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  Table of Contents  
BRIEF COMMUNICATION
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 526-528
 

Improved detection of Shigella using Escherichia coli medium enrichment: Polymerase chain reaction from stool samples


1 Department of Microbiology, Dr. RPGMC, Kangra, Himachal Pradesh, India
2 Department of Microbiology, SDM Medical College, Dharwad, Karnataka, India
3 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission20-Jun-2015
Date of Acceptance24-Oct-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
N Taneja
Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.195355

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 ~ Abstract 

Laboratory diagnosis of shigellosis using conventional culture technique is limited by lower sensitivity and higher turnaround time. Here, we have evaluated the role of polymerase chain reaction from stool samples after enrichment in Escherichia coli medium for detection of Shigellae. The technique not only increased the sensitivity but also decreased the turnaround time.


Keywords: Escherichia coli medium, polymerase chain reaction, Shigella


How to cite this article:
Gupta P K, Appannanavar S B, Mohan B, Taneja N. Improved detection of Shigella using Escherichia coli medium enrichment: Polymerase chain reaction from stool samples. Indian J Med Microbiol 2016;34:526-8

How to cite this URL:
Gupta P K, Appannanavar S B, Mohan B, Taneja N. Improved detection of Shigella using Escherichia coli medium enrichment: Polymerase chain reaction from stool samples. Indian J Med Microbiol [serial online] 2016 [cited 2017 Mar 26];34:526-8. Available from: http://www.ijmm.org/text.asp?2016/34/4/526/195355



 ~ Introduction Top


The etiological agents responsible for acute dysentery vary according to patient population and geographic location.[1] Worldwide, Campylobacter, Shigella and Salmonella are the most commonly isolated bacterial agents from dysenteric patients.[1] At our centre, shigellosis is responsible for 3.58% of all cases of diarrhoea, and Shigella flexneri is the predominant agent accounting for 58.3% of all cases of shigellosis.[2] Diagnosis of shigellosis using conventional stool culture is often met with lower sensitivity and the minimum turnaround time is 48–72 h.[3] In majority of cases, diagnosis remains obscured due to inappropriate sample collection, delay in transportation, prior antimicrobial therapy, presence of low number of organisms and competition from other commensal flora. In recent years, molecular techniques have shown a high sensitivity and specificity compared to the conventional culture technique.[4],[5] The most widely used target for Shigella identification is invasive plasmid antigen H (ipaH) locus, which is also present in enteroinvasive Escherichia coli (EIEC).[4] No suitable enrichment medium for Shigella has consistently provided greater recovery rate than direct plating of stool samples.[6] Since molecular studies show a closer relationship between Escherichia coli (EC) and Shigella spp., EC medium (Difco, Sparks, MD, USA) enrichment that is commonly used for E. coli may be beneficial for Shigella also.[7],[8] Thus, in the current study, we have evaluated the role of enrichment with EC medium followed by ipaH polymerase chain reaction (PCR) to improve the diagnosis of shigellosis.


 ~ Materials and Methods Top


This study was carried out at the Enteric Laboratory of Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India, a Tertiary Care Centre in North India. Stool specimens of patients suffering from diarrhoea/dysentery admitted in the wards or presented to the Outpatient Departments (OPDs) of PGIMER from August to December 2012 were evaluated. The stool specimens were inoculated onto MacConkey agar (MA), xylose lysine deoxycholate agar (XLD), selenite F broth and into EC medium. EC medium and selenite F broths were subcultured after 12–15 h onto XLD and MA plates. Non-lactose-fermenting and red colonies from the MA and XLD plates, respectively, were provisionally identified biochemically as Shigella using conventional biochemicals (Himedia Laboratories Pvt. Ltd. Mumbai, Maharashtra, India).[3] The isolates were further confirmed by serotyping (Shigella Antisera Set 1 Denka-Seiken Co. Ltd. Tokyo, Japan).

