Dinesh S. Chandel

A randomized synbiotic trial to prevent sepsis among infants in rural India

Sepsis in early infancy results in one million annual deaths worldwide, most of them in developing countries. No efficient means of prevention is currently available. Here we report on a randomized, double-blind, placebo-controlled trial of an oral synbiotic preparation (Lactobacillus plantarum plus fructooligosaccharide) in rural Indian newborns. We enrolled 4,556 infants that were at least 2,000 g at birth, at least 35 weeks of gestation, and with no signs of sepsis or other morbidity, and monitored them for 60 days. We show a significant reduction in the primary outcome (combination of sepsis and death) in the treatment arm (risk ratio 0.60, 95% confidence interval 0.48–0.74), with few deaths (4 placebo, 6 synbiotic). Significant reductions were also observed for culture-positive and culture-negative sepsis and lower respiratory tract infections. These findings suggest that a large proportion of neonatal sepsis in developing countries could be effectively prevented using a synbiotic containing L. plantarum ATCC-202195.

Fatal Case of Salmonella enterica subsp. arizonae Gastroenteritis in an Infant with Microcephaly

ABSTRACT Salmonella enterica subsp. arizonae is a common gut inhabitant of reptiles, with snakes as the most common reservoir. Though human cases due to this organism are exceedingly rare, it may infect young infants and immunocompromised individuals with a history of intimate associations with reptiles. Gastroenteritis is the most common presentation; others include peritonitis, pleuritis, osteomyelitis, meningitis, and bacteremia. We report a fatal case of S. enterica subsp. arizonae gastroenteritis in a 3-month-old child with microcephaly, with a review of earlier cases and problems encountered in identification of this rare human pathogen.

Extended-spectrum β-lactamase-producing Gram-negative bacteria causing neonatal sepsis in India in rural and urban settings

Extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacilli (GNB) are of increasing clinical concern in all age groups worldwide. Whilst sepsis continues to be the leading cause of morbidity and mortality in Indian neonates in the community, identification of microbiological attributes in this population is lacking. This population-based study enrolled 1738 infants with a diagnosis of clinical sepsis at four participating centres in India. Each study site conducted Bactec blood culture, identified bacterial species by API test and stored isolates at -70 °C. From 252 GNB isolates, 155 (113 Klebsiella species, 21 Escherichia coli and 21 other) were subjected to drug susceptibility testing, ESBL phenotyping and testing for clonal relatedness of ESBL strains by PFGE. The results demonstrated that Klebsiella species and E. coli are the most common GNB causes of neonatal sepsis in India, and over one-third are ESBL producers in both community and hospital settings. ESBL-producing strains exhibited frequent co-resistance to aminoglycosides and ciprofloxacin, but remained susceptible to imipenem. PFGE analysis revealed extensive genetic diversity within the ESBL-producing isolates, showing multiple profiles (total of 23). Over 40% of all ESBL-producing isolates formed three pulsed-field profiles (PFP I-III), with PFP-II being the largest cluster (>20% of all ESBL-producing isolates), sharing strains from two distant locations. Identification of a common clone at two geographically distant centres indicated that predominant clones with increased virulence may exist, even in the absence of any clear outbreak. The presence of ESBL-producing strains in community infants with no prior history of hospitalization or antibiotic use dictates heightened vigilance and further studies on the ecology of these organisms.

Trends and determinants of gastric bacterial colonization of preterm neonates in a NICU setting

