Multi-omics analysis identifies potential microbial and metabolite diagnostic biomarkers of bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common clinical manifestation of a perturbed vaginal ecology associated with adverse sexual and reproductive health outcomes if left untreated. The existing diagnostic modalities are either cumbersome or require skilled expertise, warranting alternate tests. Application of machine-learning tools to heterogeneous and high-dimensional multi-omics datasets finds promising potential in data integration and may aid biomarker discovery. OBJECTIVES: The present study aimed to evaluate the potential of the microbiome and metabolome-derived biomarkers in BV diagnosis. Interpretable machine-learning algorithms were used to evaluate the utility of an integrated-omics-derived classification model. METHODS: Vaginal samples obtained from reproductive-age group women with (n = 40) and without BV (n = 40) were subjected to 16S rRNA amplicon sequencing and LC-MS-based metabolomics. The vaginal microbiome and metabolome were characterized, and machine-learning analysis was performed to build a classification model using biomarkers with the highest diagnostic accuracy. RESULTS: Microbiome-based diagnostic model exhibited a ROC-AUC (10-fold CV) of 0.84 ± 0.21 and accuracy of 0.79 ± 0.18, and important features were Aerococcus spp., Mycoplasma hominis, Sneathia spp., Lactobacillus spp., Prevotella spp., Gardnerella spp. and Fannyhessea vaginae. The metabolome-derived model displayed superior performance with a ROC-AUC of 0.97 ± 0.07 and an accuracy of 0.92 ± 0.08. Beta-leucine, methylimidazole acetaldehyde, dimethylethanolamine, L-arginine and beta cortol were among key predictive metabolites for BV. A predictive model combining both microbial and metabolite features exhibited a high ROC-AUC of 0.97 ± 0.07 and accuracy of 0.94 ± 0.08 with diagnostic performance only slightly superior to the metabolite-based model. CONCLUSION: Application of machine-learning tools to multi-omics datasets aid biomarker discovery with high predictive performance. Metabolome-derived classification models were observed to have superior diagnostic performance in predicting BV than microbiome-based biomarkers.

Dissemination of methicillin-resistant Staphylococcus aureus SCCmec type IV and SCCmec type V epidemic clones in a tertiary hospital: challenge to infection control.

Two-hundred MRSA strains from inpatients with healthcare-associated (HA) and 100 MRSA strains from outpatients with community-associated (CA) skin and soft tissue infections (SSTIs) were tested for antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) typing, Panton-Valentine leucocidin (PVL) toxin, seh and arcA genes. Based on SCCmec typing, HA-MRSA isolates were further divided into HA-SCCmec I/II/III MRSA and HA-SCCmec IV/V MRSA, and CA-MRSA isolates into CA-SCCmec I/II/III MRSA and CA-SCCmec IV/V MRSA. SCCmec types were further characterized by pulsed-field gel electrophoresis, spa typing and multi-locus sequence typing. Seventy-five (37·5%) HA-MRSA isolates and 83/100 CA-MRSA isolates were SCCmec IV/V genotype. HA-SCCmec IV/V MRSA was associated with malignancy (P = 0·03) and bone fractures (P = 0·02) compared to CA-SCCmec IV/V MRSA. HA-SCCmec IV/V MRSA was associated with PVL gene carriage compared to HA-SCCmec I/II/III MRSA (P < 0·001). ST22-MRSA-IV (EMRSA-15), ST772-MRSA-V, and ST36-MRSA-IV and ST239:EMRSA-I:III were the major clones identified. Our study documents the emergence of SCCmec IV and SCCmec V MRSA clones in an Indian hospital.

Incidence and characterization of Clostridium perfringens isolated from antibiotic-associated diarrhoeal patients: a prospective study in an Indian hospital.

Clostridium perfringens has been reported as causing between 2-15% of all cases of antibiotic-associated diarrhoea (AAD), and may be diagnosed by detection of enterotoxin in faeces. A prospective study comprising 150 diarrhoeal patients and 100 non-diarrhoeal controls was undertaken to assess the incidence of C. perfringens-associated diarrhoea in an Indian hospital. Methods used included C. perfringens culture, reverse passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) for detection of enterotoxin, and polymerase chain reaction (PCR) assay for the presence of enterotoxin gene. Attempts were made to type the isolates by multiplex PCR. Of the 150 diarrhoeal stool samples tested, 13 were culture positive. Of these, four were positive for C. perfringens enterotoxin by RPLA, two were positive by PCR and two were positive by RPLA and ELISA. Twenty-seven samples were positive for culture of C. perfringens in non-diarrhoeal controls but none were positive for enterotoxin either by RPLA or by PCR. The average incidence of C. perfringens AAD using these methods was 2.6%. Toxin typing showed that all the isolates belonged to type A. To conclude, the relatively low incidence of toxigenic C. perfringens suggests that enterotoxigenic C. perfringens is not a major cause of AAD in this population.

