The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants

AIMS: The changes in structure and composition of faecal coliforms and enterococcal populations in sewage from different treatment plants, and the elimination of vancomycin- and erythromycin-resistant enterococci (VRE and ERE, respectively) in these treatment plants was analysed to determine any selective reduction. METHODS AND RESULTS: Faecal coliforms, enterococci, VRE, ERE and spores of sulphite-reducing bacteria were enumerated using standard methods. Samples were enriched where necessary in order to isolate antibiotic resistant strains. The structure and composition of these bacterial populations were determined by biochemical fingerprinting and clustering analysis. High diversity and similarity indexes were detected among all the bacterial populations in raw and treated sewage, independently of their origin and the treatment processes employed. Antibiotic resistant strains were detected in all sewage tested and no selective reduction was observed. CONCLUSIONS: The faecal coliforms and enterococci populations did not differ in the sewage samples studied. The vancomycin and erythromycin resistances of the enterococcal populations were similar in the sewage samples. Resistance to both antibiotics persisted after the treatment process independently of raw sewage flow, faecal origin or size of the human population contributing to sewage. However, sewage of mixed origin (human and animal) presented a lower similarity index for the two bacterial populations compared with that of the other human sewage analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Although a significant reduction in bacterial populations was observed, the persistence of VRE and ERE strains in the same proportions in sewage suggests that there is no selective elimination of bacterial populations during the treatment processes. The ability of antibiotic resistance strains to survive sewage treatment systems should be considered in certain water reuse programmes.     

Prevalence and antibiotic resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in tropical seafood

Summary The occurrence and antibiotic resistance of Escherichia coli in tropical seafood was studied. A 3-tube MPN method was used for determining the level of faecal contamination of fresh and processed seafood. Of the 188 samples tested which included finfish, shellfish, water and ice, 155 were positive for the presence of faecal coliforms following incubation at 44.5 °C. However, E. coli was isolated from only 47% of the samples positive for faecal coliforms. The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different antibiotics including ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamycin, nalidixic acid, streptomycin and vancomycin. Seven strains were resistant to more than five antibiotics of which one was resistant to eight antibiotics. The multiple drug resistant strains harboured plasmids of varying sizes. Antibiotic susceptibility studies revealed that seafood from India contains multiple antibiotic resistant strains of E. coli which may serve as a reservoir for antibiotic resistance genes in the aquatic environment. All the strains used in this study did not harbour any virulence genes commonly associated with pathogenic E. coli, when tested by polymerase chain reaction (PCR).     

Antibiotic resistant bacteria in Windermere and two remote upland tarns in the English Lake District

The incidence of antibiotic resistance was determined in over 2000 bacteria which were divided into the following groups: faecal streptococci, coliforms (excluding Escherichia coli ), E. coli, Pseudomonas spp. and aquatic bacteria (i.e. bacteria predominant in the lake water which were excluded from the previous four categories). The isolates were obtained from the water of Windermere (English Lake District) and from a sewage effluent which entered the lake. With the exception of the faecal streptococci, the incidence of antibiotic resistance was higher in the bacteria isolated from the lake water than in those from the effluent, and ranked according to groups Pseudomonas spp. > E. coli > aquatic bacteria > coliforms > faecal streptococci. The highest incidence of multiple resistance was found among the pseudomonads. When corrected for the relative size of each population the pool of antibiotic resistance in the aquatic bacteria was by far the largest. The incidence of antibiotic resistance in aquatic bacteria isolated from Windermere was, however, lower than in those isolated from two remote upland tarns. This finding may have been due to differences in the species composition of the three sites except that the same results were obtained when only fluorescent pseudomonads were tested. The upland tarns were not totally isolated from man and other animals but did not receive any sewage or other effluents and therefore the results were surprising. Possible explanations include a lack of susceptibility in aquatic bacteria and increased resistance associated with growth in nutrient poor environments.     

Antibiotic resistant bacteria in Windermere and two remote upland tarns in the English Lake District

The incidence of antibiotic resistance was determined in over 2000 bacteria which were divided into the following groups: faecal streptococci, coliforms (excluding Escherichia coli), E. coli, Pseudomonas spp. and aquatic bacteria (i.e. bacteria predominant in the lake water which were excluded from the previous four categories). The isolates were obtained from the water of Windermere (English Lake District) and from a sewage effluent which entered the lake. With the exception of the faecal streptococci, the incidence of antibiotic resistance was higher in the bacteria isolated from the lake water than in those from the effluent, and ranked according to groups Pseudomonas spp. greater than E. coli greater than aquatic bacteria greater than coliforms greater than faecal streptococci. The highest incidence of multiple resistance was found among the pseudomonads. When corrected for the relative size of each population the pool of antibiotic resistance in the aquatic bacteria was by far the largest. The incidence of antibiotic resistance in aquatic bacteria isolated from Windermere was, however, lower than in those isolated from two remote upland tarns. This finding may have been due to differences in the species composition of the three sites except that the same results were obtained when only fluorescent pseudomonads were tested. The upland tarns were not totally isolated from man and other animals but did not receive any sewage or other effluents and therefore the results were surprising. Possible explanations include a lack of susceptibility in aquatic bacteria and increased resistance associated with growth in nutrient poor environments.     

Multiple antibiotic resistances of <Emphasis Type="Italic">Enterococcus</Emphasis> isolates from raw or sand-filtered sewage

Fifty antibiotic-resistant Enterococcus strains were isolated from raw sewage of a wastewater treatment plant and from the same sewage after trickling through a 25-cm sand column, which retained >99% of the initial population. All 50 Enterococcus isolates were resistant against triple sulfa and trimethoprim/sulfamethoxazole and none were resistant against vancomycin. Most of the isolates from raw sewage were resistant to more antibiotics than the isolates from sand column effluent. One Enterococcus isolate from raw sewage (no. 61) and one Enterococcus isolate from sand column effluent (no. 95) had ten antibiotic resistances each. Isolate no. 95 maintained its resistances in the absence of antibiotics during the whole study. It was compared with isolate no. 70, which was one of the isolates, being resistant only against the two sulfonamides. Phenotypically and biochemically, the two organisms were strains of Enterococcus faecalis. Sequence analysis of partical 16S rDNA allowed alignment of isolate no. 95 as a strain of Enterococcus faecium and of isolate no. 70 as a strain of E. faecalis. E. faecium strain no. 95 carried at least six different plasmids, whereas for E. faecalis strain no. 70, no discrete plasmid band was seen on the gels.     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10³/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa ) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5–27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10(3)/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5-27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

The Effect of Cadmium on Indicator Bacteria in Sewage

The effect of cadmium (Cd) on the number of different faecal indicator bacteria in sewage, and on species composition of different indicator bacteria, was studied. Different amounts of Cd were added to aliquots of a sewage sample, and after 0, 4% and 24 h of Cd exposure at 20°C conforms, faecal coliforms, faecal streptococci and Clostridium perfringens were enumerated by the membrane filter method. The Cd-induced reduction in the number of coliforms and faecal coliforms during exposure was found to be greater than the decrease in the number of faecal streptococci. In the case of C. perfringens the Cd concentrations used produced no observable effect on the cell number. The addition of Cd changed the faecal coliforms and faecal streptococci density relationship. Escherichia coli seems to be more resistant to Cd than other coliforms and Streptococcus faecalis var. liquefaciens and Streptococcus durans more resistant to Cd than other faecal streptococci. No influence of Cd on gas production by faecal coliforms was observed. Faecal streptococci and C. perfringens seem to be better indicator bacteria than coliforms and faecal coliforms in evaluating the hygienic quality of Cd polluted sewage.     

Pseudomonads from wild free-living sea turtles in Príncipe Island,Gulf of Guinea

Dissemination of antibiotic resistance is a major concern, especially in aquatic environments, where pollution contributes for resistant bacteria selection. These strains may have serious health implications, especially for endangered species, including the sea turtles’ hawksbill Eretmochelys imbricata and green turtles Chelonia mydas.We aimed to evaluate the presence of antibiotic resistant pseudomonads in wild sea turtles from Príncipe Island, São Tomé and Príncipe, Guinea Gulf. Isolates were obtained from oral and cloacal swabs of free-living turtles by conventional techniques. Pseudomonads screening was performed by multiplex-PCR (oprI/oprL) and biochemical identification and antibiotic resistance profiling were achieved using Vitek2. All pseudomonad isolates were genotyped by Rep-PCR.Thirteen isolates were oprI-positive and classified as pseudomonads, eight from the genus Pseudomonas with the species P. aeruginosa, P. stutzeri, and P. mendocina, and five co-isolated Alcaligenes faecalis. The P. aeruginosa isolate was also oprL-positive. Regarding isolates susceptibility profile, 38.5% were susceptible to all antibiotics tested, and multidrug resistant (MDR) strains were not identified. DNA fingerprinting did not show any specific clonal-cluster similarity.Data on the worldwide incidence of antibiotic resistance among wildlife is still very scarce, especially concerning remote tropical areas. Since Pseudomonas genus has emerged as a group of increasingly reported opportunistic microorganisms in human and veterinary medicine with high resistance levels, it could be used as a tool for environmental resistance surveillance, particularly considering their ubiquity.     

Evaluation of Selected Membrane Filtration and Most Probable Number Methods for the Enumeration of Faecal Coliforms,Escherichia coli and Enterococci in Environmental Waters

Selected methods recommended in national and international water quality guidelines were compared in tests on environmental waters with different levels of faecal pollution. The following methods yielded no statistically significant differences in counts of faecal coliforms and Escherichia coli in raw sewage, semi-treated effluent, polluted urban run-off and stored potable water: Membrane filtration (MF) using MFc Agar or Chromocult Coliform Agar containing X-glucuronide, or a miniaturised microtitre-plate Most Probable Number (MPN) assay using a liquid growth medium containing chromogenic 4-methyl-umbelliferyl--D-glucuronide. Significant differences were, however, found between the Chromocult and the other methods for unpolluted river water. Counts of faecal enterococci in raw sewage, semi-treated effluent and polluted urban run-off, obtained by the following methods did not differ significantly: MF using M-Enterococcus Agar, Bile-Esculin Agar or Enterococcus Selective Agar, or a microtitre-plate MPN method with a liquid growth medium containing chromogenic 4-methyl-umbelliferyl--D-glucoside. Significant differences were, however, found between the MPN and the other methods for unpolluted river water and stored potable water. MF using Chromocult Coliform Agar had useful benefits for the simultaneous enumeration of coliforms and E coli. However, in view of cost and practical considerations, MF using MFc Agar or M-Enterococcus Agar proved the methods of choice for respectively enumerating faecal coliforms and E coli, or faecal enterococci, in analyses for general water quality surveillance purposes.     

In vitro antibiotic susceptibility of bacteria isolated from EUS-affected fishes in India

AIMS: Twelve antibiotics were evaluated for in vitro sensitivity against 16 bacterial strains isolated from surface lesions of fishes affected with epizootic ulcerative syndrome (EUS). METHODS AND RESULTS: Disc diffusion assay in Mueller-Hinton agar showed that the pseudomonads and aeromonads were mainly resistant to penicillin, ampicillin and erythromycin. Additionally, some were resistant to gentamycin and amoxycillin. However, resistance towards antibiotics previously recommended for EUS treatment, such as oxytetracycline and chloramphenicol, was not observed. Four aeromonads and two pseudomonads were found to induce ulcers when injected intramuscularly in healthy Anabas testudineus. CONCLUSIONS: All six pathogenic isolates were sensitive towards oxytetracycline, chloramphenicol and nalidixic acid. Oxytetracycline seems to be an effective antibiotic, and further investigations to determine the mode of treatment and dose appear to be worthwhile.     

The Influence of Antimicrobial Agents on the Percentage of Tetracycline resistant Bacteria in Faeces of Humans and Animals

The effect of antimicrobial administration on the percentage of tetracycline-resistant facultative and anaerobic bacteria in the intestines of humans and animals was studied. Tetracycline was chosen because it is an antibiotic commonly used both as a feed supplement for animals, and therapeutically for humans and animals. More than 200 faecal specimens of bovine, porcine, equine, canine, feline and human origin were collected into pre-reduced anaerobic media. Ninety-one subjects from groups receiving antimicrobials as feed supplements, therapeutically or with no recent antibiotic exposure were included in the study. Counts of both facultative and anaerobic bacteria were determined from media with and without tetracycline. The percentage of normal faecal specimens harbouring tetracycline-resistant coliforms was 56 for humans. 100 for calves. 100 for pigs. 36 for horses, 90 for dogs and 67 for cats. All samples contained Gram negative anaerobes resistant to tetracycline. Subjects from all species receiving antimicrobials showed higher percentages of tetracycline-resistant coliforms and anaerobes than subjects not receiving antibiotics. Greater increases in the percentage of tetracycline-resistant facultative organisms were noted in subjects which received antibiotics as feed supplements compared with subjects which were given therapeutic doses of antibiotics. Percentages of tetracycline-resistant anaerobes showed the opposite trend with more increase among those subjects receiving therapeutic levels of antimicrobial agents.     

Bacteriological indicators of faecal contamination: result of a loading experiment with untreated urban wastewater

Some observations were made on the behaviour of total coliforms, faecal coliforms, enterococci, numbers of aerobic bacteria, salmonellas and sulphur-reducing clostridia as bacterial indicators of faecal contamination of groundwater. A controlled irrigation experiment was carried out with untreated residual water in the alluvial aquifer of the Vega of Granada (Spain). The results obtained confirm the value of these parameters as useful indicators of very recent faecal contamination; and changes were detected as the level of the freatic layer increased and the chemical composition of the groundwater changed. These groups of micro-organisms persisted for about 200 h, with the exception of the aerobes which survived for much longer. Salmonellas were present at levels too low to calculate the extent of faecal contamination and sulphur-reducing clostridia were not detected. The results obtained show that irrigation with untreated wastewater offers a lower risk of microbiological contamination of groundwater compared with the direct addition of waters decanted and/or previously filtered.     

The effect of a sewage treatment plant effluent on the faecal coliforms and enterococci populations of the reception river waters

AIMS: A rural sewage treatment plant and the effect of its effluent on the enterococci and faecal coliforms populations of the receiving river waters was evaluated. METHODS AND RESULTS: The enumeration of bacteria was performed by membrane filtration. Diversity and population similarity were analysed using the PhP-plates system. The treatment plant reduces the number of enterococci and faecal coliforms to values similar to those observed upstream. All water samples showed a high diversity for both bacterial populations. A high similarity in the composition and structure, was detected among all the samples. CONCLUSIONS: The impact of the disposal of treated sewage on the river did not modify the composition of either bacterial populations in the river water. SIGNIFICANCE AND IMAPCT OF THE STUDY: The biochemical phenotyping of bacterial populations is a reliable tool for ecological and biodiversity studies. The obtained results provide a better understanding of the sewage treatment process and the impact of the treated sewage effluents in the environment.     

