Antibiotic exposure elicits the emergence of colistin- and carbapenem-resistant Escherichia coli coharboring MCR-1 and NDM-5 in a patient

Department of Neurosurgery, Tianmen First People’s Hospital, Tianmen, China; Clinical Laboratory of Lishui People’s Hospital, Lishui, China;; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, South China Agricultural University, Guangzhou, China; Department of Medical Microbiology & Parasitology and Department of General Intensive Care Unit of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China;; College of Animal Sciences, Zhejiang University, Hangzhou, China

Antimicrobial resistance (AMR) is a big challenge threatening public health worldwide. Every year, AMR-associated bacterial infections result in over 70,000 people deaths in the United States [1,2]. Worryingly, it is estimated that AMR will claim for 10,000,000 lives globally per year by 2050 [3]. The major reasons for this macabre scenario are i) The rapid appearance of new AMR determinants; ii) The global transmission of epidemic pathogens with multi-drug resistance (MDR) or even extensive-drug resistance (XDR); and iii) The failure to invent new antimicrobial agents. In general, carbapenems are used as a last line of defense antibiotics against severe infections with MDR-possessing Enteriobactericeae. Colistin, a cationic antimicrobial polypeptide, is clinically regarded as a final line of defense to treat lethal challenges with carbapenem-resistant pathogens [4,5]. Unfortunately, it seems likely that the clinical use of carbapenems and colistin has been significantly challenged by the occurrence of mobilized colistin resistance determinant (MCR-1) in clinical carbapenem-resistant isolates producing New Delhi metallo-β-lactamase 1 (NDM-1) [6,7] or its variant NDM-5 [8,9]. In addition to the intrinsic determinants of colistin resistance (e.g., EptA [10,11] and ICR-Mo [12]), the transferable determinants (MCR-1 and MCR-2 [10,13,14]) consistently encode members of phosphoethanolamine (PEA)-lipid A transferases, which adopt a 'ping-pong' catalysis reaction in transferring of the PEA moiety to the 4ʹ-phosphate position of the lipopolysaccharide (LPS)-lipid A species anchored on bacterial surface [4,5].
In the two years since its first discovery in Southern China, in late of 2015 [15], mcr-1 has been detected to coexist with bla NDM-1 -like genes in migratory birds (like the Muscovy duck [16] plus chickens [17]), in healthy people [18] and also in patients with bloodstream infections [6,7,9,19]. This indicates that the possible route of mcr-1/bla NDM -like genes is through the food-chain transmission. With the exception of Cronobacter sakazakii that carries NDM-9 [20], E. coli seems to be the predominant reservoir for the coexistence of MCR-1 and NDM-like enzymes [6][7][8][9][16][17][18][19]21,22]. Surprisingly, mcr-3, a new mcr-like gene, was found in an XDR-E. coli clinical isolate which harbored both mcr-1 and bla NDM-5 [23]. This might further highlight the importance of 'one health' approach in active surveillance of mcr-like genes in human, animal and environmental sectors. However, current knowledge on how MDR-bacterial pathogens emerge and evolve is relatively limited.
Here we report that antibiotic exposure elicits the emergence of a carbapenem-and colistin-resistant E. coli clinical isolate coharboring MCR-1 and NDM-5 in a hospitalized patient. On 3 June 2017, a 58-year-old man was admitted to the Lishui People's Hospital of Zhejiang Province, China (Figure 1(A)), and was diagnosed to be suffering from Small Intestinal Perforation with Peritonitis (SIPWP). A routine examination of this patient suggested that neither immune-suppression nor specific comorbidity were present (Table S1). While in hospital, the inflammatory markers (temperature and Leukocyte count) of this patient were normal (Figure 1(B)). The patient was subjected to anti-infection therapy through 0.5 h of intravenous guttae containing 2.0g q6h of Piperacillin/Tazobactam (TZP) prior to surgical operation. To probe the suspected bacterial agent, the specimen of peritoneal fluid from the patient (Figure 1(A)) was inoculated on a petri plate with MacConkey agar. Antibiotic susceptibility assays revealed that all of the acquired E. coli isolates (designated as S-E. coli) are sensitive to most of the antibiotics including TZP (Table S2).
Following 2-weeks of antibiotic therapy, which proved to be a clinically-successful anti-infection treatment ( Figure 1(A)), the abdominal drainage and the blood of the patient were sampled for bacterial cultivation. Although there were no E. coli isolates recovered from the patient's blood samples during the entire treatment period, a MDR-E. coli isolate was unexpectedly detected in the abdominal drainage sample (Table S2). This purulent culture was not only found to be highly-resistant to imipenem and meropenem, two widely-used carbapenems, but also it displayed an appreciable level of resistance to colistin (Table S2). Thus, it was appropriately designated as CR-E. coli LSB54 (Figure 1(A)). In addition, we favored to speculate that the LSB54 strain is a representative of a small sub-population of local microbiota on this hospitalized patient, and it was elicited by an over-exposure to antibiotics. After the evaluation of the patient's inflammatory index and the infective wound, the antibiotic treatment was immediately discontinued, but surgical debridement and drainage were resumed. As a result, the patient received a suitable course of treatment and was discharged after being hospitalized for 30 days.
