16S rRNA Gene PCR Research Articles

Best Practices for the Analytic Phase of Anaerobic Bacteriology

The analytic process for identification of anaerobic bacteria involves direct specimen Gram stains and workup of growth in culture. Direct Gram stains help to develop the initial differential, aiding in guiding both the extent of laboratory workup and clinician decision making for patient management. Workup of anaerobic growth should involve an aerotolerance test to assess aerobic growth and to compare growth rates in anaerobic versus aerobic environments. While matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has become a routine means of anaerobe identification for many clinical laboratories, spot biochemical tests, such as indole and catalase, should be available in every laboratory that identifies anaerobes. If MALDI-TOF MS and biochemical methods are unsuccessful at providing an identification, 16S rRNA gene PCR and/or sequencing may be performed if the information impacts clinical care. The extent of culture workup to the level of presumptive identification versus definitive identification depends upon several factors, including the specimen source, direct Gram stain results, and the presence or absence of mixed microbiota. In addition to discussing the current best practices for anaerobic culture workup, commonly encountered anaerobic organism Gram stains and culture growth are detailed.

Detection of 16S rRNA gene for rapid identification of bacterial pathogens causing peritonitis in patients on continuous ambulatory peritoneal dialysis

PurposePeritonitis is the most important complication with high rate of morbidity and mortality in patients on continuous ambulatory peritoneal dialysis (CAPD) despite the success and advances. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early targeted treatment. The aim of the study was to evaluate the role of 16S rRNA gene and ITS region PCR and sequencing in detecting bacterial and fungal pathogens from the dialysate of patients undergoing CAPD. MethodsFifty eight peritoneal dialysate from suspected cases of peritonitis on CAPD were subjected to conventional culture as per the ISPD guidelines and automated culture system. A conventional PCR was performed to detect the 16S rRNA gene and ITS region. Sequencing and analysis were performed to identify the etiological agent from the remaining dialysate. ResultsAmong the 58 dialysate fluid, the etiological agents were identified in 8(14%) samples by conventional culture, 28(48%) by automated culture and 47(81%) by 16S rRNA sequencing and analysis. In 8 samples there was discordance in the results of the culture and 16S rRNA PCR. BLAST search of nine sequences obtained from 16S rRNA PCR revealed that these sequences matched best with uncultured bacterial clones. In eleven samples the sequence failed. ConclusionThe molecular tool 16S rRNA gene and ITS region PCR and sequencing cannot be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in GenBank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries.

The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations’ dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents.

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis.

Gut dysbiosis induces 'leaky gut,' a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium which causes periodontal tissue breakdown, and often enters the systemic blood flow. Oral administration of P. gingivalis induced gut dysbiosis in mice model, but no systemic administration of P. gingivalis has been reported thus far. In the present study, we investigated the effect of P. gingivalis-derived lipopolysaccharide (Pg-LPS) on the intestinal flora of our established mouse model. Eight-week-old C57BL/6J mice were intraperitoneally administered Pg-LPS. Three months later, DNA was extracted from stool, and RNA from the small and large intestines. After euthanizing the mice, pathological sections of the intestinal tract were prepared and stained with hematoxylin and eosin (H&E). Tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 expression levels were evaluated using quantitative PCR. 16S rRNA gene PCR amplicon analysis data were acquired using NGS. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. Furthermore, alterations in microbial function were performed by PICRUSt2. No significant inflammatory changes were observed in the H&E. No significant differences in the mRNA levels of IL-1β, IL-6, and TNF-α were observed between the groups. Pg-LPS administration decreased the abundance of Allobacterium in the gut. A predictive metagenomic analysis by PICRUSt2 and STAMP showed that 47 pathways increased and 17 pathways decreased after Pg-LPS administration. Systemic application of periodontal pathogens may cause changes in the intestinal flora which may affect the physiological functions of the intestinal tract.

Diverse Bathyarchaeotal Lineages Dominate Archaeal Communities in the Acidic Dajiuhu Peatland, Central China.

