Abstract
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-1 was predominantly seen followed by the blaTEM-1B and blaEC genes. For quinolone resistance, the qnrS gene was widely seen. Novel mutations in gyrB, parC and parE 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.
Keywords: Antimicrobial resistance gene, blaCTX-M-15, IncF plasmid, qnr, Shigella spp., virulence
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 minimal6,7. 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 (http://www.patricbrc.org), and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html)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 (https://cge.cbs.dtu.dk//services/ResFinder/)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 (https://cge.cbs.dtu.dk//services/PlasmidFinder/) 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) |
Shigella flexneri: 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).
Shigella boydii: 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.
Shigella sonnei: 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).
Virulence gene analysis: 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 survival2,19. 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.
Footnotes
Financial support & sponsorship: This work was supported by the Indian Council of Medical Research, New Delhi (Ref. No: AMR/TF/55/13ECDII dated 23/10/2013).
Conflicts of Interest: None.
References
- 1.Aggarwal P, Uppal B, Ghosh R, Krishna Prakash S, Chakravarti A, Jha AK, et al. Multi drug resistance and extended spectrum beta lactamases in clinical isolates of Shigella: A study from New Delhi, India. Travel Med Infect Dis. 2016;14:407–13. doi: 10.1016/j.tmaid.2016.05.006. [DOI] [PubMed] [Google Scholar]
- 2.da Cruz CB, de Souza MC, Serra PT, Santos I, Balieiro A, Pieri FA, et al. Virulence factors associated with pediatric shigellosis in Brazilian Amazon. Biomed Res Int. 2014;2014:539697. doi: 10.1155/2014/539697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.The HC, Thanh DP, Holt KE, Thomson NR, Baker S. The genomic signatures of Shigella evolution, adaptation and geographical spread. Nat Rev Microbiol. 2016;14:235–50. doi: 10.1038/nrmicro.2016.10. [DOI] [PubMed] [Google Scholar]
- 4.Anandan S, Muthuirulandi Sethuvel DP, Gajendiren R, Verghese VP, Walia K, Veeraraghavan B. Molecular characterization of antimicrobial resistance in clinical Shigella isolates during 2014 and 2015: Trends in South India. Germs. 2017;7:115–22. doi: 10.18683/germs.2017.1116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Shakya G, Acharya J, Adhikari S, Rijal N. Shigellosis in Nepal: 13 years review of nationwide surveillance. J Health Popul Nutr. 2016;35:36. doi: 10.1186/s41043-016-0073-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kumar P, Bag S, Ghosh TS, Dey P, Dayal M, Saha B, et al. Molecular insights into antimicrobial resistance traits of multidrug resistant enteric pathogens isolated from India. Sci Rep. 2017;7:14468. doi: 10.1038/s41598-017-14791-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Laxminarayan R, Chaudhury RR. Antibiotic resistance in India: Drivers and opportunities for action. PLoS Med. 2016;13:e1001974. doi: 10.1371/journal.pmed.1001974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Rahman M, Haque AF, Deeba IM, Ahmed D, Zahidi T, Rimu AH, et al. Emergence of extensively drug-resistant Shigella sonnei in Bangladesh. Immunol Infect Dis. 2017;5:1–9. [Google Scholar]
- 9.Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr. 2016;4 doi: 10.1128/microbiolspec.VMBF-0016-2015. doi: 10.1128/microbiolspec.VMBF-0016-2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Nair S, Ashton P, Doumith M, Connell S, Painset A, Mwaigwisya S, et al. WGS for surveillance of antimicrobial resistance: A pilot study to detect the prevalence and mechanism of resistance to azithromycin in a UK population of non-typhoidal salmonella. J Antimicrob Chemother. 2016;71:3400–8. doi: 10.1093/jac/dkw318. [DOI] [PubMed] [Google Scholar]
- 11.Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 1997. Colour atlas and textbook of diagnostic microbiology. [Google Scholar]
- 12.CLSI Document M100. 27th ed. Wayne, PA: CLSI; 2017. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. [Google Scholar]
- 13.Dhiviya Prabaa MS, Naveen Kumar DR, Yesurajan IF, Anandan S, Kamini W, Balaji V. Identification of nonserotypeable Shigella spp. using genome sequencing: A step forward. Future Sci OA. 2017;3:FSO229. doi: 10.4155/fsoa-2017-0063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016;44:6614–24. doi: 10.1093/nar/gkw569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640–4. doi: 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Joensen KG, Scheutz F, Lund O, Hasman H, Kaas RS, Nielsen EM, et al. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol. 2014;52:1501–10. doi: 10.1128/JCM.03617-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, et al. In silico detection and typing of plasmids using plasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother. 2014;58:3895–903. doi: 10.1128/AAC.02412-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Beceiro A, Tomás M, Bou G. Antimicrobial resistance and virulence: A successful or deleterious association in the bacterial world? Clin Microbiol Rev. 2013;26:185–230. doi: 10.1128/CMR.00059-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Yaghoubi S, Ranjbar R, Dallal MMS, Fard SY, Shirazi MH, Mahmoudi M, et al. Profiling of virulence-associated factors in Shigella species isolated from acute pediatric diarrheal samples in Tehran, Iran. Osong Public Health Res Perspect. 2017;8:220–6. doi: 10.24171/j.phrp.2017.8.3.09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Medeiros PHQ, Lima AÂM, Guedes MM, Havt A, Bona MD, Rey LC, et al. Molecular characterization of virulence and antimicrobial resistance profile of Shigella species isolated from children with moderate to severe diarrhea in Northeastern Brazil. Diagn Microbiol Infect Dis. 2018;90:198–205. doi: 10.1016/j.diagmicrobio.2017.11.002. [DOI] [PubMed] [Google Scholar]
- 21.Anderson M, Sansonetti PJ, Marteyn BS. Shigella diversity and changing landscape: Insights for the twenty- first century. Front Cell Infect Microbiol. 2016;6:45. doi: 10.3389/fcimb.2016.00045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Pazhani GP, Niyogi SK, Singh AK, Sen B, Taneja N, Kundu M, et al. Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India. J Med Microbiol. 2008;57:856–63. doi: 10.1099/jmm.0.2008/000521-0. [DOI] [PubMed] [Google Scholar]
- 23.Kotloff KL. Shigella infection in children and adults: A formidable foe. Lancet Glob Health. 2017;5:e1166–7. doi: 10.1016/S2214-109X(17)30431-X. [DOI] [PubMed] [Google Scholar]
- 24.World Health Organization. Geneva: WHO; 2015. Global antimicrobial resistance surveillance system: Manual for early implementation. [Google Scholar]
- 25.Ogbolu DO, Daini OA, Ogunledun A, Terry Alli OA, Webber MA. Dissemination of IncF plasmids carrying beta-lactamase genes in gram-negative bacteria from Nigerian hospitals. J Infect Dev Ctries. 2013;7:382–90. doi: 10.3855/jidc.2613. [DOI] [PubMed] [Google Scholar]