Molecular characterization and evaluation of the emerging antibiotic-resistant Streptococcus pyogenes from eastern India.

BACKGROUND: Group A Streptococcus strains causing wide variety of diseases, recently became noticeable in eastern India, are not amenable to standard treatment protocol thus enhancing the possibility of disease morbidity by becoming antibiotic resistance. METHODS: The association of Lancefield group A Streptococcal variation with degree of vir architectural diversity was evaluated using emm typing and restriction fragment length polymorphism analyses. The antibiotic sensitivity patterns were examined by modified Kirby-Bauer method of disk diffusion. Percentage calculations, 95% confidence interval and one-way ANOVA were used to assess differences in proportions. RESULTS: Our observations revealed 20 different emm types and 13 different HaeIII vir typing patterns. A 1.2 kb fragment was found in all HaeIII typing pattern. Fragments of 1.2 kb and 550 bp were conserved in majority of the isolates. HinfI digestion was found proficient in differentiating the strains of same vir typing patterns. Strong predominance of speC (85%) and speF (80%) genes have been observed encoding exotoxins production. 4 isolates were found to be erythromycin resistant and were of genotype emm49. High degree of tetracycline resistance was shown by 53.57% isolates which belonged to 12 different emm genotypes. CONCLUSIONS: These findings suggested that in addition to emm typing, sequential application of HaeIII and HinfI restriction enzymes in vir typing analysis is an effective tool for group A streptococcal molecular characterization associated with antibiotic resistance.

Antibacterial potential of hydroalcoholic extracts of triphala components against multidrug-resistant uropathogenic bacteria--a preliminary report.

"Triphala", the Ayurvedic wonder is used traditionally for the treatment of different types of diseases since antiquity. The hydroalcoholic extracts of the three components of Triphala powder demonstrated varying degrees of strain specific antibacterial activity against multidrug-resistant uropathogenic bacteria. Terminalia chebula fruit extract was active against all the test isolates followed by Terminalia belerica and Emblica officinalis. There was a close association between antibacterial activity and total phenolic content of Triphala components.The test plant extracts were also found to be non-toxic on human erythrocyte membrane at recommended and even higher doses. The preliminary results of the present study may help in developing effective and safe antimicrobial agents from Triphala components for the treatment of urinary tract infections caused by multidrug-resistant bacteria.

Anti-inflammatory, anti-lipid peroxidative, antioxidant and membrane stabilizing activities of hydroalcoholic extract of Terminalia chebula fruits.

CONTEXT: Arthritis is inflammation of one or more joints. Terminalia chebula Retz. (Combretaceae) fruit is mentioned in Ayurveda as useful in treating arthritic disorders. OBJECTIVE: This work was undertaken to evaluate the anti-inflammatory, antioxidant, anti-lipid peroxidative and membrane-stabilizing effects of hydroalcoholic extract of Terminalia chebula fruits and also to establish a possible association between them. MATERIALS AND METHODS: In vivo anti-inflammatory activity of T. chebula fruit extract at different doses ranged from 50 to 500 mg/kg, p.o. was evaluated against carrageenin-induced inflammation in rats. Human erythrocyte hemolytic assay was used for in vitro anti-inflammatory activity testing with 50 to 500 µg/ml fruit extract. Antioxidant potential of test fruit extract (10 to 100 µg/ml) was evaluated using TBARS and DPPH methods. The fruit extract was standardized for total phenolic content using Folin-Ciocalteu method. RESULTS: The standardized extract at 250 mg/kg, p.o. dose caused 69.96% reduction in carrageenin-induced rat paw edema and demonstrated 96.72% protective effect on human RBC membrane stability. Besides, T. chebula fruit extract significantly reduced the in vivo formation of TBARS in carrageenin-induced rat liver with IC50 94.96 mg/kg, p.o. and also in vitro radical scavenging activities in DPPH assay method with IC50 42.14 µg/ml. The standardized extract contains phenolics 118.5 mg gallic acid equivalent/g of extract. DISCUSSION AND CONCLUSION: These promising findings support the traditional use of T. chebula fruits in the treatment of arthritic disorders and suggest that radical quenching may be one of the mechanisms for its anti-inflammatory activity.

