Intraspecific variation in immune gene expression and heritable symbiont density.

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These 'biotypes' have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system's complex dual role in interacting with both beneficial and harmful microbes.

Effect of different cleaning procedures on water use and bacterial levels in weaner pig pens.

Pork is one of the most globally eaten meats and the pig production chain contributes significantly to the water footprint of livestock production. However, very little knowledge is available about the on-farm factors that influence freshwater use in the pig production chain. An experiment was conducted to quantify the effect of three different washing treatments on freshwater use, bacterial levels [(total bacterial counts; TBC), Enterobacteriaceae and Staphylococcus] and cleaning time in washing of pens for weaning pigs. Three weaner rooms were selected with each room having 10 pens and a capacity to hold up to 14 pigs each. Pigs were weaned and kept in the pens for 7 weeks. Finally, the pens were cleaned before the next batch of pigs moved in. The washing treatments used were power washing and disinfection (WASH); presoaking followed by power washing and disinfection (SOAK), and presoaking followed by detergent, power washing and disinfection (SOAK + DETER). A water meter was used to collect water use data and swab samples were taken to determine the bacterial levels. The results showed that there was no overall effect of washing treatments on water use. However, there was an effect of treatment on the washing time (p<0.01) with SOAK and SOAK+DETER reducing the washing time per pen by 2.3 minutes (14%) and 4.2 minutes (27%) compared to WASH. Nonetheless, there was an effect of sampling time (before or after washing) (p<0.001) on the levels of TBC and Staphylococcus, but no effect was seen on Enterobacteriaceae levels. Thus, the washing treatments used in this study had no effect on the water use of the pork production chain. Although there was no difference in both water use and bacterial load, from a producer perspective, presoaking and detergent use can save time and labour costs, so this would be the preferred option.

Correlation Between the Clinical Severity, Bacterial Load, and Inflammatory Reaction in Children with Mycoplasma Pneumoniae Pneumonia.

Given the lack of defining features in the clinical manifestations and radiographic findings for children with mycoplasma pneumoniae pneumonia (MPP), quantitative polymerase chain reaction (qPCR) has become a useful diagnostic method. This study was performed to explore the relationship between the qPCR findings, clinical symptoms, and inflammatory markers in children with MPP. Four hundred children with MPP have been enrolled in this retrospective analysis. All clinical and analytical information, including mycoplasma pneumoniae (MP) PCR results, has been collected. Based on the PCR results, the patients were divided into groups with load values (copy number) < 105 (54 cases), ≥105 and <106 (71 cases), ≥106 and <107 (112 cases), ≥107 and ≤108 (114 cases), and >108 (49 cases). The clinical features (including symptoms and signs) and inflammatory indicators were compared among the groups. The incidence of high fever (above 39°C), thermal peak during the entire hospitalization period, fever duration, days of hospitalization, and plasma lactate dehydrogenase (LDH) levels were statistically correlated with the MP PCR load value in children with MPP. The analysis of relevance degree showed the correlative order as a thermal peak of hospitalization > duration of fever > period of hospitalization > LDH value > C-reactive protein value. The host immune response was significantly greater in the complication group than in the non-complication group.

Robust estimation of bacterial cell count from optical density.

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals  <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

The clinical significance of IL-6 s and IL-27 s in Bronchoalveolar lavage fluids from children with mycoplasma pneumoniae pneumonia.

