Survival of Campylobacter Jejuni and Escherichia Coli in Groundwater During Prolonged Starvation at Low Temperatures K.L

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged starvation at low temperatures K.L. Cook and C.H. Bolster

USDA-ARS, AWMRU, Bowling Green, KY, USA

Keywords Abstract Campylobacter jejuni, 5-cyano-2,3,-ditolyl tetrazolium chloride, Escherichia coli, Aims: To evaluate the survival of Campylobacter jejuni relative to that of groundwater, nutrient, real-time polymerase Escherichia coli in groundwater microcosms varying in nutrient composition. chain reaction, survival. Methods and Results: Studies were conducted in groundwater and deionized water incubated for up to 470 days at 4C. Samples were taken for culturable Correspondence and total cell counts, nutrient and molecular analysis. Die-off in groundwater K.L. Cook, AWMRU USDA-ARS, 230 Bennett microcosms was between 2Æ5 and 13 times faster for C. jejuni than for E. coli. Lane, Bowling Green, KY 42104, USA. E-mail: Campylobacter jejuni had the lowest decay rate and longest culturability in [email protected] ) microcosms with higher dissolved organic carbon (4 mg l 1). Escherichia coli )1 2006/1078: received 25 July 2006, revised 27 survival was the greatest when the total dissolved nitrogen (12Æ0mgl ) was October 2006 and accepted 15 November high. The transition of C. jejuni to the coccoid stage was independent of cultu- 2006 rability. Conclusion: The differences in the duration of survival and response to water doi:10.1111/j.1365-2672.2006.03285.x nutrient composition between the two organisms suggest that E. coli may be present in the waters much longer and respond to water composition much differently than C. jejuni. Signiﬁcance and Impact of the Study: The data from these studies would aid in the evaluation of the utility of E. coli as an indicator of C. jejuni. This study also provided new information about the effect of nutrient composition on C. jejuni viability.

ing that the organism does, in fact, persist for some time Introduction in aquatic and agricultural environments. Campylobacter jejuni is an enteric pathogen, which is a Waterborne outbreaks of Campylobacter occur occa- leading cause of gastroenteritis worldwide (Mead et al. sionally and are normally associated with faecal con- 1999). Most infections are sporadic and are associated tamination of the water source from agricultural waste with eating or handling undercooked poultry or con- run-off, bird droppings or sewage outﬂow (Bobb et al. sumption of unpasteurized milk or contaminated water 2003; Clark et al. 2003; Hanninen et al. 2003). Contamin- (Clark et al. 2003). Campylobacter jejuni is an obligate ated groundwater may serve as a source of inoculation of microaerophile that is extremely sensitive to light and drinking waters with C. jejuni or the introduction of the desiccation (Jones 2001). Owing to its fastidious nature, organism into livestock populations where groundwater is it is commonly assumed that C. jejuni is unable to survive used as a source of drinking and irrigation (Pearson et al. in the environment for long periods of time. However, 1993; Stanley et al. 1998; Hanninen et al. 2003). The sur- outbreaks have been associated with C. jejuni isolated vival of C. jejuni in groundwater is not surprising as the from river water and groundwater (Stanley et al. 1998; environmental conditions (e.g. low redox potential, low Hanninen et al. 2003), and C. jejuni has been isolated temperatures, lack of UV exposure and desiccation) often from sewage efﬂuent, lagoon waters and bedding on farm favour its maintenance (Stanley et al. 1998; Jones 2001). sites (Hanninen et al. 2003; Murinda et al. 2004), suggest- Detection of C. jejuni in groundwater suggests that this

