Building a Framework to Curb the Rising Colistin Resistance Among Gram-Negative Bacteria from Wastewater

In Gram-negative bacteria where colistin (polymyxin E) exhibits its bactericidal effect, it acts as a membrane disruptor by interacting with Lipopolysaccharide (LPS) and phospholipids in the outer cell membrane. Both cell membranes are damaged due to the competitive displacement of divalent cations Mg2+ and Ca2+ from the phosphate groups of membrane lipids [1], leading to intracellular content leakage and eventual bacterial mortality. Most members belonging to the order Enterobacterales, including Salmonella, Klebsiella, Shigella, E. coli and Enterobacter, as well as other medically significant Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii can be controlled using polymyxins. This class of antibiotics on the other hand, has no known effect against Gram-positive and Gram-negative cocci. Similarly, polymyxins show no activity against Gram-positive bacilli. Furthermore, these drugs are non-efficacious against intrinsically resistant bacteria such as Neisseria, Providencia, Serratia, Stenotrophomonas and Proteus spp., Morganella morganii, Burkholderia, pseudomallei, and Edwardsiella tarda, as well as anaerobic bacteria [2,3].

Colistin products are provided to a wide range of animal species in Europe, including poultry, pigs, cattle, sheep, goats, rabbits and laying hens as well as to milk-producing species such as cattle, sheep and goats to treat gastrointestinal illnesses resulting from Gram-negative bacteria such as diarrhea in pigs caused by E. coli and Salmonella spp. as well as colibacillosis prevalent in poultry [4]. This practice increases the possibility of the presence of colistin in wastewater from livestock sources. The high prevalence of colistin-resistant Gramnegative bacteria in livestock feces, process waters and wastewater from slaughterhouses and eventually wastewater treatment plants implies that these sites represent potential reservoirs with the possibility of contributing to the resistance progressing to other natural environments, especially surface water [5]. The rising prevalence of colistin-resistant isolates from wastewater points to possible selection pressure by colistin residues in the wastewaters, since colistin has been continuously and extensively used in the global livestock enterprise for preventive, medicinal, and even growth enhancement, which has been already prohibited in Europe from 2006 [6].

For the last decades, the use of colistin in animal production has been extensive [7]. Additionally, it was often used as a feed additive in low doses until the prohibition of antimicrobial growth promoters in the European Union (EU) in 2006 [8]. Inspite of its adverse effects relating to nephrotoxicity and neurotoxicity, colistin was re-introduced into human therapy to control ailments resulting from multidrug-resistant A. baumannii and P. aeruginosa or carbapenemase-Producing Enterobacterales (CPE) [9]. Following its significant impact, the World Health Organization (WHO) categorized colistin among the “highest priority critically important antimicrobials” for human medicine [10], alongside other premium antibiotics including amikacin, tigecycline and the newly produced combinations of ceftazidime-avibactam and ceftozolane-tazobactam. Earlier on in 2016, colistin had also been recognized and classified as a highly critical antimicrobial (VHIA) in the field of veterinary medicine by the World Organization for Animal Health [11].

Colistin resistance among multi-resistant Gram-negative bacteria from wastewater is undoubtedly in the upward trend considering the recent research reports and critical reviews. Recently, bacteria and associated genes that confer resistance to colistin were detected at the Los Angeles county’s two largest wastewater plants, marking the first time traces of this particular antibiotic resistance in Los Angeles has been detected [12]. Mutations in chromosomal genes were primarily linked to colistin resistance initially. However, the discovery of the first plasmidencoded, mobilizable colistin resistance gene (mcr-1) in E. coli from Chinese livestock and locally supplied meat, as well as clinical Klebsiella pneumoniae isolates from China [13,14], drew the attention of public health stakeholders as a major concern following the emergence of colistin-resistant bacteria. Further research into the genetic basis of colistin-resistant bacteria resulted in the identification of nine more mcr genes (mcr-2 to mcr-10). Furthermore, the detection of the mobile colistin resistance gene, (mcr-10) in K. quasipneumoniae in Italy following wastewater-based surveillance suggests an increasing colistin resistance trend among Gram-negative bacteria facilitated by mobile genetic elements [15]. The mcr-1 variant remains the most widespread globally [16].

