Ecology of bla_NDM and mcr-1

Antibiotic resistance is one of the most worrying threats to global public health today. Numerous common infections are becoming harder, and even impossible, to treat due to the loss of effectiveness of these molecules against resistant bacteria. This is a complex scenario involving multiple elements, and must therefore be confronted with a multidisciplinary approach (1).

Carbapenem-resistant enterobacteriaceae (CRE) are not susceptible to one of the last line antibiotics, carbapenems (2). Consequently, these resistant bacteria are included in the critical group of WHO Priority Pathogen List for Research and Discovery of New Antibiotics (3). New Delhi metallo-β-lactamase (NDM) variants are considered a particularly hazardous resistance mechanism because of its wide range of activity against β-lactams, including carbapenemas, and its high dissemination capacity (4). However, despite the worldwide spread of these genes, the scarce use of carbapenems in veterinary medicine, together with the low prevalence of CRE from animal sources, suggests that the dissemination of this resistance mechanism occurs in a human context (5).

Many of CRE are multidrug-resistant (MDR) organisms, since they tend to accumulate additional genetic determinants that confer resistance to other classes of antibiotics, even other last resort molecules, increasing the morbidity and mortality of the infections caused by these organisms (6). Polymyxins, and specifically colistin due to its clinical importance, is one of these last line antibiotics against MDR Gram negative bacteria, and were included in the Critically Important Antimicrobials group (7).

Resistance to colistin has been usually associated to chromosomal mutations, with vertical transmission and slow evolution (8). However, since the discovery of mcr-1, the first colistin resistance mechanism able to be mobilized between bacteria, the effectiveness of this antibiotic is at risk (9). Due to the increasing spread of mcr-1-positive Enterobacteriaceae (MCRPE) detected in numerous countries and from diverse sources, colistin was moved to the Highest Priority group of the Critically Important Antimicrobials list (10). Moreover, colistin has been commonly used in veterinary medicine, but not in human infections, where its renal toxicity lead to a limited prescription. For that reason, the possible transmission of mcr-1 from animal origin has been suggested, although there are not conclusive studies (11).

Considering these facts, the association of mcr-1 with bla_NDM in CRE constitutes an alarming situation for both human and animal health, regarding the drastically reduction of available effective treatments against infections caused by these bacteria (12).

Surveillance of these two relevant resistance mechanisms and the elements involved in its dissemination is essential to introduce accurate control measures and reduce their impact in public health. This surveillance must be done under a ‘One Health’ approach, including samples from human, animal, both companion and production animals, food and environment (13).

The work carried out by Wang et al. is a perfect example of application of this approach to characterize the whole scenario of bla_NDM and mcr-1 in Chinese poultry production, considering multiple pieces of the puzzle: hatchery and commercial farms (including chicken, wild bird, dog, farmer, flies and sewage samples), a slaughterhouse and supermarkets (14).

In this work, all the CRE isolated, except 14 E. coli, were carriers of a bla_NDM variant, bringing to light the high prevalence (33.2%) of this resistance mechanism to carbapenems in Chinese farming. The 14 CREC that were not bla_NDM-positive, were neither positive for any known carbapenemase gene, and the underlying resistance mechanism should be deciphered. Among the 161 carbapenem-resistant Escherichia coli (CREC), the most frequent variant was bla_NDM-5, whose high prevalence in Chinese patients has been previously described (15). Interestingly, bla_NDM genes were not detected in samples from hatchery farms, indicating that the acquisition of CREC by the chicken takes place in a later phase in the production process. On the other hand, the highest occurrence of CREC and MCRPE was identified in commercial farms, which coincides with other studies, remarking the importance of this chicken production phase as a critical stage in the acquisition of these bacteria, probably due to the antibiotic pressure to which they are subjected (16). Moreover, 37 (23%) CREC were also MCRPE, showing the high coexistence of these two dangerous resistance determinants.

In addition to the isolation of CRE and MCRPE, the study included the bla_NDM and mcr-1 gene detection directly from the samples. Comparing these results with those obtained from the CREC and MCRPE isolation, the detection rates of the two genes were much higher in the direct screening, increasing a 52% in the case of bla_NDM in a slaughterhouse and 62.5% regarding mcr-1 detected in supermarket samples. Supported by this approach, Wang et al. coined the term ‘phantom resistome’ referring the resistance determinants that are present in a sample but are not able to be identified by phenotypic testing of the isolates. This ‘phantom resistome’ constitutes an important reservoir and can be part of the origin of dissemination of these resistance mechanisms, as it has been demonstrated in previous studies where the resistance gene from susceptible bacteria was successfully transferred conferring resistance to the recipient (17). Additionally, the detection of bla_NDM and mcr-1 in this ‘phantom resistome’ remains stable at extremely elevated levels throughout time, which indicates that this reservoir is a constant risk to gene dissemination and specific control measures must be taken in order to mitigate and reduce its spread.

