Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia

Background Highly pathogenic strains of Staphylococcus aureus can cause disease in both humans and animals. In animal species, including ruminants, S. aureus may cause severe or sub-clinical mastitis. Dairy animals with mastitis frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. The aim of this study was to use genotypic and immunological methods to characterize S. aureus isolates from milk-related samples collected from 7 dairy farms across Victoria. Results A total of 30 S. aureus isolates were collected from milk and milk filter samples from 3 bovine, 3 caprine and 1 ovine dairy farms across Victoria, Australia. Pulsed Field Gel Electrophoresis (PFGE) identified 11 distinct pulsotypes among isolates; all caprine and ovine isolates shared greater than 80 % similarity regardless of source. Conversely, bovine isolates showed higher diversity. Multi-Locus Sequence Typing (MLST) identified 5 different sequence types (STs) among bovine isolates, associated with human or ruminant lineages. All caprine and ovine isolates were ST133, or a single allele variant of ST133. Two new novel STs were identified among isolates in this study (ST3183 and ST3184). With the exception of these 2 new STs, eBURST analysis predicted all other STs to be founding members of their associated clonal complexes (CCs). Analysis of genetic markers revealed a diverse range of classical staphylococcal enterotoxins (SE) among isolates, with 11 different SEs identified among bovine isolates, compared with just 2 among caprine and ovine isolates. None of the isolates contained mecA, or were resistant to oxacillin. The only antibiotic resistance identified was that of a single isolate resistant to penicillin; this isolate also contained the penicillin resistance gene blaZ. Production of SE was observed at 16 °C and/or 37 °C in milk, however no SE production was detected at 12 °C. Conclusion Although this study characterized a limited number of isolates, bovine-associated isolates showed higher genetic diversity than their caprine or ovine counterparts. This was also reflected in a more diverse SE repertoire among bovine isolates. Very little antibiotic resistance was identified among isolates in this study. These results suggest maintaining the milk cold chain will minimise any risk from SE production and highlights the need to prevent temperature abuse

Quantitative effects of in-line operations on Campylobacter and Escherichia coli through two Australian broiler processing plants

Campylobacter is an important food borne pathogen, mainly associated with poultry. A lack of through-chain quantitative Campylobacter data has been highlighted within quantitative risk assessments. The aim of this study was to quantitatively and qualitatively measure Campylobacter and Escherichia coli concentration on chicken carcasses through poultry slaughter. Chickens (n=240) were sampled from each of four flocks along the processing chain, before scald, after scald, before chill, after chill, after packaging and from individual caeca. The overall prevalence of Campylobacter after packaging was 83% with a median concentration of 0.8logCFU/mL. The processing points of scalding and chilling had significant mean reductions of both Campylobacter (1.8 and 2.9logCFU/carcase) and E. coli (1.3 and 2.5logCFU/carcase). The concentration of E. coli and Campylobacter was significantly correlated throughout processing indicating that E. coli may be a useful indicator organism for reductions in Campylobacter concentration. The carriage of species varied between flocks, with two flocks dominated by Campylobacter coli and two flocks dominated by Campylobacter jejuni. Current processing practices can lead to significant reductions in the concentration of Campylobacter on carcasses. Further understanding of the variable effect of processing on Campylobacter and the survival of specific genotypes may enable more targeted interventions to reduce the concentration of this poultry associated pathogen

Clostridium perfringens associated with dairy farm systems show diverse genotypes

Clostridium perfringens is a bacterial species of importance to both public and animal health. Frequently found in food system environments, it presents a risk to food animal health such as dairy herds, and may cross contaminate associated ingredients or food products, with potential to cause sporadic and outbreaks of disease in human populations, including gastroenteric illness. In this study, we characterized C. perfringens isolated from bovine, caprine, and ovine dairy farm systems (n = 8, 11 and 4, respectively). Isolates were phenotypically screened for antimicrobial sensitivity profiling, and subjected to whole genome sequencing to elucidate related genetic markers, as well as examine virulence gene markers, mobile genetic elements, and other features. Both toxin type A and type D isolates were identified (78 % and 22 % of isolates, respectively), including 20 novel sequence types. Resistance to clindamycin was most prevalent among antibiotics screened (30 %), followed by erythromycin (13 %), then penicillin and tetracycline (4 %), although an additional 3 isolates were non-susceptible to tetracycline. Most isolates harboured plasmids, which mobilised virulence markers such as etx, cpb2, and resistance markers tetA(P), tetB(P), and erm(Q), on conjugative plasmids. The presence of type D isolates on caprine farms emphasizes the need for control efforts to prevent infection and potential enterotoxemia. Clostridium perfringens enterotoxin (cpe) was not identified, suggesting lower risk of gastrointestinal illness from contaminated foods, the presence of other virulence and antimicrobial resistance markers suggests farm hygiene remains an important consideration to help ensure food safety of associated dairy foods produced

