Subtype prevalence, plasmid profiles and growing fluoroquinolone resistance in Shigella from Kolkata, India (2001–2007): a hospital-based study

Authors


Corresponding Author Shanta Dutta, Scientist E, Bacteriology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India. Tel.: +91 33 23700448/23632398; Fax: +91 33 23705066; E-mail: shanta1232001@yahoo.co.in

Summary

Objectives  Shigellosis is a major public health problem, and increasing antimicrobial resistance has complicated its treatment. We report isolation frequency, plasmid profiles and antimicrobial resistance of Shigella subtypes in Kolkata, India, from a prospective hospital-based study.

Methods  Fresh stool or rectal swabs were collected from children (<5 years) attending the Diarrhea Treatment Unit of a governmental paediatric referral hospital in Kolkata. Samples were processed following standard methods over a 7-year period from January 2001 to December 2007.

Results  Of 4478 samples collected, 516 (11.5%) were positive for Shigella spp. S. flexneri (312; 6.9%) was the most frequently isolated serogroup, ranking before S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Although 10 subtypes of S. flexneri were identified, the common ones circulating locally were S. flexneri 2a (179), S. flexneri 6 (38) and S. flexneri 3a (36). Knowledge of Shigella subtypes is important for vaccine development. The majority of Shigella isolates (81.0%) were multidrug (two or more antimicrobial classes) resistant and showed high minimum inhibitory concentration (MIC) with commonly used drugs like ampicillin, tetracycline, co-trimoxazole and nalidixic acid. Emergence of fluoroquinolone (FQ)-resistant S. dysenteriae type 1 (100.0%) in 2002–2003 was followed by frequent isolation (>25.0%) of FQ-resistant S. flexneri 2a, and S. flexneri 3a in 2004, which restricted use of fluoroquinolones for treatment. A number of smaller plasmids (<20 kb) with distinct patterns have been observed for several years in predominant subtypes.

Conclusion  Long-term surveillance of Shigellae and their antimicrobial resistance are mandatory in endemic areas to formulate treatment policy until any suitable candidate vaccine is available to control the disease.

Prévalence des sous-types, profils plasmidiques et augmentation de la résistance de Shigella aux fluoroquinolones à Kolkata, en Inde (2001-2007): une étude en milieu hospitalier

Objectifs:  La shigellose est un problème majeur de santé publique et la résistance croissante aux antimicrobiens a compliqué son traitement. Nous rapportons la fréquence des isolements, les profils plasmidiques et la résistance aux antimicrobiens des sous-types Shigellaà Kolkata, en Inde dans une étude prospective basée sur l’hôpital.

Méthodes:  Des échantillons de selles fraîches ou des frottis rectaux ont été recueillis chez des enfants (<5 ans) fréquentant l’unité de traitement de la diarrhée d’un hôpital de référence pédiatrique gouvernemental à Kolkata. Les échantillons ont été traités suivant des méthodes standards sur une période de 7 ans, de janvier 2001 à décembre 2007.

Résultats:  Sur 4478 échantillons prélevés, 516 (11,5%) étaient positifs pour Shigella spp; S. flexneri (312; 6,9%) était le sérogroupe le plus fréquemment isolé, se classant devant S. sonnei (123, 2,7%), S. dysenteriae (48; 1,1%) et S. boydii (33; 0,7%). Bien que 10 sous-types de S. flexneri ont été identifiés, les plus courant circulant localement étaient S. flexneri 2a (179), S. flexneri 6 (38) et S. flexneri 3a (36). La connaissance des sous-types de Shigella est importante pour le développement de vaccins. La majorité des isolats de Shigella (81,0%) étaient multirésistants (à 2 ou plusieurs classes d’antimicrobiens) et montraient une CMI élevée pour des médicaments couramment utilisés tels que l’ampicilline, la tétracycline, le co-trimoxazole et l’acide nalidixique. L’émergence de S. dysenteriae de type 1 résistant aux fluoroquinolones (FQ) en 2002-2003 a été suivie par l’isolement plus fréquemment (> 25,0%) de S. flexneri 2a, 3a résistants aux FQ et la restriction de l’utilisation des médicaments. Un certain nombre de petits plasmides (<20Kb) avec des profils distincts ont été observés depuis plusieurs années dans les sous-types prédominants.

