Survival of Escherichia coli and Campylobacter jejuni in untreated and filtered lake water

L.K. KORHONEN AND P.J. MARTIKAINEN. 1991. The survival of Campylobacter jejuni and Escherichia coli in lake water was studied using viable counts. Escherichia coli survived better than C. jejuni in all the test conditions studied. Both the species survived better in filtered than in untreated water. This suggests that predation and/or competition for nutrients affect the survival of both the species in an aquatic environment. Campylobacter jejuni survived less well in filtered autoclaved water and in 0.9% NaC1 than in filtered water without autoclaving. The lack of some essential nutrients, which may be degraded by autoclaving, might explain these results.     

Survival and injury of Escherichia coli, Campylobacter jejuni, and Yersinia enterocolitica in stream water.

Experiments were done to describe the survival and injury of three strains each of enteropathogenic Escherichia coli, Yersinia enterocolitica, and Campylobacter jejuni in agricultural surface water. Bacterial suspensions within membrane diffusion chambers were immersed in two large vessels of stream water that were held at 6 and 16 degrees C and changed daily. The results of daily plate counts, using selective and nonselective media, revealed some variation among strains and genera. Injury increased rapidly in all of the bacteria examined during an initial 4-day period of population stability. Bacterial persistence was generally prolonged at 6 degrees C, while the occurrence of injury was directly related to temperature. However, both survival and injury in C. jejuni were less dependent on temperature, while Y. enterocolitica displayed the greatest survival at both 6 and 16 degrees C. These results suggest that surface water in the temperate zone might serve as a persistent vehicle for the transmission of these enteropathogenic bacteria between animals and humans.     

Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged starvation at low temperatures

AIMS: To evaluate the survival of Campylobacter jejuni relative to that of Escherichia coli in groundwater microcosms varying in nutrient composition. METHODS AND RESULTS: Studies were conducted in groundwater and deionized water incubated for up to 470 days at 4 degrees C. Samples were taken for culturable and total cell counts, nutrient and molecular analysis. Die-off in groundwater microcosms was between 2.5 and 13 times faster for C. jejuni than for E. coli. Campylobacter jejuni had the lowest decay rate and longest culturability in microcosms with higher dissolved organic carbon (4 mg l(-1)). Escherichia coli survival was the greatest when the total dissolved nitrogen (12.0 mg l(-1)) was high. The transition of C. jejuni to the coccoid stage was independent of culturability. CONCLUSION: The differences in the duration of survival and response to water nutrient composition between the two organisms suggest that E. coli may be present in the waters much longer and respond to water composition much differently than C. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from these studies would aid in the evaluation of the utility of E. coli as an indicator of C. jejuni. This study also provided new information about the effect of nutrient composition on C. jejuni viability.     

Survival and resuscitation of ten strains of Campylobacter jejuni and Campylobacter coli under acid conditions

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4',6'-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.     

Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.     

Survival of Campylobacter jejuni strains of different origin in drinking water

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.     

A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

A PCR assay has been developed for the detection of Campylobacter jejuni and Camp. coli in water samples. The sample is filtered through a membrane which is subjected to sonication to release the impacted cells. After removal of the filter from the cell suspension and a freeze/thaw cell lysis step, a semi-nested PCR is carried out on the filtrate using the primers CF02, CF03 and CF04 ( Camp. jejuni fla and flaB gene sequences). Incorporation of a sonication stage allows removal of the filter membrane since they have been shown to inhibit the PCR. In experiments with spiked water samples (20 ml) a theoretical sensitivity of 10–20 Campylobacter cells ml^-1 was achieved. Using a sample volume of 100 ml this sensitivity can be increased to approximately 2 Campylobacter cells ml^-1.     

