The Emergence of Quinolone Resistant Shigella sonnei, Pondicherry, India

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Abstract

Ciprofloxacin resistant Shigella sonnei across the globe have been increasing alarmingly. In order to understand the emergence of S. sonnei with respect to ciprofloxacin resistance in our patient population, the following study was carried out. Of the 184 Shigella sp. Isolated from 2012 to 2015, 34 S. sonnei which were confirmed by standard methods and subjected to antimicrobial susceptibility testing were selected. The minimum inhibitory concentrations (MICs) of 16/34 quinolone resistant isolates tested ranged from 4micrograms/ml to 16micrograms/ml for ciprofloxacin, from 16 micrograms/ml to 64 micrograms/ml for ofloxacin and from 16micrograms/ml to 64micrograms/ml for levofloxacin. Sequence determination of the quinolone resistance determining regions of gyrA, gyrB, parC, and parE genes showed mutations in GyrA at Gln69/Trp, Phe71/Ser, Ser72/Pro, Met75/Leu, Ser90/Cys, Met94/Leu, His106/Pro, Asn161/His, Thr163/Ala and in ParC at Ala64/Asp. Among the plasmid-mediated quinolone resistance (PMQRs) targets investigated, qnrB was the most (93.7%) prevalent followed by qnrC (18.7%). None had qnrA, qnrS and qepA. Two (0.1%) of the isolates harboured the aac(6’)-lb gene. Drug accumulation assay detected the presence of efflux pump activity in 9/15 (60%) among ciprofloxacin resistant isolates. All isolates harboured the ipaH gene followed by ial (17.6%), sen (11.7%), set1A& set1B (5.8%) genes. None had stx1 element. PCR for Enterobacterial repetitive intergenic consensus (ERIC) sequences resulted in 4 unique clusters, of which Type III was the most (44%) dominant but there was no correlation between the ERIC types and the antibiotic resistance pattern or the virulence profile. A documented increase in S. sonnei harbouring the qnrgenes and some unusual genes like set1Aand indicate an ongoing process of horizontal gene transfer. The accumulation of novel mutations in GyrA and ParC in the presence of efflux pump and PMQR genes contributed to the raised MIC to quinolones. These findings are crucial in our understanding of quinolone resistance in these isolates.

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Natural Antibiotic Resistance and Contamination by Antibiotic Resistance Determinants: The Two Ages in the Evolution of Resistance to Antimicrobials

José L. Martínez-Carrillo, Jinyun Tang (2012)