Analysis of the Listeria monocytogenes Population Structure among Isolates from 1931 to 2015 in Australia

Listeriosis remains among the most important bacterial illnesses, with a high associated mortality rate. Efforts to control listeriosis require detailed knowledge of the epidemiology of the disease itself, and its etiological bacterium, Listeria monocytogenes. In this study we provide an in-depth analysis of the epidemiology of 224 L. monocytogenes isolates from Australian clinical and non-clinical sources. Non-human sources included meat, dairy, seafood, fruit, and vegetables, along with animal and environmental isolates. Serotyping, Multi-Locus Sequence Typing, and analysis of inlA gene sequence were performed. Serogroups IIA, IIB, and IVB comprised 94% of all isolates, with IVB over-represented among clinical isolates. Serogroup IIA was the most common among dairy and meat isolates. Lineage I isolates were most common among clinical isolates, and 52% of clinical isolates belonged to ST1. Overall 39 STs were identified in this study, with ST1 and ST3 containing the largest numbers of L. monocytogenes isolates. These STs comprised 40% of the total isolates (n = 90), and both harbored isolates from clinical and non-clinical sources. ST204 was the third most common ST. The high prevalence of this group among L. monocytogenes populations has not been reported outside Australia. Twenty-seven percent of the STs in this study contained exclusively clinical isolates. Analysis of the virulence protein InlA among isolates in this study identified a truncated form of the protein among isolates from ST121 and ST325. The ST325 group contained a previously unreported novel mutation leading to production of a 93 amino acid protein. This study provides insights in the population structure of L. monocytogenes isolated in Australia, which will contribute to public health knowledge relating to this important human pathogen

Attachment of Salmonella strains to a plant cell wall model is modulated by surface characteristics and not by specific carbohydrate interactions

Background: Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface. Results: We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin. Conclusions: Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils

Prevalence and concentration of Arcobacter spp. on Australian beef carcasses

The International Commission on Microbiological Specifications for Foods (ICMSF) classified Arcobacter spp. as emerging pathogens in 2002. Arcobacter spp. have been isolated from numerous food products at retail and from animal carcasses and feces at slaughter. A survey was conducted to determine both the prevalence and concentration of Arcobacter spp. on prechill beef carcasses. Surface swab samples were collected from 130 beef carcasses at the end of processing, prior to chilling. The concentration of Arcobacter spp. was determined by a most-probable-number per square centimeter (3 by 3) method with a limit of detection of 0.12 CFU/cm(2). Of the 100 carcasses examined from export abattoirs, 20 (20.0%) were contaminated with Arcobacter spp., and 5 of these had quantifiable levels of contamination ranging from 0.12 to 0.31 CFU/cm(2). Of the 30 carcasses examined at a pet food abattoir, 25 (83.3%) were contaminated with Arcobacter spp., and 10 of these had quantifiable levels of contamination ranging from 0.12 to 0.95 CFU/cm(2). Three species of Arcobacter, A. butzleri, A. cryaerophilus, and A. skirowii, were identified by PCR. Each of the species was present in an approximately equal ratio from export abattoirs. This study demonstrates that slaughter practices at export abattoirs are sufficient to maintain both low prevalence and low levels of contamination of beef carcasses with Arcobacter spp

Comparative Genomics and Phenotypic Investigations Into Antibiotic, Heavy Metal, and Disinfectant Susceptibilities of Salmonella enterica Strains Isolated in Australia