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 No claim to original US government works 573 Campylobacter jejuni survival in groundwater K.L. Cook and C.H. Bolster organism does exhibit significant vertical movement in presence of the targeted pathogen and the presence of fae- the environment (Stanley and Jones 2003), possibly owing cal indicators has led to serious concerns regarding the to electrostatic repulsion of the bacteria and negatively adequacy of these organisms as indicators of potential charged soil particles (Bolster et al. 2006). health risks. Research suggests that C. jejuni may enter a viable but In this study, we examine the persistence of C. jejuni nonculturable or starvation-survival state in which the relative to that of E. coli in groundwater microcosms of cells become dormant and nonculturable for extended different nutrient makeup. Past research has shown that periods in adverse environments (Rollins and Colwell C. jejuni can be isolated from groundwater (Stanley 1986; Thomas et al. 1999). This may contribute to et al. 1998), and can survive in drinking and river C. jejuni’s survival for prolonged periods in surface waters waters for prolonged periods (Korhonen and Martikai- and groundwater (Stanley et al. 1998; Obri-Danso and nen 1991; Terzieva and McFeters 1991; Cools et al. Jones 1999; Thomas et al. 1999). Regardless of the nature 2003; Baffone et al. 2006) with nutrient makeup signifi- of the survival strategy, the ability of C. jejuni to enter a cantly influencing the duration of survival (Thomas suspended state permits prolonged survival in unfavour- et al. 1999). However, research has not been conducted able conditions, often long after the cells lose culturabi- to describe the differences in the survival of the same lity. Resuscitation of C. jejuni following the loss of strain of C. jejuni in water from different sources and culturability or selective enrichment of the organism from nutrient composition or how its survival compares with mixed populations has been attempted using either select- that of the faecal indicator organism E. coli under the ive enrichment broths (Stanley et al. 1998; Baylis et al. same conditions. In this study, a multifaceted approach 2000; Cools et al. 2003) or passage through mouse intes- (including nutrient analysis, molecular analysis, and total tine (Baffone et al. 2006). Cools et al. (2003) found that and culturable cell counts) was used to evaluate the abil- starved C. jejuni lost culturability more quickly on select- ity of the organisms to remain culturable and maintain ive media, and that resuscitation was also influenced by respiratory, cellular and genomic integrity in groundwa- the media type. In fact, when incubated at low tempera- ter microcosms. tures (4–6C), C. jejuni is shown to become nonculturable weeks or even months before other measures of viability Materials and methods decrease or disappear (Terzieva and McFeters 1991; Buswell et al. 1998; Lazaro et al. 1999; Thomas et al. Bacterial strains and maintenance 1999). This maintenance strategy ensures the survival of some minimal portion of the population for passage to a Campylobacter jejuni (ATCC 49943) was maintained on new host when opportunity presents. campylobacter-selective agar (CSA) (Oxoid CM0689; Current water-quality monitoring procedures require Sparks, MD, USA) supplemented with 5% horse blood the assessment of microbiological safety through the use (Hemostat, Dixon, CA, USA) and modified Preston of indicator organisms, such as total or faecal coliforms campylobacter-selective supplement (Oxoid SR0204, (Hanninen et al. 2003). Escherichia coli is the most com- Sparks, MD, USA), which contains polymixin B, rif- monly used of the specific indicators of faecal contamin- ampicin, trimethoprim and amphortericin B. Campylo- ation. The suitability of this measure has been heavily bacter jejuni was incubated at 37C in anaerobic jars debated and has, in many instances, been found to be (BBL Gaspak System, Sparks, MD, USA). Microaerophi- inappropriate (Ferguson et al. 1996; Harwood et al. lic conditions were generated with the CampyPak Plus 2005), particularly when E. coli fails to respond to envi- microaerophilic system (BD; Sparks, MD, USA). Escheri- ronmental conditions in a manner similar to the patho- chia coli O157:H7 strain L-2 was kindly provided by gen of interest (Baggi et al. 2001; Lemarchand and Greg Siragusa (USDA-ARS, Atlanta, GA, USA). L-2 was Lebaron 2003). It is unclear whether currently used indi- maintained at 37C on Luria agar or Luria broth (LB) cator organisms are sufficient for indicating the presence containing 100 lg of ampicillin per ml. The L-2 strain of C. jejuni in environmental water samples. In some contains luxCDABE (bioluminescence genes) inserted on cases, it appears that the presence of Campylobacter a plasmid as previously described (Siragusa et al. 1999). occurs concurrently with indicator organisms (Lund This strain is derived from E. coli O157:H7 strain B6- 1996; Stanley et al. 1998). On the other hand, studies 914 (ATCC 43888) which does not produce either Shi- have shown that the densities of Campylobacter sp. do ga-like toxin I or II, and does not possess the genes for not always correlate well with those of the indicator these toxins (Siragusa et al. 1999). Stock cultures of each organisms in surface waters (Arvanitidou et al. 1995; organism in broth [trypticase soy broth (TSB) and LB, Obri-Danso and Jones 1999; Schaffter and Parriaux for C. jejuni and E. coli, respectively] with 10% glycerol 2002). The lack of significant correlation between the were kept at )80C.

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 574 No claim to original US government works K.L. Cook and C.H. Bolster Campylobacter jejuni survival in groundwater