Conclusion:  La surveillance à long terme des Shigella et leur résistance aux antimicrobiens sont obligatoires dans les zones endémiques afin de pouvoir formuler une politique de traitement jusqu’à ce qu’un vaccin candidat approprié soit disponible pour lutter contre la maladie.

Mots-clés: Shigella, sous-types, résistance aux antimicrobiens, fluoroquinolone, plasmide

Estudio hospitalario de la prevalencia de subtipos, perfiles plasmídicos y aumento de la resistencia a fluoroquinolonas en Shigella spp. de Kolkata, India (2001-2007)

Objetivos:  La shigellosis es un importante problema de salud pública y el aumento en la resistencia a antimicrobianos ha complicado su tratamiento. Aquí reportamos la frecuencia de aislamiento, perfiles plasmídicos y resistencia a antimicrobianos de varios subtipos de Shigella en Kolkata, India como parte de un estudio hospitalario prospectivo.

Métodos:  Se recolectaron heces frescas y frotis rectales de niños (<5 años) que se presentaron en la Unidad para el Tratamiento de la Diarrea en el hospital pediátrico público de referencia en Kolkata. Las muestras se procesaron siguiendo métodos estándar durante un periodo de 7 años, desde enero del 2001 a diciembre del 2007.

Resultados:  De 4478 muestras recolectadas 516 (11.5%) eran positivas para Shigella spp.. S. flexneri (312; 6.9%) era el serogrupo más frecuentemente aislado, seguido por S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) y S. boydii (33, 0.7%). Aunque se identificaron 10 subtipos de S. flexneri, los más comunes eran S. flexneri 2a (179), S. flexneri 6 (38) y S. flexneri 3a (36). El conocimiento de los subtipos de Shigella es importante para el desarrollo de vacunas. La mayoría de aislados de Shigella (81.0%) eran multirresistentes (a 2 o más clases de antimicrobianos) y mostraban un CMI alta con los antibióticos más comúnmente utilizados, tales como la ampicilina, tetraciclina, cotrimoxazol y ácido nalidíxico. Trás la aparición, en 2002-03, de las primeras cepas de S. dysenteriae tipo 1 resistentes a fluoroquinolonas (FQ) el aislamiento de cepas de S. flexneri 2a y 3a resistentes ha sido cada vez más frecuente (>25.0%), teniendo que restringirse así el uso de estos antibióticos. Durante años se ha observado en algunos subtipos predominantes un cierto número de pequeños plásmidos (<20Kb) con patrones distintivos.

Conclusión:  La vigilancia a largo plazo de Shigella y de los niveles de resistencia a antimicrobianos es obligatoria en áreas endémicas para formular las políticas de tratamiento, hasta que se disponga de una vacuna que permita controlar la enfermedad.

Palabras clave: Shigella, subtipos, resistencia a antimicrobianos, fluroquinolonas, plásmidos.

Introduction

Shigellosis is a relatively common illness and one of the major causes of morbidity and mortality among children aged below 5 years in developing countries. Recent reports indicate that about 91 million people contract shigellosis each year only in Asia, of whom 410 000 children die because of malnourishment (WHO 2005b). Shigella spp., the causative agent, has high diversity among its subtypes. Occasionally, the organism has caused dysentery outbreaks affecting thousands of people and causing many deaths (Kotloff et al. 1999; Bercion et al. 2006) because of its high infectious potential and spread by direct faecal–oral contact when personal hygiene is compromised (Niyogi 2005).

Global change in epidemiology and increasing antimicrobial resistance in Shigella pose considerable challenges to public health experts for controlling the disease. While S. sonnei is the common serogroup in industrialized countries, S. flexneri continues to be the endemic strain in many parts of the developing world. In the early 1980, a dysentery epidemic caused by S. dysenteriae type 1 broke out in Eastern India, and the organism was resistant to commonly used and inexpensive antibiotics (Pal 1984). However, in the post-epidemic period, S. dysenteriae 1 was replaced by S. flexneri, which became increasingly common (Dutta et al. 1989). During 1995–2000, a hospital-based study in Kolkata reported S. flexneri (3.4%) as the predominant serogroup followed by S. sonnei (1.6%), S. boydii (0.5%) and S. dysenteriae (0.3%) (Dutta et al. 2002). Heterogeneous distribution of serogroups and shifting subtype dominance are common in Shigella and reported from India and other countries (Taneja 2007; Kansakar et al. 2007; Vinh et al. 2009). Knowledge of circulating Shigella subtypes in any region is important because protection against Shigella is serotype specific, and cross-protection between serotypes is limited (Sansonetti 2006).

