Enhancing microbial diversity as well as multi-organ health in hind-limb unloaded mice.

During space travel, the gut microbiota is changed which can lead to health-related issues. Previously, we utilized the hind-limb unloaded (HU) mouse, which is an established ground-based in-vivo model of microgravity and observed altered gut microbiota. In this study, we evaluated the beneficial effects of novel bacterial conditioned media in HU mice to understand if they can offset the effects of unloading in the HU mouse model. We aimed to explore the influence of bacterial conditioned media on diversity and quantity of intestinal microbes in HU mice, and investigated the microarchitecture of mice retinas and kidneys to evaluate the potential systemic effects of bacterial conditioned media in HU mice. Four-month-old, male C57/Bl6 mice were separated into groups: including the ground-based control group, the HU group mice fed with vehicle as placebo (HU-placebo mice), and the HU group fed with bacterial conditioned media (HU-CP mice) and kept under controlled environmental conditions for three weeks. Next, mice were sacrificed; gut dissections were conducted, and metagenomic analysis of bacterial species was performed via DNA extraction and 16S rRNA analysis. The results revealed an HU-induced reduction in intestinal microbial diversity, and an increase in pathogenic bacteria dominated by Firmicutes (45%). In contrast, supplementation with bacterial conditioned media for three weeks led to a significant increase in gut microbial diversity with noticeable changes in the OTUs abundance in the HU mice. Additionally, HU-induced muscle weakness and structural abnormalities in the retina and kidney were partially prevented with bacterial conditioned media. Moreover, a greater diversity of several bacteria in the HU-CP was observed including, Bacteriodota, Firmicutes, Proteobacteria, Actionobacteriota, Verrucomicorbiota, Cyanobacteria, Gemmatimonadota, Acidobacteriota, Chloroflexi, Myxococcota, and others. Prospective research involving molecular mechanistic studies are needed to comprehend the systemic effects of bacterial metabolites conditioned media on experimental animal models under chronic stress.

Fish collagen peptides' modulating effect on human skin microbiota against pathogenic Staphylococcus aureus.

Aim: The current research aims to design effective strategies to enhance the body's immune system against pathogenic bacteria. Methods: Skin commensals were isolated, identified and cultured in fish collagen peptides (FCPs). Results: After culturing in FCP, the skin commensals were used in a dose-dependent manner for Staphylococcus aureus in a dual-culture test, which showed significant growth inhibition of the pathogenic bacteria, which concluded that FCP induced the immune defense system of skin microbiota against pathogenic strains. Conclusion: Results have validated that fish collagen peptide plays a vital role in the growth of selected human skin flora and induces more defensive immunity against pathogenic S. aureus bacteria in dual-culture experimentation.

The Role of 4-Phenylbutyric Acid in Gut Microbial Dysbiosis in a Mouse Model of Simulated Microgravity.

The altered gut microbes of astronauts during space travel may contribute to health issues after their return to Earth. Previously, an association between the elevated endoplasmic reticulum (ER) stress and gut microbial dysbiosis has been described. Herein, we induced gut microbial changes in mice under a simulated microgravity environment in an established model of hindlimb unloaded (HU) mice. The intestinal metabolomic profiles under microgravity conditions using the HU model were examined, along with the potential role of 4-phenylbutyric acid (4-PBA), a potent ER stress inhibitor. For a microgravity environment, the mice were suspended in special cages individually for three weeks. Mice were sacrificed, and gut dissections were performed, followed by amplicon sequencing analysis of bacterial species via DNA extraction and 16S rRNA analysis. The results indicate that the gut bacterial communities of mice differed under gravity and microgravity conditions. Principal component analyses revealed differences in the bacterial community structure in all groups. Around 434 operational taxonomic units (OTUs) were specific to mice seen in controls, while 620 OTUs were specific to HU mice. Additionally, 321 bacterial OTUs were specific to HU mice treated with 4-PBA. When the relative abundance of taxa was analyzed, Bacteroidetes dominated the gut of control and HU mice treated with 4-PBA.. In contrast, the untreated HU mice were dominated by Firmicutes. At the genus level, a reduction in beneficial species of Akkermansia and Lactobacillus was observed in HU but not the unloaded-treated and control mice. Furthermore, an increase in the relative abundance of Lachnospiraceae and Enterorhabdus, associated with inflammation, was observed in HUmice but not in controls and unloaded-treated mice. Following treatment with 4-PBA, the ratio of Firmicutes to Bacteroidetes was restored in unloaded-treated mice, comparable to controls. Of note, beneficial microbes such as Akkermansia and Lactobacillus were observed in unloaded-treated mice but not or in lesser relative abundance in HU mice. Nonetheless, microbial diversity was reduced in unloaded-treated mice compared to controls, and future studies are needed to mitigate this finding. These may comprise the addition of pre-/pro- and postbiotic species in the diet to increase microbial diversity. Overall, the findings suggest that 4-PBA, a potent ER stress inhibitor, may have therapeutic value in treating patients on prolonged bed rest or astronauts during spaceflight.

