A note on the effect of different storage procedures on the ability of Preston medium to recover campylobacters

The effect of different storage procedures on the ability of Preston medium to recover campylobacters was investigated. Freshly poured media was shown to recover more campylobacters than media stored under aerobic or anaerobic conditions at room temperature or at 4 degrees C. The growth of Campylobacter laridis was greatly reduced by storage of media and although most strains of C. jejuni and C. coli were not markedly affected, the growth of one strain of C. jejuni was considerably reduced. It is recommended that freshly prepared media be used whenever possible, but if storage is necessary, then plates should be held at 4 degrees C, preferably under anaerobic conditions. These precautions may not be necessary for workers interested solely in C. jejuni or C. coli, but are essential for the optimum isolation of C. laridis.     

Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups

Eighty-four strains of catalase-positive campylobacters could be placed into seven distinct DNA homology groups (species), corresponding to Campylobacter fetus, "C. hyointestinalis," C. jejuni, C. coli, "C. laridis," "C. fecalis," and aerotolerant campylobacters. The biochemical and physiological characteristics of the strains were examined for their correlation with the homology groups. The characterization tests that provided the most reliable differentiation at the species and subspecies level were growth at 25 and 42 degrees C, sensitivity to cephalothin and nalidixic acid, growth in semisolid media containing 1% glycine and 3.5% NaCl, growth on plates containing 1.5% NaCl, growth in a semisolid minimal medium, anaerobic growth in the presence of 0.1% trimethylamine-N-oxide, hydrogen sulfide production in SIM medium and triple-sugar iron agar, hippurate hydrolysis, nitrite reduction, and growth on plates under an air atmosphere.     

Study of haemolytic activity of some Campylobacter spp. on blood agar plates

A total of 152 strains of Campylobacter jejuni, C. coli, C. laridis and C. fetus subsp. fetus were tested for haemolysis on blood agar plates. Distinct haemolysis was detected in 92.3% (96/104) of strains of C. jejuni and 21.7% (5/23) of strains of C. coli on sheep blood heart infusion agar after incubation for 4 d microaerobically at 42 degrees C. Haemolysis was also detected on horse blood heart infusion agar. Haemolysis was not detected at 37 degrees C except with one of 50 strains of C. jejuni tested at this temperature, which was weakly positive. Campylobacter laridis was not haemolytic; C. fetus subsp. fetus, which does not grow at 42 degrees C, showed no haemolysis at 37 degrees C. Blood agar (Oxoid, BA Base No. 2) was not suitable for testing for haemolysis by these organisms. A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis easier to detect. There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae. The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters.     

Study of haemolytic activity of some Campylobacter spp. on blood agar plates

A total of 152 strains of Campylobacter jejuni, C. coli, C. laridis and C. fetus subsp. fetus were tested for haemolysis on blood agar plates. Distinct haemolysis was detected in 92.% (96/104) of strains of C. jejuni and 21.7% (5/23) of strains of C. coli on sheep blood heart infusion agar after incubation for 4 d microacrobically at 42°C. Haemolysis was also detected on horse blood heart infusion agar. Haemolysis was not detected at 37°C except with one of 50 strains of C. jejuni tested at this temperature, which was weakly positive. Campylobacter laridis was not haemolytic; C. fetus subsp. fetus , which does not grow at 42°C, showed no haemolysis at 37°C. Blood agar (Oxoid, BA Base No. 2) was not suitable for testing for haemolysis by these organisms. A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis casier to detect. There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae . The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

A study of thermophilic campylobacters in a river system

Fifteen kilometres of a river system traversing rural and urban areas and subject to sewage works effluent discharge was studied during a 12 1/2 month period. A total of 312 samples was collected from 12 sites at 14 d intervals and tested by a glass microfibre filtration method and a most probable number (MPN) method. Campylobacters were found in 43% of samples by the filtration method and 21% by the MPN method. The lowest frequency of isolation and lowest counts (<10 campylobacters/100 ml) were associated with samples collected from rural sites and fast-flowing stretches of river. The greatest frequency of isolation and highest counts (< 10–230 campylobacters/100 ml) were associated with sites adjacent to or downstream of sewage works. There was an obvious seasonal trend; most isolations and highest counts were obtained in late autumn and winter, and fewest isolations and lowest counts in spring and summer. Surface water run-off from adjacent farmland following heavy rainfall also increased the counts of campylobacters in the river system. Biotyping of isolates demonstrated that the most prevalent Campylobacter sp. was Campylobacter jejuni but C. coli, C. laridis and a previously unrecognized group of campylobacters were also isolated. Serotyping differentiated 14 serotypes of C. jejuni , 11 of C. coli and two of C. laridis. Furthermore, serotypes of C. jejuni commonly isolated from enteritis in man were frequently found in river water tested during this study.     