In-depth analysis of the presence of bla_NDM and mcr-1 in different elements involved in one of the farms evaluated revealed very high rates of these genes in dogs, wild birds, farmers and flies, bringing to light that resistance gene dissemination is not a confined phenomenon that takes place in the chicken and their products, but all the elements linked to the production chain. Regarding specifically the presence of CREC in flies, the rates decrease as the distance from the chicken house increased, showing that the physical location is a focal point and the flies a vehicle responsible for gene propagation. Studies of this kind provide data about resistance gene flow, the interconnected elements and the processes between them, which is highly valuable, especially for the food chain (18).

bla_NDM and mcr-1 were not located in the same plasmid in any case, which, attending to the high rates of occurrence of both resistance mechanisms, reflects the remarkable success in the dissemination of these genes independently. This fact is supported by the elevated conjugation frequencies obtained, reaching 3.2×10⁻¹ and 5.0×10⁻³ cells per donor cell in the case of bla_NDM- and mcr-1-carrier plasmids, respectively. Furthermore, the co-transfer of both bla_NDM and mcr-1-carrier plasmids was observed in 5 strains, demonstrating the relatively high frequency (13.5%) of this threatening event, which confers resistance to many clinically important molecules. The effective association of resistance genes and the plasmids involved in its dissemination must be analysed in order to control their spread at all levels (19).

Regarding the antimicrobial susceptibility testing results, a high proportion of CREC were resistant to other important antibiotics apart from carbapenems and colistin, such as gentamicin (79.5%) or ciprofloxacin (99.4%), which emphasizes the multi-drug resistance profile that this kind of bacteria usually shows. However, all of them were susceptible to tigecycline, the only remaining effective antibiotic in many bla_NDM- and mcr-1-carrier strains (2).

To totally characterize the genomic organization of CREC, and therefore the structures involved in the mobilization and dissemination of bla_NDM and mcr-1, whole-genome sequencing (WGS) was performed by Wang et al. This approach reveals the association between the different elements and assigns the antimicrobial resistance profiles to the surveillance of these prevalent MDR clones (20). Moreover, the inclusion of carbapenem-susceptible E. coli in the WGS analysis makes it possible to compare between these strains and the CREC, which determines the molecular features that influence in the possession of these resistance mechanisms.

The most frequent sequence type (ST) identified in bla_NDM-carriers E. coli (minus mcr-1) was ST101, which has been frequently related to NDM-producer E. coli worldwide, supporting the notion of a successful association between this resistance mechanism and this specific ST (21). In addition, ST156 E. coli was detected in diverse sources, from chicken farms (including chickens, dogs, flies and wild birds), the slaughterhouse and supermarkets, showing the broad distribution of this sequence profile in the Chinese chicken production chain. This exact ST has been described in a bla_NDM-5-carrier E. coli, the most prevalent bla_NDM variant detected by Wang et al., that also harboured mcr-1, from a duck in China (22). These findings suggest the tight link between these two resistance genes and ST156, which seems to be the main responsible genotype involved in the spread of bla_NDM-5 and mcr-1 in the poultry production context. Furthermore, E. coli belonging to ST156 presented a high commonality based on core-genome single nucleotide polymorphism (SNP) analysis, strongly confirming the implication of this clone in the dissemination of bla_NDM and, also, mcr-1, being in this group the majority of bla_NDM and mcr-1 co-carrier E. coli isolates. Other global bla_NDM-carrier CREC from human infections were incorporated in the comparison and showed the same genotypes than those identified in the analysis performed by Yang Wang et. al. This demonstrates the clonal relationship between NDM-producer E. coli worldwide, even connecting chicken production with human disease.

bla_NDM-carrying mobile genetic elements (MGEs) of the 161 bla_NDM-positive CREC were clustered in three main types (I, II and III) that were well characterized in the study. These three types are present in different STs isolated from diverse sources, suggesting that there is not a specific link between these bla_NDM-associated MGEs and the STs identified. However, the type II was present in more than the half of bla_NDM-positive CREC (52.17%), belonging to 15 different STs and from all kind of samples collected, and being the most prevalent MGE connected to this gene. Interestingly, the most prevalent STs among bla_NDM-positive E. coli, ST101 and ST156, were the only two STs in which the three MGE types were detected. This indicates a special capacity of these two STs to be associated with diverse MGE responsible for this resistance gene dissemination. Additionally, the most linked MGEs related to ST156 and found in most kind of samples were type I and III, pointing out that these MGEs-ST complex can constitute high-risk combinations for the dissemination of bla_NDM in the chicken production chain.

