Exploring Microbial Contributions to Nutraceutical Production: From Natural to Designed Foods.

For ages, societies throughout the world have used fermentation as a traditional method for food processing and preservation, helping to create a wide range of staple foods and delicacies. Due to its possible health advantages, mostly attributable to the inclusion of bioactive substances known as nutraceuticals, fermented foods have attracted a lot of interest recently. This in-depth analysis examines the wide range of nutraceuticals present in fermented foods, as well as how they are made, what health benefits they may have, and how they may be used in the nutraceutical and functional food businesses. By stressing how important fermented foods are as a source of beneficial bioactive components that support human health and well-being. Numerous bioactive substances found in fermented foods have been the subject of recent scientific studies. These molecules may find use in the pharmaceutical and nutraceutical sectors. Streptococcus thermophilus, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus bulgaricus, and Lactobacillus johnsonii are just a few examples of the probiotic bacteria that live in fermented foods and formulas. This review elucidates the importance of microorganisms sourced from fermented foods as potent agents for diverse nutraceuticals and their potential role in preventing various diseases whilst serving as functional food supplements.

Development and characterization of rice bran-gum Arabic based encapsulated biofertilizer for enhanced shelf life and controlled bacterial release.

Introduction: Currently, microbe-based approaches are being tested to address nutrient deficiencies and enhance nutrient use efficiency in crops. However, these bioinoculants have been unsuccessful at the commercial level due to differences in field and in-vivo conditions. Thus, to enhance bacterial stability, microbial formulations are considered, which will provide an appropriate microenvironment and protection to the bacteria ensuring better rhizospheric-colonization. Methods: The present study aimed to develop a phosphobacterium-based encapsulated biofertilizer using the ion-chelation method, wherein a bacterial strain, Myroid gitamensis was mixed with a composite solution containing rice bran (RB), gum Arabic (GA), tricalcium phosphate, and alginate to develop low-cost and slow-release microbeads. The developed microbead was studied for encapsulation efficiency, shape, size, external morphology, shelf-life, soil release behavior, and biodegradability and characterized using SEM, FTIR, and XRD. Further, the wheat growth-promoting potential of microbeads was studied. Results: The developed microbeads showed an encapsulation efficiency of 94.11%. The air-dried beads stored at 4°C were favorable for bacterial survival for upto 6 months. Microbeads showed 99.75% degradation within 110 days of incubation showing the bio-sustainable nature of the beads. The application of dried formulations to the pot-grown wheat seedlings resulted in a higher germination rate, shoot length, root length, fresh weight, dry weight of the seedlings, and higher potassium and phosphorus uptake in wheat. Discussion: This study, for the first time, provides evidence that compared to liquid biofertilizers, the RB-GA encapsulated bacteria have better potential of enhancing wheat growth and can be foreseen as a future fertilizer option for wheat.

Exploring the potential of lactic acid bacteria and its molecular mechanism of action in the development of biosurfactants: Current finding and future outlook.

Biosurfactants generated from lactic acid bacteria (LAB) offer an advantage over standard microbial surfactants due to their antifungal, antibacterial and antiviral capabilities. Many LAB strains have been related to the manufacture of biosurfactant, an essential chemical with uses in the treatment of a number of illnesses. Furthermore, their effectiveness as anti-adhesive agents against a diverse variety of pathogens proves their utility as anti-adhesive coating agents for medical insertional materials, reducing hospital infections without the need of synthetic drugs and chemicals. LAB produces both low and high molecular weight biosurfactants. Biosurfactants from L. pentosus, L. gasseri and L. jensenii have been reported to produce glycolipopeptides that comprise carbohydrates, proteins and lipids in the ratio of 1:3:6 with palmitic, stearic acid, and linoelaidic acid as the major fatty acid component, whereas L. plantarum has been reported to make surlactin due to the presence of non-ribosomal peptide synthetase genes (NRPS) genes. Antimicrobial activity of sophorolipids and rhamnolipids generated from LAB against B. subtilis, P. aeruginosa, S. epidermidis, Propionibacterium acnes and E. coli has been demonstrated. The safety of biosurfactants is being evaluated in compliance with a number of regulatory standards that emphasize the importance of safety in the pharmaceutical industry. This review attempts, for the first time, to provide a comprehensive evaluation of several approaches for the synthesis of biosurfactant-mediated molecular modulation in terms of their biological value. Future biosurfactant directions, as well as regulatory considerations that are crucial for the synthesis of biosurfactants from novel LAB, have also been explored.

