Andrea R. McWhorter

Comparative phenotypic and genotypic virulence of Salmonella strains isolated from Australian layer farms

There are over 2500 Salmonella enterica serovars that circulate globally. Of these, serovars those classified into subspecies I are the most common cause of human salmonellosis. Many subspecies I Salmonella serovars are routinely isolated from egg farm environments but are not frequently associated with causing disease in humans. In this study, virulence profiles were generated for 10 strains of Salmonella enterica isolated directly from egg farm environments to investigate their potential public health risk. Three virulence parameters were assessed including in vitro invasion, in vivo pathogenicity and characterization of genomic variation within five specific pathogenicity islands. These 10 Salmonella strains exhibited significant differences in invasion into the human intestinal epithelial cell line, Caco2. Low, moderate, and high invasion patterns were observed and the degree of invasion was dependent on bacterial growth in a nutritive environment. Interestingly, two Salmonella strains, S. Adelaide and S. Bredeney had consistently low invasion. The S. Typhimurium definitive types and S. Virchow exhibited the greatest cell invasion following growth in Luria Bertani broth. Only the S. Typhimurium strains caused disease in BALB/c mice, yet the majority of serovars were consistently detected in feces over the 21 day experiment. Genomic comparison of the five specific pathogenicity islands has shown that variation in virulence is likely multifactorial. Sequence variability was observed primarily in strains with low virulence. In particular, genes involved in forming the structures of the SPI-1 and SPI-2 type 3 secretion systems as well as multiple effector proteins were among the most variable. This variability suggest that serovars with low virulence are likely to have both invasion and within host replication defects that ultimately limit their pathogenicity.

Pathogenicity of Salmonella Strains Isolated from Egg Shells and the Layer Farm Environment in Australia

ABSTRACT In Australia, the egg industry is periodically implicated during outbreaks of Salmonella food poisoning. Salmonella enterica serovar Typhimurium and other nontyphoidal Salmonella spp., in particular, are a major concern for Australian public health. Several definitive types of Salmonella Typhimurium strains, but primarily Salmonella Typhimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidal Salmonella isolates obtained from Australian egg farms. To achieve this, we assessed the capacity of Salmonella isolates to cause gastrointestinal disease using both in vitro and in vivo model systems. Data from in vitro experiments demonstrated that the invasion capacity of Salmonella serovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During the in vivo infection trial, clinical signs of infection and mortality were observed only for mice infected with either 103 or 105 CFU of S. Typhimurium DT9. No mortality was observed for mice infected with Salmonella serovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and either in vitro invasive capacity or in vivo pathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-related Salmonella spp.

Study of Salmonella Typhimurium Infection in Laying Hens

Members of Salmonella enterica are frequently involved in egg and egg product related human food poisoning outbreaks worldwide. In Australia, Salmonella Typhimurium is frequently involved in egg and egg product related foodborne illness and Salmonella Mbandaka has also been found to be a contaminant of the layer farm environment. The ability possessed by Salmonella Enteritidis to colonize reproductive organs and contaminate developing eggs has been well-described. However, there are few studies investigating this ability for Salmonella Typhimurium. The hypothesis of this study was that the Salmonella Typhimurium can colonize the gut for a prolonged period of time and that horizontal infection through feces is the main route of egg contamination. At 14 weeks of age hens were orally infected with either S. Typhimurium PT 9 or S. Typhimurium PT 9 and Salmonella Mbandaka. Salmonella shedding in feces and eggs was monitored for 15 weeks post-infection. Egg shell surface and internal contents of eggs laid by infected hens were cultured independently for detection of Salmonella spp. The mean Salmonella load in feces ranged from 1.54 to 63.35 and 0.31 to 98.38 most probable number/g (MPN/g) in the S. Typhimurium and S. Typhimurium + S. Mbandaka group, respectively. No correlation was found between mean fecal Salmonella load and frequency of egg shell contamination. Egg shell contamination was higher in S. Typhimurium + S. Mbandaka infected group (7.2% S. Typhimurium, 14.1% S. Mbandaka) compared to birds infected with S. Typhimurium (5.66%) however, co-infection had no significant impact on egg contamination by S. Typhimurium. Throughout the study Salmonella was not recovered from internal contents of eggs laid by hens. Salmonella was isolated from different segments of oviduct of hens from both the groups, however pathology was not observed on microscopic examination. This study investigated Salmonella shedding for up to 15 weeks p.i which is a longer period of time compared to previously published studies. The findings of current study demonstrated intermittent but persistent fecal shedding of Salmonella after oral infection for up to 15 weeks p.i. Further, egg shell contamination, with lack of internal egg content contamination and the low frequency of reproductive organ infection suggested that horizontal infection through contaminated feces is the main route of egg contamination with S. Typhimurium in laying hens.