Bathyarchaeota are believed to have roles in the carbon cycle in marine systems. However, the ecological knowledge of Bathyarchaeota is limited in peatland ecosystems. Here, we investigated the vertical distribution of Bathyarchaeota community structure using quantitative PCR and high-throughput sequencing technology of ribosomal 16S rRNA gene integrated with detailed chemical profiling in the Dajiuhu Peatland, central China. Eight archaeal phyla were observed in peat samples, which mainly composed of Bathyarchaeota with a mean relative abundance about 88%, followed by Thaumarchaeota (9%). Bathyarchaeota were further split into 17 subgroups, and some subgroups showed habitat specificity to peat horizons with distinct lithological and physicochemical properties, for example, Bathy-6 and Bathy-15 had preference for the acrotelm, Bathy-5b, Bathy-16, and Bathy-19 were enriched in the catotelm, Bathy-5a, Bathy-8, and Bathy-11 were specific for the clay horizon. This spatial distribution pattern of archaeal communities along peat profile was mainly influenced by water content as indicated by RDA ordination and permutational MANOVA, whereas organic matter content exclusively affected Bathyarchaeota distribution along the peat profile significantly. The abundance of archaeal 16S rRNA genes ranged from 105 to 107 copies per gram dry sediment, and the highest archaeal biomass was observed in the periodically oxic mesotelm horizon with more dynamic archaeal interaction relationship as indicated by the network analysis. Bathyarchaeota dominated the archaeal interaction network with 82% nodes, 96% edges, and 71% keystone species. Our results provide an overview of the archaeal population, community structure, and relationship with environmental factors that affect the vertical distribution of archaeal communities and emphasize the ecology of bathyarchaeotal lineages in terrestrial peatland ecosystems.

Identification of enterotoxigenic Bacteroides fragilis in patients with diarrhea: A study targeting 16S rRNA, gyrB and nanH genes

ObjectivesWe aimed to identify the enterotoxigenic Bacteroides fragilis (ETBF) and bft subtypes among patients with diarrhea. In addition, we assessed whether DNA gyrase subunit B (gyrB) and neuraminidase (nanH) genes are useful determinants for identification of B. fragilis compared to 16S rRNA sequencing as a reference method. MethodsThe 530 fecal specimens were cultured on BBE agar. The colonies which supposed to be a member of B. fragilis group were subjected to 16S rRNA gene sequencing and PCR assays targeting the Bacteroides fragilis group (BFG), gyrB and nanH. The B. fragilis toxin (bft) gene and its subtype was detected by PCR. The specificity of PCR assays was calculated considering the 16S rRNA gene sequencing as the reference method. ResultsA total of 111 Gram-negative anaerobic coccobacilli were isolated from 530 fecal specimens using BBE agar. Of the 111 isolates, 100 (90.09%) were assumed to be a member of Bacteroides fragilis group as they yielded an amplicon through PCR using the group-specific primers (Bfra-F/g-Bfra-R). However, only 28 isolates out of 100 were definitively identified as species of Bacteroides using16S rRNA gene sequencing; of which 15 isolates were B. fragilis and the remaining 13 isolates were identified as B. thetaiotaomicron (n = 6), Parabacteroides distasonis (n = 3), B. vulgatus (Phocaeicola vulgatus) (n = 1), B. ovatus (n = 1), B. congonensis (n = 1) and B. nordii (n = 1). Among the 15 isolates of B. fragilis, 4 were found to be ETBF. Compared to the reference method, the specificity and accuracy of the PCR targeting gyrB gene (64.7% and 65%) was higher than of nanH (36.4% and 46%, respectively. ConclusionsThis study demonstrated that more than one-fourth of B. fragilis isolates harbored bft gene and less than 1% of patients with diarrhea harbored ETBF. The slight agreement between the PCR assays -already used for identification of B. fragilis which targeting gyrB or nanH - and 16S rRNA gene sequencing as the reference method was noted.

