Enhanced enrichment and detection of thermotolerant Campylobacter ...

849

Enhanced enrichment and detection of thermotolerant Campylobacter species from water using the Portable Microbe Enrichment Unit and real-time PCR Tarja Pitka¨nen, Juliane Bra¨cker, Ilkka T. Miettinen, Anneli Heitto, Jouni Pesola, and Elias Hakalehto

Abstract: An enhanced enrichment using the Portable Microbe Enrichment Unit (PMEU) with the microaerobic bubbling of broths was applied for the detection of thermotolerant Campylobacter species from water. This PMEU enrichment was compared with the conventional static enrichment of the international standard ISO 17995:2005. In addition, Campylobacter detection after enrichment using a real-time PCR detection was compared with colony counts. The tests with stressed Campylobacter jejuni cells in drinking water indicated that the PMEU enrichment yielded a significantly higher number of Campylobacter cells in the Bolton broth compared with the conventional static incubation. Application of the real-time PCR technique shortened the Campylobacter detection time. This combination of method modifications can be used for Campylobacter detection from water and adds methodological repertoire for the rapid survey and management of waterborne outbreaks. Key words: Campylobacter, enrichment, Portable Microbe Enrichment Unit, real-time PCR, water. Re´sume´ : Un enrichissement ame´liore´ obtenu a` l’aide d’une unite´ portative d’enrichissement microbiologique (UPEM) avec bullage des milieux de culture en microae´robie a e´te´ utilise´ pour de´tecter des espe`ces de Campylobacter tole´rantes a` la chaleur dans l’eau. Cet enrichissement par l’UPEM a e´te´ compare´ a` la me´thode d’enrichissement statique conventionnelle du standard international ISO 17995 : 2005. En plus, la de´tection de Campylobacter apre`s l’enrichissement par PCR en temps re´el a e´te´ compare´e a` la de´tection par de´compte de colonies. Les tests re´alise´s avec Campylobacter jejuni soumis au stress dans l’eau de consommation ont indique´ que l’enrichissement par l’UPEM produisait un nombre significativement plus e´leve´ de cellules de Campylobacter dans le milieu Bolton comparativement au protocole d’incubation statique conventionnel. L’application de la technique de PCR en temps re´el a diminue´ le temps de de´tection de Campylobacter. Cette combinaison de me´thodes modifie´es peut eˆtre utilise´e pour de´tecter Campylobacter dans l’eau et ajoute des e´le´ments au re´pertoire me´thodologique d’outils de de´pistage rapide et de gestion des e´pide´mies hydriques. Mots-cle´s : Campylobacter, enrichissement, unite´ portative d’enrichissement microbiologique, PCR en temps re´el, eau. [Traduit par la Re´daction]

Introduction Campylobacter is a major human intestinal pathogen, which is transmitted through contaminated food and water (Butzler 2004). In the international standard method ISO 17995 (2005), the detection of thermotolerant Campylo-

bacter species from water samples consists of enrichment in broth after membrane filtration and then culturing the broth on a selective solid medium. The enrichment procedure takes 2 days, and the subsequent cultivation on modified charcoal cefaperazone deoxycholate agar (mCCDA) medium requires 2 additional days; that is, 4 days in total to confirm

Received 31 October 2008. Revision received 27 February 2009. Accepted 16 March 2009. Published on the NRC Research Press Web site at cjm.nrc.ca on 15 July 2009. T. Pitka¨nen1 and I.T. Miettinen. National Institute for Health and Welfare, Department of Environmental Health, Water and Health Unit, P.O. Box 95, FI-70701 Kuopio, Finland. J. Bra¨cker. National Institute for Health and Welfare, Department of Environmental Health, Water and Health Unit, P.O. Box 95, FI70701 Kuopio, Finland; Biofilm Centre, University of Duisburg-Essen, Geibelstraße 41, D-47057 Duisburg, Germany. A. Heitto. Finnoflag Oy, Isoharjantie 6, FI-71800 Siilinja¨rvi, Finland. J. Pesola. Finnoflag Oy, Isoharjantie 6, FI-71800 Siilinja¨rvi, Finland; Institute of Clinical Medicine, Pediatrics, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland; Department of Pediatrics, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio, Finland. E. Hakalehto. Finnoflag Oy, Isoharjantie 6, FI-71800 Siilinja¨rvi, Finland; Department of Biosciences, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland. 1Corresponding

author (e-mail: [email protected]).

Can. J. Microbiol. 55: 849–858 (2009)

