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.     

Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells

Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10(7) cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 microliter mg of protein-1 for the culturable form and 10.96 microliter mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.     

CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46–48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.     

Culturability, injury and morphological dynamics of thermophilic Campylobacter spp. within a laboratory-based aquatic model system

AIMS: To study the survival processes of thermophilic Campylobacter spp. within a modelled aquatic system and particularly the involvement and survival potential of viable but non-culturable forms. METHODS AND RESULTS: The survival and morphological characteristics of populations of thermophilic Campylobacter species exposed to simulated aquatic conditions were examined using a combination of cultural and microscopic techniques. Populations underwent progressive decay when exposed to simulated aquatic conditions. The rates of population decay were observed to be significantly greater at the higher temperature (20 degrees C) with a rapid transition of the dominant sub-populations from non-stressed to dead cells occurred within 3 days. At 10 degrees C the rate of culturability loss was much reduced with substantial development (approx. 80% of total population) of viable but non-culturable (VBNC) populations by all species within 3 days, declining to represent approximately 5-25% of the total population at day 60. Significant differences (P < 0.001) were identified between decay rates as a consequence of different species, sub-populations and temperature but not between sub-populations of different species. Morphological variants including spiral, elongated spirals and rods, short rods and coccoid forms were identified. The endpoints of morphological transition were temperature-independent and isolate-specific yet the rate of morphological transition was directly related to temperature and approximately equivalent between species. CONCLUSION: The VBNC state is a transitory stage in the degeneration of Campylobacter population within the aquatic environments simulated during this study. SIGNIFICANCE AND IMPACT OF THE STUDY: VBNC cells form the most persistent, viable, potentially pathogenic sub-population of Campylobacter populations exposed to aquatic stress conditions.     

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.     

Recovery in embryonated eggs of viable but nonculturable Campylobacter jejuni cells and maintenance of ability to adhere to HeLa cells after resuscitation.

The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.     

Recovery in Embryonated Eggs of Viable but Nonculturable Campylobacter jejuni Cells and Maintenance of Ability To Adhere to HeLa Cells after Resuscitation

The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.     

Solid phase cytometry as a tool to detect viable but non-culturable cells of Campylobacter jejuni

Solid phase cytometry (SPC) in conjunction with fluorescent viability staining has been investigated as a tool to detect viable but non-culturable Campylobacter jejuni in drinking water. Inoculated water samples were filtered over a polyester membrane filter and the retained cells were stained using a carboxyfluorescein ester as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device (ChemScan) in 3 min. In parallel, the plate count was determined on Columbia Blood Agar. The number of culturable cells decreased below the detection limit of plate counting in less than 50 days. In contrast, the number of fluorescent bacteria remained at its initial level for at least 85 days. The discrepancy between the two results can be attributed to the transition of culturable C. jejuni cells into VBNC C. jejuni cells. Furthermore, as SPC can distinguish between low numbers of dividing and non-dividing cells of Campylobacter it has the potential to monitor attempts to resuscitate VBNC cells.     

Development and application of an insertional system for gene delivery and expression in Campylobacter jejuni

The genetic investigation of Campylobacter jejuni, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for Escherichia coli and other bacteria cannot be maintained in Campylobacter cells. Additionally, gene expression in Campylobacter requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other Campylobacter strains. The presence of a Campylobacter-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of Campylobacter. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing Campylobacter allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.     

Induction and Resuscitation of the Viable but Nonculturable State in a Cyanobacteria-Lysing Bacterium Isolated from Cyanobacterial Bloom

