Shigellosis: Epidemiology in India

Abstract

Shigellosis is one of the major causes of diarrhoea in India. The accurate estimates of morbidity and mortality due to shigellosis are lacking, though it is endemic in the country and has been reported to cause many outbreaks. The limited information available indicates Shigella to be an important food-borne pathogen in India. S. flexneri is the most common species, S. sonnei and non-agglutinable shigellae seem to be steadily surfacing, while S. dysenteriae has temporarily disappeared from the northern and eastern regions. Antibiotic-resistant strains of different Shigella species and serotypes have emerged all over the world. Especially important is the global emergence of multidrug resistant shigellae, notably the increasing resistance to third generation cephalosporins and fluoroquinolones, and also azithromycin. This calls for a continuous and strong surveillance of antibiotic resistance across the country for periodic updation of the local antibiograms. The prevention of shigellosis is desirable as it will substantially reduce the morbidity associated with diarrhoea in the country. Public health measures like provision of safe water and adequate sanitation are of immense importance to reduce the burden of shigellosis, however, the provision of resources to develop such an infrastructure in India is a complex issue and will take time to resolve. Thus, the scientific thrust should be focused towards development of a safe and affordable multivalent vaccine. This review is focused upon the epidemiology, disease burden and the therapeutic challenges of shigellosis in Indian perspective.

Introduction

Shigella species, members of the family Enterobacteriaceae, are responsible for causing acute gastroenteritis which is one of the most common causes of morbidity and mortality in children in developing countries1. Shigellosis is ubiquitous in impoverished populations of Asian and African countries and antibiotic-resistant strains of different Shigella species and serotypes have emerged all over the world2. Although shigellosis is associated with a few medical complications only, adequate control of this disease may reduce the overall diarrhoea burden globally. The diagnosis of shigellosis is made by culture isolation of Shigella from faeces or rectal swabs. Antibiotic treatment is usually recommended in patients with moderate or severe symptoms as it can reduce the duration and severity of symptoms, excretion of organisms, and prevent complications3. However, empiric antimicrobial therapy requires knowledge of the local antibiogram of circulating Shigella strains. Especially important is the awareness of the global emergence of multidrug resistant (MDR) shigellae, notably the increasing resistance to third generation cephalosporins and fluoroquinolones, and most recently azithromycin3,5,6. In this review, we will focus upon shigellosis in Indian perspective, mainly addressing the epidemiological parameters, disease burden, molecular epidemiology and the therapeutic challenges of emerging MDR shigellae.

History

Hippocrates used the term dysentery to indicate a condition characterized by the frequent passage of stool containing blood and mucus, accompanied by straining and painful defecation1. At the end of the 19thcentury, epidemics of bacillary dysentery occurred periodically in Japan, when Kiyoshi Shiga examined dysenteric stools of patients and isolated a bacterium that was agglutinated by serum from convalescent patients but not with acute disease. Later, to honour Shiga, this bacterium was christened as Shigella dysenteriae type 1, the first organism of the genus Shigella7. Following this discovery, the next few decades witnessed additional groups of related organisms being discovered and placed in this genus and named to honour the lead workers, Flexner, Sonne, and Boyd8,9,10.

Classification

The genus Shigella belongs to family Enterobacteriaceae. It comprises four species, S. dysenteriae, S. flexneri, S. sonnei, and S. boydii, which are further classified into serotypes based on biochemical differences and variations in their O-antigen. Thus, S. dysenteriae (group A) has 17 serotypes, S. flexneri (group B) has 14 classical serotypes and subserotypes, S. sonnei (group C) has a single serotype and S. boydii (group D) has 20 serotypes11.

