Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

A Bayesian inference method to estimate transmission trees with multiple introductions; applied to SARS-CoV-2 in Dutch mink farms.

Knowledge of who infected whom during an outbreak of an infectious disease is important to determine risk factors for transmission and to design effective control measures. Both whole-genome sequencing of pathogens and epidemiological data provide useful information about the transmission events and underlying processes. Existing models to infer transmission trees usually assume that the pathogen is introduced only once from outside into the population of interest. However, this is not always true. For instance, SARS-CoV-2 is suggested to be introduced multiple times in mink farms in the Netherlands from the SARS-CoV-2 pandemic among humans. Here, we developed a Bayesian inference method combining whole-genome sequencing data and epidemiological data, allowing for multiple introductions of the pathogen in the population. Our method does not a priori split the outbreak into multiple phylogenetic clusters, nor does it break the dependency between the processes of mutation, within-host dynamics, transmission, and observation. We implemented our method as an additional feature in the R-package phybreak. On simulated data, our method correctly identifies the number of introductions, with an accuracy depending on the proportion of all observed cases that are introductions. Moreover, when a single introduction was simulated, our method produced similar estimates of parameters and transmission trees as the existing package. When applied to data from a SARS-CoV-2 outbreak in Dutch mink farms, the method provides strong evidence for independent introductions of the pathogen at 13 farms, infecting a total of 63 farms. Using the new feature of the phybreak package, transmission routes of a more complex class of infectious disease outbreaks can be inferred which will aid infection control in future outbreaks.

Estimating plasmid conjugation rates: A new computational tool and a critical comparison of methods.

Plasmids are important vectors for the spread of genes among diverse populations of bacteria. However, there is no standard method to determine the rate at which they spread horizontally via conjugation. Here, we compare commonly used methods on simulated and experimental data, and show that the resulting conjugation rate estimates often depend strongly on the time of measurement, the initial population densities, or the initial ratio of donor to recipient populations. Differences in growth rate, e.g. induced by sub-lethal antibiotic concentrations or temperature, can also significantly bias conjugation rate estimates. We derive a new 'end-point' measure to estimate conjugation rates, which extends the well-known Simonsen method to include the effects of differences in population growth and conjugation rates from donors and transconjugants. We further derive analytical expressions for the parameter range in which these approximations remain valid. We present an easy to use R package and web interface which implement both new and previously existing methods to estimate conjugation rates. The result is a set of tools and guidelines for accurate and comparable measurement of plasmid conjugation rates.

Second compartment widens plasmid invasion conditions: Two-compartment pair-formation model of conjugation in the gut.

Understanding under which conditions conjugative plasmids encoding antibiotic resistance can invade bacterial communities in the gut is of particular interest to combat the spread of antibiotic resistance within and between animals and humans. We extended a one-compartment model of conjugation to a two-compartment model, to analyse how differences in plasmid dynamics in the gut lumen and at the gut wall affect the invasion of plasmids. We compared scenarios with one and two compartments, different migration rates between the lumen and wall compartments, and different population dynamics. We focused on the effect of attachment and detachment rates on plasmid dynamics, explicitly describing pair formation followed by plasmid transfer in the pairs. The parameter space allowing plasmid invasion in the one-compartment model is affected by plasmid costs and intrinsic conjugation rates of the transconjugant, but not by these characteristics of the donor. The parameter space allowing plasmid invasion in the two-compartment model is affected by attachment and detachment rates in the lumen and wall compartment, and by the bacterial density at the wall. The one- and two-compartment models predict the same parameter space for plasmid invasion if the conditions in both compartments are equal to the conditions in the one-compartment model. In contrast, the addition of the wall compartment widens the parameter space allowing invasion compared with the one-compartment model, if the density at the wall is higher than in the lumen, or if the attachment rate at the wall is high and the detachment rate at the wall is low. We also compared the pair-formation models with bulk-conjugation models that describe conjugation by instantaneous transfer of the plasmid at contact between cells, without explicitly describing pair formation. Our results show that pair-formation and bulk-conjugation models predict the same parameter space for plasmid invasion. From our simulations, we conclude that conditions at the gut wall should be taken into account to describe plasmid dynamics in the gut and that transconjugant characteristics rather than donor characteristics should be used to parameterize the models.

