Antibiotic and deoxycholate resistance in Campylobacter jejuni following freezing or heating

The surviving populations of Campylobacter jejuni serotypes following freezing or heat were found to be more sensitive to rifampicin and sodium deoxycholate on subsequent culture. Thus while control cultures had an IC50 of greater than 20 micrograms/ml rifampicin those of injured cells were less than 5 micrograms/ml. Treatment with EDTA caused almost identical changes in resistance suggesting that the altered resistance pattern of injured cells was due to loss of the barrier properties of the bacterial outer membrane.     

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9mm zones of inhibition surrounding discs impregnated with 2.5 and 20 μg CdCl₂ respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 μg CdCl₂/ml of MH agar for four C. jejuni strains. In the presence of 23 μg FeSO₄/ml of MH the MIC increased to a range of 1.5–5.4 μg CdCl₂/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO₄ were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO₄ in MH broth was increased about 10000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 μg discs of CdCl₂ were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl₂. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl₂. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.     

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4 degrees C, freezing at -20 degrees C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and -20 degrees C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Injury and recovery in freeze- or heat-damaged Campylobacter jejuni

When exposed to freezing or heat, cells of Campylobacter jejuni were initially sensitized to rifampicin and, with longer exposures, subsequently unable to grow on nutrient or blood agar. Repair of all lesions occurred within 2 h at 37°C while at 43°C organisms were only able to repair the presumably less severe injury caused by short exposures to high or low temperature.     

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.     

Inhibitory effect of oregano and thyme essential oils on moulds and foodborne bacteria

The essential oil of oregano ('origanum oil'; thymol type oil from Origanum vulgare) inhibited completely the mycelial growth of Aspergillus niger and A. flaous at 400 μg/ml, while A. ochraceus was inhibited at 600 μg/ml. At 700 μg/ml, thyme oil inhibited the mycelial growth of A. flavus and A. niger but not that of A. ochraceus . Fungal spore germination was inhibited by 600 μg/ml of origanum oil and (with the exception of A. ochraceus) by 700 μg/ml of thyme oil. Under aerobic conditions, the essential oils of oregano (250 μg/ml) and thyme (350 μg/ml) inhibited to some extent the growth of Staphylococcus aureus and Salmonella typhimurium. Pseudomonas aeruginosa was not affected by either oregano or thyme oil at concentrations up to 500 μg/ml. The origanum oil was very effective against Campylohacter jejuni and Clostridiurn sporogenes and thyme oil was very effective against C. jejuni. The antagonistic effect of the two oils on Staph. aureus and Salm. typhimuriutn was greatly enhanced when those organisms were incubated in atmospheres of low oxygen tensions     

The synergistic inhibition of Campylobacter jejuni by rifampicin and hydrogen peroxide

Hydrogen peroxide was found to prevent the recovery of Campylobacter jejuni from sub-lethal injury induced by freezing. It also acted synergistically with rifampicin to inhibit both damaged and undamaged cells. these effects could be overcome by the addition to culture media of either catalase of FBP supplement (ferrous sulphat-sodium metabisulphite-sodium pyruvate).     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to a _w 0.93 were more rapid than in unsupplemented media ( a _w 0.99). Although growth in unsupplemented medium was lower at 35°C, incubation at 21°C or 35°C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 μg potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35°C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35°C than in cultures incubated at 21° in media both with and without 300 μg potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at - 18°C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 μg potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21°C. Sensitivity to freezing increased when cultures were incubated at 21°C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Prevalence, development, and molecular mechanisms of bacteriocin resistance in Campylobacter

Bacteriocins (BCNs) are antimicrobial peptides produced by bacteria with narrow or broad spectra of antimicrobial activity. Recently, several unique anti-Campylobacter BCNs have been identified from commensal bacteria isolated from chicken intestines. These BCNs dramatically reduced C. jejuni colonization in poultry and are being directed toward on-farm control of Campylobacter. However, no information concerning prevalence, development, and mechanisms of BCN resistance in Campylobacter exists. In this study, susceptibilities of 137 C. jejuni isolates and 20 C. coli isolates to the anti-Campylobacter BCNs OR-7 and E-760 were examined. Only one C. coli strain displayed resistance to the BCNs (MIC, 64 μg/ml), while others were susceptible, with MICs ranging from 0.25 to 4 μg/ml. The C. coli mutants resistant to BCN OR-7 also were obtained by in vitro selection, but all displayed only low-level resistance to OR-7 (MIC, 8 to 16 μg/ml). The acquired BCN resistance in C. coli could be transferred at intra- and interspecies levels among Campylobacter strains by biphasic natural transformation. Genomic examination of the OR-7-resistant mutants by using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic resistance and acquired resistance to the BCNs. Altogether, this study represents the first report of and a major step forward in understanding BCN resistance in Campylobacter, which will facilitate the development of effective BCN-based strategies to reduce the Campylobacter loads in poultry.     