Serogroup distribution: S. dysenteriae usually presents in epidemic and outbreak forms of the disease. S. flexneri and S. sonnei are mainly responsible for endemic disease in developing and developed nations, respectively, while S. boydii is restricted to India and neighbouring countries12. S. flexneri was found to be the most commonly isolated species (68%) in a multi-centric study from six Asian countries including China, Vietnam, Thailand, Bangladesh, Pakistan, and Indonesia, except in Thailand where S. sonnei was the commonest (84%), while S. dysenteriae, which is most often seen in southern Asia and sub-Saharan Africa, constituted only 4 per cent of the isolates2. From India, we have observed temporal shifts in prevalent serogroups of shigellae at our tertiary care referral centre which caters to patients from eight adjoining States (Chandigarh, Punjab, Haryana, Himachal Pradesh, Jammu and Kashmir, western parts of Uttar Pradesh, Uttaranchal, and some parts of Rajasthan) thus representing a large geographical area. S. flexneri stood as the most common serogroup from 1994-2002, followed by S. dysenteriae type 1 emerging as the predominant serogroup after a decade in 2003. S. flexneri again emerged as the predominant serogroup since 20044,13,14,15,16. Such cyclical changes have also been reported from National Institute of Cholera and Enteric Disease (NICED), Kolkata, in eastern India where epidemics caused by S. dysenteriae periodically occur after a gap of a decade or so17. Overall the authors have shown S. flexneri (60%) as the most prevalent serogroup, followed by S. sonnei (23.8%), S. dysenteriae (9.8%) and S. boydii (5.7%)17. Similarly, from Manipal and Puducherry in south India, S. flexneri figures as the predominant serogroup representing >90 per cent of the isolates18,19. Interestingly, a lesser prevalence of S. sonnei has been reported from these two centres (3.9-5.4%) in south India as compared to eastern India (23.8%)17,18,19. This observation was corroborated by data from Kolkata where again higher rates of S. sonnei (16.2%) were reported20. Overall, S. flexneri was most common (74.7%), with serotypes 2a (51%) and 3a (28.7%) being predominant among children less than 5 years20. In Global Enteric Multicenter Study, serogroups 2a (26.4%), 3a (12.1%), 6 (5.5%), 4a (5.5%), 7 (5.5%) and 1b (1.1%) were found in 91 Shigella isolates from Kolkata21. Another important feature was the emergence of untypeable shigellae. At our centre, over nine years, around 4 per cent isolates were untypeable4. Similar trends of emergence of untypaeble Shigella strains (13%) have been reported from Kolkata20. A peculiar observation from Kolkata was the absence of S. dysenteriae which was similar to our centre. In contrast, in Andaman islands in the Bay of Bengal over a period of seven years from 2006-2012, S. dysenteriae (12%) was recorded to be third most common, followed after S. flexneri (68%) and S. sonnei (20%)22. The heterogenous distribution of Shigella species and serogroups across the country implicates that multivalent or cross-protective Shigella vaccines are required to address the burden of shigellosis in India.

Host

Though humans and primates are the primary reservoir of Shigella23, it has been isolated from various sources viz. aquatic bodies (rivers, surface waters as well as coastal waters), free living amoebae, insects, birds and wild animals24,25,26,27,28,29,30,31. For a continuous transmission in humans, however, the bacterium must be passed from one person to another, as it does not survive in planktonic phase for long outside the body23.

Routes of transmission/reservoirs and meteorological factors

The primary mode of transmission is by faeco-oral route and as low as 10-100 bacteria can cause infection23. Such a low infective dose enables Shigella to cause large outbreaks. The high incidence of Shigella in the developing world is generally attributed to lack of clean water, poor hygiene, malnutrition and close personal contact. Outbreaks have been associated with person-to-person transmission in crowded or unhygienic environments like prisons and asylums. Environmental factors such as rainfall and temperature have been shown to affect the transmission32. The expression of virulence genes is activated when bacteria are shifted from 30 to 37°C, in medium of moderate osmotic stress and pH 7.433. Shigella infections can occur round the year, but peak prevalence in summer months has been reported34. In most communities the incidence is highest in hot and dry weather, possibly because the scarcity of water in such conditions limits hand-washing and other hygiene measures that reduce person-to-person transfer of the bacteria23. From Bay of Bengal islands, shigellosis is reported to occur mainly during rainy seasons, while low numbers of cases are recorded in winters22.

Less commonly known route of transmission is through contaminated food and water or fomites. The ready-to-eat foods and beverages prepared by street-vendors can also be a source of shigellosis. In a study from Odisha in India, panipuri (a street food) samples examined for disease causing bacteria showed around 80 per cent to be coliform-positive with isolation of pathogenic bacteria like Escherichia coli (13.6%), Klebsiella sp. (10.6%), Enterobacter sp. (28.8%), Bacillus sp. (3%), Enterococcus sp. (6.1%), Micrococcus tetragenus (3%), Salmonella Paratyphi B (1.5%), S. dysenteriae (4.5%) and Vibrio sp. (6.1%)35. Similarly, food handlers in large food service establishments often carry pathogenic microorganisms and may be a source of infection to a huge number of people. At a tertiary care centre in north India, amongst asymptomatic food handlers, Shigella was the most common bacteria isolated36.

