Campylobacters and bacteriophages in the surface waters of Canterbury (New Zealand)

Authors


J. Andrew Hudson, Food Safety Programme, Institute of ESR Ltd, Christchurch Science Centre, PO Box 29-181, Ilam, Christchurch, New Zealand. E-mail; andrew.hudson@esr.cri.nz

Abstract

Aim:  To determine the relationship between the presence of thermotolerant campylobacters and their bacteriophages (phages) in surface waters for the potential to use phages as an indicator of Campylobacter spp.

Methods and Results:  Thermotolerant campylobacters were enumerated in 53 water samples using a three tube most probable number (MPN) series in m-Exeter broth. The presence of phages in the same samples was tested using two approaches: qualitative enrichment with five different Campylobacter hosts and a quantitative membrane concentration method. Phages infecting an Escherichia coli O157:H7 isolate were also enumerated by the membrane concentration method. Campylobacter spp. were isolated from 45/53 (85%) of the samples at 0.4–110 MPN 100 ml−1. No Campylobacter phages were isolated, but coliphages were present in 43/46 (93%) of samples.

Conclusions:  The membrane concentration method recovered >80% of Campylobacter phages from spiked samples. The absence of Campylobacter phages in environmental samples, from both enrichment and concentration methods, suggests that, if present, they are at very low titres.

Significance and Impact of the Study:  Testing for Campylobacter phages as an indicator of Campylobacter spp. presence is not effective. The quantitative data for Campylobacter spp. will be useful for risk assessment purposes.

Introduction

Campylobacter is the most frequently reported cause of acute bacterial gastroenteritis in New Zealand. Notified illness rates in New Zealand are high by world standards, with the incidence for 2007 at 302.2 /100 000 (ESR 2008). The species of Campylobacter most commonly associated with human infections are Campylobacter jejuni and Campylobacter coli (Percival et al. 2004). The consumption of untreated or inadvertently contaminated drinking water is a common mode for large outbreaks of campylobacteriosis (Hänninen et al. 2003). Animal faeces are widely believed to be the primary source of water contamination as campylobacters are often isolated from the intestinal tracts of birds and farmed animals (Devane et al. 2005) and are not thought to replicate in the environment (Kemp et al. 2005). Contamination of open surface waters with animal faeces can be by either direct deposition or indirectly via run-off (Stanley and Jones 2003). Many of the surface water sources throughout Canterbury, New Zealand, are habitats for considerable populations of waterfowl which would contribute towards bacterial contamination (Abulreesh et al. 2004) and could potentially provide a source of exposure for human pathogens. A report for New Zealand estimated that approximately 4% of the cases of campylobacteriosis may be contracted through recreational use of water (McBride et al. 2002).

It has been proposed that bacteriophages (phages) could be used as indicators of the potential contamination in water and food by pathogenic bacteria and enteric viruses (Chung et al. 1998; Contreras-Coll et al. 2002; Hot et al. 2003; Moce-Llivina et al. 2005) as phages are likely to be in the environment in which their host bacterium grows. Phage assays are simpler, quicker and cheaper than many of the enteric virus and bacterial pathogen detection methods currently employed in water testing. Many techniques have been developed for the recovery of phages, mainly coliphages, from aquatic environments for potential use as indicators of the presence of faecal contamination; for example Sinton et al. (1996). Phage numbers in aquatic environments can be diluted by water volume to very low numbers requiring a concentration step for detection.

Campylobacter phages have been isolated from sources such as broiler chickens, abattoir effluent and sewage (Salama et al. 1989; Atterbury et al. 2004) but, to our knowledge, not from water. In this study, absorption to a positively charged membrane followed by elution and an enrichment method was used for the detection of Campylobacter phages. The aim was to determine the relationship between the presence and numbers of thermotolerant campylobacters and their phages in surface water sources for possible use of phages as an indicator of Campylobacter contamination.

Materials and methods

The 53 different water sources sampled in this survey were distributed throughout the Canterbury region of the South Island of New Zealand from December 2007 through to February 2008. Three litre samples from each water source were collected directly into sterile glass bottles, maintained at 4°C in darkness and transported to the laboratory prior to analysis within 2–16 h of collection.