The changing face of community-acquired methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infection, both in hospitalised patients with significant healthcare exposure and in patients without healthcare risk factors. Community-acquired methicillin-resistant S. aureus (CA-MRSA) are known for their rapid community transmission and propensity to cause aggressive skin and soft tissue infections and community-acquired pneumonia. The distinction between the healthcare-associated (HA)-MRSA and CA-MRSA is gradually fading owing to the acquisition of multiple virulence factors and genetic elements. The movement of CA-MRSA strains into the nosocomial setting limits the utility of using clinical risk factors alone to designate community or HA status. Identification of unique genetic characteristics and genotyping are valuable tools for MRSA epidemiological studies. Although the optimum pharmacotherapy for CA-MRSA infections has not been determined, many CA-MRSA strains remain broadly susceptible to several non-ß-lactam antibacterial agents. This review aimed at illuminating the characteristic features of CA-MRSA, virulence factors, changing clinical settings and molecular epidemiology, insurgence into the hospital settings and therapy with drug resistance.

Prosthetic joint infection due to Lysobacter thermophilus diagnosed by 16S rRNA gene sequencing.

We report the first case of prosthetic joint infection caused by Lysobacter thermophilus which was identified by 16S rRNA gene sequencing. Removal of prosthesis followed by antibiotic treatment resulted in good clinical outcome. This case illustrates the use of molecular diagnostics to detect uncommon organisms in suspected prosthetic infections.

Coagulase-negative staphylococci causing blood stream infection at an Indian tertiary care hospital: Prevalence, antimicrobial resistance and molecular characterisation.

INTRODUCTION: Recent years have seen a rise of coagulase-negative staphylococci (CoNS) from common contaminants to agents of nosocomial blood stream infections (BSI's). Molecular typing and establishing a correlation with antibiotic resistance is essential particularly in countries like India where genotyping studies for drug-resistant CoNS are sparse. METHODS: A prospective study was done over 18 months, wherein 42,693 blood samples were received, and 59 patients with BSI due to CoNS were evaluated. The isolates recovered were identified by a biochemical test panel and matrix-assisted laser desorption ionization - time of flight mass spectrometry followed by antimicrobial susceptibility testing by Kirby-Baur disc diffusion method and E-test strips. Staphylococcal chromosomal cassette mec (SCCmec) element was characterised by multiplex polymerase chain reaction for all methicillin-resistant (MR) isolates. RESULTS: The majority of CoNS isolated were constituted by Staphylococcus haemolyticus (47.5%) followed by Staphylococcus epidermidis (33.9%), Staphylococcus hominis (11.86%), Staphylococcus cohnii (5.08%) and Staphylococcus warneri (1.69%). Among all isolates 57.6% were MR with statistically significant higher resistance versus methicillin sensitive-CoNS. This difference was significant for erythromycin (76% vs. 44%, P = 0.011), rifampicin (50% vs. 12%,P= 0.002) and amikacin (26.5% vs. 4%, P = 0.023), ciprofloxacin (64.7% vs. 20%, P = 0.001) and cotrimoxazole (55.9% vs. 20%, P = 0.006). SCCmec type I was predominant (61.8%, P = 0.028) and exhibited multidrug resistance (76.2%). Coexistence of SCCmec type I and III was seen in 8.82% MR isolates. CONCLUSION: CoNS exhibit high antimicrobial resistance thereby limiting treatment options. The presence of new variants of SCCmec type in hospital-acquired CoNS may predict the antibiotic resistance pattern. This is the first evaluation of the molecular epidemiology of CoNS causing BSI from India and can serve as a guide in the formulation of hospital infection control and treatment guidelines.

Application of real-time quantitative polymerase chain reaction assay to detect Legionella pneumophila in patients of community-acquired pneumonia in a tertiary care hospital.

Legionella pneumophila is one of the important pathogen responsible for community -acquired pneumonia attributing for 1-5% of cases. Since early and accurate therapy reduces mortality, rapid and reliable diagnostic methods are needed. A total of 134 samples of blood, urine and respiratory tract fluids were collected. Blood was tested for IgG, IgM and IgA antibodies using commercially available kits. A total of 8 (6%) samples were found to be positive for L. pneumophila by quantitative reverse transcription polymerase chain reaction (qRT-PCR), compared to conventional PCR where 6 (4.4%) samples were positive. Serology was positive in a total of 32 (23%) cases though only 3 (2.2%) of the PCR-positive cases were positive by serology as well. These results suggest that real-time PCR can detect Legionella infection early in the course of the disease before serological response develops.

