Genetic characteristics and changing antimicrobial ...

Journal of Medical Microbiology (2011), 60, 1460–1466 DOI 10.1099/jmm.0.032920-0

Correspondence Genetic characteristics and changing antimicrobial
T. Ramamurthy resistance among Shigella spp. isolated from
[email protected] hospitalized diarrhoeal patients in Kolkata, India

Received 6 April 2011 Santanu Ghosh,13 G. P. Pazhani,13 Goutam Chowdhury,1 Sucharita Guin,1
Accepted 6 June 2011 Sanjucta Dutta,1 K. Rajendran,1 M. K. Bhattacharya,1 Y. Takeda,2
S. K. Niyogi,1 G. Balakrish Nair1 and T. Ramamurthy1

1National Institute of Cholera and Enteric Diseases, Kolkata, India
2Collaborative Research Center of Okayama University for Infectious Diseases in India, National
Institute of Cholera and Enteric Diseases, Kolkata, India

To study the prevalence pattern and trends in the phenotypic and genetic characteristics of
shigellae, we tested 212 isolates isolated from diarrhoeal patients admitted to the Infectious
Diseases Hospital, Kolkata, India, from November 2007 to October 2010. Prevalence of Shigella
spp. was higher in the .5 years age group (69 %) than in children in the ,5 years age group
(31 %). Serotypes 2a, 3a and untypable isolates of Shigella flexneri were frequently detected. An
increase in the isolation of Shigella sonnei (15 %) is a novel trend in this region. Fluoroquinolone
resistance among S. flexneri serotypes 2a, 3a and other serogroups of shigellae is another
evolving trend. The set gene was exclusively present in S. flexneri 2a, and the sen gene was
detected in all serogroups. PFGE revealed the grouping of S. flexneri isolates according to their
serotypes with approximately 80–100 % similarity, whilst Shigella dysenteriae type 2 and
S. sonnei were clonal in nature. There was no demarcation in the prevalence of serotypes,
antimicrobial resistance or clonality between the two age groups.

INTRODUCTION et al., 2010). These drugs are also effective among
immunocompromised children, including neonatal pa-
Shigellosis remains a considerable public-health problem tients, suffering from infections caused by multi-drug-
in many parts of the world. About 125 million cases of resistant strains of Salmonella and Shigella spp. (Leibovitz,
Shigella infection occur annually in Asia, of which 2006). The use of antimicrobials for the treatment of
approximately 14 000 are fatal (Bardhan et al., 2010). The shigellosis varies from country to country. Pivmecillinam is
distribution of serogroups of Shigella spp. differs from not the drug of choice for the treatment of diarrhoea in
country to country: Shigella flexneri, Shigella sonnei and many countries, but is being used in Bangladesh (Rahman
Shigella boydii serogroups are predominant in developing et al., 2007). Ceftriaxone is an ideal drug for the treatment of
countries, whilst S. sonnei is frequently reported in in- shigellosis in regions where fluoroquinolone resistance is
dustrialized countries. Prevalence of Shigella dysenteriae is common. The American Academy of Pediatrics and the
mostly reported from South Asia and sub-Saharan Africa Infectious Diseases Society of America recommend azithro-
(Kotloff et al., 1999). Unlike other acute diarrhoeal illnesses mycin as an alternative drug for the treatment of shigellosis
that require adequate fluid replacement by either oral or (Jain et al., 2005) and this drug was found to be effective
intravenous rehydration, shigellosis needs antimicrobial against multi-drug-resistant Shigella spp. in many countries.
therapy to reduce the duration of the illness and to prevent
transmission to close contacts. Shigella spp. have developed Due to the distribution of diverse serogroups/serotypes
perpetual resistance to ampicillin and trimethoprim– and the combination of resistance to several antimicrobial
sulfamethoxazole, which have been used in paediatric cases drugs, treatment failures are often seen in Shigella-mediated
during the past two decades. Fluoroquinolones, ceftriaxone infections. Hence, it is very important to understand the
and pivmecillinam are the antibiotics currently recom- serogroup(s) and antimicrobial-resistance patterns of
mended by the World Health Organization for the Shigella spp. prevailing in different geographical regions
treatment of dysentery in children, as these drugs are for instituting effective treatment.
effective at reducing mortality caused by Shigella spp. (Traa
In Kolkata, multi-drug resistance in Shigella spp. has been
3These authors contributed equally to this work. increasingly reported since the emergence of fluoroquino-
lone-resistant strains of epidemic S. dysenteriae type 1