Bioleaching of copper from chalcopyrite ore at higher NaCl concentrations

The formation of hydrolyzed ferric iron (Fe (III)) as jarosite could limit the bioleaching of copper and significantly lower the dissolution rate of low-grade chalcopyrite ore. Thus, to improve the rate through overcoming the hindered dissolution of chalcopyrite, this study investigated the influence of chloride addition into leaching lixiviant. As a microbial source, mixed mesophiles, moderately thermophilic, and thermophilic microorganisms were adapted to about 120 mM NaCl solution. After optimization of the ore bioleaching in 64 controlled mini-columns, the scaled tests in 2-m tall columns were conducted to study the overall leaching procedure and precipitation of the jarosite. Under normal aeration conditions (0.05 Nm3/m2/h) with hybrid irrigation (120 mM NaCl in H2SO4 solution at pH = 1.5), copper recovery reached 80% after 120 days while it was approximately 50% with limited one (0.003 Nm3/m2/h). The limited aeration column had a moderate oxidation–reduction potential (ORP) with values < 450 mV. This significant improvement in dissolution is explained by the reduced precipitation of passivating layer (mainly jarosite). These conclusions were also confirmed by the estimation of pyrite and natrojarosite with a modified ASTM D-2492 (MASTM D-2492). Moreover, to identify the microbial communities in the columns, DNA extraction and 16Sr RNA gene PCR amplification and sequencing were done.

Seasonal and multi-annual variation in the abundance of isoprenoid GDGT membrane lipids and their producers in the water column of a meromictic equatorial crater lake (Lake Chala, East Africa)

Isoprenoid glycerol dialkyl glycerol tetraethers (isoGDGTs) are membrane lipids of Archaea. Organic biomarker proxies associated with these lipids, such as the TEX86 paleothermometer and Branched and Isoprenoid Tetraether (BIT) index, are often used in paleoenvironmental reconstructions for the marine environment, but their general applicability in lacustrine settings is hampered by limited understanding of the biological sources and environmental drivers influencing isoGDGT production. To validate the use of isoGDGT proxies in lakes, we studied the occurrence of isoGDGTs in Lake Chala, a permanently stratified (meromictic) crater lake in equatorial East Africa. We analyzed the abundance and distribution of isoGDGTs in 17 depth profiles of suspended particulate matter (SPM) collected monthly between September 2013 and January 2015, and compared this with the abundance and composition of archaea based on 16S rRNA gene and quantitative PCR analysis. Both isoGDGTs and archaeal abundance in the SPM were exceptionally low throughout the study period. In the oxygenated part of the water column, higher fractional abundances of crenarchaeol are matched by a predominance of the ammonia-oxidizing Thaumarchaeota I.1b that are known to produce this GDGT, whereas deep anoxic water layers are characterized by high fractional abundances of GDGT-0 as well as the anaerobic heterotrophic Group C3 MCG Bathyarchaea and specific euryarchaeotal methanogens. Analysis of intact polar lipid (IPL) isoGDGTs using SPM depth profiles from three months representing distinct seasons during the study period revealed the presence of several IPLs of GDGT-0 in the anoxic lower water column, which are rarely found in natural settings. IPL GDGT-0 with a phosphatidylglycerol (PG-), monohexose-phosphatidylglycerol (MH-PG-) and dihexose-phosphatidylglycerol (DH-PG-) head-group was typically only present just above the lake bottom at 90 m depth and probably reflect specific communities of anaerobic archaea. We also determined the flux and distribution of isoGDGTs in settling particles collected monthly between November 2006 and January 2015 from a sediment trap suspended at 35 m water depth to assess seasonal and inter-annual variability in surface-water isoGDGT production, and compared this with the temporal distribution of isoGDGTs in the 25,000-year long sediment record from Lake Chala. Monthly variation of isoGDGTs in the 98-month settling-particles record did not show a strong annual pattern related to seasonal water-column mixing and stratification, likely because the oxycline was regularly situated below sediment-trap depth. Episodes of high GDGT-0 concentrations relative to crenarchaeol in the settling particles can therefore be linked to periods of exceptionally shallow oxycline depth, which suppresses the thaumarchaeotal bloom. During such intervals, TEX86-based paleotemperatures are not reliable because isoGDGT input from other archaeal sources disproportionally influences TEX86 values and creates a cold-temperature bias. Additionally, the abundance of the crenarchaeol isomer relative to crenarchaeol (f[CREN']) gradually increases during such episodes of high GDGT-0/crenarchaeol ratio, suggesting increasing dominance of Group I.1b over Group I.1a Thaumarchaeota, and might prove a good marker for prolonged shallow-oxycline conditions. Most importantly, the associated near-absence of crenarchaeol during times of strong upper-water-column stratification results in high BIT-index values. We propose that this suppression mechanism may be the principal driver of BIT-index variation in the sediment record of Lake Chala, and the main source of observed congruence between the BIT index and climate-driven lake-level variation on long time scales.

