Carriage of Plasmid-Mediated Colistin Resistance-1-Positive Escherichia coli in Humans, Animals, and Environment on Farms in Vietnam

ABSTRACT.

Plasmid-Mediated Colistin Resistance 1 (mcr-1) was first reported in 2015 and is a great concern to human health. In this study, we investigated the prevalence of mcr-1 and mcr-1-positive Escherichia coli (MCRPEC) and the association in infection status among various reservoirs connected to livestock. The study was conducted in 70 poultry and swine farms in a commune in Ha Nam province, northern Vietnam. Samples were collected from farmers, food animals, domestic animals, and farm environments (flies and wastewater) for polymerase chain reaction (PCR) screening for mcr-1 gene and species identification of PCR positive isolates. Among 379 obtained mcr-1 positives isolates, Escherichia coli was the major identified, varying from 50% (2/4) in dog feces to 100% (31/31) in humans feces isolates. The prevalence of MCRPEC was 14.4% (20/139), 49.7% (96/193), 31.3% (25/80), 36.7% (40/109), 26.9% (18/67), and 3.9% (2/51) in humans, chickens, pigs, flies, wastewater, and dogs, respectively. The study identified association between MCRPEC infection status in humans and flies (OR = 3.4), between flies and chickens (OR = 5.3), and between flies and pigs (OR = 9.0). Farmers’ age and farm livestock unit were also associated factors of MCRPEC infection status in humans (OR = 5.1 and 1.05, respectively). These findings bring new knowledge on antibiotic resistance in livestock setting and important suggestions on potential role of flies in the transmission of mcr-1 resistance gene.

INTRODUCTION

Infections caused by resistant and multidrug resistant Gram-negative bacteria pose an immense challenge in human medicine due to limited treatment options. Colistin, which was rarely used in the past due to its high toxicity, has been approved for use in humans as a “last resort” drug for severe infections caused by carbapenem resistant Enterobacterales since 2017. The recent emergence of Plasmid-Mediated Colistin Resistance 1 (mcr-1) gene is thus a serious threat to public health if colistin lost its treatment effectiveness for resistant Gram-negative bacteria infections. Mcr-1 was reported for the first time in 2015 in an Escherichia coli isolate from farm animals and raw meat samples in China. Since then, this gene was identified in Enterobacterales globally. Available data reveal the long existence of mcr-1 before its first report in 2015 in China, in animal, food, environment, and even humans although colistin had not been approved for use in human infection treatment before 2015.^1–4

Most of reported mcr-1 containing isolates in humans are E. coli, a commensal bacterium in intestinal tract of mammals. Among 21,000 Enterobacteriaceae isolates collected from 2007 to 2015 in two hospitals in China, mcr-1 was detected in 76 of the 5,332 E. coli isolates, highest proportion in comparison with the other species including Klebsiella pneumonia, Enterobacter cloacae, and Enterobacter aerogenes. This study also reported a temporal increase of the proportion of mcr-1 carrying E. coli isolates.² A livestock antibiotic resistance surveillance program in France reported 23 out of 1,696 E. coli isolates obtained between 2007 and 2014 were resistant to colistin and all carried mcr-1 gene.⁴

In Vietnam, colistin has been used in veterinary medicine for decades as a common therapy in livestock⁵ but the data regarding colistin resistance is relatively scarce and mostly from clinical settings. Data on colistin resistance in healthy humans is even scarcer with a few studies, mainly in the south of the country.^6–9 Plasmid-Mediated Colistin Resistance 1 gene locates on plasmids and its capacity to transmit horizontally among bacteria of distinct species puts humans at greater risk of getting the resistance gene from farm animals. Therefore, it is critical to understand the presence and transmission of this gene among human and reservoirs connected to livestock. This study aims to: 1) investigate the prevalence of mcr-1 and mcr-1-positive Escherichia coli (MCRPEC) in farmers and different reservoirs in farms and 2) identify the association of MCRPEC infection among various reservoirs in livestock settings.

METHODS

Setting and population.

A cross-sectional study was conducted in livestock settings of a commune in Ha Nam, a province in the north of Vietnam in 2019. The selected commune was among the communes with the largest number of animal foods in the province and had not participated in any study on antibiotic resistance before. The farming areas in this commune are geographically separated from residential areas. All farms and all their residents who participated in farming aged over 18 were invited to join the study. A list of farms and farmers was prepared by the commune veterinary collaborators in which 70 farms with 139 farmers who provided consent and were enrolled in the study.