Ureaplasma: current perspectives.

Ureaplasma species are the most prevalent genital Mycoplasma isolated from the urogenital tract of both men and women. Ureaplasma has 14 known serotypes and is divided into two biovars- Ureaplasma parvum and Ureaplasma urealyticum. The organism has several genes coding for surface proteins, the most important being the gene encoding the Multiple Banded Antigen (MBA). The C-terminal domain of MBA is antigenic and elicits a host antibody response. Other virulence factors include phospholipases A and C, IgA protease and urease. Besides genital tract infections and infertility, Ureaplasma is also associated with adverse pregnancy outcomes and diseases in the newborn (chronic lung disease and retinopathy of prematurity). Infection produces cytokines in the amniotic fluid which initiates preterm labour. They have also been reported from renal stone and suppurative arthritis. Genital infections have also been reported with an increasing frequency in HIV-infected patients. Ureaplasma may be a candidate 'co factor' in the pathogenesis of AIDS. Culture and polymerase chain reaction (PCR) are the mainstay of diagnosis. Commercial assays are available with improved turnaround time. Micro broth dilution is routinely used to test antimicrobial susceptibility of isolates. The organisms are tested against azithromycin, josamycin, ofloxacin and doxycycline. Resistance to macrolides, tetracyclines and fluoroquinolones have been reported. The susceptibility pattern also varies among the biovars with biovar 2 maintaining higher sensitivity rates. Prompt diagnosis and initiation of appropriate antibiotic therapy is essential to prevent long term complications of Ureaplasma infections. After surveying PubMed literature using the terms 'Ureaplasma', 'Ureaplasma urealyticum' and 'Ureaplasma parvum', relevant literature were selected to provide a concise review on the recent developments.

On the mechanism of potentiation of apomorphine-induced stereotypy due to electroconvulsive shock.

Electroconvulsive shock-induced changes in the intensity of stereotype induced by apomorphine, the binding of [3H]spiroperidol in the corpus striatum, the accumulation of [3H]dopamine in brain and the permeability of the blood-brain barrier, were monitored in rats 30 min after single, or 24 hr after chronic (once daily for 7 days) electroconvulsive shock. There was significant potentiation in stereotypy induced by apomorphine after chronic electroconvulsive shock. The binding of [3H]spiroperidol did not show any change in the affinity (Kd) or density (Bmax) of receptors in the striatum after acute or chronic electroconvulsive shock. The accumulation of dopamine increased significantly in the hypothalamus after acute electroconvulsive shock and in the corpus striatum and hypothalamus after chronic electroconvulsive shock. A significant increase in the entry of sodium fluorescein into the hypothalamus occurred after acute electroconvulsive shock; it increased in all the regions of the brain after chronic electroconvulsive shock. Alteration in the blood-brain barrier (BBB) by electroconvulsive shock leading to increased accumulation of dopamine in the corpus striatum may be responsible for the potentiation of stereotypy.

The mechanism of opening of the blood-brain barrier by hypertonic saline.

The permeability of the blood-brain barrier was assessed in the cat, using Evan's blue as circulant. Regional blood flow and vascular resistance in various areas of brain were measured using radioactive microspheres. Cardiac output (CO), blood pressure (BP), heart rate (HR), pH, pO2 and pCO2 were also measured. Hypertonic saline (5 M, 0.5 ml/kg), administered intravenously, increased the staining of the brain substance. It also produced a marked increase in cerebral blood flow after 5 min and a marked decrease in blood flow to all the regions of the brain after 20 min. The flow returned to normal after 40 min. The vascular resistance decreased at 5 min, increased at 20 min and returned to normal at 40 min. Cardiac output increased significantly at 5 min and decreased at 20 min, while at 40 min it returned to normal. Blood pressure decreased at 5 min, increased at 20 min, while heart rate steadily decreased. A complete recovery of cardiac output, blood pressure and heart rate occurred in 1 hr. No change was observed in pH, pCO2 and pO2. It is concluded that intravenous administration of hypertonic saline causes marked haemodynamic changes and increases the permeability of the blood-brain barrier due to transient impairment of autoregulation.

Effects of interferon in malaria infection.

