Economic assessment of an intervention strategy to reduce antimicrobial usage in small-scale chicken farms in Vietnam.

Antimicrobials are a core aspect of most livestock production systems, especially in low-and middle-income countries. They underpin the efficient use of scarce feed resources and stabilize returns on capital and labor inputs. Antimicrobial use (AMU) contributes to the production of healthy animals, yet AMU in livestock is linked to antimicrobial resistance (AMR) in animals, humans and the environment. The Vietnamese Platform for Antimicrobial Reduction in Chicken Production was implemented during 2016-2019 and was one of Southeast Asia's first interventions focused on AMU reductions in livestock production. The project targeted small-scale commercial poultry farms in the Mekong Delta region of Vietnam using a "randomized before-and-after controlled" study design. It provided farmers with a locally adapted support service (farmer training plan, advisory visits, biosecurity, and antimicrobial replacement products) to help them reduce their reliance on antimicrobials. A partial budget analysis was performed comparing the control group (status-quo) and intervention group (alternative). The median net farm-level benefit of the intervention strategies with the project's support was VND 6.78 million (interquartile range (IR) VND -71.9-89 million) per farm. Without project support the benefit was reduced to VND 5.1 million (IR VND -69.1-87.2 million) to VND 5.3 million (IR -VND 68.9-87.5 million) depending on the antimicrobial alternative product used. At the project level with a focus on AMU and its reduction, subsequently influence on the resistance reduction, our results showed that achieving resistance reduction benefits with the current knowledge and technologies required investment of at least VND 9.1 million (US$ 395.10) per farm during the project's lifetime. The results highlight the positive net profit for the majority of enrolled farms and a reasonable investments from the project. The recommendation focuses on the implementation of policies on financial support, legislation, and information as potential solutions to facilitate the application of intervention strategies to reduce AMU in poultry production.

A review and meta-analysis of non-typhoidal Salmonella in Vietnam: Challenges to the control and antimicrobial resistance traits of a neglected zoonotic pathogen.

Non-typhoidal Salmonella (NTS) is a food-borne zoonotic pathogen with important implications for human health. Despite its importance, the burden of NTS infections in Vietnam is unknown. We conducted a systematic review and a meta-analysis to describe the prevalence and phenotypic antimicrobial resistance (AMR) for NTS over time in Vietnam. Following PRISMA guidelines, we identified 72 studies from PubMed and Google Scholar containing data relevant to 'Salmonella', 'Salmonellosis', and 'Vietnam', or 'Viet Nam'. Of those, forty-two papers reporting prevalence of NTS, and twenty-six including data on phenotypic resistance were selected for meta-analysis. The prevalence of NTS ranged from 2% to 5% in humans and from 30% to 41% in samples from animals and the farm/slaughterhouse environment. Between 2000 and 2020 we observed a 27.3% (p = 0.044) increase in absolute terms in prevalence among individuals with enteric diseases and a 12.0% (p = 0.047) increase in aquaculture/seafood samples. The most commonly serovars identified across studies included S. Weltevreden (12.0%), followed by S. Typhimurium (10.1%), S. Derby (6.4%), S. London (5.8%), S. Anatum (4.4%), S. Rissen (3.9%), S. Enteritidis (3.7%), S. Albany (3.3%) and S. 4,[5],12:i:- (3.0%). Over the same period, there was an increasing trend in the pooled AMR prevalence for quinolones (15.6% relative increase), 3rd-, 4th-, and 5th-generation cephalosporins (23.7%), penicillins (16.1%), tetracyclines (12.9%), sulfonamides (8.8%), amphenicol (17.8%), and multidrug resistance (11.4%) (all p ≤ 0.014). A broad range of AMR genes (ARGs) were detected in both human and animal populations. The observed prevalence and AMR trends in Vietnam underscore the need of adopting a One Health strategy encompassing surveillance systems, research initiatives, and awareness campaigns to effectively address the challenges posed by NTS.

Molecular characterization of Campylobacter spp. isolates obtained from commercial broilers and native chickens in Southern Thailand using whole genome sequencing.