In settings where disposal of human faeces is inadequate, flies, particularly Musca domestica, the common housefly, may serve as a vector for transmission of shigellosis28. When guts of houseflies collected from various public places including a garden, public park, garbage/dump area, hospital, restaurant/canteen, and human habitation in Pune, Maharashtra, India, were cultured, 102 bacterial strains were isolated, majority of which were known potential pathogens including Shigella, along with Klebsiella, Aeromonas, Morganella, Providencia, and Staphylococcus28. Also, animals in contact with human faeces may transiently harbour diarrhoeogenic bacteria as observed in Mumbai where Shigella as well as Salmonella, Pseudomonas, Streptococcus, Proteus and Pasteurella species were isolated from 200 pigs slaughtered at an organized slaughter house30. Shigellae have also been found in avian reservoirs in a zoological garden in Madagascar29, and muscle tissue of several wild animal carcasses from wildlife species in Canada31.

Several aquatic bodies have been found to show presence of shigellae and thus another potential source of infection may be aquatic food which may play a role in transmission of Shigella if such food is harvested from sewage-contaminated water37. The distribution of major groups of enteric bacteria was explored in sediments from coastal areas in south India from different depths (5, 15, 25 and 35 meter), and the flora was found to constitute Vibrio parahaemolyticus, Shigella, Vibrio cholerae, Salmonella and E. coli with higher bacterial populations in monsoon and pre-monsoon seasons26. Water samples of the river Narmada in Madhya Pradesh in India have been found to harbour S. flexneri, S. sonnei and S. dysenteriae. The virulence gene encoding the invasive plasmid antigen H (ipaH) was found in all the isolates, while the plasmid encoded invasion-associated genes (ipaBCD) were present only in S. flexneri, and the Shiga toxin (stx1) gene was found only in S. dysenteriae, thus demonstrating not only the existence of Shigella in the river but also the presence of an environmental reservoir of virulence genes24. Similarly, shigellae have been isolated from surface waters in Bangladesh25. Shigella species have also been found to grow symbiotically inside Acanthamoeba castellanii implicating that free-living amoebae may serve as a transmission reservoir for Shigella in water27. Such environmental gene pool may contribute to horizontal transfer of genes among strains and emergence of virulent strains leading to outbreaks25, and may also help the bacteria to survive in odd conditions and re-emerge in favourable conditions.

Risk groups

No individual is immune to shigellosis, but certain individuals are at increased risk. Worldwide, the incidence of shigellosis is highest among children less than five years of age, but it has been observed that during S. dysenteriae type 1 epidemics all age groups are affected23. The incidence of shigellosis has been reported to increase steadily after the age of 40, along with bacterial load as determined by semi-quantitative real-time PCR. This suggests that young children and old people shed the highest bacterial load and may be responsible for disproportional transmission of shigellosis2. In children who are malnourished, Shigella may cause a vicious cycle of further impaired nutrition, recurrent infection and growth retardation23. In the United States of America (USA) and Europe, children in day-care centers38, migrant population, travellers to developing countries, persons in custodial institutions39, prisoners and military personnel, and homosexual men are infected most often23.

In HIV infected patients, intestinal parasitic pathogens are more common in antiretroviral therapy naive patients40, however, the association of Shigella with HIV has not been properly studied in all patient groups. Association of shigellosis in men who have sex with men (MSM) has been implicated for a long time. In the 1970s and 1980s, shigellosis was identified as a potentially sexually transmitted disease among MSM41, and increased incidence in men was attributed to the sexual practices of MSM42. A correlation between HIV infection and increased incidence of shigellosis from San Francisco area in 1996 suggested HIV as an important risk factor for shigellosis43. Another study from San Francisco in 1998-1999 linked the sexual practices of direct oral-anal contact in MSM to confer the highest risk and HIV infection likely contributing to increased host susceptibility44. Similar outbreaks of shigellosis among MSM have been reported from Canada, Australia, London, and Chicago45,46,47,48.