Microbial drug resistance per se is a major problem because of its remarkable geographical variability, global dimension and alarming magnitude. Shigella is known for acquiring multidrug resistance, and monitoring drug resistance is important for management purposes. Antimicrobial therapy is particularly important in developing countries, where prolonged diarrhoea episodes can significantly slow the growth and worsen the nutritional status of children. Antimicrobial therapy for severe shigellosis included ampicillin or co-trimoxazole or nalidixic acid. But since the worldwide progressive development of resistance in Shigella to most of these antibiotics (Bogaerts et al. 1997; Dutta et al. 2002; Agtini et al. 2005), treatment with ciprofloxacin [a fluoroquinolone (FQ)] or one of the three-second-line antibiotics, pivmecillinam, azithromycin and ceftriaxone (a third-generation cephalosporin), has been recommended (WHO 2005a). However, there are reports of FQ-resistant Shigella isolates from India and other Asian countries (Seidlein et al. 2006; Taneja 2007). Thus, frequent change in antimicrobial resistance of Shigellae causes difficulty in recommending standard drugs effective for shigellosis.

Plasmid profile analysis of Shigella has been used by many researchers as an epidemiological tool for investigating Shigella outbreaks and determining strain diversity in particular serotypes (Farrar 1983; Lin et al. 2001; Farshad et al. 2006). The role of plasmids as a base for virulence or resistance genes including gene transfer is quite well documented (Lin & Chang 1992; Antoine et al. 2010). As large plasmids tend to be lost during cell storage and subculturing or plasmid extraction, smaller plasmids (<15 kb) are more stable. Analysis of smaller plasmids of local isolates is important because it gives insight into plasmid profile patterns, diversity, virulence and antimicrobial resistance traits of those isolates. Association of distinct plasmid profiles with specific Shigella subtypes has been reported earlier (Litwin et al. 1991; Dutta et al. 2002).

This hospital-based prospective surveillance for shigellosis is the continuation of an earlier study (Dutta et al. 2002); the reporting period is from January 2001 to December 2007. The objectives were to determine any change in isolation frequency and serotype switching of Shigella, to monitor antimicrobial resistance and minimum inhibitory concentrations (MICs) of antimicrobials, to study plasmid profiles of common Shigella subtypes and their stability.

Materials and methods

Study population

From January 2001 through December 2007, consecutive children aged <5 years with symptoms of acute diarrhoea and no history of antibiotic intake were enrolled irrespective of duration and severity of the disease when they attended the outpatient-based Diarrhoea Treatment Unit (DTU) of Dr B.C Roy Memorial Children’s Hospital, Kolkata, from Monday to Friday (10.00 am to 1.00 pm). Dr B.C. Roy Memorial Hospital, Kolkata, is the largest governmental paediatric referral hospital in Eastern Kolkata and provides free treatment to patients of all socioeconomic strata from Kolkata metropolis and to referred patients from other governmental hospitals of West Bengal.

Sample collection and microbiological processing

Stool specimens or rectal swabs were collected from study children into Mc Cartney bottles or Carry Blair medium, respectively, and were transported to the Bacteriology Division of National Institute of Cholera and Enteric Diseases (NICED), Kolkata, within 2 h for isolation and identification of Shigella following standard microbiological methods (WHO 1987). The samples were confirmed for yield of Shigellae subtypes by biochemical tests and commercially available antisera (Denka Seiken, Tokyo, Japan), and positives were included for analysis.

Determination of antimicrobial susceptibility and MICs of antimicrobials

The antimicrobial susceptibility of all Shigellae was determined by Kirby Bauer’s disc diffusion method using a panel of commercially available discs (Beckton Dickenson, MD, USA), which were ampicillin (A), tetracycline (T), chloramphenicol (C), co-trimoxazole (Q), nalidixic acid (Na), ciprofloxacin (Cip), norfloxacin (Nor), gentamicin (G), amikacin (Ak), ofloxacin (Ofx) and cefotaxime (Ctx). Results were interpreted as per Clinical Laboratory Standard Institute guidelines (NCCLS 2003). MICs of the antibiotics for resistant isolates were determined by E-test strips according to manufacturer’s recommendations (AB BIODISK, Solna, Sweden). E. coli ATCC 25922 was used as a reference strain for quality control checking.