Sample collection and laboratory analysis.

Feces samples were collected from farmers, chickens, pigs, and dogs raised in these farms. Additional samples from flies and wastewater were also collected in the same farms. Flies were captured using glue boards and were then sterilely and individually transferred to a 1.5 mL tubes then pulverized using disposable plastic sticks (SPL Korea).¹⁰ Samples were stored at 4°C after collection and transferred within 14 hours to the Antimicrobial Resistance Laboratory of the National Institute of Hygiene and Epidemiology (NIHE) Vietnam for testing. All the samples were stored at −80°C until further use to make sure all the samples remained high quality for testing.

Samples were spread onto MacConkey Agar (Merck, Germany) with 0.5 mg/L Colistin (Sigma-Aldrich) and 10 mg/L Vancomycin (Sigma-Aldrich) to select isolates having antimicrobial resistance gene. We used a concentration of colistin lower than the 2 mg/L clinical breakpoint of the European Committee on Antimicrobial Susceptibility Testing (EUCAST)¹¹ to collect all the isolates which contained mcr-1 gene, including those that did not have colistin resistance phenotype.

After 24 hours incubation at 37°C, the total DNA extracted from samples was polymerase chain reaction (PCR)-screened for mcr-1 with 30 cycles (94°C—30 seconds; 57°C—90 seconds; 72°C—60 seconds). Plasmid-Mediated Colistin Resistance 1 gene was amplified using primer pairs mcr-1F (5′-CGGTCAGTCCGTTTGTTC-3″) and mcr-1R (5′-CTTGGTCGGTCTGTA GGG-3′)¹² E. coli National Collection of Type Cultures (NCTC) 13846 was used as a positive control strain.

Samples positive for the mcr-1 gene were then subcultured on MacConkey Agar (Merck, Germany) with 0.5 mg/L Colistin (Sigma-Aldrich) and 10 mg/L Vancomycin (Sigma-Aldrich). Five single colonies were selected from each sample based on differential properties such as shape, color, size, and surface to confirm the presence of mcr-1 gene by PCR, as previously described. Deoxyribonucleic acid of isolates was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The positive isolates were species identified by the MALDI Biotyper system (Bruker Daltonik GmbH, Germany).

Epidemiological data collection and analysis.

Enrolled participants were interviewed using a structured questionnaire. Investigated variables included demographic information (age, sex, farm type, number of food animals). Livestock unit and Farm scale regulated by Decree 13/2020/ND-CP Detail Guideline of Livestock Law issued by Vietnamese Government were computed by number of all types of food animals raised, including pigs, chicken, duck, geese, quails, doves, ostriches, etc., then converts the number of each type to “livestock unit” for each type of food animals. Total farm livestock unit is sum of livestock units of all food animal types raised which used to categorize farm-scales (Household: < 10 units, small: from 10 to under 30 units, medium: from 30 to under 300 units, large: ≥ 300 units).¹³

Completed interviews and laboratory results were entered using Epidata Entry v. 3.1 (Denmark). Stata statistical package v. 16.0 software was used for data analysis. χ² test, univariate and multivariate logistic regression were performed to identify the association between MCRPEC carriage and independent variables. The associations were considered significant with P value ≤ 0.05 and 95% confidence interval were presented.

Ethical consideration.

The study protocol was reviewed and approved by the Institute Ethics Committee in NIHE, Vietnam (IRB approval number: HDDD—06/2019). Informed consent was provided by all human participants aged 18 years and older as described above. Human subjects also provided consent to collect samples from other subjects in their farms.

RESULTS

Demographic characteristics.

Among the 139 enrolled farmers, males accounted for 56.1% while females accounted for 43.9%. Most participants were over 41 years old (82.1%). More than two-third of farmers (70.5%) worked in household-scale farms, 17.3% worked in small-scale farms, 12.2% worked in medium-scale farms, and no one worked in large-scale farms. Among 70 investigated farms, 72.9% were household-scale, 17.1% were small-scale, and 10% were medium-scale. Farm livestock unit ranged from 0.2 to 37.0 with mean of 9.6. Forty-six of the 70 farms were poultry farms, four were swine farms, and 20 were mixed farms (Table 1).

Table 1.

