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

G.J. MEDEMA, F.M. SCHETS, A.W. VAN DE GIESSEN AND A.H. HAVELAAR. 1992. 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; 1.8×10⁵ 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, nonculturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

Recovery of viable but non-culturable Campylobacter jejuni.

Suspensions of Campylobacter jejuni became non-culturable after storage in sterilized pond water at 4 degrees C for periods between 18 and 28 d, depending on the strain. Suspensions of four strains of C. jejuni that had been in water for 6 weeks, and shown to be non-culturable, were fed to suckling mice. Colonization of mice was established with two of the strains and failed with the other two strains. Examination of these suspensions under the electron microscope showed some cocci having the appearance of being viable, but most cocci and all remaining spiral forms showed extensive degeneration. The results indicate that non-culturable coccal forms of C. jejuni are capable of infecting mice but that this property may differ between strains.     

Colonization of chicks by non-culturable Campylobacter spp.

Six suspensions of non-culturable Campylobacter spp. were administered by gavage to day-of-hatch chicks. Four non-culturable isolates of Campylobacter spp. were found to colonize low numbers (5/79) of 1-week-old chicks, while two isolates did not (0/30). The original and recovered Campylobacter spp. isolates were serotyped and examined by restriction enzyme analysis. Evidence of clonality of two Camp, jejuni isolates was demonstrated.     

Reduction of Campylobacter jejuni colonization of chicks by cecum-colonizing bacteria producing anti-C. jejuni metabolites.

Cecum-colonizing bacteria were isolated from Campylobacter jejuni-free White Leghorn (Gallus domesticus) laying hens and screened for the ability to produce anti-C. jejuni metabolites. Nine isolates were obtained that possessed this characteristic. The peroral administration of the nine isolates as a mixture (ca. 10(9) per chick) to 1-day-old chicks was followed 1 week later by peroral inoculation of Campylobacter jejuni (ca. 10(9) per chick) to determine if the cecal isolates could protect chicks from colonization by campylobacters. The nine-strain mixture of cecal bacteria provided from 41 to 85% protection from C. jejuni colonization. The protective bacteria were reduced to a mixture of three strains on the basis of their ability to utilize mucin as a sole substrate for growth. These strains included Klebsiella pneumoniae 23, Citrobacter diversus 22, and Escherichia coli (O13:H-) 25. Four feeding trials with this three-strain mixture provided from 43 to 100% (average, 78%) protection from C. jejuni colonization. The dominant cecal bacterium of chicks treated with the three-strain mixture was consistently E. coli O13:H-. Similarly, three trials with only E. coli 25 used as the protective bacterium resulted in 49 to 72% (average, 59%) protection from C. jejuni colonization, with E. coli O13:H- being the dominant cecal bacterium in all cases. Although not completely effective, E. coli 25 substantially reduced the incidence of C. jejuni colonization of chicks. For all trials, fewer C. jejuni were present in the ceca of colonized chicks receiving the protective bacteria before exposure to C. jejuni than in chicks receiving only C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of Campylobacter jejuni colonization of chicks by cecum-colonizing bacteria producing anti-C. jejuni metabolites.

Cecum-colonizing bacteria were isolated from Campylobacter jejuni-free White Leghorn (Gallus domesticus) laying hens and screened for the ability to produce anti-C. jejuni metabolites. Nine isolates were obtained that possessed this characteristic. The peroral administration of the nine isolates as a mixture (ca. 10(9) per chick) to 1-day-old chicks was followed 1 week later by peroral inoculation of Campylobacter jejuni (ca. 10(9) per chick) to determine if the cecal isolates could protect chicks from colonization by campylobacters. The nine-strain mixture of cecal bacteria provided from 41 to 85% protection from C. jejuni colonization. The protective bacteria were reduced to a mixture of three strains on the basis of their ability to utilize mucin as a sole substrate for growth. These strains included Klebsiella pneumoniae 23, Citrobacter diversus 22, and Escherichia coli (O13:H-) 25. Four feeding trials with this three-strain mixture provided from 43 to 100% (average, 78%) protection from C. jejuni colonization. The dominant cecal bacterium of chicks treated with the three-strain mixture was consistently E. coli O13:H-. Similarly, three trials with only E. coli 25 used as the protective bacterium resulted in 49 to 72% (average, 59%) protection from C. jejuni colonization, with E. coli O13:H- being the dominant cecal bacterium in all cases. Although not completely effective, E. coli 25 substantially reduced the incidence of C. jejuni colonization of chicks. For all trials, fewer C. jejuni were present in the ceca of colonized chicks receiving the protective bacteria before exposure to C. jejuni than in chicks receiving only C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)     

空肠弯曲菌活而不可培养状态的产毒素能力及毒素基因表达的检测

目的 通过诱导空肠弯曲菌进入活的不可培养(VBNC)状态建立细胞学模型,用VeroE6细胞和HeLa细胞检测空肠弯曲菌产细胞膨胀性肠毒素(cytotoxic distendingtoxin,CDT)能力的改变,并在RNA水平上进一步验证毒素的表达。方法 采用4℃冷藏及-70℃冷冻的方法诱导空肠弯曲菌进入VBNC状态,利用细胞总数计算、活细胞染色计数及可培养细胞计数间接确证VBNC状态细菌的存在,细胞对于VBNC状态下的细菌仍然敏感,并用反转录-聚合酶链反应(RT-PCR)证实空肠弯曲菌毒素基因的表达。结果 3种不同状态的细胞计数结果表明存活细胞进入了VBNC状态,细菌经4℃冷藏及-70℃冷冻后数量减少约1个数量级,在冷冻前,细菌对细胞毒性较强,72h时70%以上细胞凋亡,冷冻后的VBNC状态细菌对VeroE6和HeLa细胞的生长仍有影响,72h时约有40%细胞进入凋亡状态。结论 低温、贫养条件下空肠弯曲菌可进入VBNC状态,用常规传统的羊血平板培养基仅能使不到10%的空肠弯曲菌复苏生长,从而可以检测,在VBNC状态时,空肠弯曲菌产毒素能力改变较大,远远低于正常状态,但仍具备产毒素的能力,也从RNA水平实验得到证实。     