The viable but nonculturable (VBNC) state has been found to be a growth strategy used by many aquatic pathogens; however, few studies have focused on VBNC state on other aquatic bacterial groups. The purpose of this study was to explore the VBNC state of cyanobacteria-lysing bacteria and the conditions that regulate their VBNC state transformation. Three cyanobacteria-lysing heterotrophic bacterial strains (F1, F2 and F3) were isolated with liquid infection method from a lake that has experienced a cyanobacterial bloom. According to their morphological, physiological and biochemical characteristics and results of 16SrDNA sequence analysis, F1, F2 and F3 were identified as strains of Staphylococcus sp., Stappia sp. and Microbacterium sp., respectively. After being co-cultured with the axenic cyanobacterium, Microcystis aeruginosa 905, for 7 days, strains F1, F2 and F3 exhibited an inhibition effect on cyanobacterial growth, which was expressed as a reduction in chlorophyll concentration of 96.0%, 94.9% and 84.8%, respectively. Both autoclaved and filtered bacterial cultures still showed lytic effects on cyanobacterial cells while centrifuged pellets were less efficient than other fractions. This indicated that lytic factors were extracelluar and heat-resistant. The environmental conditions that could induce the VBNC state of strain F1 were also studied. Under low temperature (4°C), distilled deionized water (DDW) induced almost 100% of F1 cells to the VBNC state after 6 days while different salinities (1%, 3% and 5% of NaCl solution) and lake water required 18 days. A solution of the cyanobacterial toxin microcystin-LR (MC-LR) crude extract also induced F1 to the VBNC state, and the effect was stronger than DDW. Even the lowest MC-LR concentration (10 μg L⁻¹) could induce 69.7% of F1 cells into VBNC state after 24 h. On the other hand, addition of Microcystis aeruginosa cells caused resuscitation of VBNC state F1 cells within 1 day, expressed as an increase of viable cell number and a decrease of VBNC ratio. Both VBNC state and culturable state F1 cells showed lytic effects on cyanobacteria, with their VBNC ratio varying during co-culturing with cyanobacteria. The findings indicated that VBNC state transformation of cyanobacteria-lysing bacteria could be regulated by cyanobacterial cells or their toxin, and the transformation may play an important role in cyanobacterial termination.     

Morphology and adhesion of Campylobacter jejuni to chicken skin under varying conditions

The adhesion of Campylobacter jejuni to chicken skin, along with the associated morphological changes under aerobic conditions at 4, 25, and 37 degrees C and microaerobic (O2 5%, CO2 10%, N2 85%) conditions, were investigated using confocal laser scanning microscopy (CLSM), flow cytometry, and plate counting. The morphological change of C. jejuni from a spiral shape to a coccoid form or VBNC form (viable but nonculturable form) progressed rapidly under aerobic conditions at 25, 37, and 4 degrees C. As regards adhesion, the C. jejuni cells were mostly located in the crevices and feather follicles of the chicken skin, where the cells in the feather follicles floated freely in the entrapped water, even after the skin was rinsed quite thoroughly. CLSM also revealed the penetration of some spiral-shaped C. jejuni cells into the chicken skin. Even after changing their shape at various temperatures, coccoid-form C. jejuni cells were still found in the crevices and feather follicles of the chicken skin.     

Acanthamoeba-Campylobacter coculture as a novel method for enrichment of Campylobacter species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10(4) CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states

Enterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that cause division block. In those conditions, bacteria present in the human body would hardly be in an exponentially growing phase but would mostly be in physiological states such as starvation or the viable but nonculturable (VBNC) state. The possibility that the starved and VBNC bacteria can maintain their ability to adhere to living and inanimate substrates is the first mandatory step for them potentially to cause an infection process. In this study it is shown that starved and stationary enterococcal cells are able to form biofilms on plastic material albeit with reduced efficiency as compared to growing cells. Moreover, although VBNC enterococcal forms are not capable of forming biofilms, Enterococcus faecalis and other enterococcal species of medical interest maintain their ability to synthesize the polymeric matrix for a limited period of time under adverse environmental conditions. The data presented, together with those regarding the maintenance of the division recovery potential already proved in nonculturable bacteria, further support the possibility for the VBNC and other nondividing bacterial forms to have a role as infectious agents and to constitute a risk to human health.     

Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni.

