Genomic Characterization of New Variant of Hydrogen Sulfide (H2S)-Producing Escherichia coli with Multidrug Resistance Properties Carrying the mcr-1 Gene in China

Colistin is considered to be a ‘last-resort’ antimicrobial for the treatment of multidrug-resistant Gram-negative bacterial infections. Identification of Enterobacteriaceae, carrying the transferable colistin resistance gene mcr-1, has recently provoked a global health concern. This report presents the first detection of a hydrogen sulfide (H2S)-producing Escherichia coli variant isolated from a human in China, with multidrug resistance (MDR) properties, including colistin resistance by the mcr-1 gene, which could have great implications for the treatment of human infections.


Introduction
Escherichia coli is a significant cause of diseases in animals and humans worldwide [1], resulting in diverse community and hospital acquired infections, with major clinical concerns. Specific biochemical examinations, including the hydrogen sulfide (H 2 S) test, are important for identification of the Enterobacteriaceae species. The production of H 2 S, however, is not a typical characteristic of E. coli, though the H 2 S-producing variants of E. coli have also been reported previously [2,3]. Bacteria can produce H 2 S through orthologous enzymes, and recent studies have implicated H 2 S as a significant signaling molecule by protecting the bacteria from antibiotic-induced damage [4]. H 2 S can also prevent oxidative damage through stimulation of superoxide dismutase (SOD) and catalase activities [2,4]. Recent studies have demonstrated that H 2 S can also control the expression of Staphylococcus aureus virulence genes [5]. In this study, we present the characterization of a multidrug-resistant, H 2 S-producing E. coli isolated from the fecal sample from a clinically healthy patient in China.

Case Study
An active epidemiological surveillance study for foodborne pathogens was conducted towards healthy and diarrheal patients in Guangxi province, China. The initial aim was to screen Salmonella in the human fecal samples; we suspected this sample as Salmonella, and found this isolate was a lactose fermenter and H 2 S producer, according to a previous protocol [6]. To confirm whether this isolate was Salmonella or E. coli, we plated the sample on eosin methylene blue agar, and then confirmed the results with PCR identification and whole genome sequencing. Together, this is one isolate of interest, H 2 S-producing E. coli isolated from a 32-year old female from Guangxi province, China, during occupational health examination in 2015.
The isolate was sequenced using the MiSeq platform (Illumina Inc., San Diego, CA, USA), utilizing either 500 or 600 cycles of paired-end reads. The de novo assembly, using SPAdes 3.6, resulted in a genome size of 493,599 bp with GC content of 52.1%. The genome was annotated using the Rapid Annotation using Subsystem Technology (RAST) annotation server, and 1730 coding sequences (CDS) were identified. Detection of resistance genes and multilocus sequence typing (MLST) was accomplished at the Center for Genomic Epidemiology (CGE) (https://cge.cbs.dtu.dk/services/). We used the virulence factor database (VFDB) to obtain the virulence genes in this H 2 S-positive E. coli isolate. We performed antimicrobial susceptibility testing of the E. coli isolate using the broth microdilution method, as per the Clinical and Laboratory Standards Institute (CLSI) criteria [7]. The antimicrobials used are described in Table 1. Table 1. Antibiotic phenotype with the corresponding resistance genes of H 2 S-producing E. coli.

Minimum Inhibitory Concentration (MIC) Values (mg/L)
We found H 2 S-producing E. coli belonged to sequence type (ST) 10, serotype O10:H19, fimH25-fumC11 type. The typical virulence genes found in this E. coli isolate are shown in Table 2. The screening of the H 2 S-positive E. coli isolate for susceptibility to different antibiotics revealed that this H 2 S-positive variant was resistant to aminoglycosides, β-Lactams, polymyxins, fluoroquinolones, phenicols, sulfonamides, tetracyclines, and trimethoprim. Genome analysis revealed that this isolate also carried 3-mercaptopyruvate sulfurtransferase (sseA), indicating for the H 2 S production [4], and multiple antibiotic resistance (AR) genes. The conjugation assay confirmed both sseA and the mcr gene were on the chromosome. Table 1 shows the presence of AR genes for different antibiotics. Our study findings are clinically significant, highlighting the role of H 2 S as a microbial defense mechanism, revealing resistance against different clinically relevant antibiotics, including the 'last-line' therapeutic drug colistin, and also suggests the need of bacterial H 2 S inhibition in the treatment of infections caused by E. coli. The first extensive study of H 2 S-positive E. coli strains was found in Denmark [2]. Interestingly, it has been previously reported that H 2 S-generating enzymes (sseA in E. coli), especially, as mentioned, provided defense against antimicrobial compounds only in aerobic conditions [4]. The interesting point is that the cytoprotective effect of H 2 S is a universal defense mechanism found in bacteria as well as in mammals [2,4]. Moreover, the sequence type (ST) 10 E. coli strain is one of the predominant STs in the world [8]. Cah, AIDA-I type cah EhaB, AIDA-I type ehaB Invasion:

