An improved selective/differential medium for culturing the Bacteroides fragilis group from wastewater.

The aim of this study was to develop an effective selective/differential medium for culturing environmental strains of the Bacteroides fragilis group (BFG). This goal was achieved by modifying standard commercial Bacteroides Bile Esculin Agar (BBE Agar). Bacteroides Bile Esculin Agar was combined with substances that inhibit the growth of non-BFG bacteria, mostly Klebsiella pneumoniae and Fusobacterium mortiferum. The strains isolated from standard and modified BBE Agar were identified as BFG strains by PCR and 16S rRNA gene sequencing. The supplementation of standard BBE Agar with colistin (40 mg L-1), kanamycin (400 mg L-1) and vancomycin (7.5 mg L-1) increases the effectiveness of BFG bacteria isolation from <10% to 35%, and additional Gram staining improves the effectiveness of bacterial isolation five-fold relative to standard BBE Agar. The results of the present study also suggest that the presence of the bfr gene is not a reliable indicator for the identification of BFG strains.

The emergence of antimicrobial resistance in environmental strains of the Bacteroides fragilis group.

Anaerobic bacteria of the genus Bacteroides are a large group of commensal microorganisms that colonize the human and animal digestive tract. The genus Bacteroides and the closely related genus Parabacteroides include the Bacteroides fragilis group (BFG) of potentially pathogenic bacteria which are frequently isolated from patients with anaerobic infections. The aim of this study was to assess the antimicrobial resistance of environmental strains of the Bacteroides fragilis group. Strains were isolated from human feces, hospital wastewater, influent (UWW) and effluent (TWW) wastewater from a wastewater treatment plant (WWTP), and from the feces of lab rats as a negative control to monitor the entire route of transmission of BFG strains from humans to the environment. The resistance of 123 environmental BFG strains to six antibiotic groups was analyzed with the use of culture-dependent methods. Additionally, the presence of 25 genes encoding antibiotic resistance was determined by PCR. The analyzed environmental BFG strains were highly resistant to the tested antibiotics. The percentage of resistant strains differed between the analyzed antibiotics and was determined at 97.56% for ciprofloxacin, 49.59% for erythromycin, 44.71% for ampicillin, 35.77% for tetracycline, 32.52% for amoxicillin/clavulanic acid, 26.83% for chloramphenicol, 26.01% for clindamycin, 11.38% for moxifloxacin, and 8.94% for metronidazole. The highest drug-resistance levels were observed in the strains isolated from UWW and TWW samples. The mechanisms of antibiotic-resistance were determined in phenotypically resistant strains of BFG. Research has demonstrated the widespread presence of genes encoding resistance to chloramphenicol (100% of all chloramphenicol-resistant strains), tetracyclines (97.78% of all tetracycline-resistant strains), macrolides, lincosamides and streptogramins (81.97% of all erythromycin-resistant strains). Genes encoding resistance to ß-lactams and fluoroquinolones were less prevalent. None of the metronidazole-resistant strains harbored the gene encoding resistance to nitroimidazoles. BFG strains isolated from UWW and TWW samples were characterized by the highest diversity of antibiotic-resistance genes and were most often drug-resistant and multidrug-resistant. The present study examines the potential negative consequences of drug-resistant and multidrug-resistant BFG strains that are evacuated with treated wastewater into the environment. The transmission of these bacteria to surface water bodies can pose potential health threats for humans and animals; therefore, the quality of treated wastewater should be strictly monitored.

Microbiological quality of carbonated and non-carbonated mineral water stored at different temperatures.

