Plasmid-mediated Colistin Resistance: What Do We Know?

Tanise V. Dalmolin^1,2, Daiana de Lima-Morales¹, Afonso L. Barth^1,2*

¹LABRESIS – Laboratório de Pesquisa em Resistência Bacteriana, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

²Programa De Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

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Abstract

Polymyxins (polymyxin E/colistin and polymyxin B) are considered the last-resort therapy against carbapenem-resistant Enterobacterales; however, the resistance of Enterobacterales to polymyxins is increasing worldwide. Until 2015, this resistance was related to chromosomal mutations, but in November 2015, it was described in China the transferable colistin resistance in animals and humans isolates of E. coli and K. pneumoniae, mediated by the mcr-1 gene (mobile colistin resistance), located in a plasmid. Following the first description of the mcr-1 gene, it has been reported in several regions of the world, in different bacterial species, from different sources and others mcr variants have been described. Moreover, the co-occurence of the mcr gene and other antimicrobial resistance genes was reported. This discovery changed the scenario of resistance to polymyxins, as this gene could be promptly disseminated among Gram negative bacilli becoming a major concern for public health. This review summarizes recent data about the plasmid-borne mcr colistin resistance gene.

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Introduction

Antimicrobial resistance is considered as a major public health threat to human health mainly due to the widespread of carbapenem-resistant Enterobacterales (CRE) in the last decade. The colistin (polymyxin E) and polymyxin B belong to the class of polymyxins, which are considered the last-resort therapy against CRE; however, the resistance of Enterobacterales to polymyxins is increasing worldwide^1,2,3. Overall, colistin resistance in Enterobacterales (excluding species with intrinsic resistance) is around 0.67–1.6%, with low rates in Escherichia coli (0.2–0.6%), moderate rates in Klebsiella pneumoniae (1.5–6.8%), and much higher rates in Enterobacter spp. (13.9–20.1%). Considering other resistance mechanisms associated with colistin resistance such as ESBLs the percentage of co-producers is: 2.3–5.5% for Enterobacterales(>11.5% are K. pneumoniae); for carbapenem-non-susceptible isolates the number is higher: 4.5–16.3% for Enterobacterales (32% are K. pneumoniae) and even higher is the number found for carbapenemase-producers, 6.2–12.0% for Enterobacterales (14–36.6% are K. pneumoniae)⁴.

As mentioned above, polymyxins are active against most Enterobacterales and also have significant activity against nonfermentative Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter spp. Some Gram-negative species are naturally resistant to polymyxins: Proteus spp., Morganella morganii, Providencia spp., Serratia marcescens, Burkholderia mallei, Burkholderia cepacia complex, Chromobacterium spp., Edwardsiella spp., Brucella, Legionella, Campylobacter and Vibrio cholerae⁵. Additionally, the polymyxins are not active against Gram-negative cocci, Gram-positive and anaerobic bacteria^5,6. The natural resistance mechanism is hypothesized to be promoted by constitutive gene expression which adds cationic molecules to the LPS, leading to decreased affinity at the polymyxin site of action on the LPS, differently in Gram-positive bacteria lack of an LPS containing outer cell membrane promotes the resistance⁷.

The colistin was obtained in 1947 from a soil bacteria ^{Paenibacillus polymyxa} subsp. colistinus⁸ and has been cleared to use since 1959 by the Food and Drug Administration (FDA) for the treatment of infectious diseases caused by Gram-negative bacteria. However, in clinical use, colistin proved to be nephrotoxic and their use has declined from the early 1970s to the early 2000s, being replaced by more active and less-toxic antibiotics such as the aminoglycosides, quinolones and beta-lactams. However, due to increasing prevalence of multiresistant Gram-negative bacteria, the colistin were re-introduced into clinical practice as a valuable therapeutic option^5,9.

Unlike in human medicine, in veterinary medicine, colistin has been used extensively, and without interruption, for decades for the treatment and prevention of infectious diseases by Enterobacterales, as well as growth promoter in poultry and pigs^2,5,9. However, due to the description of the colistin resistance mediated by a mobile genetic element and its widespread in animals in China in 2015, the Ministry of Agriculture of China decided to ban colistin as a feed additive for animals in 2016. Other countries also decided to ban colistin as growth animal promoter, due to the imminent impact on polymyxin resistance in human health¹⁰.