BACKGROUND: IL-6 was associated with the severity of mycoplasma pneumoniae pneumonia (MPP). But the relationship between IL-27 and MPP was unknown. METHODS: Ninety-eight patients with MPP < 14 years old were enrolled in this study and divided into groups by severity (mild cases and severe cases), infection types (MP single infection group and MP mixed infection group) and DNA loads (low MP DNA loads group and high MP DNA loads group), respectively. Fifteen children with foreign bodies in bronchus were also enrolled as control. IL-6 s and IL-27 s in bronchoalveolar lavage fluids (BALFs) from these children were measured by ELISA. RESULTS: There were significant differences in IL-6 s of BALFs from patients between mild cases and severe cases, MP single infection group and MP mixed infection group, and low MP DNA loads group and high MP DNA loads group, respectively (P < 0.05). Compared with IL-6 s of BALFs from control, IL-6 s in BALFs from the 6 patient groups were significantly higher (P < 0.05). IL-27 s in BALFs from MP mixed infection group were significantly lower than those from MP single infection group and control (P < 0.05) respectively. CONCLUSION: IL-6 was firmly associated with MPP and had potential application in clinical practice while IL-27 was not related to MP infection.

A hydrosilver gel for plaque control in adults affected by chronic periodontitis: Effects on the 'red complex' bacterial load. A prospective longitudinal pilot study using polymerase chain reaction analysis.

In subjects affected by chronic periodontitis, the chemical control of plaque is a strategy aiming primarily at controlling infection and bacterial loading. The aim is to evaluate the bacterial loading of the so-called 'red complex' associated with a short-term use of a hydrosilver gel (HSG) by using an in vivo model in adult subjects affected by chronic periodontitis. This prospective short-term clinical trial involved 10 adult volunteers using a 15-day in vivo model. After receiving professional prophylaxis at baseline (t0), each volunteer performed daily applications of HSG at home. After 15 days (t1) from the first application, subgingival plaque samples were collected, and the bacterial loading of species belonging to the red complex was evaluated using polymerase chain reaction (PCR) analyses. The bacterial loading of the red complex showed no statistically significant difference between t0 and t1, although it tended to decrease. HSG can be used at home as an adjunct to domestic oral care because it seems a promising tool, but further studies are needed to involve a larger sample and a longer follow-up.

Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of "Candidatus Liberibacter asiaticus" in planta.

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide.

Carious status and supragingival plaque microbiota in hemodialysis patients.

OBJECTIVE: The aim of this study was to evaluate the carious status and the microbial profiles of supragingival plaque in patients with chronic kidney disease undergoing hemodialysis. METHODS: This study included 30 patients with chronic kidney disease undergoing hemodialysis as well as 30 control subjects. Dental examination was performed and the decayed-missing-filled-teeth was recorded. Supragingival plaque was taken and analyzed using 16S rRNA gene amplicon by Illumina MiSeq sequencing to detect microbial composition and community diversity and structure. RESULTS: The level of decayed-missing-filled-teeth was higher in the hemodialysis group than that in the control group. Microbial analysis showed a decrease in α diversity and a increase in relative abundance and prevalence of many acidogenic and aciduric caries related species in the supragingival plaque samples of the hemodialysis patients, including Streptococcus mutans, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus vaginalis, Scardovia wiggsiae F0424, and Actinomyces naeslundii. CONCLUSION: Our results suggested that the hemodialysis patients were more susceptible to caries. More attentions for caries prevention and treatment should be paid to improve their life quality, and even to reduce their cardiovascular events and survival.

Macrophage-derived LTB4 promotes abscess formation and clearance of Staphylococcus aureus skin infection in mice.

The early events that shape the innate immune response to restrain pathogens during skin infections remain elusive. Methicillin-resistant Staphylococcus aureus (MRSA) infection engages phagocyte chemotaxis, abscess formation, and microbial clearance. Upon infection, neutrophils and monocytes find a gradient of chemoattractants that influence both phagocyte direction and microbial clearance. The bioactive lipid leukotriene B4 (LTB4) is quickly (seconds to minutes) produced by 5-lipoxygenase (5-LO) and signals through the G protein-coupled receptors LTB4R1 (BLT1) or BLT2 in phagocytes and structural cells. Although it is known that LTB4 enhances antimicrobial effector functions in vitro, whether prompt LTB4 production is required for bacterial clearance and development of an inflammatory milieu necessary for abscess formation to restrain pathogen dissemination is unknown. We found that LTB4 is produced in areas near the abscess and BLT1 deficient mice are unable to form an abscess, elicit neutrophil chemotaxis, generation of neutrophil and monocyte chemokines, as well as reactive oxygen species-dependent bacterial clearance. We also found that an ointment containing LTB4 synergizes with antibiotics to eliminate MRSA potently. Here, we uncovered a heretofore unknown role of macrophage-derived LTB4 in orchestrating the chemoattractant gradient required for abscess formation, while amplifying antimicrobial effector functions.