Background Newborn gastrointestinal (GI) tract is considered sterile but rapidly acquires a diverse microbiota from its intimate environment. Early acquisition of a bacterial species in the upper GI tract may play a role in establishing the colonic microbiota. There is paucity of molecular data on the upper GI tract microbiota in preterm neonates. Methods Gastric aspirates from 22 neonates with an average gestational age 27.7 weeks (±2.8), weighing 973.2 grams (±297.9) admitted to a neonatal intensive care unit were collected prospectively from weeks 1-4 of life. All samples were evaluated for microbiota using 16S rRNA-based Denaturing Gradient Gel Electrophoresis. Bacterial species colonization and its association with maternal and neonatal demographics, and neonatal clinical characteristics were analyzed. Results Bacteroides spp. was the predominant species in all four weeks. Bifidobacterium spp. colonization was significantly higher in exclusively breast milk fed compared to partially breast milk (PBM) fed neonates in first (p = 0.03) and third (p = 0.03) week of life. Anaerobic bacteria colonization decreased from first through fourth week of life (p = 0.03). Aerobic bacteria colonization was highly dynamic throughout the four week period. Premature rupture of membrane (p = 0.05) and birth outside of study hospital (p = 0.006) influenced the acquisition of bacteria in the first week of life. Birth weight was positively correlated with total number of bacterial species (p = 0.002) and anaerobes (p = 0.004) in PBM-fed neonates during the fourth week of life. H. pylori and Ureaplasma were not detected in any of our samples. Conclusion Gastric bacterial colonization in preterm neonates is unstable during early weeks of life. Delayed oral feeding and use of antibiotics may be responsible for paucity of bacterial species. Monitoring of the gastric microbiota and concurrent examination of stool microbiota may yield important information on the utility of gastric signature patterns for predicting colon microbiota that may drive GI and immune dysfunctions.

Rapid diagnosis of typhoid fever by an in-house flagellin PCR

Enteric fever is an important public-health problem in developing countries. Effective control of the disease requires early detection of cases, definitive treatment and elimination of the source of infection, as well as improvement of the public-health system. Symptoms and signs of the disease are non-specific and laboratory testing is essential for diagnosis. Accurate diagnosis of cases is often not possible as empirical antibiotic therapy decreases the sensitivity of isolation procedures and serological tests lack specificity in endemic areas. Enteric fever is a common cause of morbidity in the Indian subcontinent and Salmonella Typhi accounts for approximately 90 % of infections. Salmonella Paratyphi A is less common but has shown a trend of rising incidence over the past few years, whereas S. Paratyphi B is exceedingly rare. Typhoid fever is an acute systemic infection caused by the bacterium S. Typhi. Worldwide an estimated 21.5 million people develop typhoid fever annually. The diagnosis of typhoid fever still relies on conventional serology and blood culture identification systems. The isolation procedures are very time consuming and expensive. PCR assays facilitate prompt and appropriate treatment of confirmed cases and also help in the detection of carriers thereby limiting spread.

Predictors and respiratory depositions of airborne endotoxin in homes using biomass fuels and LPG gas for cooking

Recent studies have highlighted the presence of endotoxin in indoor air and its role in respiratory morbidities. Burning of household fuels including unprocessed wood and dried animal dung could be a major source of endotoxin in homes. We measured endotoxin levels in different size fractions of airborne particles (PM10, PM2.5, and PM1), and estimated the deposition of particle-bound endotoxin in the respiratory tract. The study was carried out in homes burning solid biomass fuel (n=35) and LPG (n=35). Sample filters were analyzed for endotoxin and organic carbon (OC) content. Household characteristics including temperature, relative humidity, and carbon dioxide levels were also recorded. Multivariate regression models were used to estimate the contributing factors for airborne endotoxin. Respiratory deposition doses were calculated using a computer-based model. We found a higher endotoxin concentration in PM2.5 fractions of the particle in both LPG (median: 110, interquartile range (IQR) 100–120 EU/m3) and biomass (median: 350, IQR: 315–430 EU/m3) burning homes. In the multivariate-adjusted model, burning of solid biomass fuel (β: 67; 95% CI: 10.5–124) emerged as the most significant predictor followed by OC (β: 4.7; 95% CI: 2.7–6.8), RH (β: 1.6; 95% CI: 0.76–2.4), and PM2.5 (β: 0.45; 95% CI: 0.11–0.78) for airborne endotoxin (P<0.05). We also observed an interaction between PM organic carbon content and household fuel in predicting the endotoxin levels. The model calculations showed that in biomass burning homes, total endotoxin deposition was higher among infants (59%) than in adult males (47%), of which at least 10% of inhaled endotoxin is deposited in the alveolar region of the lung. These results indicate that fine particles are significant contributors to the deposition of endotoxin in the alveolar region of the lung. Considering the paramount role of endotoxin exposure, and the source and timing of exposure on respiratory health, additional studies are warranted to guide evidence-based public health interventions.