Chicken gut microbiome members limit the spread of an antimicrobial resistance plasmid in Escherichia coli.

Plasmid-mediated antimicrobial resistance is a major contributor to the spread of resistance genes within bacterial communities. Successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission. While these key parameters are readily quantified in vitro, the influence of interactions with other microbiome members is largely unknown. Here, we investigated the influence of three genera of lactic acid bacteria (LAB) derived from the chicken gastrointestinal microbiome on the spread of an epidemic narrow-range ESBL resistance plasmid, IncI1 carrying blaCTX-M-1, in mixed cultures of isogenic Escherichia coli strains. Secreted products of LAB decreased E. coli growth rates in a genus-specific manner but did not affect plasmid transfer rates. Importantly, we quantified plasmid transfer rates by controlling for density-dependent mating opportunities. Parametrization of a mathematical model with our in vitro estimates illustrated that small fitness costs of plasmid carriage may tip the balance towards plasmid loss under growth conditions in the gastrointestinal tract. This work shows that microbial interactions can influence plasmid success and provides an experimental-theoretical framework for further study of plasmid transfer in a microbiome context.

Effect of donor-recipient relatedness on the plasmid conjugation frequency: a meta-analysis.

BACKGROUND: Conjugation plays a major role in the transmission of plasmids encoding antibiotic resistance genes in both clinical and general settings. The conjugation efficiency is influenced by many biotic and abiotic factors, one of which is the taxonomic relatedness between donor and recipient bacteria. A comprehensive overview of the influence of donor-recipient relatedness on conjugation is still lacking, but such an overview is important to quantitatively assess the risk of plasmid transfer and the effect of interventions which limit the spread of antibiotic resistance, and to obtain parameter values for conjugation in mathematical models. Therefore, we performed a meta-analysis on reported conjugation frequencies from Escherichia coli donors to various recipient species. RESULTS: Thirty-two studies reporting 313 conjugation frequencies for liquid broth matings and 270 conjugation frequencies for filter matings were included in our meta-analysis. The reported conjugation frequencies varied over 11 orders of magnitude. Decreasing taxonomic relatedness between donor and recipient bacteria, when adjusted for confounding factors, was associated with a lower conjugation frequency in liquid matings. The mean conjugation frequency for bacteria of the same order, the same class, and other classes was 10, 20, and 789 times lower than the mean conjugation frequency within the same species, respectively. This association between relatedness and conjugation frequency was not found for filter matings. The conjugation frequency was furthermore found to be influenced by temperature in both types of mating experiments, and in addition by plasmid incompatibility group in liquid matings, and by recipient origin and mating time in filter matings. CONCLUSIONS: In our meta-analysis, taxonomic relatedness is limiting conjugation in liquid matings, but not in filter matings, suggesting that taxonomic relatedness is not a limiting factor for conjugation in environments where bacteria are fixed in space.

Dynamics of faecal shedding of ESBL- or AmpC-producing Escherichia coli on dairy farms.

