Characteristics of Salmonella Recovered From Stools of Children Enrolled in the Global Enteric Multicenter Study

Abstract Background The Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates. Methods Salmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313. Results Of 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics. Conclusions Our data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa.

about the prevalence of NTS serovars that cause gastroenteritis in Africa and South Asia.
Invasive NTS (iNTS) causes bacteremia in sub-Saharan Africa, occurring predominantly in infants, toddlers, as well as in malnourished or malaria-infected adults and/or those with human immunodeficiency virus (HIV) [8,9]. Although the incidence of iNTS has declined in many sites across Africa [10], it is still one of the most common causes of bloodstream infections in African children [11]. Interestingly, unique clades of serovars Typhimurium and Enteritidis are associated with bacteremia in this region [8,12,13]. Most of the Typhimurium strains isolated from blood in sub-Saharan Africa belong to multi-locus sequence type (ST) 313 [14]. In contrast, the most common genotype isolated worldwide is ST19, which is generally associated with gastroenteritis [15] but has recently been reported as a primary cause of invasive infections in a study in Uganda [16]. Both ST19 and ST313 genotypes have been isolated from patients with either gastroenteritis or bacteremia in Kenya, although the number of diarrhea cases was low [17].
The use of antibiotics to treat uncomplicated NTS gastroenteritis in children is not recommended, except where progression to invasive disease is a risk [18,19]. However, information about the antimicrobial susceptibility of NTS is useful as this knowledge contributes to our overall understanding of resistance markers that are circulating in specific geographic locations. In fact, NTS harboring antimicrobial resistance traits in the gastrointestinal tract could serve as a reservoir for iNTS [20]. Presently, countries with the highest burden of iNTS disease report 48-75% multidrug resistance to commonly used antibiotics, a major concern given that more-effective thirdgeneration cephalosporins or fluoroquinolones may be less available or more costly in these settings [11,21].
During 2007-2010, the Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-tosevere diarrhea (MSD) in children 0-59 months old living in The Gambia, Mali, Mozambique, Kenya, India, Bangladesh, and Pakistan [22]. This large, prospective, case-control study determined that NTS was significantly associated with MSD in infants (0-11 months) from the Bangladesh site and toddlers (12-23 months) and young children (24-59 months) from the Kenya site [22]. Here, we determined the prevalence of Salmonella serovars isolated in GEMS, evaluated antimicrobial susceptibility, identified Typhimurium sequence types, and examined the phylogenetic relatedness of Typhimurium ST313 isolates.

GEMS Study Participants
The methods and main findings from GEMS have previously been described [22][23][24]. Briefly, GEMS participants were recruited from censused populations during 2007-2010 in The Gambia, Mali, Mozambique, Kenya, Bangladesh, India, and Pakistan. Study participants included children aged 0-59 months of age with MSD who presented to a sentinel health facility (see Supplementary Methods for additional details). Children were recruited into 0-11-, 12-23-, and 24-59month age groups. For each child with MSD (case) enrolled, 1-3 children without diarrhea during the previous week (controls) were recruited. Scientific and ethics committees and institutional review boards of participating institutions in each country as well as the coordinating institution, University of Maryland, Baltimore, approved the study protocol prior to implementation. Informed consent was obtained in the local dialect from all participating caretakers before recruitment of their children into the study.

Detection of Salmonella spp.
A panel of enteropathogens was identified from stool specimens, collected at the clinic from MSD cases or obtained at home by caregivers of children in the control group, as previously described [24]. Salmonella spp. were shipped to the Center for Vaccine Development and Global Health (CVD) at the University of Maryland School of Medicine for additional characterization.

Characterization of Salmonella Serovars From Stools
At CVD, Salmonella spp. were agglutinated using polyvalent O and O1 antisera followed by . Serovars Typhimurium, Typhi, Enteritidis, and Paratyphi B were fully serotyped (using O and H typing antisera) and additionally confirmed by polymerase chain reaction (PCR) [25,26].

Sequence Typing of Typhimurium Isolates
Sequence types were determined for all 87 Typhimurium isolates using multi-locus sequence typing (MLST) by PCR and sequencing and/or by examining whole-genome sequences. Sequence typing by MLST followed methodology described previously [15].

Whole-Genome Sequencing and Phylogenetic Analysis
The majority of the Salmonella isolates (355 out of 370) were subjected to whole-genome sequencing. Following sequencing, 120 isolates were excluded from subsequent analyses as they did not meet the quality-control criteria. Details of sequencing and phylogenetic analyses are described in the Supplementary Methods.