Neutral barcoding of genomes reveals the dynamics of Salmonella colonization in cattle and their peripheral lymph nodes.

Feedlot cattle often contain Salmonella. The number of bacteria that initiate colonization of different cattle organs and the bacterial migration within these large animals are poorly understood. To investigate these questions, we constructed wild-type isogenic tagged strains (WITS) of Salmonella by inserting 21-base barcodes flanked by Illumina sequencing primers into a neutral genome location. We then delivered several different pools of uniquely barcoded clones orally and into multiple intradermal sites, in individual Holstein steers, and subsequently performed Salmonella-directed sequence tag-based analysis of microbial populations (STAMP). Using high-throughput sequencing of the barcodes of Salmonella grown from steer lymph nodes, organs and feces, we monitored how individual barcoded clones travel from different entry sites within animals. Data showed that gastrointestinal colonization was established by up to hundreds of Salmonella founder cells, whereas peripheral lymph nodes were usually colonized by very low numbers of founding bacteria, often originating from the nearest draining intradermal delivery site. Transmission of Salmonella from the gastrointestinal tract to the lymphatic system was frequently observed, whereas entry of intradermally delivered bacteria into the gut was rare. Bacteria undergo limited extraintestinal proliferation within or prior to arrival at peripheral lymph nodes. Overall, the application of the STAMP technique facilitated characterization of the migration routes and founder population size of Salmonella within feedlot cattle and their organs and lymph nodes in unprecedented detail.

Infection Density Dynamics and Phylogeny of Wolbachia Associated with Coconut Hispine Beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), by Multilocus Sequence Type (MLST) Genotyping.

The intracellular bacterium Wolbachia pipientis is widespread in arthropods. Recently, possibilities of novel Wolbachia-mediated hosts, their distribution, and natural rate have been anticipated, and the coconut leaf beetle Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), which has garnered attention as a serious pest of palms, was subjected to this interrogation. By adopting Wolbachia surface protein (wsp) and multilocus sequence type (MLST) genotypic systems, we determined the Wolbachia infection density within host developmental stages, body parts, and tissues, and the results revealed that all the tested samples of B. longissima were infected with the same Wolbachia strain (wLog), suggesting complete vertical transmission. The MLST profile elucidated two new alleles (ftsZ-234 and coxA-266) that define a new sequence type (ST-483), which indicates the particular genotypic association of B. longissima and Wolbachia. The quantitative real-time PCR analysis revealed a higher infection density in the eggs and adult stage, followed by the abdomen and reproductive tissues, respectively. However, no significant differences were observed in the infection density between sexes. Moreover, the wsp and concatenated MLST alignment analysis of this study with other known Wolbachia-mediated arthropods revealed similar clustering with distinct monophyletic supergroup B. This is the first comprehensive report on the prevalence, infection dynamics, and phylogeny of the Wolbachia endosymbiont in B. longissima, which demonstrated that Wolbachia is ubiquitous across all developmental stages and distributed in the entire body of B. longissima. Understanding the Wolbachia infection dynamics would provide useful insight to build a framework for future investigations, understand its impacts on host physiology, and exploit it as a potential biocontrol agent.

C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain.

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, ß-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, ß-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca2+-dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain.

Transcriptomic profiles of post-smolt Atlantic salmon challenged with Piscirickettsia salmonis reveal a strategy to evade the adaptive immune response and modify cell-autonomous immunity.

