Highly Colistin-resistant Aeromonas jandaei from a Human Blood Sample

Aeromonas species are Gram-negative rods known to cause infections such as gastroenteritis, bacteremia and wound infections. Colistin is one of few treatments for multidrug-resistant Gram-negative bacteria. However, colistin-resistant bacteria carrying the mobilized colistin resistance (mcr) gene are a threat in healthcare settings worldwide. In recent years, colistin-resistant Aeromonas species have been detected in environmental and clinical samples. We analyzed the genomic characteristics of one highly colistin-resistant A. jandaei isolated from a blood sample in Nepal, which harbored four novel mcr-like genes on its chromosome. Our study strongly suggests that A. jandaei is a reservoir of colistin-resistant genes. Inappropriate use of drugs in medicine and food production should be reduced and continued global surveillance for colistin-resistant bacteria is necessary.


Taxonomy of the Aeromonas genus
The Aeromonas genus are Gram-negative, straight, rigid, nonsporeforming rods which belong to the family Aeromonadaceae.They are facultatively anaerobic and widely distributed in aquatic environments and food samples.They are frequently isolated from drinking water, wastewater, seawater, livestock, vegetables, seafood and fish 1) .Although not generally considered marine organisms, they grow naturally in marine systems in contact with freshwater and are found at all salinities except extreme 2) .The etymology of the word "Aeromonas" is that "Aero" means "gas(-producing)" and "monas" means "monad."The word "monad" is derived from the Greek word "monos," meaning "one."Type species of the genus is A. hydrophila, and "hydrophila" means "water-loving."Historically, species in this genus were classified in the family Pseudomonadaceae or the family Vibrionaceae [3][4] . Ater the analysis of 16S ribosomal RNA of the Ganmamaproteobacteria, the Aeromonas genus became independent from the family Vibrionaceae and the family Aeromonadaceae was founded in 1992 5) .
A Phylogenetic dendrogram of the major type strains of the family Aeromonadaceae and clinically important Gram-negative rods is shown (Figure 1).and Vibrio parahaemmolyticus cause gastrointestinal symptoms, and Vibrio vulnificus causes wound infections, especially necrotizing soft tissue infection.Similarly, 19 of 36 Aeromonas species are pathogenic to humans, causing a broad spectrum of infections including gastroenteritis, bacteremia, and wound infections 6) .A. jandaei, named after J. Michael Janda, was originally isolated from clinical samples including blood, wounds, and stools in the USA in 1991 7) .

Emergence of mobilized colistin resistance (MCR)
The emergence of bacterial antimicrobial resistance (AMR) is a widespread problem.According to a report by the UK government, 10 million people could be killed by AMR every year 8) .In this context, colistin is once again gaining attention as a "last resort" antimicrobial agent against infections with multidrug-resistant Gram-negative bacteria including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii, by interacting with lipid A to disrupt the outer membrane of Gram-negative bacteria 9) .
Colistin was discovered from Bacillus polymyxa var.colistinus, which was from soil of Fukushima, Japan in 1947 and approved by the U.S. Food and Drug Administration (FDA) in 1959 10) .However, due to its kidney toxicity, colistin has long been restricted for use in humans.On the other hand, it is used in livestock.As a result, colistin resistant Escherichia coli and Klebsiella pneumoniae encoding the mobilized colistin resistance gene mcr-1 on a plasmid were discovered in livestock and humans in China in 2015 11) .] .Transmission of mcrpositive bacteria may occur by contact with reservoirs of mcr, ingestion of products associated with contaminated animals or plants, and international food trade and travel 13) .A meta-analysis of colistinresistant E. coli estimated mcr prevalence rates of 15.8%, 14.9%, 7.4%, and 4.2% among chickens, pigs, healthy humans, and clinical isolates, respectively 14) .In Southeast Asia, colistin use as an antibiotic in livestock was so routine that in one rural village survey in Vietnam in 2017, 29 of 36 households tested had colistin-resistant E. coli harboring mcr-1 or mcr-3 15) .

Emergence of colistin-resistant Aeromonas jandaei
Previous studies have reported that mcr related genes were detected in environments, animals, and clinical samples of Aeromonas spp.including A. allosaccharophila, A. bivalvium, A. caviae, A. hydrophila, A. jandaei, Aeromonas media, A. salmonicida and A. veronii [16][17][18][19][20] . Acording to the U.S. Department of Agriculture (USDA) study of mcr prevalence in 2018, 15 of 5,169 samples were positive for mcr: one for E. coli, nine for A. hydrophila, and five for A. jandaei.Of these, all the Aeromonas species harbored mcr-3-like genes, but only A. jandaei harbored mcr-7-like gene 16) .An A. jandaei strain isolated from retail fish in China harbored two genes encoding phosphoethanolamine transferase eptAv3 and eptAv7 similar to mcr-3 and mcr-7, respectively 21) .These findings suggest that Aeromonas spp. was a reservoir of colistin-resistant Gram-negative bacteria.

Discussion and Conclusion
This is the first report of a highly colistin-resistant A. jandaei JUNP479 with four novel genes encoding MCR-like phosphoethanolamine transferases isolated in a medical setting in Nepal.Infections caused by A. jandaei are less common than those caused A. caviae, A. veronii, A. hydrophila and A. dhakensis in clinical situations [8][9] . Hwever, when the genomic environments were compared in this study, it turned out that other Aeromonas spp.also harbored colistin-resistant genes.Moreover, mcr-3-like genes are also commonly found in Aeromonas spp.and E. coli, suggesting the mcr genes may transmit between Aeromonas spp.and Enterobacteriaceae.On the other hand, mcr-7-like genes will mainly spread among Aeromonas spp.Our study strongly suggests that Aeromonas spp. is a reservoir of colistin-resistant genes, including mcr-3-like genes and mcr-7-like genes.Aeromonas spp.have the possibility to become a threat in healthcare settings, therefore, it is necessary to continue global surveillance of colistin-resistant Aeromonas spp.If we humans continue to routinely use colistin in livestock, agriculture, aquaculture, and medicine, we could lose our "last defense" against multidrugresistant Gram-negative bacteria.

Figure 1
Figure 1 Phylogenetic dendrogram of 16S rRNA of the Gram-negative rods including the family Aeromonadaceae.This Figure is created using the CLUSTAL OMEGA program (https://www.ebi.ac.uk/Tools/msa/clustalo/). GenBank accession numbers were indicated in parenthesis.