Campylobacter jejuni strains compete for colonization in broiler chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal(r) strain) and one streptomycin-resistant strain (the 02-833L Str(r) strain). In vitro binding assays revealed that the C. jejuni F38011 Nal(r) and 02-833L Str(r) strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str(r) strain relative to that of the C. jejuni F38011 Nal(r) strain competitively inhibited the binding of the C. jejuni F38011 Nal(r) strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str(r) strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal(r) strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

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.     

Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni.

Bacterial enumeration and histologic examination of organs and tissues of 8-day-old chicks 7 days after peroral inoculation with Campylobacter jejuni revealed that the organism colonized primarily the lower gastrointestinal tract. The principal sites of localization were the ceca, large intestine, and cloaca, where densely packed cells of C. jejuni were observed in mucus within crypts. Examination of C. jejuni-colonized crypts by transmission electron microscopy revealed that the campylobacters freely pervaded the lumina of crypts without attachment to crypt microvilli. Understanding the mechanism of colonization may lead to approaches that will reduce the incidence of C. jejuni carriage by poultry.     

Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni

Bacterial enumeration and histologic examination of organs and tissues of 8-day-old chicks 7 days after peroral inoculation with Campylobacter jejuni revealed that the organism colonized primarily the lower gastrointestinal tract. The principal sites of localization were the ceca, large intestine, and cloaca, where densely packed cells of C. jejuni were observed in mucus within crypts. Examination of C. jejuni-colonized crypts by transmission electron microscopy revealed that the campylobacters freely pervaded the lumina of crypts without attachment to crypt microvilli. Understanding the mechanism of colonization may lead to approaches that will reduce the incidence of C. jejuni carriage by poultry.     

Therapeutic supplementation of caprylic acid in feed reduces Campylobacter jejuni colonization in broiler chicks

Poultry colonized with Campylobacter species are a significant source of human food-borne illness. The therapeutic use of the medium chain fatty acid caprylic acid consistently reduced enteric C. jejuni colonization in chicks by 3 to 4 logs in three separate trials. These results support caprylic acid's potential to reduce Campylobacter carriage in poultry.     

Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants.

Campylobacter jejuni, an important cause of human gastrointestinal infection, is a major food-borne pathogen in the United States and worldwide. Since poultry becomes colonized and/or contaminated during the early stages of production and is a major food-borne source for this organism, we studied the role of C. jejuni flagella on the ability of the bacterium to colonize the chicken gastrointestinal tract. Three-day-old chicks were orally challenged with a motile wild-type strain of C. jejuni IN9 or with flagellar mutants created from IN9 by disrupting the flagellin genes with a kanamycin resistance cassette by using shuttle mutagenesis (A. Labigne-Roussel, P. Courcoux, and L. Tompkins, J. Bacteriol. 170:1704-1708, 1988). One mutant, IN9-N3, lacked flagella and was nonmotile. The other, IN9-N7, produced a truncated flagellum and was partially motile. Three-day-old chicks were orally challenged with different doses of the wild-type strain and the two mutants. At challenge doses ranging from 3.0 x 10(4) to 6.6 x 10(8) CFU per chick, only the fully motile, wild-type strain colonized the chick ceca. Our results show that intact and motile flagella are important colonization factors for C. jejuni in chicks.     

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.     

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.     

Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

Study of the infectivity of saline-stored Campylobacter jejuni for day-old chicks

The culturability of three Campylobacter jejuni strains and their infectivity for day-old chicks were assessed following storage of the strains in saline. The potential for colonization of chicks was weakened during the storage period and terminated 3 to 4 weeks before the strains became nonculturable. The results from this study suggest that the role of starved and aged but still culturable campylobacters may be diminutive, but even more, that the role of viable but nonculturable stages in campylobacter epidemiology may be negligible. Even high levels of maternally derived anti-campylobacter outer membrane protein serum antibodies in day-old chicks did not protect the chicks from campylobacter colonization.     

Molecular strain typing of Campylobacter jejuni by pulsed-field gel electrophoresis in a single day

Rapid molecular strain typing is critical for effective outbreak investigation and implementation of infection control measures. Pulsed-field gel electrophoresis is a highly discriminatory technique for Campylobacter jejuni, but generally requires 3-5 days. We describe a simplified protocol for pulsed-field gel electrophoresis that provides high quality typing of C. jejuni isolates in a single day.     

The Campylobacter jejuni response regulator, CbrR, modulates sodium deoxycholate resistance and chicken colonization

Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of Campylobacter jejuni strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the C. jejuni F38011 wild-type strain. Complementation of the C. jejuni cbrR mutant in trans restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as cbrR (Campylobacter bile resistance regulator). While the MICs of a variety of bile salts and other detergents for the C. jejuni cbrR mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the C. jejuni cbrR mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that bile resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates resistance to bile salts in C. jejuni.