution in marine waters

Seawater samples collected from two beaches with different levels of pollution were studied for the presence of classically and newly proposed faecal indicators such as, total and faecal coliforms, faecal streptococci, coliphages, F-specific phages and bacteriophages of Bacteroides fragilis. Total and faecal coliforms showed lower survival rates in seawater than faecal streptococci, F-specific bacteriophages and coliphages. On the other hand, total coliform concentrations were only higher than those of faecal coliforms in heavily polluted seawater, although in samples with a low level of pollution, faecal streptococci and Escherichia coli C phage counts were generally greater than those showed by faecal coliforms. The low concentration in which F-specific and B. fragilis bacteriophages were detected in marine waters compared to the E. coli bacteriophage levels, is an important shortcoming for the general use of the former microorganisms as universal indicators of faecal pollution. From the results obtained in this study, it may be concluded that faecal streptococci and E. coli C bacteriophages are the most appropriate indicators of the remote pollution in marine waters.

Survival of pathogenic microorganisms in seawater.

The influence of different marine self-purifying factors on the survival of several indicator and pathogenic microorganisms under controlled laboratory conditions was studied. Pathogenic microorganisms showed inactivation rates similar to those obtained by indicators under the same conditions. It was observed that the visible light and biotic components of seawater were the most important inactivating factors.

Viability of Salmonella spp and indicator microorganisms in seawater using membrane diffusion chambers

Diffusion chambers with polycarbonate membrane-filter side walls were used to study the comparative survival of fecal indicators (Escherichia coli and Streptococcus faecalis) and enteric pathogens (Salmonella enteritidis, S. postdam, S. typhimurium, S. london and S. infantis) in natural seawater. It was observed that the percentages of sublethal injury increased with exposure to the marine environment, and that these environmental injuries depended on the microorganism considered. A large proportion of cells lost their ability to produce colonies on the selective media, but retained this capability on a nonselective medium. All microorganisms showed low survival percentages (less than 11%) after 48 hrs of exposure to seawater, but there is not a high difference among the microbial species studied.The results obtained in the present study showed that there were no differences in the survival rates between the serotypes of Salmonella tested. Moreover, Salmonella spp exhibited a similar persistence to E. coli in the marine environment.

Bacteriophages presence in human faeces of healthy subjects and patients with gastrointestinal disturbances

The variation of the content of enteric bacteria and their bacteriophages in faeces from the different types of diarrhoeal processes has been studied. A total of 122 samples of human faeces from both healthy individuals and patients with diarrhoeal diseases of functional or infectious origin were tested. Detection rates for all microbial parameters tested decreased in the faeces of individuals with functional gastrointestinal disturbances. On the contrary, no significant differences of the microbial detection frequency was observed in faeces containing pathogenic microorganisms compared to faeces of healthy subjects. Human faeces were a poor source of F-specific, Salmonella, and Bacteroides bacteriophages, whereas specific Escherichia coli phages were isolated in most samples tested. Coliphage concentrations in faeces of healthy individuals were not directly correlated with levels of faecal coliforms. On the basis of their high correlation, faecal streptococci and coliphages were the most adequate indicators of the intestinal ecosystem variations in subjects with diarrhoeal processes.

An intercalibration study of the use of 4-methylumbelliferyl-β-D-glucuronide for the specific enumeration of Escherichia coli in seawater and marine sediments

Summary A fluorogenic assay for the specific detection of Escherichia coli on the basis of its β-glucuronidase activity (MUG method) was applied to seawater and marine sediments with different contamination levels. The study was carried out in three Mediterranean areas (Malaga-Spain, Nice-France and Palermo-Sicily), using strictly standardized methods (membrane filtration), media (mFC and Chapman-TTC agars) and reagents, to evaluate statistically its sensitivity and specificity according to the origin and contamination of samples, the workers performing the tests and the selected culture media. The results obtained indicate that the MUG method is highly specific (94.5%) and sensitive (90.8%) for the detection of E.coli in marine samples. Its reliability however closely depends on the macroscopic differentiation of colonies typical of the species on agar plates, and on the number of colonies on the filters. Of the 798 typical colonies with a positive MUG reaction, more than 95.5% were identified as E.coli , and the false-positive results mainly corresponded to Citrobacter freudii (1.75%) and Shigella spp. (1.63%). Only 0.56% of total isolates (14.9% of non-typical MUG negative isolates) were confirmed as E. coli from non- typical colonies on both selective media with a negative MUG reaction (false-negative). It was concluded that the MUG method, in conjunction with the membrane filtration technique using mFC agar is faster, easier and cheaper than the standard procedure for the examination of seawater and sediment samples.

Modified most-probable-number technique for the specific determination of Escherichia coli from environmental samples using a fluorogenic method

Abstract A specific and senstive modification of the most-probable-number (MPN) technique by addition of 4-methylumbelliferyl-β-D-glucuronide (MUG) to both presumptive and confirmatory media was performed. The use of this modification allows the precise determination of Escherichia coli from marine samples (seawater, sediment and shellfish) within 7 days compared to 10–12 days required by using of the standard methodology. No false-positive isolates for fluorescence reaction have been observed, although one E. coli strain fluorescent-positive on agar was isolated from nonfluorescent tubes. Klebsiella pneumoniae was the species most frequently detected from tubes with gas and fluorescence production and typical morphology on mFC agar, but failed on nutrient-MUG agar. Using a unifactorial variance analysis of the mean, fluorescence in tube and on agar has been determined as the factor which allows the detection of E. coli presence in the three sample types with high accuracy. The incorporation of MUG to the selective broth in the confirmatory test has been shown to avoid false-positive results due to shellfish tissue β-glucuronidase activity. Specificity of the MUG-MPN method was high ( Se = 0.94 and Sp = 0.79). The results obtained using laboratory strains showed that although E. coli, Citrobacter freundii, Enterobacter cloacae and K. pneumoniae produce acid and gas in MacConkey-purple broth, β-glucuronidase activity in this selective broth is specific to E. coli .