OBJECTIVES: To explore the dynamics of faecal ESBL/AmpC shedding in dairy cattle and farmers, a study was conducted to examine changes in shedding by individual animals, as well as environmental exposure, and to study the association between antimicrobial use (AMU) and ESBL/AmpC shedding. METHODS: The study comprised a cross-sectional survey of 20 farms and a 1 year follow-up of 10 farms. Faecal samples were cultured by both direct inoculation on MacConkey agar + 1 mg/L cefotaxime (MC+) and enrichment in LB-broth + 1 mg/L cefotaxime with subsequent inoculation on MC+. Dust samples were collected using electrostatic dustfall collectors (EDCs). Human faecal samples were collected by the farmers. Presence of ESBL/AmpC genes was screened for by PCR and sequencing. Using mixed effects logistic regression, ORs were determined and population-attributable fractions (PAFs) calculated subsequently. RESULTS: In Phase 1, 8/20 farms were positive for ESBL/AmpC and, with 2 negative farms, were selected for Phase 2. Transient shedding of dominant allele variants was observed in the animals. EDCs and human faecal samples did not reflect what was observed in the animals. AMU was related to shedding of ESBLs in the next sampling moment [OR 14.6 (95% CI 3.0-80.0)] and the PAF of AMU was 0.36 (95% CI 0.08-0.77). Calves fed with colostrum from cows on dry-off therapy was not a risk factor [OR 1.7 (95% CI 0.7-4.9, P = 0.28)]. CONCLUSIONS: The presence of ESBL/AmpC could only be partly explained by AMU. No link was shown between shedding in cattle and humans or the environment. Interventions should focus on prevention of introduction.

Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut.

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.

Competitive Exclusion Reduces Transmission and Excretion of Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Broilers.

Extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpC) are enzymes able to hydrolyze a large variety of ß-lactam antibiotics, including third-generation cephalosporins and monobactams. Broilers and broiler meat products can be highly contaminated with ESBL- and pAmpC-producing Escherichia coli strains, also known as extended-spectrum cephalosporin (ESC)-resistant E. coli strains, and can be a source for human infections. As few data on interventions to reduce the presence of ESC-resistant E. coli in broilers are available, we used transmission experiments to examine the role of competitive exclusion (CE) on reducing transmission and excretion in broilers. A broiler model to study the transmission of ESC-resistant E. coli was set up. Day-old chickens were challenged with an ESBL-producing E. coli strain isolated from healthy broilers in the Netherlands. Challenged and not challenged chicks were housed together in pairs or in groups, and ESBL-producing E. coli transmission was monitored via selective culturing of cloacal swab specimens. We observed a statistically significant reduction in both the transmission and excretion of ESBL-producing E. coli in chicks treated with the probiotic flora before E. coli challenge compared to the transmission and excretion in untreated controls. In conclusion, our results support the use of competitive exclusion as an intervention strategy to control ESC-resistant E. coli in the field.IMPORTANCE Extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases are a primary cause of resistance to ß-lactam antibiotics among members of the family Enterobacteriaceae in humans, animals, and the environment. Food-producing animals are not exempt from this, with a high prevalence being seen in broilers, and there is evidence pointing to a possible foodborne source for human contamination. We investigated the effect of administration of a commercial probiotic product as an intervention to reduce the amount of ESBL-producing Escherichia coli in broilers. Our results showed a substantial reduction in the level of colonization of broiler intestines by ESBL-producing E. coli after administration of commercial probiotic product. The protective effect provided by these probiotics could be implemented on a larger scale in poultry production. Reductions in the levels of ESBL-producing Enterobacteriaceae in the food chain would considerably benefit public health.

The IncI1 plasmid carrying the blaCTX-M-1 gene persists in in vitro culture of a Escherichia coli strain from broilers.