doi:10.1139/W09-040

Published by NRC Research Press

850

a negative Campylobacter result. The presumptive positive result demands further confirmation that may take several days. Numerous attempts have been made to develop faster Campylobacter detection methods. In some recent works, nucleic acid sequence-based amplification (NASBA) assays (Cook 2003; Cools et al. 2006) and the fluorescent in situ hybridization (FISH) technique (Lehtola et al. 2006) have been applied, but most of the recently developed Campylobacter detection methods have been based on PCR technology (Waage et al. 1999; Moore et al. 2001; Bang et al. 2002; Sails et al. 2002; Moreno et al. 2003). It is known that the real-time application of PCR can speed up these types of methods (Josefsen et al. 2004; Abu-Halaweh et al. 2005; Nam et al. 2005), and enrichment strategies have been applied to densify the bacterial concentration in a sample before the PCR detection (Hernandez et al. 1995; Sails et al. 2003). Most of the new methods have focused on improving Campylobacter detection, and few attempts have been made to improve the enrichment step itself. Recently, a new innovative enrichment technique, called the Portable Microbe Enrichment Unit (PMEU; Finnoflag Oy, Siilinja¨rvi, Finland), has been developed and utilized in aerobic and anaerobic conditions in a Salmonella study (Hakalehto et al. 2007), as well as for the metabolic studies of intestinal Escherichia coli and Klebsiella sp. (Hakalehto et al. 2008). It was demonstrated that the diffusion of nutrients and gaseous components was sufficiently fast in the PMEU and did not significantly limit the growth of the facultatively anaerobic Salmonella group. In the PMEU, enrichment is enhanced by adding gas bubbles to the enrichment broth. It is presumed that this physical improvement of incubation makes nutrients more available to the dividing bacterial cells. In this paper, the PMEU has been applied for the first time in microaerophilic conditions in Campylobacter enrichment. The performance of the PMEU was compared with the performance of the international standard method ISO 17995 (2005). Additionally, in this work, the performance of conventional plate counting after enrichment was compared with a real-time modification of the PCR method with restriction enzyme analysis (Fermer and Engvall 1999) to study the feasibility of using these techniques to achieve fast and species-specific Campylobacter detection.

Materials and methods Bacterial strains Two strains were used during the whole study, including the preliminary tests: the environmental strain of Campylobacter jejuni isolated in 2004 from water associated with fecal contamination of a municipal drinking water system (Pitka¨nen et al. 2008), and the environmental strain of Campylobacter coli isolated from eastern Finland in 1987 (Laboratory of Environmental Microbiology, National Public Health Institute, Kuopio, Finland). The strains were stored in nutrient broth containing 15% glycerol at –70 8C or lower and cultured under microaerobic conditions (CampyGen; Oxoid, Basingstoke, UK) on mCCDA medium (Oxoid) at 42 8C. Preparation of inoculates In the growth experiments, cultures after 3 days of incu-

Can. J. Microbiol. Vol. 55, 2009

bation on mCCDA were used. Colonies were transferred to a tube with a sterile 10 mL loop, weighted, and then suspended in 5 mL of sterile deionized water, targeting an approximate concentration of 1  105 to 1  107 colonyforming units (CFU)/mL. To achieve reproducibility between the tests, in addition to the weighting of the colonies, the absorbance of the suspension at 420 nm was also measured. Optimizations of the method were performed by inoculating the dilutions of C. jejuni and C. coli cells directly to the enrichment broth and then dividing the broth to the PMEU and conventional STATIC enrichments. In drinking water and bathing water tests, the water samples were first inoculated by focusing on low counts (1– 100 CFU per 1000 mL) and then membrane filtered. After filtration, the membranes (EZ-PAK; Millipore S.A.S., Molsheim, France) were placed into the broth aliquots in PMEU syringes and in STATIC enrichment bottles. The CFUs of the inoculum were counted on mCCDA after incubation for 48 h at 42 8C under microaerobic conditions, and the weighted mean of the CFUs was calculated. Optimization of test conditions Optimization tests consisted of (i) the comparison between microaerobic and aerobic incubations, (ii) the optimization of antibiotic concentrations in the PMEU, (iii) the testing of the suitability of antifoam drops for the PMEU enrichment, and (iv) the testing of optimal subsampling times. The growth of duplicate inoculums of C. jejuni and C. coli strains in Bolton broth were tested using 4 different procedures: microaerobic (85% N2, 5% O2, and 10% CO2) and aerobic (ambient air) bubbling of the PMEU enrichment, and microaerobic (CampyGen; Oxoid) and aerobic incubation of the STATIC enrichment. The optimization of the antibiotic concentrations was needed, since in the PMEU enrichment, in addition to nutrients, selective substances may also be more readily available for microbes than in the STATIC enrichment case. The environmental C. jejuni strain (positive control) and E. coli ATCC 8739 (negative control) were utilized. The test of Bolton broth was done at 2 different C. jejuni concentrations, using full, half, and zero concentrations of selective supplement X132 (LabM, Bury, UK) according to the manufacturer’s instructions and ISO 17995 (2005), with the PMEU and STATIC enrichments. The test of Preston broth was done likewise, using selective supplement SR0204E (Oxoid), and duplicates were used instead of 2 different C. jejuni concentrations. The C. jejuni counts were enumerated by plating on mCCDA medium after 24 and 48 h enrichments in Bolton broth and after 16, 24, and 48 h enrichments in Preston broth. The CFUs of E. coli were analyzed after 48 h enrichment in Bolton and Preston broths by plating on tryptone soya agar (Oxoid) incubated aerobically for 24 h at 37 8C. The bubbling of broths in the PMEU caused foaming, especially in the Preston broth. The foam blocked the gas flow and bubbling of the PMEU syringe. In an attempt to solve this problem, antifoam drops (IDEXX Laboratories, Westbrook, Maine) were used in the Preston broth enrichments in the PMEU. The possible effects of antifoam drops on Campylobacter growth was studied, using 3 replicates with and without addition of the drops (present at a concentration Published by NRC Research Press

Pitka¨nen et al.