Characteristics of investigated population and farms

Characteristic	n	%	95% CI
Sex (N = 139)
Male	78	56.1	47.7–64.2
Female	61	43.9	35.8–52.3
Age (years, N = 139)
18–40	25	17.9	12.4–25.4
41–50	39	28.1	21.1–36.2
51–60	40	28.8	21.8–36.9
> 60	35	25.2	18.6–33.2
Farm livestock unit	Min 0.2	Max 37.0	SD 11.8
Farm scale (N = 139 farmers)
Household	98	70.5	62.3–77.6
Small	24	17.3	11.8–24.6
Medium	17	12.2	7.7–18.9
Farm scale (N = 70 farms)
Household	51	72.9	61.0–82.2
Small	12	17.1	9.9–28.1
Medium	7	10.0	4.8–19.8
Farm type (N = 70 farms)
Poultry	46	65.7	53.6–76.1
Swine	4	5.7	2.1–14.6
Mixed	20	28.6	19.0–40.5

Prevalence of MCRPEC.

A total of 639 samples from 70 farms were collected, including feces samples from humans (N = 139), chickens (N = 193), pigs (N = 80), and dogs (N = 51); samples from wastewater (N = 67) and flies (N = 109). Out of 639 PCR-tested samples, 215 (33.6%) samples were carrying mcr-1 gene and 201 (31.46%) samples were containing MCRPEC. A total of 379 mcr-1-positive isolates were obtained, in which E. coli was the major with 330 isolates (overall rate: 87.1%, ranging from 50.0% [2/4 isolates] in dog to 100% [31/31 isolates] in humans). The MCRPEC prevalence among collected samples was highest in chicken with 49.7% (96/193 samples) and lowest in dogs with 3.9% (2/5 samples). The prevalence in humans was 14.4% (20/139 samples). Fly samples showed MCRPEC prevalence at 36.7% (40/109 samples) (Figure 1A, Supplemental Tables 1 and 2).

Figure 1.

Detection of MCRPEC. (A) PCR detection rate of mcr-1 from collected samples and Isolation rate of MCRPEC from collected samples. (B) Isolation rate of MCRPEC from investigated farms. Data and estimation for (A) and (B) are presented in Supplemental Tables 1 and 3, respectively.

At farm level, prevalence of MCRPEC was also highest in chicken feces samples with 55 farms having MCRPEC samples over 66 investigated farms (83.3%). The lowest prevalence was still in dog feces samples (3.9%). The prevalence in human feces was 20% of investigated farms, higher than prevalence at individual level (14.4%) (Figure 1B, Supplemental Table 3).

In humans, overall prevalence of MCRPEC was 14.4% with 20 positive samples over 139 samples. Prevalence in male and female were similar with 11 out of 67 (14.1%) and nine out of 52 (14.8%), respectively. For age, nearly half of the infected cases were over 60 years old (9/20 cases), the rest of the cases distributed equally among age groups of 18–40 (four cases), 41–50 (four cases) and 51–60 (three cases). Prevalence in age group of over 60 was the highest at 25.7% while the lowest prevalence was of the age group of 51–60 with 7.5% (Table 2).

Table 2.

MCRPEC infection in humans and associated factors

Factor	Positive		Negative		OR (95% CI, P)	Adjusted OR (95% CI, P)
	n	%	n	%
Sex
Male	11	14.1	67	85.9	1	–
Female	9	14.8	52	85.2	1.1 (0.4–2.7, P = 0.91)	–
Age group (years)*
18–40	4	16.0	21	84.0	2.3 (0.5–11.5, P = 0.29)	2.2 (0.4–11.8, P = 0.36)
41–50	4	10.3	35	89.7	1.4 (0.3–6.8, P = 0.67)	1.2 (0.2–6.3, P = 0.81)
51–60	3	7.5	37	92.5	1	1
> 60	9	25.7	26	74.3	4.3 (1.1–17.3, P = 0.04)	5.1 (1.2–22.5, P = 0.03)
Farm livestock unit* (N = 139)					1.04 (1.00–1.08, P = 0.03)	1.05 (1.01–1.09, P = 0.01)
Farm scale (N = 139)
Household	13	13.3	85	86.7	1	–
Small	1	4.2	23	95.8	0.28 (0.04–2.29, P = 0.24)	–
Medium	6	35.3	11	64.7	3.6 (1.1–11.3, P = 0.03)	–
MCRPEC in flies*
Positive	14	21.5	51	78.5	3.1 (1.1–8.7, P = 0.03)	3.4 (1.1–10.0, P = 0.03)
Negative	6	8.1	68	91.9	1

MCRPEC = mcr-1-positive Escherichia coli.

