The effect of growth conditions on survival and recovery of Klebsiella oxytoca after exposure to chlorine

Pergamon PII: S0043-1354(96)00220-5

Wat. Res. Vol. 31, No. 1, pp. 135-139, 1997 Copyright © 1996ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/97$17.00+ 0.00

THE EFFECT OF GROWTH CONDITIONS ON SURVIVAL AND RECOVERY OF KLEBSIELLA OXYTOCA AFTER EXPOSURE TO CHLORINE KAYE N. POWER @, RENE P. SCHNEIDER @* and KEVIN C. MARSHALL School of Microbiology and Immunology, University of New South Wales, Sydney 2052, N.S.W., Australia (First received October 1995; accepted in revised form June 1996) Abstract--A strain of Klebsiella oxytoca isolated from within Sydney's water distribution system was grown under ~Lrange of physiological conditions including batch cultures and continuous culture under both carbon and nitrogen limitation. Cells were then exposed to varying concentrations of total chlorine. The initial growth conditions affected the ability of cells to survive. Cells grown under batch conditions in a medium containing mineral salts and yeast extract were the most resistant. The resistance to chlorine was not related to the presence of polysaceharide or differences in protein levels but to the formation of aggregates. The initial growth conditions also influencedthe recovery of cells after the removal of chlorine stress. Copyright © 1996 Elsevier Science Ltd Key words--regrowth, chlorine injury, recovery, drinking water, coliforms

(Milbauer and Grossowicz, 1959; Matin and Harakeh, 1990). One of the principal groups of organisms involved in regrowth within chlorinated drinking water systems is a range of Klebsiella spp. (Camper et al., 1986; Geldreich and Rice, 1987; Power and Nagy, 1989), which often display mucoid colony types on isolation (Reilly and Kippin, 1983; Wang et al., 1985). The regrowth of this organism is of particular importance since as a coliform it will make compliance to water quality guidelines difficult. Species of the genus Klebsiella may also be involved in opportunistic infections (Brown and Seidler, 1973; Geldreich, 1987; Hudson et al., 1983). The aims of the following work were to determine the effect of growth conditions on the survival of KI. oxytoca after exposure to chlorine, whether KI. oxytoca was able to recover after exposure to chlorine and whether the initial growth conditions influenced subsequent recovery.

II~TRODUCTION The occurrence of bacterial regrowth within drinking water distribution systems could be due to a variety of reasons, including the presence of sediments and biofilms and tlhe recovery of cells following injury (Le Chevallier, 1990). The presence of sediments and biofilms will contribute to regrowth by constantly seeding the aqueous phase with organisms. This is most often manifested as a sudden increase in bacterial numbers. The recovery of cells after disinfection will conl:ribute to regrowth by allowing cells to grow on the selective media used in enumeration, on which they were unable to grow immediately after t~:eatment or disinfection. This results in a constant higher bacterial count at the ends Of the system than immediately after disinfection. Factors influencing the rate and extent of cell recovery following injury have not been fully elucidated, but the initial susceptibility of cells to disinfection may be one of these. The susceptibility of an organism to disinfection may be due to a wide range of conditions including the physiological state of the organism (L,'yval et al., 1984; Russel and Gould, 1988; Stewart and Olson, 1992a), the presence of excessive amounts of polysaccharide, changes in growth temperat~Lre (Berg et al., 1982), growth rate (Harakeh et al., 1985) and nutritional status

MATERIALS AND METHODS Isolate The KI. oxytoca strain used in this study was isolated within Sydney's drinking water distribution system and was identified using the API 20E system (Carter and Wallace, Sydney N.S.W.).

*Author to whom all correspondence should be addressed [Phone: +61 2 9385 2105. Fax: +61 2 9385 1591. E-mail: R.schneider @unsw.edu.au].

Growth conditions KI. oxytoca was grown under both batch and continuous culture conditions. Growth in continuous culture was carried out in a CHEMAP AG 141 fermentor run at a 61 capacity. Cells were grown in AKC + YE medium which consisted of AK salts containing MgSO4.7H20, 1 g,

135

136

Kaye N. Power et al.