of 4 drops / 100 mL), with the STATIC enrichment of Preston broth inoculated with the environmental C. jejuni strain. The subsamples of broths were taken after 0, 16, 24, and 48 h. The optimal sampling times of enrichments in Bolton broth were estimated with environmental strains of C. jejuni and C. coli. The subsampling times of 0, 6, 16, 24, 30, 40, and 48 h were tested. Additionally, the growth curve of the C. jejuni strain was compared with that of C. coli. Enrichment procedures All enrichments were conducted in parallel using the PMEU and conventional STATIC enrichments following the principles of ISO 17995 (2005). The PMEU is a portable incubator, where the microbes are enriched in specific syringes containing the samples in the enrichment broth (Fig. 1). To the bottom of each syringe, ambient air or a desired gas mixture was funneled through a sterile filter (0.2 mm SY13TF-S; Advanced Microdevices Pvt. Ltd., Ambala Cantt, India) and a needle (0.80 mm  120 mm Sterican; B. Braun, Melsungen, Germany). The gas was bubbled through the broth, agitating the broth and the bacterial cells. Microaerobic Campylobacter enrichment using the PMEU was conducted with a gas mixture of 85% NO2, 5% O2, and 10% CO2 (Specialty Gases, Oy Aga Ab, Espoo, Finland). The gas flow before the syringes was measured and adjusted to be around 4 mL/min, enabling a constant flow of bubbles in the broths but still avoiding the excessive formation of foam. The total volume of the syringe was 50 mL. In spite of the careful adjustment of gas flow, some foam still formed. To avoid blockage of the sterile filters, the volume of broth used was 30 mL in each syringe. In addition to the Preston broth, 4 drops / 100 mL of an antifoam solution (IDEXX Laboratories) were added to prevent the blockage of the air membranes in the PMEU. In the conventional STATIC enrichment, the microaerobic conditions were obtained using gas-generating pouches (CampyGen; Oxoid) in incubation jars. The volume of the broth used was 30 mL, in accordance with the volume of broth in the PMEU syringe. The incubation temperature for the PMEU and STATIC enrichments was 37 8C. The total incubation time was 48 h, and 2 mL subsamples from the broths were taken several times during the incubation. Bolton broth (LabM, Bury, UK) was used for drinking water samples and Preston broth (Oxoid) for bathing water samples (ISO 17995 2005). Enumeration of enrichment cultures The Campylobacter viable count in the PMEU and STATIC enrichment broths were counted from broth subsamples after the predetermined incubation times. This was done by plating the broths and serial dilutions onto mCCDA medium. The swarming of Campylobacter colonies was prevented by drying the medium before plating for 30 min in a laminar flow cabinet. Typical Campylobacter colonies were counted after 48 h of microaerobic incubation at 42 8C. In case of any doubt, especially in bathing water analyses, the cultures were confirmed by tests for motility, aerobic growth, oxidase, catalase, hippurate hydrolysis, and by Gram staining. In addition, Campylobacter detection and quantification

851

were performed using quantitative real-time PCR of the DNA extracted (UltraClean, Microbial DNA isolation kit; Mo Bio Laboratories, Inc., Carlsbad, California) from 1 mL subsamples of the broths enriched using the PMEU and STATIC methods. In PCR, 12.5 pmol of the primers THERM1 and THERM4 (Fermer and Engvall 1999) and the hot start DyNAmo HS SYBR Green qPCR kit containing 5 mmol/L MgCl2 (Finnzymes, Espoo, Finland) were used. The 25 mL reactions with a DNA template volume of 1 mL were analyzed using the Rotorgene 3000 (Corbett Research, Sydney, Australia), programmed at first to hold for 15 min at 95 8C, followed by 45 repeats of cycling of 10 s at 95 8C, 20 s at 56 8C, and 30 s at 72 8C, acquiring on FAM/SYBR at the end of each cycle, and finally for 7 min at 72 8C. The melting point analysis was conducted on the PCR products using a ramp from 72 to 95 8C, rising by 1 8C each step. The first step was 45 s, and then 5 s. The melting peak that occurred approximately at 83 8C confirmed the correct product. Finally, after a 2 h run, the temperature was held for 7 min at 72 8C to prepare the PCR products for the possibility of post-PCR analysis. In case of an unspecific melting peak, the correct size (491 bp) and purity of the PCR product were verified using agarose gel electrophoresis. For naturally contaminated bathing water samples, PCR – restriction enzyme analysis (REA) using the AluI enzyme (Fermer and Engvall 1999; Engvall et al. 2002) was done to determine the detected Campylobacter species. The quantification of the real-time PCR detection was done with Rotor-Gene 6 software (Corbett Research) by comparing the results from the samples to a standard curve. The initial standard curve was made using serial dilutions of DNA extracted from a known concentration of C. jejuni ATCC 33291, diluted in sterile deionized water to 6 orders of magnitude, and by analyzing them as 4 replicates on the Rotorgene 3000. The Campylobacter concentrations in the dilutions for the standard curve were analyzed before DNA extraction, using plate counting as described earlier. In each run after the standardization, an internal amplification control was not used, but a DNA extract of the known concentration of the C. jejuni was used as a quantitative control, and the previously made standard curve was fixed with this control. In addition, in each run, sterile distilled water was used as a no-template control, E. coli ATCC 8739 as a negative control, and C. jejuni ATCC 33291 as a positive control. Tests using inoculated drinking water samples The efficiencies of the PMEU and STATIC enrichments in drinking water were studied with tap water inoculated with an environmental strain of C. jejuni. The drinking water used in the tests was tap water from the city of Kuopio, Finland. The water sample was taken into 2 clean plastic canisters. The tap water contained 0.18 mg/L chlorine, analyzed using the Palintest Micro 1000 chlorometer (Palintest Ltd., Gateshead, UK) at the time of sampling. From the other canister, chlorine was deactivated with 0.02 mol/L sodium thiosulfate (at a concentration of 50 mL / 100 mL). Both canisters were inoculated with equal amounts of the same suspension of C. jejuni. Triplicate 1000 mL samples (multiple samples from the same canister) were taken immediately after spiking and concentrated onto membrane filters. Published by NRC Research Press

