Significance of the rdar and bdar morphotypes in the hydrophobicity and attachment to abiotic surfaces of Salmonella Sofia and other poultry-associated Salmonella serovars.

AIMS: To investigate the relative role of the red dry and rough (rdar) and brown dry and rough (bdar) morphotypes on hydrophobicity and ability to attach to abiotic surfaces of poultry-associated Salmonella strains with a focus on S. Sofia. METHODS AND RESULTS: Cellulose synthase gene null mutants were constructed in five Salmonella strains converting them from rdar to bdar morphotypes. One S. Sofia null mutant displayed reduced hydrophobicity and attachment to Teflon® relative to its parent strain. The S. Virchow and S. Infantis null mutants attached less well to glass relative to their parent strains. CONCLUSIONS: The rdar or bdar morphotype may influence S. Sofia persistence but did not explain why bdar strains predominate in this serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides some insight into why some Salmonella strains survive in poultry environments and may ultimately contribute to their control.

Concentration and prevalence of Escherichia coli O157 and Salmonella serotypes in sheep during slaughter at two Australian abattoirs.

OBJECTIVE: This study aimed to determine the presence and concentration of Escherichia coli O157 and Salmonella spp. on fleece, faeces and carcases of sheep during slaughter. PROCEDURE: Faeces, fleece and pre-chill carcase samples were collected from 164 sheep slaughtered at two Australian abattoirs. The presence of E. coli O157 and Salmonella spp. were determined by use of automated immunomagnetic separation (AIMS) with enumeration by use of the 'most probable number' (MPN) method. RESULTS: Escherichia coli O157 was isolated from 5% of faeces, 3% of fleeces and 0.6% of pre-chill carcases. The mean log(10) count of E. coli O157 positive faecal samples was 2.32 MPN/g, but counts on fleeces and carcases were below the countable limit (-1 log(10) MPN/cm(2) ). Salmonella spp. were isolated from 20% of faeces, 13% of fleeces and 1.3% of pre-chill carcases. The mean log(10) count of Salmonella spp. in faeces was 1.43 MPN/g and on fleece was -0.24 MPN/cm(2) , but counts on carcases were below the countable limit (-1 log(10) MPN/cm(2) ). CONCLUSION: The prevalence and concentration of pathogens were low in the sheep tested in this study, indicating a low risk of human infection from products derived from these animals.

Relative prevalence of Salmonella Sofia on broiler chickens pre- and postprocessing in Australia.

A survey was conducted to determine the relative prevalence of Salmonella serovars on whole chicken carcasses before and after processing in 3 Australian poultry abattoirs. Ninety and 180 whole chicken carcasses were tested for Salmonella serovars before and after processing, respectively. Each carcass was subjected to a buffered peptone water rinse according to Australian Standard methodologies and Salmonella prevalence was determined using Australian Standard methodologies. After isolation, Salmonella isolates were serotyped and results were analyzed to determine the relative percentage of each serovar at both processing points. Salmonella Sofia was shown to significantly increase its relative prevalence (P < or = 0.05) after processing and proved to be the dominant serovar accounting for 45/89 (51%) isolations before processing and 51/69 (74%) isolations after processing. The reasons for the increased relative prevalence of Salmonella Sofia are currently unknown and require further investigation but may involve factors related to prevalence and numbers on chickens and the ability of Salmonella Sofia to respond to environmental stressors and attach to surfaces.

Distribution, prevalence and persistence of Cronobacter (Enterobacter sakazakii) in the nonprocessing and processing environments of five milk powder factories.

