Pinaki Panigrahi

Effects of oral Lactobacillus GG on enteric microflora in low-birth-weight neonates

Background Colonization patterns, especially by anaerobic flora, may play an important role in neonatal gut function. Probiotics could affect disease risk either directly through colonization or indirectly by promoting changes in gut microbial ecology. Methods To study the ability of Lactobacillus GG (LGG) to colonize the neonatal gut and modify its microbial ecology, a prospective, randomized study was performed in 71 preterm infants of less than 2000 g birth weight. Infants less than 1500 g (24 treated, 15 control) received 109 LGG orally twice daily for 21 days. Those infants weighing 1500 to 1999 g (23 treated, 9 control) were treated for 8 days. Stools were collected before treatment and on day 7 to 8 (and day 14 and 21, in the infants weighing less than 1500 g) for quantitative aerobic and anaerobic cultures. Results Colonization with LGG occurred in 5 of 24 (21%) infants who weighed less than 1500 g versus 11 of 23 (47%) in larger infants. Colonization was limited to infants who were not on antibiotics within 7 days of treatment with LGG. There was a paucity of bacterial species at baseline, although larger infants had more bacterial species (1.59 ± 0.13 (SEM) vs 1.11 ± 0.12;P < 0.03) and higher mean log colony forming units (CFU) (8.79 ± 0.43 vs 7.22 ± 0.63;P < 0.05) compared with infants weighing less than 1500 g LGG. Treatment in infants weighing less than 1500 g resulted in a significant increase in species number by day 7, with further increases by day 21. This increase was mainly the result of increased Gram (+) and anaerobic species. No difference in species number was noted in controls. Mean log CFU of Gram (−) bacteria did not change in treated infants weighing less than 1500 g. However, Gram (+) mean log CFU showed a significant increase on day 21 (6.1 ± 0.9) compared with day 0 (3.5 ± 0.9) (P < 0.05). No significant changes in species number or quantitative counts were noted after LGG treatment in the infants weighing 1500 to 1999 g LGG was well tolerated in all infants. Conclusion The neonatal response to a probiotic preparation is dependent on gestational and post-natal age and prior antibiotic exposure. Although LGG is a relatively poor colonizer in infants, especially those infants weighing less than 1500 g at birth, it does appear to affect neonatal intestinal colonization patterns.

Clinical Manifestations and Predictors of Severe Malaria in Indian Children

OBJECTIVE. Although the greatest morbidity and mortality attributable to malaria occurs among children in Africa, up to one third of the worlds malaria burden is borne by non-African countries, where levels of endemicity are lower. Because there are few published criteria for managing life-threatening malaria in children in these countries, we conducted a study of major syndromes and predictors of death among critically ill Indian children to identify factors that could be used to improve the approach to their treatment. METHODS. A prospective study was conducted at the pediatric ward of SCB Medical College in eastern India (Orissa). Baseline demographic data were collected on all of the patients with confirmed slide-positive falciparum malaria. Patients satisfying any 1 of the 2000 World Health Organization criteria for severe malaria were included in the analysis. Prevalence of and mortality as a result of major symptoms were calculated followed by multiple regression modeling to identify major predictors of death. RESULTS. Of 1682 confirmed cases of malaria during a 32-month period, 374 subjects met the World Health Organization criteria for severe malaria. The case fatality rate was 12% in this series. Multiple regression analysis identified respiratory distress, coma, multiple organ dysfunctions, and hyperparasitemia as major predictors of death. Anemia and jaundice did not emerge as important markers of mortality. Many patients presented with multiple major complications, and the mortality rate was consistently high when >1 major predictor was present in a patient. CONCLUSIONS. Clinical features in Indian children differed from those reported in most studies that involved an African population. Multiple organ dysfunctions emerged as an important presenting feature and a new predictor of death in childhood malaria.

Occurrence of Necrotizing Enterocolitis May Be Dependent on Patterns of Bacterial Adherence and Intestinal Colonization: Studies in Caco-2 Tissue Culture and Weanling Rabbit Models

ABSTRACT: Necrotizing enterocolitis (NEC) is one of the leading causes of death in neonatal intensive care units. The underlying pathophysiology of NEC is poorly defined, although there is a suggestion that bacterial agents play an important role in the process. In this study, we evaluated bacterial isolates from 17 NEC cases and matched asymptomatic control infants. Isolates from NEC patients were no more likely than control isolates to be adherent to enterocytes, as assessed by a Caco-2 cell tissue culture model. Adherent Escherichia coli isolates, from both NEC cases and controls, were able to cause pathologic changes typical of NEC in a weanling rabbit ileal loop model. Adherence of E. coli strains to Caco-2 cells, and subsequent production of disease in weanling rabbits, could be blocked by coinfection with Gram-positive isolates from control children. In contrast, in three of four instances, adherent E. coli from NEC cases retained their adherence and caused illness in rabbits when coinfected with Gram-positive isolates from the homologous child. Our data suggest that patterns of intestinal adherence, as influenced by the underlying intestinal microbial ecology, play a role in the pathophysiology of NEC.