Salmonella enterica is recognized as a major contributor of gastrointestinal illness worldwide. Concerns have been raised over the increasing prevalence of antibiotic resistant strains of Salmonella isolated from animals and food, and the role of antibiotics and other antimicrobial agents such as biocides and heavy metals in the selection and dissemination of antibiotic resistant bacteria to human hosts. In this study the antibiotic, heavy metal and disinfectant resistance genotypes and phenotypes of 19 S. enterica isolates from food-producing animals were established using whole genome sequence analysis, disc diffusion, as well as broth or agar dilution methods. This study also investigated the genomic environment of resistance genes on mobile genetic elements and chromosomal DNA. An ampicillin and streptomycin resistant S. Infantis isolate in this study harbored a β-lactamase (blaTEM–1), and two streptomycin resistance conferring genes (strA and strB) on a class 1 integron mobilized on a large conjugative plasmid. This plasmid also harbored two arsenic resistance gene cassettes. The arsenic resistance cassette, arsRCDAB, was also observed in two S. Singapore isolates with high tolerance to arsenate. A nalidixic acid resistant S. Typhimurium isolate was found to possess a mutation in gyrA resulting in amino acid change Asp87Gly and tetracycline resistant S. Typhimurium isolate was found to harbor efflux pump gene, tetA. No resistance (genotypic or phenotypic) was recorded to the disinfectants screened in this study. Taken together, results of this study showed a good correlation between predicted and measured resistances when comparing genotypic and phenotypic data, respectively. The findings of this study do not suggest resistance to clinically relevant antibiotics are widespread among Salmonella isolated from Australian food-producing animals

Expression and putative roles in attachment of outer membrane proteins of Escherichia coli O157 from planktonic and sessile culture

Many strains of Shiga toxigenic Escherichia coli (STEC), particularly the serotype O157:H7, are foodborne pathogens causing disease in many countries throughout the world. E. coli O157:H7 is able to attach and survive on various surfaces such as stainless steel (SS) found within the food processing environment. We examined the outer membrane protein (OMP) profiles of four E. coli O157 (three toxigenic O157:H7 and one nontoxigenic O157:HR) and one non-STEC strain (O1:H7), previously reported to have different abilities to attach to SS following growth in planktonic (nutrient broth) and sessile (nutrient agar) culture. The OMPs of the five E. coli strains grown in planktonic and sessile culture were extracted using N-lauroyl sarcosine and the OMP profiles were separated using two-dimensional (2D) gel electrophoresis. Qualitative and quantitative variations in the total number of OMPs expressed between planktonic and sessile cultures were found for all E. coli isolates tested. A number of differentially expressed protein spots were selected from 2D gels and were identified. FlgE was found to be expressed in planktonic culture but not sessile culture. MipA and OmpX had higher expression in sessile culture than planktonic culture, while expression of OmpA did not differ between E. coli strains or between the two modes of growth. Although differential expression of OMPs was found between isolates grown in planktonic and sessile culture, further investigations are required to determine a role of some of these identified proteins during growth of E. coli in planktonic and sessile culture and their influence during the attachment process

Expression and putative roles in attachment of outer membrane proteins of Escherichia coli O157 from planktonic and sessile culture

Many strains of Shiga toxigenic Escherichia coli (STEC), particularly the serotype O157:H7, are foodborne pathogens causing disease in many countries throughout the world. E. coli O157:H7 is able to attach and survive on various surfaces such as stainless steel (SS) found within the food processing environment. We examined the outer membrane protein (OMP) profiles of four E. coli O157 (three toxigenic O157:H7 and one nontoxigenic O157:HR) and one non-STEC strain (O1:H7), previously reported to have different abilities to attach to SS following growth in planktonic (nutrient broth) and sessile (nutrient agar) culture. The OMPs of the five E. coli strains grown in planktonic and sessile culture were extracted using N-lauroyl sarcosine and the OMP profiles were separated using two-dimensional (2D) gel electrophoresis. Qualitative and quantitative variations in the total number of OMPs expressed between planktonic and sessile cultures were found for all E. coli isolates tested. A number of differentially expressed protein spots were selected from 2D gels and were identified. FlgE was found to be expressed in planktonic culture but not sessile culture. MipA and OmpX had higher expression in sessile culture than planktonic culture, while expression of OmpA did not differ between E. coli strains or between the two modes of growth. Although differential expression of OMPs was found between isolates grown in planktonic and sessile culture, further investigations are required to determine a role of some of these identified proteins during growth of E. coli in planktonic and sessile culture and their influence during the attachment process