Injured Coliforms Research Articles

Viability of sublethally injured coliform bacteria on fresh-cut cabbage stored in high CO2 atmospheres following rinsing with electrolyzed water

The extent of sublethally injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, was evaluated during storage in air and high CO2 controlled atmospheres (5%, 10%, and 15%) at 5°C and 10°C using the thin agar layer (TAL) method. Sublethally injured coliform bacteria on nonrinsed shredded cabbage were either absent or they were injured at a 64–65% level when present. Rinsing of shredded cabbage with electrolyzed water containing 25ppm available chlorine reduced the coliform counts by 0.4 to 1.1 log and caused sublethal injury ranging from 42 to 77%. Pantoea ananatis was one of the species injured by chlorine stress. When shredded cabbage, nonrinsed or rinsed with electrolyzed water, was stored in air and high CO2 atmospheres at 5°C for 7days and 10°C for 5days, coliform counts on TAL plates increased from 3.3–4.5 to 6.5–9.0 log CFU/g during storage, with the increase being greater at 10°C than at 5°C. High CO2 of 10% and 15% reduced the bacterial growth on shredded cabbage during storage at 5°C. Although injured coliform bacteria were not found on nonrinsed shredded cabbage on the initial day, injured coliforms at a range of 49–84% were detected on samples stored in air and high CO2 atmospheres at 5°C and 10°C. Injured cells were detected more frequently during storage at both temperatures irrespective of the CO2 atmosphere when shredded cabbage was rinsed with electrolyzed water. These results indicated that injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, exhibited different degrees of injury during storage regardless of the CO2 atmosphere and temperature tested.

Injured coliforms in swimming pools : How big a threat?

A swimming pool is an important leisure facility, but it can harbor injured cells creating potential health hazards. Disinfection of swimming pools can cause bacterial injury, when cells are exposed to a suboptimal concentration of disinfectants. Possible pathogenic bacteria can enter into an injured state, for example, Escherichia coli, Klebsiella spp, and Enterococcus faecalis. Injured bacteria can retain their pathogenicity and virulence and they may recover causing diseases. To assess the presence of injured coliforms in swimming pools based on differential plating media. This study compared the difference in recovery of coliforms between two differential media, one designed for recovery of injured coliforms (HiCrome ECC selective agar and the other is CHROMagar ECC). A total of 120 samples were collected from 10 semi-public swimming pools with sporadic distribution around Alexandria, Egypt, and included in this study. Five pools were used for swim training, 4 were used for both training and recreational swimming and one was used for children only. The recovery medium (HiCrome ECC selective agar) detected 1.47 and 2.54 times total coliforms and E. coli, respectively, as CHROMagar ECC. The compliance of samples per fresh water swimming pool Egyptian standard in total coliforms and E. coli fell from 54.10% when examined by CHROMagar ECC medium to 35% by HiCrome ECC selective medium. The current findings may not be universal to all swimming pools but may be applicable to ones where the physicochemical properties of their water induce coliform injury. Results suggest that the use of media that detect injured yet viable coliforms will give a more sensitive and representative guidance about the quality of examined water and will assist in the treatment and decontamination of swimming pools.

Injured coliforms in swimming pools : How big a threat?

Injured coliforms in swimming pools : How big a threat?

Injured coliforms in swimming pools : How big a threat?

A swimming pool is an important leisure facility, but it can harbor injured cells creating potential health hazards. Disinfection of swimming pools can cause bacterial injury, when cells are exposed to a suboptimal concentration of disinfectants. Possible pathogenic bacteria can enter into an injured state, for example, Escherichia coli, Klebsiella spp, and Enterococcus faecalis. Injured bacteria can retain their pathogenicity and virulence and they may recover causing diseases.To assess the presence of injured coliforms in swimming pools based on differential plating media.This study compared the difference in recovery of coliforms between two differential media, one designed for recovery of injured coliforms (HiCrome ECC selective agar and the other is CHROMagar ECC). A total of 120 samples were collected from 10 semi-public swimming pools with sporadic distribution around Alexandria, Egypt, and included in this study. Five pools were used for swim training, 4 were used for both training and recreational swimming and one was used for children only.The recovery medium (HiCrome ECC selective agar) detected 1.47 and 2.54 times total coliforms and E. coli, respectively, as CHROMagar ECC. The compliance of samples per fresh water swimming pool Egyptian standard in total coliforms and E. coli fell from 54.10% when examined by CHROMagar ECC medium to 35% by HiCrome ECC selective medium.The current findings may not be universal to all swimming pools but may be applicable to ones where the physicochemical properties of their water induce coliform injury. Results suggest that the use of media that detect injured yet viable coliforms will give a more sensitive and representative guidance about the quality of examined water and will assist in the treatment and decontamination of swimming pools.

