Antimicrobial resistance, virulence & plasmid profiles among clinical isolates of Shigella serogroups.

Background & objectives: Bacillary dysentery caused by Shigella spp. remains an important cause of the crisis in low-income countries. It has been observed that Shigella species have become increasingly resistant to most widely used antimicrobials. In this study, the antimicrobial resistance, virulence and plasmid profile of clinical isolates of Shigella species were determined.
Methods: Sixty clinical Shigella isolates were subjected to whole-genome sequencing using Ion Torrent platform and the genome sequences were analyzed for the presence of acquired resistance genes, virulence genes and plasmids using web-based software tools.
Results: Genome analysis revealed more resistance genes in Shigella flexneri than in other serogroups. Among β-lactamases, blaOXA-1was predominantly seen followed by the blaTEM-1B and blaEC genes. For quinolone resistance, the qnr S gene was widely seen. Novel mutations in gyr B, par C and par E genes were observed. Cephalosporins resistance gene, blaCTX-M-15 was identified and plasmid-mediated AmpC β-lactamases genes were found among the isolates. Further, a co-trimoxazole resistance gene was identified in most of the isolates studied. Virulence genes such as ipaD, ipaH, virF, senB, iha, capU, lpfA, sigA, pic, sepA, celb and gad were identified. Plasmid analysis revealed that the IncFII was the most commonly seen plasmid type in the isolates. Interpretation & conclusions: The presence of quinolone and cephalosporin resistance genes in Shigella serogroups has serious implications for the further spread of this resistance to other enteric pathogens or commensal organisms. This suggests the need for continuous surveillance to understand the epidemiology of the resistance.

Shigella is an important cause of diarrhoea, particularly in children less than five years of age. Shigella spp. is highly contagious due to its low infective dose and high transmission rate in areas with overcrowding and poor sanitary conditions1. Depending on the virulence potential of the strain and the nutritional status of the individual, shigellosis can progress to severe disease2. The Global Enteric Multicenter Study, a case-control study of moderate-to-severe paediatric diarrhoeal disease, identified enterotoxigenic Escherichia coli and Shigella spp. as the most common bacterial pathogens in Sub-Saharan Africa and South Asia3.

Although Shigella infection is mostly self-limiting disease, antibiotics are recommended to reduce the clinical course of illness and to prevent transmission. However, antimicrobial resistance (AMR) is an emerging concern among Shigella spp. particularly in Asia and Africa4. Over the past decades, Shigella species have become increasingly resistant to most widely used antimicrobials5. Despite the alarming increase in the AMR in bacterial pathogens in India, publicly available information concerning the molecular identity of resistance traits is minimal67. According to the WHO report, AMR pattern for Shigella varies with geographic location and with time5. The continuing changing patterns of prevalent species and resistance of Shigella isolates indicate the need for monitoring antimicrobial susceptibility profiles8. The mobile genetic elements play a significant role in transferring resistance genes horizontally to non-resistant isolates. These elements are believed to be responsible for the acquisition and dissemination of AMR among clinically relevant organisms9.

The recent advancement in whole-genome sequencing technologies for routine microbiology is well documented10. However, there is limited information on the surveillance of diarrhoeagenic pathogens and their AMR pattern in developing countries. The availability of whole-genome sequences of antimicrobial-resistant pathogens enhances our knowledge of the molecular identity of resistance traits and their mechanism of dissemination within the microbial population. This study was aimed to generate the base line data of resistance, virulence and plasmid profiles of Shigella species isolated from clinical specimens through whole-genome sequencing.

Material & Methods

Shigella strains isolated from stool specimen from patients with diarrhoea or dysentery during the year 2011-2017 at Christian Medical College, Vellore, India were included in the study. Culture and biochemical identification of isolates was done using standard protocol11. Serologic confirmation was done by slide agglutination test using polyvalent somatic (O) antigen grouping sera, followed by monovalent antisera (Denka, Seiken, Japan) for Shigella-specific serotype identification. Antimicrobial susceptibility testing of isolates against ampicillin (10 μg), trimethoprim/sulphamethoxazole (1.25/23.75 μg), nalidixic acid (30 μg), norfloxacin (10 μg), cefotaxime (30 μg), cefixime (5 μg) and azithromycin (15 μg) was performed using Kirby-Bauer disc diffusion method12. The results were interpreted using breakpoints recommended by the Clinical and Laboratory Standards Institute guidelines 201712. Quality control strains used were E. coli ATCC 35218 and E. coli ATCC 25922 for the antibiotics tested.