pticase soy agar (TSA) and incubating under microaero- Water collection and nutrient analysis philic conditions at 37C for 48 h. The E. coli inoculum Water for the microcosm study was collected from four was prepared by adding one or two colonies from a fresh locations in south central Kentucky. The locations were LB-ampicillin plate into 25 ml of LB and incubating at chosen to reflect typical water quality found in karst areas 37C for 24 h. On the first day of the experiment, (e.g. high in alkalinity and calcium). Previous studies indi- C. jejuni or E. coli cells were harvested by centrifugation cated that these locations have been impacted by agricul- at 9000 g for 10 min. The cultures were washed tural activities based on elevated nutrient content (Groves twice with pH 7Æ2 phosphate buffered saline (PBS) ) ) ) et al. 2005). Samples CSC1 and CSC2 were collected from (137-mmol l 1 NaCl, 2Æ7-mmol l 1 KCl, 4Æ3-mmol l 1 )1 two different waterfalls within Cave Springs Cavern, located Na2HPO4, and 1Æ4-mmol l KH2PO4). After the final in Smiths Grove, Kentucky. The other two water samples wash, C. jejuni and E. coli inocula were prepared by resus- were collected from two underground streams, Logsdon pending pelleted cells in 25-ml PBS. Twenty millilitres of River (LR) and Hawkins River (HR), located within Mam- the C. jejuni or E. coli inocula was then added to separate moth Cave National Park. Water from all four locations 2-l flasks containing 1200 ml of filter-sterilized groundwa- was collected in sterile, acid-washed high density polyethy- ter or DI water. The flasks were mixed thoroughly to dis- lene (HDPE) bottles, transported back to the lab on ice, perse the inoculum. Twenty 50-ml plastic polypropylene and filtered through a 0Æ2-lm filter. Filter-sterilized water centrifuge tubes (microcosms) were filled completely was stored at 4C until used (less than 1 week). Filter-steril- (50 ml) with C. jejuni or E. coli inoculated water. All ized deionized (DI) water was also used as a control. microcosms were placed in the dark at 4C. Water samples were analysed for a suite of parameters (Table 1) commonly found in karst regions (e.g. Ca+2 +2 þ Microcosm sampling and Mg ) and agricultural areas (e.g. NO3 and NH4 ). The cations were analysed in triplicate using inductively HR, CSC1, CSC2 and DI microcosms were started at 2- coupled plasma optical emission spectroscopy (ICP-OES; to 4-week intervals, and were sampled for 270, 351, 470 Varian, Walnut Creek, CA, USA). Nitrate, phosphate and and 379 days, respectively. The LR study was started later, ammonia were measured in triplicate using a Lachat 500 and therefore was sampled for only 140 days. Sampling of Series ‘‘QuickChem’’ method (Hach, Loveland, CO, the HR, CSC1 and DI water microcosms was stopped USA). The total nitrogen and dissolved organic carbon (for E. coli and C. jejuni microcosms), when E. coli con- (DOC) were determined on combustion using a Liquitoc centrations went below the limits of detection (approxi- ) analyzer (Elementar, Hanau, Germany). mately 20 cells ml 1). Culturable cell numbers for E. coli ) were still above the detection limits (6Æ5 · 102 cells ml 1) when sampling from the CSC2 microcosm was stopped Inoculum preparation on day 470. Duplicate microcosms were destructively The C. jejuni inoculum was prepared by adding one or sampled on days 0, 1, 2 and every 7 days thereafter for two colonies from a fresh CSA plate into 500 ml of try- the following 2–3 months. Afterwards, duplicate micro-

) Table 1 Chemical properties (mg l 1) of groundwater at the time of sampling

HR CSC1 CSC2 LR Description Underground river (Hawkins River) Recharge water (WF1) Recharge water (WF3) Underground river (Logsdon River)

Location Mammoth Cave Cave Springs Cavern Cave Springs Cavern Mammoth Cave

DOC 4Æ0(0Æ088) 0Æ58 (0Æ11) 0Æ12 (0Æ025) 3Æ7(0Æ17) TDN 2Æ1(0Æ28) 5Æ9(0Æ34) 12Æ0(0Æ090) 2Æ3(0Æ010) NO3)-N 2Æ5(0Æ016) 4Æ7(0Æ016) 10Æ8(0Æ047) 2Æ0(0Æ025) þ NH4 -N 0Æ0179 (0Æ0031) <0Æ001 <0Æ001 0Æ0041 (0Æ0065) Al3+ 0Æ10 (0Æ0010) 0Æ0079 (0Æ00018) 0Æ0081 (0Æ00034) 0Æ18 (0Æ0062) Ca2+ 61Æ0(0Æ37) 32Æ0(0Æ16) 34Æ0(0Æ12) 48Æ0(0Æ58) Fe3+ 0Æ072 (0Æ00972) BDL 0Æ0012 (0Æ00028) 0Æ12 (0Æ0032) K+ 2Æ1(0Æ053) 1Æ1(0Æ0092) 1Æ1(0Æ0051) 2Æ6(0Æ029) Mg2+ 6Æ2(0Æ031) 6Æ2(0Æ027) 5Æ1(0Æ015) 4Æ2(0Æ067)

Values shown are the mean (standard deviation). HR, Hawkins River; CSC, Cave Springs Cavern; LR, Logsdon River; DOC, dissolved organic carbon; TDN, total dissolved nitrogen; Mammoth Cave, Mammoth Cave National Park; BDL, below detection limits.