The study of antibiotic resistance has been historically concentrated on the analysis of bacterial pathogens and on the consequences of acquiring resistance for human health. The development of antibiotic resistance is of course extremely relevant from the clinical point of view, because it can compromise the treatment of infectious diseases as well as other advanced therapeutic procedures as transplantation or anticancer therapy that involve immunosuppression and thus require robust anti-infective preventive therapies. Nevertheless, the studies on antibiotic resistance should not be confined to clinical-associated ecosystems. It was evident soon after introducing antibiotics for human therapy, that bacteria were able to develop resistance, not just as the consequence of mutations in the targets of antibiotics, but by acquiring genes conferring resistance to antimicrobials (Abraham and Chain, 1940). Since those genes were not present before in the human bacterial pathogens, the only suitable source for them was the environmental microbiota, and indeed the presence of R-factors (resistance plasmids) in pristine environments without any record of contact with antibiotics was described in the first studies of antibiotic resistance in the field (Gardner et al., 1969). Given that the origin of antibiotic resistance is the environmental microbiota, it would be necessary to study resistance in natural, non-clinical habitats in order to fully understand the cycle of acquisition of resistance by human pathogens. However, until recently the studies on antibiotic resistance in natural ecosystems have been fragmentary. The availability of metagenomic tools as well as high-throughput sequencing techniques is allowing describing in depth the presence of resistance genes in different ecosystems. Indeed, the use of functional genomic and metagenomic techniques has served to show that natural ecosystems, including not just soils but human gut as well, contain a large number of elements that, upon transfer to a new host, can confer resistance to any type of antimicrobial (D'Costa et al., 2006; Sommer et al., 2009). These include natural antibiotics, which are produced by the environmental microbiota, and synthetic antimicrobials, as quinolones. One important question from an evolutionary point of view is the function of these resistance genes in their natural environmental hosts (Davies and Davies, 2010). Whereas for naturally produced antibiotics a protective role for resistance genes in the producers organisms (or those coexisting with producers Laskaris et al., 2010) might be foreseen (Benveniste and Davies, 1973), this explanation is not suitable for synthetic antibiotics as quinolones. Indeed, it has been described that the origin of the quinolone resistance gene QnrA, which is now widespread in plasmids present in human pathogens is the environmental non-antibiotic producer Shewanella algae (Poirel et al., 2005). This means that a gene that confers resistance in a human pathogen does not necessary play the same role in its original host (Martinez et al., 2009a). The finding that several proteins, involved in basic processes of the bacterial physiology, contribute to intrinsic resistance to antibiotics (Fajardo et al., 2008; Laskaris et al., 2010; Linares et al., 2010), further supports the concept that resistance genes, acquired through horizontal gene transfer by human pathogens, might have evolved in their original host to play a different role than resisting the activity of antimicrobials in natural ecosystems. We can thus distinguish two ages in the evolution of antibiotic resistance genes. For billions of years (until the use of antibiotics by humans), these genes have been usually chromosomally encoded and had evolved for different purposes. Some of them, as those found in antibiotic producers, likely evolved for detoxifying the original host from the antibiotic it produces, although a role in the biosynthesis of the antibiotic itself has been proposed as well for some of them (Benveniste and Davies, 1973; Doyle et al., 1991). Others, as beta-lactamases might be involved in the biosynthesis of the cell wall (Jacobs et al., 1994; Massova and Mobashery, 1998), whereas others as multidrug efflux pumps might serve for different purposes including the trafficking of signaling molecules, detoxification of metabolic intermediates, or extrusion of plant-produced compounds among others (Martinez et al., 2009b). Like in the case of antibiotics, which do not necessarily have an inhibitory function at the concentrations in which they are present in natural ecosystems (Linares et al., 2006; Yim et al., 2007; Fajardo and Martinez, 2008), the fact that a plasmid-encoded gene produces resistance to antibiotics upon its expression in a new host, is not an unequivocal prove that it confers resistance as well in its original host. This reflection serves to show the relevance of the second age in the evolution of antibiotic resistance determinants. Once a gene is introduced in a new host in which it lacks its original biochemical and genetic context, its function is limited to antibiotic resistance (Baquero et al., 2009). This change of function without changing the sequence of the gene itself, has been named as exaptation (Gould and Vrba, 1982), and is the consequence of the strong selective pressure exerted by antibiotics in the last decades from the time they were introduced for therapy. Two important aspects are emerging from the studies of natural resistome. First, the environmental microbiota contains a much larger number of resistance genes than those seen to be acquired by bacterial pathogens (Wright, 2007; Davies and Davies, 2010). Furthermore, different ecosystems contain different resistance genes, which means that we are still far away to have a consistent estimation on the number of potential resistance genes present in natural ecosystems. Finally, the origin of most resistance genes currently found in transferrable elements is still ignored, despite genes (and genetic structures) belonging to the same families are regularly found in different ecosystems, including deep terrestrial subsurface (Brown and Balkwill, 2009), ice (Miteva et al., 2004), and even the permafrost (D'Costa et al., 2011), which have not been in contact with human contaminants. Second, those genes present in mobile elements in human bacterial pathogens can be found nearly everywhere, including pristine ecosystems or wild animals not supposed to be in contact with antibiotics (Martinez, 2009). This indicates that pollution with antibiotic resistance genes is widely spread and that resistance genes can persist even in the absence of a positive selection pressure. The analysis of historical soil archives has shown a consistent increase on the presence of antibiotic resistance genes since 1940 (Knapp et al., 2010), which is a clear prove of the contamination by antibiotic resistance elements of natural ecosystems and the resilience of those elements for their elimination. In this situation, which type of studies are needed to analyze in depth the role that natural ecosystems may have on the development of resistance in human bacterial pathogens? In my opinion, these studies have two faces (Martinez, 2008). One consists on the analysis of the genes already present in bacterial pathogens. In other words, we will study mainly contamination by antibiotic resistance determinants and how this contamination might increase the risks for the dissemination of those elements (Martinez, 2009). These studies might serve to define reservoirs, elements for enrichment and dissemination of resistance (as wild birds Simoes et al., 2010) or hotspots for the transfer of resistance as waste-water treatment plants (Baquero et al., 2008). For instance, a recent study has shown that soil composition and in particular the presence of heavy metals might enrich for the presence of antibiotic resistance genes in natural ecosystems (Knapp et al., 2011). The other type of studies consists on the analysis, using functional assays, of novel resistance genes in different ecosystems (D'Costa et al., 2006, 2011; Sommer et al., 2009). These studies are useful for defining novel mechanisms of resistance, but making risks assessments on whether those novel antibiotic resistance genes will be transferred to new hosts is likely unsuitable (Martinez et al., 2007). On the other hand tracking the source of currently known resistance gene has demonstrated to be a very difficult task. We have to be extremely careful for assigning the origin of resistance determinants. Only when the genes are nearly identical (as QnrA) and the gene is present in several strains of the original host, with the same synteny and without any sign of a recent acquisition event, we can firmly establish this host being the origin. The report of genes that are highly similar (even above 90%) to antibiotic resistance genes demonstrate their belonging to the same phylogenetic group, not that one is the origin of the other. Does it mean that we will be unable of tracking the source of resistance genes and to propose from this information valuable strategies for reducing antibiotic resistance? I do not believe that. It has been already determined that QnrA was originated in S. algae (Poirel et al., 2005) and that chromosomally encoded qnr genes are mainly present in water-dwelling bacteria (Sanchez et al., 2008). This suggests that the source of transferrable quinolone resistance is the water microbiota and puts a focus on the effect that the use of quinolones in aquaculture might have had for the emergence and dissemination of these resistance elements (Cabello, 2006). The study on antibiotic resistance in natural ecosystems and its role on the maintenance and spread of clinically relevant resistance determinants is still in its infancy. It is surprising that large efforts have been used to study the risks for the dissemination of resistance that may have the release of genetic modified organisms containing resistance genes in their chromosomes, whereas the study of the effect of the discharge of human wastes, which contain bacterial pathogens harboring the resistance genes that have demonstrated to be really relevant, in the elements that are important for their dissemination has received few attention if any. Studies in this new field are needed in order to understand the mechanisms involved in the emergence, spread, maintenance, and evolution of antibiotic resistance.