Quantitative Estimation of Aflatoxin Level in Poultry Feed in Selected Poultry Farms.

Statement of Novelty. Poultry feed contamination due to mycotoxins is one of the major threats to the growing poultry industry. Surveillance of different mycotoxins, including aflatoxin, is very important to control economic and health hazards associated with these toxins. Studies reporting aflatoxin levels in poultry feed are limited. Therefore, this study was conducted to examine the occurrence of total aflatoxin in poultry feed. This study is the first-ever documentation about the frequency and quantitative estimations of total aflatoxin levels in poultry feed consumed to provide solid feedback to the poultry industrialists and researchers involved in studying the mycotoxins. Objective. Contamination of poultry feed with mycotoxins such as aflatoxin is a major concern for the poultry industry that results in a significant economic loss and directly affects consumers. Monitoring the aflatoxin levels in poultry feed is crucial for controlling economic loss and decreasing the health hazards to the population. This study was conducted to examine the occurrence of total aflatoxin in poultry feed in a high consumption area. Three different poultry feeds, i.e., starter, grower, and finisher, were assessed through continuous sampling from farms. The incidence of positive samples for aflatoxin contamination was 92.5%. Grower feed had the highest frequency (100%) of aflatoxin positive samples and aflatoxin levels with a mean value of 56.34 ppb. Further, the range of moisture content was around 6.8%-10.98%. No significant correlation between humidity and aflatoxin contamination was revealed when analyzed by Pearson's correlation coefficient with r 2 of 0.05 and p value of 0.13. The results warrant the need for constant monitoring programs for the prevention of aflatoxin contamination in local poultry farms.

In-vivo toxicity and therapeutic efficacy of Paeonia emodi-mediated zinc oxide nanoparticles: In-vitro study.

This study was planned to explore the in-vitro and in-vivo therapeutic significance of Paeonia emodi-mediated zinc oxide nanoparticles (ZnO NPs) against the Staphylococcus aureus and Escherichia coli. The texture parameters were derived from nitrogen adsorption-desorption data using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, and the surface area (SBET ) was found to be 214 m2 /g with a pore size of 2.3 nm. The crystallographic parameters were investigated through X-ray diffraction analysis, and the calculated crystallite size is 29.13 nm. The microstructure was examined through transmission and scanning electron microscopies (TEM and SEM, respectively), and the average particle size estimated from a TEM image is 44.40 nm. The chemical composition and attached function groups were identified through energy-dispersive X-ray and Fourier transform infrared spectroscopies. The in-vitro minimum inhibitory concentration (MIC) for both bacterial species results was found less than 2 µg/ml. The tolerance limit of mouse models was evaluated by the inoculation of different concentrations of ZnO suspension where the concentration above 23 ppm was proved lethal. The maximum infection was caused in mouse models by inoculation of 3 × 107 CFUs (Colony forming unit) of the both bacterial species. The concentration higher than 3 × 107 CFUs led to the ultimate death of the mice. The histopathological and hematological studies reveal that the after simultaneous inoculation of both ZnO NPs and bacterial suspensions (tolerated amount), no/negligible infection was found in the mice model.

In-Vitro and In-Vivo Tolerance and Therapeutic Investigations of Phyto-Fabricated Iron Oxide Nanoparticles against Selected Pathogens.