Prediction of outcome in adults with severe falciparum malaria: a new scoring system

BackgroundMortality of falciparum malaria is related to the presence of severe complications. However, no scoring system is available to predict outcome of these patients. The aim of this paper was to devise a simple and reliable malaria prognosis score (MPS) to predict the outcome of adults with severe malaria.MethodsAll slide-positive severe falciparum malaria patients admitted to Ispat General Hospital were studied. Eight clinical parameters that may potentially differentiate or influence the outcome were identified to predict recovery or deathResultsOf 248 severe malaria cases, 35 died. There were 212 adults (34 deaths) and 36 children (one death). The malaria score for adults was (MSA) = 1(severe anaemia) + 2 (acute renal failure) + 3(Respiratory distress) +4 (cerebral malaria). The MSA ranges from 0 to 10. The mortality was 2% for MSA 0 – 2; 10% for MSA 3–4, 40% for MSA 5–6 and 90% for MSA 7 or more. The sensitivity is 89.9% and positive predictive value is 94.1% when 5 is taken as the cut off value.ConclusionMSA is a simple and sensitive predictor. It can be administered rapidly and repeatedly to prognosticate the outcome of severe malaria in adults. It can help the treating doctor to assess the patient as well as to communicate to the relatives of the patients about prognosis. The score needs revalidation in other geographical areas.

Endemic necrotizing enterocolitis: lack of association with a specific infectious agent.

We conducted a comprehensive analysis of bacterial, parasitic and viral agents present in stool samples of 23 necrotizing enterocolitis cases and 23 matched and 10 random controls. Enterococcus spp., Staphylococcus epidermidis, and Escherichia coli were the most common aerobic bacterial species isolated. Astrovirus was identified in a stool sample from one control. Eight infants were bacteremic; in 7 of 8 the same organism was also present in the stool. No one bacterial species or strain (as identified by plasmid profile analysis) was associated with occurrence of illness. Plasmid analysis further suggested that each infant was colonized with his or her own distinctive aerobic bacterial flora. With the exception of isolates from two control patients which hybridized with a probe for diffuse adherence, no diarrheagenic E. coli was identified. Five (45%) of 11 case infants were colonized with coagulase-negative staphylococci (all S. epidermidis) that produced delta-hemolysin in vitro, as compared with 13 (87%) of 15 control infants. Necrotizing enterocolitis was not associated with an increased ability to ferment carbohydrate, as measured by in uitro beta-galactosidase activity. Our data do not support the hypothesis that endemic necrotizing enterocolitis in our institution is caused by a single infectious agent, nor was there evidence that previously proposed virulence mechanisms such as production of delta-hemolysin or increased in vitro carbohydrate fermentation play a critical role in disease occurrence.

Escherichia coli transcytosis in a Caco-2 cell model: Implications in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a serious gastrointestinal disorder of preterm infants. Other than an association with prematurity and gastrointestinal feeding, no single factor or mechanism has been consistently linked to this disease. We have previously demonstrated that Escherichia coli isolates obtained from the stool of infants with NEC caused NEC-like injury in a weanling rabbit ileal loop model; this injury, in turn, could be blocked by coinfection with selected Gram(+) bacteria(Enterococcus faecium) isolated from asymptomatic controls. Using Caco-2 cells in a trans-well system, we now demonstrate that the same E. coli isolates can cross epithelial cell monolayers in the absence of ultrastructural change or damage. These results with E. coli contrast with those seen with Salmonella typhimurium, which passed through the monolayer at a higher rate and were associated with striking ultrastructural damage. Transcytosis of E. coli was reduced 3-5-fold in the presence of E. faecium previously shown to block NEC-like injury in the loop model. There was a mild increase in the rate of E. coli transcytosis when studies were conducted with younger, undifferentiated cells; these immature cells had no brush border, had decreased production of brush border-specific enzymes, but retained well defined tight junctions, as demonstrated by transepithelial electrical resistance and electron microscopy. A further reduction/complete blockage of E. coli transcytosis was observed when E. faecium was used as the coinfectant in studies with these undifferentiated cells. We hypothesize that the ability of E. coli to cross epithelial cell layer is a critical initial step in the cascade of events which lead ultimately to NEC; blockage or reduction in E. coli transcytosis in the presence of certain Gram(+) organisms may play a significant role in prevention of NEC.