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 No claim to original US government works 575 Campylobacter jejuni survival in groundwater K.L. Cook and C.H. Bolster cosms were destructively sampled periodically (usually Fixed AO and CTC samples were sonicated for 20 s at monthly). For microcosms sampled after 150 days, only 100 W to disrupt cell clumps, and the dilutions were fil- half of the sample in the tube was used, and the remain- tered onto 0Æ22-lm black polycarbonate filters (Poretics, der of the tube was returned to the dark and held at 4C. Livermore, CA, USA), and then viewed using epifluores- This was done in an effort to increase the number of cence microscopy. AO- and CTC-stained cells were visu- possible samplings when survival appeared to be extend- alized using an Olympus BX-41 microscope (Melville, ing beyond 150 days. NY, USA) equipped with a 100-W mercury lamp, a 460- to 490-nm excitation filter, and a 590-nm cut-off filter. The length and width measurements for individual cells Respiring, total and culturable cell counts were obtained using ProImage (Silver Spring, MD, USA) Samples containing C. jejuni were serially diluted and pla- software. ted onto TSA with 5% sheep’s blood (TSAB), and sam- The survival of both organisms was quantified in two ples containing E. coli were serially diluted and plated ways: calculation of a decay rate and the time required to onto LB agar for the estimation of culturable cell density. obtain a 99% reduction, t99, in culturable cells. The decay The TSAB plates were incubated at 37C for 48 h under rates were calculated from the culturable plate counts microaerophilic conditions for C. jejuni enumeration. The once a log-linear decrease was observed and statistically LB plates were incubated at 37C for 24 h for E. coli enu- compared using the MIXED procedure in SAS 9Æ1 (SAS meration. Once the culturable cell numbers dropped Institute, Cary, NC, USA). The parameter t99 was deter- ) below 100 colonies ml 1,0Æ1 and 0Æ5 ml of the micro- mined by observation rather than calculation from the cosm water was plated directly (i.e., no dilution) in all decay rate, as is often done. As a result, t99 values are subsequent samplings. When the cell numbers decreased reported as a range rather than a single value, i.e. the ) below detectable limits (approximately 20 cells ml 1) for range represents the sampling time before and after a C. jejuni samples, 1 or 5 ml of water sample was enriched 99% reduction in the culturable cells was observed. in 5 ml of nutrient broth No. 2 (Remel, Lenexa, KS, USA) with campylobacter growth supplement (Oxoid, Le- Quantitative, real-time PCR nexa, KS, USA) and 5% horse blood by incubating for 5 days at 37C under microaerophilic conditions. After Quantitative, real-time PCR (QRT-PCR) assays were run 5 days, a swab of the enrichment culture was streaked on the DNA Engine Opticon 2 (MJ Research, Inc., Walt- onto the TSAB plates. After incubating under microaero- ham, MA, USA). The primers were obtained from Sigma philic conditions for 48 h at 37C, the plates were Genosys (St. Louis, MO, USA), and the dual-labeled screened for the presence or absence of C. jejuni colonies. Black Hole Quencher probes were prepared by Biosearch The culturable plate counts were discontinued when col- Technologies, Inc. (Novato, CA, USA). The assays were ony forming units dropped below the limits of detection carried out in Qiagen HotStart Taq Master Mix (Qiagen, and resuscitation was unsuccessful. Valencia, CA, USA) in a total volume of 25 ll. (i) For The total cell concentrations in water samples were C. jejuni QRT-PCR quantification, a modification of the determined by staining the appropriate dilutions with a real-time PCR method, developed by Nogva et al. (2000), 0Æ1% solution of acridine orange (AO; Sigma, St. Louis, was used. The reaction targeted an 86-bp fragment of the MO, USA) and filtering onto 0Æ22-lm black polycar- published C. jejuni strain NCTC 11168 genome sequence bonate filters (Poretics, Livermore, CA, USA), using a (Nogva et al. 2000). The primers targeted positions modification of the AO direct count method (Hobbie 381121 to 381145 (forward) and 381026 to 381185 et al. 1977). For LR samples, respiring cell numbers (reverse). The probe targeted positions 381147 to 381181 were determined by using a modification of the 5-cy- of the C. jejuni NCTC11168 genome sequence. The ) ano-2,3,-ditolyl tetrazolium chloride (CTC) method, as amplification mixture contained 3Æ5-mmol l 1 MgCl2, ) ) described by Rodriguez et al. (1992). Samples from each 300-nmol l 1 each primer, 200 nmol l 1 of probe and microcosm (8 ml) were treated with 1% TSB supple- 35 ± 25 ng of sample DNA or dilutions of plasmid PCR ) ment and 2Æ5-mmol l 1 CTC (Polysciences, Inc., War- 2Æ1 vector (Invitrogen, Carlsbad, CA, USA) carrying the rington, PA, USA). The control samples were treated 86-bp insert sequence as standard (from 102 to 108 cop- with 2 ml of filtered formalin for 5 min before CTC ies). The PCR program was 15 min at 95C, 39 cycles at was added. Escherichia coli samples were incubated at 95C for 20 s and 61C for 20 s. (ii) For E. coli QRT- 37C on a shaker for 4 h, while C. jejuni samples were PCR quantification, a modification of the method of incubated at 37C under microaerophilic conditions for Frahm and Obst (2003) was used to target the uidA gene 4 h. The CTC reaction was stopped using 1 ml of fil- of E. coli. The reaction targeted an 82-bp fragment of the tered formalin. of the E. coli uidA gene sequence. The amplification

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 576 No claim to original US government works K.L. Cook and C.H. Bolster Campylobacter jejuni survival in groundwater

)1 )1 mixture contained 3Æ5-mmol l MgCl2, 600-nmol l (a) 8 ) 10 each primer, 200 nmol l 1 of probe and 19 ± 21 ng of 107

sample DNA or dilutions of plasmid pCR 2Æ1 vector (In- ) vitrogen, Carlsbad, CA, USA) carrying the 82-bp insert –1 106 2 8 sequence as standard (from 10 to 10 copies). The cycle 105 conditions were as follows: 15 min at 95C, 39 cycles at 4 10 95 C for 15 s and 60 C for 60 s, and 72 C for 45 s. For (log CFU ml both reactions, the baseline values were set as the lowest 103 fluorescence signal measured in the well over all cycles. 102 The baseline was subtracted from all values and the C. jejuni 20 cells = limit of detection 1 threshold was set to one time the standard deviation. 10 Assays were performed using duplicate samples, and all 100 PCR runs included standards and control reactions with- 020406080100 out template. Time (days) Annealing temperature, primer and probe concentra- No C. jejuni viable after 84 days tions were optimized for each of the QRT-PCR assays. (b) 108 The amplification efficiencies and optimum template concentrations were determined for the assays using total 107

