Plasmidic resistance to colistin mediated by mcr-1 gene in Escherichia coli clinical isolates in Argentina: A retrospective study, 2012–2018

ABSTRACT

Objective.

To describe the resistance profile and the genetic characteristics of Escherichia coli isolates that harbor the mobilizable colistin resistance gene mcr-1 in Argentina.

Methods.

This was a retrospective study of 192 E. coli isolates positive for mcr-1 obtained from 69 hospitals of Buenos Aires City and 14 Argentinean provinces in 2012 – 2018. The antimicrobial susceptibility was performed by agar diffusion, broth macrodilution, and/or agar dilution. Standard polymerase chain reaction (PCR) was performed to detect resistance genes and incompatibility groups; specific PCR was applied to discriminate between blaCTX-M allelic groups and mcr-1.5 variant. The genetic relatedness among isolates was evaluated by XbaI-pulsed field gel electrophoresis and multilocus sequence typing in a subset of isolates.

Results.

All E. coli isolates showed minimal inhibitory concentrations to colistin ≥ 4μg/mL; nearly 50% were resistant to third-generation cephalosporins, with CTX-M-2 being the main extended-spectrum β-lactamase detected. Five E. coli were carbapenemase-producers (3 NDM, 2 KPC). The mcr-1.5 variant was detected in 13.5% of the isolates. No genetic relationship was observed among the mcr-1-positive E. coli clinical isolates, but a high proportion (164/192; 85.4%) of IncI2 plasmids was detected.

Conclusions.

The presence of IncI2 plasmids among highly diverse E. coli clones suggests that the mcr-1 gene’s wide distribution in Argentina may be driven by the horizontal transmission of IncI2 plasmids.

Polymyxins, including polymyxin B and colistin, are “last-line” treatment options against multidrug-resistant (MDR) gram-negative bacteria, such as carbapenem-resistant Enterobacterales. Until November 2015, the main colistin resistance mechanisms reported were chromosome-mediated mutations involving alterations in the PmrAB or PhoPQ, a two-component regulatory system (1). The situation changed with the report of mobile colistin resistance mediated by mcr-1 gene, revealing for the first time the horizontal spread of a colistin resistance determinant (2). This gene encodes a plasmid-borne phosphoethanolamine transferase that has been reported in Escherichia coli isolates from animal, food, environment, and human samples worldwide (3, 4). At this time, nine mcr genes have been described; but, mcr-1 is, by far, the most prevalent (3 - 5). Mcr-1 has been described in almost all countries in the Region of the Americas, while mcr-3 and mcr-5 genes have been sporadically described in only Brazil and Colombia, respectively (3, 5 – 7). Although detected in other Enterobacterales, the mcr-1 gene has been associated mainly with E. coli isolates, including Klebsiella pneumoniae and Salmonella spp. (5). Regardless of species, mcr-1 has been associated with a ~2609 bp DNA fragment containing the mcr-1 and pap2 genes, and has mobilized into an ISApI1-based composite transposon and different plasmid replicons, of which IncI2, IncX4, and IncHI2 are the most common incompatibility groups described so far (8 - 10).

Mcr-1-positive E. coli isolates causing bloodstream infections are still rare (1.0%), and generally, they remain susceptible to many antimicrobial agents (11). However, it is especially worrisome that mcr-1 gene acquisition by extended-spectrum β-lactamase- (ESBL) or carbapenemase-producing Enterobacterales, render extensively- or pan-drug resistant strains (3, 10 – 15). NDM has been the main carbapenemase reported in mcr-1-positive isolates, not only in hospitalized patients where it colonizes or causes infections, but also in healthy individuals within the community; sporadic cases of mcr-1-producers harboring KPC or OXA-48 carbapenemases have also been described (10 - 15).

After the description of mcr-1 in November 2015, the National Reference Laboratory on Antimicrobial Resistance in Argentina (NRL) set national and regional alerts for detection of mcr-1 gene in E. coli clinical isolates (16). Through December 2018, a total of 192 E. coli clinical isolates were confirmed as positive for mcr-1 at the NRL. The present study aims to describe the resistance profiles and the genetic characteristics of mcr-1-producing E. coli clinical isolates in Argentina.