Isolates used and culture conditions

The recovery efficiency of the membrane concentration method was assessed using Campylobacter phage NCTC 12678 enumerated on host C. jejuni NCTC 12662. Host C. jejuni NCTC 12662 was chosen for the phage concentration method as it is known to be sensitive to a broad range of phages (Frost et al. 1999). In enrichments for the detection of Campylobacter phages the hosts used were; C. jejuni isolates NCTC 12660, NCTC 12662, NCTC 12664, NCTC 11351 and Ccoli ATCC 33559. Campylobacter jejuni isolates NCTC 12660, NCTC 12662 and NCTC 12664 are propagating hosts for different phages (Frost et al. 1999). Isolates NCTC 11351 (Biotype, Penner type 23) and Ccoli ATCC 33559 were used as they are the type strain for those two species. Testing for phages infecting Escherichia coli O157:H7 was undertaken using the nontoxigenic culture NZRM 3614.

To prepare inocula for use in Campylobacter overlays, five Columbia Blood Agar (Oxoid) plates containing 5% (v/v) defibrinated sheep blood were streaked and incubated at 42°C under 10% CO2 (Fraser et al. 1992) for 24 h before being harvested and resuspended in Nutrient Broth No. 2 (Oxoid). This suspension was used as the host inoculum for both the phage detection overlays and to inoculate the qualitative phage enrichment water samples. Escherichia coli O157:H7 was grown in Bacto™ Trypticase Soy Broth (Becton Dickinson).

Quantitative analysis of Campylobacter phages

The concentration method used was based on phage adsorption to positively charged membrane filters followed by elution as previously described (Sinton et al. 1996; Mendez et al. 2004). Recovery efficiency was tested by inoculating sterilized surface water samples with suspensions of Campylobacter phages followed by their enumeration before and after the concentration method using five replicates each time. Phage enumeration was achieved by determining the number of plaque forming units (PFU) using the double layer agar technique (Carlson 2005). Spiked samples were also tested periodically throughout the study.

Conditions of the assay were varied so that the recovery of Campylobacter phage was optimized. The final optimized method used was as described here. Two 1 L samples from each water source were filtered through a No.4 filter paper (Whatman) to remove particulates and MgCl.6H2O was added to a final concentration of 0.5 mol l−1 and mixed. The samples were then filtered through an acetate–nitrate cellulose ester membrane filter (0.22-μm pore size, 47-mm diameter, Millipore). The membrane was placed into a glass Petri dish containing 2.5 ml of eluting solution (1% w/v beef extract, 1.0 mol l−1 NaCl and 3% v/v Tween 80, pH 9.0) and gently swirled on an orbital shaker at 60 rpm for 30 min.

The eluent was then added to molten tubes of Nutrient Broth No. 2 (Oxoid) soft agar overlay (final agar concentration 0.4%) pre-equilibrated to 45°C and containing 0.001 mol l−1 CaCl2 and 1 ml of host C. jejuni NCTC 12662. The tubes were held molten at 45°C for 20 min prior to pouring onto a prepoured base plate of Nutrient Broth No. 2 (Oxoid) solidified with 1.5% (w/v) agar prior to microaerophilic incubation, one plate at 37°C and the other at 42°C. Plates were checked daily for plaques and host inhibition, up to 5 days.

To verify the filter concentration method, two 0.1 ml volumes of eluent were also enumerated in LB agar (Invitrogen) soft agar overlays containing 0.1 ml of E. coli O157:H7 and one plate each was incubated at 22 and 37°C for 24 h.

Enrichment of Campylobacter phages

A 225 ml water sample was added to a sterile glass bottle containing 25 ml of 10× concentration Nutrient Broth No. 2 (Oxoid) and inoculated with 1 ml each of five Campylobacter hosts (each in the order of 107 CFU ml−1). The sample was then incubated under 10% CO2 at 37°C for 48 h, after which a 40 ml subsample of the enrichment was centrifuged at 3000 g for 10 min and the supernatant filtered to remove bacterial cells. The filtrates were then tested for the presence of Campylobacter phages by adding 1 ml volumes to duplicate tubes of soft agar containing each of the five hosts before overlaying preformed base layers. The plates were then incubated under 10% CO2 at 37 and 42°C. Plates were checked daily for plaque formation and host inhibition, up to 5 days.