Development of Zn biofertilizer microbeads encapsulating Enterobacter ludwigii-PS10 mediated alginate, starch, poultry waste and its efficacy in Solanum lycopersicum growth enhancement.

In the present farming era, rhizobacteria as beneficial biofertilizers can decrease the negative effects of Zinc (Zn) agrochemicals. However, their commercial viability and utility are constrained by their instability under field conditions. Thus, to enhance their stability, microbial formulations are considered, which will not only offer an appropriate microenvironment, and protection but also ensure a high rate of rhizospheric-colonization. The goal of this study was to create a new formulation for the Zn-solubilizing bacteria E. ludwigii-PS10. The studied formulation was prepared using the extrusion technique, wherein a composite solution containing alginate, starch, zinc oxide, and poultry waste was uniformly mixed with the bacterial strain PS10 to develop low-cost, eco-friendly, and slow-release microbeads. The produced microbead was spherical, and characterized by SEM, FTIR, and XRD. Further, the microbeads were analyzed for their survival stability over 3 months of storage at room temperature and 4 °C. The effect of the microbead on the vegetative growth of tomato plants was investigated. Results showed that 94 % of the encapsulated microbial beads (EMB) matrix was able to encapsulate the bacterial strain PS10. The dried EMB demonstrated a moisture content of 2.87 % and was able to preserve E. ludwigii-PS10 survival at room temperature at the rate of 85.6 %. The application of the microbead to the tomato plants significantly increased plant biomass and Zn content. As a result, our findings support the use of this novel EMB prepared using an alginate/poultry waste/starch mixture to increase bacterial cell viability and plant growth.

Comparison of diversity and zinc solubilizing efficiency of rhizobacteria obtained from solanaceous crops under polyhouse and open field conditions.

Zinc-solubilizing bacteria (Zn-SB) play a crucial role in regulating soil fertility and plant health by maintaining Zn availability in the rhizosphere. It is uncertain how the Zn-SB population fluctuates across various cultivation systems since varied land-use patterns for agricultural aims may affect microbial activity and plant development effectiveness. The current study aims to examine the Zn-SB potential of various farming systems using Solanum lycopersicum, Solanum melongena, and Capsicum annuum grown in polyhouse soil (PS) and open fields (OF). Only twenty rhizobacterial isolates from PS and two isolates from OF out of 80 showed a strong ability to solubilize Zn, which was evaluated using Atomic Absorption Spectroscopy. Bacterial strain-PS4 solubilized 253.06 ppm of ZnO and produced a high quantity of lactic acid (168.62 g/ml) and acetic acid (470.5 g/ml), whereas bacterial strain OF1 solubilized 16.02 ppm of ZnO by releasing glycolic acid (42.89 g/ml), lactic acid (22.30 g/ml), formic acid (106.03 g/ml), and acetic acid (48.5 µg/ml). Further, in vitro studies demonstrated higher production of auxin, gibberellic acid and siderophore by PS1 as compared to OF1 strain. A large diversity of Zn-SB in the soil was indicated by biochemical analysis, which revealed that isolates belonged to the families Enterobacteriaceae, Bacillaceae, Burkholderiaceae, Streptococcaceae, Paenibacillaceae, Micrococcaceae, Morganellaceae, and Dietziaceae. The isolates PS4 and OF1 were identified as Bacillus cereus and Enterobacter hormaechei, respectively, using 16S rRNA sequencing. The findings show that soil from polyhouses has a greater diversity of Zn-solubilization rhizobacteria than soil from open areas. The findings suggested a potential land-use method for enhancing crop yields by employing microorganisms and polyhouse technology, which could be useful in the future study.

Plant Growth-Promoting Attributes of Zinc Solubilizing Dietzia maris Isolated from Polyhouse Rhizospheric Soil of Punjab.