MATERIALS AND METHODS

This retrospective study comprised the entire NRL collection: 192 E. coli clinical isolates (one per patient) previously confirmed as mcr-1 positive, submitted by 69 hospitals from 14 of Argentina’s provinces and Buenos Aires City (Figure 1). Isolates were recovered mainly from urine (n = 117; 60.9%), blood (n = 26; 13.6%), and other samples (n = 49; 25.5%). The first 10 isolates were recovered in July 2012 – January 2016, as previously described by Rapoport and colleagues (17) and Martino and colleagues (18). The remaining 182 isolates were submitted from February 2016 – December 2018, with minimal inhibitory concentration (MIC) to colistin > 2µg/ml and/or a positive growth on Mueller-Hinton screening agar plates containing 3 µg/mL colistin (19).

FIGURE 1. Geographical distribution of 192 mcr-1 positive Escherichia coli clinical isolates, by province and capital city, Argentina, 2012 – 2018.

Source: Prepared by the authors from the study results.

Data on sample type and patient age and sex were obtained from the clinical laboratory documentation that accompanied each isolate. Patient data were anonymized to preserve the patient’s identity.

Susceptibility profiles were determined by the agar diffusion method, with the exception of colistin which was tested by broth macrodilution and/or agar dilution according to the Clinical and Laboratory Standards Institute guidelines (CLSI; 20). CLSI criteria were used to interpret all results, except for colistin and tigecycline, for which the 2018 European Committee on Antimicrobial Susceptibility Testing guidelines (21) were used. ESBL- or plasmidic-AmpC-phenotype were defined as resistant to third-generation cephalosporins with clavulanic acid or phenyl boronic acid inhibition, respectively.

PCR was performed to detect mcr-1, plasmidic-AmpC, broad-spectrum and ESBL, and carbapenemases using the primers and conditions described in Table 1. Briefly, DNA templates were prepared by boiling for 10 min a suspension of one or two colonies of each isolate in 100 µL of Milli-Q water; 2.5µL were used for the PCR reactions. A final volume of 25µL containing 10 pmol of each primer, 25 µM of each dNTP, 1.5 mM MgCl2, 1X Taq buffer, and 2.5 U of Taq polymerase (Invitrogen,^TM ThermoFisher Scientific Inc., Waltham, MA, United States) was used. Amplifications were performed using a 2720 Thermal Cycler™ (Applied Biosystems, ThermoFisher Scientific Inc., Waltham, MA) following a standard program: pre-denaturation for 5 min at 94°C; 35 cycles of 94°C for 30 sec, T°C annealing (Table 1) for 30 sec, 72°C for 30 sec, and final extension at 72°C for 5 min. If the expected amplicon was ≥ 700 bp, the elongation step of cycling was increased to 1 min and the final elongation to 10 min. PCR products were run on 1% agarose gel for 60 min and stained with ethidium bromide. PCR was also used to discriminate between bla_CTX-M-2, bla_CTX-M-1/15, bla_CTX-M-8/25, and bla_CTX-M-9/14 groups (Table 1). Identification of incompatibility groups IncI2, IncX4, and IncHI2 was analyzed by PCR using previously described conditions (22, 23). To identify the mcr-1.5 variant, we developed an allele specific PCR that detected the C1354T modification (H452Y). A degenerated primer, MCR-1.5-F, with a mismatch at the third base from the 3’extreme was designed to increase the PCR specificity (Table 1).

TABLE 1. Primers for PCR analysis of antimicrobial resistance mechanisms.