BACKGROUND: Commensal bacteria are a reservoir for antimicrobial-resistance genes. In the Netherlands, bacteria producing Extended Spectrum Beta-Lactamases (ESBL) are found on chicken-meat and in the gut of broilers at a high prevalence and the predominant ESBL-gene is the bla(CTX-M-1) located on IncI1 plasmids. We aim to determine the fitness costs of this plasmid for the bacterium.We investigated the conjugation dynamics of IncI1 plasmids carrying the bla(CTX-M-1) gene in a batch culture and its impact on the population dynamics of three E. coli populations: donors, recipients and transconjugants. The intrinsic growth rate (ψ), maximum density (K) and lag-phase (λ) of the populations were estimated as well as the conjugation coefficient. Loss of the plasmid by transconjugants was either assumed constant or depended on the effective growth rate of the transconjugants.Parameters were estimated from experiments with pure culture of donors, recipients and transconjugants and with mixed culture of donors and recipients with a duration of 24 or 48 hours. Extrapolation of the results was compared to a 3-months experiment in which a mixed culture of recipient and transconjugant was regularly diluted in new medium. RESULTS: No differences in estimated growth parameters (ψ, K or λ) were found between donor, recipient and transconjugant, and plasmid loss was not observed. The conjugation coefficient of transconjugants was 104 times larger than that of the donor. In the 3-months experiment, the proportion of transconjugants did not decrease, indicating no or very small fitness costs. CONCLUSIONS: In vitro the IncI1 plasmid carrying the blaCTX-M-1 gene imposes no or negligible fitness costs on its E. coli host, and persists without antimicrobial usage.

Quantifying health risks from ESBL-producing Escherichia coli in Dutch broiler production chains and potential interventions using compartmental models.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) in animals are considered a human health threat, because this type of bacteria can serve as a reservoir of antibiotic resistant genes and act as a continuous threat of the emergence of new resistant bacteria, in addition to the direct effect of making infection untreatable. Although the prevalence of ESBL producing bacteria in broilers was drastically reduced in the Netherlands, chicken meat still has the highest prevalence among meat products. Therefore, further control of the ESBL-producing E. coli in the broiler production chain is important to reduce public health risks. The main objectives of this study were to evaluate the effectiveness of intervention scenarios to reduce the transmission of ESBL-producing E. coli in the broiler production chain and to quantitatively estimate the risk to public health. In this study, we developed two different types of transmission models that described the observed time-related decline in prevalence during a production round: one with time-dependent decline in susceptibility and one with partial immunity to phylogenetic groups. Both models incorporated the environmental contamination effect between production rounds and within flocks. The parameter values, including transmission rate and recovery rate, were estimated by Approximate Bayesian computation (ABC) method using data from a longitudinal study in a Dutch organic broiler farm. We applied the models to the three production stages in the broiler production chain, beginning from the Parent Stock (PS) farms, the hatcheries, and to the broiler farms. In our models, eggs were collected from different parent stock farms and transported to the hatchery and from there to a broiler farm.The size of a flock and the number of farms were adjusted to the Dutch situation. Both models were able to describe the observed dynamics within and between the production stages equally well, with estimated ESBL-producing E. coli prevalence of 8.98% and 11.47% in broilers at slaughter and 0.12% and 0.15% in humans due to chicken consumption. Both models indicated that improving farm management to eliminate the bacteria from the environment was the most effective intervention, making this outcome robust. Although chicken meat consumption is not a major risk factor for human carriage of the bacteria according to our models, reducing the bacteria in the PS and broiler farm environment to at least one percent can further decrease the prevalence in humans.

Transmission rates of veterinary and clinically important antibiotic resistant Escherichia coli: A meta- ANALYSIS.