CaCI2.2H20, 0.2 g; NaC1, 1.2 g; KCI, 1 g in I 1 of distilled water, supplemented with 10mM of NaH2PO4-2H20, 200 mg of yeast extract l -~ and 1 ml l -~ each of vitamin and trace element solutions. The pH was maintained at 6.0 with 1 M HCI. The vitamin solution contained per l of distilled water: p-aminobenzoic acid, 50 mg; pyridoxine-HCl, 100 mg; thiamine-HCl, 50 mg; riboflavin, 50 rag; nicotinic acid, 50 mg; D-Ca panthotenate, 50 mg; lipoic acid, 50 mg; nicotinamide, 50mg; vitamin Bl2, 50mg; biotin, 20mg; folic acid, 20 mg. The trace element solution contained per l of distilled water: FeCI3"6H20, 0.8 g; ZnCl2, 70 mg; MnCh.4H20, 100 mg; COC12'6H20, 120 rag; NiCI2.6H20, 25mg; CuCI2.2H20, 15mg; HCl (25%), 6.Sml; Na2MoO4-2H20, 25 mg (Schneider and Marshall, 1994). Carbon, as citric acid, and nitrogen, as NH4CI, were added to obtain carbon-limited (C:N; 6.54:1) or nitrogen-limited (C:N; 30: l) growth conditions. Growth was at 20°C, under conditions of saturated oxygen with an air flow rate of l0 l min -~, a rotor speed of 400 r.p.m, and a dilution rate of 0.067 h -~. Validation of nitrogen and carbon limitation was carried out by determining the amount of citric acid and ammonia remaining within the supernatant at each steady state. Biomass was determined, at each steady state, by filtering a 5 ml sample through a 0.22/~m Millipore HA membrane to form a cake. Membranes, with the biomass cake, were dried overnight at 100°C, stored in a desiccator and weighed when cool. The filtrates from the collection of biomass were frozen for later carbon and nitrogen analysis. Citric acid and ammonia levels present in filtrates were determined using biochemical food analysis kits (Boehringer and Mannheim Aust. P/L, Castle Hill, N.S.W.). Batch cultures were grown, shaking at 27°C, in either AKC + YE medium or tryptone soya broth (TSB) (Oxoid). Growth over time for all conditions was determined by measuring absorbance at 600 nm (A600) (Spectronic 20, Bausch and Lomb (Aust) P/L, Sydney).

Chlorine assays Cells were harvested from growth media by centrifugation at 5900g for 10min, washed twice in phosphate buffer, pH 7.2 and resuspended in phosphate buffer to an Am of 0.5. A 50 ml subsample was then added to 450 ml of phosphate buffer, pH7. 2. The chlorine reaction was commenced with the addition of chlorine at a concentration of either 0.25, 0.5, 0.75 or 1.0 mg 1-~ total chlorine. Cells were left in contact with chlorine and stirred for 10 min, after which time any remaining chlorine was neutralized by the addition of 0.1 ml of 20% sodium thiosulphate. After neutralization, apparent viable cell numbers were enumerated on tryptone soya agar (TSA) (Oxoid) using the Miles and Misra drop plate method (Miles and Misra, 1938). An unchlorinated control was conducted to determine the effect of reaction components on the apparent viability of cells. All glassware and reagents for chlorine exposure work were prepared as described in Standard Methods (APHA, 1985). The chlorine demand of glassware and magnetic fleas used in the assays was removed by soaking them in a strong chlorine solution for 48 h, and then the glassware was heated overnight at 160°C. Chlorine demand free water (CDFW) was used in all assay solutions and for diluting the chlorine stock. Chlorine demand was removed from the water by adding sufficient chlorine to distilled water to result in a final concentration of 5 mg 1-L of free chlorine. This solution was left for 2 days after which time the remaining chlorine was dissipated by exposure to ultraviolet light. Commercial grade chlorine (4%) for use in assays was purchased once a month and stored at 4°C. The chlorine solution used in the assays was made fresh each day, from the commercial stock, to a final concentration of 5 mg 1-~ in CDFW.

