- Campylobacter members of clinical relevance comprise C. jejuni, C. coli, C. lari, and C. upsaliensis
- most Campylobacter species are microaerophilic
- enter surface waters through fecal contamination.
- the receiving waters tend to reflect the species composition of the contaminating source.
- Most clinical cases of campylobacteriosis occur in summer months and decline in colder months.
- need temperatures of app. > 31°C to grow, but they persist longer at lower temperatures
- planktonic cells are sensitive to disinfection, but internalization by protozoa (e.g. by Tetrahymena pyriformis and Acanthamoeba castellani) can result in substantially increased resistance and survival
The family Campylobacteraceae includes the genera Campylobacter
. Most members of this family are considered a public health threat and can cause disease in humans and domestic animals. This document focuses on Campylobacter
is regarded as an emerging foodborne pathogen (for a review: Cervenka L 2007
members of clinical relevance comprise C. jejuni
, C. coli
, C. lari
, and C. upsaliensis
with the first two being considered the most common human enteric pathogens ( Frost JA 2001
). Due to their growth at 37°C (body temperature of humans and animals) or 42°C (body temperature of poultry) this group is also referred to as ‘thermophilic campylobacteria’. C. jejuni
is the most common bacterial cause of gastroenteritis in the developed countries ( Sails et al. 2002
). Surveys suggested that the annual incidence of campylobacter infections is as high as 1% in Europe and the United States ( Wassenaar and Newell 2000
). Typical symptoms include nausea, abdominal cramps, diarrhea, and fatigue with infections generally being self-limited ( Hiett et al. 2002
). The primary source of infection is considered to be handling and consumption of undercooked meat, especially poultry and poultry-related products ( Hiett et al. 2002
). Nevertheless, outbreaks have been associated with consumption of other contaminated food (e.g. inadequately pasteurized milk or contaminated salad vegetables) and water, including bottled water ( Morgan et al. 1994
; Evans et al. 2003
can be acquired from untreated well water, groundwater ( Kuusi et al. 2005
), or surface waters (from lakes and streams). Also outbreaks from consumption of tap water have been reported. Suspected sources of contamination were surface water from nearby pasture land ( Richardson et al. 2007
), insufficient disinfection ( Sacks et al. 1986
), contamination of water in open-top treatment towers by bird excrements ( Sacks et al. 1986
), or entire system contamination ( Vogt et al. 1982
). A Finish case-control study identified swimming in natural waters as a novel risk factor ( Schönberg-Norio et al. 2004
). Most clinical cases of campylobacteriosis occur in summer months and decline in colder months ( Vereen et al. 2007
). Members of the genus Campylobacter
are typically motile bacteria with an S-shaped spiral morphology (see image gallery). When exposed to unfavorable conditions, cells transform into a VBNC state and adopt a coccoid morphology ( Rollins and Colwell 1986
). The oxygen tolerance is variable with a few species being nearly anaerobic and the majority (including C. jejuni
) being microaerophilic ( Thomas et al. 1999
spp. are ubiquitous in the environment and have been isolated from streams, rivers, lakes, ponds, estuaries and bays. Although more frequent in surface waters due to their susceptibility to fecal contamination, C. jejuni
has also been isolated from groundwater ( Close et al. 2008
; Stanley et al. 1998
). Especially intense dairying on irrigated pastures was mentioned as a concern if shallow groundwater is used for drinking water purposes ( Close et al. 2008
). Agricultural use, human influence, and wildlife typically contribute to higher prevalence of waters. Contamination of fecal origin can occur by sewage discharge, agricultural run-off, or direct contamination by bird droppings and feces from other animals ( Richardson et al. 2007
). As different point- and non-point sources contain different Campylobacter
species, the receiving waters tend to reflect the species composition of the contaminating source. In the north-west of England, freshwater sections of rivers were reported to contain mainly C. jejuni