The Evolution of Campylobacter jejuni and Campylobacter coli

The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.Campylobacter jejuni and Campylobacter coli remain among the most common causes of human bacterial gastroenteritis worldwide (Friedman et al. 2000). In high-income countries, Campylobacteriosis is much more common than gastroenteritis caused by Escherichia coli, Listeria, and Salmonella, and accounts for an estimated 2.5 million annual cases of gastrointestinal disease in the United States alone (Kessel et al. 2001). Infection with these bacteria is also a major cause of morbidity and mortality in low- and middle-income countries, although it is almost certainly underreported in these settings, especially as culture confirmation remains challenging. Poor understanding of the transmission of these food-borne pathogens to humans in all income settings has contributed to the failure of public health systems to adequately address this problem. As a consequence, over the past 20 years, much investment has been directed at understanding how these bacteria are transmitted from reservoir hosts to humans through the food chain.Although the disease was first recognized by Theodor Escherich in 1886, who described the symptoms of intestinal Campylobacter infections in children as “cholera infantum” (Samie et al. 2007) or “summer complaint” (Condran and Murphy 2008), difficulties in the culture and characterization of these organisms precluded their recognition as major causes of disease until the 1970s. Campylobacteriosis is usually nonfatal and self-limiting; however, the symptoms of diarrhea, fever, abdominal pain, and nausea can be severe (Allos 2001), and sequelae, including Guillain–Barre syndrome and reactive arthritis, can have serious long-term consequences. Subsequently, recognition of the very high disease burden of human Campylobacter infection stimulated research on these bacteria and their relatives. Since the 1970s, C. coli and C. jejuni have been isolated from a wide range of wild and domesticated bird and mammal species, in which, typically, they are thought to cause few if any disease symptoms. Humans are usually infected by the consumption of contaminated food (especially poultry meat), water, milk, or contact with animals or animal feces (Niemann et al. 2003).Most of what is known about these species comes from isolates obtained from humans with disease, the food chain, and the agricultural environment. It is, however, important to note that such isolates are by no means representative of natural Campylobacter populations, and it is becoming increasingly apparent that much of the diversity present among the Campylobacters is in strains that colonize wild animals. Increasing numbers of novel genotypes are being found as Campylobacter populations are analyzed in different animal species, especially wild birds (Carter et al. 2009; French et al. 2009); these populations undoubtedly contain many as-yet-undescribed lineages. Most human disease isolates from cases of gastroenteritis in countries, such as the United Kingdom and the United States, are C. jejuni, which typically accounts for 90% of cases in these settings, with the remaining ∼10% of cases mostly caused by C. coli. The majority of the genotypes isolated from human disease have also been isolated as commensal gastrointestinal inhabitants of domesticated and, especially, food animals. Furthermore, clinical isolates are a nonrandom subset of these strains. Asymptomatic carriage of C. jejuni and C. coli is thought to be rare in humans, especially among people in industrialized countries, suggesting that humans are not a primary host for these organisms in these settings and that people are sporadically, and frequently pathologically, infected via the food chain from animal reservoir hosts.An understanding of the relatively short history of coevolution between humans and pathogenic Campylobacters can be obtained by examining their population structure and ecology. This approach has formed the basis of many recent investigations of the cryptic epidemiology of these organisms (Lang et al. 2010; Müllner et al. 2010; Thakur et al. 2010; Hastings et al. 2011; Jorgensen et al. 2011; Kittl et al. 2011; Magnússon et al. 2011; Sheppard et al. 2011a,b; Sproston et al. 2011; Read et al. 2013) and will be the focus of this review. Such studies have included molecular epidemiological and evolutionary analyses and, in the past 15 years or so, the application of high-throughput DNA sequencing technologies of increasing capacity has enhanced the integration of these two areas of investigation to their mutual benefit.     

Survival of Escherichia coli in lake bottom sediment.

The survival of Escherichia coli in bottom sediment (Lake Onalaska, navigation pool no. 7, Mississippi River) was studied by using in situ dialysis culture of sterile (autoclaved) and unsterile sediment samples. Bags made from dialysis tubing were filled with either course sand sediment (28.8% fine) or organic, silty clay sediment (77.2% fine) and placed at the sediment-water interface. Bags representing sterile controls, unsterile uninoculated controls, autoclaved inoculated sediment, and unsterile inoculated sediment were studied during a 5-day period for each sediment type. Daily most-probable-number determinations indicated that E. coli populations in unsterile inoculated sediment fluctuated between 5.3 X 10(2) and 2.2 X 10(3) bacteria per g of silty clay and between 3.0 X 10(3) and 1.4 X 10(4) bacteria per g of sand. Autoclaved silty clay sediment inoculated with 1.0 X 10(6) bacteria per g increased to 2.2 X 10(8) bacteria per g in 3 days. During the same period, autoclaved sand sediment inoculated with 1.2 X 10(5) cells per g increased to 5.4 X 10(7) bacteria per g. By day 5, populations in both cultures had decreased by 1 log. The ability of E. coli to survive for several days in aquatic sediment in situ suggests that fecal coliforms in water may not always indicate recent fecal contamination of that water but rather resuspension of viable sediment-bound bacteria.     

