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.     

Salmonella reduction in manure by the addition of urea and ammonia

Animal manure may contain pathogenic microorganisms and disinfection is suggested to avoid reinfection of animal herds and introduction of zoonotic disease into the food chain. Ammonia and urea were tested for disinfection of bovine manure and Salmonella was found to be rapidly eliminated by the addition of 0.5% aqueous ammonia or 2% w/w urea (s). Treatments (2% urea and 0.5% ammonia), temperature (4 degrees C or 14 degrees C) and combinations of these factors significantly affected the inactivation rate. Decimal values (T(90)) were reduced from 8.3 days in the control to 2.0 days and 0.4 days at 14 degrees C after the addition of urea and ammonia, respectively. At 4 degrees C, the decimal values were reduced from 34 to 4.8 and 1.1 days, respectively. Recommended treatments of bovine manure based on Monte Carlo simulations are 0.5% ammonia followed by storage for one week or 2% urea followed by storage for two weeks at 14 degrees C, one month at 4 degrees C. Storage without additives should include at least one summer in temperate regions. Enterococci were evaluated as indicators for Salmonella but significantly slower decay rate and different behaviour in the material made them unsuitable as indicators for Salmonella in manure disinfected by ammonia or urea. Free ammonia treatment of Salmonella-contaminated manure disinfects the material and raises its fertilizer value.     

Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.     

Bacteriophage phiX174 growth in an Escherichia coli dnaIts mutant, KS810.

A bacteriophage phiX174-sensitive Escherichia coli dnaIts mutant, KS810, was constructed and growth of phiX174 in the cells was investigated. phiX174 and phiX174am3trD could grow normally at 43 degrees C as well as 27 degrees C, therefore we conclude that the growth of bacteriophage phiX174 is not dependent upon the host dnaI gene product.     

Effect of freezing modes on the survival of Escherichia coli bacteriophages

The object of this work was to study the effect of freezing down to--196 degrees C at different cooling and warming rates on the survival of T3, T4 and phiX174 phages. Phage particles survived when T3 phage was frozen at a rate of 20-400 degrees/min and phiX174 phage at a rate of 20-45 degrees/min. The survival rate of T4 phage was highest when it was frozen at a rate of 45 degrees/min. The survival of the phages depended also on the regime of warming. The susceptibility of the phages to freezing correlated with their sensitivity to osmotic shock in NaCl and sucrose solutions.     

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

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

Enteropathogenic bacteria in frozen chicken.

Eighty-two samples of frozen chicken from retail stores were examined for the presence of Campylobacter, Yersinia enterocolitica, and salmonellae. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were determined. Campylobacter fetus subsp. jejuni was found in 22% of the samples, Y. enterocolitica was found in 24.5% and Salmonella typhimurium was found in one sample (1.2%). The isolated strains of Y. enterocolitica belonged to serotypes 4, 5b, 6, and 8. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were not found to be noticeably higher in samples containing pathogens than in pathogen-free samples. This investigation showed that chicken does contain other pathogenic bacteria than salmonellae. Campylobacter and Y. enterocolitica were isolated in much higher frequencies than Salmonella.     

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.     

Evaluation of the effect of temperature and nutrients on the survival of Campylobacter spp. in water microcosms

Batch microcosms containing various water types (de-ionized and river water with or without sediment), incubated at a range of temperatures (5-37 degrees C), were used to facilitate a comparative evaluation of the significance of such variables and their interactions upon the collective and individual survival of four species of thermophilic Campylobacter. All variables significantly influenced (P < = 0.031) population decay rates. Minimal decay for the group was identified at low temperatures (5 degrees C) in river water, i.e. nutrient-containing microcosms. Collective decay rates within river water microcosms were significantly decreased (P = 0.03) from those observed in de-ionized water, particularly at environmental temperatures (5 and 15 degrees C). However, the increased nutrient levels observed in sediment-containing microcosms did not significantly (P = 0.41) reduce population decay rates. Overall, Camp. jejuni populations demonstrated the most resilience to the environmental stressors evaluated, with the exception of 15 degrees C where Camp. lari was the most persistent. Campylobacter coli and Camp. upsaliensis demonstrated comparable survival characteristics but were less resilient than Camp. jejuni and Camp. lari. These observations identify the suitability of water systems as a reservoir and medium for Campylobacter infection, and potentially identifies Camp. jejuni and Camp. lari as the main protagonists of water-mediated campylobacteriosis.     

Profiles of adaptation in two similar viruses.