Prevalence of campylobacters and arcobacters in ducks at the abattoir

Ten duck carcasses, five from each of two different flocks, and four pairs of pooled duck caecal contents, each pair from a separate flock, were examined by a variety of techniques for arcobacters and campylobacters. Campylobacter coli, C. jejuni ssp. jejuni , C. upsaliensis, Arcobacter cryaerophilus and A. butzleri were isolated from duck caecal contents. Campylobacter coli, C. jejuni ssp. jejuni, A. cryaerophilus, A. butzleri and A. skirrowii were isolated from carcasses. The most effective methods for isolating these bacteria from carcasses involved selective enrichment in campylobacter enrichment broth, containing a cefoperazone, amphotericin, teicoplanin supplement, followed by plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA), or plating onto non-selective blood agar after filtration through a 0·65 μm pore size cellulose acetate filter. In contrast, recovery from caecal contents was most effective by direct plating onto mCCDA. API test strips performed poorly, failing to identify A. skirrowii or A. butzleri (which are not included in the scheme), or even many common campylobacters. The Preston biochemical characterization scheme was more helpful, though it did not distinguish between Arcobacter species. The species of most isolates of campylobacter, identified using the Preston scheme, was confirmed by the use of SDS-PAGE of whole cell proteins and this technique was also used successfully to speciate arcobacters.     

Comparison of Four Enrichment Media in the Recovery of Campyloracter Jejuni

The effectiveness of 4 enrichment media for the recovery of low levels of inoculated cells of Campylobacter jejuni was evaluated. The media contained antibiotics or antibiotics and bile acids as selective compounds. Three of the media recovered most of the inoculated low numbers of 6 C. jejuni strains. In the 3 media the growth rate of 3 strains, indicated by the increase in the log number of cells during 24 h or 48 h incubation at 42 ° C, was about the same as in the control medium without selective compounds. The same 3 media also recovered a low number of Campylobacter cells from artificially contaminated raw milk or ground meat samples. The enrichment medium B containing 40 I.U. Colistin, 5 μg novobiocin, 2 mg Na-cholic acid and 50 mg cycloheximide per ml was inhibitory for most Campylobacter strains studied.     

Survival of Campylobacter jejuni on beef trimmings during freezing and frozen storage

AIMS: To investigate the survival of two animal isolates of Campylobacter jejuni on beef trimmings during freezing and frozen storage. METHODS AND RESULTS: Meat packs inoculated with 10(3) or 10(6) cfu g(-1) of either strain of C. jejuni were frozen to -18 degrees C, and sampled at regular intervals over 112 d storage to determine Campylobacter numbers and sublethal injury. For both strains and inoculation levels the numbers of Campylobacter decreased in the first 7 d of storage by ca. 0.6-2.2 log cfu g(-1) and then remaining constant over the remainder of the storage trial, with neither isolate exhibiting sublethal injury. CONCLUSIONS: Despite an initially significant decrease in number, these pathogens were able to survive standard freezing conditions in meat, but did not exhibit sublethal injury. SIGNIFICANCE AND IMPACT OF THE STUDY: Strict hygiene and/or the implementation of decontamination technologies are recommended as a means to assure the safety of meat with respect to this pathogen.     

Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment.

A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter from foods. The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h. After incubation, the medium is plated directly onto Campy-BAP agar plates (M. J. Blaser et al., Ann. Intern. Med. 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests. The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g. The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g. The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively. This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.     

Survival of Campylobacter Jejuni/Coli in Ground Refrigerated and in Ground Frozen Beef Liver and in Frozen Broiler Carcasses

Survival of 5 strains of Campylobacter jejuni/coli in ground beef liver stored at 4° C and at –20° C was studied. After 6 days of storage at 4° C the beef liver was spoiled, which was indicated by APG log 7.25 and lactobacilli count log 7.0. During this storage Campylobacter counts decreased only slightly. After 12 weeks of storage at –20° C Campylobacter counts decreased by 2–3 logs in frozen ground beef liver. Survival of 4 strains of C. jejuni/coli on frozen broiler carcasses was also studied. Two inoculation levels, 103–104/g and 104–105/g were used. On frozen broiler carcasses Campylobacter counts decreased by 0.5–2.0 logs during 12 weeks at –20° C.     

Latex agglutination for the detection of Campylobacter species in water

A commercially available sensitized latex suspension could detect viable (10²organisms/ml) or heat-killed Campylobacter jejuni. Cross-reactions with other Campylobacter spp. and Helicobacter pylori were only seen with suspensions > 10⁵organisms/ml. In laboratory microcosms, C. jejuni remained viable for 16 d at 4°C but latex detected antigen for at least 6 months. In water from a sewagecontaminated lake, the latex gave results comparable with culture for C. jejuni but much more rapidly. Culture-negative water supplies in chicken sheds containing campylobacter-colonized birds were shown to be sometimes latex-positive.     

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.     

Survival of Campylobacter jejuni inoculated into ground beef.

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent.     

Survival of Campylobacter jejuni in chicken meat at frozen storage temperatures

The aim of this study was to determine the survival of Campylobacter jejuni in chicken meat samples at frozen temperatures and given length of incubation and to determine the impact of aerobic bacteria on the survival of C. jejuni. The chicken meat samples were inoculated with C. jejuni NCTC 11351 suspensions and stored in bags at temperatures of -20°C and -70°C. The mean value of C. jejuni from meat samples decreased from 7.52 log10 CFU/g after 30 minutes of incubation at ambient temperature, to 3.87 log10 CFU/g on the eighth week of incubation at -20°C, and to 3.78 log10 CFU/g at incubation at -70°C after the same incubation period. Both freezing temperatures, -20°C and -70°C, decreased the number of campylobacters. The presence of aerobic mesophilic bacteria did not influence the survival of C. jejuni in chicken meet samples. Keeping poultry meat at freezing temperatures is important for the reduction of C. jejuni, which has a strong influence on the prevention of occurrence of campylobacteriosis in humans.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。