Protein and polysaccharide levels in ceils To determine if there was any difference in the level of protein or polysaccharide produced by the cells when grown under different conditions, 5 ml subsamples were removed from the unchlorinated control and centrifuged at 6000 g for 10 min. Polysaccharide analysis was carried out using the method of Dubois et al. (1956). The cell pellet was washed twice in phosphate buffer, pH 7.2 and resuspended to an Am of 0.5. A 1 ml sample was mixed with 1 ml of 5% phenol and 5 ml of concentrated H2SO4, shaken and reacted for 10 rain with periodic mixing. This mixture was then incubated for 10-20 min in a waterbath at 30°C, after which time the absorbance of the solution was measured at 490 nm (A49o). A control blank consisting of 1 ml of water was prepared as above. A standard curve for glucose was determined each time a new assay was conducted. Samples were measured in duplicate and results expressed as #g ml -~ of glucose. Protein levels were measured after pellets were washed once in phosphate buffer and lysed by resuspending cells in l ml of 0.01% SDS and 0.025ml of chloroform. This mixture was incubated at 28°C for 30 min following which time the cells were centrifuged, at maximum speed, for 5 min in a bench microcentrifuge to remove cellular debris. The subsequent determination of protein levels was performed on the supernatant using a commercial protein assay kit (Biorad).

Recovery of cells exposed to chlorine and effect of initial growth conditions on subsequent recovery Cells grown in continuous culture under conditions of carbon limitation, and in TSB under batch conditions were exposed to 0.5 or 1.0 mg 1-~ of total chlorine, as described above. After the neutralization of chlorine, cells were diluted 1:10 into phosphate buffer, pH 7.2. Cultures were incubated statically at 27°C and apparent viable cell numbers were determined every 2 h for the first 10 h and then at 24 h and 48 h using the Miles and Misra drop plate method (Miles and Misra, 1938) onto a non-selective media (TSA). The possibility of cryptic growth by KI. oxytoca on the organic matter supplied by dead cells was also determined. KI. oxytoca was grown overnight in TSB, cells were harvested by centrifugation at 5900g for 10 min, washed twice in phosphate buffer, pH 7.2 and resuspended to an A~0 of 0.5. A subsample of the bacterial suspension was autoclaved at 103.4 kPa for 15 rain to produce 100% dead cells and equivalent organic matter. Two more subsamples of the bacterial suspension were diluted l:100 (cryptic growth l:100) and l:1000 (cryptic growth l:1000). One ml of each was added to 9 ml of phosphate buffer, pH 7.2 which also contained approx. 1.12 x l08 autoclaved cells ml-k The addition of the autoclaved cells was to mimic the potential dead cells available for growth after chlorine exposure. Viable cell numbers were determined over the same time frame as for the original recovery assays on TSA using the Miles and Misra drop plate method (Miles and Misra, 1938).

Statistical analysis All assays were carried out in duplicate and on two separate occasions. Data points are averages and error bars represent standard error of the mean. RESULTS

Effect o f growth conditions on cell survival after exposure to chlorine The a p p a r e n t viability o f cells exposed to 0.25 mg 1-t o f total chlorine declined by 3 orders o f magnitude, irrespective o f growth conditions o f the cells (Fig. 1). A t 0.5 mg 1-t, 0.75 mg I -t and 1.0 mg 1-t significantly more cells grown in A K C + Y E

Klebsiella oxytoca and exposure to chlorine

--

137

lO 8

i o.1|[ T -/

"

~"

102, --'~ vJ 0

Time (h) 0.25

0.S

0.75

1

Total chlorine (mg/I) Fig. 1. Apparent viable cell numbers of KI. oxytoca grown under different conditions after exposure to varying concentrations o f chloldne. *Significant at the 5% level. I1: AKC+YE (batch), m : TSB (batch), Iq: C-limited (continuous) and I~, N-limited (continuous).