A study of thermophilic campylobacters in a river system

Fifteen kilometres of a river system traversing rural and urban areas and subject to sewage works effluent discharge was studied during a 12 1/2 month period. A total of 312 samples was collected from 12 sites at 14 d intervals and tested by a glass microfibre filtration method and a most probable number (MPN) method. Campylobacters were found in 43% of samples by the filtration method and 21% by the MPN method. The lowest frequency of isolation and lowest counts (less than 10 campylobacters/100 ml) were associated with samples collected from rural sites and fast-flowing stretches of river. The greatest frequency of isolation and highest counts (greater than 10-230 campylobacters/100 ml) were associated with sites adjacent to or downstream of sewage works. There was an obvious seasonal trend; most isolations and highest counts were obtained in late autumn and winter, and fewest isolations and lowest counts in spring and summer. Surface water run-off from adjacent farmland following heavy rainfall also increased the counts of campylobacters in the river system. Biotyping of isolates demonstrated that the most prevalent Campylobacter sp. was Campylobacter jejuni but C. coli, C. laridis and a previously unrecognized group of campylobacters were also isolated. Serotyping differentiated 14 serotypes of C. jejuni, 11 of C. coli and two of C. laridis. Furthermore, serotypes of C. jejuni commonly isolated from enteritis in man were frequently found in river water tested during this study.     

DNA base compositions and base sequence relatedness of atypical Campylobacter jejuni strains from clinical material

Abstract The relationships of nitrate-negative campylobacters (NNC) resembling Campylobacter jejuni were investigated by DNA base composition estimation (T _m method) and DNA-DNA hybridization (S1 endonuclease assay). The 8 NNC strains which were from clinical material formed a homogeneous DNA group with a high level of relatedness (approx. 70%) to typical (nitrate-positive) C. jejuni , but were less similar (approx. 35%) to Campylobacter coli , and only slightly related (≥ 10%) to Campylobacter fetus, Campylobacter laridis and Campylobacter sputorum . The NNC strains showed small but consistent genome differences from typical C. jejuni . As these differences can be correlated with several bacteriological test differences, we conclude that the NNC strains constitute a distinct subspecies within C. jejuni .     

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39 degrees C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50 degrees C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.     

The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide

B olton , F.J. C oates D. & H utchinson , D.N. 1984. The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. Journal of Applied Bacteriology 56 , 151–157.
Nutrient agar plates stored in light and air for 48 h became inhibitory for Campylobacter jejuni, C. coli and nalidixic acid-resistant, therrnophilic campylobacter (NARTC) strains. All five campylobacter test strains showed > 5 log reduction in counts on media which had been stored in light and air. Media stored in the dark and/or in a reduced atmosphere did not become inhibitory and supported the growth of campylobacters. Ferrous sulphate, sodium pyruvate, blood, charcoal or sodium metabisulphite, compounds frequently used as supplements in campylobacter media, were added to nutrient agar prior to storage of media in light and air. All additives except sodium metabisulphite prevented the accumulation of photochemically generated toxic oxygen derivatives and allowed growth of test strains. In qualitative tests to determine the ability of supplements to neutralize hydrogen peroxide, blood was the most active, charcoal and sodium pyruvate slightly less active and ferrous sulphate and sodium metabisulphite the least active. The results of this study confirm that supplements in campylobacter media act as quenching or detoxifying agents and not as enrichment factors.     

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.     