Enumeration and Identification of Ethanol-Injured Coliform Bacteria Found on Harvest Equipment and its Cross-Contamination with Cabbage

Enumeration and Identification of Ethanol-Injured Coliform Bacteria Found on Harvest Equipment and its Cross-Contamination with Cabbage Although disinfection by sanitizers used to produce packing sheds which helps in preventing cross-contamination between farm equipment and produces, sanitizing agents like ethanol are capable of causing sanitizer-injured bacteria. Using the thin agar layer (TAL) method, the proportion of ethanol-injured coliform bacteria resulting from the use of an alcohol agent containing 1.6% and 47% ethanol was evaluated in pure cultures and in a harvest environment of cabbage, respectively. In pure cultures of Enterobacter cloacae, Escherichia coli, and E. coli O157:H7, ethanol-injured cells were observed at a range of 68-95% by treatment with an alcohol agent. When an alcohol agent was sprayed on harvest equipment including knife, container and gloves, 60% of the total coliforms detected on the knife were injured, but no injured coliforms were detected on the container and gloves. The isolation and identification of coliforms on selective and TAL media from the knife suggested that the ethanol stress caused injury to Enterobacter amnigenus, E. asburiae, and E. kobei. Injured bacteria were not detected on cabbage after harvest using the alcohol spray-treated knife because the species of bacteria found in cabbage (14 species belonging to 11 genera) were different from those on the knife. These results indicate that the sanitizer-injured coliforms would not be present on cabbage when transfer of the bacteria from harvest equipment to the cabbage is prevented or when a suitable sanitizer is used that causes death of the bacteria rather than sanitizer-induced cell injury.

Enumeration and Identification of Coliform Bacteria Injured by Chlorine or Fungicide Mixed with Agricultural Water

Chemical sanitizers may induce no injury (bacteria survive), sublethal injury (bacteria are injured), or lethal injury (bacteria die). The proportion of coliform bacteria that were injured sublethally by chlorine and fungicide mixed with agricultural water (pond water), which was used to dilute the pesticide solution, was evaluated using the thin agar layer (TAL) method. In pure cultures of Enterobacter cloacae , Escherichia coli , and E. coli O157:H7 (representing a human pathogen), the percentage of chlorine-injured cells was 69 to 77% for dilute electrolyzed water containing an available chlorine level of 2 ppm. When agricultural water was mixed with electrolyzed water, the percentage of injured coliforms in agricultural water was 75%. The isolation and identification of bacteria on TAL and selective media suggested that the chlorine stress caused injury to Enterobacter kobei . Of the four fungicide products tested, diluted to their recommended concentrations, Topsin-M, Sumilex, and Oxirane caused injury to coliform bacteria in pure cultures and in agricultural water following their mixture with each pesticide, whereas Streptomycin did not induce any injury to the bacteria. The percentage of injury was 45 to 97% for Topsin-M, 80 to 87% for Sumilex, and 50 to 97% for Oxirane. A comparison of the coliforms isolated from the pesticide solutions and then grown on either TAL or selective media indicated the possibility of fungicide-injured Rahnella aquatilis , Yersinia mollaretii , and E. coli . These results suggest the importance of selecting a suitable sanitizer and the necessity of adjusting the sanitizer concentration to a level that will kill the coliforms rather than cause sanitizer-induced cell injury that can result in the recovery of the coliforms.