EspL1 espL1
EspL4 espL4 EspR1 espR1 EspR4 espR4 EspX4 espX4 AdeFGH efflux pump/transport autoinducer adeG We found aminoglycosides resistance genes aadA1, aadA2, trimethoprim resistance gene dfrA12, β-Lactams resistance gene bla TEM-1B , polymyxins resistance gene mcr-1, fluoroquinolones resistance genes oqxA, oqxB, phenicols resistance genes floR, cmlA1, sulfonamides resistance gene sul3, and tetracyclines resistance gene tet(A) in the H 2 S-positive E. coli isolate. The isolate was susceptible to carbapenems and cephalosporins ( Table 1). The study by Jones et al. [3] and Harnett et al. [9] demonstrated previously that an H 2 S-producing variant of Escherichia coli isolated from a urinary tract infection (UTI) was also found to be resistant to different clinically relevant antibiotics. A previous study by Bailey et al. [1] reported the presence of dfrA12, sul3, tet(A), and cmlA1, including other AR genes in E. coli of healthy adults. It is interesting that E. coli of healthy humans represented a significant reservoir for several AR genes, as found in our study. The presence of aadA1, aadA2, and dfrA12 genes were also reported previously in E. coli isolated from clinical samples in Malaysia [10]. Since the first report of colistin-resistant E. coli carrying the mcr-1-gene in China in 2016 [11], the existence and prevalence of the mcr gene and their variants has been reported in the E.coli across different continents. The bacterial cell membrane is the initial site of action for colistin. Colistin binds to lipopolysaccharide (LPSs) and phospholipids in the outer cell membrane of Gram-negative bacteria [12]. Colistin resistance facilitated by the mobile mcr-1 gene has raised concerns during the last few years [13,14]. Fluoroquinolones (FQs), such as ciprofloxacin, have been the most commonly used antibiotics to treat UTIs caused by E. coli. However, the extensive use of fluoroquinolones has led to increasing fluoroquinolone resistance. The genes for multidrug efflux pump OqxAB, which are active on fluoroquinolones, were found for the first time in clinical isolates on a plasmid in E. coli in the USA in 2009 [15]. A recent study demonstrated the prevalence of plasmid-mediated quinolone resistance genes oqxA and oqxB, including other genes in clinical isolates of E. coli, obtained from UTIs in Azerbaijan and Iran [16]. Recently, bla TEM-1B and tet(A) were found, including other AR genes in E. coli isolated from a patient in Lebanon, and linked to a bloodstream infection. Interestingly, previous studies reported that among sul1, sul2, and sul3 genes responsible for sulfonamide resistance, both sul1 and sul2 are highly prevalent, and sul3 has rarely been found [17,18]. Therefore, the presence of a rare sulfonamides resistance gene, sul3, could be an interesting characteristic of this H 2 S-producing E. coli strain. Antibiotic resistance genes found in this study were also reported previously in E. coli isolated from humans in various studies in Australia [19], Argentina [20], Tunisia [21], Croatia [22], Sweden [23], Spain [24], Bolivia and Peru [25], Algeria [26], Nigeria [27], and Lithuania [28].

Conclusions
This is the first report to describe H 2 S-producing colistin-resistant E. coli carrying the mcr-1 gene, which also possesses the rare sulfonamide resistance gene sul3. The emergence and spread of the colistin resistance gene mcr-1 in E. coli has attracted considerable attention worldwide. As endogenous H 2 S reduces the efficacy of many clinically used antimicrobials, the inhibition of this gas should be considered an effective therapy against a wide range of bacteria, including pathogenic E. coli. Continuous surveillance and molecular characterization of H 2 S-producing mcr-carrying E. coli is needed to shed light upon all of the transmission pathways. It is important to strengthen the hygiene practices in the hospital to reduce the environmental contamination by H 2 S-producing MDR E. coli. Our results require future extensive study and follow-up evaluations in order to understand the trends of the AR gene's epidemiology in H 2 S-producing E. coli, in clinical settings and in the community, with time, and ultimately anticipate the detection of bacteria that can possibly cause serious public health concerns. In the future, it would be an interesting study to determine the H 2 S production by E. coli, in both aerobic and anaerobic conditions, to understand its contribution to antibiotic resistance. As a representative case, the H 2 S-producing E. coli isolate with AR genes observed in the study emphasizes the importance of rational use of antibiotics in future clinical practices.

Data Availability
Raw sequencing reads have been deposited in the NCBI BioProject database under accession number PRJNA576077.