After 12–15 h of enrichment in EC medium, 2 ml of the broth was used for DNA extraction by centrifugation and boiling in TE buffer (20 mmol Tris-HCl, 2 mmol EDTA, pH 8.0) as described previously.[7] PCR was performed targeting ipaH gene as described previously.[9] Positive (DNA from S. flexneri serotype 6 strain) and negative control (distilled water instead of template DNA) were included with each run.

To determine the analytical sensitivity of the protocol, pooled stool samples from healthy adults were spiked with a serologically confirmed S. flexneri serotype 6 or Shigella sonnei or Shigella dysenteriae type 1 or Shigella boydii to achieve a final concentration ranging from 2.5 to 2.5 × 107 colony forming units (CFU)/ml. Briefly, 0.5 ml of pooled stool sample from a healthy adult and 100 µl of suspension of control strain of serial dilutions was spiked into 4.5 ml of EC medium. To know the inhibitory effect of the stool samples, a parallel set of normal saline tubes was inoculated with control strain. These were incubated and subcultured on MA and XLD, and the DNA was extracted as described above. All the data were analysed using SPSS Inc. (Released 2008. SPSS Statistics for Windows, Version 17.0. Chicago: SPSS Inc.) (significance level of P < 0.05)


 ~ Results and Discussion Top


The analytical sensitivity (in CFU/ml) of culture was 2.5 × 104 for S. flexneri serotype 6 and 2.5 × 105 CFU/ml for S. sonnei, S. dysenteriae type 1 and S. boydii. The analytical sensitivity (in CFU/ml) of IpaH PCR was 2.5 × 102 for S. flexneri serotype 6 and 2.5 × 103 CFU/ml for S. sonnei and dysenteriae type 1 while S. boydii was not detected [Table 1]. A similar detection limit (2 × 102 CFU/ml) was earlier observed by Dutta et al. using IpaH PCR target.[4] The culture isolation has lower sensitivity and longer turnaround time. Use of molecular techniques directly on the stool samples overcomes these problems but can lead to false negative PCR results due to the presence of PCR inhibitors in the stool.[10] In the current study, we were able to show that the enrichment in EC medium was a useful technique in increasing the sensitivity of the PCR by allowing bacterial multiplication as well as dilution of PCR inhibitors. Similar observation was recently made by Ojha et al. in which they saw that 4 h enrichment was better than a 0 h inoculation in detecting Shigella spp. byPCR.[5]
Table 1: Correlation of culture and polymerase chain reaction after Escherichia coli medium enrichment with spiked stool sample

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The ipaH PCR and culture isolation for Shigella spp. performed in 302 consecutive stool samples received in the Enteric Laboratory. The mean age group of these patients was 32.6 (±standard deviation 21.3) years ranging from 1 month to 86 years. The male to female ratio was 7:3 and a ward to OPD ratio was 4.6:5.4. Majority of these patients presented with acute diarrhoea (70.9%) followed by ulcerative colitis (10.3%), acute dysentery (4.6%) and pyrexia of unknown origin (3%) as shown in [Table 2]. Culture detected Shigella spp. in 7 cases (2.32%) while PCR using ipaH was positive in 15 (4.97%) cases (P < 0.001). Of the 15 cases detected by PCR, culture grew S. flexneri serotype-6 in 6 cases while in the remaining 9 PCR positive cases there was no growth of Shigella spp. Among these 9 PCR positive and culture negative cases, the pooled E. coli DNA from culture plates was subjected for ipaH to rule out PCR positivity due to EIEC. However, the PCR was negative in all these 9 pooled E. coli samples. We noted false negative ipaH PCR in enrichment broth in one patient whose stool culture showed growth of Shigella boydii. The PCR from culture isolate was also negative and this case could be due to the absence of ipaH plasmid in the strain.[11]
Table 2: Clinical profile of study subjects and their correlation with polymerase chain reaction and culture

