Application of molecular epidemiology to understanding campylobacteriosis in the Canterbury region of New Zealand.

Pulsed-field gel electrophoresis genotypes of Campylobacter isolates from 603 human patients were compared with 485 isolates from retail offal (primarily chicken and lamb) to identify temporal clusters and possible sources of campylobacteriosis. Detailed epidemiological information was collected from 364 of the patients, and when combined with genotyping data allowed a putative transmission pathway of campylobacteriosis to be assigned for 88% of patients. The sources of infection were 47% food, 28% direct animal contact, 7% overseas travel, 4% person-to-person transmission and 3% water-related. A significant summer increase in campylobacteriosis cases was primarily attributed to an increase in food-related cases. Genotyping of isolates was essential for identifying the likely cause of infection for individuals. However, a more rapid and cheaper typing tool for Campylobacter is needed, which if applied to human and animal isolates on a routine basis could advance greatly our understanding of the ongoing problem of Campylobacter infection in New Zealand.

Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef.

A number of outbreaks of Escherichia coli O157:H7 infections involving beef have been reported. Options for controlling bacterial pathogens in raw foods are limited, but one is to use bacteriophages (phages). We describe the isolation and characterisation of phage FAHEc1, which infects E. coli O157, and its ability to kill its host in vitro and on beef. The phage belonged to the family Myoviridae and lysed 28 of 30 E. coli O157 (:H7, :HNM and :H not specified) isolates, only one other non-O157 E. coli serotype (O162:H7), and none of the other 13 bacterial species tested. The phage did not contain stx1, stx2, eae or ehxA virulence genes as assessed by PCR. An approximate 4 log10 inactivation of E. coli O157:H7 occurred at 5 °C in the presence of phage FAHEc1 at >107 PFU/ml in broth in vitro. On thinly sliced beef pieces incubated at 37 °C, a > 2.7 log10 reduction occurred with 3.2 × 107 PFU/4 cm² meat piece. At lower phage concentrations (10³-104 PFU/4 cm² piece) phage replication occurred on beef at 37 °C. When the phage was applied to beef pieces under conditions simulating hot boning and conventional carcass cooling, inactivation of E. coli O157:H7 of approximately 2 log10 was measured under optimal conditions with phages applied at 3.2 × 107 PFU/4 cm² meat piece.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Inactivation of Escherichia coli O157:H7 using ultraviolet light-treated bacteriophages.

Escherichia coli O157:H7 causes serious foodborne infections warranting the development of effective control measures. One control option is to use bacteriophages (phages), which are regarded as safe to humans and an environmentally friendly alternative to chemical antimicrobials. One of the few remaining safety concerns is the potential for phages to facilitate genetic exchange between bacteria so resulting in undesirable mobilisation of genes. UV treatment of phages causes a rapid loss in their ability to replicate, while maintaining their antibacterial activity, and so the use of UV-treated phages could be an alternative to the use of viable phages. Data presented here show the inactivation of E. coli O157:H7 by UV-treated phages in milk and on the surface of raw and cooked meat. A minimum concentration of approximately 10(5) PFU cm(-2) (pre-UV treatment titre) of UV-treated phages was required before inactivation of E. coli O157:H7 on the surface of meat was measurable, and 1-2 log10 CFU cm(-2) reductions were typically obtained at concentrations of around 10(7) UV-treated phages cm(-2) (pre-UV treatment titre). Inactivation of E. coli O157:H7 by UV-treated phages was less than that for untreated phages. The production of UV-treated phages was not optimised and it is possible that better reductions in pathogen concentration could be achieved for the same input UV-treated phages concentrations.

Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef.

A previously described phage infecting Escherichia coli O157:H7 was added to raw and cooked beef pieces at concentrations ranging from 10(1)-10(8) plaque forming units/cm(2) to either low (<100 CFU/cm(2)) or high (10(4) CFU/cm(2)) concentrations of host bacterial cells. Incubation for up to 24 h was performed at 5 ℃ and 24 ℃ to simulate refrigerated and room temperature storage/temperature abuse. Surviving bacteria were enumerated during the incubation period, with phages being counted at the first and last sampling times. Significant reductions of E. coli O157:H7 of the order of >4 log10 CFU/cm(2) at both temperatures could be achieved compared to phage-free controls. There was a trend for greater inactivation to occur with increasing phage concentration. While re-growth of surviving cells occurred in nearly all samples incubated for 24 h at 24 ℃, these conditions are not typical of those experienced by perishable foods. It was concluded that phages can be used to reduce the concentration of a bacterial pathogen on meat, but the concentration of phages needs to be high (>4-5 log10 plaque forming units/cm(2)) for reductions to occur. A concentration of the order 8 log10 plaque forming units/cm(2) was needed to achieve a 4 log10 CFU/cm(2) reduction.