, whereas estuaries tend to have a mixture of C. jejuni
, C. coli
, and C. lari
as well as urease-positive thermophilic campylobacters (UPTC) ( Obiri-Danso et al. 2001
). Coastal seawater tends to have a mixture of C. lari
and UPTG. In the marine environment, Campylobacter
spp. have been found associated with zooplankton ( Maugeri et al. 2004
The incidence of isolation from surface waters ranges from 16 to 82% ( Sails et al. 2002). Campylobacter levels cannot be predicted by standard indicator bacteria ( Arvanitidou et al. 1995; Hörman et al. 2004, Kemp et al. 2005). Concentrations in various surface waters were reported to be between <10 and 102 cfu/100 ml ( Theron and Cloete 2002; Höller et al. 1998), in raw sewage between 102 and 104 cfu/100ml, and in treated sewage around 10 – 102 CFU/100 ml. ( Höller and Schomakers-Revaka 1994; Höller et al. 1998). Concentrations in raw sewage can be significantly higher if chicken farms or abattoirs are connected to the sewer system ( Höller et al. 1998). Their occurrence follows a strong seasonal trend with higher detection prevalence in water in winter months (lower temperatures favor survival), which contrasts the fact that human infections peak in the late spring-summer period ( Skirrow MB 1991; Abulreesh et al. 2006). The higher epidemiological risk in summer might be related to food-transmitted infection, however waters is also a significant source of human infection. Like for other waterborne pathogens, a significant link can exist between heavy rainfall, warmer temperatures, and melting snow and disease outbreak ( Auld et al. 2004; Thomas et al. 2006b). A well-studied outbreak of Campylobacter and E. coli O157:H7 in Walkerton, Canada, in May 2000 with at least 2,300 cases of gastrointestinal illness was preceded by several days of heavy rainfall ( Auld et al. 2004).
Selected publications about abundance in water are summarized in the following:
- A study investigated the prevalence of culturable campylobacters in rivers in a mixed-use watershed with various degrees of agricultural and human influence in Southern Georgia, USA, for one year (biweekly to monthly sampling). The watershed had a consistently higher rate of clinical cases (17 infections per 100,000 people in Coffee County) than the state-wide average (6.8 infections per 100,000 people in 2003). The study found highest levels directly downstream of a WWTP that handled both human and poultry slaughterhouse waste. Mean Campylobacter counts were 158 CFU per ml (maximum of 595 CFU per ml) with all samples (n=13) testing positive. Another site under the influence of a WWTP showed a mean concentration of 65 CFU per ml with 75% of samples (n=12) testing positive. Sampling sites which were not influenced by the WWTP showed significantly lower mean concentrations in the range between 2 and 30 CFU per ml with 17 to 69% of samples (n=12 or 13) testing positive. Counts significantly correlated with the number of fecal coliforms, conductivity, pH, and concentrations of NO3-, PO43-, and NH3. Larger numbers were found during summer months, coinciding with the occurrence of clinical cases of campylobacteriosis ( Vereen et al. 2007).
- Water from a 100-km2 dairy farming area in northwestern England with considerable recreational use was systematically examined for Campylobacter spp. using cultivation, PCR, and PFGE-RFLP. Samples were taken from a variety of sources including ponds, streams, ditches, and cattle water troughs. On average, Campylobacter spp. were isolated from 40.5% (n = 119) of water samples with C. coli, C. jejuni and C. lari being found in 18.5%, 14.3% and 4.2% of the samples, respectively. C. jejuni was most commonly isolated from trough water and running water sources, whereas C. coli was more common in standing water. No correlation with the presence of E. coli was found ( Kemp et al. 2005).
- In a systematic study looking at the presence of different pathogens and indicators in seven lakes and 15 rivers in southwestern Finland, 17.3% of samples (n=139) tested positive for Campylobacter spp. Among the positive samples, the most prevalent species were C. jejuni (45.8%), C. lari (25%), C. coli (4.2%), apart from unidentified Campylobacter isolates (25%). No significant correlation between presence of pathogens and indicators was found ( Hörman et al. 2004).
