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.

Development and application of a new scheme of phages for typing and differentiating Salmonella strains from different sources.

A set of 25 phages for typing and differentiating Salmonella strains from different origins (food, water, and disease outbreaks) is described. All the strains were typeable by use of the phages, whereas by the serological method more than 5% of the strains could not be classified. By using the phage typing scheme, 75 phage types were established, and all the results were reproducible 1 and 6 months later. Some phages were serotype and serogroup specific, which may be useful in additional tests for the identification of strains of some Salmonella serotypes. In addition, the strains responsible for an outbreak possessed the same phage type, which implies the potential epidemiological use of these phages.

Development and application of new positively charged filters for recovery of bacteriophages from water.

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.

Application of direct plaque assay for detection and enumeration of bacteriophages of Bacteroides fragilis from contaminated-water samples.

The direct double-agar-layer plaque assay for the detection and enumeration of specific bacteriophages of Bacteroides fragilis from contaminated-water samples was performed. Several factors that affect the methods, such as conditions of the bacterial culture, composition of the assay medium, addition of divalent cations, and decontamination techniques applied to the sample, were evaluated. The results obtained show that the direct assay technique proved to be more efficient than the most-probable-number technique. A higher recovery of bacteriophages was obtained from 17 of 24 samples with the direct assay. The two methods only showed similar results from samples with a low degree of pollution.

Antibiotic resistance of Salmonella strains isolated from natural polluted waters.

Resistance to 14 antibiotics was tested in 270 Salmonella strains isolated from different aquatic environments. All the strains were sensitive to nalidixic acid (30 micrograms) and cephalothin (30 micrograms) but more than 90% were resistant to tetracycline (30 micrograms). The percentage of strains resistant to other antimicrobial substances depended on the antibiotic and on the isolation source. Twenty-four resistance patterns were recorded in strains isolated from three environmental sources. The only multi-resistance detected in the three ecosystems was that of sulphadiazine and tetracycline (about 20%). The serotypes most frequently detected with multi-resistance to different antibiotics were Salmonella typhimurium and Salm. blockley.

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.

Evaluation of different plating media used in the isolation of salmonellas from environmental samples.

Different serotypes of salmonellas were compared for selectivity and efficiency of recovery using 11 plating media. No optimal growth was obtained after 24 h incubation in any of the media, but after 48 h, brilliant green, brilliant green-phenol red-lactose-sucrose, bismuth sulphite, xylose-lysine-deoxycholate and Hektoen enteric agars showed optimal recovery of all the salmonella serotypes. Xylose-lysine-deoxycholate and brilliant green-phenol red-lactose-sucrose agars were the most selective media for all salmonella serotypes. Addition of 10 micrograms/ml of sodium novobiocin to the tryptic soy-xylose-lysine and tryptic soy-brilliant green agars significantly improved their selectivity but reduced or inhibited the growth of some salmonella serotypes, including Salmonella typhi. Xylose-lysine-deoxycholate agar gave the highest recovery percentage of stressed salmonellas with a double-agar layer technique. Good recovery was also obtained on brilliant green-phenol red-lactose-sucrose, tryptic soy-brilliant green, tryptic soy-brilliant green-novobiocin, tryptic, soy-xylose-lysine and tryptic soy-xylose-lysine-novobiocin agars. Salmonella-shigella agar was the least efficient medium for the recovery of salmonellas under stress-induced or non-stressed conditions.