A sustained outbreak of Clostridium difficile in a general hospital: persistence of a toxigenic clone in four units.

OBJECTIVE: To evaluate the endemicity and epidemiology of toxigenic Clostridium difficile in a sustained outbreak of antibiotic-associated diarrhea. SETTING: University-affiliated, 465-bed tertiary care teaching hospital with adjacent cancer clinic in Hamilton, Ontario. DESIGN: From August 8, 1991, through August 31, 1993, a total of 187 cases were investigated for epidemiologic analysis of toxigenic C difficile from stool cultures, to identify the endemic clone(s). To assess the nature of contamination, cultures of inanimate surfaces in the patient environment from the four most affected units (medical teaching, nonteaching medical, hematologic oncology, and the intensive care unit) were processed for C difficile. The 229 clinical strains and 24 environmental strains isolated were typed by numerical analysis of SDS-PAGE protein patterns. RESULTS: A majority (81%) of cases in the epidemiologic analysis were associated with a toxigenic electrophoretic (EP) type 1 C difficile that was identical to the strain first isolated from an index case that occurred 18 months before the start of this study. Culture and typing of the C difficile strains from the inanimate surfaces in the four most affected units showed that the patient environment was contaminated with the toxigenic EP type 1 organism. Six other strains that occurred infrequently among cases also were found in the environment. CONCLUSIONS: A single predominant toxigenic clone has been implicated in a sustained outbreak of antibiotic-associated diarrhea that affected elderly patients. The "endemic" clone transmitted for the 25-month study period was linked to an index case shedding a toxigenic EP type 1 strain that occurred 21 months prior to the initial outbreak on the medical teaching unit. The patient environment in the affected units was found to be contaminated with the same clone, possibly due to shedding of organisms by fecally incontinent symptomatic patients. The extrinsic factors contributing to the endemic transmission of this one clone still are not well understood.

In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA profiling: implications for epidemiological studies.

Six isolates of Campylobacter coli from different pig herds were subcultured up to 50 times over a 6-month period and DNA samples suitable for pulsed-field gel electrophoretic (PFGE) profiling prepared at regular (1, 20, 40 and 50 passages) intervals. In 5/6 strains, changes in the banding patterns of Sma1, Sal1 and/or BamH1 digests were observed. In one such strain the differences were considered artifactual. However, significant alterations in PFGE profiles between subcultures of four strains were seen, irrespective of the restriction enzyme used. Spontaneous intramolecular genomic rearrangements were considered the most likely mechanism for the changes observed. A numerical analysis based upon the combined distribution of Sma1- and sal1-derived fragments clustered most strain subcultures together, with the exception of those from one isolate which were divided into two clusters. The effect of spontaneous genetic change on PFGE profiles must be considered when evaluating strain relationships. Numerical techniques may aid data interpretation but results must be evaluated cautiously.

Identification of taxonomic and epidemiological relationships among Campylobacter species by numerical analysis of AFLP profiles.

Amplified fragment length polymorphism (AFLP)-based profiling was performed on 138 strains representing all named Campylobacter species and subspecies. Profiles of 15/16 species comprised 6 to greater than 100 fragments and were subjected to numerical analysis. The mean similarity of 48 duplicate, outbreak and/or 'identical' strain profiles exceeded 94%. Species were clearly distinguished at the 17.90% similarity (S-) level in the dendrogram. Subspecies of Campylobacter jejuni and Campylobacter hyointestinalis, and biovars of Campylobacter lari and Campylobacter sputorum were distinguished at higher S-levels. All outbreak or 'genetically identical' strains of C. jejuni subsp. jejuni, Campylobacter coli, C. hyointestinalis and C. sputorum clustered at S-levels >92% and were distinguished from unrelated strains. Numerical analysis of AFLP profiles is useful for concurrent identification of taxonomic and epidemiological relationships among most Campylobacter species.

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms.

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated from unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coli var. hyoilei strains, were identified. These relationships corresponded to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp.

Genetic heterogeneity of Campylobacter concisus determined by pulsed field gel electrophoresis-based macrorestriction profiling.

In order to investigate the genetic diversity of Campylobacter concisus to assist molecular typing studies, the use of macrorestriction profiling was examined. A suitable protocol was developed that included the use of formaldehyde pretreatment to prevent DNA degradation, and restriction enzyme NotI for pulsed field gel electrophoresis-based genotyping. Subsequently, 53 strains of C. concisus, principally from cases of diarrhoea in children, were examined. Fifty-one distinct patterns were obtained, indicating the high discriminatory potential of the method. Patterns comprised between one and 14 restriction fragments, with type and reference strains of two well-defined genomospecies of oral and faecal origin containing six and 12 fragments respectively. Our results show that C. concisus is genetically diverse and suggest the species as currently defined to be a taxonomic continuum comprised of several genomospecies. The pulsed field gel electrophoresis typing method described here has considerable potential for molecular epidemiological studies of C. concisus and may be a useful adjunctive method for helping to resolve key taxonomic issues for this species.

Isolation of Arcobacter butzleri from a neonate with bacteraemia.

We describe the case of a neonate with bacteraemia from whom the recently described organism Arcobacter butzleri was isolated. This appears to be the first report of the organism causing neonatal infection. Clinical details suggest that the infection was contracted in utero, although the mother showed no evidence of disease before delivery. Treatment of the preterm infant was ultimately successful in resolving the infection but the organism proved resistant to a wide range of antibiotics. Similar patterns of antibiotic resistance were also observed in 39 reference and field strains of the genus Arcobacter. These findings, combined with available data on the distribution of Arcobacter species, suggest that these organisms may be important human pathogens. Optimized methods for their isolation and identification are therefore required so as to ascertain their role in human disease.

Isolation of Campylobacter sputorum biovar sputorum from an axillary abscess.

Campylobacter sputorum biovar sputorum is a rarely isolated organism, particularly from human clinical specimens. Its pathogenic potential is unknown. We present here what we believe to be the first report of this organism being isolated from a clinically significant source, an axillary abscess. To our knowledge, this organism has not been reported previously as one of clinical relevance in the U.K.

Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks.

We investigated the genotype and serotype diversity of Campylobacter coli and C. jejuni in two parent flocks of adult hens and their offspring over two rotations in order to evaluate the role of hatchery mediated transmission and/or vertical transmission of campylobacters in broiler flocks. In total, 314 C. jejuni and 32 C. coli isolates from parent and broiler flocks and from the surroundings of broiler houses were typed by flagellin gene PCR/RFLP (fla-typing), and selected isolates were also typed by serotyping and macrorestriction profiling using PFGE (MRP/PFGE). The combined typing results showed that the broiler flocks could be colonised by 1-3 different Campylobacter clones and parent flocks could be colonised by 2-6 different clones. C. coli was isolated from up to 36% of birds in one parent flock, whereas only C. jejuni was isolated from broiler flocks. C. jejuni clones from different flocks were clearly discriminated by fla-typing as well as by MRP/PFGE, except for a few cases where individual isolates belonging to two different clones were found to have altered fla-types. Similarly, one C. coli clone showed pronounced fla-type variation. The present results lead to the conclusion that vertical transmission or horizontal transmission via the hatchery are not significant transmission routes of C. jejuni to broiler chickens under Danish conditions. In the cases where more than one Campylobacter clone simultaneously colonised flocks, we found that the different clones coexisted in flocks rather than excluding each other.

An outbreak of infectious hepatitis in commercially reared ostriches associated with Campylobacter coli and Campylobacter jejuni.

A disease causing high morbidity and mortality was observed in young ostriches from six properties in southeast Queensland, Australia. The disease affected birds from 2-8 weeks of age and was characterised clinically by bright-green urates and pathologically by severe necrotic hepatitis. The liver lesions resembled those of vibrionic hepatitis in other avian species. Campylobacter coli was isolated from the livers of affected ostriches from five of the six properties. Campylobacter jejuni subsp. jejuni was isolated from birds from the remaining property. Pulsed-field gel electrophoresis-based (PFGE) typing of representative isolates indicated that trade of infected birds between farms was an important factor in the spread of C. coli. Phenotypic and genotypic data suggest a clonal variant of the principal outbreak type may account for the remaining cases from which C. coli was found. Conventional biochemical test results and PFGE clearly distinguished the C. jejuni strain isolated from the geographically remote farm from the outbreak of C. coli type. We believe this to be the first definitive report of avian hepatitis associated with C. coli.

Arcobacter butzleri strains from poultry abattoir effluent in Nigeria [corrected].

OBJECTIVE: To investigate the prevalence, species distribution and genetic diversity of zoonotic Arcobacter species. DESIGN: Prospective study. SETTING: Drainage system of a cosmopolitan chicken abattoir in Lagos, Nigeria. METHODS: One hundred and fifty drainage water samples were enriched in a minimal antibiotics-containing medium at room temperature and bacteria then isolated by use of a membrane filtration method. RESULTS: Twenty six (14%) of samples were positive for Arcobacter spp. Of these, 20 were examined by a comprehensive probabilistic identification scheme for Epsilobacteria and all strains identified as A. butzleri. AFLP analysis of these strains revealed considerable genetic diversity among the strains, with 12 genotypes defined at the 90% similarity level. CONCLUSION: The prevalence of A. butzleri in Nigerian poultry abattoir effluent indicates this species may constitute a public health problem in this country. AFLP profiling could be a useful tool for molecular epidemiological and population genetic studies of this organism. This is the first known report of A. butzleri in Nigeria, and first application of AFLP analysis for genotyping the species.

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies.

OBJECTIVE: To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. DESIGN: Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis. PROCEDURE: The results of identification of 99 bacterial strains as determined by conventional phenotyping or by polymerase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis; the remaining strains were field isolates putatively identified as C fetus. In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling. RESULTS: The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories. CONCLUSION: Our results indicate that misidentification of C fetus in routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.

Epidemiology of Shiga toxin-producing Escherichia coli O157 in very young calves in the North Island of New Zealand.

AIMS: To study the occurrence and spatial distribution of Shiga toxin-producing Escherichia coli (STEC) O157 in calves less than 1-week-old (bobby calves) born on dairy farms in the North Island of New Zealand, and to determine the association of concentration of IgG in serum, carcass weight, gender and breed with occurrence of E. coli O157 in these calves. METHODS: In total, 309 recto-anal mucosal swabs and blood samples were collected from bobby calves at two slaughter plants in the North Island of New Zealand. The address of the farm, tag number, carcass weight, gender and breed of the sampled animals were recorded. Swabs were tested for the presence of E. coli O157 using real time PCR (RT-PCR). All the farms were mapped geographically to determine the spatial distribution of farms positive for E. coli O157. K function analysis was used to test for clustering of these farms. Multiplex PCR was used for the detection of Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), E. coli attaching and effacing (eae) and Enterohaemolysin (ehxA) genes in E. coli O157 isolates. Genotypes of isolates from this study (n = 10) along with human (n = 18) and bovine isolates (n = 4) obtained elsewhere were determined using bacteriophage insertion typing for stx encoding. RESULTS: Of the 309 samples, 55 (17.7%) were positive for E. coli O157 by RT-PCR and originated from 47/197 (23.8%) farms. E. coli O157 was isolated from 10 samples of which seven isolates were positive for stx2, eae and ehxA genes and the other three isolates were positive for stx1, stx2, eae and ehxA. Bacteriophage insertion typing for stx encoding revealed that 12/18 (67%) human and 13/14 (93%) bovine isolates belonged to genotypes 1 and 3. K function analysis showed some clustering of farms positive for E. coli O157. There was no association between concentration of IgG in serum, carcass weight and gender of the calves, and samples positive for E. coli O157, assessed using linear mixed-effects models. However, Jersey calves were less likely to be positive for E. coli O157 by RT-PCR than Friesian calves (p = 0.055). CONCLUSIONS: Healthy bobby calves are an asymptomatic reservoir of E. coli O157 in New Zealand and may represent an important source of infection for humans. Carriage was not associated with concentration of IgG in serum, carcass weight or gender.

Species diversity of campylobacteria in a wild bird community in Sweden.

AIMS: To analyse the occurrence and host species distribution of campylobacteria species in shorebirds, geese and cattle on grazed coastal meadows in Sweden. METHODS AND RESULTS: Species identification was performed through a polyphasic approach, incorporating Amplified Fragment Length Polymorphism (AFLP) profiling, 16S RNA gene sequence analysis together with extensive phenotypic characterization. From 247 sampled birds and 71 cattle, we retrieved 113 urease positive thermophilic Campylobacter (UPTC) and 16 Campylobacter jejuni ssp. jejuni isolates. Furthermore, 18 isolates of Helicobacter canadensis, and five isolates that potentially represent a new genus of micro-aerophilic, spiral and Gram-negative bacteria were isolated. The distribution of bacterial species on hosts was uneven: all H. canadensis isolates were retrieved from geese, while all but one of the Campylobacter lari UPTC isolates were found in shorebirds. AFLP type distribution of Camp. lari UPTC isolates among individual, resampled and breeding-paired Redshank birds generally indicated a constant shift in strain populations over time and absence of geographical clustering. CONCLUSIONS: The large number of isolated campylobacteria, including species that are zoonotic enteropathogens, indicates that these wild birds potentially may serve as reservoirs of human infections. However, despite a common environment, the different host species largely carried their own campylobacteria populations, indicating that cross-species transmission is rare. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study is one of few that provide data on the occurrence of campylobacteria in wild animals, adding information on the ecology and epidemiology of micro-organisms that are of public health concern.