A summary document about waterborne pathogens suggested that effective water treatment and a well-maintained distribution system should reduce Campylobacter
to a level that should not cause human illness ( Health Canada 2006
spp. are believed to be as effectively inactivated by chlorine and chloramine as E. coli
( Blaser et al. 1986
). They are probably also more susceptible to ozone than fecal coliforms (Jack Schijven, http://www.rivm.nl/carma/overcarma/bijeenkomsten/SYMPOSIUM170604/Schijven.pdf
). Apart from chemical disinfection, UV disinfection is considered an efficient way of reducing Campylobacter
levels and might be a good idea for routine disinfection for communities whose drinking water supply is susceptible to contamination. C. jejuni
is extremely sensitive to light and UV ( Jones K 2001
). The implementation of UV disinfection was for example implemented in a rural community in Austria, which is situated in a karst area and where a Campylobacter
outbreak in 2006 was attributed to fecal contamination after heavy rainfall ( Meusburger et al. 2007
Resistance to chemical disinfection might be partially due to their internalization by waterborne protozoa. C. jejuni was shown to remain significantly longer viable in drinking water when internalized by Tetrahymena pyriformis and Acanthamoeba castellani compared to the planktonic state ( Snelling et al. 2005). C. jejuni strains have been reported to aggregate in amoebic vacuoles after ingestion ( Axelsson-Olsson et al. 2005). King et al. showed already in 1988 that ingested C. jejuni were > 50 times more resistant to free chlorine than freely suspended cells ( King et al. 1988).
Examples of studies investigating susceptibility to disinfection:
- Blaser et al. compared the inactivation of three C. jejuni strains and E. coli by chlorine and monochloramine by plate counting. Cells (at an initial concentration of around 105 CFU per ml) were exposed to 0.1 mg chlorine and 1.0 mg of monochloramine per liter at pH 6 and pH8. Two temperatures (4°C and 25°C) were studied. For chlorine disinfection was as expected more rapid at pH 6 than at pH8, whereas the loss of culturability was similar for the two temperatures. For the C. jejuni strains, loss of culturability was more than 2 log10 units within 30 sec to 5 min of contact time and was comparable or greater than the inactivation of E. coli. A C. jejuni strains passaged through mice was as susceptible to chlorine as the laboratory-passaged strains. For monochloramine, inactivation was faster at the lower pH and at 25°C. For the C. jejuni strains a 2 log10 reduction was obtained within 5 to 15 min for all conditions tested and inactivation tended to be faster than for E. coli. ( Blaser et al. 1986)
- Sinton et al. examined the sunlight inactivation of Campylobacter jejuni, Salmonella enterica and E. coli in 100 l chambers of seawater and river water. The water was supplemented with waste stabilization pond effluent, seeded with laboratory-grown bacteria, and exposed to sunlight in summer and winter experiments. C. jejuni was found to be most significantly more susceptible (probably due to its high susceptibility to photooxidative damage) than Salmonella enterica and E. coli. ( Sinton et al. 2007
- Chaveerach et al. examined the survival of 10 strains of C. jejuni and C. coli exposed to acidic conditions. Acidified water is commonly used in food industries. When incubated in MH broth acidified with formic acid to pH4, the bacteria lost culturability within 2 hours whereas the number of total cells remained constant over the observation time of 4 hours. Numbers of viable cells capable of CTC reduction decreased in a strain-dependent manner, but were still significant after the study period indicating the adoption of a VBNC state. Although bacterial growth could not be recovered in enrichment broth, injection into fertilized chicken eggs showed that some strains could be resuscitated after a 2 hour exposure to acid. ( Chaveerach et al. 2003)
spp. survive in untreated and inadequately treated aquatic environments with differences among strains ( Buswell et al. 1998
). Persistence in the aquatic environment depends on a variety of factors including temperature, nutrients, water type, oxygenation, light exposure, presence of other microorganisms, and predation. Temperature and light are considered to be among the most significant determinants ( Thomas et al. 2002
). Although Campylobacter
spp. are fastidious and do not grow at temperatures lower than app. 31°C, they persist at lower temperatures ( Chan et al. 2001
