Colistin Resistance (cid:0) Enterobacteriaceae Isolated from Arthropods in Gifu City, Japan

The emergence of antimicrobial-resistant (AMR) bacteria is an important concern for public and livestock health. Arthropods may serve as vectors that disseminate AMR bacteria across different environments. We examined the phenotype and genotype of antimicrobial resistance in Enterobacteriaceae collected from arthropods in Gifu city, Japan. A total of 186 Enterobacteriaceae from 94 arthropods were obtained and tested for antimicrobial susceptibility. All isolates were susceptible to the antimicrobial agents tested, except for colistin (39 isolates) and kanamycin (one isolate). The aph(3')-Ia gene and amino acid substitutions in the two-component system were responsible for the kanamycin and colistin resistance, respectively.


Conclusion
Overall Enterobacteriaceae isolated from the arthropods were susceptible to most of the antimicrobial agents. However, a high prevalence of colistin resistance was observed in the isolates from the arthropods. We suspect that this was a result of the production of antimicrobial peptides by the arthropods rather than selective pressure or exposure to colistin in the environment. Thus, arthropods maybe a potential reservoir of colistin resistant bacteria. These ndings could be bene cial to public and livestock health management.

Background
The wide occurrence of antimicrobial-resistant (AMR) bacteria in various elds such as medicine, veterinary, and agriculture is of great concern and necessitates investigation into emerging AMR bacteria in various environments. Arthropods are a group of ubiquitous creatures including Hexapoda (insects in a broad sense), Crustacea such as pill bugs, Chelicerata such as spiders and Myriapoda such as centipedes that interact extensively with their environment [1]. They generally harbor complex microbial communities, and both gram-negative (Enterobacter spp., Klebsiella spp.) and gram-positive bacteria (Enterococcus spp., Staphylococcus spp.) resistant to antimicrobial agents have been isolated from them [2].
Colistin (CST) is used as a last resort for the treatment of multi-drug-resistant gram-negative bacteria.
The initial mode of action for CST is to attach to a negatively charged component of the lipopolysaccharides (LPS) found in gram-negative bacteria cell membranes. It competitively displaces divalent cations (Ca 2+ and Mg 2+ ) from the phosphate groups of membrane lipids, resulting in the disruption of the outer cell membrane that leads to a leakage of intracellular contents and bacterial cell death [3]. Gram-negative bacteria use different strategies, including genetic mutations in the PhoPQ/PmrAB two-component system and acquisition of the plasmidic mobile colistin resistant gene (mcr), to achieve modi cation of the LPS [4]. The mutations in the PhoPQ/PmrAB two-component system result in the reduction of the binding a nity of CST through the addition of either phosphoethanolamine or 4-amino-4-deoxy-L-arbinose to the lipid A moiety of the LPS which increases the net positive charge, whereas the mcr can only lead to addition of phosphoethanolamine to the LPS resulting in an increase in net positive charge but this can confer high CST resistance [5].
Arthropods produce antimicrobial peptides to perform various functions including prevention of bacterial infections and mediating symbiotic relationships between the host and bene cial bacteria [6]. It could therefore be expected that the symbiotic bacteria of arthropods have developed resistance to antimicrobial peptides. Antimicrobial peptides exhibit a similar mode of action to CST against gramnegative bacteria as described above. The difference is that antimicrobial peptides can also translocate into the cytoplasm and interrupt essential intracellular processes resulting in bacteria death [7].
Arthropods may serve as vectors that disseminate AMR bacteria across different environments. For example, house ies have been associated with the maintenance and dissemination of cephalosporin and colistin resistant Enterobacteriaceae [8]. In another study, multi-drug-resistant bacterial species were isolated from cockroaches [9]. Enterobacteriaceae are prevalent in the intestinal tract of humans and animals [10]. Members of this family have been opportunistically implicated in blood-stream, intraabdominal, skin, soft-tissue, and urinary tract infections [11]. We examined the phenotype and genotype of antimicrobial resistance in Enterobacteriaceae from arthropods in order to better understand the emergence of AMR bacteria in the environment.