852

Can. J. Microbiol. Vol. 55, 2009

Fig. 1. (a) A schematic presentation of the Portable Microbe Enrichment Unit (PMEU) and (b) the construction of the incubation syringe. Illustrations by U. Korff (Du¨sseldorf, Germany).

(a)

(b)

Sterile filters

Needle

Sample + culture medium

The filters were placed into the Bolton broths and subjected to the PMEU and STATIC enrichments. The canisters were stored in the dark, at 4 8C, and sampled again after 3 and 7 days of storage. The broths were sampled, after 0, 16, 24, 40, and 48 h enrichments prior to Campylobacter detection, with plate counting and real-time PCR. Tests using inoculated and natural bathing water samples For the inoculation experiment with environmental C. jejuni strain, a bathing water sample was taken from a bathing

site at a lake in eastern Finland. A total of 3500 mL of the sample was inoculated. Triplicate 500 mL aliquots were filtered through the membrane filters that were placed into the Preston broths, followed by PMEU and STATIC enrichments. In addition, 1000 mL volumes of the bathing water sample without the inoculum were analyzed with the PMEU and STATIC enrichments to detect the possible natural contamination of the analyzed water with Campylobacter. The broths were sampled after 0, 16, 24, and 48 h enrichments for Campylobacter detection with plate counting and realtime PCR. Published by NRC Research Press

Pitka¨nen et al.

Statistical analyses The growth curves were drawn using geometric means and geometric standard deviations of replicates. Before the statistical analyses, the counts were converted to their natural logarithms. In the statistical analyses, SPSS 14.0 for Windows software was used. The significance of the difference between the PMEU and STATIC enrichments was evaluated utilizing univariate analysis of variance including a time factor. One-way analysis of variance was performed in the comparison between real-time PCR results and colony counts. The differences were evaluated as statistically significant in cases of p £ 0.05.

Results Method optimization Both C. jejuni and C. coli were enriched in PMEU and STATIC cultures using a microaerobic atmosphere. No growth was detected in the broths bubbled aerobically in the PMEU, and with aerobic static enrichment, C. jejuni did not grow at all and the growth of C. coli was slower than that seen with the microaerobic setup. The results of the optimization of suitable antibiotic concentrations and of the addition of antifoam drops are presented in Table 1. Inclusion of half concentrations of the antibiotics in the Bolton broth seemed to be better for C. jejuni enrichment in the PMEU. However, in the STATIC enrichment, the full concentration seemed to work better. With the Preston broth, the use of half or full antibiotic concentrations did not change the C. jejuni CFUs. It was decided to use the full concentration of antibiotics according to ISO 17995 (2005) in further tests in this study with both broths and with both methods to allow comparison between the PMEU and STATIC methods. The addition of antifoam drops in Preston broth with the STATIC method did not have an effect on the C. jejuni CFUs, so the antifoam drops were added to the Preston broths enriched with the PMEU in the subsequent tests. In the optimization of the subsampling times, with a Campylobacter concentration of ~20 CFU/mL at the beginning of the enrichment, the growth of C. jejuni was observed in the PMEU already after 6 h and the growth of C. coli after 16 h (Fig. 2). In contrast, with STATIC enrichment, the growth of both strains was observed for the first time after 16 h of enrichment. In the 6 sampling times (from 6 to 48 h) using the PMEU enrichment, the counts of both strains were higher than those obtained using the STATIC enrichment. The difference between the PMEU and STATIC enrichments was more extensive with C. jejuni than with C. coli, and a decision was made to use C. jejuni in the subsequent enrichment tests. In the next series of tests, the sampling times of 0, 16, 24, 40, and 48 h were chosen based on these results and for practical reasons. Campylobacter counts from water samples after enrichments The membrane-filtered tap water samples after 0, 3, and 7 day storage times were enriched using the PMEU and STATIC methods in Bolton broth. The inoculated C. jejuni was not detected from any of the tap water samples containing chlorine (0.18 mg/L), but positive results were achieved

853 Table 1. Geometric (geom.) means and geometric standard deviations of the Campylobacter jejuni concentrations in the optimization tests with different starting concentrations and incubation times (consisting of 4–9 separate enrichments) using Portable Microbe Enrichment Unit (PMEU) and conventional STATIC enrichments. Antibiotic concn. (%) Bolton 0 50 100 Preston 0 50 100

Enrichment type

Geom. mean (CFU/mL)

Geom. SD (CFU/mL)