Antimicrobial resistance of non-typhoidal Salmonella isolates from egg layer flocks and egg shells.

This study was conducted to examine the antimicrobial resistance (AMR) of Salmonella spp. isolated from commercial caged layer flocks in New South Wales and South Australia. All Salmonella isolates (n=145) were subjected to phenotypic and genotypic characterisation of AMR and carriage of integrons. The majority of Salmonella isolates (91.72%) were susceptible to all antimicrobials tested in this study. Limited resistance was observed to amoxicillin and ampicillin (5.51%), tetracycline (4.13%), cephalothin (2.06%) and trimethoprim (0.68%). None of the isolates were resistant to cefotaxime, ceftiofur, ciprofloxacin, chloramphenicol, gentamycin, neomycin or streptomycin. A low frequency of Salmonella isolates (4.83%) harboured antimicrobial resistance genes and a class 1 integron. The most commonly detected AMR genes among the Salmonella isolates were blaTEM (2.07%), tet A (1.38%) and dhfrV (0.69%). Overall, Salmonella enterica isolates exhibited a low frequency of AMR and represent a minimal public health risk associated with the emergence of multidrug resistant Salmonella spp. from the Australian layer industry.

Multimeric Recombinant M2e Protein-Based ELISA: A Significant Improvement in Differentiating Avian Influenza Infected Chickens from Vaccinated Ones

Killed avian influenza virus (AIV) vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA) in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e) of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e) for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006) was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.

Salmonella Typhimurium in the Australian Egg Industry: Multidisciplinary Approach to Addressing the Public Health Challenge and Future Directions

ABSTRACT In Australia, numerous egg-related human Salmonella typhimurium outbreaks have prompted significant interest among public health authorities and the egg industry to jointly address this human health concern. Nationwide workshops on Salmonella and eggs were conducted in Australia for egg producers and regulatory authorities. State and national regulators represented Primary Production, Communicable Disease Control, Public Health and Food Safety, and Food Standards Australia and New Zealand. All attendees participated in discussions aimed at evaluating current evidence-based information, issues related to quality of egg production, and how to ensure safe eggs in the supply chain, identifying research gaps and practical recommendations. The perceptions from egg producers and regulatory authorities from various states were recorded during the workshops. We presented the issues discussed during the workshops, including Salmonella in the farm environment, Salmonella penetration across eggshell, virulence in humans, food/egg handling in the supply chain, and intervention strategies. We also discussed the perceptions from egg producers and regulators. Recommendations placed emphasis on the future research needs, communication between industry and regulatory authorities, and education of food handlers. Communication between regulators and industry is pivotal to control egg-borne S. typhimurium outbreaks, and collaborative efforts are required to design effective and appropriate control strategies.

Salmonella enterica isolates from layer farm environments are able to form biofilm on eggshell surfaces.

Abstract This study examined the eggshell biofilm forming ability of Salmonella enterica isolates recovered from egg farms. Multicellular behaviour and biofilm production were examined at 22 and 37°C by Congo red morphology and the crystal violet staining assay. The results indicated that the biofilm forming behaviour of Salmonella isolates was dependent on temperature and associated with serovars. Significantly greater biofilm production was observed at 22°C compared with 37°C. The number of viable biofilm cells attached to eggshells after incubation for 48 h at 22°C was significantly influenced by serovar. Scanning electron microscopic examination revealed firm attachment of bacterial cells to the eggshell surface. The relative expression of csgD and adrA gene was significantly higher in eggshell biofilm cells of S. Mbandaka and S. Oranienburg. These findings demonstrate that Salmonella isolates are capable of forming biofilm on the eggshell surface and that this behaviour is influenced by temperature and serovar.

In vitro invasive capacity of Salmonella strains into sections of the layer hen oviduct.

Raw or undercooked eggs and egg products are frequently identified as the source of Salmonella following outbreaks of foodborne gastrointestinal disease. Some Salmonella serovars, such as Salmonella Enteriditis, have a high tropism for the oviduct of laying hens. Oviduct colonization with S. Enteriditis can result in both internal and external contamination of an egg. While oviduct invasion is not limited to S. Enteriditis, the invasive capacities of other serovars is not widely known. In this study, the in vitro invasive ability of eighteen Salmonella isolates of representative serovars into different segments of the oviduct was assessed. All Salmonella isolates tested were invasive and the highest bacterial invasion was observed in segments of the isthmus and vagina. S. Bredeney consistently exhibited the lowest invasion into all sections of the oviduct. Interestingly, the S. Typhimurium definitive types included in this study did not exhibit significantly greater invasion capacity than other serovars. In this study, the genomic capacity of the selected isolates of representative Salmonella serovars to colonize the layer hen oviduct was also investigated. Previous studies have identified several genes upregulated during oviduct colonization by S. Enteriditis. Single gene comparison of 107 genes from eleven Salmonella isolates was conducted to determine whether these oviduct colonization genes were present within each bacterial genome. The degree of homology with corresponding sequences in S. Enteriditis P125109 was also determined for each gene. Genes encoding the O-antigen as well as phage and virulence plasmid genes were among the most highly variable and may serve specific roles in oviduct invasion.

Anti-bacterial and anti-biofilm activity of commercial organic acid products against Salmonella enterica isolates recovered from an egg farm environment

ABSTRACT This study evaluated the antibacterial activity of commercially available organic acid water additives against Salmonella enterica isolates and examined the susceptibility of Salmonella Typhimurium biofilms to these products. Three commercial organic acid products (A, B, and C) were evaluated for minimum inhibitory and bactericidal concentrations against isolates of S. enterica serovars. Three- and five-day-old S. Typhimurium biofilms were formed at 22 ± 2°C using an MBEC™ assay system and exposed for 30 min or 90 min at 0.2% and 0.4% concentrations. No significant difference among serovars for inhibitory and bactericidal concentrations was detected. Two products (A and C) significantly reduced viable cells from biofilms of both ages in a dose- and time-dependent manner. Increased biofilm age did not enhance resistance towards organic acid treatments. None of the products completely eliminated biofilm cells at any concentration or exposure time. Product composition, exposure time, and concentration of organic acid products were important factors in reducing viable biofilm cells. This study has expanded our understanding about the susceptibility of Salmonella biofilms to commercial organic acid products. These findings have implications in the usage, development, and optimization of organic acid products.

Egg Production Systems and Salmonella in Australia

Abstract Undercooked eggs and egg-related products are frequently implicated as primary sources of Salmonella during food poisoning outbreaks. Globally, Salmonella Enteriditis is most commonly identified from reported salmonellosis cases, but it is not endemic in commercial poultry industry in Australia. Salmonella Typhimurium has filled this niche and over the past decade has been the most frequently isolated serovar during Australian outbreaks. In 2015, the Department of Health recorded 16,952 notified Salmonella cases; of these many were epidemiologically linked with the consumption of raw egg products. Eggs can become contaminated with S. Typhimurium during lay when they come into contact with Salmonella-positive fecal material. Small defects in the egg shell may subsequently influence bacterial penetration across the shell leading to contamination of egg internal contents. Farm-specific Salmonella intervention strategies are not sufficient for bacterial control and combined efforts through the entire supply chain are essential to limiting Salmonella.