Antimicrobial Susceptibility Testing
The susceptibility of the 370 Salmonella isolates to chloramphenicol, ampicillin, ciprofloxacin, trimethoprim/sulfamethoxazole (TMP/SMX), gentamicin, and ceftriaxone was determined using the Kirby-Bauer disk-diffusion method and interpreted according to Clinical and Laboratory Standards Institute guidelines. Multidrug resistance was defined as resistance to ampicillin, chloramphenicol, and TMP/SMX. To assess whether the high resistance of NTS to antimicrobials was associated with antibiotic prescription rate, we determined the percentage of children with MSD (and Salmonella isolated in stools) who had been prescribed (but may or may not have been given) antimicrobial agents after visiting any of the sentinel health facilities that participated in GEMS.

Statistical Analysis
To determine which individual Salmonella serovars were driving the association between Salmonella and MSD that was found in the original GEMS analyses [22], we used the same conditional logistic regression model as previously described [27]. Instead of including Salmonella species in the model we included variables for each Salmonella serogroup/serovar. The association of each serovar with MSD was adjusted for other co-pathogens. The rationale for this approach, generally, and in the unique context of GEMS, has been discussed previously [27]. Analyses were conducted using R version 3.3.2 (R Foundation for Statistical Computing). P values less than .05 were considered statistically significant.

Characteristics of Study Participants With Moderate-to-Severe Diarrhea
Of the cases with Salmonella identified, 86 (44%) were in the 0-11month age group, 55 (29%) were in the 12-23-month age group, and 49 (26%) were in the 24-59-month age group (Table 1). Cases experienced severe signs of MSD. Approximately 20% of infants with Salmonella spp. detected had bloody diarrhea, while 100% of children aged 12-59 months old with Salmonella spp. produced watery diarrhea. Of note, a lower proportion of children with MSD who had Salmonella isolated tended to be female in all age groups.

Geographical Distribution and Prevalence of Salmonella Serovars
The serovar distribution of the 370 Salmonella isolates (190 from cases and 180 from controls) collected from stools of study participants is shown in Figure 1. Of these, 361 were NTS. Additionally, we recovered 8 Typhi from Asia and 1 Paratyphi A isolate from Bangladesh. The most frequent NTS serovars identified were Typhimurium, serogroup O:8 (C 2 -C 3 ), serogroup O:6,7 (C 1 ), Paratyphi B Java, and serogroup O:4 (other than Typhimurium or Paratyphi B). Serovar Typhimurium predominated in Africa, whereas serogroup O:6,7 (C 1 ) and O:8 (C 2 -C 3 ) serovars were the most common in Asia.

Prevalence of Salmonella Serovars by Site and Age Stratum
The prevalence of the most abundant serovars isolated in GEMS, as well as serovar Enteritidis (due to its importance in iNTS disease in Africa), is shown in Table 2. The individual serovars of isolates are listed in Supplementary Table 1. In general, we found that the rates of NTS isolation were low (≤5.3%) in both cases and controls regardless of age groups, although some site-to-site variation was apparent. At the Kenya site, serovar Typhimurium, the most prevalent serovar, was recovered in stools of MSD cases at a rate of 3.3% for infants, 3.7% for toddlers, and 4.3% for young children. In Bangladesh, Paratyphi B Java, the most prevalent serovar there, was recovered from 2.0% of infants with MSD. Serogroup O:8 (C 2 -C 3 ) organisms were most prevalent in stools at the Pakistan site. The prevalence of NTS in cases and controls in The Gambia, Mali, Mozambique, and India was less than 1.5%.

Salmonella Serovars Significantly Associated With Moderate-to-Severe Diarrhea
Previously, 3.2% and 3.7% of MSD episodes in toddlers (12-23 months) and children (24-59 months) at the Kenya site, respectively, and 4.6% of MSD episodes in infants at the Bangladesh site were shown to be attributable to Salmonella [22]. We determined the serovars driving the associations by using a conditional logistic regression model (Supplementary Table 2