Molecular identification of Acanthamoeba genotypes isolated from oral cavity of heart transplant patients in Iran

Heart transplant is one of the accepted treatments for some patients with advanced heart failure. Of note, transplant surgeries may cause different infections and complications for patients during the post-transplant period. A wide variety of opportunistic organisms caused these infections including bacteria, fungi, viruses, and protozoa particularly Free-living amoebae (FLA). This study aims to study the presence of pathogenic FLA from the oral cavity of post-heart transplant recipients. Throat swabs were collected from 80 patients who underwent post-heart transplant surgery. All swabs were immediately cultured in non-nutrient agar (2%). PCR and sequencing of 18S rRNA gene (DF3 region) of Acanthamoeba isolates were performed using genus-specific primers. Genetic associations among sequenced genotypes inferred by the 18S rRNA gene obtained by MEGA X and a phylogenetic tree were constructed using the maximum likelihood algorithm and Kimura 2-parameter model. Out of 80 samples collected from post-heart transplant patients, six (7.5%) samples showed positive outgrowth of Acanthamoeba based on the page key and sequencing of the DF3 region. Sequence similarity of ASA1 by basic local alignment search tool(n) showed that five isolates (ANHT1, ANHT2, ANHT3, ANHT4, and ANHT5) belonged to Acanthamoeba T5 genotype corresponding to A. lenticulata and one strain (ANHT6) belonged to the T4 genotype. To the best of our knowledge for the first time, a comprehensive study of Acanthamoeba genotypes isolated from throat samples of heart transplant recipients is described. Heart transplantation patients can be colonized by FLA and are therefore at risk of developing an invasive infection. Physicians' awareness of central nervous system infections related to FLAs and preventive and control measures of patients with compromised immune status due to heart transplant surgery are of utmost importance.

Case report of the first cured patient with Mycobacterium Chimaera infection following cardiac valve replacement in the mainland of China

BackgroundMycobacterium chimaera infections subsequent to cardiac surgery are related to contaminated heater-cooler devices, with high mortality. Nevertheless, few studies have been reported in Asia.Case presentationWe described the case of a 55-year-old man with Mycobacterium chimaera infection following cardiac surgery in the mainland of China. He was diagnosed with endocarditis caused by Mycobacterium chimaera subsequent to open heart surgery. Metagenomic next-generation sequencing (mNGS) and 16S rRNA gene PCR analysis were used to identify potential pathogens. The patient underwent redo valve replacement surgery and received combination therapy with azithromycin, ethambutol, linezolid, and amikacin. No signs of relapse were observed during the 11-month follow-up visit.ConclusionsThis is the first documented case of Mycobacterium chimaera infection following cardiac surgery in the mainland of China and the first documented transnational imported case worldwide. Moreover, mNGS is a novel diagnostic technology that can guide antimicrobial therapy prior to obtaining fluid/tissue culture results for Mycobacterium chimaera, providing a new approach for the detection of potential Mycobacterium chimaera infection.

Screening of Acidophilic Actinobacteria That Show Activity against Paddy Pest Fungi

This study aimed to isolate and identify acidophilic actinobacteria. Acidophilic actinobacteria isolates were had from a paddy field soil in Osmancık placed near Çorum province in Turkey. The dilution plate technique on seven selective media with pH 5.5 was used for isolation. 16S rRNA gene PCR amplification of acidophilic actinobacteria was performed. Three different algorithms were used in the phylogenetic analyzes made with MEGA 7.0 software. Twenty-two isolates were obtained from seven selective media, and according to 16S rRNA gene sequence analysis of 22 isolates, twenty-one Streptomyces isolates and one Rhodococcus isolate were identified. The antifungal activities of isolated acidophilic actinobacteria against Fusarium moniliforme and Rhizoctonia solani, the rice pathogenic fungi were evaluated. The isolates with antifungal activity have the potential to be used as biological control agents against rice pathogens.