The data for higher incidence of Shigella in association with HIV infection are not so convincing from India. The common enteric pathogens detected in 331 HIV infected patients presenting with diarrhoea in Pune were Cystoisospora belli (28%) and Cryptosporidium parvum (12%), whereas in HIV uninfected individuals S. flexneri (4.9%) was the second most common pathogen, only after Entamoeba histolytica (7.1%)49. In another study from New Delhi, Shigella was only found in 2 per cent cases of diarrhoea in HIV infected patients showing a similar incidence as in immunocompetent individuals50. In Senegal, Shigella was implicated as the most common cause (12.4%) of diarrhoea in immunocompetent adults but in immunocompromised individuals Shigella was fifth most common (7.6%)51. Shigellosis, presents as a more severe infection in HIV infected individuals, may cause bacteraemia and unusual features like keratitis and pneumonia, fail to respond to appropriate therapy thus requiring prolonged treatment, and may recur after completion of treatment52,53,54.

Clinical features and complications

The clinical presentation ranges from watery, loose stools to severe symptoms such as fever, abdominal pain, tenesmus, and bloody diarrhoea. Severity of the disease varies by the infecting species - S. dysenteriae infections usually cause dysentery, which may also occur in infections caused by S. flexneri, whereas S. boydii and S. sonnei generally often self-limited watery diarrhoea3. Acute complications such as toxic megacolon, peritonitis, and septicaemia are mostly observed in severely malnourished children, though may occur in absence of early antibiotic treatment23. Shigella dysentery may also lead to dangerous complications such as persistent diarrhoea, severe anorexia, weight loss and malnutrition, dilation of the large intestine, seizures, kidney damage, and haemolytic-uremic syndrome55. Bacteraemia may be reported in infants and immunocompromised adults56. Pneumonia associated with S. sonnei has also been described in malnourished children, in HIV infected patients, and in patients with chronic diseases54,57.

Mortality

The advent of oral rehydration therapy to correct dehydration due to diarrhoea has greatly decreased the number of deaths due to diarrhoea of any aetiology. Severe dehydration is uncommon in shigellosis, and with proper hydration, shigellosis is generally a self-limiting disease. However, in severe cases, without antimicrobial treatment the mortality of shigellosis is greatly increased23. The availability of age-specific estimates of case fatality due to shigellosis is limited. The death rate due to Shigella infection in developed countries is low (0.05 to 0.4%)58. The earlier data from developing countries over a period (1974-1988) have indicated that 13.9 per cent of infants and 9.4 per cent of 1-4 year-olds die annually due to shigellosis as estimated in admitted patients at the International Centre for Diarrhoeal Disease Research (ICDDRB), Bangladesh59. However, there is a change in this situation in recent years where a decrease in mortality due to shigellosis has been observed as compared to the previous reports. In a multi-centric study from six Asian countries, no deaths were recorded in 845 patients2. The authors suggested adequate treatment to study participants and absence of S. dysenteriae type 1 as possible reasons for unexpectedly low morbidity and mortality in the survey2.

Epidemiology

Shigellosis occurs worldwide, in endemic and epidemic forms. Majority of cases are children <5 yr of age. The annual number of shigellosis episodes throughout the world is estimated to be 164.7 million, with 69 per cent of all episodes and 61 per cent of all deaths attributable to shigellosis involving children <5 yr of age58. The multi-centric study from six Asian countries2 estimated Shigella as the causative agent in 5 per cent of the diarrhoeal cases, indicating an overall incidence of treated shigellosis to be 2.1 episodes per 1,000 residents per year in all ages, with higher rates in children and people more than 40 yr of age2. Among children <5 yr old, the incidence was 13 new cases per 1,000 children per year2. From Bangladesh a trend of increasing number of patients ≥60 yr has been reported in an analysis of patients from 2001-2012 than children <5 yr and adults aged 5-5959. Reports of shigellosis from various parts of the country have shown an overall isolation rate of shigellae varying from 3-6 per cent of all stool samples with diarrhoea4,20. At our tertiary care centre in north India, shigellae have been detected from 3 per cent of diarrhoeal stool samples4, whereas in Kolkata, Nair et al20 isolated 6.1 per cent Shigella spp. from hospitalized diarrhoeal patients. In paediatric age group, again, higher isolation rates have been reported, 9.5 per cent in Bay of Bengal islands and 11.5 per cent in Kolkata22,60.