AIMS: To characterise the occurrence of Cronobacter in milk powder factories. METHODS AND RESULTS: Cronobacter was isolated from 32% of 298 environmental samples from five factories. More isolations occurred in nonprocessing (49%) than processing areas (29%), although the greatest occurrence was in a single milk powder area during shutdown maintenance (81%) and the lowest after reinstatement of production hygiene practices (6%). Clonal analysis using PFGE placed 129 isolates into 49 groups. Most clones (45) were unique to each factory and seven were isolated in both milk powder and other areas of the same factory including tanker bays, evaporator rooms, an employee's shoes and external roofs. Cronobacter was not isolated from raw milk processing areas. Within powder areas, 17 clones occurred at more than one and up to eight locations and six occurred more than once at the same location. Between four and seven clones were in the powder areas at each factory. The most prevalent and persistent clones were isolated from external roofs above spray driers, in air treatment areas and where high foot traffic occurs. CONCLUSIONS: Cronobacter is dispersed widely at milk powder factories. This study suggests that distribution is assisted by movement of air, milk powder and personnel and that new hygiene strategies will be needed to reduce prevalence. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of occurrence is essential for the development of strategies to control dissemination of Cronobacter within factories and reduce risk of entry into powdered milk products.

Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology.

The exploration of new source materials and the use of alternative isolation and identification methods have led to rapid expansion in the knowledge of diversity; in Lysobacter, 11 new species having been described since 2005, and in Stenotrophomonas with six new species since 2000. The new species of Lysobacter, isolated by dilution and direct plating on standard media, differ in several key phenotypic properties from those obtained by enrichment on complex polysaccharides in the original description of the genus. Revision of the definition of the genus will be required. Both culture-dependent and culture-independent methods to assess community structure, in a variety of host and nonhost environments, have established that some species of Lysobacter are a dominant component of the microflora, where previously their presence had not been suspected. Culture-independent studies have generally not added new information on the occurrence and distribution of Stenotrophomonas maltophilia and other members of the genus, which are readily isolated on standard media from source materials. Lysobacter enzymogenes and Sten. maltophilia produce similar antibiotics and share some enzyme activities which, subject to safety considerations, may make them attractive candidates for use in biological control of plant diseases and of nematodes.

Attachment of different Salmonella serovars to materials commonly used in a poultry processing plant.

Salmonella can adhere to poultry and food contact surfaces and persist to cause diseases. Adhesion of Salmonella Sofia (n = 14), S. Typhimurium (n = 6), S. Infantis (n = 3) and S. Virchow (n = 2) to Teflon, stainless steel, glass, rubber and polyurethane were assayed using epifluorescence microscopy. Surface free energies of bacteria and materials were calculated using contact angle values and interfacial free energy between isolates and materials determined. Surface roughness of the materials was analysed using atomic force microscopy. S. Sofia isolates adhered in higher numbers (P < 0.05) to all materials compared to other serovars. The mean number of cells of S. Sofia isolates attaching to Teflon were significantly higher (P < 0.05) compared to all materials except stainless steel (P > 0.05). Mean roughness values ranged from 82.26 nm (Teflon) to 1.34 nm (glass). Correlations between the apolar component of the surface free energy of materials (gamma(S)(LW)) and bacterial adhesion (R(2) = 0.80), and between gamma(S)(LW) and the surface roughness of the materials (R(2) = 0.71) were found. Materials more positive in interfacial free energies had the highest number of adhering bacteria. Generalised surface property measurements were found to be useful in characterising Salmonella attachment but the degree of variability in results suggests that other factors, such as flagella or membrane proteins, could also contribute.

Integron-containing bacteria in faeces of cattle from different production systems at slaughter.

AIMS: To determine the prevalence and characteristics of integron-containing bacteria in faeces of cattle from grass-fed, lot-fed, or organically produced cattle. METHODS AND RESULTS: Faecal samples from grass-fed (n = 125), lot-fed (n = 125) and organic (n = 135) cattle were tested for the presence of class 1 and class 2 integrons by using PCR and colony hybridisation. The prevalence of class 1 and class 2 integrase were higher in lot-fed cattle (71% and 62%) than grass-fed cattle (52% and 30%) which in turn were higher than organic cattle (25% and 11%). Isolation rates of integron-containing bacteria were reflective of PCR prevalence results. CONCLUSIONS: The antimicrobial resistance genes harboured by the integrons differed little across the three systems and were typically to antimicrobials that would rarely be used therapeutically or for growth promotion purposes. The differences in prevalence observed between the systems may be a function of the intensiveness of each system. SIGNIFICANCE AND IMPACT OF THE STUDY: Integron-containing bacteria may be present in all cattle production systems regardless of the amount of antimicrobial use and confirms that the prudent use of antimicrobials is required so that the development of integrons harbouring genes significant to human medicine is avoided.

