Gram-negative Bacterial Community Research Articles

Symbiotic Bacteria Modulate Lymantria dispar Immunity by Altering Community Proportions after Infection with LdMNPV.

The symbiotic bacteria-insect interaction is considered to be associated with immunity and drug resistance. However, the wide variety of insect species and habitats is thought to have a significant impact on the symbiotic community, leading to disparate results. Here, we demonstrated that symbiotic bacteria regulated the immune response by changing the proportion of the Gram-positive and the Gram-negative bacterial community in Lymantria dispar (L. dispar) after infection with its viral pathogen, L. dispar Nucleopolyhedrovirus (LdMNPV). After oral infection, the immune deficiency pathway was activated immediately, and the expression of Relish was up-regulated to promote the secretion of antimicrobial peptides. Meanwhile, the abundance of the Gram-negative bacterial community increased at the same time. Moreover, the Toll pathway was not regulated in the same way as the Imd pathway was after infection. However, the change in the Toll pathway's expression remained positively correlated to the abundance of Gram-positive bacteria. This finding implied that the ratio of Gram-negative to Gram-positive bacteria in the LdMNPV infected larvae had an effect on the immune response. Our findings revealed that the immune regulation of L. dispar was regulated by the relative abundance of its symbiotic bacteria at different infection times with LdMNPV, which provides a new way to understand symbiotic bacteria-insect interactions.

Antibiotic resistance in a predominantly occurring Gram-negative bacterial community from treated sewage to assess the need for going beyond coliform standards

Abstract Antibiotic resistance surveillance is an objective of global action plan on antimicrobial resistance endorsed by the World Health Organization. The current study reports the identification of frequently occurring Gram-negative bacterial community (GNBC) previously isolated from municipal treated wastewater and their antibiotic resistance profiles. Further, the genes responsible for extended-spectrum beta lactamases (ESBL) activity were identified in ESBL-positive organisms. The isolates were characterized using biochemical assays and identification was confirmed by VITEK®2 automated system. Antibiotic susceptibility testing against seven different classes of antibiotics was also performed on the same system using AST-N280 cards. The most dominant isolates identified were Acinetobacter baumannii, Morganella morganii, Kluyvera intermedia, Stenotrophomonas maltophilia, Escherichia coli, Aeromonas hydrophila/caviae, Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii. The isolates were observed to be significantly resistant against the antibiotics amoxicillin-clavulanic acid, cefuroxime, cefuroxime axetil and colistin. Two of the isolates, E. cloacae sp. dissolvens and S. maltophilia, were found to be positive for ESBL activity encoded by blaCTX-M gene. The possible intrusion of hospital wastewater in domestic sewage is also discussed. This study may help assess the risk of wastewater reuse by detecting dominant bacteria as a step towards the development of new microbiological standards.

Appraisal of paleoclimate indices based on bacterial 3-hydroxy fatty acids in 20 Chinese alkaline lakes

Bacterial 3-hydroxy fatty acids (3-OH-FAs) show potential for terrestrial paleoclimate reconstruction; however, the possibility of using 3-OH-FAs as proxies in lacustrine environments remains to be investigated. We analysed the composition and distribution of 3-OH-FAs in surface sediments from 20 Chinese alkaline lakes and investigated the development of climate proxies based on 3-OH-FAs in these lacustrine environments. The results show that lake sediments contain higher relative abundances of anteiso 3-OH-FAs compared to soils. Analysis of the gene community composition from Lake Liangzi reveals that Gram-negative bacterial communities in that lake environment are distinct from marine sediments from the South China Sea but share many characteristics with soils from Mt. Yujia at the phylum level. At the class level, the Lake Liangzi samples yield distinctly higher proportions of Deltaproteobacteria (23%), Betaproteobacteria (13%), Gammaproteobacteria (14%), and Bacterioidia (10%). pH proxies based on 3-OH-FAs (Branching Ratio, Branched index, RIN and RIAN) yield weak linear relationships with lake water pH, while RAN13 (previously proposed as a sea surface temperature proxy) shows a linear relationship with mean annual air temperatures (MAAT; ranging from 5 °C to 17 °C). The soil temperature proxies RAN15 (previously applied to a stalagmite) and RAN17 do not appear to be suitable for temperature calibration in lacustrine environments. We propose a novel temperature proxy (RIN17, ratio of iso to normal C17 3-OH-FA) for lake environments. These field-based correlations demonstrate that the physiological response of Gram-negative bacterial cell membranes to external environmental changes are recorded in lake sediments on a large regional scale. This further highlights the potential use of bacterial 3-OH-FAs as proxies for paleoclimate reconstruction in both aquatic and terrestrial environments.