Target	Primer	Oligonucleotide sequence	Amplicon size (bp)	T °C annealing
mcr-1	CLR5-F CLR5-R	5’ CGGTCAGTCCGTTTGTTC 3’ 5’ CTTGGTCGGTCTGTAGGG 3’	309	45
mcr-1.5	MCR-1.5-F MCR-1 Full-R	5’ TCCAGTGGCTGCAGAAGT 3’ 5’ TCAGCGGATGAATGCGGT 3’	288	62
blaNDM	NDM-F NDM-R	5’ AGCACACTTCCTATCTCGAC 3’ 5’ GGCGTAGTGCTCAGTGTC 3’	512	50
blaIMP	IMP-UF1 IMP-UR1	5’ GGYGTTTWTGTTCATACWTCKTTYGA 3’ 5’ GGYARCCAAACCACTASGTTATCT 3’	404	50
blaVIM	VIM-F VIM-R	5’ AGTGGTGAGTATCCGACAG 3’ 5’ ATGAAAGTGCGTGGAGAC 3’	261	50
blaCTX-M	CTX-MU1 CTX-MU2	5’ ATGTGCAGYACCAGTAARGT 3’ 5’ TGGGTRAARTARGTSACCAGA 3’	593	52
CTX-M-G2	CTXM2G-F CTXM2G-R	5’ GCCGCTCAATGTTAACGGTGA 3’ 5’ ACCGTGGGTTACGATTTTCGC 3’	851	55
CTX-M-G9	CTXM9G-F CTXM9G-R	5’ ATGGTGACAAAGAGAGTGCAACG 3’ 5’ GCGGCTGGGTAAAATAGGTCACC 3’	808	56
CTX-M-G1/15	CTXM1/15G-F CTXM1/15G-R	5’ CAGTTCACGCTGATGGCGACG 3’ 5’ CGGCGCACGATCTTTTGGCCA 3’	756	60
CTX-M-G8/25	CTXM8/25G-F CTXM8/25G-R	5’ CTGGAGAAAAGCAGCGGGGG 3’ 5’ CGCTGCCGGTTTTATCCCCGAC 3’	604	58
blaPER	PER-U-Fw PER-U-Rv	5’ GTGTGGGAGCCTGACGATCT 3’ 5’ CTSTGGTCCTGTGGTGGTTTC 3’	524	59
blaTEM	OT-1 OT-2	5’ TTGGGTGCACGAGTGGGTTA 3’ 5’ TAATTGTTGCCGGGAAGCTA 3’	504	55
blaSHV	OS1 OS2	5’ TCGGGCCGCGTAGGCATGAT 3’ 5’ AGCAGGGCGACAATCCCGCG 3’	626	59
blaCMY	CITMF CITMR	5’ TGGCCAGAACTGACAGGCAAA 3’ 5’ TTTCTCCTGAACGTGGCTGGC 3’	462	64

Source: Prepared by the authors from the study results.

The genetic relatedness between the isolates was evaluated by XbaI-digested pulsed-field gel electrophoresis (PFGE) using a Chef-DR^® III System (Bio-Rad^TM, Hercules, CA, United States) as previously reported (24). DNA fragments were resolved in 1% agarose gel applying a switch time of 2.4 to 54.2 seconds during 20 hr at 14°C. Those PFGE patterns showing > 6 bands of difference were considered to be non-genetically related. Selected isolates were also genotyped by multilocus sequence typing (MLST) and the sequence types (STs) were analyzed according to the E. coli MLST (available from http://enterobase.warwick.ac.uk/species/index/ecoli).

RESULTS

All 192 strains from the E. coli clinical isolates harboring mcr-1 strains grew on Mueller-Hinton screening agar plates containing 3 µg/mL colistin and showed MIC to colistin ≥ 4μg/mL. According to Figure 2, the percentage of resistance to other antimicrobials was as follows: ampicillin (85.9%), ciprofloxacin (74.9%), tetracycline (65.2%), trimethoprim-sulfamethoxazole (52.7%), third-generation cephalosporins (cefotaxime and/or ceftazidime; 48.9%), minocycline (39.7%), fosfomycin (23.9%), gentamicin (17.2%), carbapenems (ertapenem and/or imipenem; 3.6%), nitrofurantoin (5.0%), amikacin (2.6%), and tigecycline (2.3%). Nine isolates were resistant only to colistin, while 80.2% showed an MDR phenotype (resistance to > 2 families of antimicrobials).

FIGURE 2. Resistance profile of 192 mcr-1-positive Escherichia coli clinical isolates in Argentina, 2012 – 2018.

Note: AMP = ampicillin; CIP = ciprofloxacin; TET = tetracycline; SXT = trimethoprim-sulfamethoxazole; 3GC = third-generation cephalosporins (cefotaxime and/or ceftazidime); MIN = minocycline; FOS = fosfomycin; GEN = gentamicin; CBP = carbapenem (ertapenem and/or imipenem); NIT = nitrofurantoin; AKN = amikacin; TGC = tigecycline.