DNA extracted from microcosm samples containing ) 6

–1 10 either E. coli or C. jejuni. There was a strong linear rela- 105 tionship between the logarithm of the template concentration and threshold cycle values (r2 >0Æ99), suggesting 104 that there was no PCR inhibition when between 1Æ00 and (log CFU ml 103 100 ng of template DNA was added per 25-ll reaction 102 mixture. The amplification efficiencies for the samples E. coli 20 cells = limit of detection were 1Æ00 and 0Æ89 for the E. coli and the C. jejuni assays, 101 respectively. The amplification efficiencies for the stand- 100 ards were 0Æ95 and 0Æ91 for the E. coli and the C. jejuni 0 50 100 150 200 250 300 350 400 450 500 assays, respectively. Time (days)

Figure 1 Survival of Campylobacter jejuni (a) or Escherichia coli (b) in Results groundwater or deionized (DI) water. (d) Hawkins River (HR); ( ) Cave Springs Cavern (CSC)1; (r) CSC2; ( ) Logsdon River (LR) and Culturability of Campylobacter jejuni and Escherichia coli ( ) DI. Values are the mean ± standard deviation of four replicate in filter-sterilized groundwater samples. The culturability of E. coli greatly exceeded that of C. jejuni in the filter-sterilized groundwater microcosms to 13Æ5 times greater than for E. coli. The time required incubated in the dark at 4C. The culturability of for the population to exhibit exponential die-off (i.e. the ) C. jejuni decreased below detection limits (20 cells ml 1) lag time) for C. jejuni was 0, 14, 21, 2 and 0 days for HR, within 85 days, regardless of the source or nutrient com- CSC1, CSC2, LR and DI microcosms, respectively. The position of the water (Fig. 1a). Escherichia coli incubated lag times for E. coli were 63, 35, 127 and 50 days for HR, under the same conditions was culturable for up to CSC1, CSC2 and DI microcosms, respectively. 470 days (Fig. 1b). No C. jejuni could be resuscitated Owing to the differences in the lag times, the die-off after the cell numbers decreased below the limits of detec- rates did not necessarily correspond to the overall survival tion. during the course of the experiment. For example, while To compare the survival of C. jejuni and E. coli in dif- the die-off rate for C. jejuni was greatest in CSC2, the ferent microcosms, the die-off rate, k, was calculated from time in which the cells remained culturable was the short- the exponential portion of the die-off curve (Table 2). est in DI (Table 2; Fig. 1) owing to the greater lag time The best-fit lines applied to the log-linear portion of the for CSC2. In most cases, however, survival and die-off die-off curve resulted in r2 values that were 0Æ90 or rates were correlated. For C. jejuni, the lowest die-off rate greater. For each microcosm, the die-off rate for C. jejuni occurred in HR, which also had the longest time of cultu- was significantly higher (P <0Æ01) than for E. coli rability. For E. coli, the greatest die-off rate and the lowest (Table 2); the die-off rates for C. jejuni ranged from 2Æ5 overall survival occurred in the HR microcosm.

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 No claim to original US government works 577 Campylobacter jejuni survival in groundwater K.L. Cook and C.H. Bolster

Table 2 Decay constants for decrease in )1 Decay constant (k day )* Campylobacter jejuni and Escherichia coli HR CSC1 CSC2 LR DI culturable cell populations

C. jejuni )0Æ25 ± 0Æ022a ) 0Æ34 ± 0Æ027b ) 0Æ54 ± 0Æ044c ) 0Æ35 ± 0Æ027b ) 0Æ42 ± 0Æ030b E. coli ) 0Æ10 ± 0Æ014d ) 0Æ05 ± 0Æ005e ) 0Æ04 ± 0Æ009e N/A ) 0Æ04 ± 0Æ003e

*Decay constant calculated by applying the best-ﬁt slope to plot with x ¼ time in days and y ¼ natural log of C/Co ± standard error. N/A, no decay in the population had occurred as of day 140 (last day of sampling). Similar letters denote statistically similar k values. HR, Hawkins River groundwater; CSC1, recharge water 1; CSC2, recharge water 2; LR, underground river; DI, deionized water.

