Antibiotic resistance genes of public health importance in livestock and humans in an informal urban community in Nepal

Our results highlight risk factors related to the occurrence of seven resistance genes (bla_SHV-1 (SHV(238G240E) strain), QnrS, ermC, tetA, tetB, aacC2, and aadA1) associated with antibiotic-resistant bacteria of public health importance in samples from humans, animals, and the environment in an urban informal settlement community setting in Kathmandu, Nepal. Genes chosen for this analysis encompass resistance to a broad range of drug classes, including aminoglycosides, beta-lactams, tetracyclines, macrolides, and fluoroquinolones. All seven genes in this analysis were detected across all species and sources sampled in this study, with the exception of Rattus spp. samples, for which none of the genes were detected (Fig. 2). In multivariate analyses, factors for detection of these genes differed across genes and resistance classes. This study focused on associations related to detection across the seven genes in order to generate hypotheses for potential transmission pathways in an urban, informal settlement community setting.

By assessing which species and sample types were associated with detection of a gene in our study, we can begin to explore the complex transmission routes of AMR in the broader community and thus prioritize where to focus further research and prevention/intervention efforts. Prior studies have assessed indicators for carriage of AMR genes in agricultural settings. For instance, in research conducted on livestock farms in Vietnam, E. coli was found across chicken, duck, and pig farms, as well as in wild small mammals including Rattus spp. and Suncus murinus shrews²². Additionally, Nhung et al. (2015) found that pig farms, the size of the farm, and well water were significantly associated with multidrug resistance in E. coli²². In a 2017 study investigating the role of mobile genetic elements in the spread of resistant E. coli from chickens to humans in rural Ecuador, Moser et al. found that isolates from small-scale chicken operations contributed to higher levels of resistance in the community sampled via selection for isolates carrying mobile genetic elements²³.

To our knowledge, this is the first study to date to have concurrently sampled across an entire community and across a broad range of antibiotic resistance genes. Additionally, our study is the first to show that while associations with specific risk factors were not uniform across resistance genes tested, poultry (chickens and ducks) and fecal samples were indicators for detection of six out of seven of the genes investigated here, suggesting that surveillance and intervention efforts should focus on poultry and feces for optimal detection and reduction of these resistance genes in this community. We also present results of novel detection of several AMR genes in previously unstudied wild small mammal species, including detection of all seven genes in Crocidura lasiura, and detection of six of the seven genes (excluding ermC) in Suncus murinus. The detection of these genes of public health importance in multiple wild small mammal species underscores the need for further research into wildlife reservoirs of antibiotic resistance.

Risk factors for detection of resistance genes of importance

In our analyses, both pairs of genes belonging to the same antibiotic classification groups (tetA/tetB and aacC2/aadA1) had similar predictors for detection. For the genes in the tetracycline efflux pump resistance group (tetA and tetB), poultry were at high risk of carrying both genes. All chickens and ducks sampled in our study were positive for tetA, and so were excluded from the tetA regression model; chickens and ducks were significantly more likely to have tetB in our modeling results (Fig. 1, Table 2). Because of the high rates of tetA and tetB in poultry in this community, use of antibiotics in this drug class (e.g., tetracycline or doxycycline) should be limited to only where absolutely necessary, in order to limit the spread of tetracycline efflux pump resistance among poultry and from poultry into the environment or to humans. The second grouping of genes in our study, aacC2 and aadA1 (both conferring aminoglycoside resistance), were also similar in their risk factors for detection. Both were more likely to be detected in chickens and ducks and had high odds of being found in fecal samples (Fig. 1, Table 2). As with tetracycline resistance, to limit the potential for AMR gene transmission among poultry and between sources sampled, limiting the usage of aminoglycoside antibiotics and limiting exposure to chicken feces might be beneficial in limiting transmission of AMR genes in this community.

For genes that were more likely to be found in humans than other species (bla_SHV-1 and QnrS), the possibility exists that beta-lactamase and fluoroquinolone resistance is actively circulating in commensal and/or pathogenic bacteria in the human population, and participants should be tested further to assess the magnitude of AMR risk based on carriage of these genes. Interestingly, AMR genes did not cluster by household except for tetB, so household was not a strong factor related to detection of the same resistance genes. This is likely because AMR genes found in humans were widespread and apparently shared across the community, regardless of household. Animals and humans lived in close quarters in this community with minimal boundaries, even for owned animals. Chickens and pigs were located in separate pens adjacent to the housing structure to which they belonged. Ducks were marked by their owners and kept penned for part of the day, but also allowed to roam free at other times. Close contact and occasional co-mingling between animals and humans is a likely reason why the household affect was not significant in regression modeling, and clustering of genes by household was not detected for most AMR genes. Improved animal biosecurity, sanitation and hygiene practices would help prevent the spread of resistance genes via fecal transmission, especially for QnrS and bla_SHV-1, which were both more likely to be found in fecal samples than oral swabs (Fig. 1, Table 2). Examples of hygiene interventions include human behavioral changes such as hand-washing routines after contact with animals and before food preparation. Potential sanitation interventions could focus on implementing community-wide sewage infrastructure (where there currently is none) and upgrading toilets to latrines with drainage.