Source: Prepared by the authors from the study results.

Nearly one-half of the isolates (n = 94; 48.9%) showed resistance to third-generation cephalosporins, with ESBLs being the main mechanism (n = 77; 81.9%), followed by AmpC (n = 12; 12.8%) and carbapenemases (n = 5; 5.3%). The ESBLs detected were CTX-M (n = 73; 94.8%), SHV (n = 3; 3.9%), and PER-2 (n = 1; 1.3%). Bla_CTX-M genes were grouped by sequence-similarity-based PCR as follow: CTX-M-2 (n = 37; 50.7%); CTX-M-9/14 (n = 21; 28.8%); CTX-M-8/25 (n = 11; 15.0%), and CTX-M-1/15 (n = 4; 5.5%). Nine of 12 isolates showing AmpC-phenotype were positive for bla_CMY-2 plasmidic-AmpC gene. Carbapenemases were detected in 5 isolates from five hospitals in four cities that had been recovered from screening (n = 2), blood (n = 2), and urine samples (n = 1). As shown in Table 2, these carbapenemases were characterized as bla_NDM-1 (n = 3) and bla_KPC-2 (n = 2). Those isolates harboring bla_NDM-1 were also positive for bla_CMY-6 variant and rmtC genes. The presence of the mcr-1.5 variant was confirmed in 26 isolates (13.5%). Other mcr-1 variants or mcr-genes (mcr-2 to mcr-9) were not evaluated.

TABLE 2. Epidemiological data of five carbapenemase-producing mcr-1 positive Enterobacterales clinical isolates.

INEI ID	Species	Date	Sample	Hosp.	Province	Genes	CIM COL	Resistance profile	ST
M17386	E. coli	May 2014	Blood	A	CABA	blaNDM-1; blaCMY-6; mcr-1	≥ 4μg/mL	3GC, CBP, GEN, AMK, COL	10
M19637	E. coli	September 2015	Blood	B	Córdoba	blaKPC-2; mcr-1	≥ 4μg/mL	3GC, CBP, CIP, MIN, SXT, COL	156
M21069	E. coli	April 2016	Screening	C	Santa Cruz	blaKPC-2; mcr-1	≥ 4μg/mL	3GC, CBP, TET, MIN, COL	5208-like
M23101	E. coli	March 2018	Screening	D	CABA	blaNDM-1; blaCMY-6; mcr-1	≥ 4μg/mL	3GC, CBP, CIP, TET, MIN, GEN, AMK, SXT, NIT, COL	354
M23335	E. coli	May 2018	Urine	E	Entre Ríos	blaNDM-1; blaCMY-6; mcr-1	≥ 4μg/mL	3GC, CBP, CIP, GEN, AMK, COL	8492


Note: Hosp = hospital; CABA = Ciudad Autonoma de Buenos Aires; 3GC = third generation cephalosporins (cefotaxime and/or ceftazidime); CBP = carbapenem (ertapenem and/or imipenem); GEN = gentamicin; AKN = amikacin; COL = colistin; CIP = ciprofloxacin; MIN, minocycline; SXT = trimethoprim-sulfamethoxazole; TET = tetracycline; NIT = nitrofurantoin; NA = not applicable; ST = sequence type. ST5208-like is a SLV of ST5208 (adk: 10; fum: 7; gyr: 265; icd: 8; mdh: 12; purA: 8; recA: 194).


Source: Prepared by the authors from the study results.

Among 110 E. coli analyzed by PFGE (57.3%), a high genetic diversity was observed defining 103 pulsotypes, while 7 isolates were repeatedly non-typeable. MLST was analyzed in the 5 carbapenemase-producing E. coli isolates identifying 5 unrelated STs: ST10, ST156, ST354, ST8492, and a SLV-ST5208 (adk: 10; fum: 7; gyr: 265; icd: 8; mdh: 12; purA: 8; recA: 194).