Enumeration of Campylobacter

The three-tube most probable number (MPN) membrane filtration technique was employed for the detection of campylobacters in water samples, following the standard methods for the examination of water as used by the Public Health Laboratory Service, London (American Public Health Association 1998). The water samples (100, 10 and 1 ml volumes) were filtered through 0.45 μm, 47-mm membrane filters (Millipore). The filters were then individually transferred into 25 ml m-Exeter enrichment broth (Wong et al. 2004). Modified Exeter (m-Exeter) enrichment broth contained 25 g Nutrient Broth No. 2 (Oxoid) dissolved in 950 ml of distilled water. After autoclaving, the following supplements were added: 50 ml lysed horse blood, 5 ml filter-sterilized Exeter supplement containing 4% (w/v) sodium metabisulphite, 4% (w/v) sodium pyruvate and 4% (w/v) FeSO4·7H20 (stored frozen); 15 mg cefaperazone and two vials of Oxoid antibiotic supplements SR204E (containing 2500 IU polymixin B, 5 mg rifampicin, 5 mg trimethoprim and 5 mg amphotericin B). The enrichment broths were incubated in a 10% CO2 atmosphere at 37°C for 24 h and then transferred to 42°C for a further 24 h. A loopful each of the enrichment broths in the MPN series was streaked onto an m-Exeter agar plate (m-Exeter broth plus 1.5% w/v agar) and further incubated at 42°C for 48 h. Presumptive isolates were confirmed by cell morphology, Gram stain, presence of oxidase and catalase and by PCR performed by the method of Wong et al. (2004).

Results

Validation of the phage membrane filter concentration method efficiency

The recovery efficiency of the concentration method ranged from 80% to 104% with a mean of 88% (Table 1) when the optimal conditions described above were used. This is equivalent to the recovery of coliphages obtained by Sinton et al. 1996. Small numbers of phages were used in these recovery experiments as it was felt that river water would likely contain only small numbers of Campylobacter phages as, although river water is frequently contaminated by Campylobacter spp., it is usually at low numbers (Savill et al. 2001).

Table 1.   Recovery efficiency of a Campylobacter phage using the membrane filtration method
Inoculum
PFU 100 ml−1
Recovery
PFU 100 ml−1
Recovery %Standard deviation %
1.3 × 1021.1 × 10286.311.5
1.3 × 1021.1 × 10280.313.2
1.4 × 1021.2 × 10285.324.7
1.3 × 1021.2 × 10289.214.4
1.4 × 1021.2 × 10289.819.0
1.3 × 1021.0 × 10281.617.5
1.3 × 1021.3 × 10298.315.3
1.7 × 1011.3 × 10180.03.1
1.2 × 1011.1 × 10190.03.6
1.8 × 1011.5 × 10183.33.5
1.1 × 1011.1 × 101103.61.7
1.6 × 1011.3 × 10183.82.6
 Mean87.6 

Isolation of Campylobacter phages and coliphages infecting an isolate of Escherichia coli O157:H7

No Campylobacter spp. phages were isolated by either the filter concentration method or by enrichment. In contrast, coliphages were common in the samples tested and were present at concentrations up to 98 PFU 100 ml−1 (Table 2). The isolation of phages infecting a single isolate of E. coli O157:H7 does not indicate that these phages were specific for E. coli O157:H7 as presumably, phages infecting generic E. coli may also infect this isolate. There was no correlation between Campylobacter spp. MPN values and coliphage counts as quantified with the E. coli O157:H7 isolate (r = −0.007). Coliphage counts were therefore of no value in indicating the presence of Campylobacter spp. Counts of coliphages made at two different temperatures (Table 2) were, however, positively correlated (r = 0.660) with each other. The temperature of incubation did not therefore seem to influence the plating efficiency.

Table 2.   Water source locations, MPN result, campylobacters isolated and coliphage counts
NameMPN (100 ml−1)Campylobacter PCR identificationColiphages* at 22°C (100 ml−1)Coliphages* at 37°C (100 ml−1)
  1. *Phages infecting Escherichia coli O157:H7 NZRM 3614.