The microbiological quality of five brands of carbonated and non-carbonated mineral water sold in Poland was studied. The study was carried out on the survival of heterotrophic bacteria at 22 and 37 degrees C (pour plate technique) in the samples of mineral waters stored at 4 and 22 degrees C. The one hundred ten bottles (twenty two bottles of each of the five brands) of carbonated and uncarbonated mineral waters with different levels of dissolved solids and organic content were chosen to microorganisms study. Ten bottles of mineral water were studied initially. Fifty bottles were stored at 4 degrees C, the other fifty were kept at 22 degrees C. The haemolysing bacteria in 1 cm3; E. coli, P. aeruginosa and A. hydrophila in 250 cm3 of mineral water were unidentifiable. Total viable count of heterotrophic bacteria at 22 and 37 degrees C in 1 cm3 of mineral water was the highest respectively for brand T and for brands T and M; the lowest for brand Z. Initially, approximately 29% of 110 water samples (respectively 4% of carbonated and 55% of uncarbonated) had bacterial counts greater than Ministry of Health's standards, notwithstanding the number of water samples which doesn't perform requirements grew up to 47% (respectively 36% of carbonated and 58% of uncarbonated) when the time of TVC 37 and 22 degrees C incubation was elongated from 1 and 3 days to 3 and 14 days respectively. The temperature of storage was inessential for the numbers of studied microorganisms. The most important factors were the brand, time of storage and the carbonating or non-carbonating of water. The highest numbers of the bacteria analysed were detected in non-carbonating water, irrespective of the water brand and temperature of storage.

Survival of Escherichia coli and Aeromonas hydrophila in non-carbonated mineral water.

The study was carried out on the survival of Escherichia coli and Aeromonas hydrophila in samples of mineral waters. Enumeration of the bacteria was performed by spread inoculation of samples (0.1 cm3) over the surface of selected media in Petri plates. Twenty bottles (four bottles of each of the five brands) of non-carbonated mineral waters with different levels of dissolved solids and organic content were chosen to study every strain. Ten bottles were stored at 4 degrees C, the other ten were kept at 22 degrees C. Half of the samples of mineral water was filtered, the remaining water was unfiltered. The resulting growth curves depended on the time of storage. The number of E. coli increased during the first two weeks (except the seventh day) and decreased during the next days. E. coli was detected in 70% of samples of water after 182 days. The number of A. hydrophila decreased during the first three days, increased on the seventh day and decreased during the next days. A. hydrophila was detected in 15% of the samples of water after 182 days. The temperature of storage was inessential for growth. The most important factors were the brand and the filtering or unfiltering of water. The highest numbers of the bacteria analysed were detected in filtered water, irrespective of the water brand and temperature of storage.

Sanitary and bacteriological aspects of sewage treatment.

A study into the removal of contamination load and indicator bacteria was carried out in 1992-1996 in the mechanical, biological and chemical waste-water treatment plant WTP in Lezany, in the County of Reszel, in the Province of Warmia and Mazury in Poland. The results of chemical analyses found a high efficiency of removal of carbon compounds, COD (90%) and BOD (98%), in the process of purification of household sewage. In addition, a high effectiveness of total nitrogen, on average 71%, and unsatisfactory removal of ammonia nitrogen and phosphorus compounds were found. The results of microbiological analyses confirmed the high efficiency of removal of indicator bacteria in the process of sewage treatment from 94 to 97%. In the sewage after the final phase of purification in stabilization ponds, the following pathogenic bacteria were identified with the use of the EPL 21tests: Escherichia coli, Enterobacter agglomerans, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter georgoriae, Citrobacter freundii, Klebsiella pnemoniae, Klebsiella oxytoca, Klebsiella ozaenae, Ervinia herbicola, Edwardsiella tarda, Serratia odoriefra, Serratia marcescens, Providencia alcalifaciens, Hafnia alvei, Yersina pestis, Yersina pseudotuberculosis, Yersinia fredericksenii, Salmonella spp., Shigella dysenteriae, Aeromons hydrophila, Pseudomonas aerulginosa. The obtained results show that although the sewage purification system is efficient and reduces the contamination load to the level required by the regulations (Ministry of Environmental Protection, Natural Resources and Forestry from 20 September 1991) and removes a great percentage of indicator bacteria, the purified sewage may be a source of pathogenic bacteria in inland waters.