Whole-genome sequencing: Genomic DNA was extracted using the QiaSymphony DNA extraction platform (Qiagen, Hilden, Germany). Genome sequencing was performed using Ion Torrent (PGM, Life Technologies, Carlsbad, CA, USA) with 400 bp read chemistry (Life Technologies) as previously described13.

Assembly & annotation: The raw data were assembled de novo using AssemblerSPAdes v.5.0.0.0 embedded in Torrent suite server v.5.0.4. The genome sequence was annotated using PATRIC, the bacterial bioinformatics database and analysis resource (), and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) ()14.

Downstream genome analysis: The whole-genome data were analyzed using open access tools at Centre for Genomic Epidemiology web-based server. AMR and virulence genes were identified using ResFinder 2.1 ()16, respectively, with 90 per cent threshold for identity and with 60 per cent of minimum length coverage, where reads were mapped to a reference database of acquired genes. Furthermore, the transferable resistance genes and chromosomal mutation in the quinolone-resistant determining region were studied through PATRIC database. The presence of plasmids was analyzed using PlasmidFinder 1.3 () with 95 per cent threshold for identity17. These whole-genome shotgun sequences were deposited in DDBJ/ENA/GenBank (Table I for accession numbers).

Table I

Characteristics of Shigella isolates analyzed in this study (n=60)