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Since multidrug-resistant Shigellae have emerged as a therapeutic challenge, early and accurate diagnosis of shigellosis along with accurate antimicrobial therapy will help in reducing the duration of illness, bacillary shedding and ultimately leading to decrease in morbidity and mortality associated with shigellosis.[12],[13],[14],[15] Currently, the major limitation of PCR is its inability to provide antibiogram. Future studies must be aimed at evaluating resistance markers for commonly used antibiotics by PCR and incorporate the same in the form of multiplex PCR along with primary identification.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Pfeiffer ML, DuPont HL, Ochoa TJ. The patient presenting with acute dysentery – A systematic review. J Infect 2012;64:374-86.  Back to cited text no. 1
    
2.
Taneja N. Changing epidemiology of shigellosis and emergence of ciprofloxacin-resistant Shigellae in India. J Clin Microbiol 2007;45:678-9.  Back to cited text no. 2
    
3.
3.WHO. Manual for Laboratory Investigation of Acute Enteric Infections. Programme for Control of Diarrhoeal Diseases. 2003;WHO/CDD/83.3. Geneva, Switzerland: WHO; 2003.  Back to cited text no. 3
    
4.
Dutta S, Chatterjee A, Dutta P, Rajendran K, Roy S, Pramanik KC, et al. Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India. J Med Microbiol 2001;50:667-74.  Back to cited text no. 4
    
5.
Ojha SC, Yean Yean C, Ismail A, Singh KK. A pentaplex PCR assay for the detection and differentiation of Shigella species. Biomed Res Int 2013;2013:412370.  Back to cited text no. 5
    
6.
Sansonetti PJ. Shigella. In: Borriello SP, Murray PR, Funke G, editors. Topley and Wilson's Microbiology and Microbial Infections. 10th ed., Vol. 2. London: Edward Arnold Ltd.; 2005. p. 1386-97.  Back to cited text no. 6
    
7.
Kumar A, Taneja N, Kumar Y, Sharma M. Detection of shiga toxin variants among shiga toxin-forming Escherichia coli isolates from animal stool, meat and human stool samples in India. J Appl Microbiol 2012;113:1208-16.  Back to cited text no. 7
    
8.
Perry CA, Hajna AA. Further evaluation of EC medium for the isolation of Coliform bacteria and Escherichia coli. Am J Public Health Nations Health 1944;34:735-8.  Back to cited text no. 8
    
9.
Aranda KR, Fagundes-Neto U, Scaletsky IC. Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp. J Clin Microbiol 2004;42:5849-53.  Back to cited text no. 9
    
10.
Rossen L, Nørskov P, Holmstrøm K, Rasmussen OF. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol 1992;17:37-45.  Back to cited text no. 10
    
11.
Schuch R, Maurelli AT. Virulence plasmid instability in Shigella flexneri 2a is induced by virulence gene expression. Infect Immun 1997;65:3686-92.  Back to cited text no. 11
    
12.
Taneja N, Mewara A, Kumar A, Verma G, Sharma M. Cephalosporin-resistant Shigella flexneri over 9 years (2001-2009) in India. J Antimicrob Chemother 2012;67:1347-53.  Back to cited text no. 12
    
13.
Njuguna HN, Cosmas L, Williamson J, Nyachieo D, Olack B, Ochieng JB, et al. Use of population-based surveillance to define the high incidence of shigellosis in an urban slum in Nairobi, Kenya. PLoS One 2013;8:e58437.  Back to cited text no. 13
    
14.
Rowe JS, Shah SS, Motlhagodi S, Bafana M, Tawanana E, Truong HT, et al. An epidemiologic review of enteropathogens in Gaborone, Botswana: Shifting patterns of resistance in an HIV endemic region. PLoS One 2010;5:e10924.  Back to cited text no. 14
    
15.
von Seidlein L, Kim DR, Ali M, Lee H, Wang X, Thiem VD, et al. A multicentre study of Shigella diarrhoea in six Asian countries: Disease burden, clinical manifestations, and microbiology. PLoS Med 2006;3:e353.  Back to cited text no. 15
    



 
 
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