MCR-5-Producing Colistin-Resistant Cupriavidus gilardii Strain from Well Water in Batna, Algeria

ABSTRACT This paper presents the first description of the mcr-5.1 gene in a colistin-resistant Cupriavidus gilardii isolate from well water that supplies a maternity hospital in Algeria. The whole-genome sequence of this strain showed the presence of putative β-lactamase, aac(3)-IVa, and multidrug efflux pump-encoding genes, which could explain the observed multidrug resistance phenotype. Our findings are of great interest, as we highlight a potential contamination route for the spread of mcr genes. IMPORTANCE Colistin resistance mediated by mcr genes in Gram-negative bacteria has gained significant attention worldwide. This is due to the ability of these genes to be horizontally transferred between different bacterial genera and species. Aquatic environments have been suggested to play an important role in the emergence and spread of this resistance mechanism. Here, we describe the first report of an mcr-5-positive Cupriavidus gilardii aquatic isolate through its isolation from well water in Algeria. The significance of our study is in shedding the light on an important environmental reservoir of mcr genes.

IMPORTANCE Colistin resistance mediated by mcr genes in Gram-negative bacteria has gained significant attention worldwide. This is due to the ability of these genes to be horizontally transferred between different bacterial genera and species. Aquatic environments have been suggested to play an important role in the emergence and spread of this resistance mechanism. Here, we describe the first report of an mcr-5-positive Cupriavidus gilardii aquatic isolate through its isolation from well water in Algeria. The significance of our study is in shedding the light on an important environmental reservoir of mcr genes.
KEYWORDS Cupriavidus gilardii, mcr-5, colistin resistance, groundwater, Algeria S ince the first detection of the plasmid-mediated colistin resistance mechanism in December 2015, 10 mcr genes and several variants have been identified worldwide from different sources (1,2). Being transferable, this mechanism has received more attention than any of the colistin resistance mechanisms previously described. Indeed, the origin of this mechanism has long preoccupied researchers, and different studies have suggested an environmental origin, particularly an aquatic one (3)(4)(5), which could participate significantly in its dissemination to pathogenic bacteria. Likewise, aquatic environments can act as an important vehicle for the spread of such resistance mechanisms to humans either in the community or, more worryingly, in hospital settings.
In this paper, we present the first report of the mcr-5 gene in an unusual bacterial isolate, Cupriavidus gilardii, recovered from well water that supplies a maternity hospital in the Batna province, Algeria.
During September and October 2019, 38 water samples were obtained from a maternity hospital in Batna city, Algeria. The hospital is located in an urban region where no agricultural activity is near the study site. One liter of water was collected in sterile glass bottles from the well that supplies the hospital with tap water, from water tanks, and from taps with the hospital's various wards. Each water sample was filtered through a cellulose membrane (0.45 mm pore size), and the filter was placed on a MacConkey agar plate (HiMedia, India). Plates were incubated overnight aerobically at 37°C. Cultures were purified and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (6). Thereafter, isolates were screened by real-time PCR for the occurrence of mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, and mcr-8 genes as previously described (7,8). The mcr-5 gene was detected in one isolate (strain Q4897) which was identified as Cupriavidus gilardii. Cupriavidus gilardii is a glucose-nonfermenting Gram-negative bacterium (GNB) that belongs to the Burkholderiaceae family. It was previously classified as Ralstonia gilardii and Wautersia gilardii (9). The gene was fully amplified by standard PCR and sequenced using BigDye terminator chemistry on an ABI 3500xl automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequence analysis confirmed an mcr-5.1 variant.