Mcr-1-bearing plasmids previously characterized from Argentina belonged to the IncI2 incompatibility group with ca. 60 Kb in size (26 - 28). Considering these previous results, a PCR to detect the IncI2 group was performed on all 192 isolates. A high prevalence (164/192; 85.4%) of IncI2 group was observed among the strains. In the 28 IncI2-negative isolates, the presence of IncX4 and IncHI2 incompatibility groups was evaluated, with IncX4 being detected in 18 isolates (9.4%). The remaining 10 isolates (5.2%) were negative for IncI2, IncX4, and IncHI2 incompatibility groups.

DISCUSSION

The global distribution of mcr-1 gene shows that most belong to E. coli and only a few belong to other bacterial species (7, 25). This low frequency of mcr-1 in non-E. coli species is an epidemiological characteristic of the mcr-1 gene dissemination (5, 7, 25). The prevalence of mcr-1 gene in E. coli and K. pneumoniae recovered from bloodstream infections is still low, ~1.0% and ~0.2%, respectively; nevertheless, the clinical impact of this mechanism is not fully understood (10, 26). However, in two recent global surveillance studies, a high proportion of mcr-1 gene was detected among colistin-resistant E. coli clinical isolates, ranging from 32.2% – 42.2% (3, 6). Analyzing data collected in 2012 – 2018 through the National Antimicrobial Resistance Surveillance Network WHONET-Argentina (91 hospitals), among Enterobacterales clinical isolates (excluding community onset infections), an incremental 4.3-fold in colistin resistance was observed in E. coli (0.3% to 1.3%; P < 0.0001) and 2.7-fold in K. pneumoniae (3.3% to 8.9%; P < 0.0001); but no significant difference was observed for E. cloacae (2.4% to 2.1%; P = 1). The rise of colistin resistance in hospital settings may be associated with its increased use in treating human infections caused by MDR or extensively-drug resistant Enterobacterales, particularly KPC-producing K. pneumoniae, and carbapenem-resistant Pseudomonas aeruginosa or Acinetobacter baumannii.

The present study observed a wide distribution of mcr-1 E. coli clinical isolates, most recovered from urine samples, in several provinces and Buenos Aires City. A high proportion of the isolates showed an MDR profile, and nearly one-half were resistant to third-generation cephalosporins, which reduces therapeutic options for systemic infections mainly to carbapenems, aminoglycosides, or combined therapies. The main mechanism of resistance to third-generation cephalosporins was mediated by ESBLs, with CTX-M being the more relevant, as observed by Wise and colleagues in a global analysis (3).

Infections caused by carbapenemase-producing bacteria have high morbidity and mortality rates, so colistin has become a last-resort option for treatment. In Argentina, bla_KPC-2 is the main carbapenemase among Enterobacterales; however, during recent years, an increase in detection of bla_NDM-1 has been observed, especially in Providencia spp. (14, 18, 24, 27). In the present collection, 5 carbapenemase-producing mcr-1-positive E. coli isolates were detected, yielding an extensively drug-resistance phenotype for which the unique therapeutic option was tigecycline. These 5 carbapenemase-producing E. coli isolates were assigned to unrelated STs; nevertheless, one of them was ST10, the predicted founder of clonal complex 10 (CC10). The CC10 lineage has been defined as an epidemic clone, presents intrinsic ability to acquire antimicrobial resistance genes (including mcr-1), and has been detected in human and animal samples (8). In a previous study, we reported the case of a pediatric patient infected or colonized with 5 NDM-1-producing Enterobacterales, including E. coli M17386 mcr-1 isolate (Table 2). This study provides evidence of intra-patient dissemination of a bla_NDM-1 harboring plasmid among 5 Enterobacterales species (18). Additionally, a C. amalonaticus clinical isolate—a species rarely reported to cause human infections, harboring 16 resistance genes including bla_NDM-1 and mcr-1 determinants borne on different plasmids—was recently described in our country (14). Worldwide, detection of carbapenem- and mcr-1 colistin-resistant Enterobacterales clinical isolates has also been reported, with NDM, KPC, and OXA-48 being the main enzymes described (10 - 15).