To further address the purulent culture (CR-E. coli LSB54), we integrated multiple approaches ranging from microbial genetics, molecular biology, to comparative genomics. The diversity of genetic backgrounds was observed among the antibiotic-susceptible clinical strains from patients with similar durations of hospitalization (Table S3). In contrast, sequence typing showed that CR-E. coli belongs to ST2179 (Table S3). This data  The plasmid map was generated using GenomeVx [28] with automatic coloring. The plasmid map was generated using GenomeVx [28] with automatic coloring. seems to rule out the possibility that the MDR-E. coli was acquired in the hospital. Of note, a ST2179 E. coli isolate with multi-drug resistance was recently found to claim for extra-intestinal infections in horses in Brazil [24]. Therefore, it seems likely that a possible transmission route for the zoonotic agent ST2179 is present between animals and humans. As we recently described with mcr-1-bearing plasmids carried by commensal E. coli [25], whole genome sequencing was performed, giving evidence that two unique plasmids coexist in the MDR-E. coli strain LSB54 (Figure 2-4). Among the two plasmids, pLSB54-mcr-1 is the mcr-1-harboring plasmid (Figures 2  and 4(A)), and pLSB54-NDM-5 is the NDM-5-carrying plasmid (Figures 3 and 4(A)). Subsequent plasmid replicon typing (https://cge.cbs.dtu.dk/services/ PlasmidFinder/) revealed that pLSB54-mcr-1 (Acc. no.: MG773376) is a IncHI2-type plasmid with a genome size of 251.657 kb (Figure 2), whereas pLSB54-NDM-5 (Acc. no.: MG773377) is a IncX3-like plasmid whose genome is 46.19 kb long (Figure 3).
with origin of swine feces, it raised the possibility that origin of pLSB54-mcr-1 might lie in animal gut microbiota. Intriguingly, p2474-MCR1 was also detected to coexist with the bla NDM-1 -carrying plasmid in a single isolate of E. coli [6]. Like most of the known IncHI2type plasmids, pLSB54-mcr-1 is also positive for other antibiotic resistance genes (Table S4). Evidently, this is consistent with the phenotype of being resistant to multiple antibiotics which was observed with the strain LSB54 (Table S2).
Fine mapping of the genetic environment surrounding the mcr-1(blaNDM-5) gene further illustrated that i) In the IncHI2-type plasmid pLSB54mcr-1, the insertion sequence ISApl1 is located upstream of the mcr-1 gene ( Figure 5(A)) and the ISApl1-mcr-1-pap2 fragment is inserted downstream of the terF gene, just like the plasmids pHNSHP45-2 (Acc. no.: KU341381) and p2474-MCR (Acc. no.: CP021209) ( Figure 5(A)); ii) The NDM-5-containing fragment is identical in all three IncX3-like plasmids, including pLSB54-NDM-5 ( Figure 5(B)), and the bla NDM-5 is adjacent to the insertion sequence IS5, which is flanked by a truncated ISAba125 sequence ( Figure 5(B)). Unlike the scenario where MCR-1 and NDM-5 is co-transferred by a single hybrid IncX3-X4 plasmid in E. coli [8], mcr-1 and bla NDM-5 are separately encoded by two distinct plasmids in the clinical E. coli isolate LSB54 (Figure 4(A)). In this clinical case, the MDR-E. coli which possesses resistance to colistin and carbapenem (two lines of last-resort antibiotics), emerges in a patient undergoing antibiotic treatment with TZP. This observation somewhat undermines rationality of anti-infection therapies (via inappropriate usage and/or overuse of antibiotics) to some extent. Very recently, we observed an increase in the antimicrobial activity of colistin against E. coli coproducing NDM-5 and MCR-1, when it was supplied with amikacin, which could be a promising A. The genetic environment of mcr-1 and its neighboring lociB. The genetic environment of bla NDM-5 and its neighboring lociThe arrows indicate open reading frames, resistance genes, insertion sequences and accessory genes are separately shown with red, green and gray arrows.
therapeutic option for the treatment of lethal infections caused by MDR-bacterial pathogens [21].
In summary, our findings report the first clinical case that antibiotic exposure elicited the emergence of MDR-E. coli coharboring MCR-1 and NDM-5 in a patient. The fact that the co-existence of MCR-1 and NDM variants has been found in bacterial species isolated from humans, animals and environments [6,8,16,19,27], reminded us to employ the approach of 'one health' in combating potential challenges with MDR-pathogens. Given that inappropriate use of antibiotics is a potential threat for the rapid development of drug resistance and therapeutic failure, it is prerequisite to reconsider (and/or screen) the subpopulations of MDR-pathogens persisting in human microbiota prior to the formulation of appropriate antibiotic-based effective anti-infection therapies.