Plasmid DNA profiles

Plasmid DNA of Shigella isolates was extracted by alkaline lysis method (Brinboin 1979) and separated by 0.8% agarose gel electrophoresis in 1× TAE buffer. DNA bands were visualized and photographed under UV light after staining with ethidium bromide (1.0%). Molecular masses of plasmid DNA of test isolates were determined by using super coiled DNA ladder (2–10 kb; Bangalore Genei, India) as molecular marker.

Data management and analysis

Data were entered into a pre-designed format in the excel spread sheet using data entry programmes and were randomly checked. Statistical analysis was carried out using spss version 12.0 for Windows (SPSS Inc., Chicago, IL, USA). A P-value of <0.05 was considered statistically significant.

Ethical clearance

Written consent was obtained from parents (or guardians) of all study children after explaining to them the purpose of this study. The study received approval from institutional ethical committee and scientific advisory committee, NICED, Kolkata. Patients were treated as per routine clinical management guideline of the hospital.

Definitions

Diarrhoea was defined as three or more loose bowel movements during a 24-h period, dysentery as one or more loose bowel movements with mucous or visible blood. A shigellosis episode was defined as a diarrhoea episode during which any Shigella species was isolated from a faecal specimen.

Results

A total of 4478 samples (3045, 68.0% rectal swabs; 1433, 32.0% stool) were collected from children (<5 years) with a clinical diagnosis of acute diarrhoea over a span of 7 years (January 2001–December 2007). Macroscopically, 1103 (77.0%) stool samples contained blood mucus, 115 (8.0%) were watery in nature, and only mucus was present in 215 (15.0%) samples. After processing all specimens, 516 (11.5%) samples yielded Shigella with distribution as follows: S. flexneri (312, 7.0%) being the major serogroup followed by S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Relatively higher isolation of Shigella was noted during the second half (2004–2007) of the study period [OR 1.90 (1.44–2.50); P < 0.001].

Of the 516 Shigella-positive children, 439 (85.0%) complained of blood-tinged mucoid stool, 35 (6.8%) had watery diarrhoea and 39 (7.5%) had mucus diarrhoea. On examination, 335 (65.0%) children had no dehydration, 176 (34.0%) had mild dehydration and one was moderately dehydrated. Children aged 1–5 years had significantly higher isolation of Shigella than infants (63.0%vs. 35.0%; P < 0.05). Boys (53.0%) were marginally more affected than girls (46.0%). Most (85.0%) patients belonged to low socioeconomic class and presented with features of malnutrition.

Figure 1 shows that S. flexneri was the most predominant serogroup throughout the study period. Relative isolation frequency of S. flexneri (60.0–70.0%) was significantly higher than that of S. sonnei (20.0–30.0%), S. dysenteriae (5.0–15.0%) and S. boydii (4.0–9.0%) (P < 0.05). No specific seasonal variation was observed for Shigella isolation, although increased attendance of diarrhoeal children in the hospital was noted during pre-monsoon and monsoon months (April to August).

Figure 1.

 Yearwise isolation frequency (%) of Shigella serogroups in Kolkata (2001–2007). SD, S. dysenteriae; SF, S. flexneri; SB, S. boydii; SS, S. sonnei.