Isolate ID	Organism	Resistant pattern	Acquired resistance genes	Chromosomal mutation				Plasmid (Inc type)	Accession no.
				gyrA	gyrB	parC	parE
FC1882	S. boydii	SXT-NAL	strA, strB, aadA1, sulII, dfrA1	D87-Y	-	-	-	IncFII	MDDI00000000
FC1764	S. boydii	AMP-SXT	strA, strB, blaTEM-1B, qnrS1, sulII, tetA, dfrA14	-	-	-	-	IncFII, IncFIB	MDDH00000000
FC1661	S. boydii	SXT-NAL-FIX	aadA1, sulI, tetA, dfrA1, dfrA4, blaEC	S83-L	-	-	*E135-V	IncA/C2, IncFII	MDGW00000000
FC2833	S. boydii	ALL SUSCEPTIBLE	-	-	-	-	-	IncFII	MDJL00000000
FC1567	S. boydii	AMP-SXT-NAL	dfrA3, blaEC	-	-	-	-	IncFII	MIIV00000000
FC2117	S. boydii	AMP-SXT	strA, strB, blaTEM-1B, qnrS1, sulII, tetA, dfrA14	-	-	-	-	IncFII, IncFIB	MINP00000000
FC2125	S. boydii	SXT-NAL-NX	aadA1, dfrA1, blaEC	-	-	-	-	IncFII	MINQ00000000
FC2175	S. boydii	SXT	aadA1, dfrA1	-	-	-	-	IncFII	MINR00000000
FC2710	S. boydii	AMP-SXT-NAL (MS)	strA. strB, blaTEM-1B, qnrS1, sulII, dfrA14	-	-	-	-	IncFII, IncFIB	MINU00000000
FC1180	S. flexneri	AMP-SXT-NAL-NX (MS)	strA, strB, aadA1, blaOXA-1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	-	MDJJ00000000
FC1139	S. flexneri	AMP-SXT	dfrA3, blaEC	-	-	-	-	-	MECX00000000
FC1172	S. flexneri	AMP-SXT-NAL-NX (MS)	strA, strB, blaOXA-1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	-	MDJI00000000
FC1056	S. dysenteriae serotype 3	NAL-TAX	strA, strB, aadA1, sulII, tetB, dfrA1, blaEC	-	*Q776-L	*C435-G, *S694-P	-	IncFII	MECW00000000
FC1708	S. dysenteriae serotype 3	SXT-NAL	aadA1, blaOXA-1, tetB, dfrA1	-	*Q776-L	*C435-G, *S694-P	-	IncFII	MIIX00000000
FC1737	S. dysenteriae serotype 3	NAL	tetB, dfrA1	-	*Q776-L	*C435-G, *S694-P	-	IncFII	MIIY00000000
FC2531	S. dysenteriae serotype 3	AMP-NAL-TAX	aadA1, blaOXA-1, tetB, dfrA1, blaEC	-	*Q776-L	*C435-G, *S694-P	-	IncFII	MINS00000000
FC2541	S. dysenteriae serotype 3	AMP-NAL-TAX	aadA1, blaOXA-1, tetB, dfrA1, blaEC	-	*Q776-L	*C435-G, *S694-P	-	IncFII	MINT00000000
FC2383	S. boydii	AMP-SXT-NAL	strA, strB, aadA1, blaTEM-1B, qnrS1, sulII, dfrA1	-	-	-	-	IncN, IncFII	MDJK00000000
FC1544	S. boydii	AMP-SXT-NAL	strA, strB, blaTEM-1B, qnrS1, sulII, dfrA14	D87-Y	-	-	-	IncFII, IncFIB	MECT00000000
FC3196	S. boydii	AMP-SXT-NAL	strA, strB, aadA1, blaOXA-1, sulII, tetB, dfrA1	S83-L	-	-	-	IncFII	MINV00000000
FC288	S. sonnei	AMP-SXT-NAL-NX	strA, strB, blaEC, sulII, dfrA1	S83-L	-	S80-I	-	Col (BS512)	NGWI00000000
FC1373	S. sonnei	AMP-SXT-NAL-NX	strA, strB, blaEC, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	NGWH00000000
FC1417	S. flexneri 4	AMP-SXT-NAL-NX-TAX-FIX	aadA1, blaOXA-1, blaCTX-M-15, qnrS1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII, Col (MP18)	NGWG00000000
FC1846	S. flexneri 6	AMP-SXT-NAL-TAX-FIX	blaEC, aadA1, tetB, dfrA1	D87-Y	*Q776-L	*Q506-L	-	IncFII	NGWF00000000
FC2615	S. flexneri 6	AMP-SXT-NAL	aadA1, blaEC, sulII, tetB, dfrA1	D87-Y	*Q776-L	*Q506-L	-	IncFII	NGWE00000000
FC906	S. flexneri 2	AMP-SXT-NAL-NX-TAX-FIX	strA, strB, blaEC, blaOXA-1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII	NGWD00000000
FC1182	S. flexneri 1	AMP-SXT-NAL	strA, strB, aadA1, sulII, blaTEM-1B, tetA, dfrA1	-	-	-	-	Col (BS512), IncFIB (K)	NGWC00000000
FC1772	S. sonnei	AMP-SXT-NAL-NX-TAX-FIX	blaEC, sulI, dfrA5	S83-L	-	S80-I	-	Col 156	NGWB00000000
FC1659	S. sonnei	SXT-NAL	strA, strB, aadA1, blaOXA-1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I, *S542-P	-	IncFII, IncI2	NGWA00000000
FC470	S. flexneri 2	AMP-SXT-NAL-NX-TAX-FIX	strA, strB, blaTEM-1B, blaDHA-1, qnrB4, qnrS1, mphA, sulI, sulII, tetA, dfrA17	-	-	-	-	IncFII, IncFIB (K)	NGVZ00000000