The mcr-5-positive isolate was examined for its susceptibility to antibiotics using the disc diffusion method, and inhibition zone diameters were interpreted according to the antibiotic committee of the French Society for Microbiology (Société Française de Microbiologie) breakpoints (https://www.sfm-microbiologie.org/wp-content/uploads/2020/04/CASFM2020 _Avril2020_V1.1.pdf). In addition, the colistin MIC was determined using the broth microdilution (BMD) method. Our isolate was resistant to ticarcillin, ticarcillin-clavulanate, aztreonam, ertapenem, meropenem, imipenem, gentamicin, fosfomycin, rifampin, and colistin (MIC = 8 mg/ml). The isolate was negative for carbapenemase production using the b-CARBA test (Bio-Rad, Marnes-la-Coquette, France). For whole-genome sequencing (WGS), genomic DNA was extracted using the EZ1 biorobot with the EZ1 DNA tissue kit (Qiagen, Hilden, Germany) and then sequenced on a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) with the Nextera Mate Pair sample preparation kit and Nextera XT Paired End (Illumina). The assembly was performed using a Shovill pipeline (https://github.com/ tseemann/shovill). Scaffolds of ,800 bp and scaffolds with a depth value lower than 25% of the mean depth were removed. The assembly generated 66 contigs with a total length of 5,335,421 bp and a GC content of 67.3%. The occurrence of antibiotic resistance genes was investigated through the ABRicate function of the Galaxy web platform (https:// usegalaxy.org.au/) using ARG-ANNOT, NCBI, CARD, and ResFinder as reference databases with minimum of 70% for identity and coverage. All detected hits are presented in Table 1. In addition to the mcr-5.1 colistin resistance gene, we identified a class D b-lactamase which was highly similar (90.84% similarity with the reference sequence) to the OXA-837 enzyme and a putative aminoglycoside inactivation enzyme, "aac(3)-IVa." Interestingly, these two antibiotic resistance genes have been found to be well conserved in C. gilardii genomes (9). Furthermore, several conserved multidrug efflux pumps were detected, which could explain the multidrug resistance phenotype observed in our isolate.
In parallel, the mcr-5 protein reference sequence (WP_053821788.1) from Proteobacteria was used to query its presence in all available complete and WGS genomes of Cupriavidus from the NCBI database. The in silico analysis showed that, of the 127 Cupriavidus genomes, five mcr-5 chromosomic sequences (4% of analyzed genomes) exhibited an identity value at 100% and 100% alignment with the reference sequence. Indeed, it has been suggested that the mcr-5 gene might have been transferred from environmental C. gilardii to Salmonella enterica (10); nevertheless, this gene was identified only in three out of the eight available C. gilardii genomes (Table 2) and in two genomes of Cupriavidus sp. However, we do not know the susceptibility of these strains to colistin, which could have provided us with more information on the resistance mechanism. In addition, Easyfig v2.2.5 software was used to investigate the genetic environment surrounding the mcr-5 gene from the five selected genomes as well as from our isolate (Fig. 1).
Our mcr-5-positive isolate was recovered from the well supplying the hospital with tap water. Except for drinking, this water is used in all applications requiring water use in the hospital, including cooking, bathing of newborns, cleaning, and hand washing. It is worth mentioning that well water is directly used without any treatment.
The mcr-5 gene was first described in Salmonella enterica subsp. enterica serovar Paratyphi B var. Java dTa1 from Germany, where the authors confirmed that the mcr-5 gene was located on a 7,337-bp Tn3-family transposon harbored by a ColE-type  plasmid (10). Interestingly, by using BLASTn search a Tn3-family transposon harboring the mcr-5 gene was also detected in chromosome 1 of a C. gilardii strain (CR3) recovered in the United States (10).
mcr variants have been previously detected in aquatic environments. mcr-5 and mcr-5.4 have been detected by culture-independent methods in a wastewater treatment plant in Germany and in hospital tap water in the Netherlands, respectively (11,12). In addition, the mcr-5 gene has been detected in an Enterobacter sp. isolated from hospital sewage in China (13), and an MCR-5.3-producing Stenotrophomonas sp. has been isolated from animal waste in China (14). Recently, mcr-5 has been detected in a Cupriavidus sp. closely related to C. gilardii isolated from the blood of an immunocompromised patient in South Korea (15). Members of the Cupriavidus genius are known for their resistance to copper and other metals. This might be due to the presence of several metal resistance loci such as cop genes, as shown in Fig. 1.
C. gilardii is gaining increasing attention as an emerging pathogen, and several studies have reported its role in human infections, including perirectal inflammation, bloodstream infection, muscular abscess, and catheter sepsis (15). In terms of antibiotic resistance, it has been suggested that C. gilardii is intrinsically resistant to ertapenem, meropenem, ampicillin, amoxicillin-clavulanate, gentamicin, tobramycin, and streptomycin, while it is susceptible to imipenem and cefotaxime and intermediately resistant to spectinomycin (9). In a study carried out on 39 Cupriavidus clinical isolates, including six C. gilardii strains, the authors tested the MICs of these strains against 20 antibiotics by BMD, and the results showed that two C. gilardii strains were resistant to colistin and four were imipenem resistant. However, the resistance mechanisms were not characterized (16).
Our findings are of great interest, as we present here a potential route for the spread of such resistant organisms in the community, where further investigations and actions are required in order to contain this problem.
Data availability. This whole-genome sequence has been deposited at GenBank under accession no. JAGFTW000000000.