  2. ND, not done; NI, not isolated; MPN, most probable number.

Albert lake2.3Campylobacter jejuniNINI
Albert stream0.4C. jejuni32
Ashburton river0.4Campylobacter coli5153
Ashley river<0.3NI2718
Avon river 1-city15C. jejuni, thermotolerant Campylobacter5523
Avon river 2-mouth7.5C. jejuni945
Avon river 3-headwater4.3C. jejuniNDND
Bullock creek0.9C. jejuniNINI
Cam river0.9C. jejuni4925
Cashmere stream46C. jejuni4876
Courtenay stream21C. jejuni7598
Dudley creek4.3C. jejuni2841
East stream0.4C. jejuni2954
Eyre river0.9C. jejuni311
Garry river<0.3NI20NI
Glentui river0.9C. jejuni8963
Groynes0.4C. jejuni7984
Halswell river0.4C. jejuni (weak)3567
Hawkins river0.4C. coli1651
Heathcote river 1-headwater110C. jejuni, C. coli3NI
Heathcote river 2-mouth24C. jejuniNDND
Hewlings stream110C. jejuniNDND
Hororata river0.9C. jejuni7577
Horseshoe lake2.3C. jejuni6880
Ilam stream1.5C. jejuni3063
Irwell river1.5C. jejuni8185
Jackson stream2.3C. jejuni629
Kaiapoi river4.3C. jejuni638
Kaikainui stream0.9C. jejuni2731
Kaituna river0.4C. jejuni (weak)5698
Kaputone creek110Thermotolerant Campylobacter1230
Knights stream<0.3N I7351
L 1 creek0.4C. jejuni3972
Lake hood0.4C. jejuni438
North brook0.4C. jejuni248
Nottingham stream24C. coli (weak), C. jejuni3243
Okeover stream0.9C. jejuniNDND
Okuku river<0.3NI2271
Otukaikino0.9C. jejuni5575
Rakaia river0.4C. jejuni8256
Selwyn river0.4C. jejuni8478
South brook0.4C. jejuniNINI
Spring creek<0.3NINI1
Styx river2.3C. jejuni2410
Upper Selwyn river<0.3NI8295
Victoria lake<0.3NI8371
Wai-iti stream46C. jejuniNDND
Waikanui creek0.4C. jejuni3732
Waikoko stream0.4C. jejuni5330
Waimairi stream46C. jejuniNDND
Waimakariri 1-head water<0.3NI176
Waimakariri 2-mouth0.4C. jejuni5726
Wairarapa stream4.3C. jejuniNDND

Isolation of campylobacters

Campylobacter spp. were isolated from 45 of 53 (85%) water samples tested, with a range of 0.3–110 MPN 100 ml−1. The distribution of MPN values is shown in Fig. 1. PCR results showed that C. jejuni was isolated from 42/53 (79%) of the samples. Three of 53 (6%) water samples contained C. coli and thermotolerant campylobacters that were neither C. coli nor C. jejuni were isolated at the same prevalence.

Figure 1.

 Distribution of Campylobacter most probable number values in Canterbury surface water sources.

Discussion

Water samples from lakes, streams, ponds and rivers from 49 different locations were tested for thermotolerant campylobacters. Locations varied from rural mountainous areas, rural agricultural areas and urban sites. While campylobacters could be isolated frequently, the numbers present were usually quite low.

There are possibly numerous faecal inputs into many of these surface waters and it would be assumed that these inputs would contain both Campylobacter spp. and their phages, as Campylobacter spp. are readily isolated from the faeces of ruminants and birds (Devane et al. 2005). Given that the membrane filtration concentration method gave good recoveries of inoculated phages and that several hosts were used in enrichments, the data suggests that Campylobacter phages occur infrequently in surface water samples.

We have previously reported that we were unable to isolate Campylobacter phages from poultry-related samples, with the exception of whole chickens (Tsuei et al. 2007). Our ability to isolate Campylobacter phages has been restricted to samples of chicken faeces or surfaces likely to be contaminated by this source, i.e. whole chickens sampled immediately postkill.

The lack of Campylobacter phage isolation from water may reflect the limited availability of the host, as Campylobacter spp. numbers in our water samples were low when compared with the bacterial concentrations in samples, such as poultry faeces, from which Campylobacter phage isolations have been reported (Atterbury et al. 2004). Phages require an actively metabolizing host in order to propagate and it is not likely that Campylobacter spp. would be actively growing in the environmental conditions from which the samples were obtained, as the temperature is too low. The absence of phage may also have been because of the methods used i.e. a limited number of hosts were used for their isolation. The data obtained in this study could be combined with previous data to refine exposure assessments and resulting risk estimates.

Acknowledgement

We would like to acknowledge the New Zealand Foundation for Research, Science and Technology (Contract No. CO3X0701) for its financial support.

Ancillary