The transmission rate per hour between hosts is a key parameter for simulating transmission dynamics of antibiotic-resistant bacteria, and might differ for antibiotic resistance genes, animal species, and antibiotic usage. We conducted a Bayesian meta-analysis of resistant Escherichia coli (E. coli) transmission in broilers and piglets to obtain insight in factors determining the transmission rate, infectious period, and reproduction ratio. We included blaCTX-M-1, blaCTX-M-2, blaOXA-162, catA1, mcr-1, and fluoroquinolone resistant E. coli. The Maximum a Posteriori (MAP) transmission rate in broilers without antibiotic treatment ranged from 0.4∙10-3 to 2.5∙10-3 depending on type of broiler (SPF vs conventional) and inoculation strains. For piglets, the MAP in groups without antibiotic treatment were between 0.7∙10-3 and 0.8∙10-3, increasing to 0.9∙10-3 in the group with antibiotic treatment. In groups without antibiotic treatment, the transmission rate of resistant E. coli in broilers was almost twice the transmission rate in piglets. Amoxicillin increased the transmission rate of E. coli carrying blaCTX-M-2 by three-fold. The MAP infectious period of resistant E. coli in piglets with and without antibiotics is between 971 and 1065 hours (40 - 43 days). The MAP infectious period of resistant E. coli in broiler without antibiotics is between 475 and 2306 hours (20 - 96 days). The MAP infectious period of resistant E. coli in broiler with antibiotics is between 2702 and 3462 hours (113 - 144 days) which means a lifelong colonization. The MAP basic reproduction ratio in piglets of infection with resistant E. coli when using antibiotics is 27.70, which is higher than MAP in piglets without antibiotics between 15.65 and 18.19. The MAP basic reproduction ratio in broilers ranges between 3.46 and 92.38. We consider three possible explanations for our finding that in the absence of antibiotics the transmission rate is higher among broilers than among piglets: i) due to the gut microbiome of animals, ii) fitness costs of bacteria, and iii) differences in experimental set-up between the studies. Regarding infectious period and reproduction ratio, the effect of the resistance gene, antibiotic treatment, and animal species are inconclusive due to limited data.

The transmission potential of Rift Valley fever virus among livestock in the Netherlands: a modelling study.

Rift Valley fever virus (RVFV) is a zoonotic vector-borne infection and causes a potentially severe disease. Many mammals are susceptible to infection including important livestock species. Although currently confined to Africa and the near-East, this disease causes concern in countries in temperate climates where both hosts and potential vectors are present, such as the Netherlands. Currently, an assessment of the probability of an outbreak occurring in this country is missing. To evaluate the transmission potential of RVFV, a mathematical model was developed and used to determine the initial growth and the Floquet ratio, which are indicators of the probability of an outbreak and of persistence in a periodic changing environment caused by seasonality. We show that several areas of the Netherlands have a high transmission potential and risk of persistence of the infection. Counter-intuitively, these are the sparsely populated livestock areas, due to the high vector-host ratios in these areas. Culex pipiens s.l. is found to be the main driver of the spread and persistence, because it is by far the most abundant mosquito. Our investigation underscores the importance to determine the vector competence of this mosquito species for RVFV and its host preference.

Evaluation of foot and mouth disease control measures: Simulating two endemic areas of Thailand.

Foot and mouth disease (FMD) is an important livestock disease in Thailand, with outbreaks occurring every year. However, the effects of FMD control measures in Thailand have received little research attention. Epidemiological models have been widely used to evaluate FMD outbreak control, but such a model has never been developed for Thailand. We constructed a stochastic between-farm transmission model to evaluate FMD control measures. The epidemiological unit of the model was the farm, which could be in different states: susceptible, latent, undetected infectious, detected infectious and recovered. The between-farm transmission was calculated by the sum of distance-dependent transmission and trade network transmission using parameters derived from FMD outbreaks in 2016-2017. We used this model to simulate the outbreaks with and without the implementation of the following control measures: culling all animals on infected farms, ring vaccination, animal movement restrictions and isolation of infected farms. The control measures were evaluated by estimating the number of secondarily infected farms and the outbreak duration for each scenario. The model was simulated in two study areas located in the Lamphaya Klang subdistrict (high farm density) and the Bo Phloi district (low farm density). The effects of control measures differed between the two study areas. When farm density was high, rigid control measures were required to prevent a major outbreak. Among all options, culling the animals on infected farms resulted in the lowest number of infected farms and the shortest outbreak duration. In contrast, for an area with a low farm density, less stringent control measures were sufficient to control the usually minor outbreaks. The results indicate that different areas require a different approach to control an outbreak of FMD.