A molecular approach to identification and profiling of first-line-drug-resistant mycobacteria from sputum of pulmonary tuberculosis patients.

Conventional and molecular techniques were applied to detect and characterize drug resistance of mycobacteria in the sputum samples of clinically confirmed tuberculosis. The sensitivities of mycobacterium detection by ZN staining, culture, multiplex PCR, and restriction fragment length polymorphism (RFLP) were 27.7%, 19.9%, 92.9%, and 95.7%, respectively, but all were 100% specific. The conventional and multiple-allele-specific PCR (MAS-PCR) methods enabled establishment of the drug resistance in 19.3% and 86.9% cases, respectively. We demonstrated that molecular techniques have potential in the accurate diagnosis of tuberculosis.

Antibiotic resistance of Salmonella enterica serovar Typhi in Kolkata, India, and in vitro experiments on effect of combined chemotherapy.

This communication states the changing patterns of Salmonella enterica serovar Typhi (S. Typhi) isolates causing enteric fever in and around Kolkata, India. Among the isolates resistance to ampicillin (A), chloramphenicol (C), cotrimoxazole (Co) and tetracycline (T) were plasmid mediated; the plasmid was unstable in S. Typhi, and the other enteric bacteria like Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris were found to be the potential source of dissemination of such plasmids into S. Typhi. The infection with such S. Typhi strains were successfully treated with ciprofloxacin (Cp: MICs 0.0075-0.075 µg mL⁻¹) and/or ofloxacin (Ofx: MICs 0.0125-0.075 µg mL⁻¹), but in the later course, the S. Typhi strains, showing resistance to nalidixic acid, developed low level of resistance to Cp and Ofx, causing the treatment failure. Thus, the treatment regimen was shifted to the third generation cephalosporins like ceftriaxone (Ct) and cefotaxime (Cf). Keeping in mind the anticipation of development of resistance to Ct/Cf, we prepared the treatment regimen for MDR enteric fever, based on the double-drug synergy tests in vitro; Cp-gentamycin (FICI 0.121-0.216) and Cp-trimethoprim (FICI 0.14-0.483) combinations were found effective against S. Typhi isolates having decreased sensitivity to cp (MICs: 0.5-1.25 µg mL⁻¹).

Design and synthesis of some new quinoline-3-carbohydrazone derivatives as potential antimycobacterial agents.

A series of 26 new quinoline derivatives carrying active pharmacophores has been synthesized and evaluated for their in vitro antituberculosis activity against Mycobacterium tuberculosis H37Rv (MTB), Mycobacterium smegmatis (MC(2)), and Mycobacterium fortuitum following the broth micro dilution assay method. Compounds 13e, 13i, 13k, 14a, 14c, 14i, and 14k exhibited significant minimum inhibition concentrations, when compared with first line drugs isoniazid (INH) and rifampicin (RIF) and could be ideally suited for further modifications to obtain more efficacious compounds in the fight against multi-drug resistant tuberculosis.

Dual cross-linked honey coupled 3D antimicrobial alginate hydrogels for cutaneous wound healing.

We report potentiation of healing efficacy of alginate by value addition at its structural level. Dual crosslinked (ionically and covalently) sodium alginate hydrogel coupled with honey (HSAG) brings about an intermediate stiffness in the fabric, confers consistent swelling property and limits erratic degradation of the polymer which ultimately provides conducive milieu to cellular growth and proliferation. In this work honey concentrations in HSAGs are varied from 2% to 10%. FTIR, XRD and nanoindentation studies on the HSAGs exhibited physicochemical integrity. In vitro degradation study provided the crucial finding on 4% HSAG having controlled degradation rate up to 12 days with a weight loss of 87.36 ± 1.14%. This particular substrate also has an ordered crystalline surface morphology with decent cellular viability (HaCaT and 3T3) and antimicrobial potential against Methicillin Resistant Staphylococcus aureus (MRSA) and Escherichia coli. The in vivo wound contraction kinetics on murine models (4% HSAG treated wound contraction: 94.56 ± 0.1%) has been monitored by both invasive (histopathology) and noninvasive (Swept Source Optical Coherence Tomography) imaging and upon corroborating them it evidenced that 4% HSAG treated wound closure achieved epithelial thickness resembling to that of unwounded skin. Thus, the work highlights structurally modified alginate hydrogel embedded with honey as a potential antimicrobial healing agent.