Metagenomic Exploration of Bacterial Community Structure of Earthworms’ Gut

Living organisms are naturally bestowed with unique and imitable qualities for maintaining ecological balance and earthworms are no exceptions. These so-called keystone species of terrestrial ecosystems are equipped with wonderful machinery, allowing them to nurture soil beautifully. Earthworm gut represents a potential microbial reservoir, having a complex interdependence with the host. The study aimed to profile bacterial community structure of three earthworm species belonging to two different life forms; Perionyx excavatus and Eudrilus eugeniae (epigeic), Polypheretima elongata (endogeic) respectively. Diversity analysis using 16S amplicon sequencing revealed that the dominant phyla were Proteobacteria (34.17-77.88) followed by Actinobacteria (13.43-35.54%), Firmicutes (1.69-15.45%) and Bacteroidetes (0.51-8.12%). The alpha diversity indices explicit similar gut microbiota of Perionyx excavatus and Eudrilus eugeniae and while higher alpha diversity was recorded in comparison to Polypheretima elongata gut. The taxonomic to the phenotypic annotation of 16S rRNA metagenomes revealed that dominance of Gram-negative bacterial community in all earthworm species while, Polypheretima elongata comprises higher percentage (78%) of Gram-negative bacterial community to Perionyx excavatus (32.3%) and Eudrilus eugeniae (38.3%). The oxygen requirement phenotypic analysis showed that all earthworm species were abundant with aerobic followed by anaerobic bacterial groups. Furthermore, functional metabolism phenotypic analysis revealed that a high abundance of ammonia oxidizers (29.3-80.2%), the gut microbiomes showed the relative abundance of sulphate reducer (22.6-78.7%), nitrite reducer (19.8-73.2%), dehalogenators (12.6-25.1%), illustrating in the role of these microbial communities in various degradation and bioremediation processes. The present study signifies the intrinsic gut microbiota of earthworm species for intensified biodegradation.

The selective pressure of quorum quenching on microbial communities in membrane bioreactors

In conventional membrane bioreactor (MBR) treatment systems, Gram-negative bacterial population appears to be always outnumbered Gram-positive community. Thereby, acyl homoserine lactones (AHLs), major signaling molecules utilized by Gram-negative bacteria, have been targeted for biofouling control in quorum quenching (QQ) based studies. This study investigated the impact of AHL and autoinducer-2 (AI-2)-degrading QQ consortium on the selective accumulation of microbial communities in a QQ MBR (MBR-QQb). The results show that addition of the QQ consortium (in the form of beads) increased the filtration time of MBR-QQb by 3.5 times. The distribution of mixed liquor extracellular polymeric substances (EPS), especially the tightly bound (TB) proteinous EPS and the floc size were strongly affected by the QQ activity, and the endless ‘battle’ between QQ and quorum sensing (QS). More importantly, QQ induced the significant suppression of Gram-negative bacterial community. The average abundance of Gram-positive bacteria at the genus level in the biocake of MBR-QQb (51%) was significantly higher than that of the control MBR (11%) and the MBR with vacant beads (28%). These findings suggest that an unintended condition is created to favor the growth of Gram-positive bacteria in QQ MBRs, resulting in a distinct microbial social network in both bulk sludge and biocake.