^*Variables with asterisk were included in multivariate analysis because univariate analysis identified an association with MCRPEC in human. In this paper, Farm livestock unit and Farm scale are two variables used to describe the size of livestock. Farm livestock unit is a continuous variable, presenting number of food animals while Farm scale is a category variable generated by categorize values of Farm livestock unit. We assessed association between MCRPEC infection in human with both Farm livestock unit and Farm scale in univariate analysis but in multivariate analysis of MCRPEC and associated factors, Farm livestock unit but not Farm scale was included because basically the two variables the same and inclusion of both of them in the multivariate analysis may lead to incorrect result.

Associated factors of MCRPEC carriage in humans.

Age was identified as an associated factor of MCRPEC carriage. Farmers at the age group of over 60 were 4.3 times more likely to be infected with MCRPEC than those who were 51–60 years old. Age was not an associated factor. Farm scale was also an associated factor of MCRPEC infection in this study. Farmers working in medium farms were 3.6 times more likely to get infected than those working in household farms. Farm livestock unit also effected the MCRPEC carriage in human when farm livestock unit increase of one unit lead to MCRPEC increase of 4%. People living in farms with MCRPEC-positive files were 3.1 times higher at risk of being infected with MCRPEC. Multivariate analysis strengthened the association of age (adjusted OR = 5.1 for over 60 age group), farm livestock unit (adjusted OR = 1.05), and infection status in flies (adjusted OR = 3.4) with infection status in humans (Table 2).

None of the associations between MCRPEC infection status in humans and infection status in other sources of samples other than flies were significant. However, significant associations between MCRPEC infection status in flies and MCRPEC infection status in chickens, and pigs were found (Table 3). Flies collected in farms having chicken feces samples carrying MCRPEC were five times more likely to be positive with this pathogen (OR = 5.3) while the likelihood was nine times for the flies in farms having pig feces samples carrying MCRPEC (OR = 9.0) (Table 3).

Table 3.

Association between MCRPEC infection status in flies and in other sources

MCRPEC infection status in other sources		MCRPEC infection status in flies				OR (95% CI, P)
		Positive		Negative
		N	%	N	%
Chicken feces	Positive	28	53.8	24	46.2	5.3 (1.03–26.7, P = 0.046)
	Negative	2	18.2	9	81.8	1
Pig feces	Positive	9	69.2	4	30.8	9.0 (1.3–63.0, P = 0.03)
	Negative	2	20.0	8	80.0	1
Dog feces	Positive	1	50.0	1	50.0	1.1 (0.1–19.3, P = 0.93)
	Negative	22	46.8	25	53.2	1
Wastewater	Positive	9	52.9	8	47.1	1.4 (0.5–4.2, P = 0.56)
	Negative	21	44.7	26	55.3	1

MCRPEC = mcr-1-positive Escherichia coli.

DISCUSSION

Escherichia coli is a ubiquitous bacterium in the intestinal tract of mammals and also a significant pathogen in foodborne infections and in antibiotic resistant infections. Multiple-drug resistant (MDR) E. coli is reported worldwide and presents an increasing trend in both hospital and community.^14–23 Multiple-drug resistant E. coli is also reported in animal food and farming settings.^24–28 Escherichia coli was also the first to be reported harboring mcr-1 resistance gene recovered from food and food animal samples in China in 2015.¹² Since then, detection of this gene in humans and animals including food animals and companion animals has been reported in many countries and Vietnam in both clinical and community settings.^8,12,29–33 While research on antibiotic resistance in food animals is more abundant, only a few studies on healthy humans in the community have been conducted. This study is one among a few studies in Vietnam utilizing the One Health approach in investigating the situation and transmission of antibiotic resistance in livestock settings.

In contrast to the study of Yang Wang and colleagues which identified the dominance of NDM gene in poultry settings in China,¹² this current study revealed that mcr-1 was the principal resistance gene with 33.6% of collected samples carrying this gene while only some isolates carrying NDM detected in this study population (unpublished data). The study observed 14.4% of healthy farmers infected with bacteria carrying mcr-1 gene which is lower than 20.6% reported in Tien Giang in 2012–2013⁸ and the 88% reported in Ha Nam (same province as our study) in 2014–2015.³⁴ A total of 31 MCRPEC isolates was obtained from 139 farmers in this study which was also lower than a study in Thai Binh province with 64 isolates from 98 humans feces samples.³³