The Paeonia emodi (P. emodi)-mediated iron oxide nanoparticles (Fe2O3 NPs) were screened for in-vitro and in-vivo antibacterial activity against the Staphylococcus aureus (S. aureus) (ATCC #: 6538) and Escherichia coli (E. coli) (ATCC #:15224). The synthesized Fe2O3 NPs were characterized via nitrogen adsorption-desorption process, X-ray diffractometer (XRD), transmission and scanning electron microscopies (TEM and SEM), energy dispersive X-ray (EDX) and Fourier transform infrared (FTIR) spectroscopies. The SBET was found to be 94.65 m2/g with pore size of 2.99 nm, whereas the average crystallite and particles size are 23 and 27.64 nm, respectively. The 4 µg/mL is the MIC that inhibits the growth of E. coli, whereas those for S. aureus are below the detection limit (<1.76 µg/mL). The tolerance limit of the mice model was inspected by injecting different concentration of Fe2O3 NPs and bacteria suspensions. The 14 ppm suspension was the tolerated dose and the concentration above were proved lethal. The most severe infection was induced in mice with injection of 3 × 107 CFUs of both bacteria, while the inoculation of higher concentrations of bacterial suspensions resulted in the mice's death. The histopathological and hematological studies reveals that the no/negligible infection was found in the mice exposed to the simultaneous inoculation of Fe2O3 NPs (14 ppm) and bacterial suspensions (3 × 107 CFUs).

Preparation, characterization and stability studies of cross-linked α-amylase aggregates (CLAAs) for continuous liquefaction of starch.

In current study, α-amylase of fungal origin was immobilized using cross-linking strategy. The influence of precipitant (ammonium sulphate) and cross-linker (glutaraldehyde) concentration revealed that 60% (w/v) precipitant and 1.5% (v/v) cross-linker saturation was required to attain optimum activity. Cross-linked amylase aggregates (CLAAs) were characterized and 10-degree shift in optimum temperature (soluble enzyme: 50 °C; cross-linked: 60 °C) and 1-unit shift in pH (soluble enzyme: pH -6; cross-linked: pH -7) was observed after immobilization. The Vmax for soluble α-amylase and its cross-linked form was 1225 U ml-1 and 3629 U ml-1, respectively. The CLAAs was more thermostable than its soluble form and retained its 30% activity even after 60 min of incubation at 70 °C. Moreover, cross-linked amylase retained its activity after two months while its soluble counterpart lost its complete activity after 10 and 20 days at 30 °C and 4 °C storage, respectively. Reusability test showed that cross-linked amylase could retain 13% of its residual activity after 10 repeated cycles. Therefore, 10 times more glucose was produced after cross-linking than soluble amylase when it was utilized multiple times. This study indicates that amylase aggregates are highly effective for continuous liquefaction of starch, hence have strong potential to be used for different industrial processes.

A review on recent advances and applications of fish collagen.

During the processing of the fishery resources, the significant portion is either discarded or used to produce low-value fish meal and oil. However, the discarded portion is the rich source of valuable proteins such as collagen, vitamins, minerals, and other bioactive compounds. Collagen is a vital protein in the living body as a component of a fibrous structural protein in the extracellular matrix, connective tissue and building block of bones, tendons, skin, hair, nails, cartilage and joints. In recent years, the use of fish collagen as an increasingly valuable biomaterial has drawn considerable attention from biomedical researchers, owing to its enhanced physicochemical properties, stability and mechanical strength, biocompatibility and biodegradability. This review focuses on summarizing the growing role of fish collagen for biomedical applications. Similarly, the recent advances in various biomedical applications of fish collagen, including wound healing, tissue engineering and regeneration, drug delivery, cell culture and other therapeutic applications, are discussed in detail. These applications signify the commercial importance of fish collagen for the fishing industry, food processors and biomedical sector.

Mycoremediation: a treatment for heavy metal-polluted soil using indigenous metallotolerant fungi.