). Survival in water environments ranges from a few hours (37°C) to several weeks (4°C) ( Buswell et al. 1998
; Theron and Cloete 2002
). Temperature-dependent changes in the fatty acid composition, morphology, and concentration of excreted metabolites were reported ( Höller et al. 1998
). C. jejuni
was found to survive longer in culturable form than C. coli
in water both at 4°C and 20°C which might explain its more frequent isolation from surface water ( Korhonen and Martikainen 1991
). In a sterile water microcosm, mean survival times of different isolates were 202 h (4°C), 167 h (10°C), 43 h (22°C) and 22 h (37°C) with the greatest change between 10°C and 22°C ( Buswell et al. 1998
). Survival in this experiment was significantly enhanced by close presence of other organisms and in biofilms. For example, survival of strains 9753 and CH1 in the microcosm at 4°C increased 2.3 and 3 fold, respectively in the presence of autochthonous water microflora ( Buswell et al. 1998
spp. may enter a VBNC state when suspended in water or when subjected to starvation or stress ( Hege et al. 2000
). VBNC cells have been found to be metabolically active and to persist at lower temperatures ( Chan et al. 2001
). The infectivity of cells once they have entered such a state is, however, subject of controversy. C. jejuni
became non-culturable in sterilized pond water at 4°C between 18 and 28 days, depending on the strain ( Jones et al. 1991
). Two out of four strains maintained the potential to colonize mice after being in water for 6 weeks although parts of the suspended cells showed signs of degradation using electron microscopy. In a different experiment, Ziprin et al. inoculated day-of-hatch chicks with a culture of normal cecal organisms to establish a normal gut microflora. The same animals were administered 2 days later with a high dose (> 109
CFU) of VBNC C. jejuni
( Ziprin and Harvey 2004
). Cecal contents were collected seven days after the pathogen challenge and examined for the presence of culturable C. jejuni
. The negative result led to the conclusion that VBNC C. jejuni
cells were unable to revert to a culturable form. These and similar other observations are in contrast to other studies where VBNC cells were reported to successfully colonize mice (see study summary below, Baffone et al. 2006
). In addition to strain-dependent differences, also the strain origin might be important for survival of C. jejuni
in water: Poultry isolates survived longer on average than human isolates in sterilized drinking water at 4°C ( Cools et al. 2003
). Also the choice of culture conditions was found to be an important factor for survival studies with some media supporting growth longer than others ( Cools et al. 2003
). Another factor to be considered is the temperature at which cultures used for survival studies were grown. Duffy and Dykes showed that C. jejuni
grown at 37°C (body temperature of humans and cattle) and subsequently spiked into water (stored at 4°C) could be cultured longer than the same strain cultured at 42°C (body temperature of poultry) ( Duffy and Dykes 2006
It has to be noted that data on survival in water greatly depends on the detection method chosen. This was illustrated by Lehtola et al. who detected C. jejuni only for one day by cultivation after spiking potable water biofilms, whereas the bacteria could be detected from for at least 1 week and from using FISH ( Lehtola et al. 2006). Even when using cultivation, the choice of the medium and pre-culture conditions have a great impact on results ( Guillou et al. 2008, for example see below). Passage in embryonated chicken eggs was suggested as an efficient way to resuscitate Campylobacter cells in a VBNC state ( Cappelier et al. 1999; Chaveerach et al. 2003). Interestingly, resuscitation of cells previously negative in culturability cells was also successful after inoculation into fresh amoeba cultures ( Axelsson-Olsson et al. 2005). Microscopic examination of resuscitated samples showed an abundance of amoebae with ruptured cell walls and the emergence of large numbers of planktonic cells with Campylobacter-like morphology. Also survival of culturable Campylobacter spp. was reported to be greatly enhanced when Campylobacter become ingested by protozoa ( Snelling et al. 2005). On the other hand, grazing by planktonic crustaceans and predation might contribute to Campylobacter reduction ( Schallenberg et al. 2005; Korhonen and Martikainen 1991).