A new sea surface temperature proxy based on bacterial 3-hydroxy fatty acids

3-Hydroxy fatty acids (3-OH-FAs), derived from Gram-negative bacterial outer membranes, have received recent attention for their potential as new terrestrial pH and temperature proxies for palaeoclimate studies. However, it is not known whether 3-OH-FA based proxies can be developed and applied to the marine environment. Here we analyze 3-OH-FAs from a latitudinal transect of marine surface sediments from the North Pacific Ocean (12°N to 61°N with a annual mean sea surface temperature (SST) range of 28.1–1.3 °C). The results show that distributions of 3-OH-FAs in marine sediments yield overall higher abundances of anteiso 3-OH-FA homologues compared to soils. Furthermore, 16S rRNA gene sequencing of the marine sediments and soils shows the Gram-negative bacterial community is dominated by Proteobacteria (ca. 94%) at the phylum level. In contrast, in regional soils the Gram-negative bacterial community is more diverse with significant contributions from Proteobacteria (ca. 50%), Acidobacteria (ca. 24%), Verrucomicrobia (6%), Bacteroidetes (6%) and other phyla. These distinct genomic and molecular differences suggest distinctly different aggregate compositions of bacteria that produce 3-OH-FAs in the marine realm vs soils. Moreover, we find that the ratio of anteiso to normal C13 3-OH-FA (RAN13) measured in surface sediments is highly correlated with annual mean SST throughout the temperature transect. When applied to a short sediment core from the East China Sea, the SST changes reconstructed by the RAN13 proxy are comparable to instrumental SST data. These findings demonstrate that RAN13 and potentially other, so far undiscovered, proxies based on 3-OH-FAs have potential for environmental and palaeoclimate applications in marine environments.

Impact of Nitriles on Bacterial Communities

Nitriles are organic molecules with −C≡N as functional group and often toxic for living organisms. Detoxification can occur via nitrilases that degrade nitriles directly to carboxylic acids and ammonia, or with nitrile hydratases and amidases that convert nitriles to amides and subsequently to carboxylic acids and ammonia. Despite the knowledge of enzymatic degradation pathways, the influence of these compounds on the composition of bacterial communities is unknown. The tolerances of four phylogenetically different bacterial strains without known nitrile detoxification systems (Agrobacterium tumefaciens, Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli) to the toxic effects of nine nitriles and the corresponding carboxylic acids were determined. Based on these results, the effect of nitriles on diversity and composition of compost-derived bacterial communities was monitored over time by 16S rRNA gene amplicon-based and metagenome analyses. Acetone cyanohydrin, 2-phenylpropionitrile, and pyruvonitrile exhibited a lethal, phenylacetonitrile, 4-hydroxybenzonitrile, and cyclohexanecarbonitrile a growth-suppressing and succinonitrile, acetonitrile, and crotononitrile a growth-promoting effect on the studied communities. Furthermore, each nitrile had a specific community-shaping effect, e.g. communities showing growth-suppression exhibited high relative abundance of Paenibacillus. In general, analysis of all data indicated a higher resistance of Gram-positive than Gram-negative bacterial community members and test organisms to growth-suppressing nitriles. More than 70 putative nitrilase-encoding and over 20 potential nitrile hydratase-encoding genes were identified during analysis of metagenomes derived from nitrile-enrichments, underlining the high yet often unexplored abundance of nitrile-degrading enzymes.

Evaluation of 3-hydroxy fatty acids as a pH and temperature proxy in soils from temperate and tropical altitudinal gradients