Zinc solubilizing rhizobacteria (ZSR) enhance the phyto-availability of Zn by converting its insoluble forms into usable forms that are essential for the growth and nutritional quality of crops. In the present study, a potential ZSR, hereafter referred to as strain N14, was isolated from the polyhouse rhizospheric soil of Punjab, India. The isolated rhizobacteria was found to be Gram-positive, aerobic, rod-shaped, and demonstrated a solubilization index of 63.75 on the Bunt Rovira (BR) medium. The 16S rRNA gene sequence analysis revealed that isolated strain N14 matches substantially with type strain Dietzia maris DSM 43672 T. In its ZnO broth assay, a significant amount of soluble Zn was detected along with a simultaneous decrease in pH of the broth. Ultra-performance liquid chromatography analysis revealed the release of organic acids, specifically, lactic acid and acetic acid by D. maris strain N14 which could be the reason for the decrease in broth pH. The production of indole acetic acid (29.91 µg/ml), gibberellic acid (4.72 µg/ml), ammonia (38.87 µg/ml), siderophore (0.89%), along with the release of HCN and appearance of phosphate solubilization zone (14.4 mm) with this strain suggested its possible plant growth-promoting (PGP) characteristics. Therefore, this strain was employed in the formulation of pellets which were applied for in vivo PGP studies using tomato plants. The developed bioformulated pellets showed a significant enhancement in plant growth as compared to control and vermicompost treated plants. To the best of our knowledge, this is the first report describing the Zn solubilizing and PGP characteristics of D. maris.

Microbial endophytes: application towards sustainable agriculture and food security.

Microbial endophytes are ubiquitous and exist in each recognised plant species reported till date. Within the host plant, the entire community of microbes lives non-invasively within the active internal tissues without causing any harm to the plant. Endophytes interact with their host plant via metabolic communication enables them to generate signal molecules. In addition, the host plant's genetic recombination with endophytes helps them to imitate the host's physicochemical functions and develop identical active molecules. Therefore, when cultured separately, they begin producing the host plant phytochemicals. The fungal species Penicillium chrysogenum has portrayed the glory days of antibiotics with the invention of the antibiotic penicillin. Therefore, fungi have substantially supported social health by developing many bioactive molecules utilised as antioxidant, antibacterial, antiviral, immunomodulatory and anticancerous agents. But plant-related microbes have emanated as fountainheads of biologically functional compounds with higher levels of medicinal perspective in recent years. Researchers have been motivated by the endless need for potent drugs to investigate alternate ways to find new endophytes and bioactive molecules, which tend to be a probable aim for drug discovery. The current research trends with these promising endophytic organisms are reviewed in this review paper. KEY POINTS: • Identified 54 important bioactive compounds as agricultural relevance • Role of genome mining of endophytes and "Multi-Omics" tools in sustainable agriculture • A thorough description and graphical presentation of agricultural significance of plant endophytes.

Exploration of phytate-mineralizing bacteria with multifarious plant growth-promoting traits.

Phytate-mineralizing bacteria (PMB) with plant growth-promoting activity can be considered as a potential biofertilizer for plant nutrition. PMB catalyzes the conversion of insoluble sugar phosphates, inositols, nucleic acids, phospholipids, nucleotides, phytate, and phytin into soluble forms that can be assimilated by plants. The present study aimed to isolate potential PMB from rhizospheric soils and to study their plant growth-promoting potential for the possible development of a potential phosphobacterium biofertilizer. For this purpose, 34 PMB isolates were isolated that showed potent phytate-mineralizing potential. These isolates were tested for their potential to solubilize tricalcium phosphate (TCP) and for various other plant growth-promoting activities. Significant differences were found among the isolates with regard to phytate mineralization and other plant growth-promoting characteristics. The bacterial isolates biochemically identified as Bacillus, Paenibacillus, Arthrobacter, and Burkholderia exhibited high/medium P solubilization, medium/high phytohormone production, and medium/low siderophore and ammonia production. Among all these isolates, isolate A14 (Burkholderia cenocepacia strain FDAARGOS_7) was the promising isolate with high TCP solubilization, medium phytate mineralization, high enzyme production, medium/high phytohormone production, and medium ammonia production. This strain also showed nitrogen fixation activity, zinc solubilizing potential, potassium solubilization, ACC deaminase production, and catalase production. Hence, it can be concluded that B. cenocepacia can be the potential candidate for biofertilizer development. Future studies are planned for exploring the role of PMB in biofertilizer formulations.

Plant growth-promoting potential of 'Myroides gitamensis' isolated from virgin soils of Punjab.

Phosphate-solubilizing (PS) and phosphate-mineralizing (PM) bacteria are considered vital for augmenting the plant growth through phosphorus mobilization and plant growth-promoting attributes. In the present study, a rhizospheric bacterium was isolated from the virgin land of Punjab, India and identified as 'Myroides gitamensis' BSH-3 through 16S rRNA sequencing. 'M. gitamensis' showed potential halo zone on Pikovskaya agar. The novelty of the study lies in the fact that plant growth-promoting potential of 'M. gitamensis' has not been studied earlier. It was able to solubilize 17.53-106.66 µg/mL of tricalcium phosphate and demonstrated a promising potential of mineralizing sodium phytate corresponding to 44.6-94.70 µg/mL at 28 °C. Variable PS and PM activity was observed at temperature range of 15-42 °C with the maximum activity observed at 28 °C after 96 h of incubation. The nitrogen fixation ability, hydrogen sulfide production, cellulose hydrolysis test and chitin degradation was found to be negative. High indole acetic acid (42.82 µg/mL), gibberellic acid (72.93 µg/mL), ammonia (22.58 µg/mL) production, phytase activity (0.49 pi/mL/min) and comparable amount of siderophore (28.55%) and acid phosphate activity (0.606 µM p-nitrophenol/ml/min) was shown by 'M. gitamensis'. Inoculation of wheat with 'M. gitamensis' in pot experiment showed increased shoot and root length by 30.58% and 38.32%. Fresh weight and dry weight was increased by 45.74% and 67.81%, respectively, compared to uninoculated control. These results demonstrate that 'M. gitamensis' has promising PS, PM and plant growth-promoting attributes to be used as a bio-inoculant to enhance plant growth and soil fertility.

Variation in the Phosphate Solubilizing Bacteria from Virgin and the Agricultural Soils of Punjab.

Bacteria with phosphorus (P) solubilization potential are considered vital in promoting bioavailability of phosphorus in soil. The present study was conducted to isolate and study the variation of phosphate solubilizing potential of bacteria isolated from virgin and agricultural soils. Total 30 isolates from virgin soil and 4 isolates from agricultural soil which retained their activity on repeated subculturing were selected. Among the isolates, there was insignificant difference in the total bacterial count from virgin and agricultural soils, however, a significant difference was found in the phosphate solubilizing bacteria (PSB) count and their P solubiling potential. Soil organic matter and available P content were correlated with PSB count. The mean solubilization index (SI) was higher from the isolates from virgin soils. Equal distribution method was employed to categorize the bacterial isolates into low, medium, and high P solubilizers which depicted H ≥ 89.44 and L ≤ 68. Among all the isolates, 23.53% were high P solubilizers (P-89.44-110.88 µg/ml), 55.88% were medium P solubilizers (P- 68-89.44 µg/ml), and 20.58% isolates produced low soluble P (46.56-68 µg/ml). Analysis of the data showed that all the isolates categorized under high P solubilizers belonged to the virgin soil. The isolates were characterized based upon biochemical characterization and belonged to Pseudomonadaceae, Enterobacteriaceae, Bacillaceae, Paenibacillaceae, Micrococcaceae, Burkholderiaceae, Flavobacteriaceae, and Streptococcaceae families. 16 sRNA sequencing of the two isolates showing maximum P solubilization were characterized as Enterobacter hormaechi. However, they differ appreciably in their P solubilization at different temperatures.

Effect of Lactobacillus acidophilus & epidermal growth factor on experimentally induced Clostridium difficile infection.

BACKGROUND & OBJECTIVES: Clostridium difficile-associated disease (CDAD) remains an important nosocomial ailment. Antimicrobial therapy used for CDAD gives inconsistent results. This experimental study was planned to investigate the beneficial effects of Lactobacillus acidophilus and epidermal growth factor (EGF) for CDAD management. METHODS: Among 10 groups of BALB/c mice (6 in each), group 1 served as controls receiving no inoculum. Animals in groups 2-10 received C. difficile, those in groups 3, 6 and 9 received L. acidophilus and those in groups 4, 7 and 10 received EGF after C. difficile inoculation. Animals in groups 5-7 were pre-treated with ampicillin and those in groups 8-10 with lansoprazole prior to C. difficile. The animals were killed and investigated for colonisation by C. difficile and toxin production, myeloperoxidase (MPO) activity and histopathology. RESULTS: Colonisation by C. difficile was found to be significantly different (P<0.001) in the various groups. C. difficile toxin titres and MPO activity were significantly lower in animals given L. acidophilus and EGF after ampicillin (groups 6 and 7) and lansoprazole (groups 9 and 10). The severity of acute inflammation was also significantly less (P<0.05) in caecal and colonic segments of animals in groups 6 and 7 compared to those in group 5. Although the severity of acute inflammation was less in the caecal and colonic segment of animals in groups 9 and 10, the reduction was not significant compared to group 8. INTERPRETATION & CONCLUSIONS: Our findings showed that the administration of L. acidophilus and EGF reduced the severity of C. difficile infection in the experimental animals.

Phenotypic and molecular characterization of clinically isolated Escherichia coli.

All diarrheagenic Escherichia coli carry at least one virulence-related property. Stool samples from 244 patients having acute or persistent diarrhea received after the exclusion of routine enteropathogens were investigated. Purely or predominantly isolated E. coli (n = 100) were subjected to serotyping, of which only 25 were typable. They belonged to 14 different O-serogroups comprising 5 O153, 4 O102, 3 O25, 2 each of O130 and O169, and 1 each of O1, O8, O15, O37, O86, O101, O127, O143, and O160. The typable E. coli isolates along with 5 other untypable isolates were investigated for molecular markers, such as intimin (eae), enterohemolysin (EhlyA), a-hemolysin, heat-labile enterotoxins (LT), heat-stable enterotoxins (STa), verotoxins (VT1 and VT2), invasivity (ial), enteroaggregative E. coli (EAEC) gene (EAGG), and enterotoxin (EAST). Two of the isolates (O153 and O86) were positive for enterohemolysin phenotypically and 5 for beta-hemolysin both phenotypically and genotypically. Interestingly, 16.6% of the randomly isolated E. coli were O153, a serogroup common in cattle, and 10% belonged to EAEC pathotype of which two-thirds had the EAST gene, which is quite frequent in these strains. Additionally, there was one strain (O153) that was positive for EAST only. Between the two 0130:H6 strains isolated, one belonged to EAEC serogroup. None of the E. coli isolated were positive for verotoxins, eae, LT1, STa, and ial. Data obtained emphasize the need for additional research into the role of eae gene and other putative factors affecting the virulence of diarrheagenic E. coli in India.

Effect of biotherapeutics on cyclosporin-induced Clostridium difficile infection in mice.

BACKGROUND AND AIM: Immunosuppressive therapy may precipitate Clostridium difficile associated disease (CDAD). We evaluated the role of cyclosporin in the development of CDAD in the experimental mouse model and studied the effect of probiotic and epidermal growth factor (EGF) as biotherapeutics measures. METHODS: BALB/c mice (n = 24) were divided into four groups. Group I animals not given any inoculum served as controls. Animals in the remaining three groups (Group II, III and IV) were given cyclosporin daily from days 1-7 followed by C. difficile inoculum on day 8. Additionally, the animals received Lactobacillus acidophilus (Group III) and EGF (Group IV) for one-week post C. difficile challenge. The animals were evaluated for colonization and toxin production by C. difficile, myeloperoxidase (MPO) activity and histopathological changes. RESULTS: Clostridium difficile was colonized and elaborated its toxins in animals receiving cyclosporin and C. difficile. MPO activity was significantly higher (P < 0.05) and histopathological epithelial damage, cryptitis and acute inflammatory changes were seen in the cecum and colon. C. difficile count, toxins A and B titers and MPO activity were significantly lowered (P < 0.05) in animals receiving probiotic and EGF. Histopathologically, mucodepletion and inflammatory infiltrate were decreased in the biotherapeutic receiving animals. CONCLUSIONS: Cyclosporin led to the development of mild to moderate CDAD in animals. Administration of biotherapeutics reduced the severity of CDAD. Future clinical trials are needed for further investigation of these potential biotherapeutic measures.

Clostridium perfringens enterotoxin in antibiotic-associated diarrhea.

Clostridium perfringens type A is associated with 5-20% cases of antibiotic-associated diarrhea (AAD) even though Clostridium difficile is implicated in the most severe cases. Fecal specimens from one hundred hospitalized patients, who developed diarrhea regardless of antibiotic intake and who were negative for C. difficile toxin assay, were investigated for C. perfringens enterotoxin (CPE). Simultaneously, cultures were set up for other possible aetiological factors. Ten healthy controls were also similarly investigated. CPE was positive in 2/100 (2%) of the patients and the samples were also positive for the organism in culture. Other organisms isolated were non-toxigenic C. difficile (4%), staphylococci (6%), Candida (18%) and Klebsiella pneumoniae (1%). Stool samples from healthy controls grew mixed growth of no significance and CPE was negative in all of them. Detection of CPE is not part of routine laboratory investigation due to resource implication. Criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens-associated AAD by further research.

Speciation of fecal Candida isolates in antibiotic-associated diarrhea in non-HIV patients.

Candida is the most frequently encountered fungal infection of the gastrointestinal tract after antibiotic exposure. The pathogenesis of Candida probably varies with each species. The speciation of fecal Candida after antibiotic use is not well investigated. One hundred and eleven fecal samples negative for Clostridium difficile toxin and for other enteric pathogens formed the basis of our investigation. The diarrheic samples came from patients receiving antibiotics in a hospital setting. In addition, samples from 30 age-matched healthy participants who did not receive antibiotics and did not have diarrhea were also studied. Initially, a Gram stain identification for yeasts was performed for each fecal sample, then each sample was cultured on Sabouraud's dextrose agar. Candida was isolated as pure growth (>10(5) cfu/ml) from the stools of 32 (28.8%) patients. The identification of the yeast was done based upon a combination of morphological, physiological and biochemical criteria. The predominant isolates were C. tropicalis (n=16), C. albicans (n=14) and C. krusei (n=2). Candida isolated from healthy participants (n=4) was sparse and therefore not speciated. Different Candida spp. may play an important role in precipitating antibiotic-associated diarrhea.

Comparative role of antibiotic and proton pump inhibitor in experimental Clostridium difficile infection in mice.

Clostridium difficile inoculated BALB/c mice were investigated to assess the comparative role of antibiotic and proton pump inhibitor. They were examined for colonization and toxin production by C. difficile as well as myeloperoxidase activity and histopathological changes in the intestinal tract. The C. difficile count, toxin A and B titres and myeloperoxidase activity were significantly higher (P>0.05) in ampicillin and lansoprazole receiving groups as compared to the control and the C. difficile receiving groups. Similarly they showed significant difference (P >0.05) for epithelial damage, oedema and neutrophilic infiltrate in colons. In addition to antibiotic, PPI also acts as an independent risk factor for C. difficile infection in experimental studies.

Amplification of mecA gene in multi-drug resistant Staphylococcus aureus strains from hospital personnel.

BACKGROUND: Antibiotic resistance is common among bacterial pathogens associated with both community acquired and nosocomial infections. In view of the present problem of drug resistance we investigated the prevalence of methicillin resistant Staphylococcus aureus (MRSA) and amplified the mecA gene in the isolates from the hand swabs of the hospital personnel. METHODOLOGY: The nuc gene was amplified to characterize these isolates at species level. The S. aureus isolates were analyzed for their susceptibility to different classes of antibiotics using the disk diffusion method. The spot inoculation test was performed to detect methicillinase production in these isolates. RESULTS: In the screened isolates of S. aureus, 14.2 and 15 kb of plasmids were present. These isolates showed pronounced resistance against beta-lactam antibiotics including second- and third-generation cephalosporins, aminoglycosides, macrolides and fluoroquinolone. Some of the isolates included in this study were resistant to three or more antibiotics. Expression of methicillinase was detected by spot inoculation test, and a few of the isolates were found to produce methicillinase. Moreover, mecA gene was also amplified. Of 17 isolates only 7 showed presence of mecA gene. CONCLUSION: This study highlights the emerging trend of multiple drug resistance in S. aureus strains isolated from hospital personnel working in a premier hospital in North India.