The mechanism of action of polymyxins (colistin and polymyxin B) occurs by the electrostatic binding of the molecule to the lipopolysaccharide (LPS) and phospholipids in the outer membrane of Gram-negative bacteria. The polymyxins destabilize the LPS through the exchange of ions (Ca⁺² and Mg⁺²), increasing the permeability of the bacterial membrane, promoting the extravasation of the cytoplasmic content and consequent bacterial death. Despite the fact that the initial target for polymyxin is the LPS, the exact mode of action of polymyxins is still not totally clear. There are other possible mechanisms of action described for polymyxins: the endotoxin effect (during cell lysis the endotoxin molecule - the lipid A which is the main component of the LPS) is released and the polymyxins are able to bind and neutralize it and the inhibition of respiratory enzymes (type II NADH-quinone oxidoreductases [NDH-2]) in the bacterial membrane^5,11.

Until 2015, the reports of polymyxin resistance were all due to chromosomal mutations. These chromosomal mutations lead to modification of the LPS via cationic substitution that alter the lipid A, decreasing its affinity for the polymyxins, similarly to that observed in bacteria with intrinsic resistance to polymyxins. There are several mutation in genes and operons which are involved in the modification of the LPS: (i) mutations in genes (pmrC, pmrE and pmrHFIJKLM) responsible for synthesis of cationic groups and their addition to the LPS; (ii) mutations in regulatory genes that encode proteins involved in the PmrAB system in K. pneumoniae, Enterobacter aerogenes and Salmonella enterica (pmrA and pmrB genes) and the PhoPQ system in K. pneumoniae and E. coli (phoP and phoQ genes); (iii) and mutations in the regulators of these systems as the mgrB gene in Klebsiella spp. (regulates the PhoPQ system) and crrAB operon in K. pneumoniae (regulates the PmrAB system)¹².

In November 2015, Liu and colleagues described the transferable colistin resistance in animals and humans isolates of E. coli and K. pneumoniae recovered in China, mediated by the mcr-1 gene (mobile colistin resistance). The mcr-1 gene is located in a plasmid and its discovery changed the scenario of resistance to polymyxins, as this gene could be promptly disseminated among Gram-negative bacilli becoming a major concern for public health. MCR-1 protein is a member of the phosphoethanolamine transferase family, its acquisition promotes the addition of phosphoethanolamine (PEtN) to lipid A, similarly to the chromosomal mutations, resulting in reduction of polymyxin affinity¹.

Epidemiology of Plasmid-mediated Colistin Resistance

Following the first description of the mcr-1 gene, it has been reported in several regions of the world, including countries of Asia, Africa, Europe and America^2,12,13. Further studies indicated that bacteria carrying the mcr-1 gene were identified in the 1980´s in China. The earliest report of the mcr-1 gene among isolates from humans is from 2008 and it was described in a Shigella sonnei from Vietnam. These findings indicate that mcr-1 gene has existed in Enterobacterales but remained not identified for a long time^9,14.

The mcr-1 gene has been reported in several species of Enterobacterales but mostly in E. coli. The occurrence in Salmonella, Klebsiella, Shigella, Vibrio and Enterobacter was only sporadically reported, but indicates interspecies gene transfer^2,12.

The mcr-1 gene has been mainly detected in E. coli isolates obtained from animals (livestock, wild animals and food of animal origin). Farm animals, in particular pigs and chickens, have been considered as reservoirs for E. coli isolates carrying the mcr-1 gene. In humans, Enterobacterales carrying the mcr-1 gene have been obtained from clinical samples, as well as from asymptomatic patients, including international travellers. The possible dissemination of the mcr-1 gene from bacteria obtained from animals to bacteria from humans is a serious concern, mainly whether this gene could be transferred into carbapenemase producing Enterobacterales^2,9,12.

MCR-1 producers may exhibit low level resistance to colistin or polymyxin B (minimum inhibitory concentration [MIC] of 4µg/mL) and a few cases of isolates carrying the mcr-1 gene which are susceptible to colistin have already been described^15,16. This may contribute to the silent dissemination of mcr-1 gene harbouring isolates. Other aspects may also play an important role in the dissemination of the mcr-1 gene: the technical problems related to the detection of resistance to polymyxins and the fact that most laboratories only evaluate the susceptibility profile to polymyxins among carbapenem non-susceptible isolates¹⁵.