Earlier, we reported that prophylactic treatment with human interferon gamma (rHuIFN-gamma) protected monkeys against Plasmodium cynomolgi B malaria infection. We have tested the efficacy of rHuIFN-gamma on relapsing stage of experimental P. cynomolgi B malaria infection in rhesus monkeys. No effect of rHuIFN-gamma was seen against experimental relapsing stage compared with controls; however, it appears that chloroquine (CHL) may have interfered with the antimalarial effect of IFN, since treatment with CHL inhibits the antiviral activity of mouse alpha/beta IFN and polyinosinic-polycytidylic acid (poly I:C) against Semliki forest virus (SFV) in mice. These results may have clinical implications especially with the use of IFN against virus infection, cancer and in parasitic infections in malaria endemic areas where CHL is one of the most widely used antimalarial drugs. Our result also shows that CHL treatment enhances the virus replication in mice and suggest a possible connection between AIDS and malaria infection, since the spread of AIDS has been rapid in parts of tropical Africa that have a high incidence of malaria, and chloroquine has been frequently used in the chemotherapy of malaria.

Facilitation of the flexor reflex in the cat by intrathecal injection of catecholamines.

1. Effects of some alpha- and beta-adrenoceptor stimulants and antagonists were investigated on flexor reflex (FR) in chloralosed cats.2. Noradrenaline (NA) produced facilitation of FR which was dose-dependent and reproducible and was blocked by alpha-adrenoceptor blocking agents.3. Strychnine also produced facilitation of FR but the response was unaffected by alpha-adrenoceptor blocking agents.4. Metaraminol and alpha-methyl-noradrenaline had little effect on FR but blocked the NA response.5. beta-adrenoceptor stimulants and antagonists had neither any effect on FR nor modified the NA response.6. Vasopressin and histamine also failed to modify FR.7. Possibility of alpha-adrenoceptors in the neurones integrating FR is suggested.

Evidence for the presence of -adrenoceptors in the central thermoregulatory mechanism of rabbits.

1. The effects of intracerebroventricular administration of some alpha- and beta-adrenoceptor stimulants and antagonists on the body temperature of rabbits were investigated.2. Noradrenaline produced a dose dependent rise in body temperature. Other catecholamines were less active.3. The noradrenaline response was blocked by alpha-adrenoceptor blocking agents while beta-adrenoceptor antagonists had no effect.4. alpha-Methyl-noradrenaline and metaraminol had some hyperthermic effect, but significantly reduced the response of noradrenaline.5. The possible presence of alpha-adrenoceptors in the central thermoregulatory mechanisms is suggested.

Anti-inflammatory and some other pharmacological effects of 3,4-trans-2,2-dimethyl-3-phenyl-4-(p-(beta-pyrrolidinoethoxy)-phenyl)-7-methoxy-chroman (Centchroman).

1. Anti-inflammatory, analgesic, antipyretic and other pharmacological activities of a new chroman derivative, Centchroman, have been described.2. Centchroman was found to possess significant anti-inflammatory action in the carrageenin-induced oedema test in mice and rats. It inhibited granuloma formation in the cotton pellet test. The anti-arthritic activity was also evident in formaldehyde-induced arthritis and adjuvant-induced arthritis in rats.3. Centchroman had a lower ulcerogenic index than phenylbutazone.4. The mechanism of the anti-inflammatory action of Centchroman seemed to be independent of endogenous adrenocortical hormones or of its weak oestrogenic activity.5. Centchroman antagonized phenylquinone writhing and bradykinin-induced bronchospasm but was devoid of any antipyretic activity.

Analysis of histamine receptors in the central thermoregulatory mechanism of Mastomys natalensis.

1 The effect of intracerebroventricular (i.c.v.) injection of histamine on the rectal temperature of Mastomys natalensis at ambient temperatures of 10, 24 and 33 degrees C has been studied. 2 Low doses (0.1-1.0 microgram) of histamine produced hypothermia while larger doses (5-20 micrograms) produced dose-dependent hyperthermia. The hypothermic effect was significantly antagonized by mepyramine while the hyperthermia was blocked by cimetidine. 3 Histamine H1-receptor agonists, 2-methyl-histamine and 2-pyridyl-ethylamine, also produced hypothermia which could be blocked by mepyramine. 4 Histamine H2-receptor agonists, impromidine and dimaprit, produced hyperthermia which was antagonized by cimetidine. 5 Pretreatment of the animals with a beta-adrenoceptor antagonist, MJ1999, did not affect the response to histamine. 6 The hyperthermic effect of histamine (10 micrograms) was most marked at 10 degrees C and was attenuated at 33 degrees C. 7 It is concluded that both H1 and H2-histamine receptors are present in the brain of Mastomys. The H1-receptors mediate hypothermia and H2-receptors hyperthermia.

Selective inhibition by glycine of some somatic reflexes in the cat.