An Hepatozoon americanum-like protozoan in crab-eating (Cerdocyon thous) and grey pampean (Lycalopex gymnocercus) foxes from Uruguay.

In South America, apicomplexan parasites of the genus Hepatozoon have been sporadically detected in mammals. Previous studies in wild canids from Brazil and Argentina demonstrated infections by species genetically related to Hepatozoon americanum. The aim of the present work was to detect the presence of Hepatozoon in road-killed foxes encountered in Uruguayan highways. Blood samples from 45 crab-eating (Cerdocyon thous) and 32 grey pampean (Lycalopex gymnocercus) foxes were analyzed by PCR for Hepatozoon 18S rRNA gene. Eight foxes (10.4%) were found to be infected with an H. americanum-like protozoan, an Hepatozoon closely related to H. americanum. Bayesian and maximum-likelihood phylogenetic analyses revealed that the sequences obtained in this study cluster with H. americanum from the United States, and with an H. americanum-like species from dog and foxes from Brazil and Argentina. In the Unites States, H. americanum causes severe disease in dogs. In addition to this, an increasing habitat overlap between dogs and foxes makes the presence of H. americanum-like protozoan in foxes acquires veterinary relevance. This work represents the first report of L. gymnocercus infected with an H. americanum-like protozoan, and of wild canids infected with Hepatozoon in Uruguay.

Influence of Environmental Factors on the Variability of Archaeal Communities in a Karst Wetland.

Archaea are ubiquitous and play an important role in elemental cycles in Earth’s biosphere; but little is known about their diversity, distribution, abundance, and impact in karst environments. The present study investigated the effect of environmental factors on the variability of archaeal communities in the sediment of the Huixian karst wetland, the largest karst wetland in South China. Sediment cores were obtained from four sampling sites with different water depths and macrophyte inhabitants in both the winter of 2016 and the summer of 2018. The community analysis was based on PacBio sequencing and quantitative PCR of the archaeal 16S rRNA gene. The results showed that Euryarchaeota (57.4%) and Bathyarchaeota (38.7%) were dominant in all the samples. Methanogenic Methanosarcinales (25.1%) and Methanomicrobiales (13.7%), and methanotrophic archaea ANME-2d (9.0%) were the dominant Euryarchaeota; MCG-11 (16.5%), MCG-6 (9.1%), and MCG-5b (5.5%) were the dominant Bathyarchaeota. The community composition remained stable between summer and winter, and the vertical distributions of the archaeal phyla conformed to two patterns among the four sampling sites. In the winter samples, the archaeal 16S rRNA gene abundance was approximately 1.0E+10 copies/g of wet sediment and the Shannon index was 7.3±5, which were significantly higher than in the summer samples and in other karst environments. A correlation analysis showed that the moisture content and pH were the factors that mostly affected the archaeal communities. The prevalence of nitrate in the summer may be a key factor causing a significant decrease in archaeal abundance and diversity. Two features specific to karst environments, calcium-richness and weak alkalescence of the water supplies, may benefit the prevalence of bathyarchaeotal subgroups MCG-11, MCG-5b, and MCG-6. These results suggest that in karst wetlands, most of the archaea belong to clades that have significant roles in carbon turnover; their composition remains stable, but their abundance and diversity vary significantly from season to season.

PCR, in-situ hybridization, and phylogenetic analysis suggest that 'big belly' disease in barramundi, Lates calcarifer (Bloch), is caused by a novel Vibrio species.

'Big belly' disease is a chronic, granulomatous bacterial enteritis and peritonitis, first reported in 3- to 4-week-old Asian seabass or barramundi, Lates calcarifer Bloch fry. Affected fry are emaciated and have a swollen abdomen, and the condition is referred to as 'skinny pot-belly' or 'big belly' disease. In this study, histopathological examinations of diseased fish from a batch of 2-month-old, 6- to 8-cm L.calcarifer fingerlings, kept in seawater recirculating aquaculture systems, showed pathology resembling 'big belly' disease. Ethanol-fixed tissues were tested positive using specific PCR primers based on 16S rRNA genes. In situ hybridization using dioxygenin-labelled positive PCR products on formalin-fixed paraffin-embedded tissues showed positive reactions with intralesional, clusters of the large, 'big belly' coccobacilli. A phylogenetic tree constructed based on analyses of these 16S rRNA gene PCR products from five positive fish suggests that the 'big belly' bacterium is most likely a novel Vibrio species.