One explanation for the reported mcr-1 positive but colistin susceptible isolates was that the gene was truncated as described in a Shigella sonnei isolate¹⁷. Interestingly, the truncated mcr-1 gene could be re-activated after conjugation experiments resulting in a colistin resistant phenotype¹⁸. Conversely, the report of an E. coli colistin susceptible and mcr-1 positive with an intact gene indicates that the gene truncation is not the only mechanism related to the susceptibility to polymyxins among mcr-1 harboring isolates¹⁹.

According to an search assessment of the data at the GenBank (https://www.ncbi.nlm.nih.gov/genbank/ – 17^th of June 2018), 13 mcr-1 subgroups were already described in several countries, differing from mcr-1 by only one nucleotide: mcr-1.2 (K. pneumoniae from Italy)²⁰, mcr-1.3 (E. coli from China)²¹, mcr-1.4 (E. coli from China), mcr-1.5 (E. coli from Argentina), mcr-1.6 (Salmonella typhimurium from China)²² , mcr-1.7 (E. coli from China), mcr-1.8 (E. coli from Brunei)²³, mcr-1.9 (E. coli from China)²⁴, mcr-1.10 (Moraxella spp. from Great Britain)²⁵, mcr-1.11 (KY853650.1), mcr-1.12 (LC337668.1) and mcr-1.13 (E. coli from Italy)²⁶.

To date, eight mcr variants have been described: mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7 and mcr-8 (Table 1). The mcr-2 gene is 1,617 bp long, nine bases shorter than mcr-1 (1,626 bp) and it has 76.7% nucleotide identity to mcr-1²⁷. The mcr-3 gene presented 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively. MCR-3 also showed 94.1% to 94.8% amino acid identity with proteins found in three Aeromonas species. Moreover, a truncated transposon element (TnAs2), which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3. These findings suggest that mcr-3 gene in Enterobacterales might have originated from Aeromonas species²⁸. Unusually, the mcr-4 is inserted in a small and not self-conjugative plasmid. However, the addition of an auxiliary plasmid can promote conjugation²⁹.

Table 1: mcr variants available at the GenBank database (update in July 17, 2018- https://www.ncbi.nlm.nih.gov/genbank/)

Gene	Country	Origin	Species	Reference	Variants
mcr-1	China	Retail meat from chicken and pig, and inpatientsa	E. coli and K. pneumoniae	1	mcr-1.2 – mcr-1.13
mcr-2	Belgium	Pig and bovine	E. coli	27	mcr-2.2
mcr-3	China	Pig	E. coli	28	mcr-3.2-mcr-3.12
mcr-4	Italy, Spain and Belgium	Pig	Salmonella enterica and E. coli	29	mcr-4.2
mcr-5	Germany	Faecal content of pigs and faecal samples from pigs	S. enterica subsp. enterica serovar Paratyphi B	30	mcr-5.2
mcr-6b	Great Britain	Faecal contents of healthy pigs	Moraxella sp.	25
mcr-7	China	Chickens	K. pneumoniae	31
mcr-8	China	Pig, chicken and inpatient	K. pneumoniae	32

^aThe gene mcr-1 was isolated from several other origin, after its first publication from Liu et al., 2016¹.

^b mcr-2.2 (1617 bp) has been renamed as mcr-6.

Co-occurrence of mcr and Other Resistance Genes

There have been reports of co-production of the mcr-1 gene and other resistance genes in several bacterial species. The presence of the mcr-1 gene has been associated with ESBLs (CTX-M, SHV and TEM type) and AmpC cephalosporinase (CMY type). Moreover, there are descriptions of co-occurrence of the mcr-1 gene and quinolone resistance genes (qnrS and aac(6=)-Ib-cr)^9,15,33,34.

