Potential and use of bacterial small RNAs to combat drug resistance: a systematic review

Figures & data

Table 1 Summary of the included studies

References	Methods for sRNA detection	Sample size	Small RNA	Bacterial species	Target site of sRNA	Antibiotics	Key findings
Molina-Santiago et al^Citation26	RNA sequencing genome analysis Gene ontology analysis	NA	NA	Pseudomonas putida (DOT-T1E)	NA	Chloramphenicol Rifampicin Tetracycline Ciprofloxacin Ampicillin Kanamycin Spectinomycin Gentamicin	Small RNA fine-tuned the expression of antibiotic resistance genes
Howden et al^Citation27	RNA-seq	Not mentioned	Not mentioned	Staphylococcus aureus	NA	Vancomycin Linezolid Ceftobiprole Tigecycline	Antimicrobial-responsive small RNA associated with the ribosomal functions and protein synthesis
Yu et al^Citation28	Quantitative real-time PCR (qPCR) Northern blots	3	sYJ75	Salmonella enterica serovar Typhimurium SL1344	Iron-enterobactin transporter periplasmic binding protein related genes	Tigecycline	sYJ75 regulated enterobactin transport and metabolism
Chen et al^Citation29	qRT-PCR Northern blots	≥5	sCAC610	Clostridium genus	ABC transporter genes	Clindamycin	sCAC610 adjusted downstream of ABC transporter genes and efflux pump functions
Kim et al^Citation30	DNA sequencing qRT-PCR Northern blots	3	RyeB	Escherichia coli	Muts TolC DnaK LpxL	Levofloxacin	RyeB enhanced the therapeutic effect of levofloxacin against MDR strains
Ramos et al^Citation31	Northern blots Electrophoretic mobility shift assays Western blots Reverse transcription- PCR	≥12	MtvR	Escherichia coli Pseudomonas aeruginosa	uhpA uhpT	Tetracycline Chloramphenicol Ciprofloxacin Tobramycin Gentamycin Ampicillin	MtvR increased the sensitivity of antibiotics
Liu et al^Citation35	β-lactamase assay	3	Riboswitch with theophylline-specific aptamer sequence	Escherichia coli	β-lactamase gene	Ampicillin Rifampin Carbenicillin Ciprofloxacin Erythromycin Amoxicillin Cefamandole Cefoxitin Ceftazidime Cephalexin Piperacillin Chloramphenicol Nalidixic acid Fusidic acid Gentamicin	Regulated β-lactamase gene expression to decrease the antimicrobial susceptibility
Sharma et al^Citation36	qPCR Northern blots RACE	NA	AbsR25	Acinetobacter baumannii	AIS_1331	Excluded	AbsR25 negatively regulated the efflux pump and transporter
Johnson et al^Citation37	Northern blots qRT-PCR Western blots Random mutagenesis	≥3	Anti-Q	Enterococcus faecalis	prgQ conjugation operon	Excluded	Anti-Q repressed the conjugation among bacteria
Jia et al^Citation38	5′ leader RNA sequence Native gel electrophoresis Crosslinking analysis	3	Aminoglycoside-binding riboswitch	Five aminoglycoside resistance genes sequence was used including acetyl transferase, phospho transferase, adenyl transferase, rRNA methyl transferase, and efflux pump genes	Aminoglycoside acetyl transferase (AAC) and glycoside adenyl transferase (AAD) resistance genes	Kanamycin B Sisomycin Ribostamycin Neamine	Aminoglycoside-binding riboswitch involved in encoding two aminoglycoside antibiotics degrading enzymes AAC and AAD
Baisa et al^Citation39	PCR d-alanine dehydrogenase assays	3	GcvB	Escherichia coli	cycA	D-cycloserine	GcvB involved in the repression of cycA gene which encoded an enzyme for antibiotics D-cycloserine
Knopp et al^Citation41	qRT-PCR In-gel digestion LC-MS	3	ChiX	Escherichia coli	chiP	Amikacin Ampicillin Azteronam Cefaclor Cefpirome Cefuroxime Chloramphenicol Ciporfloxacin Colistin Erythromycin Kanamycin Mecillinam Mupirocin Nalidixic acid Tetracycline Sulfamethoxazole	ChiX increased the loss of Chip porins which conferred the antibiotic resistance.
Khan et al^Citation42	Northern blots Large-scale liquid culture	3	GlmY and GlmZ	Escherichia coli	glmS	Bacilysin	GlmY/GlmZ system shaped the antibiotic resistance by reactivation of glmS enzyme in cell envelope synthesis
Chou et al^Citation43	Northern blots	3	micF encoded sRNA	Escherichia coli	ompF	Bleomycin Kanamycin	The soxRS mutations were controlled by the micF-encoded sRNA
Allen et al^Citation44	qPCR	3	small RNA regulator micF	Escherichia coli	acrD mdtA mdtB	Carbenicillin Tetracycline Kanamycin	Small RNA regulator micF was irrelevant to the carbenicillin resistance in E. coli.
Parker et al^Citation45	Northern blots Western blots Plasmid extraction	3	SdsR	Escherichia coli Vibrio cholera Pseudomonas aeruginosa	tolc	Ampicillin Novobiocin Rifampicin Erythromycin	sdsR repressed the tolC gene expression for encoding multidrug-resistance efflux pumps SdsR involved in the minimization of mismatch-repair mutation which resulted in antibiotics resistance
Eyraud et al^Citation46	Western blots Northern blots Toeprint assays	4	SprX	Staphylococcus aureus	SpoVG	Vancomycin Teicoplanin	CU-rich domain of SprX resulted in vancomycin and teicoplanin glycopeptides resistance by inactivation of stage V sporulation protein G (SpoVG)
Jeeves et al^Citation47	Transcriptomic analyses qRT-PCR	Not mentioned	G2 MTS1082	Mycobacterium tuberculosis	efpA	Excluded	Antibiotic repressed the expression of sRNAs G2 and MTS1082 over time at a slow growth rate
Jackson et al^Citation48	Microarray qRT-PCR	3	NrrF	Neisseria meningitidis Neisseria gonorrhoeae	sdhAB	Excluded	NrrF promoted mRNA degradation which encoded antibiotic efflux pump
Kim et al^Citation49	qRT-PCR	3	RyhB-1	Salmonella typhimurium	acnA sodB ftn STM1273.1n acnB	Ampicillin Norfloxacin	RyhB-1 repressed the target gene to affect the sensitivity to antibiotics