Chickens are the primary reservoirs of Campylobacter spp., mainly C. jejuni and C. coli, that cause human bacterial gastrointestinal infections. However, genomic characteristics and antimicrobial resistance of Campylobacter spp. in low- to middle-income countries need more comprehensive exploration. This study aimed to characterize 21 C. jejuni and 5 C. coli isolates from commercial broilers and native chickens using whole genome sequencing and compare them to 28 reference Campylobacter sequences. Among the 26 isolates, 13 sequence types (ST) were identified in C. jejuni and 5 ST in C. coli. The prominent ST was ST 2274 (5 isolates, 19.2%), followed by ST 51, 460, 2409, and 6455 (2 isolates in each ST, 7.7%), while all remaining ST (464, 536, 595, 2083, 6736, 6964, 8096, 10437, 828, 872, 900, 8237, and 13540) had 1 isolate per ST (3.8%). Six types of antimicrobial resistance genes (ant(6)-Ia, aph(3')-III, blaOXA, cat, erm(B), and tet(O)) and one point mutations in the gyrA gene (Threonine-86-Isoleucine) and another in the rpsL gene (Lysine-43-Arginine) were detected. The blaOXA resistance gene was present in all isolates, the gyrA mutations was in 95.2% of C. jejuni and 80.0% of C. coli, and the tet(O) resistance gene in 76.2% of C. jejuni and 80.0% of C. coli. Additionally, 203 virulence-associated genes linked to 16 virulence factors were identified. In terms of phenotypic resistance, the C. jejuni isolates were all resistant to ciprofloxacin, enrofloxacin, and nalidixic acid, with lower levels of resistance to tetracycline (76.2%), tylosin (52.3%), erythromycin (23.8%), azithromycin (22.2%), and gentamicin (11.1%). Most C. coli isolates were resistant to all tested antimicrobials, while 1 C. coli was pan-susceptible except for tylosin. Single-nucleotide polymorphisms concordance varied widely, with differences of up to 13,375 single-nucleotide polymorphisms compared to the reference Campylobacter isolates, highlighting genetic divergence among comparative genomes. This study contributes to a deeper understanding of the molecular epidemiology of Campylobacter spp. in Thai chicken production systems.

The characterization and correlation between the phenotypic and genotypic resistance of Campylobacter spp. isolates from commercial broilers and native chickens in the south of Thailand.

RESEARCH HIGHLIGHTS: High Campylobacter prevalence in chickens; C. jejuni more prevalent than C. coli.Susceptibility to macrolides but resistance to quinolones/tetracyclines in isolates.Homogeneous resistance patterns within farms; higher in broilers than in native birds.Partial association between phenotypic and genotypic resistance among isolates.

Impact of antimicrobial use on abundance of antimicrobial resistance genes in chicken flocks in Vietnam.

Objectives: We investigated longitudinally Vietnamese small-scale chicken flocks in order to characterize changes in antimicrobial resistance gene (ARG) content over their life cycle, and the impact of antimicrobial use (AMU) on an intervention consisting of veterinary advice provision. Methods: AMU data and faecal samples were collected from 83 flocks (25 farms) at day-old, mid- and late-production (∼4 month cycle). Using high-throughput real-time PCR, samples were investigated for 94 ARGs. ARG copies were related to 16S rRNA and ng of DNA (ngDNA). Impact of AMU and ARGs in day-olds was investigated by mixed-effects models. Results: Flocks received a mean (standard error, SE) animal daily dose (ADD) of 736.7 (83.0) and 52.1 (9.9) kg in early and late production, respectively. Overall, ARGs/16S rRNA increased from day-old (mean 1.47; SE 0.10) to mid-production (1.61; SE 0.16), further decreasing in end-production (1.60; SE 0.1) (all P > 0.05). In mid-production, ARGs/16S rRNA increased for aminoglycosides, phenicols, sulphonamides and tetracyclines, decreasing for polymyxins ß-lactams and genes that confer resistance to mutiple classes (multi-drug resistance) (MDR). At end-production, aminoglycoside resistance decreased and polymyxin and quinolone resistance increased (all P < 0.05). Results in relation to ngDNA gave contradictory results. Neither AMU nor ARGs in day-olds had an impact on subsequent ARG abundance. The intervention resulted in 74.2% AMU reduction; its impact on ARGs depended on whether ARGs/ngDNA (+14.8%) or ARGs/16S rRNA metrics (-10.7%) (P > 0.05) were computed. Conclusions: The flocks' environment (contaminated water, feed and residual contamination) is likely to play a more important role in transmission of ARGs to flocks than previously thought. Results highlight intriguing differences in the quantification of ARGs depending on the metric chosen.