Influencing factors in the occurrence of injured coliforms in the drinking water distribution system in the city of La Plata, Argentina

The objective of this study was to evaluate the influencing factors in the occurrence of coliforms in the drinking water in La Plata (Argentina) from June 1999 to June 2001. A total of 180 samples were collected from Rio de La Plata (102 samples) and Puelche Aquifer (78 samples); 45 samples were collected for each of the four seasons. The membrane filter procedure was used for isolating bacteria, and each sample was tested for chlorine and pH. The highest percentage of samples positive for coliforms in the two tested media was obtained in summer while the highest percentage of negative samples was obtained in winter. No Escherichia coli was isolated. The percentage of injured coliforms fluctuated between 70 and 100%. The most frequently isolated bacteria was Enterobacter cloacae in summer, Enterobacter agglomerans in autumn and Klebsiella oxytoca in winter and spring. Significant correlations were observed between coliforms and the distance from the initial treatment point, and with the level of free chlorine. We conclude that drinking water contamination in La Plata occurs in the distribution system due to increased temperatures and reduced disinfectant levels, which result in bacterial regrowth.

Evaluation of Chromogenic Enzyme Substrate Mediums, Chromocult Coliform Agar(CCA) and XM-G, by Detection of Freeze-, Heat-, High-Pressure-Injured Coliforms, and Coliforms in Food Samples

The two chromogenic enzyme substrate mediums of chromocult coliform agar (CCA) and XM-G were applied to detect freeze-, heat-, and high-pressure-injured coliforms (Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Klebsiella ozaenae, and Klebsiella pneumoniae) as well as coliforms in various food items. Their detection abilities were then compared with the following three conventional media: tryptic soy agar (TSA), violet red bile agar (VRBA), and VRBA/TSA. The enumerated results of injured coliforms showed that the ability to detect injured cells was in the following descending order: non-selective medium TSA>VRBA/TSA>XMG≥CCA≫VRBA. The recovery rate of injured coliforms, when compared with selective agars, was higher in CCA and XM-G than in VRBA. Investigation of the total coliform counts from 100 food samples showed that the enumeration results of the three selected media (CCA, XM-G, and VRBA) were quite similar. The correlation coefficients were 0.89 for CCA vs. VRBA, 0.91 for XM-G vs. VRBA, and 0.91 for CCA vs. XM-G; indicating that CCA and XM-G are recommendable and can substitute for the conventional selective medium VRBA. In addition to the advantage of simultaneous detection of coliforms and Escherichia coli by CCA and XM-G, their superiority in detecting injured coliforms reveals that these two methods were highly effective and suitable to monitor total coliforms and E. coli including injured cells in food samples.

Development of a Thin Tube Cultivation Method for Determining Coliform Counts in Milk

A new rapid coliform-monitoring method using thin tube cultivation (TTC) was developed. One hundred microliters of modified selective medium (1.5% agar) and 100μl of sample were mixed in thin cultivation tubes, yielding a semisolid mixture of 0.75% agar. The tubes were incubated at 37°C and checked for a positive reaction (gas production from lactose fermentation and color changes from acid production) at 30-min intervals. The positive reaction times for TTC were compared with actual coliform numbers. The first experiment was performed to validate the TTC method using pure coliform cultures (the Escherichia coli group, the Enterobacter cloacae group, and the Klebsiella pneumoniae group). The results indicated that the TTC method saved about 10 to 14 h of incubation time compared with the conventional plating method. When coliforms were present in small numbers, such as 1 to 1.5 log10 CFU/ml, the TTC method detected their presence within 10 h. When highly contaminated samples were assayed (6 log10 CFU/ml), the TTC method detected the presence of coliforms within 4 to 5 h. In a food system experiment, pasteurized skim milk inoculated with a coliform mixture was assayed by the TTC method. The coliforms in the milk were quickly detected, resulting in a savings of 11 to 14 h compared with the conventional plating method. Cold-injured coliforms were studied to evaluate the TTC system. The recovery and estimation of injured coliforms by the TTC method strongly agree with those of the conventional plating method. The TTC system was also applied to ascertain the levels of natural coliforms in noninoculated milk. The TTC system reduced incubation times by about 11 to 14 h compared with the conventional plating method. According to these data, this method is a rapid, simple, accurate, and economical way to monitor and estimate total coliforms in milk.

Resistance to antibiotics in injured coliforms isolated from drinking water.