The distributions of Shigellae serogroups and subtypes of S. flexneri are shown in Figure 2. Although 10 subtypes of S. flexneri were isolated, the predominant circulating subtypes in the order of descending frequency were S. flexneri 2a (179/312; 57.4%), S. flexneri 6 (38/312; 12.2%) and S. flexneri 3a (36/312; 11.5%). Seven strains became non-viable and were lost during the study period. Six isolates, biochemically identical with Shigella, were agglutinable with S. flexneri polysera (Group B) (Denka Seiken), but untypable with monosera. Those strains were sent to the Metropolitan Research Laboratory of Public Health, Tokyo, Japan, for further serotyping and were identified as S. flexneri 88-893, a provisional serotype, reported earlier (Ueda et al. 2001). Among 48 S. dysenteriae isolates, S. dysenteriae type 1 (27 isolates) was the most frequently isolated subtype during 2002–2004. Interestingly, no S. dysenteriae 1 has been isolated since 2005. Other dysenteriae subtypes were S. dysenteriae 2 (six isolates), S. dysenteriae 3 (three isolates), S. dysenteriae 12 (three isolates), S. dysenteriae 4 (one isolate), S. dysenteriae 6 (two isolates) and S. dysenteriae 9 (two isolates). Four isolates belonged to S. dysenteriae 204/96, a new serovar as reported earlier (Dutta et al. 2003a). Among S. boydii (33 isolates), major subtype was S. boydii 14 (nine isolates) followed by S. boydii 12 (seven isolates), S. boydii 8 (six isolates), S. boydii 2 (four isolates), S. boydii 4 (two isolates), S. boydii 5 (two isolates) and one new serovar of S. boydii E16553. Two isolates became non-culturable and could not be revived. Most of the S. boydii isolates were resistant to tetracycline and cotrimoxazole.

Figure 2.

 Frequency distribution of Shigella serogroups and S. flexneri subtypes in Kolkata (n = 516). SD, S. dysenteriae; SF, S. flexneri; SB, S. boydii; SS, S. sonnei.

Table 1 shows that of 516 Shigella strains only nine isolates were susceptible to all tested antimicrobials. Resistance to common drugs like ampicillin, chloramphenicol, cotrimoxazole and tetracycline was observed in approximately 46.0% (238 of 516), 47.0% (242/516), 90.0% (465/516) and 87.0% (447/516) isolates, respectively. Subsequently, amoxicillin–clavulanic (AmC) discs were introduced since 2005 expecting better result with the combination drugs, but 40.6% (70 of 174) of the isolates turned out to be resistant to AmC.

Table 1.   Antimicrobial resistance of Shigella isolates in Kolkata during 2001–2007
Antimicrobial disc (symbol)Disc potency (μg)2001 (n = 89)2002 (n = 101)2003 (n = 55)2004 (n = 97)2005 (n = 56)2006 (n = 59)2007 (n = 59)
  1. *P < 0.0001; significantly increasing trend of antimicrobial resistance of Shigella over the years.

  2. †Appearance of fluoroquinolone-resistant S. dysenteriae type 1.

  3. ‡Appearance of fluoroquinolone-resistant S. flexneri 2a strains.

Ampicillin (A)1034 (38.20)41 (40.59)27 (49.09)55 (56.70)25 (44.64)30 (50.84)26 (44.06)
Tetracycline (T)3077 (86.51)80 (79.20)44 (80.00)87 (89.69)49 (87.50)56 (94.91)54 (91.52)
Chloramphenicol (C)3033 (37.07)37 (36.63)24 (43.63)58 (59.79)27 (48.21)33 (55.93)30 (50.84)
Co-trimoxazole (Q)2583 (93.25)88 (87.12)44 (80.00)86 (88.65)55 (98.21)58 (98.30)51 (86.44)
Nalidixic acid (Na)*3055 (61.79)72 (71.28)36 (65.45)88 (90.72)51 (91.07)55 (93.22)55 (93.22)
Ciprpfloxacin (Cip)*51 (1.12)7 (6.93)†8 (14.54)†32 (32.98)‡26 (46.42)32 (54.23)28 (47.45)
Norfloxacin (Nor)*100 (0)7 (6.93)†8 (14.54)†32 (32.98)‡25 (44.64)32 (54.23)27 (45.76)
Gentamicin (G)101 (1.20)12 (11.88)3 (5.45)0 (0)1 (1.78)1 (1.69)0 (0)
Amikacin (Ak)301 (1.12)7 (6.93)2 (3.63)1 (1.03)3 (5.35)0 (0)0 (0)
Cefotaxime (Ctx)300 (0)0 (0)0 (0)0 (0)0 (0)0 (0)0 (0)
Ofloxacin (Ofx)*50 (0)6 (5.94)†6 (10.90)†29 (29.89)‡22 (39.28)32 (54.23)28 (47.45)

Shigella also showed resistance to quinolone-group drugs like Na (80.0%), Cip (26.0%), Nor (25.0%) and Ofx (24.0%). A significantly increasing trend of resistance was noted against these drugs over the years (P < 0.0001). FQ-resistant S. dysenteriae type 1 emerged in Kolkata in 2002 followed by the appearance of FQ-resistant S. flexneri 2a in 2004, which completely replaced the susceptible isolates since 2005. Six of 36 S. flexneri 3a isolates were FQ resistant and four of them were isolated in 2007. We did not isolate any FQ-resistant S. sonnei until 2007. All (100%) Shigellae were susceptible to cephalosporins when tested with cefixime (5 μg), ceftriaxone (30 μg), ceftazidime (30 μg), cefpodoxime (10 μg) and cefotaxime (30 μg).

Comparisons of antibiotic resistance of common serogroups S. flexneri, S. dysenteriae and S. sonnei are shown in the bar diagram (Figure 3). Average resistance was more common in S. dysenteriae and S. flexneri isolates than S. sonnei. Significantly higher resistance was observed in S. sonnei (>80.0%) isolates towards T, Q and Na than towards other antibiotics.

Figure 3.

 Comparison of antimicrobial resistance among three Shigella serogroups. SD, S. dysenteriae; SF, S. flexneri; SS, S. sonnei.

The median MIC90 (i.e. minimum concentrations of antibiotics at which growth of 90.0% of a population of Shigella isolates were inhibited) of antimicrobials was determined for most common subtypes like S. flexneri 2a (50 isolates), S. dysenteriae type 1 (20 isolates) and S. sonnei (30 isolates). The isolates showed increased MICs towards most of the antimicrobials like A (126 μg/ml), T (96 μg/ml), C (64 μg/ml), Na (256 μg/ml) and Q (32 μg/ml). FQ-resistant S. flexneri 2a and S. dysenteriae 1 had MICs of ≥8, ≥24 and ≥12 μg/ml for Cip, Nor and Ofx, respectively. On an average, the MIC90 for S. dysenteriae 1 was higher than that for S. flexneri 2a.

Table 2 shows the distribution of common resistance (R) profiles among Shigella subtypes in Kolkata. Most (417/516; 81.0%) of the Shigella isolates were multi-drug resistant (MDR, resistant to two or more antibiotic classes) and 220 (42.6%) isolates were resistant to at least five or more drugs. TRQRNaR was the most frequent resistance profile seen in S. sonnei (100/123; 81.0%) and S. flexneri 6 (15/38; 39.5%). Before 2004, an ARCRTRQRNaR profile was common in S. flexneri 2a (35/179, 19.5%) and S. flexneri 3a (6/36, 16.6%) isolates. Concurrent resistance to a large number (≥8) of antimicrobials (ARCRTRQRNaRCipRNorROfxR) was noted after 2002 in the emerging S. dysenteriae 1 (20/20; 100.0%), S. flexneri 2a (81/179; 45.0%) and 3a (6/36; 16.6%) isolates, which was alarming.

Table 2.   Yearwise distribution of common antimicrobial resistance profiles of Shigella isolates in Kolkata
Common resistance profilesSerotypeYearwise distribution
2001200220032004200520062007
  1. R, resistance; A, ampicillin; C, chloramphenicol; T, tetracycline; Q, cotrimoxazole; Na, nalidixic acid; Cip, ciprofloxacin; Nor, norfloxacin; Ofx, ofloxacin; SF, S. flexneri; SD, S. dysenteriae; SS, S. sonnei.

ARCRTRQRNaRSF 2a 2 9 618 0 0 0
SF 3a 3 3 0 0 0 0 0
Other SF 5 2 2 5 2 0 0
SF X
SF Y
SF 4a
SF 2b
       
ARCRTRQRNaR
CipRNorROfxR
SF 2a
SD 1
0
 0
0
 7
0
 5
20
 7
16
 1
27
 0
18
 0
TRQRNaRSS
SF 6
Other
SD
21
 0
 0
19
 2
 0
10
 1
 0
13
 2
 2
10
 3
 1
13
 2
 0
14
 5
 0
CRQRNaRSF 2a1112111

Plasmid profiles of some representative strains of predominant Shigella subtypes are shown in Figure 4 (Panel a, S. dysenteriae 1; Panel b, S. sonnei; and Panel c, S. flexneri 2a). Large plasmids (approximately 220 kb) were present in >90% MDR S. flexneri 2a and S. dysenteriae 1 isolates, but absent in S. sonnei (data not shown). All common subtypes possessed at least two or more copies of smaller plasmids (<20 kb), revealing specific patterns for subtypes with a common resistance profile (Figure 4).

Figure 4.

 0.8% Agarose gel electrophoresis of plasmid DNA from three common Shigella serotypes in Kolkata: Panel a, S. dysenteriae type 1; Panel b, S. sonnei; Panel c, S. flexneri 2a. In each panel, supercoiled plasmid DNA ladder was used as marker (lane M).

All MDR S. dysenteriae type 1 isolates harboured at least three smaller plasmids of sizes 11.7, 9.1 and 3.0 kb. Plasmids of 5.7, 3.9 and 3.1 kb were observed in FQ-resistant S. flexneri 2a isolates. Shigella sonnei with TRQRNaR profile showed the presence of four plasmids of 1.8, 2.7, 4.6 and 6.5 kb. The plasmid profiles were compared with the profiles obtained in earlier study (Dutta et al. 2002).

Discussion

In this study, we report the isolation frequency, serotype switch and antimicrobial resistance pattern of Shigella strains isolated from Kolkata, India, over a period of 7 years from 2001 to 2007. We used passive surveillance for case detection, which largely depends on health care seeking behaviour of the study population. Therefore, a significant number of cases may have been missed and the actual rate of Shigella infection may be much higher than reported. The current practice of indiscriminate use of antibiotics against diarrhoea limited the collection of stool samples as per the inclusion criteria of the study, causing a considerable drop in the number of examined stool samples during 2005–2007.

Overall, Shigella were isolated by classical microbiology procedure from 11.5% of under-fives with diarrhoea who attended the DTU of a governmental referral hospital, almost double the overall rate (6.0%) observed during 1995–2000 (Dutta et al. 2002). The situation is similar in neighbouring Asian countries like China, Thailand, Indonesia, Vietnam, Pakistan and Bangladesh, where culture-confirmed cases varied from 2.0% in Thailand to 13.0% in Bangladesh during 2000–2004 (Seidlein et al. 2006). Studies from other regions of India also confirmed our findings (Pazhani et al. 2005; Taneja 2007; Srinivasa et al. 2009).

Shigella flexneri constituted the predominant (312/516; 60.5%) serogroup and S. flexneri 2a (179/312; 57.3%), the frequently isolated subtype in Kolkata, and this trend remained unchanged for the last 14 years (1994–2007). This distribution of Shigella strains is frequently observed in many other developing countries (Ahmed et al. 2006; Seidlein et al. 2006; Bercion et al. 2008). Throughout the study period, an association was observed between periodic increase in isolation of total Shigellae and that of total S. flexneri (Figure 1). The major serogroup isolated following S. flexneri was S. sonnei (123/516; 24.0%) and both serogroups together constituted almost 85.0% of total Shigellae isolates. Other common subtypes were S. flexneri 6 (38/312; 12.1%) and S. flexneri 3a (36/312; 11.5%). These findings contradict the aim of developing serotype-based vaccine and emphasize the development of cross-protective vaccine candidates.

Although serotype shift was not obvious during the study period, increased admission of dysentery patients to the hospitals and increased isolation of S. dysenteriae 1 was reported during 2002–2004, which re-emerged after a gap of 18 years (Dutta et al. 2003a,b) since its first appearance in 1984 (Pal 1984). The emerged organism was MDR including FQs e.g. ciprofloxacin, norfloxacin, ofloxacin and had the potential to cause several dysentery outbreaks in different parts of India (Sarkar et al. 2003; Niyogi et al. 2004). Similar FQ-resistant S. dysenteriae type 1 strains were also isolated from sporadic outbreak cases in Nepal, Pakistan and Bangladesh, leading to a regional alert in South East Asia (Bhattacharya et al. 2003; Kansakar et al. 2007). Surprisingly, after 2004, there was no report of dysentery outbreak and isolation of S. dysenteriae 1 also remained low. But FQ-resistant S. flexneri 2a started appearing for the first time in Kolkata and growing FQ resistance in Shigella isolates became a cause of concern regarding treatment of the disease. Although FQ-resistant S. sonnei was not detected during the study period, such strains may be presumed to be isolated during the years to come. The sudden disappearance of S. dysenteriae 1 since 2005 after causing the menace is interesting and warrants further investi-gation.

The Shigella subtypes follow one cyclical pattern and a change in the trend is evident from reports of other countries. In recent years (2001–2005), a serotype switch from S. flexneri to S sonnei was noticed in Iran, Thailand, Taiwan, Sri Lanka and later in Vietnam (Chompook et al. 2005; Kuo et al. 2008; Vinh et al. 2009). Some researchers reported that predominant Shigella serogroup in a country predicts the economic development of that country (Seidlein et al.2006). Emergence of a new serotype is common in Shigella species (Matsushita et al. 1998; Coimbra et al. 2001; Dutta et al. 2003a). In this study, we report six strains of S. flexneri 88-893, four strains of S. dysenteriae 204/96 and four strains of S. boydii E16553, provisional serovars isolated from Kolkata. Recently, a study from Pakistan reported 20.0% of isolates suggestive of Shigella remained untypable (Zafar et al. 2009). All these observations emphasize the need for surveillance of Shigella species in endemic regions for detailed understanding of epidemiology of shigellosis.

Mild symptoms are self-limiting but antibiotics are recommended for the cure of severe dysentery cases to reduce the duration of diarrhoea. The antibiotic resistance traits of Shigellae are diverse and regionally different. The choice of antibiotics becomes limited because of worldwide increase in MDR strains. Gradual increase in resistance towards ampicillin, tetracycline, chloramphenicol, co-trimoxazole was observed in Kolkata isolates during the study period. Emergence of growing numbers (>25.0%) of FQ-resistant Shigella strains was worrying and restricted the use of these drugs. Pulsed field gel electrophoresis showed >80% similarity and high clonality among the emerging FQ-resistant S. dysenteriae 1 (Dutta et al. 2003b; Talukder et al. 2004) and S. flexneri 2a (S. Nandy, U. Mitra, S. Dutta, unpublished data) isolates. CipR-resistant S. flexneri was also identified in other Asian countries like China (6.0%), Pakistan (3.0%), Vietnam (2.0%) (Seidlein et al. 2006) and recorded from other parts of India (Taneja 2007). Fortunately, all isolates were susceptible to third-generation cephalosporin, which is consistent with other international data.

Profiles of smaller plasmids of Shigella followed a specific pattern and the correlation between plasmid patterns and subtypes may be useful for species or subtype identification (Dutta et al. 2002; Lin & Chang 1992; Litwin et al. 1991). The smaller plasmids (5.7, 3.9, 3.1 kb) of S. flexneri 2a were similar to those obtained in earlier study conducted during 1995 to 2000 (Dutta et al. 2002). Acquisition of plasmids (4.6 and 1.8 kb) was observed in S. sonnei in addition to the plasmids (6.5 and 2.7 kbs) reported earlier. This indicated that each of the common Shigella subtypes was associated with a distinct plasmid pattern and this association has remained uniform for the past 13 years (1995–2007). Smaller plasmids were seen to have a high rate of persistence for several years in almost all Shigella strains, which suggests that they may be important for virulence or survival. But this hypothesis is yet to be proved.

In the light of our findings, we strongly recommend that continuous monitoring, periodic analysis and reporting of antibiotic susceptibility of local Shigella isolates are necessary for appropriate selection of drugs and development of treatment guidelines for shigellosis in any endemic region. Uncontrolled use of any antibiotic is likely to provoke a major crisis by enhancing the emergence of resistant isolates, which is a major threat to diarrhoea disease control. Although good hygiene, improved sanitation and provision of safe water can effectively reduce the burden of shigellosis, implementing these measures in resource-poor settings within a short period is unrealistic. Therefore, development of a reliable cross-protective candidate Shigella vaccine remains the best option to control the disease.

Acknowledgements

Part of the study was carried out during the tenure of JSPS RONPAKU fellowship awarded to Dr S. Dutta (2001–2005) from the Japan Society for the Promotion of Science. We thank Dr Shigeru Matsushita, Metropolitan Research Laboratory of Public Health, Tokyo, Japan, for typing the new serovars of Shigella. Technical work performed by Mr D. K. Banerjee and Mr A. Ganai is gratefully acknowledged.

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