Chlorogenic Acid Improves NAFLD by Regulating gut Microbiota and GLP-1.

Our previous studies have shown that chlorogenic acid (CGA) could significantly improve acute and chronic liver injury through antioxidant and anti-inflammatory activities. However, its effect on non-alcoholic fatty liver disease (NAFLD) are not entirely clear. This study aims to explore the effect of CGA on NAFLD induced by high-fat diet (HFD) and whether it regulates the gut microbiota and Glucagon-like peptide-1 (GLP-1). NAFLD mice were established by HFD and treated with or without CGA. Serum transaminase, fasting blood glucose (FBG), blood lipids, insulin, GLP-1 and lipopolysaccharide (LPS) were detected. Liver histology was evaluated with Hematoxylin-eosin staining. Toll like receptor 4 (TLR4) signaling pathway was analyzed with western blot and inflammatory cytokines were detected with real-time PCR. The content of gut microbiota were determined with real-time PCR of the bacterial 16S rRNA gene. Expressions of intestine tight junctional protein were examined with immunohistochemistry. CGA could alleviate HFD-induced hepatic steatosis and inflammation, reduce serum transaminase, FBG and blood lipids, increase insulin sensitivity. CGA also could reverse HFD-induced activation of TLR4 signaling pathway and expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in liver. Meanwhile, CGA increased the content of Bifidobacterium and reduced the content of Escherichia coli in feces. Furthermore, CGA could increase the expression of tight junction proteins Occludin and zonula occludens-1 (ZO-1) in intestinal tissue. Moreover, CGA could the level of LPS and increased the level of GLP-1 in portal vein. These results indicated that CGA protected against HFD-induced hepatic steatosis and inflammation probably through its anti-inflammatory effects associated with regulation of gut microbiota and an increase of GLP-1 secretion and thus could be used as a potential drug for prevention and treatment of NAFLD.

Effect of treatment of pneumonia and otitis media with tildipirosin or florfenicol + flunixin meglumine on health and upper respiratory tract microbiota of preweaned Holstein dairy heifers

The objective of this randomized clinical study was to compare the effect of 2 antimicrobial interventions, tildipirosin or florfenicol + flunixin meglumine, used for treatment of pneumonia and extralabel treatment for otitis on health parameters and upper respiratory tract (URT) microbiota of preweaned Holstein calves. Housed preweaned Holstein heifers diagnosed with either otitis or pneumonia were assigned into 1 of 2 treatment groups, receiving a single subcutaneous injection of either 4 mg/kg of tildipirosin (TLD; n = 444) or 40 mg/kg of florfenicol combined with 2.2 mg/kg of a nonsteroidal anti-inflammatory, flunixin meglumine (FLF; n = 442). Calves were enrolled and treated on the day of diagnosis of the first case of pneumonia or otitis. If a calf had a recurrent case, the opposite drug was administered, respecting an interval of 5 d between drug injections. Blood samples for leukocyte counts were collected at 0, 2, 4, and 6 d after treatment, and rectal temperature was measured daily during the 5 d after treatment. Ear scores were observed from calves with otitis. Additionally, swabs of the URT were collected from a subset of 20 calves in each treatment group at d 0, 3, 6, 9, and 11 following enrollment for analysis of URT microbiota through next-generation sequencing of the 16S rRNA gene and quantitative PCR. Swabs were also collected from a comparative group of 20 healthy calves that did not receive any drug. No differences were observed between groups for recurrence risk of either pneumonia (TLD = 32.4%; FLF = 29.7%) or otitis (TLD = 72.7%; FLF = 73.6%). Similarly, no differences were observed for the total number of treatments for pneumonia (TLD = 1.45; FLF = 1.42) or otitis (TLD = 2.96; FLF = 3.07). On the other hand, both drugs reduced rectal temperature, ear scores, and leukocyte counts, with FLF calves having a greater reduction in rectal temperature within 4 d after treatment. Both TLD and FLF reduced the total bacterial load when compared with healthy untreated calves, but no differences were observed between treatment groups. Furthermore, compared with the untreated group, treated calves had lower mean relative abundances (MRA) of the genera Mannheimia, Moraxella, and Pasteurella within 11, 9, and 3 d after treatment, respectively; however, no significant differences were observed between TLD and FLF. On the other hand, MRA of Mycoplasma was not decreased by both treatments compared to untreated animals, and a higher MRA was observed in the TLD group during 11 d after treatment in comparison to FLF and untreated calves. Based on this data, we concluded that both drugs used in the study were effective in reducing rectal temperature, ear scores, leukocyte counts, and MRA of the genera Mannheimia, Pasteurella, and Moraxella in the URT, and calves treated with FLF had a greater reduction in rectal temperature.

Differential Impact of Plant Secondary Metabolites on the Soil Microbiota.

Plant metabolites can shape the microbial community composition in the soil. Two indole metabolites, benzoxazolinone (BOA) and gramine, produced by different Gramineae species, and quercetin, a flavonoid synthesized by many dicot species, were studied for their impacts on the community structure of field soil bacteria. The three plant metabolites were directly added to agricultural soil over a period of 28 days. Alterations in bacterial composition were monitored by next generation sequencing of 16S rRNA gene PCR products and phospholipid fatty acid analysis. Treatment of the soil with the plant metabolites altered the community composition from phylum to amplicon sequence variant (ASV) level. Alpha diversity was significantly reduced by BOA or quercetin, but not by gramine. BOA treatment caused a decrease of the relative abundance of 11 ASVs, while only 10 ASVs were increased. Gramine or quercetin treatment resulted in the increase in relative abundance of many more ASVs (33 or 38, respectively), most of them belonging to the Proteobacteria. Isolation and characterization of cultivable bacteria indicated an enrichment in Pseudarthrobacter or Pseudomonas strains under BOA/quercetin or BOA/gramine treatments, respectively. Therefore, the effects of the treatments on soil bacteria were characteristic for each metabolite, with BOA exerting a predominantly inhibitory effect, with only few genera being able to proliferate, while gramine and quercetin caused the proliferation of many potentially beneficial strains. As a consequence, BOA or gramine biosynthesis, which have evolved in different barley species, is accompanied with the association of distinct bacterial communities in the soil, presumably after mutual adaptation during evolution.

Diversity and abundance of soil microbial communities decline, and community compositions change with severity of post-logging fire.

Understanding the effects of logging and fire on forest soil communities is integral to our knowledge of forest ecology and effective resource management. The resulting changes in soil biota have substantial impacts on forest succession and associated ecosystem processes. We quantified bacterial and fungal abundance, diversity and community composition across a logging and burn severity gradient, approximately one month after fire, in temperate wet eucalypt forests in Tasmania, Australia. Using amplicon sequencing and real-time quantitative PCR of the bacterial 16S rRNA gene and fungal ITS1 region, we demonstrate that (i) burn severity is a strong driver of soil microbial community composition, (ii) logging and high severity burning substantially reduce the biomass and diversity of soil bacteria and fungi, and (iii) the impacts of logging and burning on soil microbial communities are largely restricted to the top 10cm of soil, with weak impacts on the subsoil. The impacts of disturbance on microbial community composition are greater than the effects of site-to-site edaphic differences. Fire also drives more divergence in community composition than logging alone. Key microbial taxa driving differences in severely burnt soils include bacterial genera implicated in plant-growth promotion and producing antifungal compounds as well as saprotrophic fungi that are also capable of forming ectomycorrhizal associations. Our research suggests that low-moderate severity burns are important for maintaining diversity and biomass in soil microbial communities but having a range of burn severities across a site contributes to the overall diversity of habitat conditions providing for both microbial and plant diversity.

