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

Abstract

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.

1. 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 (H₂S) test, are important for identification of the Enterobacteriaceae species. The production of H₂S, however, is not a typical characteristic of E. coli, though the H₂S-producing variants of E. coli have also been reported previously [2,3]. Bacteria can produce H₂S through orthologous enzymes, and recent studies have implicated H₂S as a significant signaling molecule by protecting the bacteria from antibiotic-induced damage [4]. H₂S can also prevent oxidative damage through stimulation of superoxide dismutase (SOD) and catalase activities [2,4]. Recent studies have demonstrated that H₂S can also control the expression of Staphylococcus aureus virulence genes [5]. In this study, we present the characterization of a multidrug-resistant, H₂S-producing E. coli isolated from the fecal sample from a clinically healthy patient in China.

2. 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 H₂S 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, H₂S-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 H₂S-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. Antibiotic phenotype with the corresponding resistance genes of H₂S-producing E. coli.

Classes	Antibiotics	Minimum Inhibitory Concentration (MIC) Values (mg/L)	Interpretation	Antibiotic Resistance Genes
Aminoglycosides	Gentamicin	>32	R	aadA1, aadA2
	Kanamycin	64	R
	Streptomycin	>64	R
β-Lactams	Ampicillin	>128	R	blaTEM-1B
Polymyxins	Colistin	4	R	mcr-1
Fluoroquinolones	Ciprofloxacin	2	R	oqxA, oqxB
	Nalidixic acid	64	R
Phenicols	Chloramphenicol	128	R	floR, cmlA1
Trimethoprim /Sulfonamides/	Trimethoprim/ Sulfamethoxazole	32/608	R	dfrA12, sul3
Tetracyclines	Tetracycline	>128	R	Tet(A)
Carbapenems	Imipenem	<0.5	S
	Meropenem	0.5	S
Cephalosporins	Cefotaxime	<0.5	S
	Ceftiofur	<0.5	S

R = Resistant; S = Susceptible.

.

We found H₂S-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 virulence genes found in H₂S-producing E. coli isolate.

Virulence Factors	Related Genes
Adherence:
E. coli laminin-binding fimbriae (ELF)	elfA
E. coli laminin-binding fimbriae (ELF)	elfC
E. coli laminin-binding fimbriae (ELF)	elfD
E. coli laminin-binding fimbriae (ELF)	elfG
EaeH	eaeH
Hemorrhagic E. coli pilus (HCP)	hcpA
Hemorrhagic E. coli pilus (HCP)	hcpB
Type I fimbriae	fimD
Type I fimbriae	fimF
Type I fimbriae	fimG
Type I fimbriae	fimH
Autotransporter:
Cah, AIDA-I type	cah
EhaB, AIDA-I type	ehaB
Invasion:
Invasion of brain endothelial cells (Ibes)	ibeB
Invasion of brain endothelial cells (Ibes)	ibeC
Non-LEE encoded TTSS effectors:
EspL1	espL1
EspL4	espL4
EspR1	espR1
EspR4	espR4
EspX4	espX4
Secretion system:
ACE T6SS	aec15
ACE T6SS	aec17
ACE T6SS	aec18
ACE T6SS	aec19
ACE T6SS	aec22
ACE T6SS	aec24
ACE T6SS	aec25
ACE T6SS	aec26
ACE T6SS	aec27/ clpV
ACE T6SS	aec28
Toxin:
Hemolysin/cytolysin A	hlyE/clyA
Biofilm formation:
AdeFGH efflux pump/transport autoinducer	adeG

. The screening of the H₂S-positive E. coli isolate for susceptibility to different antibiotics revealed that this H₂S-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 H₂S 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 H₂S 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 H₂S inhibition in the treatment of infections caused by E. coli. The first extensive study of H₂S-positive E. coli strains was found in Denmark [2]. Interestingly, it has been previously reported that H₂S-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 H₂S 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].

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 H₂S-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 H₂S-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 H₂S-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].

3. Conclusions

This is the first report to describe H₂S-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 H₂S 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 H₂S-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 H₂S-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 H₂S-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 H₂S production by E. coli, in both aerobic and anaerobic conditions, to understand its contribution to antibiotic resistance. As a representative case, the H₂S-producing E. coli isolate with AR genes observed in the study emphasizes the importance of rational use of antibiotics in future clinical practices.

4. Data Availability

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