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Nucleic acid-based approaches to investigate microbial-related cheese quality defects

Daniel J. O’Sullivan, Linda Giblin, Paul L. H. McSweeney … (2013)

The microbial profile of cheese is a primary determinant of cheese quality. Microorganisms can contribute to aroma and taste defects, form biogenic amines, cause gas and secondary fermentation defects, and can contribute to cheese pinking and mineral deposition issues. These defects may be as a result of seasonality and the variability in the composition of the milk supplied, variations in cheese processing parameters, as well as the nature and number of the non-starter microorganisms which come from the milk or other environmental sources. Such defects can be responsible for production and product recall costs and thus represent a significant economic burden for the dairy industry worldwide. Traditional non-molecular approaches are often considered biased and have inherently slow turnaround times. Molecular techniques can provide early and rapid detection of defects that result from the presence of specific spoilage microbes and, ultimately, assist in enhancing cheese quality and reducing costs. Here we review the DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses and, in the process, highlight how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.

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A Multicentre Study of Shigella Diarrhoea in Six Asian Countries: Disease Burden, Clinical Manifestations, and Microbiology

Lorenz von Seidlein, Deok Kim, Mohammad Ali … (2006)

Introduction The burden of shigellosis is greatest in resource-poor countries where the disease may cause as many as 167 million episodes of diarrhea and over a million deaths annually [1]. Previously efficacious drugs such as sulphonamides, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole have become largely ineffective against prevalent Shigella strains. The recently reported emergence of ciprofloxacin resistance further narrows the choice of effective antimicrobials [2–4]. S. flexneri species are known to have 15 serotypes and subtypes, S. dysenteriae 13 serotypes, S. boydii 18 serotypes, and S. sonnei a single serotype. Several vaccine candidates are under development [5–10], but these may need to be tailored according to prevalent species and serotypes, since only type-specific immunity has been demonstrated in humans [11–13] and cross-serotype protection is controversial [14]. A detailed understanding of the epidemiology of shigellosis is essential for the rational development of potential vaccine candidates to control shigellosis. Because of the lack of recent, reliable data on shigellosis, especially from nonindustrialized countries, we conducted a prospective, multicentre, population-based study of the burden and patterns of shigellosis in six developing countries of Asia. Methods To allow pooling of data and comparisons across study sites, standardized epidemiologic, clinical, and laboratory methods were employed. Prior to the project start, methods were agreed upon by the principal investigators during workshops. The studies were monitored by epidemiologic and laboratory coordinators during regular visits to the study sites. A full description of healthcare systems and healthcare utilization patterns in the study sites [15–20] as well as epidemiologic and microbiologic methodology in the study sites in Indonesia [21], China [22], and Thailand [23] have been presented elsewhere. Study Sites and Population Surveillance was conducted at study sites in six developing countries of Asia: three rural or semirural areas (China, Vietnam, and Thailand) and three urban slums (Bangladesh, Pakistan, and Indonesia) (Table 1). The population size in each study site was estimated through existing, recent census data (China, Thailand, Indonesia) or a census specially conducted for the purpose of the study (Bangladesh, Pakistan, Vietnam). Table 1 Study Population, Disease Episodes, and Incidence at Six Study Sites The research site in China was in Hebei Province, approximately 270 km south of Beijing. The catchment area consisted of 29 villages in four rural townships in Zhengding County with a total population of 75,630 in 2000, of which 2,997 (4%) were children under age 60 mo. About 80% of the study population were agricultural workers. Surveillance was conducted in 101 village clinics and four township hospitals. The Thailand study area was in Kaengkhoi District, Saraburi Province, approximately 100 km north of Bangkok. The area includes a small city surrounded by rural villages that depend on agriculture for income. In 2001 80,141 people lived in the catchment area, including 5,686 (7%) children under age 60 mo. Surveillance was conducted in 20 community health centres and the district hospital. In Indonesia, two adjacent districts (kecamatans) in North Jakarta, Tanjung Priok, and Koja, formed the study area. Many homes are temporary structures without running water, and more than one-third of households have no access to tap water. The area is prone to flooding during the rainy season. The main occupations include harbour labour, small business, and clerical work. In 2000 the population in the catchment area was 160,257, of whom 15,741 (10%) were under age 60 mo. Surveillance was conducted in eight public health care centres (puskesmas) and two hospitals (the Infectious Disease Hospital and Koja Hospital). In Vietnam, the study was conducted in the coastal city of Nha Trang, the provincial capital of Khanh Hoa Province in the central part of the country. The population in the catchment area was 200,410, of which 13,970 (7%) were under age 60 mo. This population is mostly employed in fisheries, agriculture, and tourism. Surveillance was conducted in community health centres of 16 communes, in four polyclinics, and in the general hospital. In Pakistan the research site included four low-income communities. Rehri Goth, a suburban fishing village, and Sherpao Colony, where most people earn a living as labourers, are both southeast of Karachi. Hijrat Colony and Sultanabad, two contiguous urban slums, are near the business centre and port of Karachi. The catchment area in 2002 had a population of 59,584, of which 8,381 (14%) were children under age 60 mo. In each of the four communities a treatment centre was established for the purpose of surveillance. In Bangladesh the study catchment area was in Kamlapur, a densely populated settlement of rural immigrants in the southeastern sector of Dhaka city. The population in the catchment area was 29,309, of which 3,741 (13%) were children under age 60 mo. The rapid growth of the settlement has resulted in a mixture of permanent structures and temporary squatter dwellings. The most common occupations are trading, clerical work, and rickshaw puller. One central treatment centre was used for surveillance. Project Design Before and during surveillance, information campaigns were conducted to encourage all residents in each of the catchment areas to visit a participating health care centre and provider for all diarrhoea episodes. Individuals of all ages presenting with diarrhoea or dysentery were enrolled in the study. The clinical history and physical findings of each patient were documented on standardized case report forms. A rectal swab or bulk stool was obtained from each patient who provided verbal informed consent. The rectal or bulk stool swabs were inserted into 1.5 ml of buffered glycerol saline or Cary Blair medium, refrigerated until collection by a courier, transported in a cool box to the central laboratory by motorcycle or car, and plated on the day of collection. Participants received treatment according to national guidelines. 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 visible blood. A diarrhoeal episode was defined as new if the diarrhoea definition was met after three or more days free of diarrhoea or dysentery [24]. A shigellosis episode was defined as a diarrhea episode during which a faecal specimen was obtained from which any Shigella species was isolated. An episode of persistent diarrhea was defined as an episode of diarrhea lasting for 14 d or more. Laboratory Procedures The swabs were inoculated in MacConkey agar and Salmonella-Shigella agar. After overnight incubation at 37 °C, the MacConkey agar and Salmonella-Shigella agar plates were checked for nonlactose-fermenting colonies. Three colonies characteristically resembling Shigella were differentiated from other nonlactose-fermenting enteropathogens by inoculating into Kligler's iron agar for typical reaction, mannitol fermentation, citrate utilization, urease and indole production, and lysine decarboxylation. After incubation for 18–24 h at 37 °C, the test media were read for characteristic Shigella reactions. Serologic identification was performed by slide agglutination with polyvalent somatic (O) antigen grouping sera, followed by testing with monovalent antisera for specific serotype identification (antisera from Denka Seiken, Japan, were used in all sites). In cases where no agglutination occurred with live bacteria, the test was repeated with boiled suspensions of bacteria. S. flexneri isolates that were not typeable with commercial antisera were evaluated at Centre for Health and Population Research, Dhaka, Bangladesh using a panel of monoclonal antibodies specific for S. flexneri group and type factor antigen [25–28]. Antimicrobial susceptibility testing of Shigella isolates against ampicillin, cotrimoxazole, nalidixic acid, and ciprofloxacin was performed by disk diffusion following standardized National Committee for Clinical Laboratory Standards methods. Specimens were processed in local laboratories (Bangladesh: Centre for Health and Population Research, Dhaka; China: Preventive Health Laboratory, Zhengding; Pakistan: Aga Khan University, Karachi; Indonesia: United States Navy Medical Research Unit 2, Jakarta; Vietnam: Institute Pasteur, Nha Trang; and Thailand: Kaengkhoi Hospital, Saraburi). The species, serotypes, subtypes, and antimicrobial resistance patterns of a sample of Shigella strains from the study sites in Vietnam, Bangladesh, and Pakistan were confirmed at the United States Armed Forces Research Institute for Medical Sciences, Bangkok during the initial months of each study. All Shigella isolates from the site in Thailand were confirmed at the World Health Organization National Salmonella and Shigella Centre, Ministry of Public Health, Nonthaburi, and all isolates from the study site in China were confirmed in Fudan University, Shanghai. Real-Time PCR PCR can be used to amplify the gene coding for the invasion plasmid antigen H (ipaH), a gene nearly exclusively derived from the four Shigella spp. in Asia. The only alternative source of ipaH are enteroinvasive E. coli, an organism that is exceedingly rare in the Asian region [29]. Studies using ipaH-based PCR have been published from several Asian countries, including Thailand, Bangladesh, and more recently, India [30–32]. These studies suggest that ipaH can be detected in a large percentage of patients with diarrhoea who are culture-negative for Shigella. It is likely that ipaH detection rates differ not only between stool specimens from culture-positive and culture-negative diarrhoea patients but also between age groups and patients with dysentery and patients with diarrhoea without visible blood. Stool specimens from diarrhoea patients were therefore classified into eight categories as follows: by culture status (Shigella spp. isolated yes/no), presentation (dysentery/nondysentery), and age group (under 60 mo, 60 mo and older). Because it was not feasible to test all culture-negative stool specimens, a sample of specimens was selected from each of the above categories at each site. The sample size required to detect a 95% prevalence of ipaH within a 95% confidence interval (CI) of 86%–99% was 60, and the sample size required to detect a 35% prevalence of Shigella DNA within a 95% CI of 26%–43% was 125. If more specimens had been collected than were required for testing, samples were randomly selected using a computer algorithm. 560 specimens each from Vietnam, Pakistan, and Indonesia were tested. For logistic reasons, a smaller sample was tested from China (n = 337) and Thailand (n = 320). The Bangladesh site provided 980 specimens from diarrhoea patients, of which 39 were culture-confirmed shigellosis cases. A real-time PCR targeting ipaH was employed to detect Shigella DNA in faecal specimens [33]. Briefly, the fluorogenic probe (FAM-CGC CTT TCC GAT ACC GTC TCT GCA-TAMRA) and its flanking primer pair (forward primer ipaH U1, 5′- CCT TTT CCG CGT TCC TTG A-3′; reverse primer ipaH L1, 5′- CGG AAT CCG GAG GTA TTG C-3′) were designed on the basis of ipaH gene sequences. For real-time PCR detection, faecal swabs were washed in 0.8 ml of PBS, of which 0.5 ml was pipetted into 1.5 ml microcentrifuge tubes. The tubes were incubated in boiling water for 30 minutes to lyse bacterial cells. The lysate was subjected to centrifugation at 10,000 rpm for 1 min. The lysate was either used directly for real-time PCR or stored at −70 °C. The working cocktail for the detection contained 1 μl of DNA template, 1× TaqMan