A systematic review and meta-analysis of integrated studies on antimicrobial resistance in Vietnam, with a focus on Enterobacteriaceae, from a One Health perspective.

Vietnam is a low- and middle-income country (LMIC), a primary food producer, and an antimicrobial resistance (AMR) hotspot. AMR is recognized as a One Health challenge since it may transfer between humans, animals and the environment. This study aimed to apply systematic review and meta-analysis to investigate the phenotypic profiles and correlations of antimicrobial-resistant Enterobacteriaceae across three compartments: humans, animals and the environment in Vietnam. A total of 89 articles found in PubMed, Science Direct, and Google Scholar databases were retrieved for qualitative synthesis. E. coli and non-typhoidal Salmonella (NTS) were the most common bacterial species in studies of all compartments (60/89 studies). Among antimicrobials classified as critically important, the resistance levels were observed to be highest to quinolones, 3rd generation of cephalosporins, penicillins, and aminoglycosides. Of 89 studies, 55 articles reported the resistance prevalence of E. coli and NTS in healthy humans, animals and the environment against ciprofloxacin, ceftazidime, ampicillin, gentamicin, sulfamethoxazole-trimethoprim, chloramphenicol was used for meta-analysis. The pooled prevalence was found highest in E. coli against ampicillin 84.0% (95% CI 73.0-91.0%) and sulfamethoxazole-trimethoprim 66.0% (95% CI 56.0-75.0%) while in NTS they were 34.0% (95% CI 24.0-46.0%), 33.0% (95% CI 25.0-42.0%), respectively. There were no significant differences in the pooled prevalence of E. coli and NTS to these antimicrobials across healthy humans, animals and the environment, except for ceftazidime-resistant E. coli (χ2 = 8.29, p = 0.02), chloramphenicol-resistant E.coli (χ2 = 9.65, p < 0.01) and chloramphenicol-resistant NTS (χ2 = 7.51, p = 0.02). Findings from the multiple meta-regression models indicated that the AMR levels in E. coli (ß = 1.887, p < 0.001) and the North (ß = 0.798, p = 0.047) had a higher fraction of AMR than NTS and other regions of Vietnam. The outcomes of this study play an important role as the baseline information for further investigation and follow-up intervention strategies to tackle AMR in Vietnam, and more generally, can be adapted to other LMICs.

Impact of Freeze Storage on the Estimation of Phenotypic Antimicrobial Resistance Prevalence in Escherichia coli Collected from Faecal Samples from Healthy Humans and Chickens.

Many studies on phenotypic antimicrobial resistance (AMR) of bacteria from healthy populations are conducted on freeze-stored samples. However, the impact of this practice on phenotypic AMR is not known. We investigated the prevalence of phenotypic AMR in Escherichia coli from chicken (n = 10) and human (n = 11) faecal samples collected from healthy subjects, subject to freeze storage (-20 °C and -80 °C) for 1, 2, 3, and 6 months. We compared counts of E. coli and prevalence of phenotypic resistance against five antimicrobials commonly used in chicken farming (ciprofloxacin, enrofloxacin, doxycycline, gentamicin, and florfenicol) with samples processed within 24 h of collection. Prevalence of phenotypic AMR was estimated by performing differential counts on agar media with and without antimicrobials. At -20 °C, there was a considerable reduction in E. coli counts over time, and this reduction was greater for human samples (-0.630 log10 units per 100 days) compared with chicken samples (-0.178 log10 units per 100 days). For most antimicrobials, AMR prevalence estimates decreased in freeze-stored samples both in humans and chickens over time. Based on these results, we conclude that results on the prevalence of phenotypic AMR on samples from freeze-stored samples are unreliable, and only fresh samples should be used in such studies.