Isolates carrying mcr-1 and carbapenemase resistance genes have been described in Enterobacterales. Specifically, the co-production of mcr-1 and carbapenem resistance genes is of great concern, since polymyxins represent the “last-line” therapeutic option for an infection caused by a carbapenemase-producing isolate. In the literature the mcr-1 gene has been found mainly in isolates that produce the carbapenemase NDM in particular among E. coli isolates from animal and human origin^35,36,37. However, it was also detected in Cronobacter sakazakii³⁸ from animal origin and K. pneumoniae³⁹ from human origin. Only one report about co-producer OXA-48 carbapenemase and mcr-1⁴⁰ and VIM carbapenemase and mcr-1⁴¹ have been related in the literature, both from human sources. There are a few KPC and mcr-1 co-producers reported, in particular those genes were identified in E. coli^{42,43,44,45,46} and less frequently in K. pneumoniae isolates^20,47,48.

Genetic Context of the mcr Gene

The first plasmid carrying the mcr-1 gene belonged to the plasmid incompatibility type Incl2¹. Others studies indicated that mcr-1 gene is not restricted to the IncI2 plasmid group and various plasmids may carry the mcr-1 gene including those belonging to the IncX4, InHI2, IncF, IncHI1, IncY and IncP^33,49. These incompatibility groups of plasmids have been implicated in the global spread of others resistance genes among Enterobacterales from human and animal sources^50,51. As the mcr-1 gene is not associated with a specific incompatibility group of plasmids, it can be considered that this molecular flexibility promotes global dissemination of the gene⁵².

Noteworthy, there are intriguing descriptions of coexistence of two mcr bearing plasmids in a single bacterial isolate. Nevertheless, the MIC for colistin of the isolates presenting more than one mcr gene in different plasmids was not increased^52,53.

Often the mcr gene is flanked by an insertion sequence designated ISApI1 and the nikB gene, in this order. It is suggested that the ISApl1 is a key component in the mobilization of the mcr gene. ISApl1 belongs to the IS30 family and was first identified in Actinobacillus pleuropneumoniae, a Gram-negative bacterium of the Pasteurellaceae family, causing pig necrotic pleuropneumonia^42,49,54.

It is also suggested that the mcr-1 gene had been initially mobilized by two copies of ISApl1 from an unknown progenitor. Over the course of evolution, this complex transposon has lost one or both copies of ISApl1, since it is common for IS30 family member to lose a copy of the IS element by transposition or illegitimate recombination events, in order to stabilize the mcr-1 bearing plasmid. Sometimes the mcr-1 gene can be mobilized by only a single copy of ISApl1 upstream the gene in combination to an incomplete sequence of the ISApl1 downstream. ISApl1 is presents in multiple copies in the genome and these insertion sequence sites are notable for their high AT content49,52,55,56.

As already mentioned, the mcr-1 gene was originally described mostly among E. coli isolates from animal sources and this indicates that this gene is of animal origin. The genetic context of the mcr-1 gene reinforces the theory that the gene was originally from isolates obtained from animals. The first evidence is that the mcr-1 gene is very often associated to the insertion sequence ISApl1 and this insertion sequence is usually identified in bacteria of animal origin. Another indicative of the animal origin and the genetic context of the mcr-1 is the presence of other antibiotic resistance genes specific to veterinary medicine, such as the floR gene which confers resistance to florfenicol⁵⁷.

Moreover, the Moraxella genus was identified as potential reservoirs of mcr-like genes that might be mobilized from their original host to become an acquired resistance mechanism in clinically significant species. The mcr-1 and mcr-2 genes present a significant identity with intrinsic chromosomal genes of the Moraxella species. The exact species of the progenitors of mcr-1 and mcr-2 genes remains to be determined, but the most closely-related variant compared to MCR-1 was identified in Moraxella porci, while the most closely-related variant of MCR-2 was identified in Moraxella osloensis⁵⁷.

Detection of MCR-Producers

Broth microdilution (BMD) is considered the reference test for determining the susceptibility profile of polymyxins regardless the fact that BMD do not establish the underlying mechanism of resistance⁵⁸. There are a few phenotypic tests which are supposedly able to detect MCR producers, including: the Colistin MAC Test (CMT)⁵⁹; Combined Disc Test (CDT); Colistin MIC Reduction (CMR); Modified Rapid Polymxin NP Test (modified-RPNP) and alteration of Zeta potential tests⁶⁰. Nevertheless, the molecular tests are considered the reference tests for the detection of the mcr gene; the molecular techniques using sequencing can also specify the gene variants.

