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ORIGINAL ARTICLE
Year : 2019  |  Volume : 149  |  Issue : 2  |  Page : 247-256

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


1 Department of Clinical Microbiology, Christian Medical College, Vellore, India
2 Division of Epidemiology & Communicable Diseases, Indian Council of Medical Research, New Delhi, India

Date of Submission29-Dec-2017
Date of Web Publication3-Jun-2019

Correspondence Address:
Dr Balaji Veeraraghavan
Department of Clinical Microbiology, Christian Medical College, Vellore 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmr.IJMR_2077_17

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   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-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.

Keywords: Antimicrobial resistance gene - blaCTX-M-15- IncF plasmid - qnr - Shigella spp. - virulence


How to cite this article:
Sethuvel DP, Perumalla S, Anandan S, Michael JS, Ragupathi NK, Gajendran R, Walia K, Veeraraghavan B. Antimicrobial resistance, virulence & plasmid profiles among clinical isolates of Shigella serogroups. Indian J Med Res 2019;149:247-56

How to cite this URL:
Sethuvel DP, Perumalla S, Anandan S, Michael JS, Ragupathi NK, Gajendran R, Walia K, Veeraraghavan B. Antimicrobial resistance, virulence & plasmid profiles among clinical isolates of Shigella serogroups. Indian J Med Res [serial online] 2019 [cited 2019 Jun 18];149:247-56. Available from: http://www.ijmr.org.in/text.asp?2019/149/2/247/259582

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 conditions [1]. Depending on the virulence potential of the strain and the nutritional status of the individual, shigellosis can progress to severe disease [2]. The Global Enteric Multicenter Study, a case-control study of moderate-to-severe paediatric diarrhoeal disease, identified enterotoxigenic  Escherichia More Details coli and Shigella spp. as the most common bacterial pathogens in Sub-Saharan Africa and South Asia [3].

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 Africa [4]. Over the past decades, Shigella species have become increasingly resistant to most widely used antimicrobials [5]. Despite the alarming increase in the AMR in bacterial pathogens in India, publicly available information concerning the molecular identity of resistance traits is minimal [6],[7]. According to the WHO report, AMR pattern for Shigella varies with geographic location and with time [5]. The continuing changing patterns of prevalent species and resistance of Shigella isolates indicate the need for monitoring antimicrobial susceptibility profiles [8]. 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 organisms [9].

The recent advancement in whole-genome sequencing technologies for routine microbiology is well documented [10]. 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 Top


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 protocol [11]. 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 method [12]. The results were interpreted using breakpoints recommended by the Clinical and Laboratory Standards Institute guidelines 2017[12]. 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 described [13].

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/)[15] and VirulenceFinder 1.5 (https://cge.cbs.dtu.dk//services/VirulenceFinder/)[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 identity [17]. These whole-genome shotgun sequences were deposited in DDBJ/ENA/GenBank ([Table 1] for accession numbers).
Table 1: Characteristics of Shigella isolates analyzed in this study (n=60)

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   Results Top


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 1].

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 1].

Species-wise antimicrobial resistance (AMR) gene analysis

Shigella dysenteriae: Of the five S. dysenteriae isolates, three were found to carry blaOXA-1β-lactamase gene. All the isolates carried tetracycline (tet) and trimethoprim (dfr A1) resistance genes, whereas only one isolate carried sulphonamidegene (sul II). An aminoglycoside resistance gene such as str A/B and aad A1 was also identified. No mutations were observed in gyr A and par E genes, but novel mutations were observed in gyr B (Gln776 - Leu) and par C (Cys435 - Gly) genes. None of the isolates harboured cephalosporin resistance gene [Table 1] & [Table 2].
Table 2: Antimicrobial resistance genes distribution among Shigella serogroups % (n)

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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, qnr B4 (n=1) and qnr S1 (n=7) genes were identified. Fifteen isolates showed two identical mutations in the gyr A and par C genes. The mutations were observed at codon 83 in the gyr A gene and at codon 80 in the par C gene which resulted in the replacement of serine by leucine and isoleucine, respectively. Two isolates had an additional mutation at codon 87 in gyr A gene, resulting in the replacement of aspartic acid by tyrosine. Novel mutations were observed in gyr B (Gln776 to Leu) and par C (Gln506 to Leu and Arg86 to Cys) genes. No mutation was seen in the par E gene [Table 1]. Genes encoding trimethoprim (dfr A1, dfr A14, dfr A17) and sulphonamide (sul I and sul II) resistance were identified. Most of the isolates carried genes such as str A/B, aad A1, tet A/B and cat A1, conferring resistance to aminoglycosides, tetracycline and chloramphenicol [Table 2].

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 qnr S1 gene was identified in eight isolates [Table 1] & [Table 2]. Four isolates showed mutations in gyr A (S83-L and D87-Y), two in par C (Q506-L) and a single isolate had a mutation in the par E (E135-V) gene. No mutation was seen in the gyr B gene. Resistance genes such as dfr A1, dfr A14, dfr A4, sul I, sul II, str A/B, aad A1 and tet A/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 gyr A and par C genes, S83-L and S80-I, respectively. One isolate had additional mutation in par C (S542-P) gene [Table 1]. The isolates also carried resistance genes for sulphonamides, aminoglycoside, tetracycline and chloramphenicol [Table 2].

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 3].
Table 3: Virulence genes observed among Shigella serogroups % (n)

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Plasmid analysis: Plasmid distribution among Shigella species is given in [Table 4]. 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 4: Plasmids prevalence among Shigella serogroups % (n)

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   Discussion Top


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 characteristics [18]. 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 survival [2],[19]. However, only a few studies have attempted to illustrate its molecular virulence profile. A recent study by Medeiros et al[20] 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 sep A 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 mechanism [21]. 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-trimoxazole [21].

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 dhfr 1A gene followed by the sul II gene. The resistance to chloramphenicol, tetracycline and streptomycin was due to the presence of cat A1, tetA/B and of either str A/B or aad A1 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 byblaTEM-1. The predominance of OXA-1 in Shigella has been reported earlier [22]. 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 therapy [23].

The WHO has listed fluoroquinolone-resistant Shigella as one of its top concerns in the current international focus on AMR [24]. In general, quinolone resistance involves the accumulation of mutations in DNA gyrase and DNA topoisomerase IV; and plasmid-mediated quinolone resistance (PMQR) determinants like qnr A, qnr B, qnr Sand aac(6)-Ib-cr genes which confer low-level resistance to quinolones. In this study, the plasmid-mediated qnr S gene was widely distributed among S. flexneri and S. boydii isolates. qnr B4 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 gyr B, par C and par E 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 determinants [25]. The present study showed the dominance of IncFII plasmid among the tested isolates. Beceiro et al[18] 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.

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.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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