Survival of Campylobacter jejuni in Waterborne Protozoa

The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers.     

Survival of Campylobacter jejuni in waterborne protozoa

The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers.     

Substrate utilization by Campylobacter jejuni and Campylobacter coli

An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four 14C-labeled substrates. Campylobacter jejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, alpha-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with alpha-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, CO2 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and alpha-ketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.     

Comparison of the survival of Campylobacter jejuni and Campylobacter coli in culturable form in surface water

Six Campylobacter jejuni and six Campylobacter coli strains were isolated from cows and pigs, and their survival in lake water was compared by viable counts. Campylobacter jejuni survived longer in culturable form than C. coli in untreated and membrane-filtered water both at 4 and 20 degrees C. This difference in survival time may be a reason why C. jejuni is generally isolated from surface waters more frequently than C. coli. Both species survived better in filtered than in untreated water. This suggests that predation and competition for nutrients affect the survival of both Campylobacter species in the aquatic environment.     

Genome maps of Campylobacter jejuni and Campylobacter coli.

Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.     

High-throughput cloning of Campylobacter jejuni ORfs by in vivo recombination in Escherichia coli

A rate-limiting and costly step in many proteomics analyses is the cloning of all of the ORFs for an organism into technique-specific vectors. Here, we describe the generation of a Campylobacter jejuni expression clone set using a high-throughput cloning approach based on recombination in E. coli. The approach uses native E. coli recombination functions and requires no in vitro enzymatic steps or special strains. Our results indicate that this approach is an efficient and economical alternative for high-throughput cloning.     

A multiple logistic model for predicting the occurrence of Campylobacter jejuni and Campylobacter coli in water

A multiple logistic regression model was established to predict the occurrence of Campylobacter jejuni/coli , related to index bacteria such as faecal coliforms, faecal streptococci, and sulphite-reducing clostridia, in a water source in southern Norway. The fitted model indicated that faecal coliforms were strong predictors for C. jejuni/coli , although the water temperature also had a strong influence on results. Sulphite-reducing clostridia, faecal streptococci, and season of the year had no significant influence on the results, in spite of their apparent predictor value as separate variables. The model employed offers a new approach to the relationship between index bacteria and the occurrence of pathogenic bacteria in water. Similar models can also be established in general food microbiology.     

Substrate utilization by Campylobacter jejuni and Campylobacter coli.

An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four 14C-labeled substrates. Campylobacter jejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, alpha-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with alpha-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, CO2 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and alpha-ketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.     

Survival of Campylobacter jejuni in different gas mixtures

Campylobacter jejuni in fresh chilled chicken meat is known to be a major risk factor for human gastrointestinal disease. In the present study, the survival under chilled conditions of different C. jejuni strains exposed to different gas mixtures usually used for gas packaging of food was examined. Bolton broth and fresh, skinless chicken fillets were inoculated with six and four strains, respectively, and exposed to the gas mixtures 70/30% O(2)/CO(2), 70/30% N(2)/CO(2), and 100% N(2) (the latter only investigated in broth) at refrigeration temperature (4-5 degrees C). In broth culture, the strains survived significantly longer when exposed to 100% N(2) and 70/30% N(2)/CO(2) than in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001). For the two anaerobic gas mixtures, the reductions only reached 0.3-0.8 log(10) CFU mL(-1) within the same period. In the presence of oxygen, the numbers of C. jejuni were reduced by a minimum of 4.6 log(10) CFU mL(-1) over 21 days. When inoculated onto chicken fillets, the C. jejuni strains also died significantly faster in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001), reaching reductions of 2.0-2.6 log(10) CFU g(-1) after 8 days. In the gas mixture without oxygen (70/30% N(2)/CO(2)), no reductions were observed.