buffer A (Applied Biosystems, Foster City, California, United States), 2 mM MgCl2, 100 nM each of dNTPs, 200 nM of primers (ipaH-U1 and ipaH-L1), 40 nM of fluorogenic probe, ipaH-P1 (TET-labelled), and 1.25 units of AmpliTaq Gold (Applied Biosystems) in 25 μl of total reaction volume. The TaqMan assays were conducted using an ABI 7700 Detection System (Applied Biosystems). The amplification profile consisted of heat activation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 30 s; and annealing, extension, and fluorogenic probe hybridization at 60 °C for 1 min. The assay was considered positive when the number of cycles to detection was 38 or less. Real-time PCR-negative samples found to contain inhibitors were further purified using Qiagen Stool Kit (Qiagen, Valencia, California, United States). All PCR assays were conducted at the USAFRIMS, Bangkok, Thailand, and technicians were blinded as to the culture status and clinical presentation of the participants from which the faecal specimens were obtained. Data Management and Analysis Data were double entered into a custom data entry program (FoxPro, Microsoft, Redmond, Washington, United States) with error- and consistency-check programs. Annualised incidence per thousand population was calculated by using age-specific denominators from the baseline census at each site. The observation periods ranged from 12 to 36 mo at each site, and we assumed that each person residing in the study area at the time of the census contributed the respective months to the denominator. The number of all age-specific disease episodes including repeat episodes was used as the numerator. We calculated 95% CIs for incidence rates by the Wilson score method [34]. For intergroup comparisons, we used Chi-square tests for comparison of categorical variables. For the analysis of continuous variables, Student's t-test was used for normally distributed and Wilcoxon rank sum, and Kruskal-Wallis test for non-normally distributed data. Rate ratios were used to detect statistically significant differences between incidence rates. Because the presentations varied by age, species, and site, adjusted logistic regression models were used to assess whether symptoms were more frequent in shigellosis patients compared to diarrhoea patients from whom no Shigella species were isolated. The models took the presence of a sign or symptom in each analyzed diarrhoea episode as the dependent variable and fitted the shigellosis status (shigellosis or a diarrhoea episode during which no Shigella spp. could be isolated) and biologically plausible potential confounders (age and site) as independent variables. Interactions were tested to estimate the effect of age, site, and Shigella species on the frequency of the presenting signs and symptoms. In a secondary analysis, the shigellosis status was replaced as the independent variable with individual Shigella species to evaluate the association between individual species and presentations. The analysis of factors associated with persistent diarrhoea was conducted in an analog fashion. Coefficients of independent variables in the models were exponentiated to estimate the odds ratio of symptoms associated with shigellosis status. Standard errors for the coefficients were used to estimate two-tailed p-values and associated 95% CIs for the odds ratios. The disproportionate sampling of specimens for PCR analysis was accounted for in the analysis which based on weighted averages. A p-value of less than 0.05 (two-tailed) was considered significant. Stata/SE 8.0 (Stata Corporation, College Station, Texas, United States) was used for the analysis. Ethics and Informed Consent After the project's purpose was explained, patients—or in the case of minors, their parents or guardians—gave verbal consent prior to participation in the study. Participation consisted of providing faecal specimens and the information required to complete the case report forms. All studies were approved by each site's local ethics review committees. International approval for all studies, except the study in Bangladesh, was obtained from the Secretariat Committee for Research Involving Human Subjects, WHO, Geneva, Switzerland. The study in Bangladesh was approved by the Bangladeshi Government and by the ICDDR,B ethics review board, which has international membership. Results In the six sites, a total of 605,331 people were studied between 1 and 3 years, resulting in 1,415,583 person-years of shigellosis surveillance (Table 1). The project detected 62,266 diarrhoea episodes, of which 56,958 (91%) fulfilled the eligibility criteria, which consisted of diarrhoea for 1 d or more and patient consent to participate in the study (Figure 1). Figure 1 Assembly of Cases * Eligibility criteria: three or more bowel movements per 24 h or at least one loose stool with blood, and consent from patient or parent/guardian. Incidence The overall diarrhoea incidence was 40 episodes per 1,000 patients per year in all age groups and 254/1,000/y among those under age 60 mo. Shigella was isolated from 2,927/56,958 (5%) of diarrhoea episodes. The overall incidence of treated shigellosis was 2.1 episodes/1,000/y in all ages and 13.2/1,000/y in children under age 60 mo. The shigellosis incidence increased after age 40 y (test for trend, p < 0.001; Figure 2). The shigellosis rates of the overall population as well as in children under 5 y in the site in Bangladesh were statistically significantly higher than the shigellosis rates in China, Pakistan, and Indonesia (p < 0.001), which in turn were significantly higher than those in the two countries with the lowest shigellosis rates, Vietnam and Thailand (p < 0.001; Table 1). Figure 2 Overall Shigellosis Incidence by Age Group at Study Sites in Six Asian Countries Note: Shigellosis incidence in the age group 0–4 y is 13.2/1,000/y. Clinical Presentations of Shigellosis Episodes Culture-confirmed shigellosis cases frequently reported more than one clinical sign or symptom, ranging from watery stools through mucoid stools to dysentery. During 1,807 (65%) of 2,766 culture-confirmed shigellosis episodes, watery diarrhoea was reported; in 1,518 (54%) of 2,802 episodes patients reported mucoid stools; and 790 (27%) of 2,925 episodes patients reported dysentery (Figure 3). In multiple logistic regression models adjusted for study site and age of the patient four clinical signs and symptoms correlated positively with the detection of Shigella spp in stool specimens: dysentery, mucoid diarrhoea, fever, and abdominal cramps (Table 2). In contrast, watery diarrhoea and vomiting were significantly more frequently reported during