The related bacteriophages phiX174 and G4 were adapted to the inhibitory temperature of 44 degrees and monitored for nucleotide changes throughout the genome. Phage were evolved by serial transfer at low multiplicity of infection on rapidly dividing bacteria to select genotypes with the fastest rates of reproduction. Both phage showed overall greater fitness effects per substitution during the early stages of adaptation. The fitness of phiX174 improved from -0.7 to 5.6 doublings of phage concentration per generation. Five missense mutations were observed. The earliest two mutations accounted for 85% of the ultimate fitness gain. In contrast, G4 required adaptation to the intermediate temperature of 41.5 degrees before it could be maintained at 44 degrees. Its fitness at 44 degrees increased from -2.7 to 3.2, nearly the same net gain as in phiX174, but with three times the opportunity for adaptation. Seventeen mutations were observed in G4: 14 missense, 2 silent, and 1 intergenic. The first 3 missense substitutions accounted for over half the ultimate fitness increase. Although the expected pattern of periodic selective sweeps was the most common one for both phage, some mutations were lost after becoming frequent, and long-term polymorphism was observed. This study provides the greatest detail yet in combining fitness profiles with the underlying pattern of genetic changes, and the results support recent theories on the range of fitness effects of substitutions fixed during adaptation.     

Nucleotide clusters in deoxyribonucleic acids. Comparison of the sequences of the large pyrimidine oligonucleotides of bacteriophages S13 and phiX174 deoxyribonucleic acids.

The large pyrimidine oligonucleotides from the DNAs of the two related bacteriophages phiX174 and S13 have been sequenced. The largest pyrimidine oligonucleotide present is unique to S13 DNA and is the undecanucleotide C5T6, sequence C-T-T-C-C-T-C-T-T-C-T. Considerable sequence homology has been found between the pyrimidine oligonucleotides of the two phage DNAs. Out of 14 oligonucleotide sequences from S13 DNA (120 bases) at least ten are identical with sequences of oligonucleotides from phiX174 DNA (92 bases) and two are closely related (17 bases), the only difference being a single thymine to cytosine transition in each sequence (a total of 107 identical bases). The pyrimidine oligonucleotides of each phage DNA show extensive internal sequence homology among each other with up to eight bases identical in sequence in pairs of different oligonucleotides. Another interesting observation is the occurrence of symmetrical sequences (true palindromes) which read the same forwards as backwards. The longest symmetrical sequence is the nonanucleotide C4T5 sequence, C-T-C-T-T-T-C-T-C, present in both S13 and phiX174 DNAs. The extensive sequence homology observed between the pyrimidine oligonucleotides of S13 and phiX174 supports the close relationship of the two phages and provides further evidence that they were derived from recent common ancestors.     

Ultraviolet-Induced Cross-Links in the Deoxyribonucleic Acid of Single-Stranded Deoxyribonucleic Acid Viruses as a Probe of Deoxyribonucleic Acid Packaging

Deoxyribonucleic acid (DNA) from ultraviolet (UV)-irradiated phiX174 sediments in alkali at rates up to 1.7 times that of unirradiated phiX174 DNA and is observed as a condensed, cross-linked structure when examined in the electron microscope by the formamide spreading technique. This structure appears to result from multiple cross-links induced in the tightly coiled DNA contained within the spherical phiX174 capsid. In contrast, the DNA extracted after UV irradiation of the filamentous bacteriophage M13 is not strikingly altered in its sedimentation properties and appears by electron microscopy to be rod-shaped as a result of side-to-side association of the circular DNA. The differences in these UV-induced structures reflect the differences in the packaging of the single-stranded DNA in the two virions.     

Decline of zoonotic agents in livestock waste and bedding heaps

AIMS: To measure the rates of decline of zoonotic agents introduced into heaps of spent bedding and faecal wastes generated by commercially farmed livestock and managed in a similar way to that of a working farm. METHODS AND RESULTS: Livestock isolates of Salmonella, pathogenic Listeria, Campylobacter and Escherichia coli O157 were laboratory cultured and used to inoculate 5 m3 heaps of cattle, sheep or pig wastes mixed with bedding materials. Decline of each of the infectious agents was monitored with time as was the temperature inside each heap. Temperatures of >50 degrees C were typically achieved at the core of the heaps. Pathogen decline was rapid, typically <3 days for a 1-log reduction in levels. The longest time that zoonotic agents were isolated from the heaps was 93 days. CONCLUSIONS: Movement of heaps of livestock bedding waste from animal pens to a secondary store, and storing them under conditions conducive for increased temperature is a simple and cost-effective treatment for rapidly lowering levels of zoonotic agents in solid farm wastes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a simple and cheap treatment that can be used to help prevent the spread of zoonotic agents through agricultural environments.     

In vivo methylation of bacteriophage phi X174 DNA.