FC1247	S. flexneri 2	AMP-SXT-NAL-NX-TAX-FIX	strA, strB, aadA1, blaEC, blaTEM-1B, qnrS1, sulII, tetA, dfrA1	S83-L	-	*Q506-L	-	IncFII, IncFIB (K)	NGVY00000000
FC1607	S. flexneri 4	AMP-SXT-NAL-NX-TAX-FIX	aadA1, strA, strB, blaEC, blaCTX-M 15, qnrS1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII, IncFIB (K)	NGVX00000000
FC1481	S. flexneri 4	AMP-SXT-NAL-NX-TAX-FIX	strA. strB, aadA1, blaTEM-1B, blaOXA-1, blaCTX-M-15, qnrS1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII, IncFIB (K)	NGVW00000000
FC3278	S. sonnei	AMP-SXT-NAL	strA, strB, blaTEM1B, sulII, dfrA5	S83-L	-	S80-I	-	Col 156, IncB/O/K/Z	NMYB00000000
FC1244	S. sonnei	SXT-NAL	strA, strB, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	NMYA00000000
FC3433	S. flexneri 2	AMP-SXT-NAL-TAX	aadA1, blaEC, blaOXA-1, catA1, tetB, dfrA1	S83-L	-	S80-I	-	IncFII	NMXZ00000000
FC653	S. sonnei	AMP-SXT-NAL	blaEC, strA, strB, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	NMXY00000000
FC1170	S. flexneri 2	AMP-SXT-NAL	blaOXA-1, catA1, tetB, dfrA1	S83-L	-	S80-I	-	IncFII	NMXX00000000
FC1824	S. flexneri 2	AMP-SXT-NAL	strA, strB, blaOXA-1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII	NMXW00000000
FC601	S. flexneri 1	AMP-SXT-AZM	strA, strB, aadA1, blaTEM1B, qnrS1, sulII, tetA, dfrA1	-	-	-	-	Col 156	NMXV00000000
FC3209	S. sonnei	SXT-NAL-NX	strA, strB, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	NMXU00000000
FC666	S. boydii	SXT-NAL	aadA1, sulII, tetB, dfrA1	S83-L, D87-Y	-	*Q506-L	-	IncFII	NMXT00000000
FC1747	S. sonnei	SXT-NAL	strB, strA, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	NMXS00000000
FC15	S. sonnei	AMP-SXT-NAL-NX-TAX-FIX	strB, strA, blaCTX-M-15, blaEC sulII, dfrA1	S83-L	-	S80-I	-	Col (BS512), Col 156, Incl1	NMXR00000000
FC401	S. flexneri 1	AMP-SXT-NAL-NX	strA, strB, aadA1, blaTEM-1B, qnrS1, sulII, dfrA1, dfrA14	-	-	-	-	IncFII, IncFIB (K)	NMXQ00000000
FC420	S. flexneri 2	AMP-SXT-NAL-NX	strA, strB, blaOXA-1, sulII, tetB, dfrA1, catA1	S83-L	-	S80-I	-	IncFII	NMXP00000000
FC248	S. flexneri	AMP-SXT-NAL-NX	blaOXA-1, tetB, dfrA1, catA1	S83-L	-	S80-I	-	IncFII	NMXO00000000
FC1642	S. boydii	SXT-NAL	aadA1, tetB, dfrA1, sulII	S83-L, D87-Y	-	*Q506-L	-	IncFII	PDYE00000000
FC1655	S. boydii	AMP-SXT-TAX-FIX	strA, strB, aadA1, blaEC, blaCTX-M-15, qnrS1, sulII, dfrA1	-	-	-	-	IncFII	PDYD00000000
FC1676	S. boydii	AMP-SXT	strA, strB, blaTEM-1B, qnrS1, sulII, tetA, dfrA14	-	-	-	-	IncFII, IncFIB (K)	PDYC00000000
FC1706	S. sonnei	SXT-NAL	dfrA1	S83-L	-	S80-I	-	Incl1, Col 156	PDYB00000000
FC1628	S. sonnei	SXT-NAL	strA, strB, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	PDYA00000000
FC1667	S. sonnei	NAL	dfrA1	S83-L	-	S80-I	-	Col 156, ColpVC	PDXZ00000000
FC1717	S. boydii	AMP-SXT	strA, strB, blaTEM-1B, qnrS1, tetA, sulII	-	-	-	-	IncFII, IncFIB (K)	PDXY00000000
FC1653	S. sonnei	SXT-NAL	strA, strB, sulII, dfrA1	S83-L	-	S80-I	-	Col 156	PDXX00000000
FC1677	S. sonnei	AMP-SXT-NAL-TAX-FIX	strA, strB, blaEC, blaCTX-M-15, sulII, dfrA1	S83-L	-	S80-I	-	Col 156, Incl1	PDXW00000000
FC1405	S. flexneri	AMP-SXT-TET-NAL-NX	strA, strB, aadA1, blaOXA-1, catA1, sulII, tetB, dfrA1	S83-L	-	S80-I, *R86-C	-	IncFII	PDXV00000000
FC2101	S. flexneri 2	AMP-SXT-NAL-TAX-FIX	aadA1, blaEC, blaCMY-4, dfrA1	S83-L	-	S80-I	-	IncB/O/K/Z	PDXU00000000
FC2414	S. flexneri 2	AMP-SXT-NX	strA, strB, blaOXA-1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII	PDXT00000000
FC1954	S. flexneri 2	AMP-SXT-NAL-NX	strA, strB, blaOXA-1, sulII, tetB, dfrA1	S83-L	-	S80-I	-	IncFII	PDXS00000000