According to the genetic context of mcr-1, the analysed E. coli were classified into four types (A, B, C and D). Type A was the most prevalent genetic structure associated to mcr-1 (88.64%), sharing a high identity with the mcr-1-adjacent elements in the pHNSHP45 characterized by Liu et al. (9) However, this structure does not contain ISApl1, a mobilization component that has been frequently linked to mcr-1 (23). Type A genetic context is present in all MCRPE analysed belonging to ST156, which are from different kind of samples recovered throughout Chinese chicken production chain. These finding alerts about the spread capacity of this resistance gene associated to this genetic structure and contained in this specific ST. Remarkably, the type C genetic environment showed the integration of mcr-1 in the chromosome, including the ISApl1 element. This event has been described before and supports the idea that ISApl1 is one of the main causes implicated in the global dissemination of mcr-1 (24).

Altogether, the results from Wang et al. reveal the presence of bla_NDM integrated in a type III MGE that is found in numerous ST156 E. coli that are disseminated in diverse elements of the Chinese chicken production chain. Furthermore, most of these ST156 E. coli also harbour the mcr-1 gene, making this resistance determinants-genetic contexts- ST association a very worrying concern that menaces the clinical effectiveness of most last resort antibiotics.

Commercial farms were one of the main critical points in the dissemination of bla_NDM and mcr-1 in the Chinese poultry industry, where chicken and dogs are the major sources of these genes, but also flies suppose a focus for mcr-1 dissemination. The potential role of flies in the dissemination of these resistance genes is a particularly alarming issue, since it constitutes a link between a broad range of elements of the process, including the environment, as it has been shown previously (25). Beyond this local spread, the detection of bla_NDM and mcr-1 in wild birds with migratory behaviour to long distance destinies implies the possibility of dissemination of these resistance mechanisms to completely new areas, including new elements in this transmission chain, making it a problem on a larger scale (26).

From commercial farms, the levels of bla_NDM and mcr-1 keep relatively constant in the subsequent stages of chicken production chain, reaching supermarket products. This fact exposes the relevance of this kind of studies to look into resistance gene dissemination routes and the involved elements. The application of adequate control measures at diverse levels based on this knowledge can reduce and prevent the spread of these resistance mechanisms, safeguarding the clinical effectiveness of last resort antibiotics such as carbapenems and colistin (27).