Assays based on colistin MIC reduction in the presence of dipicolinic acid (DA) or ethylene diamine tetra-acetic acid (EDTA) have been evaluated to detect mcr-producing Enterobacterales^59,60. These tests are based on the fact that the catalytic domains of the MCR enzymes resemble that of zinc metalloproteins⁶¹. DA or EDTA are able to chelate zinc (necessary for the enzymatic activity of the PEtN transferase) and consequently can inhibit the enzymatic activity of MCR-1 and finally reduce colistin resistance in MCR-producing strains.

The CMT method is a broth microdilution method, in which colistin MIC (0.125 to 8.0 µg/mL) is tested in absence and in presence of fixed concentration of the DA (900 µg/mL). If the MIC increases ≥8-fold, in the presence of DA, the result is interpreted as mcr-positive while an MIC reduction of ≤2-fold is interpreted as mcr-negative. The test does not present satisfactory results with K. pneumoniae, probably due to a reduced permeability to DA and/or the presence of additional unknown mechanisms. In addition, the authors tested the effect of DA in increasing colistin susceptibility of mcr-1 positive isolates, in a disc-diffusion format, however no significant difference was detected as the inhibition zones between mcr-1-positive and mcr-negative isolates were very similar⁵⁹.

Similarly, Esposito et al. (2017)⁶⁰ evaluated four EDTA-based assays to detect MCR-1-positive isolates. The CDT test compare the inhibition zones of colistin (10µg) and colistin plus EDTA (100mM). An incremental difference of ≥ 3mm between the colistin impregnated disc and the colistin-EDTA-impregnated disc was interpreted as MCR-1-positive. The CDT presented sensitivity and specificity of 96.7% and 89.6%, respectively⁶⁰.

The CMR test was designed according to the BMD method with or without the addition of 80μg/ml EDTA, into the wells containing 0.06–32μg/ml of colistin. A ≥ 4-fold colistin MIC reduction in EDTA-containing wells was interpreted as MCR-1-positive. The sensitivity and specificity of this method was 96.7% and 83.3%, respectively⁶⁰.

The original RPNP test consists of detecting bacterial growth on a glucose based medium, in the presence of a defined concentration of colistin. The bacterial growth leads to acid formation in the medium which alters the pH and this can be detected visually by a pH indicator color change⁶². If the well containing colistin, change its colour from orange to yellow, it is considered positive (colistin resistance). The modified RPNP is based on the incorporation of two extra wells, one containing 80μg/mL EDTA (without colistin) and the other one containing 80μg/ml EDTA plus 5μg/ml colistin. In this case, the MCR-1 positive result is observed when the colistin containing solution supplemented with EDTA remained orange (i.e., absence of glucose metabolization due to EDTA inhibition). The sensitivity and specificity of this test are higher (96.7% and 100.0%, respectively) compared to CDT and CMR tests⁶⁰.

Another method for phenotypic detection of mcr producers is the alteration of Zeta potential in the absence and presence of 80 μg/ml EDTA, which increase the anionic charges on the surface membrane of MCR-1-positive isolates. However, according to the authors, this method can only be used in well-resourced microbiology laboratories⁶⁰.

Polymerase Chain Reaction (PCR) and Whole Genome Sequence (WGS) are considered the reference tests to identify the mcr gene from cultured bacteria as well as in clinical, fecal, environmental and food samples^58,63. While PCR can only detect known mcr genes, due to specific primers and probes, the WGS can identify all known or unknown colistin resistance mechanisms within 2 days⁵⁸. Further molecular tests are the commercial microarray that can simultaneously detect both β-lactamases and mcr-1/-2 genes, even though those tests are inaccessible to under-resourced laboratories⁶⁴.

Conclusion

Considering the importance of polymyxins in human and veterinary medicine, there is an urgent need to limit the propagation of mcr-1 harboring plasmid. Given the constant exchange of resistance genes in all microbiomes (animals, environment and human population), surveillance programs to monitor the diversity of reservoirs, plasmids and to evaluate the real dimension of resistance mediated by the mcr gene are necessary.

Funding information: This work was supported by Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) (project no.16–0559) and by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico-Brazil (CNPq).

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