A mutant (designated mec(-)) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage lambda against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec(-) mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec(+) and C mec(-) cells infected with bacteriophage phiX174. phiX. mec(-) RFI is sensitive to in vitro cleavage by R.EcoRII and is cut twice to produce two fragments of almost equal size. In contrast, phiX.mec(+) RFI is relatively resistant to in vitro cleavage by R.EcoRII. R.BstI, which cleaves mec(+)/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R. EcoRII. These results demonstrate that phiX.mec(+) RFI is methylated in vivo by the host mec(+) enzyme and that this methylation protects the DNA against cleavage by R.EcoRII. This is consistent with the known location of two mec(+)/ RII sequences (viz., [Formula: see text]) on the phiX174 map. Mature singlestranded virion DNA was isolated from phiX174 propagated in C mec(+) or C mec(-) in the presence of l-[methyl-(3)H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that phiX.mec(+) DNA had a 10-fold-higher ratio of [(3)H]5-methylcytosine to [(3)H]cytosine compared to phiX.mec(-). Since phiX.mec(+) contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of phiX174 is mediated by the host mec(+) enzyme only. These results are not consistent with the conclusions of previous reports that phiX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.     

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.     

FgoI, a Type II restriction endonuclease from the thermoanaerobe Fervidobacterium gondwanense AB39(T)

Restriction endonuclease activity was detected in 11 out of 13 Fervidobacterium isolates, including F. islandicum H21(T), F. gondwanense AB39(T), and nine other Fervidobacterium-like strains isolated from the Great Artesian Basin of Australia. The restriction endonuclease from F. gondwanense AB39(T) was partially purified and designated FgoI. FgoI recognized a 4 nucleotide sequence 5'-CTAG-3' and cleaved between nucleotides C and T to produce a 2 base 5' overhang (5'-C/TAG-3'). As predicted from the enzyme recognition and cleavage specificity, FgoI was found to cleave delta DNA 13 times, phiX174 three times, pBR322 five times, pUC18 four times, and pSK six times. FgoI exhibited a broad temperature optimum range (between 60 to 70 degrees C) and was active at pH 6.5 to 8.5, but not at pH 9.0. Manganese could replace magnesium as a cofactor for activity, but not cobalt chloride, calcium chloride, cupric chloride, or zinc chloride. The restriction endonuclease was completely inactivated by phenol/chloroform extraction and was heat inactivated at 80 degrees C for 60 min or at 100 degrees C for 15 min. FgoI has been identified as a heat stable isoschizomer of the Type II restriction endonucleases, MaeI and BfaI.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.     

Effect of temperature on growth and chemotactic behaviour of Campylobacter jejuni

AIM: To determine the effect of two physiologically important temperatures on growth and chemotaxis in Campylobacter jejuni. METHODS AND RESULTS: Growth curves of Camp. jejuni were compared at 37 degrees C and 42 degrees C. Chemotaxis was compared at 37 degrees C and 42 degrees C by the disc and capillary assays. Student's t-test was applied to the results of the capillary assay to assess the significance in the difference between chemotaxis at the two temperatures. Both, the growth rate and chemotactic ability of the isolate, were found to be greater at 37 degrees C. CONCLUSIONS: Quorum sensing (related to population density), a regulation mechanism of virulence in micro-organisms, has been reported in Campylobacter. Chemotaxis is also a known virulence factor of Campylobacter. Both, growth (in terms of population density) and chemotaxis, being greater at 37 degrees C than at 42 degrees C, suggests that the physiological temperature of humans (37 degrees C) might be more favourable for the expression of virulence in Campylobacter than that of birds (42 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: It is as yet not known why Campylobacter causes disease in humans but is avirulent in birds. This study suggests that the human body temperature is optimum for growth and chemotaxis in Campylobacter. There is scope for the study of temperature regulation of other virulence determinants of Campylobacter.     

Biocontrol of the food-borne pathogens Listeria monocytogenes and Salmonella enterica serovar Poona on fresh-cut apples with naturally occurring bacterial and yeast antagonists

Fresh-cut apples contaminated with either Listeria monocytogenes or Salmonella enterica serovar Poona, using strains implicated in outbreaks, were treated with one of 17 antagonists originally selected for their ability to inhibit fungal postharvest decay on fruit. While most of the antagonists increased the growth of the food-borne pathogens, four of them, including Gluconobacter asaii (T1-D1), a Candida sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcherrima (T1-E2), proved effective in preventing the growth or survival of food-borne human pathogens on fresh-cut apple tissue. The contaminated apple tissue plugs were stored for up to 7 days at two different temperatures. The four antagonists survived or grew on the apple tissue at 10 or 25 degrees C. These four antagonists reduced the Listeria monocytogenes populations and except for the Candida sp. (T4-E4), also reduced the S. enterica serovar Poona populations. The reduction was higher at 25 degrees C than at 10 degrees C, and the growth of the antagonists, as well as pathogens, increased at the higher temperature.     

Mechanism of Replication of φX174 Single-Stranded DNA IX. Requirement for the Escherichia coli dnaG Protein

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.