STATIC PMEU STATIC PMEU STATIC PMEU

5.6101 7.4101 2.4105 2.6107 1.4106 1.1106

1.2101 8.1100 1.7102 1.3102 1.6102 1.1103

STATIC PMEU STATIC PMEU STATIC PMEU

2.0105 1.7104 4.8106 2.1107 3.6106 2.3107

1.9101 1.9100 9.9101 4.4101 1.6102 6.2101

With Without

2.2106 1.3106

1.3102 1.3102

Antifoam

Note: The percentages represent the antibiotic concentrations in Bolton and Preston broths compared with the standard method. The addition of antifoam drops was tested in Preston broth with the STATIC method.

from the tap water sample dechlorinated with sodium thiosulfate. Immediately after inoculation (0 days, Fig. 3), C. jejuni counts in the PMEU enrichment were higher than those found with STATIC enrichment: the counts obtained using the PMEU and STATIC enrichments differed significantly from each other when, in addition to the enrichment method, the effect of incubation time of the broth was taken into consideration (p £ 0.01). Samples taken from inoculated drinking water after 3 days of storage were positive for C. jejuni after 16 and 24 h of PMEU enrichment (Fig. 3). Interestingly, with the STATIC enrichment, the growth was observed later: a few colonies were detected after 24 h but real growth was not observed until 40 h of enrichment. During the whole enrichment of samples taken after 3 days of storage, the geometric mean of counts in the STATIC broths was lower than the corresponding value for the PMEU broths (p = 0.03). The counts of C. jejuni in the enrichment broths were on average lower after 3 days of storage than immediately after inoculation. After 7 days of storage, no C. jejuni was detected. Three replicates of a bathing water sample inoculated with C. jejuni enriched in Preston broth did not display any difference between PMEU and STATIC enrichments (Fig. 4; p = 0.26). In the analysis of 1000 mL of the same bathing water sample before the inoculum, it was found that the sample already contained C. jejuni. In the PMEU, the counts from this uninoculated sample were higher after 16 and 24 h enrichments than the counts obtained using STATIC enrichment. Published by NRC Research Press

854

Can. J. Microbiol. Vol. 55, 2009

Fig. 2. The colony-forming units (CFUs) of Campylobacter jejuni and C. coli after several sampling times in Bolton enrichment broth. 1.0×10 8

1.0×10 7

PMEU C. jejuni

STATIC C. jejuni

PMEU C. coli

STATIC C. coli

CFU/mL

1.0×10 6

1.0×10 5

1.0×10 4 1.0×10 3 1.0×10 2

1.0×10 1

1.0×10 0 0

5

10

15

20

25

30

35

40

45

50

Enrichment (h)

Fig. 3. Campylobacter jejuni counts (geometric mean of 3 replicates) in Portable Microbe Enrichment Unit (PMEU) and STATIC enrichments using Bolton broth and membrane-filtered tap water sample after 0 and 3 days of storage. The error bars show the geometric standard deviation. 1.0×1010 1.0×109

PMEU 0 days

STATIC 0 days

PMEU 3 days

STATIC 3 days

1.0×108

CFU/mL

1.0×107 1.0×106 1.0×105 1.0×104 1.0×103 1.0×102 1.0×101 1.0×100 0

5

10

15

20

25

30

35

40

45

50

Enrichment (h)

Published by NRC Research Press

Pitka¨nen et al.

855

Fig. 4. Campylobacter jejuni counts (geometric mean of 3 replicates) in Portable Microbe Enrichment Unit (PMEU) and STATIC enrichments using Preston broth and a membrane-filtered bathing water sample inoculated with C. jejuni. The error bars show the geometric standard deviation. 1.0×1010 PMEU 1.0×109

STATIC

1.0×108 1.0×107

CFU/mL

1.0×106 1.0×105 1.0×104 1.0×103 1.0×102 1.0×101 1.0×100 0

5

10

15

20

25

30

35

40

45

50

Enrichment (h)

Quantitative real-time PCR in comparison with the colony counts The real-time PCR detection considerably shortened the Campylobacter detection time. Whereas the colony count detection took 48 h, the time required for PCR detection was ~5 h, depending on the number of samples. The PCR quantification was based on colony counts obtained from the control strain dilutions before DNA extraction to achieve comparability with the colony counts. Figure 5 shows the standard curve produced. During this study, a total of 27 comparable results of quantitative PCR and colony counts were obtained, with 15 of them originating from PMEU enrichments and 12 from STATIC enrichments. Three of the PCR results were negative (11%), when a colony count was obtained from the same sample. Two samples were PCR positive (7%), but their concentration remained below the determination limit of quantification. One of these 2 was culture negative and the other culture positive. In addition, 1 sample was culture negative but PCR positive. The geometric mean of all PCR results was slightly lower than the geometric mean of all colony counts, but the difference was not statistically significant (Table 2). When the results were analyzed according to the incubation time points, it was found that in extracts from broths after 16 h of enrichment, the geometric mean of PCR results was slightly higher than the geometric mean of colony counts, but at the time points of 24 and 48 h, the situation was reversed.

Discussion Microbial contamination of drinking water and also recreational water can lead to severe infections and epidemics (Neumann et al. 2005; Hrudey and Hrudey 2007). It is important in the investigation of waterborne outbreaks that there is efficient and rapid detection of pathogenic microbes including Campylobacter sp. In this study, a combination of enhanced enrichment (PMEU) and real-time PCR detection was tested as a way of attaining sensitive and rapid Campylobacter detection from water samples. The PMEU enrichment was shown to be efficient in enriching stressed Campylobacter cells: the PMEU produced higher C. jejuni counts than could be obtained with the STATIC enrichment after 3 days of storage in drinking water. This improvement may be significant in solving the source of waterborne outbreaks, where there are clear possibilities for delays in the sampling and detection of Campylobacter. As Lehtola et al. (2006) reported earlier in a biofilm study, the conventional culture method might underestimate the actual numbers of Campylobacter in water and biofilms. In that study, culturable C. jejuni was found only 1 day after spiking using conventional static enrichment, but the bacteria were detectable for at least 1 week using the FISH peptide nucleic acid probe method. In this study, PMEU technology was utilized for the first time in microaerobic enrichment of Campylobacter. There is Published by NRC Research Press

856

Can. J. Microbiol. Vol. 55, 2009

Fig. 5. Standard curve based on the concentration of Campylobacter jejuni ATCC 33291 on modified charcoal cefaperazone deoxycholate agar medium (CFU/mL) before DNA extraction. The quantification of real-time PCR analysis was done using Rotor-Gene 6 software. CT, cycle threshold. 26

Cycling A.FAM/Sybr R = 0.99607

24

R 2 = 0.99216 22

M = –3.350

20

B = 35.445

CT

Efficiency = 0.99

18 16 14 12 10 10×103

10×104

10×106

10×105

10×107

Concentration CFU/mL

Table 2. Geometric (geom.) means and geometric standard deviations of quantitative PCR results in comparison with the colony counts (CFU/mL). Geom. mean ± SD All results included Pairs with results below detection limit excluded Pairs with results below detection limit and >1.0109 excluded Results after 16 h enrichment Results after 24 h enrichment Results after 48 h enrichment

N 27 21

qPCR (CFU/mL) 4.9104±4.7102 5.2105±9.9101

Colony counts (CFU/mL) 1.0105±7.1102 9.5105±2.8102

p 0.66 0.71

19

2.8105±7.7101

4.2105±2.0102

0.79

8

4.6102±5.4101

3.4102±2.4101

0.86

10

1.0104±1.3102

5.2104±6.2101

0.43

9

1.7107±8.0101

3.5107±5.8102

0.77

Note: The significance of the difference between PCR results and colony counts was calculated from natural logarithms using one-way analysis of variance.

also one earlier publication (Heisick et al. 1984), where a constant gas flow into the C. jejuni enrichment broths was studied in comparison with an evacuation-replacement method. The data of Heisick et al. (1984) are in agreement with our findings, i.e., the constant gas flow resulted in higher recoveries. For some unknown reason, it seems that this earlier finding was ignored in the commonly used commercial applications, since to date the incubation is still conducted in jars with a modified atmosphere, not with a constant gas flow directly into the broth. The full concentration of antibiotics in the Bolton and Preston broths was used in this study. In the future for the PMEU, the use of a lower concentration of antibiotics in Bolton broth may be advantageous, but in that case, the efficiency of inhibition will need to be evaluated more carefully with several species and natural samples. Abulreesh et al. (2005) reported that the antagonistic effects of background

growth in enrichment broths may out-compete Campylobacteria during the enrichment process. This was also seen in our study during the optimization of the antibiotic concentrations: the C. jejuni counts were lower in broths where the growth of E. coli was not inhibited, compared with broths that contained antibiotics inhibiting the growth of E. coli. This antagonistic effect might also explain our results in the comparison of the PMEU and STATIC methods using Preston broth, where no difference was observed between the 2 methods when the bathing water samples were evaluated, since these types of water are known to contain large amounts of background microbes. Furthermore, we used a volume of the broth (30 mL) that was smaller than that described in the standard method (100 mL), and this might cause an excessive background growth in some naturally contaminated water samples, but it is probably not a problem with drinking water samples. Published by NRC Research Press

Pitka¨nen et al.

Although our results indicate that the PMEU could produce higher Campylobacter recoveries in drinking water analysis than the conventional STATIC method, there is still a general need to improve the PMEU technique. During our study, we encountered some difficulties owing to the varying gas flow and with the syringe holders. It would be advantageous if these technical difficulties could be solved, as the portability of the PMEU is an advantage, since the microbial analysis of water samples should be started as soon as possible after sampling. For example, the international standard ISO 19458 (2006) states that 24 h is the maximum storage time, including transport, for thermophilic Campylobacter spp. Further validation of the PMEU technique is needed, and the validation should include tests under field conditions and tests with thermotolerant Campylobacter species other than C. jejuni, which was mainly used in the present study. Quantitative real-time PCR for Campylobacter detection ideally would be applied directly to the samples when the concentration of the target organisms in the sample is determined (Yang et al. 2003). However, in Campylobacter analysis from water, direct detection has encountered problems because of the low numbers of cells in the samples and the existence of PCR inhibitors (Abulreesh et al. 2006), and consequently an enrichment step prior to real-time PCR has been used (Nam et al. 2005). The presence/absence approach using real-time PCR after enrichment was also applied in our study, but quantification was conducted for method validation purposes. In some samples of our study, the colony counts reached higher concentrations than those of PCR, indicating that the concentration in those samples had exceeded the upper limit of PCR, and dilution of the DNA for PCR should have been done. In addition, when a colony count was obtained, a slightly higher proportion of samples remained PCR negative compared with the proportion of samples remaining culture negative when PCR was positive, suggesting the possibility for a lower detection limit in colony counting than in PCR. On the whole, the calculated colony count obtained using quantitative real-time PCR did not differ significantly from the colony counts of samples cultured on mCCDA. However, culturing also is needed for the isolation of the strains, since further tests such as pulsed-field gel electrophoresis may well be required in outbreak investigations. In this study, species-level Campylobacter detection was achieved within 5 h using the PCR detection and restriction fragment analysis of PCR products. This is a remarkable advantage in comparison with the 44 ± 4 h incubation time required for culturing (ISO 17995 2005). It is noteworthy that it is possible to achieve even faster Campylobacter detection by using probe-based real-time PCR, but then a second assay may be required for species identification (Abu-Halaweh et al. 2005). In the present study, the SYBR Green technique was used, since this application allowed for species differentiation between thermotolerant Campylobacter species (C. jejuni, C. coli, C. lari, and C. upsaliensis) from the product of the assay. The specificity of the assay was improved using a hot start enzyme, and the correctness of the PCR product was confirmed by melting point analysis. There are only a few previously reported studies of realtime PCR applications for Campylobacter detection in water

857

samples (Yang et al. 2003; Nam et al. 2005), since most of the reported real-time PCR studies have dealt with the detection of foodborne Campylobacter species (Josefsen et al. 2004; Perelle et al. 2004). In the future, real-time PCR should be applied also in water analyses, to obtain the Campylobacter results after enrichment more rapidly than the period of 2 days required for plate counting. The application of the PMEU procedure in Campylobacter enrichment introduced in the present study might result in a more efficient enrichment than conventional static enrichment. Thus, by utilizing the PMEU, it might be possible to achieve the Campylobacter detection limit with a reduced time of incubation. A combined analytical tool utilizing PMEU and PCR techniques may result in very important time savings, e.g., in solving suspected waterborne outbreaks.

Acknowledgements The authors thank P. Tiittanen for help with the statistical analyses and L. Korhonen for providing the Campylobacter coli strain. M.-L. Ha¨nninen is acknowledged for a critical reading of the manuscript. This work was done as a part of a student exchange in co-operation with the University of Duisburg – Essen and the University of Kuopio. The PMEU is protected under Finnish patent (No. 106561) and corresponding international patents. This study was supported by the Ministry of Education (Graduate School in Environmental Health – SYTYKE).

References Abu-Halaweh, M., Bates, J., and Patel, B.K.C. 2005. Rapid detection and differentiation of pathogenic Campylobacter jejuni and Campylobacter coli by real-time PCR. Res. Microbiol. 156: 107–114. doi:10.1016/j.resmic.2004.08.008. PMID:15636755. Abulreesh, H.H., Paget, T.A., and Goulder, R. 2005. Recovery of thermophilic campylobacters from pond water and sediment and the problem of interference by background bacteria in enrichment culture. Water Res. 39: 2877–2882. doi:10.1016/j.watres. 2005.05.004. PMID:15979120. Abulreesh, H.H., Paget, T.A., and Goulder, R. 2006. Campylobacter in waterfowl and aquatic environments: Incidence and methods of detection. Environ. Sci. Technol. 40: 7122–7131. doi:10. 1021/es060327l. PMID:17180958. Bang, D.D., Wedderkopp, A., Pedersen, K., and Madsen, M. 2002. Rapid PCR using nested primers of the 16S rRNA and the hippuricase (hipO) genes to detect Campylobacter jejuni and Campylobacter coli in environmental samples. Mol. Cell. Probes, 16: 359–369. doi:10.1006/mcpr.2002.0434. PMID:12477440. Butzler, J.P. 2004. Campylobacter, from obscurity to celebrity. Clin. Microbiol. Infect. 10: 868–876. doi:10.1111/j.1469-0691. 2004.00983.x. PMID:15373879. Cook, N. 2003. The use of NASBA for the detection of microbial pathogens in food and environmental samples. J. Microbiol. Methods, 53: 165–174. doi:10.1016/S0167-7012(03)00022-8. PMID:12654488. Cools, I., Uyttendaele, M., D’Haese, E., Nelis, H.J., and Debevere, J. 2006. Development of a real-time NASBA assay for the detection of Campylobacter jejuni cells. J. Microbiol. Methods, 66: 313–320. doi:10.1016/j.mimet.2005.12.004. PMID: 16443295. Engvall, E.O., Brandstrom, B., Gunnarsson, A., Morner, T., Wahlstrom, H., and Fermer, C. 2002. Validation of a polymerase chain reaction/restriction enzyme analysis method for spePublished by NRC Research Press

858 cies identification of thermophilic campylobacters isolated from domestic and wild animals. J. Appl. Microbiol. 92: 47–54. doi:10.1046/j.1365-2672.2002.01491.x. PMID:11849327. Fermer, C., and Engvall, E.O. 1999. Specific PCR identification and differentiation of the thermophilic campylobacters, Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. J. Clin. Microbiol. 37: 3370–3373. PMID:10488210. Hakalehto, E., Pesola, J., Heitto, L., Narvanen, A., and Heitto, A. 2007. Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella. Pathophysiology, 14(1): 61–69. doi:10.1016/j.pathophys.2007.01.003. PMID:17434297. Hakalehto, E., Humppi, T., and Paakkanen, H. 2008. Dualistic acidic and neutral glucose fermentation balance in small intestine: simulation in vitro. Pathophysiology, 15(4): 211–220. doi:10.1016/j.pathophys.2008.07.001. PMID:18804970. Heisick, J., Lanier, J., and Peeler, J.T. 1984. Comparison of enrichment methods and atmosphere modification procedures for isolating Campylobacter jejuni from foods. Appl. Environ. Microbiol. 48: 1254–1255. PMID:6393876. Hernandez, J., Alonso, J.L., Fayos, A., Amoros, I., and Owen, R.J. 1995. Development of a PCR assay combined with a short enrichment culture for detection of Campylobacter jejuni in estuarine surface waters. FEMS Microbiol. Lett. 127: 201–206. doi:10. 1111/j.1574-6968.1995.tb07474.x. PMID:7758934. Hrudey, S.E., and Hrudey, E.J. 2007. Published case studies of waterborne disease outbreaks — evidence of a recurrent threat. Water Environ. Res. 79: 233–245. doi:10.2175/ 106143006X95483. PMID:17469655. ISO 17995. 2005. Water quality. Detection and enumeration of thermotolerant Campylobacter species. International Organization for Standardization (ISO), Geneva, Switzerland. ISO 19458. 2006. Water quality. Sampling for microbiological analysis. International Organization for Standardization (ISO), Geneva, Switzerland. Josefsen, M.H., Jacobsen, N.R., and Hoorfar, J. 2004. Enrichment followed by quantitative PCR both for rapid detection and as a tool for quantitative risk assessment of food-borne thermotolerant campylobacters. Appl. Environ. Microbiol. 70: 3588–3592. doi:10.1128/AEM.70.6.3588-3592.2004. PMID:15184161. Lehtola, M.J., Pitka¨nen, T., Miebach, L., and Miettinen, I.T. 2006. Survival of Campylobacter jejuni in potable water biofilms: a comparative study with different detection methods. Water Sci. Technol. 54: 57–61. doi:10.2166/wst.2006.448. PMID: 17037133. Moore, J., Caldwell, P., and Millar, B. 2001. Molecular detection of Campylobacter spp. in drinking, recreational and environmen-

Can. J. Microbiol. Vol. 55, 2009 tal water supplies. Int. J. Hyg. Environ. Health, 204: 185–189. doi:10.1078/1438-4639-00096. PMID:11759163. Moreno, Y., Botella, S., Alonso, J.L., Ferrus, M.A., Hernandez, M., and Hernandez, J. 2003. Specific detection of Arcobacter and Campylobacter strains in water and sewage by PCR and fluorescent in situ hybridization. Appl. Environ. Microbiol. 69: 1181– 1186. doi:10.1128/AEM.69.2.1181-1186.2003. PMID:12571045. Nam, H.M., Srinivasan, V., Murinda, S.E., and Oliver, S.P. 2005. Detection of Campylobacter jejuni in dairy farm environmental samples using SYBR Green real-time polymerase chain reaction. Foodborne Pathog. Dis. 2: 160–168. doi:10.1089/fpd.2005.2.160. PMID:15992311. Neumann, N.F., Smith, D.W., and Belosevic, M. 2005. Waterborne disease: An old foe re-emerging? J. Environ. Eng. Sci. 4: 155– 171. doi:10.1139/s04-061. Perelle, S., Josefsen, M., Hoorfar, J., Dilasser, F., Grout, J., and Fach, P. 2004. A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter. Mol. Cell. Probes, 18: 321–327. doi:10.1016/j.mcp.2004.04.005. PMID:15294320. Pitka¨nen, T., Miettinen, I.T., Nakari, U.-M., Takkinen, J., Nieminen, K., Siitonen, A., et al. 2008. Faecal contamination of a municipal drinking water distribution system in association with Campylobacter jejuni infections. J. Water Health, 6(3): 365–376. doi:10.2166/wh.2008.050. PMID:19108557. Sails, A.D., Bolton, F.J., Fox, A.J., Wareing, D.R.A., and Greenway, D.L.A. 2002. Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzymelinked immunosorbent assay. Appl. Environ. Microbiol. 68: 1319–1324. doi:10.1128/AEM.68.3.1319-1324.2002. PMID: 11872483. Sails, A.D., Fox, A.J., Bolton, F.J., Wareing, D.R., and Greenway, D.L. 2003. A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture. Appl. Environ. Microbiol. 69: 1383–1390. doi:10.1128/AEM.69.3.1383-1390. 2003. PMID:12620820. Waage, A.S., Vardund, T., Lund, V., and Kapperud, G. 1999. Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay. Appl. Environ. Microbiol. 65: 1636–1643. PMID:10103261. Yang, C., Jiang, Y., Huang, K., Zhu, C., and Yin, Y. 2003. Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk, and environmental water. FEMS Immunol. Med. Microbiol. 38: 265–271. doi:10.1016/S09288244(03)00168-8. PMID:14522462.

Published by NRC Research Press