*Novel mutations. AMP, ampicillin; SXT, trimethoprim/sulphamethoxazole; NAL, nalidixic acid; NX, norfloxacin; TAX, cefotaxime; FIX, cefixime; AZM, azithromycin

Results

Whole-genome sequences of 60 Shigella isolates were analyzed in this study, which included S. dysenteriae (n=5), S. flexneri (n=23), S. boydii (n=17) and S. sonnei (n=15). Among the study isolates, 68 per cent (n=41) were resistant to more than or equal to three antimicrobials, 30 per cent (n=18) were resistant to less than three antimicrobials and two per cent (n=1) were susceptible to all tested antimicrobials. Ampicillin susceptibility was lower in S. flexneri compared to S. sonnei, while the susceptibility profile of other antibiotics remained unchanged. The susceptibility profile of the isolates is shown in Table I.

Whole genome sequencing: The genome length for the Shigella isolates ranged from ca. 4.2 Mbp to ca. 4.6 Mbp with coverage of 36× to 100×. Genomes were screened for known acquired genes. The presence of resistance determinants conferring resistance to β-lactams, aminoglycosides, quinolones, cephalosporins, tetracycline and sulphonamides was identified, as detailed in Table I.

Species-wise antimicrobial resistance (AMR) gene analysis

Shigella dysenteriae: Of the five S. dysenteriae isolates, three were found to carry blaOXA-1 β-lactamase gene. All the isolates carried tetracycline (tet) and trimethoprim (dfrA1) resistance genes, whereas only one isolate carried sulphonamide gene (sulII). An aminoglycoside resistance gene such as strA/B and aadA1 was also identified. No mutations were observed in gyrA and parE genes, but novel mutations were observed in gyrB (Gln776 - Leu) and parC (Cys435 - Gly) genes. None of the isolates harboured cephalosporin resistance gene (Tables I & II).

Table II

Antimicrobial resistance genes distribution among Shigella serogroups % (n)

Shigella serogroup	blaOXA-1	blaTEM-1B	blaCTX-M-15	blaDHA-1	blaCMY-4	dfrA1	dfrA14	dfrA17	dfrA4	dfrA5
S. dysenteriae (n=5)	60 (3)	-	-	-	-	100 (5)	-	-	-	-
S. flexneri (n=23)	56 (13)	22 (5)	13 (3)	4 (1)	4 (1)	91 (21)	4 (1)	4 (1)	-	-
S. boydii (n=17)	6 (1)	41 (7)	6 (1)	-	-	53 (9)	29 (5)	-	6 (1)	-
S. sonnei (n=15)	7 (1)	7 (1)	13 (2)	-	-	87 (13)	-	-	-	13 (2)
	qnrB4	qnrS1	sulI	sulII	strA	strB	aadA1	tetA	tetB	catA1
S. dysenteriae (n=5)	-	-	-	20 (1)	20 (1)	20 (1)	80 (4)	-	100 (5)	-
S. flexneri (n=23)	4 (1)	30 (7)	4 (1)	74 (17)	65 (15)	65 (15)	52 (12)	17 (4)	69 (16)	43 (10)
S. boydii (n=17)	-	47 (8)	6 (1)	70 (12)	59 (10)	59 (10)	53 (9)	29 (5)	18 (3)	-
S. sonnei (n=15)	-	-	7 (1)	80 (12)	80 (12)	80 (12)	7 (1)	-	7 (1)	7 (1)

All S. flexneri isolates were multi-drug resistant except one, which was resistant to ampicillin and trimethoprim/sulphamethoxazole alone. Among the β-lactamases, blaOXA-1, blaTEM-1B, blaCTX-M-15 genes were present in 13, 5 and 3 isolates, respectively. AmpC genes such as blaDHA-1 and blaCMY-4 were found each in single isolate. For plasmid-mediated quinolone resistance, qnrB4 (n=1) and qnrS1 (n=7) genes were identified. Fifteen isolates showed two identical mutations in the gyrA and parC genes. The mutations were observed at codon 83 in the gyrA gene and at codon 80 in the parC gene which resulted in the replacement of serine by leucine and isoleucine, respectively. Two isolates had an additional mutation at codon 87 in gyrA gene, resulting in the replacement of aspartic acid by tyrosine. Novel mutations were observed in gyrB (Gln776 to Leu) and parC (Gln506 to Leu and Arg86 to Cys) genes. No mutation was seen in the parE gene (Table I). Genes encoding trimethoprim (dfrA1, dfrA14, dfrA17) and sulphonamide (sulI and sulII) resistance were identified. Most of the isolates carried genes such as strA/B, aadA1, tetA/B and catA1, conferring resistance to aminoglycosides, tetracycline and chloramphenicol (Table II).