Effect of hookworm infection and anthelmintic treatment on naturally acquired antibody responses against the GMZ2 malaria vaccine candidate and constituent antigens

BackgroundMalaria and helminths diseases are co-endemic in most parts of sub-Saharan Africa. Immune responses from each of these pathogens interact, and these interactions may have implications on vaccines. The GMZ2 malaria vaccine candidate is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP R0). GMZ2 has recently showed modest efficacy in a phase IIb multicenter trial. Here, we assessed the effect of hookworm (Necator americanus) infection and anthelmintic treatment on naturally acquired antibody responses against GMZ2 and constituent antigens.MethodsThis longitudinal cross-sectional study was conducted in the Kintampo North Municipality of Ghana. Blood and stool samples were taken from 158 individuals (4–88 years old) infected with either P. falciparum alone (n = 59) or both hookworm and P. falciparum (n = 63) and uninfected endemic controls (n = 36). Stool hookworm infection was detected by the Kato-Katz method and PCR. Malaria parasitaemia was detected by RDT, light microscopy and P. falciparum-specific 18S rRNA gene PCR. Serum samples were obtained prior to hookworm treatment with a single dose of albendazole (400 mg) and 3 weeks (21 days) after treatment. Levels of IgG1, IgG3 and IgM against GMZ2, MSP3 and GLURP R0 were measured by ELISA and compared among the groups, before and after treatment.ResultsParticipants with P. falciparum and hookworm co-infection had significantly higher IgG3 levels to GMZ2 than those with only P. falciparum infection and negative control (p < 0.05) at baseline. Treatment with albendazole led to a significant reduction in IgG3 levels against both GMZ2 and GLURP R0. Similarly, IgM and IgG1 levels against MSP3 also decreased following deworming treatment.ConclusionIndividuals with co-infection had higher antibody responses to GMZ2 antigen. Treatment of hookworm/malaria co-infection resulted in a reduction in antibody responses against GMZ2 and constituent antigens after albendazole treatment. Thus, hookworm infection and treatment could have a potential implication on malaria vaccine efficacy.

Relative contributions of bacteria and fungi to nitrous oxide emissions following nitrate application in soils representing different land uses

Bacteria and fungi have been shown to produce nitrous oxide (N2O) during denitrification, but their contribution after nitrate (NO3−) application to soil is not clearly established. In a microcosm experiment, the relative contribution of bacteria and fungi to N2O and carbon dioxide (CO2) production by four contrasting soils representing different land uses after KNO3 addition was studied. The soils were daily wetted to 80% water-filled pore space (WFPS) and kept under greenhouse conditions for 10 days. The fungicide cycloheximide and the bactericide streptomycin were used to determine the possible microbial origin of the N2O and CO2 emissions. Non-target effects of the antibiotics on the emission of N2O and CO2 were evaluated using the inhibitor additivity ratio (IAR). The abundance of the bacterial and fungal communities was estimated by quantitative PCR (qPCR) of the bacterial 16S rRNA gene and the fungal internal transcribed spacer (ITS) region, respectively. The gene copy number of bacterial denitrifiers was calculated after quantification of the nirK, nirS, norB, nosZI and nosZII genes. After 10 d, regardless of the soil type, the cumulative N2O emission from the soils treated with cycloheximide or streptomycin were similar. In all the four soils, N2O fluxes were greater (on average 1.8 ± 0.3 times) in soils amended with the fungicide than with the bactericide during incubation for the first 48–96 h. Greater N2O emissions (on average 1.7 ± 0.2 times) were detected in soils where bacteria were inhibited in comparison to those treated with the fungicide from 96 to 240 h. On average, 68.5% of the total CO2 emitted during the 10-d incubation period was produced in soils treated with the fungicide and 31.5% in those treated with the bactericide. The greater contribution of bacteria to the production of N2O than fungi during the first 48–96 h was possibly due to a faster used of nitrate. Variations in the abundance of bacterial 16S rRNA genes, the ITS region, and the nirK, nirS, norB and nosZI bacterial denitrification genes indicated that the antibiotics used to prevent the growth of bacteria and fungi were effective during incubation. These results suggest that both bacteria and fungi should be considered when designing and applying greenhouse gas mitigation strategies in soils and that their relative contribution to produce N2O and CO2 can vary with time and nitrate availability.