Studies on survival in water are numerous, examples are given in the following:
- Cook and Bolster compared the survival of C. jejuni in groundwater microcosms to that of E. coli. Groundwater might be an especially beneficial environment for survival (e.g. low redox potential, low temperatures, lack of light exposure. Microcosms with water of different sources and nutrient contents were seeded with app. 107 CFU per ml and kept in the dark at 4°C. Culturability of C. jejuni in all microcosms decreased below detection limits within 85 days, whereas E. coli could be cultured for at least 470 days. C. jejuni survival was greatest in groundwater with the highest concentration of DOC and NH4. Survival was shortest in a microcosm with deionized water (<35 days). For every microcosm the die-off rate was found to be significantly (2.5 to 13.5 fold) higher for C. jejuni compared to E. coli. Times required for a 99% decrease in culturability (t99) in different groundwaters ranged between 14 and 35 days for C. jejuni and between 91 and 186 days for E. coli. Analysis by qPCR and direct cell counts suggested that DNA and cellular integrity was maintained for both species for the duration of the experiment (up to app. one year). C. jejuni cell morphology changed from spiral to coccoid over the course of the experiment. Although at the time point when culturability was lost, cells were predominantly coccoid, a significant percentage of cells retained its spiral form. In an experiment with water from an underground stream, culturability was compared to direct cell counts and respiratory activity (using CTC). When C. jejuni cell counts dropped to zero at day 42, more than 2 x 105 cells per ml were still detected CTC-ositive. Respiratory activity was maintained for more than 100 days without change of total cell counts. For E. coli, plate counts, total cell counts and CTC positive cells remained essentially unchanged until the end of the experiment of 140 days. ( Cook and Bolster 2007).
- Guillou et al. studied the survival of C. jejuni in bottled mineral water stored at 4°C in the dark. Survival was monitored by plate counting on two different media. Starting from an inoculum of app. 107 CFU per ml, culturability dropped over time. Culturable cells were seen from 28 days until the end to the study period of 48 days. Survival was better in water with high mineral content compared to water with low mineral content. Apart from differences between strains, pre-culture conditions had a great impact on survival monitoring. Colony counts could be obtained longer using CBA medium compared to Karmali medium. Precultivation in broth significantly enhanced cultivability compared to precultivation on agar. Recovery of non-cultivatable cells after passaging in embryonated chicken eggs showed that loss of culturability was not correlated with complete cell death ( Guillou et al. 2008).
- Tatchou-Nyamsi-König et al. studied the survival of C. jejuni in experimentally contaminated bottled natural mineral water at 4 and 25°C. Complete loss of culturability at 25°C was seen around 5 – 6 days and was at least 14 times greater at 25°C than at 4°C. The presence of autochthonous flora had no significant effect on survival: At 25°C the culturability decay constant kd was -0.71 day-1 in non-filtered water and -0.76 day-1 in filtered water. Addition of biodegradable organic matter (sterile Brucella broth) was reported to slow down loss of culturability with decay rates changing from -2 day-1 (without organic matter) to -0.9 day-1 (with organic matter). However, no dose-effect relationship between the amount of organic matter and survival was observed. Addition of organic matter resulted in a multiplication of C. jejuni: the number of cells increased while culturability at the same time decreased. This discrepancy was explained by C. jejuni entering a viable-non culturable state. At the same time the proportion of bacteria taking up propidium iodide decreased from day 2 to day 8 suggesting the cells had intact membranes ( Tatchou-Nyamsi-König et al. 2007)
- Baffone et al. evaluated the survival of different C. jejuni strains (9 of clinical origin and one ATCC reference strain) in artificial seawater microcosms using plate counts, CTC-DAPI double staining and CTC-fluorescent antibody detection. Samples were seeded with bacteria to a concentration of app. 106 CFU per ml and kept at 4°C. Strains were classified into three different groups based on their different growth profiles. Viability parameters were monitored for 150 days. Whereas culturability disappeared within 12 to 35 days (depending on the strain), DAPI counts remained unchanged for the study period. Values of over 103 CTC-respiring bacteria per ml were detectable for up to 60 days. Metabolic activity was measured until 138 to 152 days and was not correlated to the culturability properties of the different strains and their entry in a VBNC state. To assess potential recovery of VBNC cells, 0.1 ml aliquots from 30-, 45-, 60-, and 90-day old microcosms were used to intragastrically inoculate Balb/C mice. With 30-and 45-day old microcosms, colonization of challenged mice with VBNC cells was nearly as efficient as a control where mice were challenged with culturable bacteria. With 60-day old microcosms, only two strains were able to colonize mice, and no colonization was seen with 90-day old microcosms. Strains from 60-day old microcosms which successfully colonized mice revealed 1 x 104 CTC-respiring bacteria/ml (in comparison, culturable cells colonized mice at 103 cells/ml). ( Baffone et al. 2006)
- Thomas et al. studied survival and morphological changes of thermophilic Campylobacter spp. (C. jejuni, C. coli, C. lari) in a modelled aquatic system. The study confirmed the positive relationship between temperature and loss of culturability seen in previous studies. The decay at 20°C was significantly greater than at 10°C with culturability being lost between 7 and 14 days. The decline in culturability was accompanied by a morphological transition from spiral or gull-wing (typical for active cells) to rod-like elongated shapes. Also the number of metabolically active cells capable of CTC reduction declined significantly faster at 20°C than at 10°C ( Thomas et al. 2002). These results correlate with an earlier study examining the survival of C. jejuni, C. coli, C. upsaliensis, and C. lari in batch microcosms containing various water types and seeded with app. 107 CFU per ml. Whereas culturability in all water types was lost rapidly within days at 25°C and 37°C, plate counts were obtained for at least 40 days at 15°C. At 5°C culturability declined only around 2-3 log10 units within 60 days. Collective decay rates in sterile river water were smaller than those in sterile de-ionized water, especially at 5 and 15°C. Additional nutrients in sterile river water microcosms with sediment did not significantly reduce decay rates. Among the four species, C. jejuni and C. lari demonstrated most resilience ( Thomas et al. 1999).
- Talibart et al. studied the persistence of 85 Campylobacter strains in microcosms, which were seeded with app. 108 CFU per ml and stored at 4°C in the dark. Cultivability declined progressively over time. Based on their persistence, strains were classified in 3 groups. Group 1 (12 C. coli and 6 C. jejuni strains) lost culturability within 14 days; group 2 (31 C. coli and 27 C. jejuni strains) produced plate counts for 14 to 21 days; group 3 (6 C. coli and 3 C. jejuni strains) could be cultured for more than 21 days. Two strains (C. jejuni ATCC 33560 and C. coli A849) were reported to still grow after 35 days in microcosms when shaken and > 60 days without shaking. The authors tested the potential of VBNC cells for resuscitation. One ml of 47 microcosms containing different strains, which were not able to grow after 30 days in liquid Preston medium, were injected in 9-day old fertilized chicken eggs: 51% of these strains could be revived under the chosen experimental conditions ( Talibart et al. 2000).
- Obiri-Danso et al. examined the effects of artificial sunlight and temperature on the survival of different Campylobacter species. When exposed to artificial sunlight (equivalent to a sunny day in June in the UK), natural C. lari populations and urease-positive thermophilic campylobacters (UPTC) in seawater and C. jejuni in river water lost culturability within 30 min. The T90 time to reduce the initial culturability by 90% was reported to be 25 min for a river water population and 15 min for a seawater population, respectively. Cultures of Campylobacter isolates lost culturability in distilled water and artificial seawater within 40 to 60 min when exposed to artificial sunlight. In darkness, survival was reported to be determined by the temperature with natural populations being cultivatable for 12 hours at 37°C and 5 days at 4°C in seawater and slightly less in river water. The results suggested that C. lari and UPTCs survive longer than C. jejuni and C. coli under the conditions chosen, especially in the dark. Due to the efficient inactivation by UVB, intense sunlight in the summer together with elevated water temperatures might explain low culturable campylobacter numbers in environmental waters ( Obiri-Danso et al. 2001).
The infectious dose of C. jejuni
is estimated to be between app. 500-800 cells based on human volunteer studies ( Black et al. 1988
; Robinson DA 1981
). A dose of 800 cells produced clinical symptoms in 10% of humans ( Thomas et al. 1999
). A correlation between ingested dose and disease rate has been suggested ( Black et al. 1992
). The observation that attack rates during outbreaks vary has been attributed to protective immunity acquired by regular exposure to low doses of Campylobacter
( Thomas et al. 1999
Many PCR methods are used in combination with enrichment cultures. The sample volumes in this respect have to be chosen carefully. In the case of turbid water, problems with cultivation can arise from processing large sample volumes. Abulreesh et al. examined the recovery of thermophilic campylobacters from volumes of 10, 100, and 1000 ml of turbid pond water or 0.1, 1.0, or 5.0 ml of sediment using selective enrichment and subsequent biochemical and genetic confirmation ( Abulreesh et al. 2005
isolates were never obtained from the highest sample volumes, but only from the smallest and the intermediate volumes. It was concluded that processing of large sample volumes was counterproductive, probably due to antagonistic effects of large numbers of background bacteria outcompeting campylobacters during the enrichment stage. Detection methods include:
- FISH: Campylobacter spp. can be detected from enrichment cultures or directly from water samples using FISH. Lehtola et al. reported specific detection of thermotolerant C. coli, C. jejuni, and C. lari using peptide nucleic acid (PNA) probes. The probes targeting 16S rRNA had higher affinity than the corresponding DNA probes. Hybridization signals depended on the physiological state of the cells and were stronger with actively growing cultures compared to aging cultures with coccoid morphologies. The method was successfully validated by detection of C. coli in spiked tap water samples (100 ml containing app. 102 and 104 CFU per ml were filtered and analyzed). ( Lehtola et al. 2005)
- PCR: A PCR assay targeting 16S and 23S rRNA genes for detection of Arcobacter and thermotolerant Campylobacter species was presented. The detection limits were determined with seeded river water samples and were 103 (without enrichment) and 1 (with 24 h enrichment) for Arcobacter and 102 (without enrichment) and 10 (with 24 h enrichment) for Campylobacter species ( Moreno et al. 2003b). Lübeck et al. compared 15 published and unpublished PCR primers targeting the 16S rRNA gene in all possible pairwise combinations, and two primers targeting the 23S rRNA gene. Only one primer pair was found to be selective. Based on the results, a standardized quantitative PCR assay (for food applications) was developed employing the Tth polymerase which proved significantly less susceptible towards inhibition than Taq polymerase or DyNAzyme. The standardized assay included an IAC and was designed to target C. jejuni, C. coli, and C. lari. The detection limit for C. jejuni was reported to be 17 genome copies per PCR reaction when amplified without IAC ( Lübeck et al. 2003).
- qPCR: A TaqMan assay for rapid detection of C. jejuni had been presented based on the amplification of the VS1 gene. Standard curves with diluted DNA suggested a detection limit of 1 CFU per reaction without prior enrichment. The assay was validated by screening 300 environmental water samples (surface and groundwater) besides 300 frozen chicken meat and 300 milk samples). A significant number of samples (13.6% of water, 30.6% of meat, and 27.3% of milk samples) tested positive ( Yang et al. 2003).
- RT-PCR: An assay was reported for specific detection of C. jejuni, C. coli, and C. upsaliensis. Primers (published earlier by Jackson et al. ( Jackson et al. 1996) targeted a 256 bp sequence of an open reading frame. An mRNA signal was obtained with heat-killed C. jejuni cells directly after heat killing, but not after 4 hours when the cells were held at 37°C. The assay thus had the ability to distinguish between live and dead ( Sails et al. 1998). No detection limit was determined for the RT-PCR assay, but a sensitivity of 25 CFU per reaction were reported for the previously published corresponding PCR assay ( Jackson et al. 1996).