diarrhoea episodes from which no Shigella spp. was isolated. The percentage of shigellosis patients admitted for hospital care varied between the sites. None of 394 shigellosis patients in Pakistan was admitted, in contrast to 71 of 390 (18%) shigellosis cases in Vietnam (Table 1). Figure 3 Clinical Presentation of Shigellosis Episodes A history of more than one clinical sign and symptom during a single episode is possible. Table 2 A Comparison of Clinical Presentation among Patients from whom Shigella spp. Was and Was Not Isolated Differences in the Age of Shigellosis Patients between Study Sites The median age of shigellosis patients in Bangladesh and Pakistan was 2 y; in Vietnam was 4 y; and in Indonesia and Thailand 5 y. China was a outlier with a median age of 32 y (Kruskal-Wallis test; 5 degrees of freedom; p < 0.001). By age 2 y, 37% and 38% of shigellosis patients in Bangladesh and Pakistan, respectively, had acquired the infection; in Indonesia and Vietnam 30% and 28%, respectively; in Thailand 12%; and in China 5% (Chi-square test; 5 degrees of freedom; p < 0.001). Outcome of Shigellosis Episodes Of 1,114 eligible shigellosis patients, 870 (18%) were followed for 14 d or longer, and 845 (76%) were followed for 90 d in the study sites in China, Vietnam, and Pakistan. Of the 870 (18%) shigellosis patients who were followed for at least 14 d, 153 (18%) reported that the total diarrhoea episode lasted for 14 d or more (Table 3); 91 (11%) stated that diarrhoea was still present 14 d after presentation. Of 845 patients with culture-confirmed shigellosis, 21 (3%) reported medical events such as pneumonia during the 90 d follow-up period. No deaths were detected following shigellosis episodes. Five clinical signs and symptoms at the time of presentation were statistically significantly associated with persistent diarrhoea in adjusted regression models age, fever, mucoid diarrhoea, vomiting, and abdominal cramps (Table 4). No statistically significant association was detected between Shigella species, antimicrobial resistance, and persistent diarrhoea. Table 3 Persistent Diarrhoea and Other Sequelae of 870 Shigellosis Episodes in China, Vietnam, and Pakistan Table 4 Factors Associated with Persistent Diarrhoea in Shigellosis Patients Microbiology S. flexneri was the most frequently isolated species (1,976 [68%] of 2,927) at all sites except in Thailand, where S. sonnei was most common (124 [85%] of 146; p < 0.001; Figure 4). S. boydii was infrequently isolated, except in Bangladesh where it was the second most-common species, accounting for 23% (106 of 464) of shigellosis episodes. In all, 110 (4%) S. dysenteriae serotypes were isolated but none was S. dysenteriae type 1. In each of the six study sites S. flexneri was significantly more frequently isolated from patients ages 60 mo or older than from children under age 60 mo (Figure 4, left bar graph; p < 0.0001). In contrast, S. sonnei was more frequently isolated from children under age 60 mo than from patients age 60 mo or older (Figure 4, right bar graph; p < 0.0001). Dysentery and cramps were significantly more frequently reported by patients with diarrhoea from whom S. flexneri was isolated (Table 5). Figure 4 The Relative Distribution of S. flexneri and S. sonnei at Study Sites in Six Asian Countries S. flexneri (left bar graph) was more frequently isolated from diarrhea patients 5 y and older (p < 0.0001). In contrast, S. sonnei (right bar graph) was more frequently isolated from children under 5 y of age (p < 0.0001). Table 5 Clinical Presentationa of Each Shigella spp. Compared with All Other Shigella spp. Isolated The eight most frequently isolated S. flexneri serotypes (in order of prevalence 2a, 3a, 1a, 1b, 2b, 1c, 6, x) were responsible for 90% of all S. flexneri episodes in all five study sites where S. flexneri is the dominant Shigella species (Table 6). There were statistically significant differences in serotype prevalence of S. flexneri isolates between study sites (Chi square; 40 degrees of freedom; p < 0.001). Table 6 The S. flexneri Serotype Distribution in Six Study Sites In Indonesia, Bangladesh, and Pakistan the S. flexneri serotype distribution was compared from one year to the next and in Thailand and Vietnam over 3 y. Statistically significant shifts in the in the relative proportions of S. flexneri serotypes were seen in each site (p < 0.001), with the exception of the site in Thailand, where over a 3 y period only 22 S. flexneri strains were isolated and no statistically significant shifts in the distribution of serotypes were observed. Antimicrobial Resistance A high percentage of Shigella strains were resistant to ampicillin and cotrimoxazole at all sites (Table 7). The highest percentage of ampicillin-resistant isolates was found in S. flexneri (84%), followed by S. dysenteriae (37%), S. boydii (25%), and S. sonnei (10%; p < 0.001). In contrast, the highest percentage of cotrimoxazole-resistant specimens were S. sonnei (92%), followed by S. flexneri (76%), S. dysenteriae (62%), and S. boydii (49%; p < 0.001). Resistance to nalidixic acid varied widely, from 100% of S. flexneri and 98% of S. sonnei in China, through 75% of S. sonnei and 48% of S. flexneri in Bangladesh, to little or no resistance at the other sites. Ciprofloxacin-resistant S. flexneri isolates were identified in China (18 [6%] of 305), Pakistan (8 [3%] of 242), and Vietnam (5 [2%] of 282). Of the 1,653 ampicillin-resistant and the 1,490 cotrimoxazole-resistant S. flexneri isolates, 1,322 were cross-resistant to ampicillin and cotrimoxazole. Eighteen isolates were resistant to all four tested antimicrobials. Table 7 Antimicrobial Resistance Pattern for Four Shigella Species Detection of ipaH using Real-Time PCR Of 427 Shigella culture-positive specimens, ipaH was detected in 385 (90%). In 1,124 culture-negative patients who reported dysentery, 564 (50%) had ipaH in the stool, in contrast to 673 of 1767 (38%) patients with nonbloody diarrhoea (p = 0.0001). The highest percentage of culture-negative, ipaH-positive specimens was detected in the two countries with the highest shigellosis incidence, China (52%; 95% CI, 45%–60%), and Bangladesh (45%; 95% CI, 42%–49%). In Thailand, one of the sites with the lowest shigellosis incidence rate, ipaH was detected in 14% (95% CI, 9%–19%) of stool samples (Table 1). In Vietnam, another country with relatively low shigellosis incidence, ipaH was detected in 38% (95% CI, 32%–43%) of stool samples. IpaH detection was lowest in children under age 6 mo, peaked between the second and tenth year of life, after which the percentage of ipaH-positive specimens declined until age 40 y, at which time the percentage of positive specimens increased again (Figure 5). Due to the semiquantitative nature of real-time PCR it is possible to compare the relative bacterial load between age groups, which is inversely related to the number of cycles required to detect ipaH (Figure 5). The bacterial load was relatively low during the first year of life and peaked during the second year of life. Between 5 and 40 years of life the bacterial load was relatively low, but it increased again after age 40 y. Figure 5 Relation of Proportion of ipaH-Positive Faecal Specimens and PCR Cycle Number to Age The percentage of Shigella culture-negative stool specimens from diarrhea patients in which ipaH was detected and the mean PCR cycle number required to detect ipaH by patient age suggests that children between ages 2 and 4 y and adults over age 40 y with diarrhoea are most likely to have ipaH in their stool specimens and the bacterial load is likely to be highest in these two age groups. Discussion This first multicentre shigellosis surveillance study found that shigellosis is more ubiquitous than previously thought. At six Asian study sites the overall culture-confirmed shigellosis annual incidence was 13.2 per 1,000 children under age 5 y and 2.1/1,000 in all ages. The shigellosis incidence in the study sites is approximately 100-fold higher than in industrialized countries. In the US in 1999 and the Netherlands 1996–2000 the estimated incidence shigellosis incidence in all ages was 3.7 and 3.2, respectively, per 100,000 per year [35,36]. Shigellosis incidence at our Asian study sites is in the same range as in Chile, where Prado and coworkers reported 9.0–12.6 shigellosis episodes per 100 children aged 12–47 mo in a semirural area between 1994 and 98 [37]. Although considerable shigellosis burden was detected, the actual burden caused by shigellosis was underestimated for two reasons. First, in using passive surveillance for case detection, we depended on the healthcare-seeking behaviour of individual patients. Studies of the care-seeking behaviour conducted in the context of the shigellosis surveillance studies in each site found significant differences in treatment uptake for diarrhoea and dysentery not only between sites but also within sites between adults and children and between patients presenting with diarrhoea and dysentery [15–20]. Shigellosis patients who treated themselves or sought healthcare from providers outside the surveillance system could not be captured. Active surveillance would have provided a more complete detection of all diarrhoea episodes at the risk of capturing trivial episodes that do not require medical care. Data collected in the mid-1980s in a poor, periurban community in Santiago, Chile, indicated that among children under one year of age, 88% of episodes of diarrhoea were mild cases that did not require health care but were detected by active household surveillance [13]. Studies using alternative designs such as active case detection could provide a more complete understanding of the shigellosis burden in the study sites. Second, Shigella spp. are highly fastidious organisms that die rapidly in an unsuitable environment, including the unavoidable temperature fluctuations encountered during transport. Therefore a sample of culture-negative stool specimens from each site was subjected to PCR analysis, which has been found to highly sensitive and specific for Shigella spp. in Asia [33]. Evidence of Shigella DNA was detected in one-third of culture-negative stool specimens. The proportion of PCR-positive stool specimens correlated with age groups, thus lending support to the PCR findings. Presently it is unknown whether the detection of genetic material related to Shigella indicates disease, colonisation, or asymptomatic carriage. The percentage of PCR-positive specimens should therefore be viewed as upper limits of diarrhoeal disease potentially caused by Shigella spp. Besides these limitations we found two additional explanations for earlier underestimates of the shigellosis burden. We found that less than one-third of culture-proven shigellosis episodes presented with dysentery. Clinical case definitions that include only patients with a history of dysentery, frequently used in government data collections, miss more than two-thirds of shigellosis cases. Lastly, in contrast to many other enteric infections, shigellosis is clearly not confined to childhood. On the contrary, the incidence of shigellosis not only increased steadily after age 40 y, but the bacterial load of shigellosis patients increased after age 40 y, suggesting that older people as well as very young children shed the highest bacterial load and may contribute disproportionally to the transmission of shigellosis. Based on these observations, we hypothesize that shigellosis is responsible for a larger proportion of the diarrhea burden in Asia than was previously inferred from culture results or clinical diagnoses. Equally surprising was the benign clinical course of the shigellosis episodes. No deaths were detected and only 21/845 (3%) of patients reported medical events during follow-up. Persistent diarrhea was seen in 18% of patients following shigellosis episodes; however, the clinical importance of these persistent episodes is not clear. Earlier reports from the Asian region have stressed the potential severity and poor outcome of shigellosis episodes [38–40]. Several explanations for the unexpectedly low morbidity and mortality following shigellosis have been considered. First, by consenting to participate in the study, patients were assured to receive adequate treatment. Second, the Shigella strains may have changed during the decade since earlier reports on high shigellosis morbidity and mortality appeared. The absence of S. dysenteriae type 1, the only Shigella species with chromosomal genes encoding the 70-kDa heterodimeric protein known as Shiga toxin, supports this suggestion [41]. Third, earlier studies reporting high morbidity and mortality were hospital-based. As only the most severe shigellosis cases are admitted, the study population is likely to differ from outpatients enrolled in our study. Overall in our study, 6% of shigellosis patients were admitted, with large differences in hospitalization rates between sites. The range of hospitalization rates is perhaps best explained by differences in hospitalization policies between countries; for example, Vietnam has a very low threshold for triggering admissions compared to Pakistan. Finally, the host characteristics have changed over the last decade. While severe malnutrition