Three hydroxy (3-OH) fatty acids (FAs), containing 10 to 18 C and a hydroxyl group in third position are characteristic components of Gram-negative bacteria. These compounds were used previously to detect and quantify Gram-negative bacterial communities in various types of samples, from terrestrial, aquatic and atmospheric environments. The relative abundance of 3-OH FAs in soils was recently shown to vary with mean annual air temperature (MAAT) and pH in soils from Mt. Shennongjia (China). In the current study, the concentrations and abundances of 3-OH FAs were determined in soils from altitudinal transects under tropical and temperate climates: Mt. Rungwe (SW Tanzania) and Mt. Majella (Central Italy), respectively. The aim was to (i) examine and compare the distributions of 3-OH FAs in soils from different climatic zones and (ii) to investigate the potential of 3-OH FAs as temperature and pH proxies. When combined with previously published data, a moderate correlation (R2 0.62) between the summed iso and anteiso to the total amount of normal 3-OH FAs (RIAN index) and pH was obtained. We show that Gram-negative bacteria respond in the same way to temperature variations in soils from different latitudes, with a relative increase of the anteiso to normal 3-OH FA ratios of the C15 and C17 compounds (RAN15 and RAN17 indices, respectively) with decreasing temperature. Nevertheless, the intercepts of the relationships between RAN15 and MAAT are mountain-dependent, suggesting that regional calibrations may be required to use RAN15 as a temperature proxy. In contrast with RAN15, the statistical similarity of the local relationships between RAN17 and MAAT leads to a combined calibration (R2 0.60, residual mean error 5.1 °C) covering a wide range of temperature (ca. 0–25 °C). As 3-OH FAs seem well-preserved in sedimentary archives, this strengthens the potential of these compounds as a temperature and pH proxy for paleoreconstructions in terrestrial settings.

N-acyl-homoserine lactone dynamics during biofilm formation of a 1,2,4-trichlorobenzene mineralizing community on clay

In Gram-negative bacteria, quorum sensing systems are based on the N-acyl-homoserine lactone (AHL) molecule. The objective of this study was to investigate the role of quorum sensing systems during biofilm formation by a microbial community while degrading the pollutant. Our model system included 1,2,4-trichlorobenzene (1,2,4-TCB) and its mineralizing Gram-negative bacterial community to investigate the relationships between AHL dynamics, cell growth and pollutant degradation. Biomineralization of 1,2,4-TCB was monitored for both the planktonic bacterial community with and without sterile clay particles in liquid cultures. The bacterial growth and production of AHLs were quantified by fluorescent in situ hybridization and immunoassay analysis, respectively. A rapid production of AHLs which occurred coincided with the biofilm formation and the increase of mineralization rate of 1,2,4-TCB in liquid cultures. There is a positive correlation between the cell density of Bodertella on the clay particles and mineralization rate of 1,2,4-TCB. 3-oxo-C12:1-HSL appears to be the dominant AHL with the highest intensity and rapidly degraded by the bacterial community via two main consecutive reactions (lactone hydrolysis and decarboxylic reaction). These findings suggest that the integrated AHLs and their degraded products play a crucial role in biofilm formation and biomineralization of 1,2,4-TCB in culture.

Assessment of anaerobic bacterial diversity and its effects on anaerobic system stability and the occurrence of antibiotic resistance genes

This study evaluated the link between anaerobic bacterial diversity and, the biodegradation of antibiotic combinations and assessed how amending antibiotic combination and increasing concentration of antibiotics in a stepwise fashion influences the development of resistance genes in anaerobic reactors. The biodegradation, sorption and occurrence of the known antibiotic resistance genes (ARGs) of erythromycin and tetracycline were investigated using the processes of UV-HPLC and qPCR analysis respectively. Ion Torrent sequencing was used to detect microbial community changes in response to the addition of antibiotics. The overall results indicated that changes in the structure of a microbial community lead to changes in biodegradation capacity, sorption of antibiotics combinations and occurrence of ARGs. The enhanced biodegradation efficiency appeared to generate variations in the structure of the bacterial community. The results suggested that controlling the ultimate Gram-negative bacterial community, especially Acinetobacter-related populations, may promote the successful biodegradation of antibiotic combinations and reduce the occurrence of ARGs.