Prevalence and serotypes of Salmonella associated with goats at two Australian abattoirs.

AIMS: This study was carried out to determine the prevalence and serotype of Salmonella in goats presented for slaughter. METHODS AND RESULTS: A total of 121 goats were examined for the presence of Salmonella in matching rumen, faecal and carcass samples. Samples were analysed for the presence of Salmonella following the Australian Standard AS 1766.2.5-1991. Salmonella was isolated from 56 (46.3%) faecal samples, 55 (45.5%) rumen samples and 35 (28.9%) carcass samples. The dominant serotypes isolated were Salmonella serotype Saintpaul (31%), Salmonella serotype Typhimurium (13%) and Salmonella serotype Chester (11%). CONCLUSIONS: Salmonella was isolated from at least one of the three sample sites in 68% of animals. Carcase contamination with faeces, compared with rumen liquor, is a greater hazard for Salmonella contamination of goat carcases. Goat meat is a potential source of Salmonella serovars associated with human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Goat carcases contaminated with Salmonella during slaughter could be a source of food-borne disease if consumed raw or inadequately cooked, or may be a source of cross-contamination to other foods.

A comparison of antibiotic resistance integrons in cattle from separate beef meat production systems at slaughter.

AIMS: To compare antibiotic resistance integrons in cattle from three separate grass-fed, grain-fed and certified organic cattle production systems at slaughter. METHODS AND RESULTS: In this study 198 samples from three separate cattle production systems were tested by PCR for the presence of class 1 and class 2 integrons. Integron-containing bacteria were readily isolated from pen faeces and hide samples regardless of production system. Lower numbers of integron-containing bacteria were isolated from the remaining sample types. Ninety-one class 1 and 34 class 2 integron-containing bacteria were isolated. Characterization of the integrons demonstrated a high degree of similarity across the three production systems with aadA1 and aadA2 routinely present. Integrons harbouring the cassette array cmlA5-bla(OXA-10)-aadA1 and the putative insertion sequence IS1066 were isolated from organic and grass-fed cattle and have not been described previously. CONCLUSIONS: Integrons carrying antibiotic resistance genes were common in cattle from differing production systems at slaughter and the likelihood of presence appears unrelated to the production system. SIGNIFICANCE AND IMPACT OF THE STUDY: Similar integron arrays are present in different cattle production systems suggesting that their presence may be independent of production practices. This is the first report of two novel integron structures present in Aeromonas.

Salmonella Sofia differs from other poultry-associated Salmonella serovars with respect to cell surface hydrophobicity.

Salmonella enterica is one of the most important foodborne pathogens. Salmonella enterica subsp. II 4,12:b:- (Salmonella Sofia) is commonly found in Australian poultry. It has been suggested that physicochemical properties such as surface charge and hydrophobicity may affect bacterial attachment to surfaces and their ability to persist in food systems. A possible link between hydrophobicity cell surface charge and persistence of Salmonella from the poultry system was examined. Hydrophobicity of Salmonella Sofia (n = 14), Salmonella Typhimurium (n = 6), Salmonella Infantis (n = 3), and Salmonella Virchow (n = 2) was assayed using hydrophobic interaction chromatography, bacterial adherence to hydrocarbons (BATH), using xylene or hexadecane, and the contact angle method (CAM). Cellular surface charge (CSC) of the isolates was determined using zeta potential measurements. The majority (12 of 14) of Salmonella Sofia isolates were found to be hydrophobic when assayed using BATH with xylene, except isolates S1635 and S1636, and the other serovars were found to be hydrophilic. Salmonella Sofia isolates were not significantly different (P > 0.05) from isolates of other serovars as measured by hydrophobic interaction, BATH with hexadecane, or the CAM. No significant differences (P > 0.05) in zeta potential measurements were observed between isolates. Principal component analysis using results from all four measures of hydrophobicity allowed clear differentiation between isolates of the serovar Salmonella Sofia (except S1635 and S1636) and those of other Salmonella serovars. Differences in physicochemical properties may be a contributing factor to the Salmonella Sofia serovar's ability to attach to surfaces and persist in a food system.