Bioleaching of heavy metals from industrial contaminated soil using metallotolerant fungi is the most efficient, cost-effective, and eco-friendly technique. In the current study, the contaminated soil samples from Hattar Industrial Estate revealed a total lead (Pb) and mercury (Hg) concentration of 170.90 mg L-1 and 26.66 mg L-1, respectively. Indigenous metallotolerant fungal strains including Aspergillus niger M1, Aspergillus fumigatus M3, Aspergillus terreus M6, and Aspergillus flavus M7 were isolated and identified by pheno- and genotyping. A. fumigatus and A. flavus of soil sample S1 showed higher efficiency for Pb removal (99.20% and 99.30%, respectively), in SDB medium. Likewise, A. niger and A. terreus of soil sample S2 showed higher efficiency for Hg removal (96% and 95.50%, respectively), in YPG medium. Furthermore, the maximum uptake efficiency for Pb removal (8.52 mg g-1) from soil sample S1 was noticed for A. fumigatus in YPG medium, while the highest uptake efficiency (4.23 mg g-1) of A. flavus M2 strain was observed with CYE medium. Similarly, the maximum uptake efficiency of 0.41 mg g-1 and 0.44 mg g-1 for Hg removal from soil sample S2 was found for A. niger and A. terreus strains, respectively, in CYE medium. Thus, in order to address the major issue of industrial waste pollution, indigenous fungal strains A. fumigatus (M1) and A. terreus (M7), isolated in this study, could be used (ex situ or in situ) to remediate soils contaminated with Pb and Hg.

Biological and biochemical characteristics of male reproductive system, serum metabolites and carcass quality of Japanese quails by the supplementation of Pinus ponderosa leaves and α-tocopherol acetate.

The aim of the current study was to evaluate the effect of dietary supplementation of Pinus ponderosa leaves (pine leaves) and α-tocopherol acetate (vitamin E) powder on male reproductive system, serum metabolites and carcass characteristics of Japanese quails. A total of 360-day-old male quails were purchased from the open market and kept at poultry shed for ninety-four days. After ten days of adaptation, all quails were randomly assigned into 4 groups, control (IC); supplemented with α-tocopherol acetate (IE) at the rate of 150 mg/L; Pinus ponderosa leaves (IP) at the rate of 150 mg/L; and 70 mg α-tocopherol acetate and 70 mg Pinus ponderosa leaves (IEP). Pinus ponderosa leaves and α-tocopherol acetate supplementation had not significantly (p > .05) effected on final body weight gain, feed intake and feed conversion ratio of quails. The high-density lipoprotein cholesterol (HDLC) and total cholesterol (TC) were significantly (p > .05) affected by IE and IP groups as compared to IC and IEP groups. Triglyceride (TG), glutathione peroxidase (GPx) and superoxide dismutase (SOD) significantly (p < .05) increased in all treatment groups except for the IC group. Aspartate transaminase (AST) significantly (p > .05) decreased in treatment groups as compared to control group. Overall, the mineral levels significantly (p < .05) increased in treatment groups as compared to control. Cloacal gland index values, the quantity of foam production and testis weight were significantly (p < .05) increased in treatment groups. It was concluded that the supplementation of Pinus ponderosa leaves and α-tocopherol acetate improved the testis weight, foam production, serum antioxidant enzymes and mineral level especially zinc in Japanese quail considered an indicative characteristic of higher sperm production rate and improved sexual activity. Further, higher gametogenesis rate, sperm production or reproductive behaviour including different hormonal level will be analysed in future study.

Contributions and Challenges of High Throughput qPCR for Determining Antimicrobial Resistance in the Environment: A Critical Review.

Expansion in whole genome sequencing and subsequent increase in antibiotic resistance targets have paved the way of high throughput qPCR (HT-qPCR) for analyzing hundreds of antimicrobial resistance genes (ARGs) in a single run. A meta-analysis of 51 selected studies is performed to evaluate ARGs abundance trends over the last 7 years. WaferGenTM SmartChip is found to be the most widely used HT-qPCR platform among others for evaluating ARGs. Up till now around 1000 environmental samples (excluding biological replicates) from different parts of the world have been analyzed on HT-qPCR. Calculated detection frequency and normalized ARGs abundance (ARGs/16S rRNA gene) reported in gut microbiome studies have shown a trend of low ARGs as compared to other environmental matrices. Disparities in the HT-qPCR data analysis which are causing difficulties to researchers in precise interpretation of results have been highlighted and a possible way forward for resolving them is also suggested. The potential of other amplification technologies and point of care or field deployable devices for analyzing ARGs have also been discussed in the review. Our review has focused on updated information regarding the role, current status and future perspectives of HT-qPCR in the field of antimicrobial resistance.