Several epidemics have been reported from many Asian countries such as Bangladesh (1972-1978, 2003), Sri Lanka (1976), Maldives (1982), Nepal (1984-1985), Bhutan (1984-1985) and Myanmar (1984-1985)61,62,63,64. In India, epidemics have been reported from southern India, Vellore (1972-1973, 1997-2001)65,66, eastern India (1984)67,68, Andaman and Nicobar islands (1986)69,70 and Chandigarh (2003) in northern India14. Epidemic dysentery caused by MDR S. dysenteriae serotype 1 has been a recurrent challenge.

Outbreaks: Shigellae have the ability to cause outbreaks involving a large number of people. In the 1984 outbreak in West Bengal and Tripura, 3,50,000 people were affected with 3500 deaths68. In the 2002 outbreak, which occurred in West Bengal and tea gardens of Siliguri, the overall attack rate was 25.6 per cent. The death rate among those admitted to hospital was 6 per cent and the overall case-fatality ratio was 0.9 per cent71. All these outbreaks were due to S. dysenteriae serotype 1. The re-emergence of S. dysenteriae type 1 with added resistance to ciprofloxacin which has epidemic potential, has also been reported from our centre12. Outbreaks caused by S. flexneri and S. sonnei have also been reported recently from various parts of the country like West Bengal (2007), Kerela (2009) and Maharashtra (2010)72,73. In 2007, in a municipality in West Bengal, S. flexneri serotypes 2a and 3a caused water borne outbreak affecting 461 persons. The sources associated with the illness were drinking, washing utensils and bathing in tap water contaminated with Shigella, and the outbreak subsided following repair of the pipeline72. Food-borne outbreaks of S. sonnei have also been reported. More than 300 people suffered in Kerala in south India in 2009 where local food made of rice, lentils, milk, and water was implicated as the source73. In Maharashtra in 2010, about 150 persons suffered from shigellosis after eating in a madrasa (a religious place)73. These reports support the extension of S. sonnei into India73. These outbreaks indicate that in India, food- and water-borne routes for transmission of shigellosis may not be so uncommon, and thus reinforces the need to provide adequate sanitation.

Traveller's diarrhoea: Shigellae are also important agents of diarrhoeal disease in travellers from developed world to other countries. As per an estimate, 15-20 million travellers to developing countries experience diarrhoea annually74. Of the 64,039 enteric infections reported to FoodNet with information about travel, 8270 (13%) were travel associated, and amongst the bacterial agents, Shigella (13%) was the third most common, after Campylobacter (42%), and non-typhoidal Salmonella (32%). The most common travel destinations were Mexico, India, Peru, Dominican Republic, and Jamaica75.

Therapeutic challenge of multidrug resistant shigellae

The emergence of MDR Shigella has been reported from all over the world, including the USA, Iran, China, Indonesia, Vietnam, Bangladesh and India2,64,76,77. The progressive development of antibiotic resistance in Shigella isolates, as in all other bacteria, is not a new phenomenon. Sulphonamides were the first drugs of choice when introduced in the early 1940s and all the Shigella strains were sensitive to this drug. In late 1940s sulphonamides became ineffective, and tetracycline followed by chloramphenicol were recommended for shigellosis. Soon, resistance to these two drugs was also observed and ampicillin and co-trimoxazole came to the rescue78. These were clinically highly effective. However, during the epidemic in eastern India in 1980s, the S. dysenteriae type 1 isolates were found to be resistant to most of the antibiotics except nalidixic acid which was found to be clinically effective78,79. Later, resistance to nalidixic acid appeared in S. dysenteriae type 1 isolates from an outbreak in Tripura in 198880. In the late 1980s, fluoroquinolones (norfloxacin, ciprofloxacin and ofloxacin) were introduced in India and were found to be very effective for shigellosis, including MDR S. dysenteriae type 181.