Materials And Methods
A total of 94 arthropods comprising Hexapoda (n = 49), Myriapoda (n = 32), Chelicerata (n = 10), and Crustacea (n = 3) were collected at Gifu university and Mount Kinka in Gifu city, Japan between 2016 and 2018 (Table 1). Flying and walking arthropods were caught using net traps and forceps, respectively, then euthanized using carbon dioxide in separate bags [12] and were returned to the laboratory for bacterial isolation from the arthropods' whole body. Bacterial isolation was performed using deoxycholate hydrogen sul de lactose agar medium and incubated at 37 ºC overnight. API20E (BioMerieux Tokyo, Japan) was used for bacterial identi cation. The minimum inhibitory concentration was determined using frozen plates (Eiken Chemical Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions.

CST-colistin
The mcr-1-5 genes were not detected in any of the CST-resistant isolates. The absence of mcr-1-5 gene in the CST-resistant isolates observed in this study prompted us to investigate the PhoPQ/PmrAB twocomponent system. We examined amino acid substitution in the PhoPQ/PmrAB two-component system in CST-resistant Enterobacter isolates and compared them to those of CST-susceptible Enterobacter strains. Our investigation showed various amino acid substitutions: three amino acid substitution in phoP (L129I, F141L, H207Q), ve in phoQ (V102I, L133I, M298L, S448A, G464S), four in pmrA (A19G, S21A, N89T, L146Q) and four in pmrB (H132S, A172T, Q271V, R276Q). The amino acid substitutions observed in phoP, phoQ, and pmrA in this study corresponded with those reported by Uechi et al. [25]. On the other hand, the amino acid substitutions observed in this study were different from those reported in another study by Nawfal Dagher et al. [26]. We could not clarify the speci c amino acid substitution responsible for the CST resistance observed. Arthropods rarely have direct contact with CST because of restrictions on CST usage for humans and pigs in Japan. Hence, the observed CST resistance was unexpected and as such it is not plausible to attribute this CST resistance to selective pressure or exposure to CST in the environment. Most arthropods have antimicrobial peptides in their hemolymph that serve as a defense mechanism and plays an important role in fostering symbiotic relationships with bene cial bacteria [6]. It therefore stands to reason that symbiotic bacteria may have developed resistance to antimicrobial peptides. The initial mode of action of antimicrobial peptides and CST against gram-negative bacteria involves binding to the LPS [6], this may have led to the development of cross-resistance to CST [27].
One isolate of K. oxytoca from a butter y in this study showed resistance to KAN. WGS analysis revealed that the resistance gene aph(3')-Ia was located on the chromosome of K. oxytoca. The aph(3′)-Ia gene was rst discovered on transposon Tn903 in Escherichia coli [28] and has subsequently been found on plasmids and chromosomes of clinical and veterinary Enterobacteriaceae isolates [29][30]. The presence of aph(3')-Ia in arthropods may suggest the possibility that arthropods received this bacterium from human and/or domestic animals or vice versa.
In previous studies, Enterobacteriaceae isolated from cockroaches showed resistance to more than two antimicrobial agents [9,31]. In Japan, Enterobacter spp. isolated from companion animals showed resistance to CTX (33.3%), GM (23.3%), TC (40%), CPFX (43.3%), and CP (46.7%) and were reported to be extended spectrum beta-lactamase (ESBL) producers [32]. In addition, Klebsiella spp. isolated from companion animals were resistant to aminoglycosides and quinolones and were reported to be extended beta-lactamase producers [33]. However, susceptibility to antimicrobial agents were high, as expected in this study. The high susceptibility observed indicate an absence or low prevalence of AMR bacteria and minimal antimicrobial agents' pollution in the immediate environment of the arthropods investigated.
The present study did not compare the isolation rate of bacteria on the external surface and alimentary tract of the arthropods. However, there was no signi cant difference between the isolation rate of bacteria on the external surface and the alimentary tract of cockroaches in a previous report [9].

Conclusion
In conclusion, the Enterobacteriaceae isolated from the arthropods were susceptible to most of the antimicrobial agents. However, high prevalence of CST resistance was observed in the isolates from the arthropods. We suspect that this was a result of the production of antimicrobial peptides by the arthropods rather than selective pressure or exposure to CST in the environment. These ndings could be bene cial to public and livestock health management as well as present novel approaches to exploring bacterial adaptations to its environment and the impact this has on the larger ecosystem. MY and MS performed sample collection and laboratory work. JOO processed and analyzed sequence data. JOO and TA wrote the manuscript. All authors read and approved the manuscript.