The survival times were also expressed as t values 99 Cellular integrity evaluation by QRT-PCR and total cell (the time taken for a two log decrease in culturable-cell counts concentration from the original inoculum concentration) (Table 3). The t99 values for C. jejuni were less The QRT-PCR analysis and direct cell counts (AO analy- than 35 days for all microcosms, while those for E. coli sis) showed that cellular and DNA integrity was main- were greater than 91 days (Table 3). The t99 value for tained by both E. coli and C. jejuni for the duration of C. jejuni in the DI water microcosms was lower the experiments (Fig. 2). The total cell numbers, as deter- (between 7 and 14 days) than in any other water mined by direct cell counting, did not change concur- sources (Table 3). The values for E. coli were lowest in rently with the decrease in culturable counts (Fig. 2a,b). HR (between 91 and 119 days), corresponding to the In fact, the total cell numbers remained relatively con- greatest die-off rates. stant in all microcosms for both E. coli and C. jejuni. The The effect of water source on survival was noticeably starting cell concentrations were slightly higher and more ) different between the two micro-organisms. For exam- variable for E. coli (4Æ4±7Æ1 · 107 cells ml 1) than for ) ple, the die-off rate was the lowest (P <0Æ05) and the C. jejuni (2Æ6±1Æ3 · 107 cells ml 1). The average total survival of C. jejuni the greatest (84 days) in the HR cell counts were approximately the same for C. jejuni ) ) microcosm, which had the highest concentrations of (2Æ1 · 107 cells ml 1) and E. coli (1Æ2 · 107 cells ml 1).

DOC and NH4, yet the die-off rate for E. coli in HR Based on the QRT-PCR data showing high cell num- ) was significantly higher than in any other microcosms bers (between 1Æ3 · 107 and 4Æ8 · 107 copies ml 1) until (P <0Æ01) (Table 1; Fig. 1). In contrast, in microcosms the end of the studies, C. jejuni cellular integrity was containing DI water, the survival of C. jejuni was the maintained for the duration of the studies (Fig. 2c). The shortest (<35 days), whereas the survival of E. coli was cellular and DNA integrity was maintained despite the 379 days (the second longest survival time observed in loss of culturability. In fact, no significant change in cell our study). The die-off rates for E. coli in CSC1 and numbers, as determined by QRT-PCR, was observed CSC2 were similar to those of DI water. CSC1 and (Fig. 2c). Similarly, E. coli uidA gene copy numbers CSC2 samples had high total dissolved nitrogen remained high (between 0Æ3 · 107 and 1Æ0 · 107 cop- ) and nitrate concentrations, but low DOC. The use of ies ml 1) until the final days of sampling (Fig. 2d). PBS as the final resuspension solution increased (greater than tenfold) the concentration of sodium and Evaluation of cellular morphology phosphate in solution. Therefore, these compounds were omitted from the nutrient analysis shown in During the first days of incubation in water microcosms, Table 1. C. jejuni cells were predominantly spiral shaped (>97%)

Table 3 Time required for 99% (t99) t99 decrease in culturability (days) HR CSC1 CSC2 LR DI

Campylobacter 15 > t <21 28>t <35 21>t <28 14>t <21 7>t <14 jejuni Escherichia coli 91 > t < 119 157 > t < 220 127 > t < 186 t > 140 147 > t < 186

HR, Hawkins River groundwater; CSC1, recharge water 1; CSC2, recharge water 2; LR, underground river; DI, deionized water.

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 578 No claim to original US government works K.L. Cook and C.H. Bolster Campylobacter jejuni survival in groundwater

(a) 109 (b) 109

108 108 E. coli

C. jejuni

7 –1 7 –1 10 10

106 106

105 105 Log total cell ml Log total cell ml

104 104 0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400 Time (days) Time (days)

Figure 2 Total cell [acridine orange (AO)] 108 108 E. coli numbers per ml for Campylobacter jejuni (a) C. jejuni

–1 or Escherichia coli (b) and gene copy [quanti- –1 7 7 tative, real-time PCR (QRT-PCR)] numbers per 10 10 ml for C. jejuni (c) or E. coli (d) in groundwater or deionized water (DI). (d) Hawkins River 106 106 (HR); ( ) Cave Springs Cavern (CSC)1; (r) CSC2; ( ) Logsdon River (LR) and ( ) DI. 105 105

Total cell numbers represent the mean of Log copy number ml Log copy number ml duplicate samples. Gene copy numbers repre- 4 4 10 0 50 100 150 200 250 300 350 400 10 0 50 100 150 200 250 300 350 400 sent the mean± standard deviation of four Time (days) replicate samples. Time (days) with average cell length of 2Æ37 ± 0Æ20 lm and width of in CSC2 microcosms, the spiral cells were only 2Æ5% of 0Æ90 ± 0Æ12 lm. When C. jejuni became nonculturable, the total C. jejuni population. However, after 375 days’ coccoid forms of the cell were predominant (average cell incubation in the DI water microcosms, the spiral forms length of 1Æ07 ± 0Æ09 lm and width of 0Æ87 ± 0Æ09 lm); were still 34% of the total C. jejuni population, despite however, a significant percentage of the population was the fact that the population had lost culturability more still in the spiral form (Table 4). In fact, the spiral form than 300 days prior to the final sampling. persisted until the final days of sampling (27%, 15% and 56% of the total C. jejuni population after 270, 126 and Comparison of culturability and respiration 140 days’ incubation at 4C in HR, CSC1 and LR microcosms, respectively). After more than 320 days incubation For LR samples, the viability was determined by direct cell counts and by measuring the respiratory activity Table 4 Percentage of spiral and coccoid forms of Campylobacter using the redox dye CTC (Fig. 3). When C. jejuni cultur- jejuni present when cells became nonculturable able cell counts in the water microcosms declined below the detection limits (day 42), more than Form (percentage) ) 2Æ0 · 105 cells ml 1 CTC-positive cells were still detected Sample Time (days)* Spiral Coccoid as respiring (Fig. 3a). In fact, the respiratory activity, as determined by CTC analysis, was maintained for more HR 84 32 68 CSC1 56 37 63 than 100 days in microcosms inoculated with C. jejuni. CSC2 49 40 60 For E. coli, cellular respiration mirrored total and cultura- LR 49 67 33 ble cell counts until the experiment was terminated after DI 35 76 24 140 days (Fig. 3b).