Antimicrobial resistance in commensal Escherichia coli from humans and chickens in the Mekong Delta of Vietnam is driven by antimicrobial usage and potential cross-species transmission.

Objectives: To investigate phenotypic antimicrobial resistance (AMR) in relation to antimicrobial use (AMU) and potential inter-species transmission among Escherichia coli from humans and chickens located in the same households in the Mekong Delta of Vietnam. Methods: We collected data on AMU and faecal swabs from humans (N = 426) and chickens (N = 237) from 237 small-scale farms. From each sample, one E. coli strain was isolated and tested for its susceptibility against 11 antimicrobials by Sensititre AST. The association between AMR and AMU was investigated by logistic regression modelling. Using randomization, we compared the degree of similarity in AMR patterns between human and chicken E. coli from the same farms compared with isolates from different farms. Results: The AMU rate was ∼19 times higher in chickens (291.1 per 1000 chicken-days) than in humans (15.1 per 1000 person-days). Isolates from chickens also displayed a higher prevalence of multidrug resistance (63.3%) than those of human origin (55.1%). AMU increased the probability of resistance in isolates from human (ORs between 2.1 and 5.3) and chicken (ORs between 1.9 and 4.8). E. coli from humans and chickens living on same farms had a higher degree of similarity in their AMR patterns than isolates from humans and chicken living on different farms. Conclusions: We demonstrated the co-influence of AMU and potential transmission on observed phenotypic AMR patterns among E. coli isolates from food-producing animals and in-contact humans. Restricting unnecessary AMU alongside limiting interspecies contact (i.e. increasing hygiene and biocontainment) are essential for reducing the burden of AMR.

Prevalence of carbapenem resistance and its potential association with antimicrobial use in humans and animals in rural communities in Vietnam.

Background: Vietnam and Southeast Asia are hotspots for antimicrobial resistance; however, little is known on the prevalence of carriage of carbapenem resistance in non-hospitalized humans and in animals. Carbapenem-resistant Enterobacteriaceae (CRE), particularly Escherichia coli (CREC) and Klebsiella pneumoniae (CRKP) and also Acinetobacter baumannii (CRAB) are emerging threats worldwide. Methods: We investigated healthy humans (n = 652), chickens (n = 237), ducks (n = 150) and pigs (n = 143) in 400 small-scale farms in the Mekong Delta of Vietnam. Samples (rectal swabs, faecal swabs) were investigated for carriage of CRE/CRAB and were further characterized phenotypically and genotypically. Results: In the Mekong Delta of Vietnam, the prevalence of CRE isolates in human rectal swabs was 0.6%, including 4 CREC and 1 CRKP. One pig was infected with CREC (prevalence 0.7%). CRAB was isolated from chickens (n = 4) (prevalence 2.1%) and one duck (prevalence 0.7%). CRKP was isolated from a human who was also colonized with CREC. The CRKP strain (ST16), from an 80 year-old person with pneumonia under antimicrobial treatment, genetically clustered with clinical strains isolated in a hospital outbreak in southern Vietnam. The prevalence of CRE was higher among humans that had used antimicrobials within 90 days of the sampling date than those had not (4.2% versus 0.2%) (P = 0.005). All CRE/CRAB strains were MDR, although they were susceptible to colistin and neomycin. The carbapenemase genes identified in study strains were bla NDM and bla OXA. Conclusions: The finding of a CRKP strain clustering with previous hospital outbreak raises concerns about potential transmission of carbapenem-resistant organisms from hospital to community settings or vice-versa.

Modelling the impact of antimicrobial use and external introductions on commensal E. coli colistin resistance in small-scale chicken farms of the Mekong delta of Vietnam.