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(Pazhani et al., 2005). This trend has been rising among 2009. After serotype 2a, prevalence of S. flexneri 3a was the
diverse serogroups of Shigella spp., not only in Kolkata but next highest in this study. Interestingly, 19 (11.8 %) S.
also in other parts of India (Srinivasa et al., 2009). In this flexneri isolates agglutinated only with polyvalent type B
study, we analysed the extent of antimicrobial resistance, antiserum; complete serotyping of these isolates was not
distribution of virulence genes and clonality of diverse accomplished with commercially available Shigella antisera
serogroups of Shigella isolates from hospitalized children and none of them reacted with the provisional S. flexneri
and adult patients with acute diarrhoea. monoclonal antiserum (# 88-893). These isolates were
designated untypable. S. dysenteriae types 1 and 3 (one
METHODS isolate each) and three isolates of type 2 were isolated in
this study. Among 14 S. boydii isolates, type 12 was
Bacterial strains. From November 2007 to October 2010, 212 relatively common (four isolates) and the others were
(6.5 %) Shigella isolates were isolated from 3262 enrolled diarrhoeal represented by serotypes 1, 11 and 15 (two isolates each)
patients admitted to the Infectious Diseases Hospital, Beliaghata, and serotypes 9, 18 and 10 (one isolate each). One strain
Kolkata, India. Stool specimens collected from these patients were agglutinated with only polyvalent S. boydii antiserum, but
examined for common enteric pathogens within 2 h, using standard not with any of the monovalent antisera.
microbiological methods (Nair et al., 2010; WHO, 1987). Bio-
chemically identified Shigella isolates were confirmed serologically by More than 75 % of Shigella-positive patients were infected
slide agglutination using commercially purchased antisera (Denka with S. flexneri, followed by S. sonnei (15.6 %). About 31 and
Seiken Co. Ltd). The untypable S. flexneri isolates were tested with a 69 % of Shigella-infected patients were children of ,5 years
provisional monoclonal antiserum (# 88-893; a gift from N. and .5 years of age, respectively. Ninety per cent of the
Strockbine, CDC, Atlanta, GA, USA). Institutional ethical clearance Shigella isolates in this study were resistant to multiple drugs
was obtained to conduct this study. and the prevalence of different resistance patterns is shown in
Table 1. Overall, the majority of the isolates were resistant
Antimicrobial-susceptibility testing and determination of MIC. to trimethoprim–sulfamethoxazole (94.3 %), nalidixic acid
Antimicrobial-susceptibility testing was performed on all Shigella (93.4 %), tetracycline (88.7 %), ciprofloxacin, norfloxacin,
isolates by a disc-diffusion method in accordance with the Clinical ofloxacin (85.8 % each), furazolidone (79.2 %) and chlor-
and Laboratory Standards Institute guidelines (CLSI, 2010), using amphenicol (62.7 %). Resistance to ampicillin (53.8 %) and
commercially available ampicillin (10 mg), azithromycin (15 mg), azithromycin (34.4 %) was moderate, whilst much lower
ceftriaxone (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), resistance was observed to the third-generation cephalosporin
co-trimoxazole (25 mg), furazolidone (100 mg), nalidixic acid (30 mg), ceftriaxone (1.9 %). Among S. flexneri, serotype 2a was totally