Seven strains of Campylobacter jejuni, isolated from various sources [human (n = 2), chicken (n = 3), water (n = 2)], were studied under starvation conditions in filter-sterilized and pasteurized surface water by acridine orange direct count (AODC), viable count (DVC) and culture methods. Plate counts showed a rapid decline (2 log-units/day) for all strains under these conditions. Only one of the seven strains (14%) showed a (prolonged) viable, non-culturable 'state'. The ability of these viable, non-culturable cells to colonize the intestine was tested on day-old chicks. The infectious oral dose of freshly cultured cells of this model was 26-260 cfu; 1.8 x 10(5) viable, non-culturable C. jejuni were introduced to day-old chicks orally. Campylobacter jejuni was not isolated from the caeca of the chicks after incubation for 7 d. Also, passage through the allantoic fluid of embryonated eggs did not recover viable, non-culturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni. The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO2 + 80% N2, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O2 + 10% CO2 + 85% N2. The packaging material in the first two treatments was PA 80/PE 100-PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37 degrees C, 20 degrees C and 4 degrees C for 48 h, 4 days and 25 days, respectively. At 37 degrees C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20 degrees C and at 4 degrees C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37 degrees C its numbers increased only in the optimal gas atmosphere; at 20 degrees C the strain was not detectable after 24 to 48 h storage and at 4 degrees C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

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.     

Biphasic culture system for rapid Campylobacter cultivation

We developed a biphasic culture system consisting of 4 ml of brucella agar (BA) and 6 ml of brucella broth (BB) in 25-cm2 tissue culture flasks, which were incubated in air (BB/BAa) or in a gas mixture of 5% O2, 10% CO2, and 85% N2 (BB/BAg). These media were also used with a supplement consisting of ferrous sulfate, sodium metabisulfite, and sodium pyruvate and incubated as above (FB/FAa and FB/FAg, respectively). Highly satisfactory growth of Campylobacter jejuni 301 was obtained with all medium-gas phase combinations provided that the number of viable cells in the inoculum was large (greater than or equal to 10(6)/ml). The use of FB/FAa permitted the inoculum to be reduced to 100 cells per ml. With an adjusted gas phase (BB/BAg and FB/FAg), near-optimal growth was obtained from an inoculum of 1 to 10 cells per ml. Under most of these conditions the generation time was approximately 90 min. During the logarithmic growth phase, the cells retained their typical spiral morphology and high motility. These media also proved to be highly satisfactory for the cultivation of fresh isolates as well as other stock strains of Campylobacter. When the broth phase of the cultures, after addition of 15% glycerol, was quickly frozen and maintained at -70 degrees C, all strains thus far examined were readily recoverable and satisfactorily cultivated without additional passage.     

Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes

A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in filtered, sterilized distilled water. We used different concentrations of ciprofloxacin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and ciprofloxacin. The mixture was incubated at different incubation times at 37 degrees C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solution or orange acridine. The filters were prepared and examined by epifluorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.     

Biphasic culture system for rapid Campylobacter cultivation.

We developed a biphasic culture system consisting of 4 ml of brucella agar (BA) and 6 ml of brucella broth (BB) in 25-cm2 tissue culture flasks, which were incubated in air (BB/BAa) or in a gas mixture of 5% O2, 10% CO2, and 85% N2 (BB/BAg). These media were also used with a supplement consisting of ferrous sulfate, sodium metabisulfite, and sodium pyruvate and incubated as above (FB/FAa and FB/FAg, respectively). Highly satisfactory growth of Campylobacter jejuni 301 was obtained with all medium-gas phase combinations provided that the number of viable cells in the inoculum was large (greater than or equal to 10(6)/ml). The use of FB/FAa permitted the inoculum to be reduced to 100 cells per ml. With an adjusted gas phase (BB/BAg and FB/FAg), near-optimal growth was obtained from an inoculum of 1 to 10 cells per ml. Under most of these conditions the generation time was approximately 90 min. During the logarithmic growth phase, the cells retained their typical spiral morphology and high motility. These media also proved to be highly satisfactory for the cultivation of fresh isolates as well as other stock strains of Campylobacter. When the broth phase of the cultures, after addition of 15% glycerol, was quickly frozen and maintained at -70 degrees C, all strains thus far examined were readily recoverable and satisfactorily cultivated without additional passage.