Antimicrobial Susceptibility at GEMS Sites
Salmonella isolates from the African and Asian sites differed in terms of their antimicrobial susceptibility ( Figure 2). Isolates from Africa were susceptible to ciprofloxacin and ceftriaxone, whereas resistance to these antibiotics was observed among Asian NTS isolates. We observed 65.4% of NTS from MSD cases in Kenya to be multidrug resistant (MDR) to ampicillin, TMP/SMX, and chloramphenicol. However, isolation of nonsusceptible NTS was less frequent at the site in The Gambia; only 6.7% of NTS from cases showed an MDR phenotype ( Figure 2A). In Asia, Indian isolates showed more resistance to the antibiotics tested than isolates from the other 2 Asian sites ( Figure 2B). We observed antimicrobial susceptibility profiles among NTS from controls that were similar to cases at each site except for Kenya and India. Serovars Typhimurium and Enteritidis from Africa and serogroup O:6,7 (C 1 ) and serogroup O:13 (G) isolates from Asia showed the highest percentage of antimicrobial resistance ( Figure 3). All Enteritidis and Paratyphi B Java isolates recovered from GEMS stools from Asia were pan-susceptible to antimicrobial agents, while the 6 serogroup O:13 (G) isolates from Africa were pan-susceptible. Five of the 8 (62.5%) serovar Typhi from Asia (Pakistan and India) were MDR.
In general, most MSD cases with Salmonella had been prescribed or given an antibiotic (except in Pakistan) ( Table 3). Trimethoprim/sulfamethoxazole was the most commonly prescribed antibiotic in Africa, whereas ciprofloxacin was the most common in Asia.

Phylogenetic Analysis of Salmonella Typhimurium
Since Typhimurium was the most important cause of iNTS disease at several GEMS sites and was the most frequent serovar isolated from stools, a phylogenetic analysis was performed. Of 87 Typhimurium isolates, 74 (85.0%) were from Africa (Kenya), while 13 (14.9%) were from Asia (Pakistan, India, and Bangladesh). Table 4 shows the sequence types (ST) of these Typhimurium isolates listed by site of origin.
A phylogeny was constructed using whole-genome sequences to determine the relationship between the Typhimurium ST313 isolates from MSD cases and controls (Figure 4). The African ST313 sequence type has been divided into the older lineage 1 isolates and the more recent lineage 2 [13,28]. Here, 50 of 55 study isolates analyzed (90.9%) clustered with the ST313 lineage 2 reference genome D23580, 49 of 55 (89%) of which showed the typical MDR phenotype associated with lineage 2, namely resistance to chloramphenicol, co-trimoxazole, and ampicillin. The lineage 2 isolates from the MSD cases and diarrhea-free controls were closely related and could not be distinguished phylogenetically. A group of 5 isolates in stools of cases and controls, regardless of age, formed a small lineage 2 subcluster associated with susceptibility to chloramphenicol. Four of the 55 isolates (7%) clustered with the ST313 lineage 1 reference genome A130, all of which were isolated from MSD cases in Kenya and were sensitive to chloramphenicol. Of note, the 1 study isolate (specimen 700477) that failed to cluster with lineage 1 or lineage 2 and demonstrated pan-susceptibility to antibiotics was isolated from a case of MSD from Pakistan.