1. The effects of intracerebroventricular or intrathecal administration of glycine on some somatic reflexes in chloralosed cats were investigated.2. Glycine produced marked inhibition of the flexor reflex (FR) and crossed extensor reflex (CER) at low doses (2.5-5.0 mg) but even large doses (up to 40.0 mg) had no effect on the facilitation or inhibition of the patellar reflex produced by stimulation of either the reticular formation or the sciatic nerve. The linguomandibular reflex (LMR) could be blocked by 10-20 mg glycine.3. Glycine antagonized strychnine-induced facilitation of FR, CER and LMR and was 5-6 times as active as gamma-aminobutyric acid in this respect.4. The possibility of glycine being a transmitter in spinal pathways is discussed.

Effect of clonidine on the excitability of vasomotor loci in the cat.

1. The effect of clonidine on the direct excitability of hypothalamic, medullary and spinal vasomotor loci has been investigated in cats anaesthetized with chloralose. 2. Clonidine inhibited the excitability of these loci when it was localized to the central sites by intracerebroventricular, intravertebral arterial or intrathecal injection in very low doses (1-2 mug). 3. Topical application of clonidine (0.01 percent and 1.0 percent) to the floor of the fourth ventricle inhibited pressor responses evoked either by stimulation of medullary or hypothalamic vasomotor areas. Inhibition of the pressor responses was accompanied by hypotension and bradycardia in many experiments. 4. It appears that effects of clonidine on the vasomotor loci of the medulla oblongata and the spinal cord contribute to its hypotensive action.

Picroliv, picroside-I and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions.

Picroliv, the active principle of Picrorhiza kurrooa, and its main components which are a mixture of the iridoid glycosides, picroside-I and kutkoside, were studied in vitro as potential scavengers of oxygen free radicals. The superoxide (O2-) anions generated in a xanthine-xanthine oxidase system, as measured in terms of uric acid formed and the reduction of nitroblue tetrazolium were shown to be suppressed by picroliv, picroside-I and kutkoside. Picroliv as well as both glycosides inhibited the non-enzymic generation of O2- anions in a phenazine methosulphate NADH system. Malonaldehyde (MDA) generation in rat liver microsomes as stimulated by both the ascorbate-Fe2+ and NADPH-ADP-Fe2+ systems was shown to be inhibited by the Picroliv glycosides. Known antioxidants tocopherol (vitamin E) and butylated hydroxyanisole (BHA) were also compared with regard to their antioxidant actions in the above system. It was found that BHA afforded protection against ascorbate-Fe(2+)-induced MDA formation in microsomes but did not interfere with enzymic or non-enzymic O2- anion generation; and tocopherol inhibited lipid peroxidation in microsomes by both prooxidant systems and the generation of O2- anions in the non-enzymic system but did not interfere with xanthine oxidase activity. The present study shows that picroliv, picroside-I and kutkoside possess the properties of antioxidants which appear to be mediated through activity like that of superoxide dismutase, metal ion chelators and xanthine oxidase inhibitors.

Atropine and naloxone sensitive stimulation produced analgesia from pretectal nucleus in rat.

Mild and brief electrical stimulation of sites in the pretectal nucleus (PTN) of rats evoked potent analgesia of long duration, without significant aversions and was unassociated with motor deficit. The present study has analysed effects of opioidergic and cholinergic neurotransmitter antagonists administered intracerebroventricularly (i.c.v.) on this analgesia. Pretreatment either with naloxone or atropine sulphate both in doses of 30 and 50 micrograms each, respectively i.c.v., 10 min prior to subsequent pretectal stimulation, significantly attenuated the increase in tailflick latency. The antagonism of pretectal stimulation produced analgesia (PSPA) by naloxone and atropine, raises the possibility of involvement of both endogenous opioids and cholinergic mechanisms in pretectal analgesia.

Analgesic effect of morphine, clonidine and serotonin microinjected into the PTN of rats.

The study was aimed to delineate the neurotransmitter receptors involved in pretectal analgesic mechanisms by direct microinjection of neurotransmitter agonists and antagonists through chronically implanted cannulae in the pretectal nucleus of rats. Morphine, clonidine and serotonin, at doses of 2.5 and 5.0 micrograms microinjected into the pretectal nucleus, produced a significant and prolonged analgesia as measured by the tail-flick test. The analgesia produced by morphine, clonidine and serotonin is significantly attenuated by pretreatment of the animals with naloxone (1 micrograms), yohimbine (5 micrograms) and methysergide (5-10 micrograms) respectively. The results indicate the possible involvement of opioid, adrenergic and serotonergic mechanisms in pretectal analgesia.