Effects of antecedent fermentative and respiratory growth on the detection of chloramine-stressed Escherichia coil and Salmonella typhimurium.

In vitro laboratory studies were performed to assess the effects of antecedent growth conditions on the recovery of Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028 following chloramine disinfection. Six- and 18-h cultures of each organism were grown under aerobic, fermentative, and nitrate-reducing conditions prior to disinfection. At predetermined time intervals during a 10-min exposure to chloramine, survivors were surface plated on nonselective recovery media to determine C(n)t values. It was observed that nitrate-reducing growth predisposed the test organisms towards an increased sensitivity to chloramine stress over cells grown under fermentation or aerobic conditions (p < 0.01).

Inhibition and reversal of Salmonella typhimurium attachment to poultry skin using zinc chloride.

A skin attachment model was used to determine if ZnCl2 would reverse or inhibit Salmonella attachment to broiler skin. In the reversal experiments, skin samples, treated first with 1 ml of Salmonella Typhimurium suspension (10(8) CFU/ml) for 30 min, were then treated with 25 or 50 mM ZnCl2 for 5 or 15 min. Zinc chloride solutions were applied while the culture was present on the skin. In the inhibition experiments, ZnCl2 solutions were added first; treatment solutions were discarded after 5 or 15 min of application, and then the culture was added. Firmly and loosely attached Salmonella were enumerated on xylose lactose tergitol plates. A duplicate section of skin, subjected concurrently to the above treatments, was observed under a scanning electron microscope to enumerate attached bacteria directly. In the reversal experiments, 25 and 50 mM ZnCl2 reduced (P < 0.01) firmly attached cells by 77 and 89%, respectively, when compared to the control (water). Micrographs indicated that 25 and 50 mM ZnCl2 reduced (P < 0.1) Salmonella attachment by 69 and 99.9%, respectively, in the reversal experiments. In the inhibition experiments, 25 and 50 mM ZnCl2 reduced (P < 0.01) firmly attached cells by 82 and 91%, respectively. Reduction of Salmonella may be attributed, in part, to the bactericidal activity of ZnCl2 in addition to bacterial cell detachment.

Detection and characterization of filterable heterotrophic bacteria from rural groundwater supplies.

AIMS: The chemical/physical environment of groundwater may contribute to the existence of a subpopulation of small-sized bacteria (filterable bacteria) that fails to be trapped on conventional 0.45 microm-pore-size membrane filters during routine bacteriological water quality analyses. Efforts were directed to determining an efficient recovery method for detection of such cells. METHODS AND RESULTS: Individual groundwater supplies in a rural setting were examined by a double membrane filtration procedure to determine the presence of heterotrophic plate count (HPC) bacteria capable of escaping detection on conventional pore size (0.45 microm) membrane filters but retained on 0.22 microm-pore-size filters. Since optimum cultural conditions for recovery of filterable bacteria are not well defined, initial efforts focused on evaluation of various media (R2A, m-HPC and NWRI) and incubation temperatures (15, 20, 28 and 35 degrees C) for specific recovery of filterable bacteria. Maximum recovery of small-sized HPC bacteria occurred on low-nutrient concentration R2A agar incubated for 7 d at 28 degrees C. Similarly, identical cultural conditions gave enhanced detection of the general HPC population on 0.45 microm-pore-size filters. A 17-month survey of 10 well water supplies conducted with the cultural conditions described above resulted in detection of filterable bacteria (ranging in density from 9 to 175 cfu ml-1) in six of the groundwater sources. The proportion of filterable bacteria in any single sample never exceeded 10% of the total HPC population. A majority of the colonies appearing on the 0.22 microm membrane filters was pigmented (50-90%), whereas the proportion of colonies demonstrating pigmentation on the larger porosity filters failed to exceed 50% for any of the samples (19-49%). CONCLUSION: A reliable recovery method was developed for the detection of filterable bacteria from groundwater. During a subsequent survey study using this procedure, filterable bacteria were detected in a majority of the groundwater supplies examined; however, the density of filterable bacteria in any single sample never exceeded 10% of the total HPC population. Identification of randomly selected isolates obtained on the 0.22 microm filters indicated that some of these filterable bacteria have been implicated as opportunistic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: We have determined the presence of small-sized HPC bacteria in ground water that may go undetected when using standard porosity membrane filters for water quality analyses. Further study is needed to assess the significance and possible health risk associated with presence of filterable bacteria in drinking water supplies from groundwater sources.