S. boydii isolates also carried the β-lactamase genes, blaOXA-1 (n=1), blaTEM-1B (n=7), and blaCTX-M-15 (n=1). AmpC genes were not detected. Among the quinolone resistant isolates, only a qnrS1 gene was identified in eight isolates (Tables I & II). Four isolates showed mutations in gyrA (S83-L and D87-Y), two in parC (Q506-L) and a single isolate had a mutation in the parE (E135-V) gene. No mutation was seen in the gyrB gene. Resistance genes such as dfrA1, dfrA14, dfrA4, sulI, sulII, strA/B, aadA1 and tetA/B were identified in S. boydii isolates.

Like other serogroups, S. sonnei isolates were also found to carry blaOXA-1 (n=1), blaTEM-1B (n=1), blaCTX-M-15 (n=2) genes. None of the isolates carried AmpC or the qnr genes. However, all S. sonnei isolates showed two identical mutations in gyrA and parC genes, S83-L and S80-I, respectively. One isolate had additional mutation in parC (S542-P) gene (Table I). The isolates also carried resistance genes for sulphonamides, aminoglycoside, tetracycline and chloramphenicol (Table II).

The presence of virulence genes was analyzed using E. coli database. Most of the isolates were found to harbour virulence genes such as ipa involved in the entry of bacteria into epithelial cells. Other virulence genes such as virF, senB, iha, capU, lpfA, sigA, pic, sepA, celb and gad were also identified in the isolates. Distribution of these genes among Shigella serogroups are given in Table III.

Table III

Virulence genes observed among Shigella serogroups % (n)

Shigella serogroup	ipaH	ipaD	senB	virF	iha	capU	lpfA	sigA	pic	sepA	celb	gad
S. dysenteriae (n=5)	-	100 (5)	100 (5)	100 (5)	100 (5)	100 (5)	100 (5)	100 (5)	-	-	-	-
S. flexneri (n=23)	4 (1)	74 (17)	9 (2)	65 (15)	9 (2)	56 (13)	69 (16)	69 (16)	48 (11)	65 (15)	-	-
S. boydii (n=17)	6 (1)	94 (16)	100 (17)	100 (17)	100 (17)	88 (15)	41 (7)	82 (14)	-	-	-	6 (1)
S. sonnei (n=15)	-	7 (1)	93 (14)	7 (1)	-	13 (2)	100 (15)	100 (15)	7 (1)	7 (1)	60 (9)	13 (2)

Plasmid analysis: Plasmid distribution among Shigella species is given in Table IV. IncFII type was the most prevalent plasmid among all four Shigella serogroups. S. dysenteriae isolates had only the IncFII type plasmid, whereas S. flexneri isolates were found to have IncFIB(K), IncFII, Col156, Col(BS512), ColMP18 and IncB/O/K/Z plasmids. S. boydii isolates were found to have plasmids such as IncFIB, IncA/C2 and IncN. Plasmids such as IncI2, IncI1 and ColpVC were identified in S. sonnei.

Table IV

Plasmids prevalence among Shigella serogroups % (n)

Shigella serogroup	IncFIB	IncFIB (K)	IncFII	IncA/C2	IncN	Col156	Col (BS512)	IncI2	Incl1	IncB/O/K/Z	ColpVC	Col MP18
S. dysenteriae (n=5)	-	-	100 (5)	-	-	-	-	-	-	-	-	-
S. flexneri (n=23)	-	26 (6)	74 (17)	-	-	4 (1)	4 (1)	-	-	4 (1)	-	4 (1)
S. boydii (n=17)	23 (4)	12 (2)	100 (17)	6 (1)	6 (1)	-	-	-	-	-	-	-
S. sonnei (n=15)	-	-	7 (1)	-	-	87 (13)	13 (2)	7 (1)	20 (3)	7 (1)	7 (1)	-

Discussion

Shigella remains a leading cause of childhood dysentery. The clones with high virulence and multidrug resistance (MDR) have spread globally where plasmids play a major role in conferring these characteristics18. The pathogenesis of Shigella is related to various virulence factors located in the chromosome or large virulent inv plasmid carrying gene responsible for functions like host cell invasion and intracellular survival219. However, only a few studies have attempted to illustrate its molecular virulence profile. A recent study by Medeiros et al20 showed that the presence of virulence genes in Shigella was associated with various clinical symptoms such as intense abdominal pain and bloody stools. They also highlighted that the higher numbers of virulence genes were associated with resistance to more antimicrobials.