Lastly, while shrews were carriers of resistance genes in this community, they had a statistically lower odds of having several of the AMR genes tested compared to humans (Fig. 1, Table 2). Furthermore, rodents (Rattus spp.) were not carriers of any of the seven resistance genes tested for in this community. Further testing should be done on a larger sample of small wild mammals including shrews and rodents to more fully characterize this population as a reservoir of resistance and to assess the extent of risk wild small mammals might pose as potential transmission sources of resistance genes.

Prevalences of resistance genes among species and environmental sources sampled

Qnr genes have been found across a wide distribution of animals and habitats, including in aquatic and waterborne organisms, suggesting an origin in the natural environment^24,25,26. The high prevalence of QnrS in chickens and ducks sampled in our study supports this, although further research is needed to evaluate where the genes resided (e.g., in commensal or pathogenic bacteria, or as part of a mobile genetic element). We also found QnrS in human, shrew, swine, and water samples, although the prevalence for each source sampled was lower than it was in poultry (36%, 41%, 36%, and 12%, respectively). The only source sampled in which we did not find QnrS were rodents.

Of note, ermC was not found in any human samples, and in only one shrew and swine sample. It was found overwhelmingly in chicken and duck samples (in 85% and 95% of samples, respectively), perhaps due to the frequent use of macrolides such as erythromycin (SI Table 1) to treat Staphylococcus aureus infections in poultry²⁷. Previous studies have found similar patterns of high resistance to erythromycin and other macrolides in poultry^28,29,30 and that ermC-related erythromycin resistance is transferable between poultry and to human isolates of Staphylococcus aureus³¹. In addition to macrolides, all but one chicken and one duck sample also carried tetracycline resistance (via tetA and tetB). This was to be expected, as tetracyclines are the most widely used antibiotics in poultry due to their wide margin of safety and broad spectrum of activity against both gram-negative and gram-positive bacteria as well as Mycoplasma bacteria^27,30. The poultry sampled in this study also had high rates of resistance to aminoglycosides (aacC2: > 79% for both species, aadA1: > 95% for both species), especially as compared to human samples (aacC2: 12% positive, aadA1: 21% positive). It is unclear why aminoglycoside resistance was low in the human population in our results, as both genes have been found in humans in previous studies^32,33,34. However, prior studies have found aminoglycoside resistance genes such as aadA1 and aacC2 in E. coli of animal origin, whereas different aminoglycoside resistance genes (e.g. aac(6’)-lb-cr) were more related to human E. coli isolates^24,35. Lastly, bla_SHV-1 was found in less than 50% of all sources sampled except for ducks, where it was positive in 14/19 samples. Despite the low prevalence across most sources sampled in this study, these results support the previous literature, in which ESBL-producing β-lactamases such as the SHV enzymes have been found across humans, animals, and wildlife sources^36,37.

Importance in veterinary medicine in Nepal

Despite only testing for resistance genes, the seven resistance genes assessed in this study belong to a broad range of antibiotic resistance classes and are associated with specific drug resistance classes that comprise common antibiotics currently in use in Nepal in animal and human populations³⁸. For example, aadA1 and aacC2 confer resistance to aminoglycosides such as streptomycin and gentamicin, both of which are categorized as extremely important for veterinary medicine by the World Organisation for Animal Health (OIE), as they treat a wide range of diseases, including septicemias and urinary, respiratory, and digestive infections in livestock and pets²¹. Additionally, gentamicin is the main medication indicated for Pseudomonas aeruginosa infections, with few alternatives available for effective treatment²¹.

ErmC, which is categorized in the macrolide-lincosamide-streptogramin B resistance group (SI Table 2), can be induced by Staphylococcus aureus³⁹. Erythromycin is an example of a macrolide drug to which ermC can provide resistance and is currently used in animals in Nepal (SI Table 1). As stated by the OIE, macrolides cover a wide range of diseases in animals, including those sampled in the current study (poultry and swine), with few alternative medications should macrolides fail²¹. The strain of β-lactamase for which this study tested (SHV(238G240E)) is linked to E. coli in GenBank (E. coli strain HB101 SHV-1 β-lactamase (bla_SHV-1) gene⁴⁰). Classified as a Class A β-lactamase (SI Table 2), bla_SHV-1 is included in the genes that are the primary cause of resistance to beta-lactam drugs among Enterobacteriaceae, including cephalosporins and penicillins such as amoxicillin, ampicillin, Cloxacillin, benzylpenicillin (penicillin G), and phenoxymethylpenicillin (penicillin V), all in use in Nepal in animals³⁸ (SI Table 1). Beta-lactams are extremely important for veterinary medicine as they are used for the treatment of septicemias as well as urinary and respiratory infections across a broad range of animal species with few economical alternatives currently available²¹. Quinolones such as nalidixic acid as well as fluoroquinolones such as ciprofloxacin are both targeted by the QnrS gene via diverse molecular mechanisms of resistance. Additionally, both drug classes are critically important in veterinary medicine in Nepal²¹. This is also true of the tetracycline drug class, with drugs such as doxycycline and tetracycline used to treat many species and a wide range of diseases²¹. Both tetA and tetB provide resistance to tetracyclines via encoding for proteins that act as tetracycline efflux pump proteins⁴¹.