Contribution of cats and dogs to SARS-CoV-2 transmission in households.

Introduction: SARS-CoV-2 is known to jump across species. The occurrence of transmission in households between humans and companion animals has been shown, but the contribution of companion animals to the overall transmission within a household is unknown. The basic reproduction number (R0) is an important indicator to quantify transmission. For a pathogen with multiple host species, such as SARS-CoV-2, the basic reproduction number needs to be calculated from the partial reproduction numbers for each combination of host species. Method: In this study, the basic and partial reproduction numbers for SARS-CoV-2 were estimated by reanalyzing a survey of Dutch households with dogs and cats and minimally one SARS-CoV-2-infected human. Results: For households with cats, a clear correlation between the number of cats and the basic reproduction number (Spearman's correlation: p 0.40, p-value: 1.4 × 10-5) was identified, while for dogs, the correlation was smaller and not significant (Spearman's correlation: p 0.12, p-value: 0.21). Partial reproduction numbers from cats or dogs to humans were 0.3 (0.0-2.0) and 0.3 (0.0-3.5) and from humans to cats or dogs were 0.6 (0.4-0.8) and 0.6 (0.4-0.9). Discussion: Thus, the estimations of within-household transmission indicated the likelihood of transmission from these companion animals to humans and vice versa, but the observational nature of this study limited the ability to establish conclusive evidence. This study's findings support the advice provided during the pandemic to COVID-19 patients to maintain distance from companion animals as a precautionary measure and given the possibility of transmission, although there is an overall relatively limited impact on the pandemic when compared to human-to-human transmission.

Comparing the transmission of carbapenemase-producing and extended-spectrum beta-lactamase-producing Escherichia coli between broiler chickens.

The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to public health, because of their resistance to clinically important carbapenem antibiotics. The emergence of CPE in meat-producing animals is particularly worrying because consumption of meat contaminated with resistant bacteria comparable to CPE, such as extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, contributed to colonization in humans worldwide. Currently, no data on the transmission of CPE in livestock is available. We performed a transmission experiment to quantify the transmission of CPE between broilers to fill this knowledge gap and to compare the transmission rates of CPE and other antibiotic-resistant E. coli. A total of 180 Ross 308 broiler chickens were distributed over 12 pens on the day of hatch (day 0). On day 5, half of the 10 remaining chickens in each pen were orally inoculated with 5·102 colony-forming units of CPE, ESBL, or chloramphenicol-resistant E. coli (catA1). To evaluate the effect of antibiotic treatment, amoxicillin was given twice daily in drinking water in 6 of the 12 pens from days 2-6. Cloacal swabs of all animals were taken to determine the number of infectious broilers. We used a Bayesian hierarchical model to quantify the transmission of the E. coli strains. E. coli can survive in the environment and serve as a reservoir. Therefore, the susceptible-infectious transmission model was adapted to account for the transmission of resistant bacteria from the environment. In addition, the caecal microbiome was analyzed on day 5 and at the end of the experiment on day 14 to assess the relationship between the caecal microbiome and the transmission rates. The transmission rates of CPE were 52 - 68 per cent lower compared to ESBL and catA1, but it is not clear if these differences were caused by differences between the resistance genes or by other differences between the E. coli strains. Differences between the groups in transmission rates and microbiome diversity did not correspond to each other, indicating that differences in transmission rates were probably not caused by major differences in the community structure in the caecal microbiome. Amoxicillin treatment from day 2-6 increased the transmission rate more than three-fold in all inoculums. It also increased alpha-diversity compared to untreated animals on day 5, but not on day 14, suggesting only a temporary effect. Future research could incorporate more complex transmission models with different species of resistant bacteria into the Bayesian hierarchical model.

Conserved developmental trajectories of the cecal microbiota of broiler chickens in a field study.