Characterization of cross-linked amyloglucosidase aggregates from Aspergillus fumigatus KIBGE-IB33 for continuous production of glucose.

Current research deals with immobilization of amyloglucosidase through carrier-free approach using cross-linking strategy. Cross-linked amyloglucosidase aggregates (CLAAs) with aggregation yield of 94% were prepared in 04 h by incorporating 40% ammonium sulfate and 1.5% glutaraldehyde in enzyme solution. CLAAs were characterized by optimizing various conditions including reaction time, pH, temperature and substrate concentration. It was noticed that after cross-linking no change in optimum reaction time and substrate concentration was observed however, a 5-degree shift in optimum temperature from 60 °C to 65 °C was obtained as compared to soluble amyloglucosidase. Activation energy (Ea) of amyloglucosidase as calculated from Arrhenius plot was 5.5 kcal mol-1 and 5.2 kcal mol-1 for soluble and cross-linked aggregates, respectively. Stability studies revealed that CLAAs can be used at higher temperatures for longer time period than soluble amyloglucosidase. Furthermore, data of recycling studies showed that CLAAs can be efficiently reused for 20 cycles with the retention of 63% of its initial activity. Due to the continuous reusability of CLAAs, the product formation is also increased 8 times from 5.71 mg ml-1 (soluble enzyme) to 46.548 mg ml-1 (CLAAs). Findings of this research show that carrier-free strategy is more effective for continuous hydrolysis of starch and production of glucose.

Age-related variations in the in vitro bactericidal activity of human sera against Pseudomonas aeruginosa.

The human serum is a vital component of the innate immunity of the host that acts as the first line of defence against invading pathogens. A key player in serum-mediated innate immune defence is a system of more than 35 proteins, collectively named as the complement system. After exposure of the pathogen, these proteins are activated in a cascade manner, ultimately forming a membrane attack complex (MAC) on the surface of the pathogen that directly lyses the bacterial cell. Formation of the MAC can be demonstrated in vitro by using serum bactericidal assay (SBA) that works in the absence of cellular components of blood after incubating the serum along with bacteria. Here, we describe the age-related differences in the bactericidal activity of human serum against Pseudomonas aeruginosa, an opportunistic human pathogen causing an array of hospital and community-acquired infections. We demonstrate that adult sera were highly effective in the in vitro killing of Pseudomonas aeruginosa as compared to children and the elderly (p < 0.0001). Sera from children were seriously compromised in the killing P. aeruginosa, whereas elderly sera showed a reduced level of killing. Data revealed a positive correlation between age and serum-killing with higher coefficient of determination values of 0.34, 0.27, and 0.58 and p values of < 0.0001, < 0.001, and < 0.0001, respectively, after 60, 90, and 120 minutes of incubation. Hence, our study highlights the age-related difference in the bactericidal activity of human sera. We conclude that sera of children are totally compromised, whereas elderly sera are only partially compromised, in the killing of P. aeruginosa.

Isolation, identification, and pathological effects of beach sand bacterial extract on human skin keratinocytes in vitro.

BACKGROUND: Beaches are recreational spots for people. However, beach sand contains harmful microbes that affect human health, and there are no established methods for either sampling and identifying beach-borne pathogens or managing the quality of beach sand. METHOD: This study was conducted with the aim of improving human safety at beaches and augmenting the quality of the beach experience. Beach sand was used as a resource to isolate bacteria due to its distinctive features and the biodiversity of the beach sand biota. A selected bacterial isolate termed FSRS was identified as Pseudomonas stutzeri using 16S rRNA sequencing and phylogenetic analysis, and the sequence was deposited in the NCBI GenBank database under the accession number MF599548. The isolated P. stutzeri bacterium was cultured in Luria-Bertani growth medium, and a crude extract was prepared using ethyl acetate to examine the potential pathogenic effect of P. stutzeri on human skin. A human skin keratinocyte cell line (HaCaT) was used to assess cell adhesion, cell viability, and cell proliferation using a morphological analysis and a WST-1 assay. RESULT: The crude P. stutzeri extract inhibited cell adhesion and decreased cell viability in HaCaT cells. We concluded that the crude extract of P. stutzeri FSRS had a strong pathological effect on human skin cells. DISCUSSION: Beach visitors frequently get skin infections, but the exact cause of the infections is yet to be determined. The beach sand bacterium P. stutzeri may, therefore, be responsible for some of the dermatological problems experienced by people visiting the beach.

Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.

In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain.

Investigation into the Antigenic Properties and Contributions to Growth in Blood of the Meningococcal Haemoglobin Receptors, HpuAB and HmbR.

Acquisition of iron from host complexes is mediated by four surface-located receptors of Neisseria meningitidis. The HmbR protein and heterodimeric HpuAB complex bind to haemoglobin whilst TbpBA and LbpBA bind iron-loaded transferrin and lactoferrin complexes, respectively. The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease. Both these receptors are subject to phase variation and 70-90% of disease isolates have one or both of these receptors in an ON expression state. The surface-expression, ubiquity and association with disease indicate that these receptors could be potential virulence factors and vaccine targets. To test for a requirement during disease, an hmbR deletion mutant was constructed in a strain (MC58) lacking HpuAB and in both a wild-type and TbpBA deletion background. The hmbR mutant exhibited an identical growth pattern to wild-type in whole blood from healthy human donors whereas growth of the tbpBA mutant was impaired. These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood. To examine immune responses, polyclonal antisera were raised against His-tagged purified-recombinant variants of HmbR, HpuA and HpuB in mice using monolipopolysaccharide as an adjuvant. Additionally, monoclonal antibodies were raised against outer membrane loops of HmbR presented on the surface of EspA, an E. coli fimbrial protein. All antisera exhibited specific reactivity in Western blots but HmbR and HpuA polyclonal sera were reactive against intact meningococcal cells. None of the sera exhibited bactericidal activity against iron-induced wild-type meningococci. These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

Phase variation of PorA, a major outer membrane protein, mediates escape of bactericidal antibodies by Neisseria meningitidis.

Several outer membrane proteins of Neisseria meningitidis are subject to phase variation due to alterations in simple sequence repeat tracts. The PorA protein is a major outer membrane protein and a target for protective host immune responses. Phase variation of PorA is mediated by a poly-G repeat tract present within the promoter, leading to alterations in protein expression levels. N. meningitidis strain 8047 was subjected to serial passage in the presence of P1.2, a PorA-specific bactericidal monoclonal antibody. Rapid development of resistance to bactericidal activity was associated with a switch in the PorA repeat tract from 11G to 10G. Phase variants with a 10G repeat tract exhibited a 2-fold reduction in surface expression of PorA protein. A mutS mutant of strain 8047, with an elevated phase variation rate, exhibited a higher rate of escape and an association of escape with 10G and 9G variants, the latter having a 13-fold reduction in surface expression of PorA. We conclude that graduated reductions in the surface expression of outer membrane proteins mediated by phase variation enable meningococci to escape killing in vitro by bactericidal antibodies. These findings indicate how phase variation could have a major impact on immune escape and host persistence of meningococci.

A PCR-RFLP based protocol for the detection of hepatitis B virus variants in some lamivudine-untreated chronic hepatitis B virus carriers in Pakistan.

Hepatitis B virus (HBV) affects more than 350 million people worldwide and is a leading cause of morbidity and mortality in developing countries like Pakistan. Lamivudine has potential to inhibit hepatitis B virus (HBV) replication but long term lamivudine treatment results in mutations in YMDD region of HBV, making this therapy ineffective. In this study, we have optimized a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based protocol to detect two mutations in HBV DNA polymerase gene (at codon 528 and 552) in chronic hepatitis patients, without any prior lamivudine treatment. HBV genome was extracted and tested by PCR-RFLP for detection of mutations in polymerase gene. Variations in HBV genome were not detected in enrolled patients confirming that lamivudine can be used to treat chronic Hepatitis B in these patients. Several studies have reported the natural occurrence of mutation in YMDD motif of polymerase gene in chronic hepatitis B patients, not treated with lamivudine, but these mutants were not detected in Pakistani lamivudine-untreated chronic hepatitis B patients.

Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence.

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin-haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains.