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.     

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.     

Identification of strains isolated as total and fecal coliforms and comparison of both groups as indicators of fecal pollution in tropical climates

This study was undertaken to better characterize the groups of total coliforms (TC) and fecal coliforms (FC) and to evaluate both groups as indicators of fecal contamination of drinking well water in a tropical climate (The Ivory Coast, West Africa). Isolated colonies obtained as TC or FC on membrane filters were identified using the API-20E system. From the well water samples, 58 golden-green colonies with a metallic sheen isolated on Endo medium (TC) were identified as Escherichia coli (55%), Enterobacter (26%), Klebsiella (14%), Proteus (3%), and Citrobacter (2%). Among 132 colonies isolated on Endo medium as non-TC (not showing the characteristic golden metallic sheen), 10% were identified as E. coli. The 196 blue colonies isolated on M-FC medium at 44.5 degrees C (FC) were identified as E. coli (66%), Klebsiella (12%), Enterobacter (10%), Citrobacter (5%), Salmonella (3%), Serratia (3%), Proteus (2%), and Yersinia (0.5%). Among 24 nonblue colonies on M-FC medium, none were identified as E. coli. Of the colonies isolated from human feces, E. coli represents 92% of the TC and 89% of the FC. Although these results are limited, they tend to confirm the greater specificity of the fecal coliform technique over that of total coliform for the detection of fecal contamination of untreated well water. From the results presented here and the observations of other workers, it is suggested that the use of FC instead of TC should be considered as the method of choice for determining drinking water pollution of untreated groundwater supplies.     

Multiple antibiotic resistance indexing of coliforms to identify high risk contamination sites in aquatic environment

Bacteriological analysis of the water samples collected from upstream, midstream and downstream points along the bank of the river revealed high populations of Escherichia coli, Citrobacter freundii, Citrobacter diversus, Enterobacter aerogens and Klebsiella species. All these isolates were screened against eight antibiotics to determine the prevalence of multiple antibiotic resistance among isolates at different sites of the river. The study revealed that multiple antibiotic resistance was prominently seen in coliforms at downstream sites (Average multiple antibiotic resistance index, MAR Index = 0.43) while it was low in coliforms at upstream sites (MAR Index = 0.15). These differences in MAR indices provide a method for distinguishing high risk contamination sites in aquatic environment.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Holding effects on coliform enumeration in drinking water samples

Standard procedures for analyzing drinking water stress the need to adhere to the time and temperature conditions recommended for holding samples collected for microbiological testing. The National Drinking Water Laboratory Certification Program requires compliance with these holding limits, but some investigators have reported difficulties in meeting them. Research was conducted by standard analytical methods to determine if changes occurred when samples were held at 5 and 22 degrees C and analyzed at 0, 24, 30, and 48 h. Samples were analyzed for coliforms by the membrane filter and fermentation-tube procedures and for heterotrophs by the pour plate method. A total of 17 treated water samples were collected from a large municipal distribution system from August to December 1981, and 12 samples were collected from January to May 1983. The samples were dosed with coliforms previously isolated from the water system, Enterobacter cloacae in 1981 and Citrobacter freundii in 1983. The coliform counts declined linearly over time, and the rates of decline were significant at both 5 and 22 degrees C. Within 24 h at 22 degrees C, levels of E. cloacae and C. freundii decreased by 47 and 26%, respectively, and at 5 degrees C, E. cloacae numbers declined by 23%. Results from these representative laboratory-grown coliforms reinforced those previously obtained with naturally occurring coliforms under the same experimental conditions. Significantly, some samples with initially unacceptable counts (greater than 4/100 ml) met the safe drinking water limits after storage at 24 h at 5 and 22 degrees C and would have been classified as satisfactory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective detection and enumeration of fecal coliforms in water by potentiometric measurement of lipoic acid reduction.

Water samples of various origins were inoculated into a specific coliform-selective lactose broth provided with lipoic (thioctic) acid, and the time evolution of the redox potential of the cultures was monitored during incubation at 41 degrees C by use of gold versus reference electrodes. Positive potential-time responses, i.e., 100-mV potential shifts recorded within 20 h of inoculation, were related to the initial number of fecal coliforms in the broth determined by control enumeration techniques, and the organisms responsible were isolated and identified by conventional procedures. A total of 30 samples of wastewater, 38 of surface water, 553 of groundwater, and 110 of drinking water were tested successively. A total of 240 natural water samples, including 172 groundwater samples, and 1 drinking water sample were found to be positive in the potentiometric test. The majority (i.e., 92.5%) of the relevant potentiometric detection times were shorter than 15 h, and 96% of these could be attributed to Escherichia coli. Fifteen hours corresponded to the limit for detecting 1 E. coli cell per 100 ml of water. About 78% of the potentiometric responses occurring after 15 h were induced by fecal coliforms other than E. coli (Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii). Calibration curves relating detection times shorter than 15 h to fecal coliform (i.e., E. coli) concentrations were constructed for the natural water samples tested. There were minor variations in the average growth rate of the organisms in the relation to the contamination level of the water tested. The number of false-positive samples in the potentiometric test was equivalent to that of false-negative samples (groundwater or drinking water).     

Failure of the most-probable-number technique to detect coliforms in drinking water and raw water supplies.

A procedure was developed to detect false-negative reactions (interference) in the standard most-probable-number (S-MPN) technique for coliform enumeration of untreated surface water and potable water supplies. This modified MPN (M-MPN) procedure allowed a quantitative assessment of the interference with coliform detection in untreated surface water and potable water supplies. Coliform interference was found to occur in the presumptive, confirmed, and completed tests of the S-MPN technique. When coliforms were present, interference with their detection occurred in over 80% of the samples. The inferior nature of the S-MPN was revealed by the 100% increase in the incidence of completed coliform-positive drinking water samples obtained with the M-MPN technique. The M-MPN procedure was also superior to the standard membrane filter technique. Eight different species of coliforms were recovered from false-negative tests, including Citrobacter, Enterobacter, Klebsiella, and Escherichia coli (in decreasing order of occurrence). The use of standard MPN techniques for monitoring potable water supplies may lead to a false security that the drinking water supply is potable, i.e., free from indicator bacteria.     

Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers

More than 750 isolates of faecal coliforms (>200 strains), enterococci (>200 strains) and pseudomonads (>340 strains) from three wastewater treatment plants (WTPs) and from four groundwater wells in the vicinity of leaking sewers were tested for resistance against 14 antibiotics. Most, or at least some, strains of the three bacterial groups, isolated from raw or treated sewage of the three WTPs, were resistant against penicillin G, ampicillin, vancomycin, erythromycin, triple sulfa and trimethoprim/sulfamethoxazole (SXT). Only a few strains of pseudomonads or faecal coliforms were resistant against some of the other tested antibiotics. The antibiotic resistances of pseudomonads, faecal coliforms and enterococci from groundwater varied to a higher extent. In contrast to the faecal coliforms and enterococci, most pseudomonads from all groundwater samples, including those from non-polluted groundwater, were additionally resistant against chloramphenicol and SXT. Pseudomonads from sewage and groundwater had more multiple antibiotic resistances than the faecal coliforms or the enterococci, and many pseudomonads from groundwater were resistant to more antibiotics than those from sewage. The pseudomonads from non-polluted groundwater were the most resistant isolates of all. The few surviving faecal coliforms in groundwater seemed to gain multiple antibiotic resistances, whereas the enterococci lost antibiotic resistances. Pseudomonads, and presumably, other autochthonous soil or groundwater bacteria, such as antibiotic-producing Actinomyces sp., seem to contribute significantly to the gene pool for acquisition of resistances against antibiotics in these environments.     

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.     

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.     

Comparison of membrane filter, multiple-fermentation-tube, and presence-absence techniques for detecting total coliforms in small community water systems

Methods for detecting total coliform bacteria in drinking water were compared using 1,483 different drinking water samples from 15 small community water systems in Vermont and New Hampshire. The methods included the membrane filter (MF) technique, a 10-tube fermentation tube (FT) technique, and the presence-absence (P-A) test. Each technique was evaluated using a 100-ml drinking water sample. Of the 1,483 samples tested, 336 (23%) contained coliforms as indicated by either one, two, or all three techniques. The FT detected 82%, the P-A detected 88%, and the MF detected 64% of these positives. All techniques simultaneously detected 55% of the positives. Evaluation of the confirmation efficiency of the P-A technique showed 94% of the presumptive positives confirming as coliforms. Thirteen different species of coliforms were identified from the 37 tests in which the P-A was positive but the MF and FT were negative. The P-A test was simple to inoculate and interpret and was considerably more sensitive than the MF and slightly more sensitive than the FT in detecting coliforms in this type of drinking water supply.     

Comparison of membrane filter, multiple-fermentation-tube, and presence-absence techniques for detecting total coliforms in small community water systems.

Methods for detecting total coliform bacteria in drinking water were compared using 1,483 different drinking water samples from 15 small community water systems in Vermont and New Hampshire. The methods included the membrane filter (MF) technique, a 10-tube fermentation tube (FT) technique, and the presence-absence (P-A) test. Each technique was evaluated using a 100-ml drinking water sample. Of the 1,483 samples tested, 336 (23%) contained coliforms as indicated by either one, two, or all three techniques. The FT detected 82%, the P-A detected 88%, and the MF detected 64% of these positives. All techniques simultaneously detected 55% of the positives. Evaluation of the confirmation efficiency of the P-A technique showed 94% of the presumptive positives confirming as coliforms. Thirteen different species of coliforms were identified from the 37 tests in which the P-A was positive but the MF and FT were negative. The P-A test was simple to inoculate and interpret and was considerably more sensitive than the MF and slightly more sensitive than the FT in detecting coliforms in this type of drinking water supply.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

Coliform species recovered from untreated surface water and drinking water by the membrane filter, standard, and modified most-probable-number techniques.

The species of total coliform bacteria isolated from drinking water and untreated surface water by the membrane filter (MF), the standard most-probable-number (S-MPN), and modified most-probable-number (M-MPN) techniques were compared. Each coliform detection technique selected for a different profile of coliform species from both types of water samples. The MF technique indicated that Citrobacter freundii was the most common coliform species in water samples. However, the fermentation tube techniques displayed selectivity towards the isolation of Escherichia coli and Klebsiella. The M-MPN technique selected for more C. freundii and Enterobacter spp. from untreated surface water samples and for more Enterobacter and Klebsiella spp. from drinking water samples than did the S-MPN technique. The lack of agreement between the number of coliforms detected in a water sample by the S-MPN, M-MPN, and MF techniques was a result of the selection for different coliform species by the various techniques.     

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.     

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 sensitivity of Corynebacterium amycolatum

C. amycolatum strains belongs to opportunistic bacteria considered as etiological factors of hospital infections. It's usually handled as a human natural flora, so antibiotic sensitivity is not checked. There's a few reports relative to antibiotic sensitivity of C. amycolatum in the world literature. So, we decided to examine antibiotic sensitivity of isolated strains. The 70 of C. amycolatum strains isolated from clinical samples from patients hospitalised at Samodzielny Publiczny Szpital Kliniczny in Bydgoszcz were analysed. Antimicrobial susceptibility testing of the strains was performed by means of a disk diffusion method. 28.6% of analysed strains were susceptible to penicillin and 38.6% to ampicillin. Susceptibility to another 16 antibiotics was from 40.0% for ceftazidime to 64.3% for ceftriaxone. Penicillinase was not produced by analysed strains. We stated higher percentage of strains susceptible to combinations of penicillin with inhibitors than to penicillin and ampicillin. The most strains were susceptible to quinupristin-dealfopristin, linezolid and glycopeptide antibiotics but resistance to mupirocin. 35.7% analysed strains were multiresistance; there were resistance to beta-lactams (approximately 100%), lincosamides (96.0%), macrolides (92.0%) and quinolones (92.0%). Multiresistant strains were the most frequently isolated from wound swabs (60.0%) and mainly came from patients treated at the departments of general surgery (28.0%) and vascular surgery (16.0%).