Drug resistance in Vibrio cholerae strains isolated from clinical specimens

Cholera is a serious epidemic and endemic disease caused by the Gram-negative bacterium Vibrio cholerae. SXT is an integrative conjugation element (ICE) that was isolated from a V. cholerae; it encodes resistance to the antibiotics chloramphenicol, streptomycin and sulfamethoxazole/trimethoprim. One hundred seven V. cholerae O1 strains were collected from cholera patients in Iran from 2005 to 2007 in order to study the presence of SXT constin and antibiotic resistance.The study examined 107 Vibrio cholerae strains isolated from cholera prevalent in some Iranian provinces. Bacterial isolation and identification were carried out according to standard bacteriological methods. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) to four antibiotics (chloramphenicol, streptomycin, sulfamethoxazole, and trimethoprim) were determined by broth microdilution method. PCR was employed to evaluate the presence of established antibiotic resistance genes and SXT constin using specific primer sets.The resistance of the clinical isolates to sulfamethoxazole, trimethoprime, chloramphenicol, and streptomycin was 97%, 99%, 99%, and 90%, respectively. The data obtained by PCR assay showed that the genes sulII, dfrA1, floR, strB, and sxt element were present in 95.3%, 95.3%, 81.3%, 95.3%, and 95.3% of the V. cholerae isolates.The Vibrio strains showed the typical multidrug-resistance phenotype of an SXT constin. They were resistant to sulfamethoxazole, trimethoprime, chloramphenicol, and streptomycin. The detected antibiotic resistance genes included dfrA for trimethoprim and floR, strB, sulII and int, respectively, for chloramphenicol, streptomycin, sulfamethoxazole, as well as the SXT element.     

Antibiotic resistance among coliforms and Pseudomonas spp. from bodies of water around Port Harcourt, Nigeria

Samples from municipal waste water, the Bonny River estuary and wells in and around Port Harcourt were examined for bacteriological quality over a 9 month period. A total of 157 Pseudomonas spp., 133 Escherichia coli and 282 other coliforms were isolated and tested for the incidence of resistance to 10 antibiotics. All of the Pseudomonas spp. were resistant to at least one of the antibiotics while 96.2% were resistant to two or more. Most (83.5%) of the E. coli and other coliforms (91.8%) were resistant to at least one antibiotic. All strains were susceptible to gentamicin. Minimal inhibitory concentrations of ampicillin and tetracycline for E. coli ranged from 6.25 to 50 and 6.25 to 12.5 μg/ml, respectively. Minimal inhibitory concentrations of ampicillin and tetracycline were 1000 and 25 μg/ml for the Pseudomonas strains. The high incidence of bacterial resistance to antibiotics is discussed in relation to the widespread use of antibiotics, and possible public health implications.     

Antimicrobial susceptibility of enterococci recovered from commercial swine carcasses: effect of feed additives

Enterococcus faecium is an important nosocomial pathogen often displaying multiple antibiotic resistance. The increase in clinical isolates can be attributed in part to hospital practices in antibiotic usage, but there is concern that antibiotic-resistant strains might also originate in animals fed rations containing antibiotic growth promoters. Ingestion of meat from carcasses contaminated with faecal enterococci might then result in human colonization or resistance gene transfer to human enterococci. Because there are few comparisons of bacteria isolated from matched animals that have, or have not, been fed a diet containing antibiotic, two such groups of pig carcasses were sampled at a commercial abattoir. Forty isolates from each group of pigs were tested for their resistance to avilamycin and tylosin. Although a modest number of pigs was examined, and the number of strains of E. faecium tested was small, there was no evidence that the feeding of a growth promoter caused selection of enterococci resistant to tylosin or avilamycin.     

Antibiotic resistance among coliforms and Pseudomonas spp. from bodies of water around Port Harcourt, Nigeria

Samples from municipal waste water, the Bonny River estuary and wells in and around Port Harcourt were examined for bacteriological quality over a 9 month period. A total of 157 Pseudomonas spp., 133 Escherichia coli and 282 other coliforms were isolated and tested for the incidence of resistance to 10 antibiotics. All of the Pseudomonas spp. were resistant to at least one of the antibiotics while 96.2% were resistant to two or more. Most (83.5%) of the E. coli and other coliforms (91.8%) were resistant to at least one antibiotic. All strains were susceptible to gentamicin. Minimal inhibitory concentrations of ampicillin and tetracycline for E. coli ranged from 6.25 to 50 and 6.25 to 12.5 micrograms/ml, respectively. Minimal inhibitory concentrations of ampicillin and tetracycline were 1000 and 25 micrograms/ml for the Pseudomonas strains. The high incidence of bacterial resistance to antibiotics is discussed in relation to the widespread use of antibiotics, and possible public health implications.