*First day of experiment in which no culturable C. jejuni were obtained. Discussion Percentage of total cells present in either the spiral or coccoid form. HR, Hawkins River groundwater; CSC1, recharge water 1; CSC2, Our results show that the survival of C. jejuni and E. coli recharge water 2; LR, underground river; DI, deionized water. in ﬁlter-sterilized water varies considerably depending on

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 No claim to original US government works 579 Campylobacter jejuni survival in groundwater K.L. Cook and C.H. Bolster

(a) 109 rather, survival is likely attributed to the synergistic effects of many compounds in the water (Bolster et al. 2005). 108 Our results support the conclusion of Thomas et al. ) 7 –1 10 (1999) that variability in C. jejuni survival is related to 106 differences in the chemical properties of the water 105 sources, and conﬁrm that the groundwater chemical com- 4 position is an important factor affecting the survival of (log cells ml 10 C. jejuni. 3 10 The variability in survival was also observed for E. coli. 2

C. jejuni 10 Based on previous studies, we expected that survival 101 would be directly related to DOC and/or nitrogen content of the groundwater (Bolster et al. 2005). The survival was 100 0 20406080100 greatest in the microcosm with the highest total dissolved nitrogen (TDN) but low DOC (CSC2), and the survival (b) 9 10 time was the least for the microcosm with the highest 108 DOC and lowest TDN (HR). This is somewhat surprising 7 ) 10 given that the presence of carbon sources has been shown –1 106 to increase coliform survival in other studies (LeCheval- lier et al. 1991; Boualam et al. 2001). However, Lim and 5 10 Flint (1989) found that nitrogen supplementation affected 104

(log cells ml E. coli survival more than phosphate or carbon supple- 103 mentation in lake water. It is unclear, however, why E. coli would survive as well in nutrient free water (DI) as in E. coli 102 water containing carbon and nitrogen (CSC1 and CSC2). 1 10 The use of PBS in the final resuspension, prior to the 100 addition to the water sources, contributed significant 0 20406080100 amounts of phosphate and sodium; It could only be spe- Time (days) culated that this might have influenced the survival of E. Figure 3 Survival of Campylobacter jejuni (a) or Escherichia coli (b) in coli in the DI microcosm. However, these nutrients were Logsdon River (LR) groundwater. (d) Culturable cells; ( ) respiring added equally to all microcosms, including the C. jejuni cells (CFU) and ( ) total cells [acridine orange (AO)]. Culturable cells microcosms. This phosphate and sodium addition did represent the mean ± standard deviation of four replicate samples. not increase the survival of C. jejuni in the DI solutions. Total and respiring cell values are the mean of duplicate samples. Furthermore, the shorter survival time for E. coli in HR and CSC1 microcosms than in the DI treatment is consis- the composition of the water. For instance, C. jejuni sur- tent with the results of Klein and Alexander (1986), who vived the longest in the microcosm with the highest DOC observed greater survival of Klebsiella pneumonia and (HR) and the shortest in the microcosm with the lowest Micrococcus flavus in distilled water as compared with fil- DOC (CSC2). This trend, however, was not consistent. ter-sterilized lake water. Their conclusion was that inhibi- Survival was significantly lower in the LR microcosm tory compounds in lake water caused the rapid decline in which had a DOC similar to that of HR. This kind of cell numbers compared with distilled water. It is possible variability in the survival of C. jejuni is not uncommon, that inhibitory compounds existed in our microcosms and has been associated with differences between C. jejuni that were not measured during our study. strains, initial culture conditions, growth temperature, pH Differences in the response of E. coli and C. jejuni to and oxygenation (Buswell et al. 1998; Federighi et al. the chemical composition of different groundwater 1998; Lazaro et al. 1999; Cools et al. 2003). In this study, sources brings into question the adequacy of using E. coli a single strain of C. jejuni was used, and inoculum pre- as an indicator of C. jejuni. Indicator organisms should paration (wash and resuspension in PBS) and incubation have survival characteristics similar to those of the patho- (in the dark at 4C) conditions were all held constant. gens of interest (Scott et al. 2002). Concerns arise when Therefore, the differences in C. jejuni survival in water the behaviour of the indicator organism in the environ- microcosms should be related to the water composition. ment is dramatically different from that of the pathogen The lack of a clear trend in the survival, based on chem- of concern, as we observed in our study and has been ical composition, suggests that a single compound was observed by others (Korhonen and Martikainen 1991; not responsible for the survival of the organisms, but Terzieva and McFeters 1991; Obri-Danso and Jones

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 580 No claim to original US government works K.L. Cook and C.H. Bolster Campylobacter jejuni survival in groundwater