- PCR-ELISA: The assay is based on PCR-amplification of ORF-C gene sequences and a colorimetric end-point detection format. DIG-labels incorporated into the PCR product during amplification were hybridized to biotinylated capture probes complementary to target gene sequences. Colorimetric detection was performed in a microtitre plate format. The sensitivity of the PCR ELISA assay was demonstrated to be 10-100-fold more sensitive than a gel-based PCR method using the same primers in an earlier publication ( Sails et al. 2001). When applied to the detection of C. jejuni and C. coli in environmental waters, detection was achieved with concentrations between 20 to >108 cfu per ml after enrichment ( Sails et al. 2002).
- Multiplex PCR: The assay targeting the lipid A gene lpxA was designed to identify and discriminate between isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis ( Klena et al. 2004).
- Multiplex PCR–microarray: Both universal bacterial (16S rRNA genes) and specific Campylobacter sequences were amplified in a multiplex PCR reaction. Specific primers targeted 16S rRNA genes for genus level detection, 16S-23S rRNA internal regions for C. coli detection, and the hippuricase gene for C. jejuni detection. Fluorescently labeled amplification products were hybridized to immobilized capture probes on a microarray. The detection limit of the PCR was reported to be 3-30 genome copies of Campylobacter. The sensitivity of the microarray hybridization reaction was app. 3 genome equivalents. The two species C. jejuni and C. coli could be successfully differentiated ( Keramas et al. 2003). The method was applied in a later study to detect Campylobacter directly from chicken feces ( Keramas et al. 2004).
- Multiplex PCR and CE (Agilent 2100 bioanalyzer): Alternatively to hybridizing the PCR products from Keramas et al. 2003 to a microarray, they were analyzed by capillary electrophoresis in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.). The microchip-based device provides gel-like images and replaces agarose gel electrophoresis. The detection limit was around 300 genome copies and thus about 100 fold less compared to hybridizing the amplicons to a microarray ( Keramas et al. 2003; Keramas et al. 2004).
- NASBA: The assay based on the amplification of 16S rRNA was established for food control and detected as few as 3-5 CFU C. jejuni in 10 g of the original food sample with a 18 h enrichment. Detection of C. jejuni was achieved up to a 10,000:1 ratio of indigenous flora to C. jejuni. 16S rRNA amplification products were analyzed using a nonradioactive hybridization procedure (enzyme-linked gel assay, ELGA). Time demand for complete procedure around 26 hours ( Uyttendaele et al. 1995)
- Real-time NASBA: The assay was based on amplification of mRNA of the tuf- and GTPase genes. A detection limit 102 cells/reaction was achieved for the tuf gene-based assay. The authors pointed out, however, that the detection of the tuf gene transcript is not a good viability indicator due to its stability, also dead cells were detected ( Cools et al. 2006).
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.
This Campylobacter fetus (C. fetus ss. jejuni) culture was grown on Skirrow's and Butzler's medium.
Photo ID: 3918
Credit: not stated
Content provider(s): Centers for Disease Control and Prevention/Sheila Mitchell
This scanning electron micrograph depicts a grouping of Gram-negative Campylobacter fetus bacteria, magnified 4,976x.
Photo ID: 5776
Credit: Janice Carr
Content provider(s): Centers for Disease Control and Prevention/ Dr. Patricia Fields, Dr. Collette Fitzgerald
This scanning electron micrograph depicts a number of Gram-negative Campylobacter jejuni bacteria, magnified 9,951x.
Photo ID: 5781
Credit: Janice Carr
Content provider(s): Centers for Disease Control and Prevention/ Dr. Patricia Fields, Dr. Collette Fitzgerald
This scanning electron micrograph depicts a number of Gram-negative Campylobacter jejuni bacteria, magnified 20,123x.
Photo ID: 5780
Credit: Janice Carr
Content provider(s): Centers for Disease Control and Prevention/ Dr. Patricia Fields, Dr. Collette Fitzgerald