In this study, vast distribution of genes was observed among all four Shigella serogroups, especially in S. flexneri. pic and sepA genes were also seen more in S. flexneri. The shiga toxin gene (stx) is an important virulence determinant related to S. dysenteriae, but none of the S. dysenteriae isolates carried this gene.

The pathogens capacity to rapidly acquire AMR is a major concern. Development of AMR was common in all Shigella species, particularly in S. sonnei which were known to acquire resistance genes from E. coli through horizontal gene transfer mechanism21. Furthermore, resistance in S. flexneri is well documented with several studies showing a high frequency of resistance to commonly used antimicrobials such as ampicillin and co-trimoxazole21.

In the present study, increased resistance was observed to first-line antibiotics such as ampicillin, trimethoprim-sulphamethoxazole and nalidixic acid. Therefore, these drugs should not be recommended for treatment unless susceptibility is known or expected based on local surveillance. In the present study, trimethoprim-sulphamethoxazole resistance was mainly due to dhfr1A gene followed by the sulII gene. The resistance to chloramphenicol, tetracycline and streptomycin was due to the presence of catA1, tetA/B and of either strA/B or aadA1 genes or both.

Among β-lactams, ampicillin resistance was usually encoded by OXA-type β-lactamase genes followed by TEM. In the present study, the resistance was predominantly due to blaOXA-1 followed by blaTEM-1. The predominance of OXA-1 in Shigella has been reported earlier22. Twenty one isolates in this study harboured blaEC gene, a class C β-lactamase conferring resistance to β-lactam antibiotics. CTX-M-type β-lactamases blaCTX-M-15, was identified in all serogroups except S. dysenteriae and plasmid-mediated AmpC β-lactamases genes were found only in S. flexneri isolates. Increasing number of reports of third-generation cephalosporins resistance in Asia left limited options for effective therapy23.

The WHO has listed fluoroquinolone-resistant Shigella as one of its top concerns in the current international focus on AMR24. In general, quinolone resistance involves the accumulation of mutations in DNA gyrase and DNA topoisomerase IV; and plasmid-mediated quinolone resistance (PMQR) determinants like qnrA, qnrB, qnrS and aac(6)-Ib-cr genes which confer low-level resistance to quinolones. In this study, the plasmid-mediated qnrS gene was widely distributed among S. flexneri and S. boydii isolates. qnrB4 gene was present only in S. flexneri isolates. Besides, mutation analysis of DNA gyrase and topoisomerase IV genes added more information in an understanding of resistance to fluoroquinolone in Shigella. Novel mutations were observed in gyrB, parC and parE genes. However, the detailed study on the impact of these mutations in conferring quinolone resistance needs to be done.

The presence of these AMR genes in most of the isolates was related with their phenotypic profile. However, phenotypic resistance in spite of the absence of genes represents that other mechanisms might be responsible for resistance, whereas the presence of resistance genes genotypically with no phenotypic expression corresponds to non-expression of AMR genes. One susceptible isolate did not carry any resistance genes but instead carried a plasmid. Another important factor involved in the spread of resistance was the presence of incompatible plasmid particularly, the IncF plasmid which was known to be associated with the worldwide emergence of clinically relevant extended-spectrum β-lactamases (ESBLs) and multiple AMR determinants25. The present study showed the dominance of IncFII plasmid among the tested isolates. Beceiro et al18 have reported that IncF is a major incompatibility group involved in the co-transfer of resistance and virulence determinants. All the isolates harbouring virulence genes also harboured either single or more than one Inc type plasmid in this study, which further highlighted the significant association of these determinants in pathogenic bacteria.

The widespread emergence of MDR Shigella and increasing incidence with changing AMR patterns makes treatment a challenge for shigellosis. As shown here, AMR in Shigella spp. was serogroup-specific.

In conclusion, screening of AMR genes among Shigella genome showed that resistant gene distribution was variable among the Shigella serogroups. The findings of the present study also showed the species ability in acquiring AMR determinants and suggested the continuous surveillance of this species and its resistance profile particularly in Shigella endemic region.