Live Colonocytes in Newborn Stool: Surrogates for Evaluation of Gut Physiology and Disease Pathogenesis

Studies of gastrointestinal pathophysiology are not feasible by biopsies in human neonates. We examined the utility of live colonocytes in stool in studying cellular markers during early neonatal life. Expression of IgA, IgG, cluster of differentiation-45 cells (CD45), and toll-like receptors-2 and 4 (TLR2 and TLR4) were analyzed by flow cytometry. Colonocyte RNA extracts were used in quantitative real-time PCR (qRT-PCR) to examine the expression of cytokeratin-19, ribosomal protein-24, and tight-junction (Tj) protein zonula occludens-1 (ZO-1). Colonocyte yield varied between 5 × 104 to 2 × 106 cells/g of stool. Meconium samples yielded a highly enriched population of viable cells. Although low, all samples showed CD45-positive cells during the initial weeks of life. Starting as early as d 2, IgA expression was observed in 69% of the cells. Low to moderate expression of IgG was observed with a linear increase as the infants grew. There was an almost total lack of TLR2 staining; however, >55% of the colonocytes showed TLR4 expression. Although high levels of IgA in gut cells may serve as a natural protectant during neonatal period, increased TLR4 may provide a niche for lipopolysaccharide (LPS)-mediated epithelial damage. Use of stool colonocytes can be a valuable noninvasive approach for studying gut pathophysiology in the neonatal period.

Molecular Typing Reveals a Unique Clone of Salmonella enterica Serotype Typhi among Indian Strains

Typhoid fever (TF) is endemic in India, and a seasonal upsurge of cases is observed annually. Although mortality from TF has been greatly reduced by effective therapies and the availability of vaccines, morbidity is still high due to the global emergence of multidrug resistance and subsequent treatment failures (4). Studies have also documented the circulation of multiple genetic variants (17) and various clinical manifestations of TF (5). In an ongoing study comprising molecular subtyping of Salmonella enterica serotype Typhi strains collected over the past 5 years, we encountered a strain cluster that had been isolated during a seasonal upsurge of TF cases. A total of 16 strains was recovered serially from cases reported during a weeks time. We highlight the emergence of a unique clone of serotype Typhi strains circulating in the Delhi region. Among the probes used for strain typing with DNA fingerprint analysis, insertion sequences (IS) have acquired great popularity (7). While originally discovered in the genome of serotype Typhimurium LT2, one such shorter element, IS200, a 707- to 710-bp sequence, is widely distributed among conserved loci on the chromosomes of most Salmonella serotypes (7, 10). The apparent copy numbers, 4 to 7 in serotype Paratyphi A and 10 to 25 in serotype Typhi, and identities among strains in the actual copy numbers and positions of IS200 indicate their evolutionary origin (6, 15). Since IS200, unlike other enterobacterial IS elements (e.g., IS1 and IS5), rarely translocates under laboratory conditions, its use has become increasingly popular for strain differentiation, including the determination of the origins of drug-resistant strains (18). Studies with sufficiently large numbers of strains have clearly shown that IS200 profiles are indeed characteristic for the serovars, where the patterns are not only of value as epidemiological markers but also sufficiently stable to allow reconstruction of molecular phylogenies (2). In the present study, salmonella-specific IS200 fingerprinting (15) and pulsed-field gel electrophoresis (17) were used in the characterization of serotype Typhi strains from this identified episode. Genome hybridization by IS200 probing revealed two distinct profiles: insertion sequence type I (IST-I) and IST-II. The IS200 probe traced either 11 to 14 hybridization bands (IST-I) or a mere 2 hybridization bands (IST-II) bearing IS200 insertions distributed over PstI-digested DNA fragments ranging from 1 to 25 kb in size. IST-II represented a unique pattern, as revealed by the presence of 2 IS200-probed bands versus the 10 to 25 apparent copies consistently observed for the global population of serotype Typhi strains. Six out of the 16 strains were assigned this unique pattern (Fig. ​(Fig.11). FIG. 1. Chemiluminescence X-ray signals generated by IS200 probing. Lanes 2 to 7, the unique IST-II pattern shared by six strains with two IS200-probed DNA fragments; lanes 1 and 8 to 16, the IST-I pattern shows a serotype Typhi-specific pattern with 11 to 14 ... PFGE analysis using XbaI showed two major profiles: pulsed-field profile I (PFP-I), with 14 to 17 genomic fragments ranging from 550 kb to 20 kb, and PFP-II, showing 11 to 12 fragments of 440 kb to 20 kb. Notably, major differences in genome sizes were observed among PFP subtypes. The absence of nearly 500 to 850 kb of the genomic portion of PFP-II resulted in reduced genome sizes ranging between 3.7 and 4.1 Mb, in comparison to the 4.4- to 4.8-Mb genomes observed among predominant serotype Typhi strains. Displaying a high degree of correlation in their discriminatory abilities, both the IS200 and the PFGE analyses discerned unique clones (IST-II and PFP-II) that were nearly identical (Fig. ​(Fig.22). FIG. 2. Comparative cluster analysis of IS200 and PFGE profiles. (A) The unique IST-II pattern (strains A2 to A7) revealed reduced apparent IS200 copies. The IST-I cluster shows a serotype Typhi-specific predominant pattern and further subtypes Ia to Ic. Serotype ... Clinically, the patients presented with the usual TF symptoms and without any significant complications. Overall, the strains did not reveal any significant phenotype variations. Based on phage group determination, the majority of the isolates grouped into a single phage type, E1; however, two strains from the unique cluster remained untypeable by available phage panels. Although several phage types linked to human disease activities have been recognized for serotype Typhi, one or a few types dominate within a geographical region for a specified period, presumably due to the spread of a successful clone or to the infrastructure of the host population (16). A long-standing and paradoxical trait of IS200s behavior is its extremely low transposition frequency, which can be viewed as a self-restraint strategy imparting evolutionary advantages (1). Only a few cases of IS200 transposition or other forms of rearrangement have been documented in salmonellae (8). For instance, serotype Typhimurium strain LT2 was shown to harbor 6 copies of the IS200 element (7), whereas recent studies have actually evidenced 7 to 16 copies (15). Recently, a new clonal line of Salmonella serotype Saintpaul which virtually lacks this IS element has also been reported to be circulating in Japan (9). It has previously been shown that the apparent number of serotype Typhi IS200 insertions ranges from 10 to 25 (7), while PstI profiling probed at least 11 to 13 bands (18). Recently, the whole-genome sequence comparisons of the serotype Typhi Ty2 and CT18 strains have actually revealed 26 copies of IS200 (6); however, earlier IS200 profiling of PstI-digested genomic DNA of the same strains yielded only 15 discrete hybridization signals (3). These studies point towards a remarkable conservation of numbers and locations of the IS200 element among serotype Typhi strains globally and also indicate that the number of hybridization fragments may not depict the actual copy number, since individual bands of IS200 profiles either may carry multiple copies of IS200 or may represent various copies over PstI fragments of similar sizes. In the present study, the new unique pattern (IST-II) showed only 2 apparent IS200 hybridizing bands compared to 11 to 14 copies consistently present among the majority of Indian serotype Typhi strains. The findings suggest that recent genomic rearrangements may have caused the emergence of newer IS200 types during an outbreak in this region. It is well known that even a few genetic changes can generate large differences in pattern or number of hybridization bands displaying an uneven distribution of IS200 insertions. Therefore, caution should be exercised in the analysis of hybridization results, especially while probing a low-copy-number IS element, since it can affect the cluster analysis by separating otherwise closely linked strains. Large-scale evolutionary genome alterations may affect the microbial ecology of this important human pathogen. Genomes of serotype Typhi are shown to undergo rearrangements due to homologous recombination among the seven copies of rrn genes (13). Therefore, even closely related strains may differ widely in gene content. Although most Salmonella serovars share over 90% genome homology, they often display drastically different properties pertaining to host range specificity and the nature of the illnesses they cause. A recent study indicated that genomic regions encoding phenotypic features required in drug resistance and virulence mechanisms tend to evolve faster than other genomic regions due to diversifying selection pressure exerted by the hosts immune response (11). Previously, seven strains of serotype Typhi have been shown to exhibit precise deletions of the entire 134 kb of salmonella pathogenicity island 7 (SPI7), all with a reduction in size of the I-CeuI (intron-encoded endonuclease) G fragment known to contain the SPI7-harboring gene for Vi polysaccharide synthesis. Such alterations are believed to render many strains nonagglutinable by antiserum to Vi antigen (12). Since, in the present study, two of six IST-II strains remained untypeable by Vi-phage grouping, these strains probably had asymmetrical genomic rearrangements. Notably, even large alterations have not substantially altered the stability or survival of this clone. This is perhaps not surprising in light of the extensive genomic diversities observed for the highly plastic serotype Typhi genomes, an intrinsic characteristic of intracellular bacteria. Whole-genome analysis of a strain with a multidrug resistance phenotype, CT18, revealed hundreds of insertions and deletions ranging from a few genes to larger islands, mainly attributed to the human-restricted host range for serotype Typhi (14). Although whole-genome analysis may incompletely depict the total gene content of any bacterial species (e.g., Salmonella enterica serotype Typhi) with circulating variants, global therapeutic and prevention strategies based on localized inferences against highly plastic serotype Typhi genomes may also yield only partial success. More sequence-based comparative analyses of such unique strains may also yield further insights into IS200s evolutionary mechanisms. Careful monitoring of larger groups of clinical and field isolates for diverse clones and their spread in the population is envisaged in light of the varying molecular epidemiology of serotype Typhi strains.

Neonatal sepsis in rural India: Timing, microbiology and antibiotic resistance in a population-based prospective study in the community setting

Objective:To examine the timing and microbiology of neonatal sepsis in a population-based surveillance in the Indian community setting.Study Design:All live born infants in 223 villages of Odisha state were followed at home for 60 days. Suspect sepsis cases were referred to study hospitals for further evaluation including blood culture.Results:Of 12 622 births, 842 were admitted with suspected sepsis of whom 95% were 4 to 60 days old. Culture-confirmed incidence of sepsis was 6.7/1000 births with 51% Gram negatives (Klebsiella predominating) and 26% Gram positives (mostly Staphylococcus aureus). A very high level of resistance to penicillin and ampicillin, moderate resistance to cephalosporins and extremely low resistance to Gentamicin and Amikacin was observed.Conclusion:The bacterial burden of sepsis in the Indian community is not high. Judicious choice of empiric antibiotics, antibiotic stewardship and alternate modalities should be considered for the management or prevention of neonatal sepsis in India.

Changes in the Gut Microbiota After Early Administration of Oral Synbiotics to Young Infants in India

Objectives: The authors examined the changes in the developing gut microbiota of Indian infants enrolled in a colonization study of an oral synbiotic (Lactobacillus plantarum and fructo-oligosaccharides) preparation. Methods: Frozen stool samples were available from a previously published clinical study of the synbiotic preparation administered daily for 7 days to full-term Indian infants delivered by C-section. 16S rRNA gene sequencing of fecal bacterial community-DNA was done in 11 infants sampled on day 7 and day 60 of life. Results: All infants showed changes in bacterial diversity with age. While Firmicutes and Proteobacteria were predominant in all, Actinobacteria and Bacteroidetes were initially low on day 7. In control infants, we observed a significant increase (P = 0.012) in the proportions of Actinobacteria on day 60. In the treated group, during the 60-day period, there was a 10-fold increase in Bacteroidetes, a somewhat smaller increase in Firmicutes, and a reduction in Proteobacteria. Compared to controls, treated infants were increasingly colonized by different Gram-positive genera including Enterococcus, Lactobacillus, and Bifidobacterium. Relatively less known taxa and some unassigned sequence reads added to enriched diversity observed in the treated group. Conclusions: There was a high level of bacterial diversity among infants examined in the present study. Synbiotic treatment induced an increase in overall taxa and Gram-positive diversity, especially in the first week of life. Changes in the microbiota during early infancy should be used as a rationale for selecting probiotics in diverse clinical settings.