norfloxacin (10 mg), ofloxacin (5 mg) and tetracycline (30 mg) discs resistant to nalidixic acid, ciprofloxacin, norfloxacin, oflox-
(BD) on Mueller–Hinton agar (Difco). MICs of nalidixic acid, acin and streptomycin. Ninety-six per cent of isolates were
ciprofloxacin, norfloxacin, ofloxacin and ceftriaxone were determined resistant to co-trimoxazole and tetracycline and 90 % of
by using an E-test (AB bioMe´rieux), following the manufacturer’s isolates were resistant to ampicillin and chloramphenicol.
instructions. Escherichia coli ATCC 25922 was used as the quality- Resistance to azithromycin was detected among 32 % of
control strain for each batch of the assay. isolates, whilst 1.3 % of isolates were resistant to ceftriaxone.
MIC ranges of S. flexneri type 2a for nalidixic acid, nor-
Detection of virulence genes. Virulence genes such as set (ShET-1), floxacin, ciprofloxacin and ofloxacin were .256, 20–24, 5–8
sen (ShET-2), ipaH, ial, sat and virF were detected by a simplex PCR and 8–16 mg ml21, respectively. This trend was more or less
assay using published primer pairs (Frankel et al., 1989; Thong et al., similar to the results for the other S. flexneri serotype, 3a. S.
2005). sonnei isolates, on the other hand, were susceptible (100 %)
to ampicillin and ceftriaxone. The majority of the S. sonnei
PFGE. PFGE of NotI- or XbaI-digested genomic DNA was performed isolates were resistant to ciprofloxacin (94 %), norfloxacin
using a CHEF-Mapper (Bio-Rad Laboratories) according to the (94 %), ofloxacin (94 %), co-trimoxazole (94 %) and
PulseNet standardized protocol for subtyping of Shigella species tetracycline (85 %), but resistance to azithromycin was
(Ribot et al., 2006). PFGE images were captured by using a Gel Doc lower (27 %). The MIC ranges of S. sonnei for nalidixic acid,
XR system (Bio-Rad). The gel images were normalized by aligning the norfloxacin, ciprofloxacin and ofloxacin were .256, 12–20,
peaks of the ‘Salmonella braenderup’ size standard and analysed by 4–6 and 8–12 mg ml21, respectively. Fluoroquinolone res-
using the BioNumerics software version 5.0 (Applied Maths). Degree istance was lower in S. boydii, with the exception of one
of banding similarity was determined by comparison of the Dice isolate (serotype 18) that was highly resistant to all
coefficient, and the clustering correlation coefficients were calculated antimicrobial agents tested and its MICs for nalidixic acid,
by UPGMA. norfloxacin, ciprofloxacin, ofloxacin and ceftriaxone were
.256, .256, .32, .32 and .32 mg ml21, respectively.
RESULTS MIC ranges of other S. boydii isolates for nalidixic acid,
norfloxacin, ciprofloxacin and ofloxacin were 7 to .256,
During this study, 212 (6.5 %) Shigella isolates were 0.38–6, 0.22–1.5 and 0.38–4 mg ml21, respectively. Only one
identified from 3262 diarrhoeal patients. The numbers S. dysenteriae type 1 isolate was identified in this study
and proportions of different Shigella serogroups detected during 2010, and it was resistant to all antimicrobials tested
were 160 (75.5 %) S. flexneri, 33 (15.6 %) S. sonnei, 14
(6.6 %) S. boydii and 5 (2.3 %) S. dysenteriae. Throughout
the study period, S. flexneri was the most common
serogroup, with a predominance of serotype 2a during

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Table 1. Antimicrobial-resistance patterns of Shigella species and their serotypes

AM, Ampicillin; AZM, azithromycin; C, chloramphenicol; CIP, ciprofloxacin; CRO, ceftriaxone; FX, furazolidone; NA, nalidixic acid; NOR, norfloxacin;
OFX, ofloxacin; SXT, co-trimoxazole; TE, tetracycline; Sf, S. flexneri; Sf UT, S. flexneri untypable; Sb, S. boydii; SS, S. sonnei; Sd, S. dysenteriae.

Resistance profile Species/serotype (n)

AM, AZM, C, CIP, CRO, FX, NA, NOR, OFX, SXT, TE Sb 18 (1), Sf UT (1)
AM, AZM, CIP, CRO, FX, NA, NOR, OFX, SXT, TE Sf UT (1)
AM, C, CIP, CRO, FX, NA, NOR, OFX, SXT, TE Sf 2a (1)
AM, AZM, C, CIP, FX, NA, NOR, OFX, SXT, TE Sf 2a (24), Sf 3a (2), Sf UT (1)
AM, C, CIP, FX, NA, NOR, OFX, SXT, TE Sf 1b (1), Sf 2a (32), Sf 3a (2), Sf Y (1), Sf 2b (1), Sd 1 (1)
AZM, C, CIP, FX, NA, NOR, OFX, SXT, TE Sf 2a (1), Sf 3a (9), Sf 3b (1), SS (1)
AM, AZM, CIP, FX, NA, NOR, OFX, SXT, TE Sf UT (2)
AM, C, CIP, NA, NOR, OFX, SXT, TE Sf 2a (11), Sf 3a (1), Sf 4 (1)
AM, CIP, FX, NA, NOR, OFX, SXT, TE Sf 2a (1), Sb 9 (1), Sf UT (1), Sb 12 (2)
AM, AZM, C, CIP, NA, NOR, OFX, SXT Sd 2 (1)
C, CIP, FX, NA, NOR, OFX, SXT, TE Sf 3a (17), Sf 3b (1), SS (1)
AZM, CIP, FX, NA, NOR, OFX, SXT, TE SS (5), Sf 6 (2), Sb 1 (1)
AZM, C, CIP, NA, NOR, OFX, SXT, TE Sf 3a (1)
AM, C, CIP, FX, NA, NOR, OFX, TE Sf 2a (2)
CIP, FX, NA, NOR, OFX, SXT, TE Sf 2a (3), SS (18), Sf Y (1)
C, CIP, NA, NOR, OFX, SXT, TE Sf 3a (10), Sf 3b (1)
AM, AZM, CIP, NA, NOR, OFX, SXT Sd 2 (2)
AZM, CIP, FX, NA, NOR, OFX, SXT SS (1), Sf 2a (1)
AM, C, CIP, NA, NOR, OFX, TE Sf 3a (1), Sf 2a (1)
AM, AZM, CIP, FX, NA, NOR, OFX, Sb 12 (1),
AZM, CIP, NA, NOR, OFX, SXT, TE SS (1),
CIP, FX, NA, NOR, OFX, SXT Sf 2a (1), SS (1)
CIP, NA, NOR, OFX, SXT, TE Sf 2a (2), SS (1), Sf 6 (1)
C, CIP, FX, NA, NOR, OFX, TE SS (1)
AM, AZM, FX, NA, SXT, TE Sf UT(1)
AZM, FX, NA, SXT, TE SS (1), Sf UT(1)
CIP, FX, NA, NOR, OFX SS (1), Sf 3a (1)
AM, AZM, FX, SXT, TE Sf UT (2)
AM, FX, NA, SXT, TE Sf UT (3), Sb 1 (1)
C, FX, NA, SXT, TE Sf 3a (1)
AM, FX, SXT, TE Sf UT (2), Sf 4 (2)
C, NA, SXT, TE Sf 3a (2)
AZM, NA, SXT, TE Sf 6 (1)
AM, AZM, SXT, TE Sb 15 (1), Sf UT (1)
AZM, FX, NA, SXT Sb 11 (1)
AM, FX, SXT Sf UT (1)
FX, NA, SXT SS (1), Sb 11 (1)
AM, AZM, FX Sb UT (1)
NA, SXT, TE Sb UT (1)
AM, SXT Sb 15 (1)
AZM, SXT Sd 4 (1)
AZM, NA Sf UT (1)
AZM, FX Sb 10 (1)
AM, FX Sf UT (1)

except ceftriaxone and azithromycin. All S. dysenteriae type 2 harboured a virulence plasmid (virF). The secreted
isolates exhibited reduced susceptibility to fluoroquino- autotransporter toxin Sat, encoded by the sat gene
lones. MIC ranges of S. dysenteriae for nalidixic acid, harboured by 73.5 % of the Shigella isolates (156 of 212),
norfloxacin, ciprofloxacin and ofloxacin were 2.5–14, 0.125– was present in three of five (60 %) S. dysenteriae, 146 of 160
1.5, 0.109–0.315 and 0.047–0.5 mg ml21, respectively, and (91.2 %) S. flexneri and seven of 14 (50 %) S. boydii isolates.
no S. dysenteriae isolates were resistant to ceftriaxone. None of the S. sonnei isolates harboured this gene. The set
gene that encodes Shigella enterotoxin 1 (ShET1), which
All isolates tested were positive for the invasive plasmid contains two subunits, A and B, was exclusively present in
antigen H (ipaH) gene, and 75 % of Shigella isolates all isolates of S. flexneri serotype 2a. One S. flexneri serotype

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Fig. 1. NotI-PFGE profiles of S. flexneri
isolates of different serotypes, with dendro-
gram showing percentage similarity.

variant Y [agglutinated with polyvalent B and group 3(4) sonnei (8.4 %), S. boydii (4.2 %) and S. dysenteriae (2.3 %).
sera] harboured only the ShET1 subunit A gene. The sen One S. dysenteriae type 1 isolate in this study harboured the
gene encoding ShET2 was present in 194 of 212 (91.5 %) stx1 gene encoding Shiga toxin.
Shigella isolates, more specifically in 144 of 160 (90.0 %) S.
flexneri, 32 of 33 (96.9 %) S. sonnei, 13 of 14 (92.8 %) S. PFGE analysis of representative serotypes of S. flexneri
boydii and all S. dysenteriae (100 %). The invasive- after digestion with NotI revealed serotype-specific
associated locus, encoded by the ial gene, was present in clusters, with approximately 80–100 % similarity within
164 of 212 (77.3 %) Shigella isolates, and was most each serotype (Fig. 1). When XbaI-digested genomic
predominant among S. flexneri (62.2 %), followed by S. DNAs of current S. dysenteriae type 1 isolates and

Fig. 2. XbaI-PFGE profiles of S. dysenteriae
serotype 1 and 2 isolates, with dendrogram
showing percentage similarity.

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S. Ghosh and others

reported from travellers in Japan with diarrhoea
(Matsushita et al., 1992) and from a restaurant-associated
outbreak in California (Trevejo et al., 1999). In Bangladesh,
about 20 % of S. flexneri isolates were untypable and some
of them cross-agglutinated with S. boydii-specific antisera
(Talukder et al., 2001). Emergence of S. flexneri serotype 1c
was identified in Bangladesh, Egypt, Indonesia and
Pakistan. In the province of Son Tay, Vietnam, this
serotype accounted for about 40 % of S. flexneri isolates
(Stagg et al., 2008). Interestingly, the untypable S. flexneri
isolates in this study did not react with provisional serovar
S. flexneri 1c monoclonal antiserum (S. flexneri # 88-893
antiserum) and also did not cross-react with other Shigella
serogroup/serotype-specific antisera. As reported pre-
viously (Pazhani et al., 2005), S. flexneri type 2a continued
to dominate the other serotypes. As seen in this study,
prevalence of S. flexneri type 3a is increasing along with Y
variants. The X variant of S. flexneri [agglutinated with
polyvalent B and group 7(8) sera] appeared in China
during 2001, replaced the predominant serotype 2a in 2003
and spread to several other provinces in 2007 (Ye et al.,
2010). In Thailand, this variant was the second-most
predominant serotype after S. flexneri type 2a and reduced
over a period of time (Bangtrakulnonth et al., 2008).

Fig. 3. XbaI-PFGE profiles of S. sonnei isolates, with dendrogram After the dysentery outbreaks in eastern India in the early
showing percentage similarity. 1980s, S. dysenteriae type 1 re-emerged in 2002 as
epidemics in several parts of India (Pazhani et al., 2004).
epidemic strains isolated previously in this region were Since 2002, isolation of this serotype has steadily declined
compared, the recent isolates were identified as belonging compared with S. flexneri, and S. dysenteriae type 1 was not
to a different clone. S. dysenteriae type 2 isolates were isolated from 2005 in Kolkata or other locations such as
identified as closely related (.90 % band similarity; Fig. Bangladesh (Talukder et al., 2006a). We have identified one
2) and the representative S. sonnei isolates were observed S. dysenteriae type 1 isolate in 2010, indicating the existence
to be clonally related (less than three bands difference; of an isolate that has epidemic potential in our setting.
Fig. 3). Prevalence of other serotypes of S. dysenteriae, including
serotypes 2 and 3, was very low. S. dysenteriae type 2
DISCUSSION occurred in Bangladesh between 1999 and 2002 and caused
an unusual outbreak (Talukder et al., 2006b). In India,
except for S. dysenteriae type 1, other Shigella serotypes
have rarely been involved in outbreaks/epidemics.
Shigellosis caused by S. sonnei is an emerging trend in
India and has been increasingly detected during the past
two decades. A similar increase in the incidence of this
serotype with greater epidemiological impact has been
reported from many Asian countries (Kuo et al., 2008;
Vrints et al., 2009).

Shigellosis remains a considerable public-health problem in In India, antimicrobial resistance in the genus Shigella is
many developing countries and one of the most common more common than in any other enteric bacteria (Taneja
enteric bacterial infections in the USA (Wong et al., 2010). et al., 2004). Subsequent to the emergence of nalidixic acid-
Shigella spp. are phenotypically variable, as evidenced by resistant isolates all over the world during the 1990s,
variations in serotypes and antimicrobial-resistance profiles fluoroquinolones, especially ciprofloxacin, have been used
in different endemic locations. Many studies have high- for the treatment of shigellosis. However, in 2002, the
lighted the heterogeneous distribution of Shigella sero- appearance of Shigella isolates resistant to fluoroquinolones
groups (Pazhani et al., 2005; Taneja et al., 2004). In was reported, first with S. dysenteriae type 1. From that
addition, an increasing number of Shigella isolates have time, fluoroquinolone resistance has increased in devel-
been identified as untypable by serotyping in many oping countries, and also among other serogroups of
countries (Isenbarger et al., 2001; Roy et al., 2006; Zafar Shigella. After S. dysenteriae type 1 acquired fluoroquino-
et al., 2009). Unusual isolates of S. flexneri were first lone resistance, its incidence began declining and finally

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Changing trends in shigellosis

this serotype was isolated rarely, if at all. The first The trend in antimicrobial resistance changes year by year,
appearance of ciprofloxacin-resistant S. flexneri type 2a especially as the previously susceptible Shigella serogroups
was identified in Kolkata, India, in 2003 and in the are now resistant to most of the effective antimicrobials
following year this resistance was 41 % (Pazhani et al., used in the treatment of shigellosis. The current anti-
2005). During 2007–2009, this resistance increased to microbial resistance towards fluoroquinolones indicates
91.6 %. The same trend has also been reported in many that these drugs will no longer be effective and emphasizes
South Asian countries (Kuo et al., 2008; von Seidlein et al., the need for using alternative drugs for the treatment of
2006). The incidence of fluoroquinolone resistance is rising shigellosis.
year by year in other serogroups of Shigella spp. Six of 19
untypable S. flexneri isolates encountered in this study ACKNOWLEDGEMENTS
showed reduced susceptibility to fluoroquinolones and all
were resistant to nalidixic acid. In contrast, S. flexneri This work was supported in part by the Japan Initiative for Global
isolates from north-west Ethiopia were found to be Research Network on Infectious Diseases, the Ministry of Education,
resistant to fluoroquinolones, but susceptible to nalidixic Culture, Sports, Science and Technology of Japan, the Ministry of
acid (Tiruneh, 2009). An upsurge in fluoroquinolone Health, Labor and Welfare of Japan (project H17, -Shinkou-3) and by
resistance among S. flexneri and S. sonnei in developed intramural grants from the Indian Council of Medical Research, New
countries has been reported, but these serogroups were Delhi.
isolated more frequently from travellers who had a history
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