medium (batch) suzvived exposed to chlorine than cells grown under C-limitation, N-limitation or in TSB (batch). Further increases in chlorine concentration did not result in a greater decrease in apparent cell viability for cells grown in AKC + YE. The apparent viable celL,; numbers of KI. oxytoca grown under the remaining three conditions declined an additional 1 order of magnitude as the concentration of total chlorine was increased from 0.25 mg 1-~ to 0.75 mg 1-', with no further decline in apparent viable numbers at higher c,hlorine concentrations. Nitrogen-limited cells produced the highest amount of polysaccharide, followed by cells grown in AKC + YE (batch), carbon-limited cells and, finally, cells grown in TSI]; (Table 1). Cells grown under carbon limitation and in TSB contained the highest concentration of protein followed by cells grown under nitrogen limilation and AKC + YE (batch).

Fig. 2. The recovery of carbon-limited grown KI. oxytoca cells in phosphate buffer, pH 7.2 after exposure to chlorine. l : untreated control. &: 0.5rag 1-~ total chlorine. O: 1.0 m g l -~ total chlorine.

cultures exposed to 1.0mg 1-~ of total chlorine showed an increase of 6 orders of magnitude over 48 h. For both chlorine concentrations, the final cell numbers attained after 48 h incubation were similar and exceeded those in the untreated control (Fig: 2). Cells grown in TSB and exposed to 0.5 mg 1-j and 1.0mg 1-~ of total chlorine showed a 1.5 log and 3 log increase in apparent viable cell numbers over 48 h. Unlike the carbon-limited cells, the number of apparent viable cells did not reach the same level as the control. Those exposed to 1 mg l-' of total chlorine remaining a log lower than those exposed to 0.5 rag 1-' of total chlorine. No change in apparent viable cell numbers over time occurred with the untreated control (Fig. 3). Cryptic growth 1:100 showed a 0.5 order of magnitude increase in viable cell numbers after 24 h incubation and cryptic growth 1:1000 showed an increase of 1 to 1.5 order of magnitude from 12 to 48 h incubation (Fig. 4).

Recovery of cells after exposure to chlorine and effect of initial growth conditions on subsequent recovery The recovery of KI. oxytoca cells, grown under carbon limited conditions and in TSB, after exposure to chlorine is illustrated in Figs 2 and 3, respectively. With carbon-limited cells, the apparent viable cell numbers, after exposure to 0.5 mg 1-~, showed a 3.5 order of magnitude increase over 48 h and those Table 1. Protein and polysaceharide levels in cells under different ~:owth conditions Polysaccharide Protein Sample growth OJg ml -t) (/zg ml -t) conditions Mean + SEM Mean + SEM Nitrogen limited Carbon limited AKC + YE (batch) TSB (batch)

5.3 ± 0.7 + 1.5 ± 0.3 ±

0.7 0.1 0.1 0.3

0.4 ± 0.4 + 0.2 ± 0.4 ±

0.0 0.1 0.0 0.1

,08] |

,0.

°

1° 5

Q

"-

-

1

1041

I

o

~.........'~j~.41

o

a

I

;e

~

2',

Tlme

3'2

,o

,'g

(h)

Fig. 3. The recovery of TSB grown Kl. oxytoca cells in phosphate buffer, pH 7.2 after exposure to chlorine. I1: untreated control. &: 0 . S m g 1-~ total chlorine. O: 1.0 m g 1-L total chlorine.

Kaye N. Power et al.

138

106i loS(~ "~ Q 104 103 Time (h) Fig. 4. Cryptic growth of KI. oxytoca cells. O: cryptic growth 1:100. I-1: cryptic growth 1:1000.