Recovery of coliform bacteria from rural groundwater supplies using reduced oxygen concentrations during incubation.

The effect of decreased oxygen concentration during incubation of M-Endo medium on detection of coliforms from rural groundwater supplies was examined. Incubation oxygen concentrations of 0¿(anaerobic GasPak), 4, 8, and 21% (atmospheric) were examined. Our findings point to several advantages of using anaerobic incubation for the isolation of coliforms: (i) higher verification rates with concomitant decreases in occurrence of false-positive coliforms; (ii) overall reduction in growth of nonsheen colonies; and (iii) reduction in colony size for nonsheen organisms, thereby minimizing crowding effects and facilitating enumeration of coliform colonies. However, these advantages were not sufficient to permit increased recovery of total coliforms as compared with aerobic incubation. In addition, the increased frequency of detecting false-negative coliforms during anaerobic incubation is a disadvantage to this method. While detection of total coliforms was reduced under conditions of anaerobiosis,the detection of fecal coliforms and (or) E. coli was not impeded.

Effect of point-of-use, activated carbon filters on the bacteriological quality of rural groundwater supplies.

The water quality of 24 rural, domestic groundwater supplies treated with point-of-use, powdered activated carbon (PAC) filters was monitored to determine how such treatment might impact the bacteriological quality of private, residential drinking water supplies. Heterotrophic-plate-count (HPC) and total coliform analyses were performed on raw, PAC-treated, and overnight or stagnant (first-draw) PAC-treated water samples. Densities of HPC bacteria were elevated by 0.86 and 0.20 orders of magnitude for spring and well water systems, respectively, in PAC-treated effluents following overnight stagnation compared with levels in untreated treated effluents. Densities of HPC bacteria in PAC-treated effluents were significantly reduced (P < 0.01) below influent levels, however, after the point-of-use device was flushed for 2 min. While PAC significantly reduced the number of coliforms in product waters (P < 0.01), these indicator organisms were still detected in some effluents. Seasonal variations were evident in microbial counts from spring but not well water systems. It appears that aside from periods following stagnant-water use, such as overnight, PAC treatment does not compromise the bacteriological quality of drinking water obtained from underground sources.

Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator.

BACKGROUND/AIMS: Human rectal epithelium in cystic fibrosis (CF) shows impaired ion transport in response to theophylline or bethanechol, although it possesses regulatory subunits of adenosine cyclic 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A). Protein kinase A-specific phosphorylation of CF transmembrane conductance regulator (CFTR) in rectal tissues of control and CF volunteers was examined in this study. METHODS: CFTR was evaluated using a polyclonal antiserum (pre-NBF) raised against a peptide corresponding to residues 415-427 of CFTR. Microsomal membranes from normal and CF rectal mucosa and from T-84 cells were incubated with [gamma 32P]-adenosine triphosphate +/- protein kinase A and subjected to immunoblotting with pre-NBF and autoradiography. RESULTS: Pre-NBF recognized a single band of 180 kilodaltons. Protein kinase A altered phosphorylation of this 180-kilodalton band 1.4-, 2.2- and 0.9-fold in T-84, normal, and CF rectal membranes, respectively. Catalytic activities of protein kinase A, Ca2+ calmodulin protein kinase, or protein kinase C in control and CF tissues were similar. CONCLUSIONS: cAMP and Ca(2+)-signaling pathways are normal up to the kinases in CF rectal mucosa. Our results suggest differences in CFTR phosphorylation in normal and CF rectal mucosal membranes.

Sheen formation and growth response of groundwater bacteria to reduced oxygen concentrations during incubation of M-Endo medium.