Comparison of Endotoxin Levels Found in Primary and Secondary Endodontic Infections

IntroductionThis clinical study was conducted to compare the levels of endotoxins (lipopolysaccharides [LPSs]) found in primary and secondary endodontic infections with apical periodontitis by correlating LPS contents with clinical/radiographic findings. In addition, the presence of target gram-negative anaerobic bacteria was also investigated. MethodsSamples were taken from 15 root canals with primary infections and 15 with secondary infections by using paper points. The limulus amebocyte lysate assay was used to quantify endotoxins, and the polymerase chain reaction technique (16S rDNA) was used for bacterial investigation. ResultsEndotoxins were detected in 100% of the root canal samples collected from primary (15/15) and secondary (15/15) infections with median values of 7.49 EU/mL and 3.96 EU/mL, respectively (P < .05). The median value of endotoxins found in the presence of clinical symptoms was significantly higher than in asymptomatic teeth with primary infections (P < .05). A positive correlation was found between endotoxin contents and a larger size of the radiolucent area (>3 mm) (P < .05). Prevotella nigrescens (10/15, 4/15), Fusobacterium nucleatum (5/15, 1/15), Treponema denticola (3/15, 1/15), and Treponema socranskii (5/15, 1/15) were detected in teeth with primary and secondary infections, respectively. P. endodontalis was present only in teeth with primary infections (5/15). ConclusionsTeeth with primary endodontic infections had higher contents of endotoxins and a more complex gram-negative bacterial community than teeth with secondary infections. Moreover, the levels of endotoxins were related to the severity of bone destruction in periapical tissues as well as the development of clinical features in teeth with primary infections.

Grape berry bacterial microbiota: Impact of the ripening process and the farming system

Wine grapes are a primary source of microbial communities that play a prominent role in the quality of grapes prior to harvesting, as well as in the winemaking process. This study investigated the dynamics and diversity of the epiphytic bacteria on the grape berry surface during maturation. The quantitative and qualitative effects of conventional and organic farming systems on this microbial community were investigated, using both cultivation-dependent and independent approaches. Analyses of grape berry bacterial microbiota revealed changes in the size and structure of the population during the berry ripening process, with levels rising gradually and reaching their highest value when the berries were over ripe. As the season progressed to maturity, Gram-negative bacterial communities (mostly Pseudomonas spp.) declined whereas Gram-positive communities (mostly Micrococcus spp.) increased. The 16S rRNA gene sequences of cultured isolates were analysed and over 44 species were identified from 21 genera in the Proteobacteria, Actinobacteria, and Firmicutes phyla. Copper concentrations originating from phytosanitary treatments varied according to the vineyard and farming system. A negative correlation between copper concentrations and cell densities provided clear evidence that copper inhibited bacterial communities. The bacterial community structure was analysed by targeting the 16S rRNA genes, using PCR-DGGE on cultivable populations and T-RFLP on whole communities in cell suspension. The results suggest that the farming system has a clear impact on the bacterial community structure.

Comparison of culturable Gram-negative bacterial community structures in the rhizosphere of three fruit plants

This research work was oriented to outlining the diversity of Gram-negative culturable portion of the bacterial community in three fruit plants rhizosphere. Rhizosphere samples were taken from European chestnut (Castanea sativa Mill), true service tree (Sorbus domestica L.) and cornelian cherry (Cornus mas L.) plants. Experiments were conducted for three years during the vegetation period, and the bacterial community structure was assessed with cultivation-dependent approach. Many Gram-negative isolates (n = 251) from the rhizosphere survived sub culturing and were identified by biochemical tests. A total of 57 species belonging to 29 genera were identified and assigned to four broad taxonomic groups (Bacteroidetes, Alpha-, Beta -a ndGamma-proteobacteria). Several specific bacterial cluster communities were identified inside all the three rhizospheres. Most of the species belonged to the genera Moraxella, Pseudomonas, Pantoea, Enterobacter and Acinetobacter. In addition, while, using the plate count analysis, large discrepancies in numbers among physiological groups of bacteria cultured from three rhizosphere samples have not been revealed, more expressive distinctions among bacterial populations were obtained concerning the relative abundance of different genera, different taxonomic groups as well as different diversity indices. Furthermore, the number of cultured bacteria and their taxonomic distribution in the rhizosphere of all three plants changed not only explicitly during vegetation period but continually during the three years of investigation. It seems that rhizosphere bacterial populations of each plant are under the influence of the specific root- released materials.