The mcr-1.5 variant was previously described only in three countries: Japan, Bolivia, and Argentina (13, 28, 29). In the present collection, 13.5% of the isolates were positive for this variant. Moreover, the mcr-1.5 variant was detected in 8 of 10 plasmids from E. coli isolates recovered from healthy chickens on commercial farms, and in 4 of 12 E. coli from diarrheic piglets and healthy fattening pigs in Argentina (30). Therefore, tracking the distribution of mcr-1.5 among E. coli isolates from food-producing animals and from humans may be a clue to understanding the dissemination of this gene among different sources.

High clonal diversity among mcr-1 E. coli isolates was observed, as it has been by other authors (2, 3), suggesting that clonal expansion is not involved in the spread of this mechanism. To date, all mcr-1 plasmids from human isolates in Argentina were characterized as a ca. 60kb IncI2 plasmid (14, 28, 31). Similarly, in the present study, a high proportion (85.4%) of IncI2 plasmids was detected. Additionally, the same IncI2 plasmids have been reported in mcr-1 E. coli isolates recovered from gulls, chicken, dogs, and pigs in Argentina (30, 32, 33). The high proportion of IncI2 plasmids among genetically diverse E. coli isolates suggests that in Argentina, these plasmids might be the main vehicle for horizontal dissemination of mcr-1 among human and animal isolates. However, finding IncX4 plasmids in 9.4% of the isolates may indicate recent changes in the genetic platforms involved in mcr-1 dissemination in our country. A different epidemiological scenario seems to occur in Brazil, where IncX4 has been the major incompatibility group reported among mcr-1 harboring plasmids, while IncA/C2 and IncHI2 are rarely reported (7, 8). Moreover, IncX4 and IncI2 were the main incompatibility groups detected in other Latin American countries as well (5, 8). Interestingly, both IncX4 and IncI2 plasmids carry mcr-1 gene as the unique determinant of resistance, while IncHI2 generally harbor multiple resistance determinants (7, 8).

The One Health approach is directed to design and implement programs, policies, legislation, and research to combat antibiotic resistance among multiple sectors, including human, animal, and environmental health. Colistin is known to be used widely to prevent infection and promote growth in food-producing animals (1, 2, 34). The use of colistin in food animals is believed to be responsible for the emergence and transmission of mcr-genes (1, 2). It has been suggested that mcr-carrying plasmids move from animals to humans, since mcr-genes are prevalent in animal food production, which is where the most colistin is consumed (1, 4, 34). According to the National Antimicrobial Resistance Surveillance (35), within the National Service for Safety and Quality of Food and Agriculture of Argentina, high levels of colistin resistance were observed in poultry (31.5%), cattle (16.5%), and pigs (15.0%). In Argentina, mcr-1 gene has been reported not only in E. coli isolates recovered from chicken and swine, but also from pets and wild birds (32, 33). This suggests that this gene is successfully circulating among different environments. Given the situation and to reserve this drug for treating human infections, in January 2019 the Ministry of Agriculture of Argentina banned the use of colistin for veterinary purposes (available from http://servicios.infoleg.gob.ar/infolegInternet/anexos/315000-319999/318811/norma.htm). This initiative aims to intensify stewardship efforts for this last-resort antibiotic.

Limitations

This study’s main limitation was that the presence of other mcr-genes (mcr-2 to mcr-9) was not evaluated, nor were mcr-genes among species other than E. coli. Even though this collection of mcr-1 E. coli isolates represents the largest and most diverse in Argentina, local and/or regional epidemiological differences are probable and expected.

Conclusions

The study findings show that mcr-1 E. coli is circulating among several provinces in Argentina. Mcr-1 E. coli is associated with MDR, with CTX-M being the main ESBL. Although infrequently, mcr-1 E. coli co-producing NDM or KPC carbapenemases have emerged. The presence of IncI2 plasmids among highly diverse E. coli clones indicates that they have driven wide distribution of the mcr-1 gene among clinical isolates through horizontal transmission.

This study provides a basic framework for understanding the molecular epidemiology of mcr-1-positive E. coli in Argentina. For comprehensive picture from the perspective of One Health, further studies are essential to understanding the dissemination of mcr-1 gene through the environment.

Funding.

This work was supported by the regular federal budget of the National Ministry of Health of Argentina and the Préstamo BID-PICT-2016-3154 to D.F. from ANPCYT. The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Disclaimer.

Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the RPSP/PAJPH and/or PAHO.