In vitro pure-culture studies were conducted to assess growth and sheen formation of groundwater bacteria on M-Endo medium incubated under reduced oxygen concentrations (0, 4, 8, 12, and 16%). Coliform and noncoliform bacteria were isolated from 17 untreated, rural groundwater supplies on M-Endo medium. All 16 coliform isolates tested were capable of sheen formation at oxygen concentrations of 4% or greater, yet some of these same isolates (Enterobacter aerogenes, Enterobacter cloacae, and Hafnia alvei) were either unable to grow or failed to produce a metallic sheen when incubated under strict anaerobiosis. Approximately 70% of the 21 noncoliform isolates examined exhibited growth inhibition at oxygen concentrations of 8% or less. The growth of a false-positive coliform isolate of Serratia fonticola was inhibited when incubated under reduced oxygen concentrations of 16% or less. Our findings suggest that the selectivity of M-Endo medium, and resultant inhibition of noncoliforms and false-positive coliforms, is enhanced by incubation in the absence of oxygen. However, the failure of strict anaerobiosis to permit detection of total coliforms such as Hafnia and Enterobacter spp. may compromise the reliability of this technique for evaluating the sanitary quality of some waters. On the other hand, oxygen concentrations of 4, 8, 12, and 16% permitted adequate sheen development of all coliforms tested while inhibiting some noncoliforms.

Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters.

Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters. Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars). Pseudomonads were the most frequently identified group of filterable bacteria detected. Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified. Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar. In addition, none of the isolates identified from nonselective media were coliforms. Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.

Improved membrane filtration method incorporating catalase and sodium pyruvate for detection of chlorine-stressed coliform bacteria.

In vitro pure culture studies were conducted on three different strains of Escherichia coli (K-12, EPA 00244, and SWEI) to determine the effect of chlorination on catalase activity. In each case, stationary-phase cells exhibited significant (P less than 0.001) reductions in enzyme activity following exposure to chlorine. Mean differences in activity between control and chlorine-stressed cells ranged from 8.8 to 20.3 U/mg of protein for E. coli SWEI and EPA 00244, respectively. Following initial enzyme studies, resuscitation experiments utilizing the membrane filtration technique were conducted on chlorinated sewage effluent. Five different amendments, including catalase (1,000 U per plate), heat-inactivated catalase (1,000-U per plate), sodium pyruvate (0.05%), a catalase-sodium pyruvate combination (1,500 U/0.01%), and acetic acid (0.05%), were tested for the ability to enhance detection of chlorine-stressed cells on M-fecal coliform (M-FC), mT7, M-Endo, and tryptone-glucose-yeast extract (TGY) media. Significant (P less than 0.001) increases in recovery of fecal coliforms on M-FC, total coliforms on mT7 and M-Endo, and total heterotrophs on TGY were obtained on plates containing catalase, pyruvate, or the combination of these compounds. Supplementation with heat-inactivated catalase and acetic acid did not improve recovery of chlorine-stressed cells compared with recovery on nonamended media. Subsequent analysis of colonies from plates containing compounds which enhanced recovery indicated coliform verification percentages of greater than 80% on M-FC, greater than 90% on mT7, and greater than 94% on M-Endo media. These data suggest that the addition of peroxide-degrading compounds to various standard recovery media may improve detection of both coliform and heterotrophic bacteria in chlorinated waters.

Improved detection of acid mine water stressed coliform bacteria on media containing catalase and sodium pyruvate.

Pure culture suspensions of two strains of exponential and stationary phase Escherichia coli exhibited significant reductions in catalase activity following exposure to acid mine water (AMW). The exogenous addition of catalase (500-2000 U) or sodium pyruvate (0.05-5%) to a nonselective recovery medium resulted in enhanced detection (12- to 465-fold) of AMW-stressed E. coli as compared with recovery on the medium lacking these supplements, whereas addition of 3,3'-thiodipropionic acid failed to improve recovery. Additional in vitro experiments utilizing selective M-FC, mT7, and M-Endo media containing 1000 U catalase or 1.0% pyruvate similarly resulted in improved detection of AMW-stressed cells, with the exception of M-Endo containing pyruvate. Appropriately modified media were then used to analyze an AMW-impacted stream by the membrane filtration technique. Addition of catalase, pyruvate, or a combination of both significantly improved recovery of fecal and total coliforms without promoting growth of noncoliforms. Supplementation of plate count agar with pyruvate and (or) catalase enhanced detection of total heterotrophs. These findings suggest that addition of catalase or pyruvate to standard recovery media may improve detection of coliform and total heterotrophic bacteria in AMW-impacted waters.

Detection of Acinetobacter spp. in rural drinking water supplies.

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.

Survival of chlorine-injured enterotoxigenic Escherichia coli in an in vitro water system.

Survival of chlorine-injured and noninjured subpopulations of enterotoxigenic Escherichia coli was compared in KH2PO4-buffered water and chlorine-neutralized tap water. Injured cells were no less persistent than noninjured cells and did not exhibit limited survival as a consequence of chlorine injury. At high inoculum densities, some injured cells were able to repair, apparently owing to the accumulation of materials arising from the chlorination procedure.

Metabolic processes involved in repair of Escherichia coli cells damaged by exposure to acid mine water.

Escherichia coli was stressed by exposure to filter-sterilized acid mine water. Synthetic processes required for repair of sublethally injured survivors were studied by the addition of specific metabolic inhibitors to a resuscitation broth. Inhibitors of protein, RNA, DNA, lipid, and peptidoglycan synthesis as well as uncouplers and inhibitors of electron transport and ATPase activity were used. Acid mine water injury was severe, causing damage to the outer and cytoplasmic membranes. Repair of sublethally injured cells required protein, RNA, and lipid synthesis as well as a proton motive force.

Effect of chlorine injury on heat-labile enterotoxin production in enterotoxigenic Escherichia coli.

Heat-labile enterotoxin (LT) production was examined in chlorine-injured and noninjured populations of enterotoxigenic Escherichia coli (ETEC) by passive immune hemolysis and Y-1 mouse adrenal tumor cell assays. Sublethally injured populations showed reduced LT production after 1, 2.5, and 4 h incubation in trypticase soy broth plus 0.25% glucose, pH 8.0. Reduction was observed during injury, resuscitation, and for at least 1.5 h following repair. LT levels comparable with that present in noninjured cells were found after 24 h incubation in the same medium, indicating delayed toxigenesis rather than permanent damage. Chlorinated populations failed to incorporate [14C]glucose until repair was completed suggesting a possible explanation for delayed toxin production. The results indicate a temporary loss of virulence among sublethally injured ETEC in chlorinated waters.

Chlorine-induced damage to surface adhesions during sublethal injury of enterotoxigenic Escherichia coli.

A comparison of the adhesive ability of noninjured and chlorine-injured enterotoxigenic Escherichia coli was made by in vitro attachment to human peripheral leukocytes. Chlorination selected for noninjured cells with greater capabilities for colonizing the small intestine. Injured populations exhibited reduced association with leukocytes. Maximum reduction was seen in populations with greater than 80% injury. These cells demonstrated less adhesive ability than nonpiliated populations. Electron micrographs suggested that reduced adhesive ability was due to the loss of surface structures as a consequence of sublethal chlorination. The data imply a reduced ability among chlorine-injured pathogens to colonize the small intestine and initiate disease.

Evaluation of recovery methods to detect coliforms in water.

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.

Statistical inferences about injury and persistence of environmentally stressed bacteria.

A standard technique for ascertaining the survival characteristics of bacteria after being environmentally stressed is to incubate the bacteria on both selective and non-selective media and count the colonies produced. Based on these colony counts, indexes of injury and persistence of the bacteria are calculated. To compare the stress of two different environments, a persistence ratio is calculated. In this paper, methods of statistical inference concerning these indexes and ratios are presented. These statistical methods use well-known procedures for analysis of binomial data and 2 times 2 table data, and are appropriate when the colony counts follow a Possion distribution.

Influence of environmental stress on enumeration of indicator bacteria from natural waters.

The problems associated with recovery of pure cultures of Escherichia coli and Streptococcus faecalis from stream environments were examined utilizing membrane filter chambers. It was observed that upon exposure to the aquatic environment a significant proportion of cells lost their ability to produce colonies on a selective medium, yet retained this capability on a nutritionally rich, nonselective medium. Discrepancies in colony-forming units between nonselective and selective media indicated that a substantial portion of bacterial cells may become physiologically injured due to the environmental stress imposed by the aquatic environment. The extent of injury was observed to vary considerably among the eight different stream environments, since the amount of injury was not uniform for all types of water environments examined. It was observed that the injury acquired by a population of E. coli, during exposure to the aquatic environment, could be rapidly repaired in a nutritionally rich, nonselective medium. As the injured population of cells was exposed to the rich, nonselective broth, increasing proportions of cells were able to repair themselves such that they became insensitive to inhibitory agents in selective media.

Comparative survival of indicator bacteria and enteric pathogens in well water.

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.