DISCUSSION

Results of the chlorine exposure work indicated that the physiological condition of the cells does have an effect on their ability to survive exposure to chlorine. KI. oxytoca grown in shake flasks in A K C + YE medium showed the greatest resistance to inactivation by total chlorine, when compared to cells grown under conditions of nitrogen or carbon limitation or in a rich medium. Characterization of polysaccharide and protein levels within cells did not give any indication of possible reasons for the increased resistance of cells grown in shake flasks in A K C + YE medium. It has been speculated that the presence of high amounts of cellular and extracellular polysaccharide may result in increased resistance to biocides by reducing the exposure of the cell membrane to biocides (Leyval et al., 1984; Seyfried and Fraser, 1980; Brown et al., 1995). Other work on the susceptibility of KI. pneumoniae has indicated that the presence of extracellular polysaccharide had no significant effect on cell survival in the presence of chlorine or chloramine (Le Chevallier et al., 1988). Protein and polysaccharide analyses did not reveal any significant difference in the levels produced by cells except for polysaccharide production by nitrogen-limited cells, yet nitrogen-limited cells did not show greater survival after exposure to chlorine. The increased resistance of cells grown in A K C + YE medium in shake flasks may have been due to the tendency of cells to form flocs. KI. pneumoniae grown in low-nutrient media was found to be more resistant to chloramine than cells grown in rich media, but KI. pneumoniae was also shown to form large aggregates of 10 to 10,000 cells ml -~ (Stewart and Olson, 1992b). The formation of flocs or aggregates of cells could have been the result of physiological alterations to the cell surfaces. Growth in A K C + YE medium in shake flasks to stationary phase resulted in an increase in the pH of the medium. The pH of the continuous culture was controlled at 6 and TSB contains sufficient buffering capacity to control the pH at 7. The increased pH of

the A K C + YE medium may have resulted in physiological changes in the cells causing them to aggregate. The results of the recovery assay indicated that KI. oxytoca could be injured by chlorine and recover such that colonies were produced on non-selective agar. The results also indicated that the recovery of cells after exposure to chlorine is influenced by the original physiological condition of the cells. The initial chlorine assay showed that there was no significant difference in the susceptibility of cells grown under carbon limitation and grown in TSB to chlorine. The variation in the recovery of cells may have been due to differences in the amount of the injury experienced by cells or the ratio of dead to injured cells. TSB-grown cells after exposure to chlorine may have contained a higher number of dead cells than carbon-limited cells. It was possible that the increase in viable cell numbers was due to the cryptic growth of remaining cells on the organic matter produced from lysed cells. The results of the cryptic growth assay indicated that, although growth does occur on the organic matter provided by dead cells, cells treated with chlorine, for both conditions, experienced a greater increase in apparent viable cell numbers than could be accounted for by cryptic growth. In water distribution systems, the recovery of cells from injury will result in an increase in cell number as the distance from the point of treatment increases. The formation of flocs and aggregates under certain conditions of growth by KI. oxytoca allows cells to survive chlorination and enter the distribution system. The recovery of cells after exposure to chlorine contributes to regrowth as does cryptic growth on organic matter from dead cells. The results presented here indicate that the control of regrowth of KI. oxytoca within drinking water systems would be best approached by the removal of large aggregates of cells or sediments before the application of chlorine, such that protection of cells from chlorination does not occur.

Acknowledgements--This work was funded by the Urban Water Research Association of Australia. Chemostat equipment was supplied by Ensight, Australia Water Technology (AWT), Sydney, Australia.

REFERENCES

American Public Health Association (1985) Standard Methods for the Examination of Water and Wastewater American Water Works Association and Water Pollution Control Federation. Washington, DC. Berg J. D., Matin A. and Roberts P. V. (1982) Effect of antecedent growth conditions on sensitivity of Escherichia coil to chlorine dioxide. Appl. Environ. Microbiol. 44, 814-819. Brown C. and Scidler R. J. (1973) Potential pathogens in the environment: Klebsiella pneumoniae, a taxonomic and ecological enigma. Appl. Microbiol. 25, 900-904.