DISCUSSION
Salmonella isolates were detected in stools of children with MSD and from diarrhea-free community controls at each of the GEMS sites. A primary finding of our analysis was that, except for Typhimurium, the prevalence of most Salmonella serovars was similar in stools of cases and controls, regardless of age and across study sites. Because children enrolled as controls in GEMS only had to have been free of diarrhea for the previous 7 days, we could not rule out asymptomatic carriage or shedding of Salmonella among controls due to persistent excretion or convalescence [29]. Nontyphoidal Salmonella are reportedly excreted for longer periods in children than adults, lasting from several weeks to months [18,30]. We found that NTS was as prevalent in cases as in controls, which suggests that NTS is endemic at the 7 GEMS sites [3,31,32].
In this study, we report the association of Typhimurium ST313 with acute diarrhea in Kenya using a conditional logistic regression model, showing that these bacteria cause diarrhea and are not just associated with invasive disease. This observation is supported by recent studies from Kenya, the Central African Republic, and Democratic Republic of Congo, which also detected Typhimurium ST313 in stool [17,33,34]. Typhimurium ST313 was identified in both MSD cases and controls, confirming that this important sequence type can be carried asymptomatically by humans. Phylogenetic analysis identified lineages 1 and 2, in accordance with previous findings [28]. We found that the same ST313 lineage (lineage 2) was prevalent in the stools of both MSD cases and controls. The fact that isolates from cases and controls are found in every part of the phylogeny suggests that the Typhimurium isolates that cause MSD are closely related to those associated with asymptomatic carriage; NTS carriage has been reported elsewhere in sub-Saharan Africa [34][35][36].
Several groups have attempted to identify the reservoir of iNTS isolates in Africa. Kariuki et al [20] were the first to suggest that these bacteria are not acquired zoonotically but are acquired by anthroponotic transmission. In this and other studies, NTS isolated from blood cultures of bacteremic index cases were highly similar to isolates from household contacts but different from NTS from animal or environmental sources taken from around the homes of index cases [20,36]. Collectively, these prior studies suggest that the reservoir for Typhimurium ST313 is indeed humans. It remains possible that the lack of detection of Salmonella spp. from animals and the environment reflects difficulties in culture from these specimen types. However, if the inference from the above-mentioned studies is correct, our data would support these findings by showing that Typhimurium strains isolated from stools of cases and controls in GEMS are highly genetically related to isolates from blood.
When we examined the antimicrobial susceptibility of GEMS NTS isolates, we detected marked regional differences in resistance. We observed similar antimicrobial susceptibility patterns in stools of cases and asymptomatic controls at all GEMS sites except for Kenya and India. Our data suggest that antibiotic-resistant NTS are circulating in the GEMS communities. Nonsusceptible NTS strains could serve as a reservoir from which antibiotic-resistance determinants can spread horizontally to other microorganisms [37]. In Africa, the majority of Typhimurium and Enteritidis isolates were MDR, which is consistent with previous findings [20,38]. Importantly, none of the isolates from GEMS African sites were resistant to ciprofloxacin or ceftriaxone, in contrast to isolates from Asia, suggesting a difference in utilization of these antibiotics. Five (of 8) Typhi from India and Pakistan were MDR but none were extensively drug resistant, as seen in the recent typhoid fever outbreak in Hyderabad, Pakistan [39].
Antibiotics are not recommended for the treatment of NTS gastroenteritis in pediatric patients due to the predisposition for extended excretion of bacteria and relapse of infection [18,40,41]. However, our data suggest that children with NTS disease are being prescribed antibiotics, which may have selected for resistant bacteria. We observed high prescription rates for ciprofloxacin and other fluoroquinolones in Asia and, not surprisingly, also high resistance of Salmonella to ciprofloxacin in Asia but not Africa (where ciprofloxacin was rarely prescribed). In contrast, we recorded high antibiotic prescription rates of TMP/SMX in Africa, which possibly led to the high resistance observed in Africa. TMP/SMX in combination with highly active antiretroviral therapy has been used routinely as prophylaxis for opportunistic infections in patients with HIV in Africa [42].
The low frequency of Salmonella in MSD cases from Mali, The Gambia, and Mozambique was somewhat unexpected given that these countries report high iNTS disease burdens [3,31,32]. However, the incidence of iNTS disease during GEMS (2007-2010) in these 3 countries decreased relative to earlier estimates, concomitant with a reduction in clinical malaria [3,31,32,43]. Indeed, there is growing evidence to suggest that iNTS disease is correlated with clinical malaria and that efforts to control malaria have resulted in reduced iNTS disease incidence [9,44]. A re-analysis of GEMS using quantitative molecular diagnostic methods showed higher attributable fractions for Salmonella in all age groups at all sites [45].
Our findings have 3 main implications: (1) the prevalence data could be used to refine incidence estimates for individual Salmonella serovars; (2) we report for the first time the a Total number of cases with moderate-to-severe diarrhea who were prescribed or given any antimicrobial agent at any participating sentinel health facility.
b Antimicrobial agents that were tested for susceptibility. association of Typhimurium ST313 with acute diarrhea, thereby showing that these bacteria are not just associated with invasive disease; and (3) our data demonstrate widespread asymptomatic carriage of ST313, a key cause of iNTS infections. Because we found that humans are carriers of MDR Salmonella strains that also cause iNTS [46], it is possible that these individuals serve as . Genetic relationship between Salmonella Typhimurium ST313 isolated from MSD cases and diarrhea-free controls. The core genome maximum likelihood tree is shown for Salmonella Typhimurium ST313 isolated from the stool of cases and controls of children aged under 5 years in Kenya and Pakistan (1 isolate), which were collected as part of the GEMS. Scale bars in SNPs are shown beneath the phylogeny. Patient group, age range, and AMR data for the isolates are displayed using color strips created using Interactive Tree of Life (iTOL; Biobyte Solutions, Heidelberg, Germany) and are labeled and colored according to the in-laid key. Isolates that cluster with the lineage 1 or lineage 2 reference genomes are indicated. The tree is rooted using ST19. Abbreviations: AMR, antimicrobial resistance; GEMS, Global Enteric Multicenter Study; MSD, moderate-to-severe diarrhea; SNP, single nucleotide polymorphism; ST, sequence type.