We studied the antibiotic sensitivity of injured coliforms isolated from drinking water of La Plata, Argentina. The antibiotic sensitivity test by the agar diffusion method were proved in: Klebsiella oxytoca (14 strains), Enterobacter aerogenes (4 strains) and Enterobacter cloacae genomic group 3 (14 strains). We found that while these impaired total coliforms were sensitive to piperacillin-tazobactam (TAZ), netilmicin (NTL), ofloxacin (OFLX), and norfloxacin (NFLX) (100%), they had resistant to aminopenicillin-sulbactam (AMS) and nitrofurantoin (NIT) (100%). The resistance to antibiotics demonstrated in these strains would point to the need to promote a rational and judicious use of antimicrobial agents while at the same time implementing a program of active vigilance aimed at ensuring the highest quality of drinking water throughout the system.

Investigation of injury of coliforms after chlorination

The extent of coliform injury after chlorination and the relationship between regrowth of injured coliforms and assimilable organic carbon concentrations (AOC) were studied. Seeded (Escherichia coli E25), sterilized tap water (AOC concentration adjusted to 400mg C/l) was incubated for a period of 3 weeks (25-28°C) after chlorination (0.5 and 1mg/l). Bacteria were enumerated using m-Endo agar LES and m-T7 agar. Chlorination resulted in a 3 log reduction in coliform counts within one minute. No viable colonies were found for 27% of the water samples enumerated with m-Endo agar LES. Injured coliforms were enumerated from all the water samples except one and were present in higher numbers than non-injured coliforms in the majority of the water samples. During the second and third weeks of incubation, only injured bacteria were observed in chlorinated samples. Additional AOC contributed little to the survival and regrowth of chlorinated bacteria.

Investigation of injury of coliforms after chlorination

The extent of coliform injury after chlorination and the relationship between regrowth of injured coliforms and assimilable organic carbon concentrations (AOC) were studied Seeded (Escherichia coli E25), sterilized tap water (AOC concentration adjusted to 400mg C/l) was incubated for a period of 3 weeks (25–28°C) after chlorination (0.5 and lmg/l). Bacteria were enumerated using m-Endo agar LES and m-T7 agar. Chlorination resulted in a 3 log reduction in coliform counts within one minute. No viable colonies were found for 27% of the water samples enumerated with m-Endo agar LES. Injured coliforms were enumerated from all the water samples except one and were present in higher numbers than non-injured coliforms in the majority of the water samples. During the second and third weeks of incubation, only injured bacteria were observed in chlorinated samples. Additional AOC contributed little to the survival and regrowth of chlorinated bacteria.

Bacteriological quality of potable water sources supplying Morogoro municipality and its outskirts: a case study in Tanzania.

Bacteriological quality of potable water supplying Morogoro municipality and its outskirts (population 135,000 people) was assessed by the determination of the most probable number (MPN) of faecal coliforms, Escherichia coli, faecal streptococci and Clostridium perfringens for a period of 6 months. River water and chlorinated pipeborne water were found contaminated with microorganisms in the order of 3.8 x 10(1) to 4.95 x 10(3); 3.2 x 10(1) to 4.5 x 10(3); 10(1) to 6.4 x 10(1) and 1.2 x 10(1) to 2.5 x 10(2)/100 ml of water respectively. Injured coliforms in treated water averaged 87%. Pollution of river water by organic matter was much above acceptable standard. These findings indicate that there is a need for further treatment of water before consumption in order to avoid potential health hazards.

Penetration of coliforms through municipal drinking water filters

The penetration of coliform bacteria through municipal drinking water filters was studied under normal operating conditions of water treatment plants. Samples of filtered water were taken during the 5 h immediately following the backwash cycle and analyzed for turbidity and coliform bacteria. Coliforms were enumerated using both m-Endo and m-T7 media to evaluate coliform passage and injury. Elevated turbidity and intermittent peaks of coliforms (as high as 60/100 ml) were observed during the post backwash period. Highest numbers of coliforms were observed during the flow through time for the interface of the backwash remnants and the influent water after backwash. In addition, most of the coliforms passing through the filters during this period were injured because they failed to grow on m-Endo but were enumerated on m-T7 medium. These results indicate that the post backwash period can be characterized by comparatively higher levels of turbidity as well as coliforms that cannot be detected using accepted enumeration methods. Furthermore, such undetected bacteria may seed the distribution system. Therefore, improved bacteriological surveillance for injured coliforms in filter effluents could guide the establishment and design of filter-to-waste periods. This would reduce the incidence of coliforms in some municipal potable water systems.