Klebsiella oxytoca and exposure to chlorine Brown M. L., Aldrich H. C. and Gauthier J. J. (1995) Relationship between glyeocalyx and providone-iodine resistance in Pseudmonas aeruginosa (ATCC 27853) biofilm. Appl. Environ. Microbiol. 61, 187-193. Camper A. K., Le Chavallier M. W., Broadway S. C. and McFeters G. A. (1986) Bacteria associated with granular activated carbon particles in drinking water. Appl. Environ. Microbiol. 52, 434-438. Dubois M., Giles K. A., Hamilton J. K., Rebers P. A. and Smith F. (1956) Colormetric methods for sugars and related substances. Anal. Chem. 28, 350-356. Geldreich E. E. (1987) Drinking water microbiology for the 1980s and beyond. Conference proceedings on microbiological diagnostic needs in the food industry. Atlanta, Ga, USA. Geldreich E, E. andt Rice E. W. (1987) Occurrence, significance, and detection of Klebsiella in water supply. J. Am. Wat. Wks. Assoc. 79, 74--79. Harakeh M. S., Berg J. D., HoffJ. C. and Matin A. (1985) Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pnenrmoniae to chlorine dioxide. Appl. Environ. Microbiol. 49, 69-72. Hudson L. D., Hankins J. W. and Battaglia M. (1983) Coliforms in a wal:er distribution system: a remedial approach. J. Am. Wat. Wks Assoc. 75, 564-571. Le Chevallier M. W. (11990) Coliform regrowth in drinking water: A review. J. Am. Wat. Wks Assoc. 82, 74-86. Le Chevallier M. W., Cawthorn C. D. and Lee R. G. (1988) Factors promoting survival of bacteria in chlorinated water supplies. Appl. Environ. Microbiol. 54, 649--654. Leyval C., Arz C., ]Block J. C. and Rizel M. (1984) Escherichia coil resistance to chlorine after successive chlorinations. Environ. Technol. Letts. 5, 359-364. Matin A. and Harakeh S. (1990) Effect of starvation on bacterial resistance to disinfectants. In Drinking Water

139

Microbiology (Edited by McFeters G. A.), pp. 88--103. Springer-Verlag. New York. Milbauer R. and Grossowicz N. (1959) Effect of growth conditions on chlorine sensitivity of Escherichia coll. Appl. Microbiol. 7, 71-74. Miles A. A. and Misra S. S. (1938) Estimations of bactericidal power of blood. J. Hyg. 38, 732-748. Power K. N. and Nagy L. A. (1989) Bacterial regrowth in drinking water supplies. Report No. 4. Urban Water Research Association, Australia. Reilly K. K. and Kippin J. S. (1983) Relationship of bacterial counts with turbidity and free chlorine in two distribution systems. J. Am. Wat. Wks Assoc. 75, 309-312. Russell A. D. and Gould G. W. (1988) Resistance of enterobacteriaceae to preservatives and disinfectants. J. Appl. Bact. Sym. Supp. 67, 167s-195s. Schneider R. P. and Marshall K. C. (1994) Retention of the Gram-negative marine bacterium SW8 on surfaces-Effects of microbial physiology, substratum nature and conditioning films. Coll. Surf. B: Biointerfaces 2, 387-396. Seyfried P. L. and Fraser D. J. (1980) Persistance of Pseudomonas aeruginosa in chlorinated swimming pools. Can. J. Microbiol. 26, 350-355. Stewart M. H. and Olson B. H. (1992a) Impact of growth conditions on resistance of Klebsiella pneumoniae to chloramines. Appl. Environ. Microbiol. 58, 2649-2653. Stewart M. H. and Olson B. H. (1992b) Physiological studies of chloramine resistence developed by Klebsiella pneumoniae under low-nutrient growth conditions. Appl. Environ. Microbiol. 58, 2918-2927. Wong S. H., Cullimore D. R. and Bruce D. L. (1985) Selective medium for the isolation and enumeration of Klebsiella spp. Appl. Environ. Microbiol. 49, 1022-1024.