Univariate analysis showed a significant association between MCRPEC infection status in human and scale of farms. Following that, increase of farm livestock unit of one unit added 4% change of acquiring MCRPEC in farmers and farmers working in medium farms were 3.6 times more likely to get infected than those working in household farms. Multivariate analysis confirmed the association of farm livestock unit (adjusted OR = 1.05). This result was consistent with studies in Thailand which identified greater prevalence of resistance in larger farm scales.^35,36 This can be explained by the difference in antibiotic use practices in livestock among farm scales. The study in Thailand reported that more types and greater amounts of antibiotics were used in medium-scale farms compared with small-scale farms.³⁵ Similarly, a study in Ghana identified antibiotic use in 100% commercial farms while the percentage in backyard farms was only one-fourth.³⁷ Higher demand on productivity in commercial farms can explain a higher use of antibiotics to maintain wellness of animals.³⁸

Plasmid-Mediated Colistin Resistance 1 prevalence in chickens (51.3%) was two times higher than what was observed in the study in the Mekong Delta area (22.2%) in 2013–2014³⁰ and similar to the study results in Tien Giang (59.4%). A study in Bangladesh published in 2019 reported a high prevalence of resistance to colistin (73.5%) among E. coli isolates obtained from cloacal swabs of broiler chicken. However, the presence of resistance genes to colistin was not identified in this study.²⁶ We did not observe the association between MCRPEC infection status in farmers and chicken while Tien Giang study reported a risk of 5.3 times for farmers raising chicken carrying mcr-1 gene compared with people living in urban areas.⁸

Regarding pigs, our study results showed that the prevalence of MCRPEC was significantly high in pigs (31.3%) comparing to the prevalence in the southern regions of the country. Among 33 mcr-1 isolates obtained from pigs in our study, 97% was E. coli while the study in Mekong Delta in 2013–2014 detected mcr-1 gene in only 18.9% E. coli isolates obtained from pigs.³⁰

This was the first study in Vietnam to investigate resistance status in dogs and flies. We observed three over 51 (5.95%) collected dog feces samples positive to mcr-1 gene and half of four mcr-1-carrying isolates were E. coli, and a very high prevalence of MCRPEC in farm flies with 49 over 109 samples collected (45%). Among 83 mcr-1 isolates recovered, 62 (74.7%) were E. coli. Farm dogs and flies have been reported as potential reservoirs of resistance genes by a study in China. They identified E. coli carrying NDM resistance gene in different sample types collected in farms, indicating their potential role as disseminators of resistance genes in farming environment and as transmitters to migratory animals such as birds to the outside environment.¹⁰ However, we did not find any association between MCRPEC infection status in dogs and in humans. This might be due to the small sample size of the investigated population. Regarding flies, no direct association between MCRPEC infection status in humans and food animals like chickens and pigs was detected, but we found an association between MCRPEC infection status in humans and infection status in flies (adjusted OR = 3.4) and an association between MCRPEC infection status in flies and food animals such as chickens (OR = 5.3) and pigs (OR = 9). These results suggest that flies could play a role in the dissemination of antibiotic resistance genes between humans and animals.

Age was identified as an associated factor of antibiotic resistance in previous studies. Data from a multinational survey in 1990–2007 reported that outpatients over 65 years old were 2.4 times more likely to be infected with ESBL-producing enterobacteria.³⁹ Our study also identified an association between age and MCRPEC carriage status in farmers with the oldest age group (over 60 years old) being at highest risk of infection compared with the age group of 51–60 (adjusted OR = 5.1). Sex was also reported an associated factor of antibiotic resistance by several studies^39,40 but was not observed in our study.

Small sample size was a limitation of the study thus we were unable to identify direct associations between MCRPEC infection status in humans and food animals. Besides, association between colistin use and presence of mcr-1 gene in this population could not be identified due to inability to investigate usage of colistin in farming activities in the scope of this study.

CONCLUSION

This study is the one among a few studies investigating antibiotic resistance utilizing the One Health approach in livestock settings in Vietnam. With prevalence of mcr-1 and MCRPEC in different reservoirs and the association between MCRPEC infection status among them, our study provided an overview of the colistin resistance in the farm setting. In addition, it supplemented important data to the poor database on antibiotic resistance in healthy people in community in Vietnam as well as in the globe. More importantly, our findings on the association of flies and farmers, and flies and farm animals provided suggestions for larger population surveys and molecular research to investigate and confirm direct transmission of resistance genes between farm animals and humans through environmental factors such as flies or birds. The next step of our study is performing molecular research to further investigate transmission of mcr-1 gene and we will report the results in later articles.

Note: Supplemental tables appear at www.ajtmh.org.