In 1990, ciprofloxacin was recommended as the drug of choice for empiric treatment of shigellosis in view of the existing high level resistance to agents like chloramphenicol, ampicillin, co-trimoxazole and nalidixic acid82. Ciprofloxacin proved to be highly effective in the treatment of shigellosis, but possibly due to overuse and misuse of this agent promoted by the easy over-the-counter availability of antibiotics without prescription, resistance emerged against this agent also. Outbreak investigations in India (Chandigarh, Siliguri, Aizawl and Diamond Harbour, Kolkata) showed high level resistance to ciprofloxacin, norfloxacin and ofloxacin71,77. At our centre, an outbreak of ciprofloxacin-resistant S. dysenteriae serotype 1 occurred in 2003. There was no mortality associated with these cases and they were managed by cefotaxime and amikacin14. Thereafter, high levels of resistance to nalidixic acid (S. dysenteriae 81.8%, S. flexneri 74.1%) and ciprofloxacin (S. dysenteriae 54.5%, S. flexneri 45.6%) have consistently been noted in various serogroups of shigellae16,83. From north eastern region, an outbreak of bacillary dysentery caused by quinolone resistant S. dysenteriae type 1 was reported84. Another study from Kolkata reported MDR shigellae most of which were found resistant to fluoroquinolones viz. ciprofloxacin (90%), norfloxacin (83%), and ofloxacin (81%), however, majority were still susceptible to ceftriaxone (94%)20. At another hospital in Kolkata, majority of Shigella isolates (81%) were MDR and emergence of fluoroquinolone-resistant S. dysenteriae type 1 (100%) in 2002-2003 was followed by frequent isolation (>25%) of fluoroquinolone resistant S. flexneri 2a and 3a in 2004, which restricted use of fluoroquinolones for treatment of shigellosis60. Fluoroquinolones are thus no longer the preferred group of drugs for managing shigellosis in India.

World Health Organization (WHO) now recommends ceftriaxone, pivmecillinam and azithromycin as alternative drugs to fluroquinolone resistant shigellae85. We found 15.1 per cent of S. flexneri isolates collected over a period of nine years (2000-2009) resistant to at least one of the third-generation cephalosporins (ceftriaxone/cefotaxime)4. The first isolate showing ceftriaxone resistance was obtained in 2001 and an increase in number of isolates resistant to third generation cephalosporins was observed 2005 onwards. This situation has now become a therapeutic challenge in this region. The minimum inhibitory concentration (MIC) values for Shigella isolates revealed a rise for ceftriaxone (MIC90: 12 mg/l) and cefepime (MIC90: 8 mg/l). MIC values for S. dysenteriae remained below 1 mg/l for ceftriaxone, however, for cefepime the MIC90 has raised to 4 mg/l. These infections caused by ceftriaxone resistant S. flexneri isolates were successfully treated by azithromycin at our center4. Emerging resistance to cephalosporins has also been reported from Puducherry and Manipal in south India18,19. Azithromycin has also now joined the exhaustive list of drugs to which Shigella has developed resistance59.

MDR shigellae have also been reported from travellers who have visited India86. At Kansai Airport Quarantine Station in Japan, stool samples were collected from overseas travellers with a history of diarrhoea over the period 2001-2005, and 53-106 Shigella strains were isolated per year (average 82.4), about 80 per cent of which were S. sonnei, and the most frequent country of origin was India86. Such widespread resistance to almost all classes of drugs presently in use for shigellae narrows down the choice of effective antimicrobial agents for shigellosis and is a matter of concern.

Molecular epidemiology

There has been an upsurging interest in exploring the molecular epidemiology of genetically encoded virulence factors and antimicrobial resistance markers of Shigella. Such insights may be valuable in understanding the transmission patterns, severity of clinical presentations and response of Shigella to antimicrobial drugs.

Molecular epidemiology of virulence genes: Several virulence factors have been reported in Shigella. Virulent genes of Shigella may be present in isolates from both symptomatic and asymptomatic people. In Kolkata, all 91 Shigella isolates from both cases and controls were positive for ipaH gene87. Other virulence genes such as virulence regulator (VirF), secreted autotransporter toxin (sat), Shigella enterotoxin 1 subunit A (setA), Shigella enterotoxin 1 subunit B (setB), Shigella enterotoxin 2 (sen) and epithelial cell penetration encoded by invasion associated locus (ial) were detected in Shigella isolates in 80.2, 49.4, 27.4, 27.4, 80.2 and 79.1 per cent of cases and in 64.7, 52.9, 17.6, 17.6, 64.7 and 64.7 per cent of controls, respectively. The Shigella pathogenicity island (SH-PAI) was detected exclusively in serotype 2a. Such asymptomatic carriers may play a crucial role in the transmission of shigellae in endemic communities87.

Molecular epidemiology of antimicrobial resistance: Shigella has adapted well to many antimicrobial agents due to its ability to carry mobile genetic elements that may facilitate inter- and intra-species dissemination of antimicrobial resistance genes88. In Kolkata, integron carriage has been detected in high numbers in Shigella isolates from cases (76.9%) than from controls (3505%). Of these, atypical class 1 integron has been exclusively detected in S. flexneri from cases but not from the controls87. The main mechanism of quinolone resistance involves accumulation of sequential mutations in DNA gyrase and DNA topoisomerase IV. The plasmid-mediated quinolone resistance (PMQR) has been described due to mutations in the aac(60)-Ib-cr gene that encodes for a variant of aminoglycoside acetyltransferase, also known to reduce ciprofloxacin activity89,90. PMQR genes in shigellae have been reported from USA, Japan, China and India87,91,92,93. From north India, we screened S. flexneri (n=139) and S. dysenteriae serotype 1 (n=38) isolated over the period 2001-2011 for PMQR determinants, and found 6.2 per cent shigellae to harbour PMQR determinants, of which two were positive for the qnrS1 gene while nine were positive for aac(60)-Ib-cr gene. A high MIC for ciprofloxacin (32 mg/l) was shown by four of the 11 PMQR-positive isolates, while majority of isolates with a ciprofloxacin MIC 1 mg/l were negative for PMQR determinants83. Although qnrB is the most common gene belonging to the qnr family, we detected qnrS1-positive Shigella from the Indian subcontinent in two strains from 2010, indicating a relatively new appearance of this PMQR determinant among Shigella in India83. After the description of qnrS1-harbouring Shigella in Japan, China and the USA, appearance of qnrS1-positive Shigella in India is a matter of concern. We also detected at least two mutations in the QRDRs of each of the 11 PMQR-positive strains by sequencing of gyrA and parC indicating that PMQR determinants may have provided selection advantage by contributing to reduced susceptibility to quinolones and thus facilitating the selection of gyrA/parC mutants. Besides, four of the PMQR-positive isolates were positive for bla_CTX-M-15, one of which was also positive for bla_CMY-285. A close association of aac(60)-Ib-cr with bla_CTX-M-15 has been detected in Shigella and is of great concern as bla_CTX-M-15 has emerged worldwide in recent years. From Andaman and Nicobar Islands also, presence of aac(60)-Ib-cr and qnrB in Shigella has been reported, indicating a widespread presence of these resistance determinants in the country22.

Resistance to cephalosporins is mediated by extended-spectrum beta lactamases (ESBLs) which hydrolyze a wide variety of penicillins and cephalosporins. The ESBL mediated resistance to third-generations cephalosporins arises from mutations which broadens the spectrum of native beta-lactamases like TEM-1, TEM-2, and SHV-1. Another resistance mechanism includes overproduction of chromosomal or plasmid-derived AmpC beta-lactamases94. We investigated the presence of antimicrobial resistance genes in 119 S. flexneri and 24 S. dysenteriae isolates over a period of nine years (2001-2009)4, and found 20 S. flexneri isolates with high MICs for cephalosporins. Nine of the 20 isolates were found to be positive for ESBL and six for AmpC production by phenotypic tests, and among the resistance determinants bla_TEM closely resembling bla_TEM-116 was the most common ESBL gene present in all 20 isolates, followed by bla_CTX-M-15 in 10, bla_OXA in eight, and bla_CMY-2 in seven, while none was positive for bla_SHV4. In contrast, of the 88 isolates from Bay of Bengal islands, 2 per cent showed the presence of the bla_SHV gene, while all 15 third generation cephalosporins-resistant isolates showed the presence of the bla_TEM, bla_OXA1, and bla_CTX-M3 genes22. A high ESBL prevalence in members of the Enterobacteriaceae family has been reported in India95 with bla_CTX-M-15 being the most common ESBL gene. The finding of a high prevalence of ESBL producing genes like bla_CTX-M-15 which spread by horizontal transfer and/or mobilization of genetic mobile elements by orofaecal route, has serious implications in terms of further spread of resistance to third generation cephalosporins to other regions96. Overcrowding and poor sanitation, and the selective pressure due to overuse of antibiotics have been considered responsible for such widespread dispersal of bla_CTX-M-1596.

From south India in Bay of Bengal islands, other resistance determinants were analyzed in 88 isolates and MDR was found to be associated with various drug-resistant genes22. Ampicillin resistance was largely associated with TEM β-lactamase genes (100% isolates), the most prevalent resistance gene in ampicillin-resistant Enterobacteriaceae while gentamicin resistance was associated with aac2 gene in 22 per cent of the isolates. Plasmid mediated resistance was commonly found for tetracycline (tetB in 92% and tetA efflux genes in 90%), chloramphenicol (catI gene in 32%) and co-trimoxazole (dfrA1 in 81% and dfrA5 in 78%)22. Thus, there is a widespread emergence of MDR Shigella in the face of rampant injudicious antimicrobial use which reinforces the need for continuous surveillance of antimicrobial resistance determinants across the country to know the molecular epidemiology of resistance, which is further essential for implementing timely intervention steps to control the disease as well as spread of these resistance genes to other parts of the world.

Prevention and control

The most effective measure to decrease transmission of shigellosis is proper washing of hands, especially after defaecation. Community health education must emphasize upon good personal hygiene, adequate disposal of faeces, as well as the imminent threat of MDR pathogens. The widespread practice of misuse of antibiotics in viral diarrhoea should be discouraged by means of education as well as legislation. Better awareness about general measures such as washing, peeling and cooking of all fruits and vegetables, avoidance of food preparation by personnel who change diapers in daycare centres, proper handling and refrigeration of food, encouraging prolonged breastfeeding in infants, and appropriate case reporting to health authorities may be helpful to prevent further transmission85. Though public health measures to reduce exposure and transmission are highly effective, the establishment of such infrastructure in developing countriesis resource intensive and thus remains challenging.

Vaccines: There is a strong need for an effective, safe and cheap vaccine against shigellosis2. The high disease burden of shigellosis in developing countries, children <5 yr of age as the main victims, difficulty in achieving adequate sanitation and personal hygiene in these regions and scarce therapeutic alternatives for emerging MDR Shigella point towards vaccination as a hope for effective and sustainable strategy against shigellosis. Shigellosis is targeted by WHO as one of those enteric infections for which new vaccines are most needed, the target populations being travellers from developed countries and military service personnel, as well as children living in endemic areas2,58.

Although the need for a Shigella vaccine is urgent, not much progress has been done due to the antigenic complexity, lack of inter-species cross-protective epitopes, and gaps in understanding of the protective immune response. Several different types of vaccines against Shigella have been experimentally tested in animal models and in volunteer trials12,97. Various live attenuated vaccines such as CVD103, CVD104, CVD107, CVD108, SC602 and WRSS1 have been developed in the past12, however, most were serotype-specific with no cross-protectivity. These vaccines progressed into phase 1/2 trials but none could go beyond12. (Ipa, B, C) subunit vaccine approach has been used for Invaplex (Shigella invasion complex) containing invasion plasmid antigens B and C, and lipopolysaccharide (LPS), which was found to induce protective immunity in experimental animals98. Similarly, outer membrane proteins are being developed as attractive vaccine options99. However, apart from a live, non-invasive S. flexneri 2a-S. sonnei bivalent vaccine used in China, there are currently no licensed vaccines available100. The current vaccine candidates are either not sufficiently attenuated or not properly immunogenic101. Thus, the goal for an effective, safe and successful multivalent vaccine targeting prevalent species and serotypes is yet to be achieved98,102. Livio et al21 have shown that a quadrivalent vaccine with O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 may be effective against these most common serotypes.

Conclusion

Shigellosis is one of the major causes of diarrhoea in India. S. flexneri is the most common species present in the country, S. sonnei and non-agglutinable shigellae are steadily surfacing, while S. dysenteriae has temporarily disappeared from northern and eastern regions. Though shigellosis appears to be endemic and has been reported to cause many outbreaks, the accurate estimates of morbidity and mortality are lacking. The limited information available indicates Shigella to be an important food-borne pathogen in India. There is a nationwide presence of MDR shigellae developing rapid resistance to most antibiotics available. Thus, judicious use of antibiotics for Shigella is amongst the most essential measures to combat shigellosis. This calls for a continuous and strong surveillance of antibiotic resistance across the country for periodic updation of the local antibiograms. The prevention of shigellosis is desirable as it will substantially reduce the morbidity associated with diarrhoea in the country. Public health measure like provision of safe water and adequate sanitation are of immense importance to reduce the burden of shigellosis, however, the provision of resources to develop such infrastructure to the huge population of a country like India is a complex issue and will take time to resolve, hence focusing the scientific thrust towards development of a safe and affordable multivalent vaccine may be the need of the hour.