1999). In this case, DOC had the opposite effect on the of Campylobacter contamination of human and livestock survival of the two organisms. It was also found that water supplies (Pearson et al. 1993; Jones 2001; Hanninen C. jejuni die-off in groundwater microcosms was between et al. 2003), little is known about the survival of this 2Æ5 and 13 times faster than that of E. coli. In all micro- important pathogen in groundwater environments. Admit- cosms, C. jejuni was culturable for less than 85 days, tedly, the aseptic conditions used in this study are not while E. coli was culturable for more than 200 days. The truly representative of natural groundwater environments; differences in the duration of survival and response to the nonetheless, using this experimental design, we were able water nutrient composition between the two organisms to determine that C. jejuni does not survive nearly as long suggest that E. coli may be present in the waters much as E. coli, and that the survival of the two micro-organisms longer and respond to water composition much differ- varied in a markedly different manner to the nutrient ently than C. jejuni. composition of the groundwater source. These results Campylobacter jejuni cell morphology was also observed should provide useful information on the survival and to vary between the microcosms. In the CSC2 micro- behaviour of C. jejuni in groundwater environments. cosms, 2Æ5% of the cells were in the spiral form after more than 320 days’ incubation at 4C. In contrast, more Acknowledgements than 30% of cells were still in the spiral form after a similar amount of time in the DI microcosm. We also found The authors wish to thank John Sorrell, Tinesha Mack that the loss of culturability of C. jejuni occurred inde- and Natalie Bills for valuable technical assistance. Thanks pendent of the formation of coccoid cells. In fact, when also to Jason Simmons and Stacy Antle for helping with culturability was lost, between 32% and 76% of cells were sample collection and analysis. We are grateful to Greg still in the spiral form. These data are in agreement with Siragusa (USDA-ARS, Atlanta, GA) for supplying the that from other studies which suggest that coccoid cells E. coli O157:H7 strain. may represent the degenerative form of the organism rather than the nonculturable form of C. jejuni (Federighi References et al. 1998; Hazeleger et al. 1998; Lazaro et al. 1999; Tho- mas et al. 2002). These results show that the process of Arvanitidou, M., Stathopoulos, G.A., Constantinidis, T.C. and coccoid cell formation is independent of culturability, Katsouyannopoulos, V. (1995) The occurrence of Salmon- and may instead reﬂect a response to nutrient conditions ella, Campylobacter and Yersinia spp. in river and lake present in the environment. waters. Microbiol Res 150, 153–158. The loss of culturability in C. jejuni did not correlate Baffone, W., Casaroli, A., Citterio, B., Pierfelici, L., Campana, with the loss in respiratory activity or cellular integrity. R., Vittoria, E., Guaglianone, E. and Donelli, G. (2006) Campyobacter jejuni cells remained viable (as measured by Campylobacter jejuni loss of culturability in aqueous CTC analysis) for more than 50 days after culturability microcosms and ability to resuscitate in a mouse model. was lost. Likewise, C. jejuni cellular and genomic integrity Int J Food Microbiol 107, 83–91. (as measured by DNA extraction and QRT-PCR analysis) Baggi, F., Demarta, A. and Peduzzi, R. (2001) Persistence of viral pathogens and bacteriophages during sewage treat- was maintained over the course of more than 300 days’ ment: lack of correlation with indicator bacteria. Res incubation at 4C. These data are in agreement with those Microbiol 152, 743–751. from other studies which have shown that the respiratory Baylis, C.L., MacPhee, S., Martin, K.W., Humphrey, T.J. and activity and many other cellular processes (e.g. protein Betts, R.P. (2000) Comparison of three enrichment media synthesis, ATP formation) are maintained long after the for the isolation of Campylobacter spp. from foods. J Appl culturability is lost (Rollins and Colwell 1986; Cappelier Microbiol 89, 884–891. et al. 1997; Buswell et al. 1998; Federighi et al. 1998; Bobb, D.J., Sauders, B.D., Waring, A.L., Ackelsberg, J., Dumas, Hazeleger et al. 1998; Lazaro et al. 1999). The long-term N., Braun-Howland, E., Dziewulski, D., Wallace, B.J., et al. survival of C. jejuni at cold temperatures occurs despite (2003) Detection, isolation and molecular subtyping of the fact that the genome of the organism lacks many of Escherichia coli O157:H7 and Campylobacter jejuni associ- the genes that are used for adaptation to environmental ated with a large waterborne outbreak. J Clin Microbiol 41, stress and are important to the success of many other 174–180. common foodborne pathogens (Park 2002; Murphy et al. Bolster, C.H., Bromley, J.M. and Jones, S.H. (2005) Recovery 2006). These ﬁndings illustrate the importance of con- of chlorine-exposed Escherichia coli in estuarine micro- tinuing research to better understand the survival mecha- cosms. Environ Sci Technol 39, 3083–3089. nisms of this perplexing organism. Bolster, C.H., Walker, S.L. and Cook, K.L. (2006) Comparison Although C. jejuni has been isolated from groundwater of Escherichia coli and Campylobacter jejuni transport in (Stanley et al. 1998), and water sources are a known route saturated porous media. J Environ Qual 35, 1018–1025.

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 573–583 No claim to original US government works 581 Campylobacter jejuni survival in groundwater K.L. Cook and C.H. Bolster

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