Evaluation of an in-house rapid diagnostic method for detection of Salmonella enterica serovar Typhi in fecal specimens.

Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. Laboratory diagnosis requires isolation and identification of the organism from the patient's blood or feces. Feces is the specimen most commonly submitted to laboratories. Detection of bacterial antigens is an important adjunct to laboratory diagnosis. We carried out an in-house diagnostic method by preparing test reagents comprising of latex beads coated with specific antisera to detect Vi, O9 and H-d antigens of S. typhi. Fecal specimens from one hundred patients with diarrhea and fever as well as from twenty healthy controls were incubated for enrichment in Selenite F broth for 6 hours or overnight. Latex agglutination tests to detect antigens of S. typhi were carried out on centrifuged broth supernatants. Parallel cultures on media selective for S. typhi were also set up. Nine of the supernatants were positive for two or more specific antigens and S. typhi grew in three of the corresponding cultures. None of the samples from 20 healthy controls were positive by either the diagnostic method or by culture. The result of the in-house diagnostic assay can be obtained overnight and may help in directing immediate antimicrobial therapy.

Estimation of endotoxin in infected bile from patients with biliary diseases.

Diseases of the biliary tract can get complicated by infection. Endotoxin may theoretically be responsible for damage to the gall bladder due to its numerous pathophysiological effects. The aim of the present study was to detect and semi-quantitate the amount of endotoxin present in the bacteriologically positive bile samples and to correlate the endotoxin levels with the clinical profile of the patients. One hundred patients with gall bladder diseases and with infected bile constituted the population for investigation. The clinical profile included presence of fever, jaundice, abdominal pain and gall bladder stones. Endotoxin detection and semi-quantitation in the bile samples were carried out using the Limulus amoebocyte assay: Of 100 infected bile samples investigated, 9 samples (9%) were positive for endotoxin ranging from 1.9 EU/ml to 15 EU/ml. Four of them had Klebsiella pneumoniae, 2 had Acinetobacter anitratus and one each of the remaining 3 samples was positive for (i) Escherichia coli and Serratia marcescens (ii) Pseudomonas aeruginosa and (iii) Salmonella enteritidis. The stool sample of the patient with S. enteritidis in the bile also grew the same microorganism. Statistical analysis showed a significant increase in the presence ofjaundice (p<0.05) and abdominal pain (p<0.01) in the endotoxin positive patients compared to the endotoxin negative ones. Hitherto this is the first report that investigated the endotoxin levels in the bile of patients with gall bladder and biliary tract diseases, along with their biliary bacterial profile. Further research is warranted on the effects of endotoxin on gall stone formation.

Is Campylobacter involved in antibiotic associated diarrhoea?

Campylobacter jejuni is an important cause of acute bacterial diarrhoea. In developing countries like India, children gain immunity early during infancy. However, the incidence is higher in non-immune hosts. Antibiotic use destabilizes the gut flora and can inhibit the local immune responses, thereby compromising resistance to a variety of infections. It is not yet known whether antibiotic intake can also precipitate C. jejuni enteritis as the infectious dose is low and attack rates are high. We made a preliminary study to determine the prevalence of C. jejuni in hospitalized patients receiving antibiotics for various ailments. One hundred and thirty eight stool samples submitted for Clostridium difficile toxin assay were additionally cultured for C. jejuni in blood-free campylobacter selectivity agar. All suspected colonies were subjected to Gram staining, oxidase, catalase and nalidixic acid sensitivity tests. Confirmation of C. jejuni was done by the hippurate hydrolysis test. Of the 138 faecal samples investigated, 14 (10.1%) grew C. jejuni and 11 of them belonged to adults. Two of these 14 samples were also positive for C. difficile toxin. Though not as yet reported, C. jejuni may also be involved in antibiotic associated diarrhoea due to lowered immunity in the host. It may cause enteritis either by itself or in synergy with C. difficile infection.