Managing safety and quality through the red meat chain.

To successfully manage food safety and quality risks in meat production, a holistic approach is required. The ideal would be a fully integrated assurance system, with effective controls applied at all stages. However, the red meat industry is by nature somewhat fragmented, and a truly integrated system is not at present achievable in all but a few operations. This paper describes a variety of assurance initiatives, and explores how targeted research and development can be used to augment assurance programmes by providing underpinning knowledge, using the Australian beef and lamb industry as an example.

Physicochemical properties of Shiga toxigenic Escherichia coli.

AIMS: To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. METHODS AND RESULTS: Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0.05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). CONCLUSIONS: Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces.

Quantification and prevalence of Salmonella in beef cattle presenting at slaughter.

AIMS: A survey to determine the prevalence and numbers of Salmonella in beef cattle presented for slaughter at abattoirs across Australia was conducted between September 2002 and January 2003. METHODS AND RESULTS: Automated immunomagnetic separation (AIMS) was used for detection and isolation of Salmonella enriched from cattle faeces. Salmonella were enumerated from positive samples using a combination of the Most Probable Number (MPN) technique and AIMS. A total of 310 faecal samples were tested, 155 were from lot-fed cattle and 155 from grass-fed cattle. Salmonella spp. were isolated from 21 (6.8%) of the cattle and the prevalence amongst grass-fed cattle (4.5%) was not significantly different to that found in lot-fed cattle (9%). Counts of Salmonella in positive faeces varied from <3 MPN g(-1) of faeces to 2.8 x 10(3) MPN g(-1) and 71% of positive samples had counts <10 MPN g(-1) faeces. There was no significant difference in the mean log10 number of Salmonella in faeces of cattle from each production system. CONCLUSION: Low numbers of beef cattle were found to shed Salmonella at the time of slaughter and the prevalence and the associated faecal concentrations did not vary significantly with the pre-slaughter production system (grass or lot feeding). The faecal concentration of Salmonella in the majority of faeces was low (<10 MPN g(-1)) with few high concentrations up to 3 x 10(3) MPN g(-1), suggesting there may be a low risk of carcase contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Beef cattle do not appear to be a major source of entry of Salmonella into the human food chain and the quantitative information contained in this study can be used in quantitative assessments of the associated risk of human salmonellosis.

Molecular epidemiology of two fowl cholera outbreaks on a free-range chicken layer farm.

Two outbreaks of fowl cholera on a multiage free-range egg farm were investigated. The outbreaks occurred in 1994 and 2002. A total of 22 strains of Pasteurella multocida were available for study, 11 from the 1994 outbreak and 11 from the 2002 outbreak. Lesions typical of acute fowl cholera were seen in the 1994 outbreak, whereas both acute and chronic fowl cholera occurred in the 2002 outbreak. The isolates were examined in an extended phenotypic typing methodology, by a P. multocida-specific polymerase chain reaction (PCR), by the Heddleston somatic serotyping scheme, and by restriction endonuclease analysis (REA) typing using the enzyme HpaII. All 22 strains had the same phenotypic properties, all were confirmed as P. multocida by PCR, all were Heddleston serovar 4, and all had the same REA pattern. The results indicate that these 2 outbreaks were caused by the same clone of P. multocida--despite the 8-year time period between the outbreaks.

The prevalence and concentration of Escherichia coli O157 in faeces of cattle from different production systems at slaughter.

AIMS: To determine the prevalence and concentration of Escherichia coli O157 shed in faeces at slaughter, by beef cattle from different production systems. METHODS AND RESULTS: Faecal samples were collected from grass-fed (pasture) and lot-fed (feedlot) cattle at slaughter and tested for the presence of E. coli O157 using automated immunomagnetic separation (AIMS). Escherichia coli O157 was enumerated in positive samples using the most probable number (MPN) technique and AIMS and total E. coli were enumerated using Petrifilm. A total of 310 faecal samples were tested (155 from each group). The geometric mean count of total E. coli was 5 x 10(5) and 2.5 x 10(5) CFU g(-1) for lot- and grass-fed cattle, respectively. Escherichia coli O157 was isolated from 13% of faeces with no significant difference between grass-fed (10%) and lot-fed cattle (15%). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g(-1)) to 1.1 x 10(5) MPN g(-1). Twenty-six (67%) of 39 O157 positive faeces had <10 MPN g(-1) and three (8%) had counts between 10(3)-10(5) MPN g(-1). There was no significant difference between concentrations of E. coli O157 in the faeces of grass-fed or lot-fed cattle. CONCLUSION: The prevalence and numbers of E. coli O157 in the faeces of cattle at slaughter were not affected by the production systems evaluated in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the prevalence and numbers of E. coli O157 can be used for formulating intervention strategies and in quantitative risk assessments.

Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation.

AIMS: To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. METHODS AND RESULTS: A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g-1 of faeces) to 2.4 x 104 MPN g-1. There was no significant difference (P = 0.06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g-1) than in pasture-fed (13 MPN g-1). CONCLUSIONS: Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia.

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

Characterisation of a novel Mannheimia sp from Australian feedlot cattle.

OBJECTIVE: To characterise eight isolates of a Gram-negative organism obtained from the upper respiratory tract of cattle showing evidence of mild upper respiratory tract disease. DESIGN: The isolates were compared with the five recognised species within the genus Mannheimia - M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena--using a range of phenotypic and genotypic methods. RESULTS: Phenotypic characterisation indicated that the isolates belonged to the trehalose-negative [Pasteurella] haemolytica complex. This complex has recently been reorganised into five species within the new genus Mannheimia. Ribotyping performed using HindIII and a computerised analysis system indicated that the eight Australian isolates formed a distinct cluster that was related to, but different from, the five recognised species of Mannheimia. The 16S rRNA sequence of one isolate (BNO311) was determined and a phylogenetic analysis performed. Isolate BNO311 was distinct from the five named Mannheimia spp but did join a larger cluster consisting of rRNA cluster IV (M varigena) and the unnamed rRNA cluster V of Mannheimia. DNA:DNA hybridisation between isolate BNO311 and M haemolytica NCTC 9380T, M granulomatis P411 and Actinobacillus ligniersii NCTC 4189T all suggested similarities of approximately 30%. CONCLUSIONS: These phenotypic and genotypic characterisation studies suggest that the eight Australian isolates represent a new species of Mannheimia. Until further characterisation studies are performed, we are unwilling to propose a name for this taxon, preferring to refer to this possible new species as Bisgaard taxon 39 of cluster V of Mannheimia.

Ancestral divergence, genome diversification, and phylogeographic variation in subpopulations of sorbitol-negative, beta-glucuronidase-negative enterohemorrhagic Escherichia coli O157.

The O157:H7 lineage of enterohemorrhagic Escherichia coli is a geographically disseminated complex of highly related genotypes that share common ancestry. The common clone that is found worldwide carries several markers of events in its evolution, including markers for acquisition of virulence genes and loss of physiological characteristics, such as sorbitol fermentation ability and beta-glucuronidase production. Populations of variants that are distinct with respect to motility and the sorbitol and beta-glucuronidase markers appear to have diverged at several points along the inferred evolutionary pathway. In addition to these variants, distinct subpopulations of the contemporary non-sorbitol-fermenting, beta-glucuronidase-negative O157:H7 clone were recently detected among bovine and human clinical isolates in the United States by using high-resolution genome comparison. In order to determine if these recently described subpopulations were derived from a regional or ancestral divergence event, we used octamer-based genome scanning, marker sorting, and DNA sequence analysis to examine their phylogenetic relationship to populations of non-sorbitol-fermenting, beta-glucuronidase negative O157:H7 and O157:H- strains from Australia. The inferred phylogeny is consistent with the hypothesis that subpopulations on each continent resulted from geographic spread of an ancestral divergence event and subsequent expansion of distinct subpopulations. Marker sorting and DNA sequence analyses identified sets of monophyletic markers consistent with the pattern of divergence and demonstrated that phylogeographic variation occurred through emergence of regional subclones and concentration of regional polymorphisms among distinct subpopulations. DNA sequence analysis of representative polyphyletic markers showed that genome diversity accrued through random drift and bacteriophage-mediated events.