There is great interest in identifying gut microbiota development patterns and underlying assembly rules that can inform strategies to improve broiler health and performance. Microbiota stratification using community types helps to simplify complex and dynamic ecosystem principles of the intestinal microbiota. This study aimed to identify community types to increase insight in intestinal microbiota variation between broilers and to identify factors that explain this variation. A total of 10 well-performing poultry flocks on four farms were followed. From each flock, the cecal content of nine broilers was collected at 7, 14, and 35 days posthatch. A total of two robust community types were observed using different clustering methods, one of which was dominated by 7-day-old broilers, and one by 35-day-old broilers. Broilers, 14-day-old, were divided across both community types. This is the first study that showed conserved cecal microbiota development trajectories in commercial broiler flocks. In addition to the temporal development with age, the cecal microbiota variation between broilers was explained by the flock, body weight, and the different feed components. Our data support a conserved development of cecal microbiota, despite strong influence of environmental factors. Further investigation of mechanisms underlying microbiota development and function is required to facilitate intestinal health promoting management, diagnostics, and nutritional interventions.

Quantitative Risk Assessment for the Introduction of Carbapenem-Resistant Enterobacteriaceae (CPE) into Dutch Livestock Farms.

Early detection of emerging carbapenem-resistant Enterobacteriaceae (CPE) in food-producing animals is essential to control the spread of CPE. We assessed the risk of CPE introduction from imported livestock, livestock feed, companion animals, hospital patients, and returning travelers into livestock farms in The Netherlands, including (1) broiler, (2) broiler breeder, (3) fattening pig, (4) breeding pig, (5) farrow-to-finish pig, and (6) veal calf farms. The expected annual number of introductions was calculated from the number of farms exposed to each CPE source and the probability that at least one animal in an exposed farm is colonized. The total number of farms with CPE colonization was estimated to be the highest for fattening pig farms, whereas the probability of introduction for an individual farm was the highest for broiler farms. Livestock feed and imported livestock are the most likely sources of CPE introduction into Dutch livestock farms. Sensitivity analysis indicated that the number of fattening pig farms determined the number of high introductions in fattening pigs from feed, and that uncertainty on CPE prevalence impacted the absolute risk estimate for all farm types. The results of this study can be used to inform risk-based surveillance for CPE in livestock farms.

SARS-CoV-2 infection in cats and dogs in infected mink farms.

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2-positive cats and dogs in 10 infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of 10 farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all 10 farms. This study shows evidence of SARS-CoV-2 infection in 12 feral cats and 2 dogs. Eleven cats (18%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10%-18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2.

Spatial model of foot-and-mouth disease outbreak in an endemic area of Thailand.

Foot-and-mouth disease (FDM) is a disease of cloven-hoofed animals with high costs in animal welfare and animal production. Up to now, transmission between farms in FMD-endemic areas has been given little attention. Between farm transmission can be quantified by distance independent transmission parameters and a spatial transmission kernel indicating the rate of transmission of an infected farm to susceptible farms depending on the distance. The spatial transmission kernel and distance-independent transmission parameters were estimated from data of an FMD outbreak in Lamphaya Klang subdistrict in Thailand between 2016 and 2017. The spatial between-farm transmission rate in Lamphaya Klang subdistrict was higher compared with the spatial between-farm transmission rate from FMDV in epidemic areas. The result can be explained by the larger size of the within-farm outbreak in the endemic area due to no culling. The inclusion of distance-independent transmission parameters improved the model fit, which suggests the presence of transmission sources from outside the area and spread within the area independent of the distance between farms. The remaining distance-dependent transmission was mainly local and could be due to over-the-fence transmission or other forms of contact between nearby farms. Farm size on the kernel positively affects the transmission rate, by increasing both infectivity and susceptibility with increasing farm size. The results showed that both distance-dependent transmission and distance-independent transmission were contributed to FMDV transmission in Lamphaya Klang outbreak. These transmission parameters help to gain knowledge about FMD transmission dynamic in the endemic area.