Validation of a polymerase chain reaction/restriction enzyme analysis method for species identification of thermophilic campylobacters isolated from domestic and wild animals

AIMS: To compare and evaluate a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method with standard phenotypic tests for the identification and differentiation of the thermophilic campylobacters Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. METHODS AND RESULTS: One hundred and eighty-two presumptive thermophilic campylobacters from 12 different animal species were tested by a recently published PCR/REA and standard phenotypic tests. By PCR/REA, 95% of the isolates were clearly identified as either one of the four thermophilic Campylobacter species or as not belonging to this group of organisms at all. By standard phenotyping, 174 of the 182 isolates were initially identified as either C. jejuni, C. coli, C. lari or C. upsaliensis. Additional genotypic tests and phenotyping showed that 52 of these identifications were either incorrect or unreliable. Of the C. jejuni isolates, 19% were identified as C. coli by initial phenotyping and 27 sheep isolates phenotyped as C. coli or C. lari were, in fact, arcobacters. CONCLUSIONS: The PCR/REA was more reliable than standard phenotyping for the identification of thermophilic campylobacters from different animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Routinely used phenotypic tests often resulted in unreliable identifications, requiring additional testing. The PCR/REA, however, gave unequivocal results and was considered useful for the routine identification of thermophilic campylobacters from different animals.     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.     

Use of PCR for direct detection of Campylobacter species in bovine feces

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at approximately 10(4) CFU g(-1), and 50 to 83% of the samples inoculated at approximately 10(3) CFU g(-1) were positive. At approximately 10(2) CFU g(-1), C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (相似文献   

Occurrence of thermotolerant campylobacters in fresh vegetables sold at farmers' outdoor markets and supermarkets.

A total of 1564 fresh samples of 10 vegetable types from two different retail levels (533 samples from farmers' outdoor markets and 1031 samples from supermarkets) were surveyed for the occurrence of thermotolerant campylobacters. In samples from the outdoor markets, campylobacters were detected on six types of vegetables; the detection rates were spinach, 3.3; lettuce, 3.1; radish, 2.7; green onions, 2.5; parsley, 2.4; and potatoes, 1.6%. Campylobacter jejuni was the predominant species (88%), with the remainder being C. lari (8%) and C. coli (4%). When the outdoor market samples were thoroughly washed with chlorinated water, all were negative for campylobacters. Of the samples from supermarkets, all were negative for campylobacters whether purchased in summer or winter. These results suggest that vegetables sold at farmers' outdoor markets are produced and (or) stored under less sanitary conditions than those sold at supermarkets, and they could constitute health hazards. Therefore, vegetables (e.g., potatoes and spinach) from farmers' markets must be decontaminated by washing with chlorinated water or cooked thoroughly before consumption.     

Enterotoxigenicity and frequency of Campylobacter jejuni, C. coli and C. laridis in human and animal stool isolates from different countries

Campylobacter jejuni and C. coli strains were collected during three different years from adult patients with enterocolitis in Sweden (n = 372) from 49 patients in Kuwait, and Campylobacter strains from hens from Mexico, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C. jejuni was the predominant species in human and hen isolates, and C. coli in pigs C. coli was significantly more common in human isolates from Sweden, and more common in hen isolates from Pakistan, than in hens from Sweden and Mexico. C. laridis was only isolated from pigs (17%) and was in no case enterotoxigenic. Both in human and hen isolates, C. jejuni strains were more enterotoxigenic than C. coli strains. C. jejuni strains from Swedish hens were less enterotoxigenic than those from Pakistan and Mexico (P less than 0.001), and strains from pigs were less enterotoxigenic than those from hens (P less than 0.001). We conclude that C. jejuni are more often enterotoxigenic and possibly more virulent than c. coli and C. laridis. The relative frequency of C. jejuni and C. coli in humans and animals differs from one country to another.     

Campylobacter in chicken livers and their destruction by pan frying

AIM: To enumerate Campylobacter spp. on the external surface and internal portions of chicken livers, and to assess the cooking required to inactivate naturally present cells. METHODS AND RESULTS: Of 30 livers tested all yielded Campylobacter spp. on their surfaces and 90% were found to contain the organism in internal tissue. Four (13%) livers contained >10(4) MPN campylobacters, and an additional seven (23%) contained >10(3) MPN campylobacters per liver. The internal temperature of pan-fried livers under the conditions used reached a maximum of 70-80 degrees C, and maintaining this temperature for 2-3 min was necessary to inactivate naturally occurring Campylobacter spp. All isolates identified were either C. jejuni or C. coli. CONCLUSIONS: Chicken livers represent a potential source of human campylobacteriosis as they contained >10(4) MPN per liver in 13% of the samples tested. Pan-frying can produce an acceptable product that is safe to eat. SIGNIFICANCE AND IMPACT OF THIS STUDY: The data provided can be used in exposure assessments of Campylobacter in poultry products in terms of both quantitative data and assessing pan-frying and its ability to destroy campylobacters.     

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 co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.     

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.