At present, carbapenems are include in the Critically Important Antimicrobials list, but not in the Highest Priority group due to the limited transmission of CRE from non-human sources (10). Considering the results observed by Wang et al., the inclusion of carbapenems in this category should be evaluated, since these resistant bacteria probably have a human origin, but the animal and environmental components of food chain are sources of dissemination of MDR bacteria that can reach the human context, being a serious global concern for Public Health.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Executive Editor Bing Gu (Department of Laboratory Medicine, the Affiliated Hospital of Xuzhou Medical University, Xuzhou, China).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jlpm.2017.07.03). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. who.int [Internet]. World Health Organization: Antimicrobial resistance, September 2016 [cited 2017 May 30]. Available online: http://www.who.int/mediacentre/factsheets/fs194/en/
2. Potter RF, D'Souza AW, Dantas G. The rapid spread of carbapenem-resistant Enterobacteriaceae. Drug Resist Updat 2016;29:30-46. [Crossref] [PubMed]
3. who.int [Internet]. World Health Organization: Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, February 2017 [cited 2017 May 30]. Available online: http://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf
4. Khan AU, Maryam L, Zarrilli R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health. BMC Microbiol 2017;17:101. [Crossref] [PubMed]
5. Poirel L, Stephan R, Perreten V, et al. The carbapenemase threat in the animal world: the wrong culprit. J Antimicrob Chemother 2014;69:2007-8. [Crossref] [PubMed]
6. Logan LK, Weinstein RA. The Epidemiology of Carbapenem-Resistant Enterobacteriaceae: The Impact and Evolution of a Global Menace. J Infect Dis 2017;215:S28-S36. [Crossref] [PubMed]
7. World Health Organization (WHO). Critically important antimicrobial agents for human medicine - 3rd rev. Geneva, 2012. Available online: http://apps.who.int/iris/bitstream/10665/77376/1/9789241504485_eng.pdf
8. Al-Tawfiq JA, Laxminarayan R, Mendelson M. How should we respond to the emergence of plasmid-mediated colistin resistance in humans and animals? Int J Infect Dis 2017;54:77-84. [Crossref] [PubMed]
9. Liu YY, Wang Y, Walsh TR, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016;16:161-8. [Crossref] [PubMed]
10. World Health Organization (WHO). Critically important antimicrobial agents for human medicine – 5th rev. Geneva, 2017. Available online: http://apps.who.int/iris/bitstream/10665/255027/1/9789241512220-eng.pdf?ua=1
11. Catry B, Cavaleri M, Baptiste K, et al. Use of colistin-containing products within the European Union and European Economic Area (EU/EEA): development of resistance in animals and possible impact on human and animal health. Int J Antimicrob Agents 2015;46:297-306. [Crossref] [PubMed]
12. Delgado-Blas JF, Ovejero CM, Abadia-Patiño L, et al. Coexistence of mcr-1 and blaNDM-1 in Escherichia coli from Venezuela. Antimicrob Agents Chemother ;2016:6356-8. [PubMed]
13. Caniaux I, van Belkum A, Zambardi G, et al. MCR: modern colistin resistance. Eur J Clin Microbiol Infect Dis 2017;36:415-20. [Crossref] [PubMed]
14. Wang Y, Zhang R, Li J, et al. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production. Nat Microbiol. 2017;2:16260. [Crossref] [PubMed]
15. Hu X, Xu X, Wang X, et al. Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China. Int J Infect Dis 2017;55:92-5. [Crossref] [PubMed]
16. Huang X, Yu L, Chen X, et al. High Prevalence of Colistin Resistance and mcr-1 Gene in Escherichia coli Isolated from Food Animals in China. Front Microbiol 2017;8:562. [Crossref] [PubMed]
17. Ovejero CM, Delgado-Blas JF, Calero-Caceres W, et al. Spread of mcr-1-carrying Enterobacteriaceae in sewage water from Spain. J Antimicrob Chemother 2017;72:1050-3. [PubMed]
18. Hu Y, Gao GF, Zhu B. The antibiotic resistome: gene flow in environments, animals and human beings. Front Med 2017;11:161-8. [Crossref] [PubMed]
19. González-Zorn B, Escudero JA. Ecology of antimicrobial resistance: humans, animals, food and environment. Int Microbiol 2012;15:101-9. [PubMed]
20. Crofts TS, Gasparrini AJ, Dantas G. Next-generation approaches to understand and combat the antibiotic resistome. Nat Rev Microbiol 2017;15:422-34. [Crossref] [PubMed]
21. Toleman MA, Bugert JJ, Nizam SA. Extensively drug-resistant New Delhi metallo-β-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012. Emerg Infect Dis 2015;21:1027-30. [Crossref] [PubMed]
22. Yang RS, Feng Y, Lv XY, et al. Emergence of NDM-5- and MCR-1-Producing Escherichia coli Clones ST648 and ST156 from a Single Muscovy Duck (Cairina moschata). Antimicrob Agents Chemother 2016;60:6899-902. [Crossref] [PubMed]
23. Li R, Xie M, Zhang J, et al. Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant. J Antimicrob Chemother 2017;72:393-401. [Crossref] [PubMed]
24. Zurfluh K, Tasara T, Poirel L, et al. Draft Genome Sequence of Escherichia coli S51, a Chicken Isolate Harboring a Chromosomally Encoded mcr-1 Gene. Genome Announc 2016;4:e00796-16. [PubMed]
25. Blaak H, van Hoek AH, Hamidjaja RA, et al. Distribution, Numbers, and Diversity of ESBL-Producing E. coli in the Poultry Farm Environment. PLoS One 2015;10:e0135402 [Crossref] [PubMed]
26. Carroll D, Wang J, Fanning S, et al. Antimicrobial Resistance in Wildlife: Implications for Public Health. Zoonoses Public Health 2015;62:534-42. [Crossref] [PubMed]
27. Nordmann P, Poirel L. Plasmid-mediated colistin resistance: an additional antibiotic resistance menace. Clin Microbiol Infect 2016;22:398-400. [Crossref] [PubMed]