Colistin is a critically important antimicrobial for human medicine, and colistin-resistant Escherichia coli are commonly found in poultry and poultry products in Southeast Asia. Here, we aim at disentangling the within-farm and outside-farm drivers of colistin resistance in small-scale chicken farms of the Mekong delta of Vietnam. Nineteen Vietnamese chicken farms were followed up along a whole production cycle, during which weekly antimicrobial use data were recorded. At the beginning, middle and end of each production cycle, commensal E. coli samples from birds were collected, pooled and tested for colistin resistance. Twelve models were fitted to the data using an expectation-maximization algorithm and compared. We further tested the spatial clustering of the occurrence of resistance importations from external sources using the local Moran's I statistic. In the best model, colistin resistance in E. coli from chickens was found to be mostly affected by importations of resistance, and, to a lesser extent, by the use of antimicrobials in the last 1.73 weeks [0.00; 2.90], but not by the use of antimicrobials in day-olds, nor their colistin resistance carriage from hatchery. The occurrence of external source importations proved to be sometimes spatially clustered, suggesting a role of local environmental sources of colistin resistance.

Minimum inhibitory concentrations of commercial essential oils against common chicken pathogenic bacteria and their relationship with antibiotic resistance.

AIMS: We investigated the antibacterial effect of seven essential oils (EOs) and one EO-containing liquid phytogenic solution marketed for poultry and pigs ('Product A') on chicken pathogens, as well as the relationship between minimum inhibitory concentration (MIC) in EOs and antibiotics commonly administered to chicken flocks in the Mekong Delta (Vietnam). METHODS AND RESULTS: Micellar extracts from oregano (Origanum vulgare), cajeput (Melaleuca leucadendra), garlic (Allium sativum), black pepper (Piper nigrum), peppermint (Mentha × piperita L.), tea tree (Melaleuca alternifolia), cinnamon (Cinnamomum zeylanicum) EOs and Product A were investigated for their MIC against Avibacterium endocarditidis (N = 10), Pasteurella multocida (N = 7), Ornitobacterium rhinotracheale (ORT) (N = 10), Escherichia coli (N = 10) and Gallibacterium anatis (N = 10). Cinnamon EO had the lowest median MIC across strains (median 0.5 mg/ml [IQR, interquartile range 0.3-2.0 mg/ml]), followed by Product A (3.8 mg/ml [1.9-3.8 mg/ml]), oregano EO (30.4 mg/ml [7.6-60.8 mg/ml]) and garlic 63.1 mg/ml [3.9 to >505.0 mg/ml]. Peppermint, tea tree, cajeput and pepper EOs had all MIC ≥219 mg/ml. In addition, we determined the MIC of the 12 most commonly used antibiotics in chicken flocks in the area. After accounting for pathogen species, we found an independent, statistically significant (p < 0.05) positive correlation between MIC of 10 of 28 (35.7%) pairs of EOs. For 67/96 (69.8%) combinations of EOs and antibiotics, the MICs were correlated. Of all antibiotics, doxycycline was positively associated with the highest number of EOs (peppermint, tea tree, black pepper and cajeput, all p < 0.05). For cinnamon, the MICs were negatively correlated with the MICs of 11/12 antimicrobial tested (all except colistin). CONCLUSIONS: Increases in MIC of antibiotics generally correlates with increased tolerance to EOs. For cinnamon EO, however, the opposite was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest increased antibacterial effects of EOs on multi-drug resistant pathogens; cinnamon EO was particularly effective against bacterial poultry pathogens.

Feasibility study of a field survey to measure antimicrobial usage in humans and animals in the Mekong Delta region of Vietnam.

BACKGROUND: Development of antimicrobial use (AMU) surveillance systems in humans and animals is a priority for many low- and middle-income countries; however accurate estimations are hampered by a diversity of animal production systems and metrics. The Mekong Delta region of Vietnam is a 'hotspot' of antimicrobial resistance and is home to a high density of humans and animal populations. OBJECTIVES: To measure and compare AMU using different metrics (standing population, biomass and population correction unit) in the Mekong Delta, and to explore the potential of field-based data collection methods in the design of AMU surveillance systems. METHODS: We collected AMU data from humans and animals (chickens, ducks, Muscovy ducks, pigs) from 101 small-scale farms in the Mekong Delta over a fixed period (90 days in humans, 7 days in animals). RESULTS: Humans used 7.1 DDDkg, or 175.9 mg of antimicrobial active ingredients (AAIs) per kg of standing body mass annually; animals consumed 60.9 ADDkg or 1324 mg. In the Mekong Delta humans represented 79.3% of the total body mass but consumed 29.6% of AAIs by weight. AAIs regarded of critical importance by WHO represented 56.9% and 50.2% of doses consumed by animals and humans, respectively. CONCLUSIONS: Using a One Health approach, we show that AMU can potentially be estimated from cross-sectional surveys, although results are hypothetical due to small sample size and are sensitive to the chosen population denominator. The methodology proposed here can potentially be scaled up be applied to design AMU surveillance in low-resource settings, allowing AMU reduction efforts to be focused on particular animal species.