Performance of SNAPPE-II score in neonatal sepsis: an experience from a tertiary care center.

BACKGROUND AND OBJECTIVES: The Score for Neonatal Acute Physiology II with Perinatal Extension (SNAPPE-II) is a vital tool for prognostication in newborns. The study was conducted with the hypothesis that the performance of the SNAPPE-II score might be affected by the presence of sepsis in newborns admitted with possible early onset septicemia and whether score performance varies between culture positive and culture negative sepsis. METHODS: The prospective observational study was conducted over a period of 1 year (January 2014 to January 2015) in neonates presenting with clinical suspicion of sepsis to the Sick Newborn Care Unit (SNCU) of a tertiary care hospital in Eastern India. RESULTS: SNAPPE-II score cut-off of ≥20 offered the highest sensitivity of 74.5% with specificity 48.3%, PPV 27.6% and NPV 87.7%. Comparison of mortality proportions between the two subgroups defined by this cut-off returned p= 0.005 with OR 3.47 (95% 1.40 to 8.64). No significant association was found between SNAPPE-II score and blood culture results; mean scores for culture positive (25.16 ± 15.6) and negative groups (24.49 ± 15.6) were comparable (p= 0.920). CONCLUSIONS: At a cut-off value of ≥20 in presence of sepsis, SNAPPE-II score offers acceptable indices to predict mortality outcome. Prediction of outcome by SNAPPE-II score is not affected by positive or negative blood culture sepsis.

A randomized controlled trial to evaluate the efficacy of lactobacillus GG in infantile diarrhea.

In a randomized, double-blind, placebo-controlled trial, 229 infants hospitalized for acute diarrhea in rural India were given a 10-day course of Lactobacillus rhammosus GG (minimum dose, 10 degrees bacteria) or placebo. There was no difference in groups in the duration of diarrhea or numbers of stool on days 3, 6, or 10 of treatment.

A typhoid fever outbreak in a slum of South Dumdum municipality, West Bengal, India, 2007: evidence for foodborne and waterborne transmission.

BACKGROUND: In April 2007, a slum of South Dumdum municipality, West Bengal reported an increase in fever cases. We investigated to identify the agent, the source and to propose recommendations. METHODS: We defined a suspected case of typhoid fever as occurrence of fever for > or = one week among residents of ward 1 of South Dumdum during February - May 2007. We searched for suspected cases in health care facilities and collected blood specimens. We described the outbreak by time, place and person. We compared probable cases (Widal positive > or = 1:80) with neighbourhood-matched controls. We assessed the environment and collected water specimens. RESULTS: We identified 103 suspected cases (Attack rate: 74/10,000, highest among 5-14 years old group, no deaths). Salmonella (enterica) Typhi was isolated from one of four blood specimens and 65 of 103 sera were > or = 1:80 Widal positive. The outbreak started on 13 February, peaked twice during the last week of March and second week of April and lasted till 27 April. Suspected cases clustered around three public taps. Among 65 probable cases and 65 controls, eating milk products from a sweet shop (Matched odds ratio [MOR]: 6.2, 95% confidence interval [CI]: 2.4-16, population attributable fraction [PAF]: 53%) and drinking piped water (MOR: 7.3, 95% CI: 2.5-21, PAF-52%) were associated with illness. The sweet shop food handler suffered from typhoid in January. The pipelines of intermittent non-chlorinated water supply ran next to an open drain connected with sewerage system and water specimens showed faecal contamination. CONCLUSION: The investigation suggested that an initial foodborne outbreak of typhoid led to the contamination of the water supply resulting in a secondary, waterborne wave. We educated the food handler, repaired the pipelines and ensured chlorination of the water.

Antibacterial activity of ciprofloxacin and trimethoprim, alone and in combinittion, against Vibrio cholerae O1 biotype El Tor serotype Ogawa isolates.