Enumeration of Enterobacter cloacae after chloramine exposure.

Growth of Enterobacter cloacae on various media was compared after disinfection. This was done to examine the effects of monochloramine and chlorine on the enumeration of coliforms. The media used were TLY (nonselective; 5.5% tryptic soy broth, 0.3% yeast extract, 1.0% lactose, and 1.5% Bacto-Agar), m-T7 (selective; developed to recover injured coliforms), m-Endo (selective; contains sodium sulfite), TLYS (TLY with sodium sulfite), and m-T7S (m-T7 with sodium sulfite). Sodium sulfite in any medium improved the recovery of chloramine-treated E. cloacae. However, sodium sulfite in TLYS and m-T7S did not significantly improve the detection of chlorine-treated E. cloacae, and m-Endo was the least effective medium for recovering chlorinated bacteria. Differences in recovery of chlorine- and chloramine-treated E. cloacae are consistent with mechanistic differences between the disinfectants.

Health Significance and Occurrence of Injured Bacteria in Drinking Water

Enteropathogenic and indicator bacteria become injured in drinking water with exposure to sublethal levels of various biological, chemical and physical factors. One manifestation of this injury is the inability to grow and form colonies on selective media containing surfactants. The resulting underestimation of indicator bacteria can lead to a false estimation of water potability. m-T7 medium was developed specifically for the recovery of injured coliforms (both "total" and fecal) in drinking water. The m-T7 method was used to survey operating drinking water treatment and distribution systems for the presence of injured coliforms that were undetected with currently used media. The mean recovery with m-Endo LES medium was less than 1/100 ml while it ranged between 6 and 68/100ml with m-T7 agar. The majority of samples giving positive results with m-T7 medium yielded no detectable coliforms with m-Endo LES agar. Over 95% of the coliform bacteria in these samples were injured. Laboratory experiments were also done to ascribe the virulence of injured waterborne pathogens. Enteropathogens including Salmonella typhimurium, Yersinia enterocolitica and Shigella spp. required up to 20 times the chlorine levels to produce the same injury in enterotoxigenic Escherichia coli (ETEC) and nonpathogenic coliforms. Similar results were seen with Y. enterocolitica exposed to copper. The recovery of ETEC was followed by delayed enterotoxin production, both in vitro and in the gut of experimental animals. This indicates that injured waterborne enteropathogenic bacteria can be virulent.

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.

Enumerating Injured Coliforms in Drinking Water

This article emphasizes the importance of enumerating injured coliforms in drinking water and reviews the sources of injury, factors influencing the extent of injury, problems in enumerating stressed coliforms, recent advances in enumeration techniques, and the health implications of injured coliforms. Recommendations regarding future research are included.

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.

Influence of diluents, media, and membrane filters on detection fo injured waterborne coliform bacteria.

Pure cultures of Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter freundii were injured ( greater than 90%) in water from a dead-end section of the Bozeman, Montana, distribution system. The effects of the following laboratory variables on the enumeration efficiency of injured and undamaged control cells were examined: (i) diluent composition, temperature, and time of exposure; (ii) media, using various formulations employed in enumerating gram-negative bacteria; and (iii) surface pore morphology of membrane filters. The addition of peptone or milk solids to diluents and low temperature (4 degrees C) maximized the recovery of injured cells, but had little effect on undamaged cells. Control cells were recovered with high efficiencies on most media tested, but recoveries of injured cells ranged from 0 to near 100%. Most of the media commonly used in water analysis recovered less than 30% of injured cells. This was explained in part by the sensitivity of injured bacteria to deoxycholate concentrations greater than 0.01%, whereas control cells were unaffected by 0.1%. Membrane filter surface pore morphology (at 35 degrees C) had a negligible effect on total coliform recoveries. Recommendations are made regarding procedures to improve the recovery of injured coliforms by routine laboratory practices.