The detection of these seven resistance genes found across animal populations in our study is of concern, as the genes could already be circulating in pathogens. Moreover, if the genes are not already found in pathogenic bacteria in the community, they might reside on mobile genetic elements or commensal bacteria as part of the community resistome and may be transferred to pathogenic bacteria in the future, thus providing a source of resistance to antibiotics that are currently in use in animals in Nepal that could spread within the community. Further, more targeted research should explore where these genes exist within the community resistome (e.g., in commensal or pathogenic bacteria or on mobile genetic elements). Additionally, we found these resistance genes in humans as well as animals and water in our study, and as the same drugs mentioned above are also indicated in humans in Nepal, the risk for AMR presence and transmission exists across populations studied.

Importance in human medicine in Nepal

In this study, we prioritized antibiotic classes that are important for use in the human population in Nepal³⁸ (SI Table 1). In addition to the OIE’s list of critically important antimicrobials for veterinary medicine, the World Health Organization (WHO) published and continually updates a list of priority pathogens with the goal of aiding in research and development of new antibiotics as well as supporting good antibiotic stewardship and furthering policies in both the human and veterinary/agricultural spheres⁴². Prior to the publication of this list, antibiotic research and development was driven by factors such as investor pressure, technological availability, and market size, and did not focus on crucial parameters such as global bacterial pathogens that have few alternative treatments and have shown an increase in resistance⁴². Because of the priority pathogen list’s publication, research and development priorities can now be better aligned with global health needs, thus supporting the fight against antibiotic resistance worldwide.

Resistance genes in this study are associated with pathogenic bacteria that are listed under all three priority levels set by the WHO in their priority pathogen list⁴² (SI Table 1). The highest priority (level 1, critical) focuses on carbapenem-resistant Enterobacteriaceae including E. coli, as well as Pseudomonas aeruginosa from non-human sources⁴². Diverse β-lactamases that confer this resistance, such as CTX-M, TEM, and SHV type genes (including bla_SHV-1, the gene in the current study), have been observed in E. coli, Salmonella spp., Klebsiella pneumoniae and other Enterobacteriaceae from non-human sources such as livestock and companion animals^{24,43,44,45,46,47}. High priority pathogens (level 2, high) include fluoroquinolone-resistant Salmonella spp., associated with Qnr resistance genes such as the one studied here, QnrS. A high prevalence of Salmonella has been documented in Nepal in humans, animals, and the environment, including in meat markets in Kathmandu^48,49. Additionally, QnrS is associated with one of the medium priority pathogens on the WHO list (priority 3), fluoroquinolone-resistant Shigella spp.⁴².

Although antibiotic use has not been historically well-documented in humans in Nepal, it is acknowledged to be widespread and is inadequately regulated⁵⁰. Additionally, prior research has found a high prevalence of resistant organisms in both hospital and community settings in Nepal⁵⁰. The lax regulations on widespread antibiotic use in both human and animal populations allow for the transfer of resistance from the animal to human population or vice versa via both direct and indirect methods. Furthermore, with common antibiotics in use such as those listed in SI Table 1, it is essential that the necessity and usage of these drugs are better prioritized and managed in both human and animal populations to help limit the spread of resistance, thus allowing for their prolonged efficacy, as all are listed as highly or critically important in both populations.

While this study assessed prevalence of a broad range of resistance genes with public health significance, we did not simultaneously characterize commensal or pathogenic bacteria in the samples to inform on specific bacteria from which these genes have been identified to provide evidence for transmission mechanisms between human and animal populations. Future work involving 16S metagenome sequencing to identify bacteria in which the AMR genes reside would provide a more comprehensive overview of all antibiotic resistant genes and mobile genetic elements in this microbial community. Additionally, it is important to note that the presence of these resistance genes does not necessarily mean clinical treatment failure, because in vitro testing with minimum inhibitory concentrations (MIC) of corresponding microorganisms was not done. As resistance genes can be found on mobile genetic elements as well as in unculturable commensal bacteria, especially in the environment (not just in pathogenic bacteria), we focused on characterizing the genetic resistome of the community. Future research should expand on our findings with studies involving metagenomic and culture-based methods. Further work could also follow the framework set up by Martínez et al. (2015) to assess AMR risk in humans and animals based on carriage of resistance genes in this community⁵¹.

In summary, this study characterized and highlighted risk at the animal-human-environmental interface for the presence of seven resistance genes associated with antibiotics and resistant pathogens of public health importance in an urban informal settlement in Kathmandu, Nepal from a One Health perspective. These seven genes (bla_SHV-1 (SHV(238G240E) strain), QnrS, ermC, tetA, tetB, aacC2, and aadA1) span a broad range of drug resistance classes, including aminoglycosides, beta-lactams, tetracyclines, macrolides, and fluoroquinolones. Animal, human, and environmental reservoirs for these genes may reside in the community as well as hospital settings, and by distinguishing parameters that increase the risk of finding a resistance gene, we can guide AMR policies in the larger community context from a one health perspective.