in childhood remains a problem in the region, the prevalence of malnutrition has declined over the last ten years with the steady increase of economic markers. The widespread increase in income has contributed to the easy access to antibiotics in each of the study sites. In general, patients may have become less vulnerable to severe disease due to the availability of better nutrition and early self-treatment with antibiotics. The over-the-counter sale of antibiotics without prescription enjoys popularity in all our study sites and may be responsible for the emergence of antibiotic resistance. The project confirmed that ampicillin and cotrimoxazole no longer have a place in the treatment of shigellosis. Nalidixic acid was recommended by the WHO as the first-line treatment against shigellosis until 2004, when it was replaced by ciprofloxacin [42]. Complete resistance to nalidixic acid in China and high levels of resistance in Bangladesh have clearly reduced the usefulness of this drug, at least in these two countries. Already 6% of S. flexneri isolates in China are resistant to ciprofloxacin. The emergence of multidrug-resistant Shigella isolates could reverse the benign course of shigellosis episodes observed in this study. The prevention of shigellosis could exert an immediate benefit by substantially reducing the diarrhea burden in the region and by preventing the spread of panresistant Shigella strains. Safe water supplies and adequate sanitation combined with improved hygiene are likely to reduce the shigellosis burden in the future. Steady economic growth is likely to overcome the barriers currently obstructing improvements in infrastructure for underserved populations. But such progress can take decades and will provide little relief to unstable and mobile populations. In this context a safe and affordable vaccine to protect against shigellosis would be a welcome public health tool. Several Shigella vaccine candidates are under development [5–10]. Our findings indicate that vaccines to prevent shigellosis may need to be tailored according to prevalent species and serotypes, since only type-specific immunity has been demonstrated in humans [11–13] and cross-serotype protection is controversial [14]. We found an unexpectedly complex landscape of circulating Shigella strains in six Asian countries. The Shigella species believed to be dominant are S. flexneri in resource-poor countries and S. sonnei in industrialized nations. Consistent with previous reports [1], S. flexneri was most frequently isolated in the study sites in the five resource-poor countries (Bangladesh, China, Pakistan, Indonesia, and Vietnam), whereas in Thailand, which is rapidly becoming industrialized, S. sonnei was the most common species. Surprisingly, S. boydii, which had been thought to be relatively rare, was responsible for nearly one-quarter of shigellosis episodes at the Bangladesh site. Relatively few S. dysenteriae were detected during this surveillance project. Amongst the S. flexneri isolates were a surprisingly wide range of serotypes that varied across the Asian sites. The large variety in Shigella species and serotypes may explain the unusual age distribution of this enteric disease: Patients may remain susceptible to serotypes to which they have not been exposed earlier. The finding that S. sonnei is more frequently isolated in younger than in older children and S. flexneri is more frequently isolated from older than from younger shigellosis patients may be evidence for the steady replacement of Shigella strains with increasing age. Not only do S. flexneri serotypes vary geographically, they vary temporally. We found statistically significant shifts in S. flexneri serotypes between observation years at each of the three sites where a comparison was possible. Temporal shifts in Shigella serotypes have been reported previously in Kolkata, India [43] but not in Santiago, Chile, where the serotype distribution was stable over prolonged periods [13]. Such shifts pose a double challenge for vaccine developers, who must choose the most relevant serotypes for inclusion in a multivalent vaccine, while knowing that replacement strains may emerge following the widespread introduction of the vaccine. To have a major epidemiological impact a shigellosis vaccine may need be a cocktail of antigens from several Shigella species and serotypes. An alternative approach would be to search for an antigen common to all Shigella species and serotypes. A vaccine that could elicit immunity against such a common antigen may be a promising future strategy to control shigellosis. In conclusion, shigellosis is a frequent cause of diarrhea in the more impoverished areas of Asia. Although there were few medical complications associated with shigellosis, control of this disease could reduce of the overall diarrhea burden globally. The development of a vaccine protective against shigellosis is a highly desirable public health goal, but the development of such a vaccine is complicated by the variation in species and serogroups between sites, years, and age groups. Supporting Information Alternative Language Abstract S1 Urdu Translation of the Abstract (1.2 MB PDF) Click here for additional data file. Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov) accession number of the gene discussed in this paper is ipaH (M32063).

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Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

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Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

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Publication date (Electronic): 5 August 2016

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Volume: 11

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Electronic Location Identifier: e0160290

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[001]Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India

Baylor College of Medicine, UNITED STATES

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Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: JM.
Performed the experiments: AD MN.
Analyzed the data: JM AD MN.
Contributed reagents/materials/analysis tools: JM AD MN.
Wrote the paper: JM AD MN.

* E-mail: drjharna@ 123456gmail.com

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Jharna Mandal http://orcid.org/0000-0002-5945-8425

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DOI: 10.1371/journal.pone.0160290

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Date received : 22 April 2016

Date accepted : 15 July 2016

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