Vibrio cholerae 01 can assume a ‘rugose’ survival form that resists killing by chlorine, yet retains virulence

Vibrio cholerae 01 is able to shift between smooth and rugose colonial morphologies. Cultures of smooth V. cholerae strains were inactivated in less than 20 s at a concentration of 1.0 mg l‐1 free chlorine. In contrast, cultures of rugose variants exposed to this concentration of chlorine showed an initial rapid drop in viable counts, followed by persistence of a protected subpopulation of cells. Viable V. cholerae could still be recovered from rugose cultures even after exposure to 2.0 mg l‐1 free chlorine for 30 min. Preliminary studies suggest that resistance to killing by chlorine was due to formation of cell aggregates enclosed in a gelatinous mucoid material. Rugose strains appeared to be fully virulent, based on their ability to adhere to Caco‐2 cells and elicit fluid accumulation in rabbit ileal loops. Our data suggest that the V. cholerae rugose phenotype represents a fully virulent survival form of the organism that can persist in the presence of free chlorine.

High Mortality Rates for Very Low Birth Weight Infants in Developing Countries Despite Training

OBJECTIVE: The goal was to determine the effect of training in newborn care and resuscitation on 7-day (early) neonatal mortality rates for very low birth weight (VLBW) infants. The study was designed to test the hypothesis that these training programs would reduce neonatal mortality rates for VLBW infants. METHODS: Local instructors trained birth attendants from 96 rural communities in 6 developing countries in protocol and data collection, the World Health Organization Essential Newborn Care (ENC) course, and a modified version of the American Academy of Pediatrics Neonatal Resuscitation Program (NRP), by using a train-the-trainer model. To test the impact of ENC training, data on infants of 500 to 1499 g were collected by using a before/after, active baseline, controlled study design. A cluster-randomized, controlled trial design was used to test the impact of the NRP. RESULTS: A total of 1096 VLBW (500–1499 g) infants were enrolled, and 98.5% of live-born infants were monitored to 7 days. All-cause, 7-day neonatal mortality, stillbirth, and perinatal mortality rates were not affected by ENC or NRP training. CONCLUSIONS: Neither ENC nor NRP training of birth attendants decreased 7-day neonatal, stillbirth, or perinatal mortality rates for VLBW infants born at home or at first-level facilities. Encouragement of delivery in a facility where a higher level of care is available may be preferable when delivery of a VLBW infant is expected.

Necrotizing enterocolitis: a practical guide to its prevention and management.

Neonatal necrotizing enterocolitis is the second most common cause of morbidity in premature infants and requires intensive care over an extended period. Despite advances in medical and surgical techniques, the mortality and long-term morbidity due to necrotizing enterocolitis remain very high.Recent advances have shifted the attention of researchers from the classic triad (ischemia, bacteria, and the introduction of a metabolic substrate into the intestine) of necrotizing enterocolitis, to gut maturation, feeding practices, and inflammation. The focus on inflammation includes proinflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-6, IL-18, and platelet-activating factor. Research related to the etiology of necrotizing enterocolitis has moved quickly from clostridial toxin to bacterial and other infectious agents. More recently, the pattern of bacterial colonization has been given emphasis rather than the particular species or strain of bacteria or their virulence. Gram-negative bacteria that form part of the normal flora are now speculated as important factors in triggering the injury process in a setting where there is a severe paucity of bacterial species and possible lack of protective Gram-positive organisms. Although the incidence of necrotizing enterocolitis has increased because of the survival of low birthweight infants, clinicians are more vigilant in their detection of the early gastrointestinal symptoms of necrotizing enterocolitis; however, radiographic demonstration of pneumatosis intestinalis remains the hallmark of necrotizing enterocolitis. With prompt diagnosis, a large proportion of infants with necrotizing enterocolitis are now able to be managed medically with intravenous fluid and nutrition, nasogastric suction, antibacterials, and close monitoring of physiologic parameters. In the advanced cases that require surgery, clinicians tend to opt for either simple peritoneal drainage (for very small and sick infants) or laparotomy and resection of the affected part. Intestinal transplantation later in life is available as a viable option for those who undergo resection of large segments of the intestine.It is becoming more evident that treatment of this devastating disease is expensive and comes with the toll of significant long-term sequelae. This has resulted in renewed interest in designing alternative strategies to prevent this serious gastrointestinal disease. Simple trophic feeding and the use of L-glutamine and arginine are novel avenues that have been examined. The use of probiotics (‘friendly’ bacterial flora) has been introduced as a promising tool for establishing healthy bacterial flora in the newborn gut to block the injury process that may ultimately lead to necrotizing enterocolitis.

The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region

BackgroundIn V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S.ResultsThe structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified.ConclusionThe co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations.