Francesca Martelli

Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain

OBJECTIVES The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured β-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to 2015

Objectives: To determine the occurrence of mcr-1-harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Methods: Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT–PCR. mcr-1-harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Results: Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%–1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1-positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1-plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1-plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%–99%. Conclusions: mcr-1-harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1-positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.

Observations on Salmonella contamination of commercial duck farms before and after cleaning and disinfection

ABSTRACT In the European Union, statutory control of Salmonella is in place in the chicken and turkey sectors, but not in the duck sector. In this study, 14 Salmonella-positive duck farms were sampled before and after cleaning and disinfection, and once the houses had been restocked with a new flock. The cleaning and disinfection programmes used were subdivided into two main categories: ones in which a final formaldehyde disinfection step was included (1) and ones in which it was not included (2). Several types of samples were collected during the study, and faecal samples were those more frequently positive (62% of faecal samples were positive for Salmonella in comparison to 2–23% of samples from all the other sample categories) (P < 0.001). Independently of the cleaning and disinfection programme used, there was a statistically significant (P < 0.001) reduction in the percentage of Salmonella-positive samples between before cleaning and disinfection (41.1%) and after cleaning and disinfection (3.1%). After restocking, the number of Salmonella-positive samples increased significantly (P < 0.001), with 65.3% of the samples tested being positive for Salmonella. Farms in which disinfection programme 1 was used were 5.34 times less likely to have samples positive for Salmonella after cleaning and disinfection than farms which implemented programme 2. Formaldehyde acts effectively against Salmonella even in the presence of some residual organic matter. Limited residual contamination on farms after cleaning and disinfection represents a risk of infection for young ducklings, and thorough cleaning and disinfection procedures should be implemented to reduce the carry-over of infection between flocks.

Observations on the distribution and control of Salmonella in commercial duck hatcheries in the UK

ABSTRACT Salmonella infection causes a significant number of cases of gastroenteritis and more serious illnesses in people in the UK and EU. The serovars Salmonella Enteritidis and Salmonella Typhimurium are most frequently associated with foodborne illness in Europe. Whilst control programmes exist to monitor these serovars in the chicken and turkey sectors, no regulatory programme is currently in place for the duck sector. A voluntary industry scheme (Duck Assurance Scheme) was launched in the UK in 2010. Hatcheries act as focal points of Salmonella contamination, in particular if Salmonella-contaminated eggs from positive breeding farms enter the hatchery. Five duck hatcheries were visited in this study and four were positive for Salmonella. S. Typhimurium DT8 and S. Indiana were isolated from hatchery 1 and S. Typhimurium DT41 and S. Senftenberg were isolated from hatchery 3. S. Kottbus, S. Bovismorbificans and S. Senftenberg were isolated from hatchery 2 and S. Kedougou was isolated from hatchery 4. Advice on the control/elimination of Salmonella was provided at each visit and a longitudinal study was undertaken to monitor its effectiveness. Extensive sampling was carried out in the hatcheries visited and the tray wash area and waste/external areas had the highest probability of being contaminated. The hatcher area was also found to be a primary focus of contamination. Improvements of farm and hatchery biosecurity standards have resulted in a reduction of hatchery contamination in this study and in previous investigations. Hatcheries 1 and 5 were cleared of Salmonella, demonstrating that elimination of Salmonella contamination from duck hatcheries is achievable.

Of Mice and Hens—Tackling Salmonella in Table Egg Production in the United Kingdom and Europe

Laying hens can become infected with a number of non–host-specific Salmonella serovars, of which Salmonella Enteritidis and Salmonella Typhimurium (including monophasic strains) are considered the most relevant threat to public health in Europe. S. Enteritidis in particular is able to persist indefinitely on layer farms unless effective interventions are implemented. In 2014, 2.5% of adult laying flocks in the European Union were reported to be infected by Salmonella (0.9% with either S. Enteritidis or S. Typhimurium). Laying hens can become infected as a result of contact with a contaminated environment (either at the hatchery or farm). Once Salmonella infection is established in a flock, it is perpetuated through a fecal–oral cycle. Rodents, particularly house mice, play a major role in the persistence of the infection, especially for flocks infected with S. Enteritidis. Rodents can readily become infected via contact with feces and dust and harbor salmonellae in their intestines and liver, where they can multiply to high numbers and persist for the whole life of a wild mouse. Contamination of feeding and water supply systems and surfaces of buildings and equipment by rodent droppings leads to infection in birds and contamination of egg collection equipment can directly contaminate eggs. Even when hens are vaccinated against Salmonella , the presence of breeding and young rodents shedding high numbers of organisms usually undermines vaccinal protection, although the occurrence of systemic infection of hens and internally contaminated eggs will still be reduced. When infected flocks are removed at the end of a production cycle, Salmonella can persist in the environment and infect replacement flocks. Intensive baiting of rodents before depopulation, and especially at the time when houses are empty and poultry feed has been removed, is essential to break the cycle of infection and avoid Salmonella carryover between flocks. Flies and litter beetles can also be infected for short periods and may be involved in the transmission and persistence of infection when downtime between flocks is short, or multiple age production is in place on the laying farm. Infestation by red mites can reduce the ability of birds to resist or clear infection, as a result of stress and anemia, and red mites are the main means of transmission of the nonzoonotic Salmonella Gallinarum biovar Gallinarum infection. Salmonella can survive for months in contaminated dust and laying hen houses tend to generate large quantities of dust during the long production cycle. Effective cleaning and disinfection between flocks is often costly to implement in laying houses, especially large cage units that are usually not designed to facilitate drainage after washing, leading to persistence of contamination between flocks. Removing all organic matter and using highly bioactive disinfectants such as aldehydes at the correct concentration and application rate is essential to avoid environmental persistence of Salmonella . Salmonella can be present at low prevalence in adult laying hens in mid lay and in older pullets and may therefore be difficult to detect. It is often assumed that new infections have entered laying farms when in reality the Salmonella has been continuously present but below the limits of detection of standard monitoring programs. Effective sampling and testing is therefore essential to the control of Salmonella on-farm. In the European Union, major progress on control of Salmonella in egg production has only been achieved after the introduction of effective vaccination programs and the application of severe financial penalties in terms of trade restrictions on the sale of fresh eggs from infected farms, which in many countries makes continued production from infected flocks economically unsustainable. Official sampling by the competent authority is also in place to validate operator sampling. Such incentives are usually required to ensure that effective control measures that come at a cost to the producer are fully implemented.

Estimation of the sensitivity of various environmental sampling methods for detection of Salmonella in duck flocks

Reports of Salmonella in ducks in the UK currently rely upon voluntary submissions from the industry, and as there is no harmonized statutory monitoring and control programme, it is difficult to compare data from different years in order to evaluate any trends in Salmonella prevalence in relation to sampling methodology. Therefore, the aim of this project was to assess the sensitivity of a selection of environmental sampling methods, including the sampling of faeces, dust and water troughs or bowls for the detection of Salmonella in duck flocks, and a range of sampling methods were applied to 67 duck flocks. Bayesian methods in the absence of a gold standard were used to provide estimates of the sensitivity of each of the sampling methods relative to the within-flock prevalence. There was a large influence of the within-flock prevalence on the sensitivity of all sample types, with sensitivity reducing as the within-flock prevalence reduced. Boot swabs (individual and pool of four), swabs of faecally contaminated areas and whole house hand-held fabric swabs showed the overall highest sensitivity for low-prevalence flocks and are recommended for use to detect Salmonella in duck flocks. The sample type with the highest proportion positive was a pool of four hair nets used as boot swabs, but this was not the most sensitive sample for low-prevalence flocks. All the environmental sampling types (faeces swabs, litter pinches, drag swabs, water trough samples and dust) had higher sensitivity than individual faeces sampling. None of the methods consistently identified all the positive flocks, and at least 10 samples would be required for even the most sensitive method (pool of four boot swabs) to detect a 5% prevalence. The sampling of dust had a low sensitivity and is not recommended for ducks.

Practical aspects of the suitability of different sampling methods for detecting Salmonella infection in duck flocks

SUMMARY While the consumption of duck eggs has gained popularity over the past few years, little is known about the prevalence of Salmonella in duck flocks and the associated potential Public Health impact. In order to detect Salmonella infection in duck flocks, sensitive sampling methods are essential; however, sampling costs and the practicalities of sampling have to be reasonable to allow testing on a large scale. In this study of 67 Salmonella‐positive duck flocks, we assessed the suitability of several sample types. Different combinations of samples were also explored to produce advice on the most sensitive, yet practical sampling method. Overall, detecting all positive flocks proved challenging, and the number of samples collected from a flock appeared to influence the outcome rather than the actual type of sample. While a number of different sampling options proved successful to detect high prevalence flocks, low prevalence flocks were only detected satisfactorily if several sample types were taken and the results combined. It was concluded that the detection of Salmonella‐positive breeding, laying or fattening duck flocks requires more samples compared to chicken or turkey flocks in order to achieve a meaningful outcome.

Maternal vaccination as a Salmonella Typhimurium reduction strategy on pig farms

The control of Salmonella in pig production is necessary for public and animal health, and vaccination was evaluated as a strategy to decrease pig prevalence.

Longitudinal study on the occurrence in pigs of colistin-resistant Escherichia coli carrying mcr-1 following the cessation of use of colistin

In 2015, colistin‐resistant Escherichia coli and Salmonella with the mcr‐1 gene were isolated from a pig farm in Great Britain. Pigs were subsequently monitored over a ~20‐month period for the occurrence of mcr‐1‐mediated colistin resistance and the risk of mcr‐1 E. coli entering the food chain was assessed.

Review of egg-related salmonellosis and reduction strategies in United States, Australia, United Kingdom and New Zealand

Abstract Globally, Salmonella enterica subsp. enterica is one of the most commonly reported causes of foodborne illness in humans. Contaminated food products of animal origin, particularly egg and egg products are frequently implicated in outbreaks of human salmonellosis. Salmonella enteritidis is frequently involved in egg and egg products-associated foodborne outbreaks in the USA and UK. However, in Australia and New Zealand, human infections caused by this serovar occur as a result of infection acquired while overseas travel, with Salmonella typhimurium being a predominant cause of local foodborne outbreaks. In this paper, an overview of Salmonella epidemiology on laying farms, egg-related Salmonella outbreaks in humans, and regulatory practises to control Salmonella across USA, UK, Australia and New Zealand is provided. Considering the estimated production of eggs in the USA, UK, Australia and New Zealand in 2015, the risk of foodborne illness in general is quite low for humans consuming eggs. Salmonella diagnostics, reporting and surveillance systems have improved over the years and will continue to improve in the years to come. However, given the number of different emerging Salmonella serovars a regular review of Salmonella control strategies from farm to fork is required.