Abbreviations: LC-MS, liquid chromatography-mass spectrometry; NA, data not available; PCR, polymerase chain reaction; qRT-PCR, quantitative real-time polymerase chain reaction; RACE, rapid amplification of cDNA ends; RNA-seq, ribonucleic acid sequencing.

Figure 1 Selection of relevant scientific literature for systematic review.

Table 2 Summary of study quality assessment

References	Selection bias			Performance bias	Detection bias		Attrition bias	Reporting bias	Other	Total score
	Q1	Q2	Q3	Q4	Q5	Q6	Q7	Q8	Q9
Molina-Santiago et al^Citation26	1	1	0	0	1	1	1	1	1	7
Howden et al^Citation27	1	1	0	0	1	1	1	1	1	7
Yu et al^Citation28	1	1	0	0	0	1	1	1	1	6
Chen et al^Citation29	1	1	0	0	1	1	1	1	1	7
Kim et al^Citation30	1	1	0	0	0	1	1	1	1	6
Ramos et al^Citation31	1	1	0	0	0	1	1	1	1	6
Liu et al^Citation35	0	1	0	0	0	1	1	1	1	5
Sharma et al^Citation36	0	1	0	0	0	1	1	1	1	5
Johnson et al^Citation37	0	1	0	0	0	1	1	1	1	5
Jia et al^Citation38	0	1	0	0	0	1	1	1	1	5
Baisa et al^Citation39	1	1	0	0	1	1	1	1	1	7
Knopp et al^Citation41	1	1	0	0	0	1	1	1	1	6
Khan et al^Citation42	1	1	0	0	0	1	1	1	1	6
Chou et al^Citation43	1	1	0	0	0	1	1	1	1	6
Allen et al^Citation44	1	1	0	0	0	1	1	1	1	6
Parker et al^Citation45	1	1	0	0	0	1	1	1	1	5
Eyraud et al^Citation46	1	0	0	0	0	1	1	1	1	5
Jeeves et al^Citation47	1	1	0	0	0	1	1	1	1	6
Jackson et al^Citation48	1	1	0	0	0	1	1	1	1	6
Kim et al^Citation49	1	1	0	0	0	1	1	1	1	6

Notes: The quality of the included studies was assessed by a modified guideline for Systematic review Center for Laboratory animal Experimentation (SYRCLE) as previously reported.^28,29 Each of the nine questions (Q1–A9) received one score if the study complied with the question concerned. Q1: Was the allocation sequence adequately generated and applied? Q2: Were the groups similar at baseline or were they adjusted for confounders in the analysis? Q3: Was the allocation adequately concealed? Q4: Were the caregivers and/or investigators blinded from knowledge of which intervention each experimental group received during the experiment? Q5: Were the experimental groups selected at random for outcome assessment? Q6: Was the outcome assessor blinded? Q7: Were incomplete outcome data adequately addressed? Q8: Are reports of the study free of selective outcome reporting? Q9: Was the study apparently free of other problems that could result in high risk of bias?

Figure 2 Small bacterial RNAs interact with canonical (in blue) and unknown mechanisms (in red) in acquisition of antimicrobial resistance phenotype. The pointed arrowheads denote putative stimulatory effect whereas the blunted arrowheads represent inhibitory actions.

Figure 3 The biological action of small bacterial RNAs. These molecules are classified into cis- and trans-encoded sRNAs. By binding to non-coding region of mRNA or a gene per se, sRNAs either activate or repress the expression of a given protein.

Abbreviation: RBS, RNA binding site.

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