Small-scale commercial chicken production: A risky business for farmers in the Mekong Delta of Vietnam.

Small-scale farming of meat chicken flocks using local native breeds contributes to the economy of many rural livelihoods in Vietnam and many other low- and middle-income countries (LMICs). These systems are also the target of high levels of antimicrobial use (AMU); however, little is known about the profitability and sustainability of such systems. Since small-scale farms are commercial enterprises, this knowledge is essential to develop successful strategies to curb excessive AMU. Using longitudinal data from 203 small-scale (100-2,000 heads) native chicken flocks raised in 102 randomly selected farms in Dong Thap province (Mekong Delta, Vietnam), we investigated the financial and economic parameters of such systems and the main constraints to their sustainability. Feed accounted for the largest financial cost (flock median 49.5 % [Inter-quartile range (IQR) 41.5-61.8 %]) of total costs, followed by day-old-chicks (DOCs) (median 30.3 % [IQR 23.2-38.4 %]), non-antimicrobial health-supporting products (median 7.1 % [IQR 4.7-10.5 %]), vaccines (median 3.1 % [IQR 2.2-4.8 %]), equipment (median 1.9 % [IQR 0.0-4.9 %]) and antimicrobials (median 1.9 % [IQR 0.7-3.6 %]). Excluding labor costs, farmers achieved a positive return on investment (ROI) from 120 (59.1 %) flocks, the remainder generating a loss (median ROI 124 % [IQR 36-206 %]). Higher ROI was associated with higher flock size and low mortality. There was no statistical association between use of medicated feed and flock mortality or chicken bodyweight. The median daily income per person dedicated to raising chickens was 202,100 VND, lower than alternative rural labor activities in the Mekong Delta. In a large proportion of farms (33.4 %), farmers decided to stop raising chickens after completing one cycle. Farmers who dropped off chicken production purchased more expensive feed (in 1000 VND per kg): 11.1 [10.6-11.5] vs. 10.8 [10.4-11.3] for farms that continued production (p = 0.039), and experienced higher chicken mortality (28.5 % [12.0-79.0 %] vs. 16 [7.5-33.0 %]; p = 0.004). The rapid turnover of farmers raising chickens in such systems represents a challenge to the uptake of messages on appropriate AMU and chicken health. To ensure sustainability of small-scale commercial systems, advisory services need to be available to farmers as they initiate new flocks, and support them in the early stages to help overcome their limited experience and skills. This targeted approach would support profitability whilst reducing risk of emergence of AMR and infectious disease from these systems.

Effects of prophylactic and therapeutic antimicrobial uses in small-scale chicken flocks.

Antimicrobials are extensively used both prophylactically and therapeutically in poultry production. Despite this, there are little data on the effect of antimicrobial use (AMU) on disease incidence rate and per cent mortality. We investigated the relationships between AMU and disease and between AMU and mortality using data from a large (n = 322 flocks) cohort of small-scale chicken flocks in the Mekong Delta, Vietnam, that were followed longitudinally from day old to slaughter (5,566 observation weeks). We developed a parameterized algorithm to emulate a randomized control trial from observational data by categorizing the observation weeks into 'non-AMU', 'prophylactic AMU' and 'therapeutic AMU'. To evaluate the prophylactic AMU effect, we compared the frequencies of clinical signs in 'non-AMU' and 'prophylactic AMU' periods. To analyse therapeutic AMU, we compared weekly per cent mortality between the weeks of disease episodes before and after AMU. Analyses were stratified by clinical signs (4) and antimicrobial classes (13). Prophylactic AMU never reduced the probability of disease, and some antimicrobial classes such as lincosamides, amphenicols and penicillins increased the risk. The risk of diarrhoea consistently increased with prophylactic AMU. Therapeutic AMU often had an effect on mortality, but the pattern was inconsistent across the combinations of antimicrobial classes and clinical signs with 14/29 decreasing and 11/29 increasing the per cent weekly mortality. Lincosamides, methenamines and cephalosporins were the only three antimicrobial classes that always decreased the mortality when used therapeutically. Results were robust respective to the parameters values of the weeks categorization algorithm. This information should help support policy efforts and interventions aiming at reducing AMU in animal production.