Piscirickettsiosis is the main bacterial disease affecting the Chilean salmon farming industry and is responsible for high economic losses. The development of effective strategies to control piscirickettsiosis has been limited in part by insufficient knowledge of the host response. The aim of this study was to use RNA sequencing to describe the transcriptional profiles of the responses of post-smolt Atlantic salmon infected with LF-89-like or EM-90-like Piscirickettsia salmonis. Enrichment and pathway analyses of the differentially expressed genes revealed several central signatures following infection, including positive regulation of DC-SIGN and TLR5 signalling, which converged at the NF-κB level to modulate the pro-inflammatory cytokine response, particularly in the PS-EM-90-infected fish. P. salmonis induced an IFN-inducible response (e.g., IRF-1 and GBP-1) but inhibited the humoral and cell-mediated immune responses. P. salmonis induced significant cytoskeletal reorganization but decreased lysosomal protease activity and caused the degradation of proteins associated with cellular stress. Infection with these isolates also delayed protein transport, antigen processing, vesicle trafficking and autophagy. Both P. salmonis isolates promoted cell survival and proliferation and inhibited apoptosis. Both groups of Trojan fish used similar pathways to modulate the immune response at 5 dpi, but the transcriptomic profiles in the head kidneys of the cohabitant fish infected with PS-LF-89 and PS-MS-90 were relatively different at day 35 post-infection of the Trojan fish, probably due to the different degree of pathogenicity of each isolate. Our study showed the most important biological mechanisms used by P. salmonis, regardless of the isolate, to evade the immune response, maintain the viability of host cells and increase intracellular replication and persistence at the infection site. These results improve the understanding of the mechanisms by which P. salmonis interacts with its host and may serve as a basis for the development of effective strategies for the control of piscirickettsiosis.

Silkworm model for Francisella novicida infection.

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida, which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm (Bombyx mori) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida. We found that silkworms died within 3-7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM+ and IgM++ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT+ and/or IgT++ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT+ B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT+ B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM+ B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT+ B lineage cells may have a protective function in the immune response to Fp.

Effect of cadexomer iodine on the microbial load and diversity of chronic non-healing diabetic foot ulcers complicated by biofilm in vivo.

Objectives: The performance of cadexomer iodine was determined against microbial populations from chronic non-healing diabetic foot ulcers (DFUs) complicated by biofilm in vivo , using molecular, microscopy and zymography methods. Methods: Chronic non-healing DFUs due to suspected biofilm involvement were eligible for enrolment. DNA sequencing and real-time quantitative PCR was used to determine the microbial load and diversity of tissue punch biopsies obtained pre- and post-treatment. Scanning electron microscopy and/or fluorescence in situ hybridization confirmed the presence or absence of biofilm. Zymography was used to determine levels of wound proteases. Results: Seventeen participants were recruited over a 6 month period. Scanning electron microscopy and or fluorescence in situ hybridization confirmed the presence of biofilm in all samples. Eleven participants exhibited log 10 reductions in microbial load after treatment (range 1-2 log 10 ) in comparison with six patients who experienced <1 log 10 reduction ( P = 0.04). Samples were tested for levels of wound proteases pre- and post-treatment. Reductions in the microbial load correlated to reductions in wound proteases pre- and post-treatment ( P = 0.03). Conclusions: To the best of our knowledge, this study represents the first in vivo evidence, employing a range of molecular and microscopy techniques, of the ability of cadexomer iodine to reduce the microbial load of chronic non-healing DFUs complicated by biofilm. Further analyses correlating log reductions to optimal duration of therapy and improvements in clinical parameters of wound healing in a larger cohort are required.

miR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila.

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. We found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Finally, we provide evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Azithromycin 1.5g Over 5 Days Compared to 1g Single Dose in Urethral Mycoplasma genitalium: Impact on Treatment Outcome and Resistance.

BACKGROUND: We evaluated the impact of extended azithromycin (1.5g over 5 days) on selection of macrolide resistance and microbiological cure in men with Mycoplasma genitalium urethritis during 2013-2015 and compared this to cases treated with azithromycin 1g in 2012-2013. METHODS: Microbiological cure was determined for men with M. genitalium urethritis treated with azithromycin 1.5g using quantitative polymerase chain reaction specific for M. genitalium DNA on samples 14-100 days post-treatment. Pre- and post-treatment macrolide resistance mutations were detected by sequencing the 23 S gene. RESULTS: There was no difference in proportions with microbiological cure between azithromycin 1.5g and 1g: 62/106 (58%; 95% confidence interval [CI], 49%, 68%) and 56/107 (52%; 95%CI 42-62%), P = .34, respectively. Also, there was no difference in the proportion of wild-type 23 S rRNA (presumed macrolide sensitive) infections cured after 1.5g and azithromycin 1g: 28/34 (82%; 95%CI 65-92%) and 49/60 (82%; 95%CI 70-90%), P=1.0, respectively. There was no difference between 1.5g and 1g in the proportions of wild-type infections with post-treatment resistance mutations: 4/34 (12%; 95%CI 3-27%) and 11/60 (18%; 95%CI 10-30%), respectively, P = .40. Pre-treatment resistance was present in 51/98 (52%; 95%CI 42-62%) cases in 2013-2015 compared to 47/107 (44%; 95%CI 34-54%) in 2012-2013, P = .25. CONCLUSIONS: Extended azithromycin 1.5g was no more effective than a single 1g dose at achieving cure of M. genitalium urethritis and importantly did not reduce the selection of macrolide resistance. Nonmacrolide and new approaches for the treatment of M. genitalium urethritis are required.

Changes in Host Cytokine Patterns of TB Patients with Different Bacterial Loads Detected Using 16S rRNA Analysis.

BACKGROUND: Tuberculosis (TB) has overtaken HIV as the biggest infectious disease killer, with the majority of deaths occurring in sub-Saharan Africa. However it is unknown how differences in bacterial load alter host immune profiles in the sputum and blood of TB patients. METHODS: 16S ribosomal RNA analysis was used to determine bacterial load in sputum samples obtained from 173 patients with active TB (57 pre-treatment and 116 post-treatment). Host analyte concentrations in sputum and Mycobacterium tuberculosis (Mtb) antigen stimulated whole blood assay supernatants were analysed using multiplex cytokine arrays. RESULTS: Multiple logistic regression adjusting for age, sex and HIV status showed highly significant correlation of bacterial load with IL1ß, IL2, IL1RA, IL4, IL6, IL8, IL9, IL15, IL17, EOTAX, FGF, IFN-γ, GCSF, MCP1, M1P1α, M1P1ß, PDGF, TNFα, VEGF in sputum. With increasing time on treatment, FGF levels in sputum displayed the most significant inverse correlation with reduction in bacterial load. CONCLUSIONS: We show that differences in bacterial load correlates with changes in several host biomarkers. These findings have implications for development of tests for TB diagnosis and treatment response.

Measurement of bacterial replication rates in microbial communities.

Culture-independent microbiome studies have increased our understanding of the complexity and metabolic potential of microbial communities. However, to understand the contribution of individual microbiome members to community functions, it is important to determine which bacteria are actively replicating. We developed an algorithm, iRep, that uses draft-quality genome sequences and single time-point metagenome sequencing to infer microbial population replication rates. The algorithm calculates an index of replication (iRep) based on the sequencing coverage trend that results from bi-directional genome replication from a single origin of replication. We apply this method to show that microbial replication rates increase after antibiotic administration in human infants. We also show that uncultivated, groundwater-associated, Candidate Phyla Radiation bacteria only rarely replicate quickly in subsurface communities undergoing substantial changes in geochemistry. Our method can be applied to any genome-resolved microbiome study to track organism responses to varying conditions, identify actively growing populations and measure replication rates for use in modeling studies.