In this communication, the ciprofloxacin-trimethoprim (Cp-Tm) combination showed synergistic (Fractional Inhibitory Concentration, FIC index 0.399) and additive (FIC index 0.665-0.83) effects against Vibrio cholerae O1 biotype El Tor serotype Ogawa isolates having Cp MICs 10 microg/ml and Cp 0.66 microg/ml, respectively, following agar dilution checkerboard method. The time-kill study results demonstrated synergy between Cp and Tm against both groups of isolates providing 2.04 log10 (for strain with Cp MIC 0.66 microg/ml) and 3.12 log10 (for strain with Cp MIC 10 microg/ml) decreases in CFU/ml between the combination and its most active compound. Thus, the findings of the present study suggest an introduction of Cp-Tm combination treatment regimen against drug resistant cholera and this in turn will help in combating the drug resistance of V. cholerae O1 biotype El Tor serotype Ogawa.

Arabinosylated lipoarabinomannan modulates the impaired cell mediated immune response in Mycobacterium tuberculosis H37Rv infected C57BL/6 mice.

Mycobacterium tuberculosis is a facultative intracellular pathogen that flourishes inside the host macrophages. This organism has the ability to deactivate the cell-mediated immune responses involving the down-regulation of pro-inflammatory cytokines, T cell proliferation, apoptosis of CD4+T cells and impairment of the expression of MHC Class II molecules. We observed that Arabinosylated Lipoarabinomannan (Ara-LAM), a glycolipid present in the cell wall of the avirulent Mycobacterium smegmatis, could effectively restrict the growth of tubercle bacilli, induced the transcription of Th1 cytokines in alveolar macrophages (AMs) and splenocytes, enhanced the frequency of CD4+T cells secreting IFN-gamma and induced the expression of MHC Class II molecules on the splenocyte membrane, compared to that of Mycobacterium tuberculosis H37Rv infected C57BL/6 mice. Collectively our findings strongly suggest that Ara-LAM had the potency to restore the impaired cell mediated immune responses in mice infected with Mycobacterium tuberculosis H37Rv, and hence could be utilized as an effective immuno-prophylactic tool in the control of tuberculosis.

A prospective study of rotavirus diarrhea in children under 1 year of age.

The aim of this study was to document the changing clinical profile and prognosis of acute diarrhea in infants. This was a prospective observational study with follow-up. Demographic, anthropometric, and clinical data were collected in children younger than 1 year with acute diarrhea. Stool was examined under the microscope, cultured, tested for presence of reducing substance and occult blood, and subjected to electrophoresis to detect rotavirus infection. Thirty-one (91.2%) of the 34 infants were breastfed, 18 exclusively and 13 partially. Twenty-three had rotavirus infection and had slower nutritional recovery than others. There was no difference in the incidence of rotavirus infection between exclusively and partially breastfed infants. Continuation of feeds containing lactose did not affect prognosis, though 23 (67.6%) infants had reducing substance in stool. We documented a high incidence of rotavirus infection, which negatively affected growth of infants by some ill-defined mechanism. Failure of exclusive breastfeeding to protect against rotavirus infection highlights the need for universal rotavirus vaccination. Lactose malabsorption detected in many infants did not affect prognosis after acute diarrhea.

Indigenous Probiotic Lactobacillus Isolates Presenting Antibiotic like Activity against Human Pathogenic Bacteria.

BACKGROUND: Indigenous lactic acid bacteria are well known probiotics having antibacterial activity against potentially pathogenic bacteria. This study aims to characterize the curd lactobacilli for their probiotic potentiality and antagonistic activity against clinical bacteria. METHODS: Four curd samples were processed microbiologically for the isolation of lactic acid bacteria (LAB). The LAB strains obtained were identified by conventional methods: cultural aspect, gram-staining, biochemical and sugar fermentation tests. The probiotic properties were justified with tolerance to low-pH, bile salt and sodium chloride, and the antagonistic activity of the lactobacilli against human pathogenic bacteria (Escherichia coli, Proteus vulgaris, Acinetobacter baumannii and Salmonella enterica serovar Typhi) was assessed. Hemolytic activity and antibiotic susceptibility were determined for the lactobacilli isolates, and the cumulative probiotic potential (CPP) values were recorded. RESULT: Four lactobacilli isolates, L. animalis LMEM6, L. plantarum LMEM7, L. acidophilus LMEM8 and L. rhamnosus LMEM9, procured from the curd samples, survived in low-pH and high bile salt conditions, and showed growth inhibitory activity against the indicator bacteria by agar-well (zone diameter of inhibition; ZDIs: 13.67 ± 0.58-29.50 ± 2.10 mm) and agar overlay (ZDIs: 11.33 ± 0.58-35.67 ± 2.52 mm) methods; the average growth inhibitory activity of lactobacilli ranged 233.34 ± 45.54-280.56 ± 83.67 AU/mL, against the test bacterial pathogens. All the lactobacilli were non-hemolytic and sensitive to most of the test antibiotics. The CPP values of the isolated LAB were recorded as 80-100%. CONCLUSION: The curd lactobacilli procured might be used as the valid candidates of probiotics, and bio-therapeutics against bacterial infection to humans.

Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling.

Mycobacterium tuberculosis infection inflicts the disease Tuberculosis (TB), which is fatal if left untreated. During M. tuberculosis infection, the pathogen modulates TLR-4 receptor down-stream signaling, indicating the possible involvement of TLR-4 in the regulation of the host immune response. Mycobacterium indicus pranii (MIP) possesses immuno-modulatory properties which induces the pro-inflammatory responses via induction of TLR-4-mediated signaling. Here, we observed the immunomodulatory properties of MIP against tuberculosis infection. We have studied the detailed signaling mechanisms employed by MIP in order to restore the host immune response against the in vitro tuberculosis infection. We observed that in infected macrophages MIP treatment significantly increased the TLR-4 expression as well as activation of its downstream signaling, facilitating the activation of P38 MAP kinase. MIP treatment was able to activate NF-κB via involvement of TLR-4 signaling leading to the enhanced pro-inflammatory cytokine and NO generation in the infected macrophages and generation of protective immune response. Therefore, we may suggest that, TLR4 may represent a novel therapeutic target for the activation of the innate immune response during Tuberculosis infection.

Antibiotic resistance pattern of Enterococci isolates from nosocomial infections in a tertiary care hospital in Eastern India.

BACKGROUND: Resistance to commonly used antibiotics by Enterococci causing nosocomial infections is of concern, which necessitates judicious, responsible and evidence-based use of antibiotics. The present study was conducted to review the prevalence and identify therapeutic options for nosocomial Enterococcal infections in our tertiary care hospital. MATERIALS AND METHODS: Isolates identified by morphological and biochemical characteristics were tested for antibiotic susceptibility using Kirby-Bauer method. RESULT: 153 of 2096 culture positive clinical samples comprised of 101 urine, 30 wound swab/pus, 13 blood and 09 high vaginal swab isolates were identified as Enterococcus faecalis (90.85%), Enterococcus faecium (8.50%) and Enterococcus gallinarum (0.65%). Enterococci accounted for 8.45%, 4.53%, 4.23%, 4.43% of urinary, wound swab or pus, blood, high vaginal swab isolates respectively, causing 7.3% of all nosocomial infections. Significant number of Enterococci isolated from nosocomial urinary tract infection (66.01%) and wound infections (19.6%) were multidrug resistant (MDR). Although all isolates were sensitive to vancomycin and linezolid, resistance to erythromycin (71.24%) and ciprofloxacin (49.67%) was frequently observed. High-level gentamicin resistance was observed in 43.88%, and 61.53% of E. faecalis and E. faecium isolates respectively. Minimal inhibitory concentration of vancomycin of all the isolates were ≤1 µg/ml. 7% of the Enterococcal isolates were MDR strains and vancomycin or linezolid were the only effective antibiotics. CONCLUSION: A combination of vancomycin and/or linezolid were effective against Enterococci causing nosocomial infections in our tertiary care facility, nevertheless continuous and frequent surveillance for resistance patterns are necessary for judicious and evidence based use of antibiotics.

Arabinosylated lipoarabinomannan (Ara-LAM) mediated intracellular mechanisms against tuberculosis infection: involvement of protein kinase C (PKC) mediated signaling.