Antimicrobial use through consumption of medicated feeds in chicken flocks in the Mekong Delta of Vietnam: A three-year study before a ban on antimicrobial growth promoters.

Antimicrobials are included in commercial animal feed rations in many low- and middle-income countries (LMICs). We measured antimicrobial use (AMU) in commercial feed products consumed by 338 small-scale chicken flocks in the Mekong Delta of Vietnam, before a gradual nationwide ban on prophylactic use of antimicrobials (including in commercial feeds) to be introduced in the country over the coming five years. We inspected the labels of commercial feeds and calculated amounts of antimicrobial active ingredients (AAIs) given to flocks. We framed these results in the context of overall AMU in chicken production, and highlighted those products that did not comply with Government regulations. Thirty-five of 99 (35.3%) different antimicrobial-containing feed products included at least one AAI. Eight different AAIs (avilamycin, bacitracin, chlortetracycline, colistin, enramycin, flavomycin, oxytetracycline, virginamycin) belonging to five classes were identified. Brooding feeds contained antimicrobials the most (60.0%), followed by grower (40.9%) and finisher feeds (20.0%). Quantitatively, chlortetracycline was consumed most (42.2 mg/kg SEM ±0.34; 50.0% of total use), followed by enramycin (18.4 mg/kg SEM ±0.03, 21.8%), bacitracin (16.4 mg/kg SEM ±0.20, 19.4%) and colistin (6.40 mg/kg SEM ± 4.21;7.6%). Other antimicrobials consumed were virgianamycin, avilamycin, flavomycin and oxytetracycline (each ≤0.50 mg/kg). Antimicrobials in commercial feeds were more commonly given to flocks in the earlier part of the production cycle. A total of 10 (9.3%) products were not compliant with existing Vietnamese regulation (06/2016/TT-BNNPTNT) either because they included a non-authorised AAI (4), had AAIs over the permitted limits (4), or both (2). A number of commercial feed formulations examined included colistin (polymyxin E), a critically important antimicrobial of highest priority for human medicine. These results illustrate the challenges for effective implementation and enforcement of restrictions of antimicrobials in commercial feeds in LMICs. Results from this study should help encourage discussion about policies on medicated feeds in LMICs.

A novel method for measuring phenotypic colistin resistance in Escherichia coli populations from chicken flocks.

Colistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methodologies to benchmark and monitor changes in resistance among mixed commensal bacterial populations in farms. We aimed to evaluate the performance of a broth microdilution method based on culturing a pooled Escherichia coli suspension (30-50 organisms) obtained from each sample. To confirm the biological basis and sensitivity of the method, we cultured 16 combinations of one colistin-susceptible and one mcr-1 encoded colistin-resistant E. coli in the presence of 2mg/L colistin. Optical density (OD600nm) readings over time were used to generate a growth curve, and these values were adjusted to the values obtained in the absence of colistin (adjusted Area Under the Curve, AUCadj). The median limit of detection was 1 resistant in 104 susceptible colonies [1st - 3rd quartile, 102:1 -105:1]. We applied this method to 108 pooled faecal samples from 36 chicken flocks from the Mekong Delta (Vietnam), and determined the correlation between this method and the prevalence of colistin resistance in individual colonies harvested from field samples, determined by the Minimum Inhibitory Concentration. The overall prevalence of colistin resistance at sample and isolate level (estimated from the AUCadj) was 38.9% [95%CI, 29.8-48.8%] and 19.4% (SD± 26.3%), respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin (OR=3.67) and other, non-colistin antimicrobials (OR=1.84). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in E. coli populations from faecal samples over time.IMPORTANCE Colistin (polymyxin E) is an antimicrobial with poor solubility in agar-based media, and therefore broth microdilution is the only available method for phenotypic resistance. However, estimating colistin resistance in mixed Escherichia coli populations is laborious since it requires individual colony isolation, identification and susceptibility testing. We developed a growth-based microdilution method suitable for pooled faecal samples. We validated the method by comparing it with individual MIC of 909 E. coli isolates; we then tested 108 pooled faecal samples from 36 healthy chicken flocks collected over their production cycle. A higher level of resistance was seen in flocks recently treated with colistin in water, although the observed generated resistance was short-lived. Our method is affordable, and may potentially be integrated into surveillance systems aiming at estimating the prevalence of resistance at colony level in flocks/herds. Furthermore, it may also be adapted to other complex biological systems, such as farms and abattoirs.

The role of animals as a source of antimicrobial resistant nontyphoidal Salmonella causing invasive and non-invasive human disease in Vietnam.

BACKGROUND: Nontyphoidal Salmonella (NTS) are associated with both diarrhea and bacteremia. Antimicrobial resistance (AMR) is common in NTS in low-middle income countries, but the major source(s) of AMR NTS in humans are not known. Here, we aimed to assess the role of animals as a source of AMR in human NTS infections in Vietnam. We retrospectively combined and analyzed 672 NTS human and animal isolates from four studies in southern Vietnam and compared serovars, sequence types (ST), and AMR profiles. We generated a population structure of circulating organisms and aimed to attribute sources of AMR in NTS causing invasive and noninvasive disease in humans using Bayesian multinomial mixture models. RESULTS: Among 672 NTS isolates, 148 (22%) originated from human blood, 211 (31%) from human stool, and 313 (47%) from animal stool. The distribution of serovars, STs, and AMR profiles differed among sources; serovars Enteritidis, Typhimurium, and Weltevreden were the most common in human blood, human stool, and animals, respectively. We identified an association between the source of NTS and AMR profile; the majority of AMR isolates were isolated from human blood (p < 0.001). Modelling by ST-AMR profile found chickens and pigs were likely the major sources of AMR NTS in human blood and stool, respectively; but unsampled sources were found to be a major contributor. CONCLUSIONS: Antimicrobial use in food animals is hypothesized to play role in the emergence of AMR in human pathogens. Our cross-sectional population-based approach suggests a significant overlap between AMR in NTS in animals and humans, but animal NTS does explain the full extent of AMR in human NTS infections in Vietnam.

Characterizing Antimicrobial Resistance in Chicken Pathogens: A Step towards Improved Antimicrobial Stewardship in Poultry Production in Vietnam.

In the Mekong Delta of Vietnam, farmers use large quantities of antimicrobials to raise small-scale chicken flocks, often including active ingredients regarded of "critical importance'" by the World Health Organization. Due to limitations in laboratory capacity, the choice of antimicrobials normally does not follow any empirical criteria of effectiveness. The aim of this study was to highlight non-critically important antimicrobials against which chicken pathogens are likely to be susceptible as a basis for treatment guidelines. Microtiter broth dilution method was performed to determine the minimal inhibitory concentration (MIC) of 12 commonly used antimicrobials for 58 isolates, including Ornithobacterium rhinotracheale (ORT) (n = 22), Gallibacterium anatis (n = 19), and Avibacterium endocarditidis (n = 17). Unfortunately, internationally accepted breakpoints for resistance in these organisms do not exist. We drew tentative epidemiological cut-offs (TECOFFs) for those antimicrobial-pathogen combinations where MIC distributions suggested the presence of a distinct non-wild-type population. Based on the observed results, doxycycline would be the drug of choice for A.endocarditidis (11.8% presumptive non-wild type) and G. anatis infections (5.3% presumptive non-wild type). A total of 13.6% ORT isolates were non-wild type with regards to oxytetracycline, making it the drug of choice against this pathogen. This study illustrates the challenges in interpreting susceptibility testing results and the need to establish internationally accepted breakpoints for veterinary pathogens.