A. Colistin is only used in clinical situations where no other treatments are available. Currently, the use of colistin in livestock in certain European countries like in the case of Hungary is restricted and only allowed for clinical use as a last resort antibiotic. As a result, very limited residues of this antibiotic if any, may be detected in wastewater or other environmental matrices. This ban would be of great value if extended to other regions.
B. In a concerted effort to promote the achievement of the One Health paradigm, it is imperative for the global regions to formulate and adopt a universal regulatory framework on the use of this salvage antibiotic in order to minimize its presence in environmental matrices as a strategy of reducing its exposure to microbes with the aim of minimizing the development of its resistance among bacteria associated with human and animal hosts.
C. At present, there is hardly any documented data investigating colistin residues in wastewater from slaughterhouses, poultry or pig farming process wastewater despite its enormous usage in animal production globally. A focus on the load of colistin residues in wastewater would be useful to correlate its presence in the environment to the development of its resistance among environmental bacteria associated with human infections.
D. There is a handful of research reports on colistin resistance in Gram-negative bacteria from wastewater with numerous articles focusing mainly on Enterobacterales and centered on clinical setting, thus the need to extend research to focus on additional multiple drug resistant Gram-negative bacteria of medical importance either transient or domiciled in the environment.
E. Besides monitoring what happens at the point-of-care, it is important to continuously monitor or “characterize” wastewater from anthropogenic activity and the immediate microbial community. This can be coupled with tracking the use of byproducts of wastewater from human and animal sources such as biosolids which supply nutrients for agricultural crops to establish the developing resistance trends which would provide useful insights.
F. There is need for all the stakeholders to remain loyal to “One Health” approach so that our focus on resistance development illuminates human hosts, animals and the environment as well as any other reservoirs. Additionally, frequent testing of more environmental sites would be valuable.

1. Falagas ME, Kasiakou SK (2005) Colistin: The revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis 40(9): 1333-1341.
2. Muyembe T, Vandepitte J, Desmyter J (1973) Natural colistin resistance in Edwardsiella tarda. Antimicrob Agents Chemother 4(5): 521-524.
3. Pogue JM, Marchaim D, Kaye D, Kaye KS (2011) Revisiting “older” antimicrobials in the era of multidrug resistance. Pharmacotherapy 31(9): 912-921.
4. Binsker U, Käsbohrer A, Hammerl JA (2022) Global colistin use: A review of the emergence of resistant Enterobacterales and the impact on their genetic basis. FEMS Microbiol Rev 46(1): fuab049.
5. Savin M, Bierbaum G, Blau K, Parcina M, Sib E, et al. (2020) Colistin-resistant Enterobacteriaceae isolated from process waters and wastewater from German poultry and pig slaughterhouses. Front Microbiol 11: 575391.
6. EU (2005) Ban on antibiotics as growth promoters in animal feed enters into effect. Europa, Brussels.
7. EMEA (2002) Committee for veterinary medicinal products. Colistin. Summary Report (2).
8. EMA (2016) Updated advice on the use of colistin products in animals within the European union: Development of resistance and possible impact on human and animal health.
9. Azzopardi EA, Boyce DE, Thomas DW, Dickson WA (2013) Colistin in burn intensive care: Back to the future? Burns 39(1): 7-15.
10. WHO (2019) No time to wait–securing the future from drug-resistant infections. J Report to the Secretary General of the Nations.
11. OIE (2018) List of antimicrobial agents of veterinary importance.
12. Wang P, Smith AL (2023) Emergence of mobile colistin resistance genes within Los Angeles County wastewater. Environ Sci Technol Lett 10(4): 316-321.
13. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, et al. (2016) 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 16(2): 161-168.
14. Sun J, Zhang H, Liu YH, Feng Y (2018) Towards understanding MCR-like colistin resistance. Trends Microbiol 26(9): 794-808.
15. Formenti NF, Guarneri C, Bertasio G, Parisio C, Romeo F, et al. (2022) Wastewater-based surveillance in Italy leading to the first detection of mcr-10-positive Klebsiella Antimicrob Resist Infect Control 11(1): 155.
16. Elbediwi M, Li Y, Paudyal N, Pan H, Li X, et al. (2019) Global burden of colistin-resistant bacteria: Mobilized colistin resistance genes study (1980–2018). Microorganisms 7(10): 461.