The 3-hydroxy fatty acids as biomarkers for quantification and characterization of endotoxins and Gram-negative bacteria in atmospheric aerosols in Hong Kong

Abstract Endotoxins from Gram-negative bacteria have received much attention because they could elicit strong pro-inflammatory responses in the human respiratory tract. In this study, 3-hydroxy fatty acids (3-OH FAs) with carbon chain lengths from 10 to 18 (C10–C18) were employed as biomarkers to quantify and characterize the endotoxins and Gram-negative bacterial community in atmospheric aerosols. Gas chromatography–mass spectrometry (GC–MS) was utilized for quantification of this biomarker in fine (PM 2.5 ) and coarse (PM 2.5–10 ) particulates collected by high volume samplers simultaneously at a rural and an urban site in Hong Kong. The geometric mean concentrations of the endotoxins were 5.5 and 1.35 ng m −3 in fine and coarse particulates at the rural site, respectively. At the urban site, the corresponding concentrations were 9.4 and 2.80 ng m −3 in fine and coarse particulates, respectively. It is found that 70–80% of the total endotoxins are associated with the fine particulates. Significant higher endotoxin levels at the urban site were observed throughout the 8-month study period. This could possibly relate to the heavier human activities in the urban areas. The distribution patterns of the 3-OH FAs with respect to carbon number are similar between the rural and urban sites regardless of particle sizes. The C10 and C16 were predominant and accounted for about 40–50% of the total 3-OH FAs. Furthermore, the odd carbon chain length 3-OH FAs constituted a non-negligible fraction (15–25%) of the total 3-OH FAs. The biologically active endotoxins estimated as the sum of C12 and C14 portions in this study ranged from 0.6–3.7 and 1.9–4.8 ng m −3 at the rural and urban sites, respectively. Applying the biomarker-to-microbial mass conversion factors, the dry mass loading of the Gram-negative bacteria are in the order of 10–10 2  ng m −3 in atmospheric aerosol. This study also demonstrates that the biomarker (3-OH FAs) approach yields much more quantitative information such as the mass loading, total endotoxin concentration and estimation of potent endotoxin level in the atmospheric aerosol.

Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes

We studied the changes in water quality and formation of biofilms occurring in a pilot-scale water distribution system with two generally used pipe materials: copper and plastic (polyethylene, PE). The formation of biofilms with time was analysed as the number of total bacteria, heterotrophic plate counts and the concentration of ATP in biofilms. At the end of the experiment (after 308 days), microbial community structure, viable biomass and gram-negative bacterial biomass were analysed via lipid biomarkers (phospholipid fatty acids and lipopolysaccharide 3-hydroxy fatty acids), and the numbers of virus-like particles and total bacteria were enumerated by SYBR Green I staining. The formation of biofilm was slower in copper pipes than in the PE pipes, but after 200 days there was no difference in microbial numbers between the pipe materials. Copper ion led to lower microbial numbers in water during the first 200 days, but thereafter there were no differences between the two pipe materials. The number of virus-like particles was lower in biofilms and in outlet water from the copper pipes than PE pipes. Pipe material influenced also the microbial and gram-negative bacterial community structure in biofilms and water.

Quantitative lipid biomarker detection of unculturable microbes and chlorine exposure in water distribution system biofilms

Biofilms in the drinking water distribution system can protect pathogens from disinfection and provide the inocula for periodic infestations. Assessing these biofilms can be difficult, as the plate counts of pelagic bacteria may bear little relationship to the biofilm load. Culturing the water at the outlet most often does not reflect the biofilm composition. Herein we show that analysis of polar lipid fatty acids recovered from biofilms on devices possessing a large surface area provides quantitative analysis of the viable biomass, community composition, and nutritional status that is independent of the recovery and culturability. Analysis of the polar lipid fatty acids indicated the biofilm contained a stressed and predominantly Gram-negative bacterial community. The composition was not significantly different whether collected in the summer or winter. Oxirane (epoxide) fatty acids were detected in the polar lipids of the biofilm, indicating exposure to chlorine and loss of viability within the biofilm. Tests with monocultures of Escherichia coli and Sphingomonas paucimobilis exposed to chlorine resulted in oxirane fatty acid generation and rendered them nonculturable.

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities.

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.