Tuberculosis causes severe immunosuppression thereby ensuring the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates TLR-2 receptor down-stream signaling, indicating the possible involvement of TLR-2 in the regulation of the host immune response. Moreover, different PKC isoforms are also involved in the course of infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immuno-modulatory properties which induce the pro-inflammatory responses via induction of TLR-2-mediated signaling. Here, we found that pretreatment of M. tuberculosis-infected macrophages with Ara-LAM caused a significant increase in the conventional PKC expression along with their active association with TLR-2. This association activated the TLR-2 -mediated downstream signaling, facilitating the activation of MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response, which was abrogated by treatment with PKC-α and P38 inhibitors. Moreover, pretreatment of macrophages with Ara-LAM abrogated the IL-10 production while restored MHC-II expression in the infected macrophages. This study demonstrates that Ara-LAM confers protection against tuberculosis via TLR-2/PKC signaling crosstalk which is responsible for the induction of host protective immune response against tuberculosis.

Immunomodulation in host-protective immune response against murine tuberculosis through regulation of the T regulatory cell function.

Tuberculosis, caused by the bacteria Mycobacterium tuberculosis, is characterized by an infection in lung and spleen. In the present study, we have elucidated the mechanism by which Mycobacterium indicus pranii renders protection in in vivo Mycobacterium tuberculosis infection. We observed that Mycobacterium indicus pranii treated infected C57BL/6 mice showed a strong host-protective Th1 immune response along with a marked decrease in immunosuppressive cytokines, TGF-ß, and IL-10-secreting CD4(+) T cells. This Mycobacterium indicus pranii mediated decrease in immunosuppressive cytokines was correlated with the reduction in the elevated frequency of CD4(+)CD25(+) T regulatory cells, along with the reduced TGF-ß production from these T regulatory cells in tuberculosis-infected mice. This reduction in the T regulatory cell population was a result of effective modulation of STAT4-STAT5 transcription factor counter-regulation by Mycobacterium indicus pranii, which in turn, reduced the immunosuppressive activity of T regulatory cells. Thus, these findings put forward a detailed mechanistic insight into Mycobacterium indicus pranii mediated regulation of the T regulatory cell functioning during experimental murine tuberculosis, which might be helpful in combating Mycobacterium-induced pathogenesis.

Scattergram analysis to explore the emerging problem related to in vitro susceptibility test for Salmonella enterica serovar Typhi to ciprofloxacin.

Ciprofloxacin susceptibility using a disk diffusion method and minimum inhibitory concentration (MIC) value determination for 421 S. Typhi isolates showed comparable results for 296 (70%) isolates with an MIC /=0.1 mg/L, signifying resistance, 123 (98.4%) showed discrepant results with the disk diffusion test (66 sensitive, 57 intermediately sensitive). This raises questions about National Committee for Clinical Laboratory Standards guidelines regarding the interpretative zone size for ciprofloxacin.

Ofloxacin minimum inhibitory concentration versus disk diffusion zone diameter for Salmonella enterica serovar Typhi isolates: problems in the detection of ofloxacin resistance.

Using the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS), a total of 421 blood culture isolates of Salmonella enterica serovar Typhi obtained during 1991-2001 were tested for susceptibility to ofloxacin (OFX) by the disc diffusion method, and for the determination of minimum inhibitory concentration (MIC) values of OFX by the agar dilution method. Among 421 isolates, 248 were fully OFX-sensitive showing MICs of 0.0125-0.075 microg/ml and inhibitory zone diameters of > or =24 mm. The remaining 173 isolates (MICs of 0.5-1.5 microg/ml) that were treated with OFX did not respond to the therapy. However, 169 (97.69%) of the 173 isolates were determined to be susceptible (zone diameter > or =16 mm) by the disc diffusion method, whereas only 3 were intermediately susceptible (zone diameter 13-15 mm) and the final isolate showed OFX-resistance (zone diameter 12 mm). Thus, following the NCCLS guidelines, OFX-resistance in S. enterica serovar Typhi was not detected by the disc diffusion test. The present data suggest a revision of the NCCLS breakpoints in selecting OFX as the preferred treatment regimen for S. enterica serovar Typhi