Low Bacterial Density Research Articles

Evaluation of an outbred mouse model for Francisella tularensis vaccine development and testing.

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity.

In situ biodegradation, photooxidation and dissolution of petroleum compounds in Arctic seawater and sea ice

In pristine sea ice-covered Arctic waters the potential of natural attenuation of oil spills has yet to be uncovered, but increasing shipping and oil exploitation may bring along unprecedented risks of oil spills.We deployed adsorbents coated with thin oil films for up to 2.5 month in ice-covered seawater and sea ice in Godthaab Fjord, SW Greenland, to simulate and investigate in situ biodegradation and photooxidation of dispersed oil.GC-MS-based chemometric methods for oil fingerprinting were used to identify characteristic signatures for dissolution, biodegradation and photooxidation. In sub-zero temperature seawater, fast degradation of n-alkanes was observed with estimated half-life times of ∼7 days. PCR amplicon sequencing and qPCR quantification of bacterial genes showed that a biofilm with a diverse microbial community colonised the oil films, yet a population related to the psychrophilic hydrocarbonoclastic gammaproteobacterium Oleispira antarctica seemed to play a key role in n-alkane degradation. Although Oleispira populations were also present in sea ice, we found that biofilms in sea ice had 25 to 100 times lower bacterial densities than in seawater, which explained the non-detectable n-alkane degradation in sea ice. Fingerprinting revealed that photooxidation, but not biodegradation, transformed polycyclic aromatic compounds through 50 cm-thick sea ice and in the upper water column with removal rates up to ∼1% per day.Overall, our results showed a fast biodegradation of n-alkanes in sea ice-covered seawater, but suggested that oils spills will expose the Arctic ecosystem to bio-recalcitrant PACs over prolonged periods of time.

Differential Effects of Chlorhexidine Skin Cleansing Methods on Residual Chlorhexidine Skin Concentrations and Bacterial Recovery.

BACKGROUND Bathing intensive care unit (ICU) patients with 2% chlorhexidine gluconate (CHG)-impregnated cloths decreases the risk of healthcare-associated bacteremia and multidrug-resistant organism transmission. Hospitals employ different methods of CHG bathing, and few studies have evaluated whether those methods yield comparable results. OBJECTIVE To determine whether 3 different CHG skin cleansing methods yield similar residual CHG concentrations and bacterial densities on skin. DESIGN Prospective, randomized 2-center study with blinded assessment. PARTICIPANTS AND SETTING Healthcare personnel in surgical ICUs at 2 tertiary-care teaching hospitals in Chicago, Illinois, and Boston, Massachusetts, from July 2015 to January 2016. INTERVENTION Cleansing skin of one forearm with no-rinse 2% CHG-impregnated polyester cloth (method A) versus 4% CHG liquid cleansing with rinsing on the contralateral arm, applied with either non-antiseptic-impregnated cellulose/polyester cloth (method B) or cotton washcloth dampened with sterile water (method C). RESULTS In total, 63 participants (126 forearms) received method A on 1 forearm (n=63). On the contralateral forearm, 33 participants received method B and 30 participants received method C. Immediately and 6 hours after cleansing, method A yielded the highest residual CHG concentrations (2500 µg/mL and 1250 µg/mL, respectively) and lowest bacterial densities compared to methods B or C (P<.001). CONCLUSION In healthy volunteers, cleansing with 2% CHG-impregnated cloths yielded higher residual CHG concentrations and lower bacterial densities than cleansing with 4% CHG liquid applied with either of 2 different cloth types and followed by rinsing. The relevance of these differences to clinical outcomes remains to be determined. Infect Control Hosp Epidemiol 2018;39:405-411.

Response to: Comment on Rohrscheib et al. 2016 "Intensity of mutualism breakdown is determined by temperature not amplification of Wolbachia genes".

Unlike other intracellular bacteria, Wolbachia genomes are highly labile largely due to the presence of repetitive sequences such as transposons, active phage and high rates of recombination [1–3]. Within the wMelPop genome a locus of considerable instability, referred to as the Octomom locus, has been described by several studies [4–8]. As this locus is one of the few variable loci between wMel (single Octomom locus, low bacterial density and non-pathogenic) and wMelPop (3–12 Octomom loci, high bacterial density and pathogenic) genomes, and no genetic transformation tools are available, there is considerable interest in understanding the link between genotype and phenotype. Chrostek and Teixeira hypothesized “that Octomom region amplification underlies wMelPop virulence” and went on to correlate increased Octomom copy number to increased bacterial density within the host and strength of pathology [5]. Absent from this study was a systematic assessment of temperature, which previous studies had shown to affect wMelPop pathology, and its effect on wMelPop density and Octomom copy number. We hypothesized that temperature would affect Octomom copy number and/or gene expression, and that such changes would affect the strength of pathology [8]. Our study showed that while Octomom copy number did vary over developmental time, no consistent trend was observed among Octomom copy number, bacterial density nor pathology [8]. We determined that temperature, not Octomom copy number or bacterial density, had the greatest effect on wMelPop infected host lifespan. The link between Octomom copy number and pathology was further challenged by our discovery of a pathogenic Wolbachia strain (wMel3562) that maintained a single copy of the Octomom locus, and supports an earlier study of a wMelPop variant that lacks the Octomom locus but which is still pathogenic [7–9]. These observations suggest that at the very least, genetic elements beyond the Octomom locus are responsible for pathology...

An early mechanical coupling of planktonic bacteria in dilute suspensions

It is generally accepted that planktonic bacteria in dilute suspensions are not mechanically coupled and do not show correlated motion. The mechanical coupling of cells is a trait that develops upon transition into a biofilm, a microbial community of self-aggregated bacterial cells. Here we employ optical tweezers to show that bacteria in dilute suspensions are mechanically coupled and show long-range correlated motion. The strength of the coupling increases with the growth of liquid bacterial culture. The matrix responsible for the mechanical coupling is composed of cell debris and extracellular polymer material. The fragile network connecting cells behaves as viscoelastic liquid of entangled extracellular polymers. Our findings point to physical connections between bacteria in dilute bacterial suspensions that may provide a mechanistic framework for understanding of biofilm formation, osmotic flow of nutrients, diffusion of signal molecules in quorum sensing, or different efficacy of antibiotic treatments at low and high bacterial densities.

The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

Does a prenatal bacterial microbiota exist?

Does a prenatal bacterial microbiota exist?

Antimicrobial effect of Al2O3, Ag and Al2O3/Ag thin films on Escherichia coli and Pseudomonas putida

The influence of Al2O3, Ag and Al2O3/Ag thin films on bacterial growth of Gramnegative bacteria Pseudomonas putida and Escherichia coli is studied. The nanostructured thin films are deposited on glass substrates without intentional heating through r.f. magnetron sputtering in Ar atmosphere of Al2O3 and Ag targets or through sequential sputtering of Al2O3 and Ag targets, respectively. The individual Ag thin films (thickness 8 nm) have a weak bacteriostatic effect on Escherichia coli expressed as an extended adaptive phase of the bacteria up to 5 hours from the beginning of the experiment, but the final effect is only 10 times lower bacterial density than in the control. The individual Al2O3 film (20 nm) has no antibacterial effect against two strains E. coli - industrial and pathogenic. The Al2O3/Ag bilayer films (Al2O3 20 nm/Ag 8 nm) have strong bactericidal effect on Pseudomonas putida and demonstrate an effective time of disinfection for 2 hours. The individual films Al2O3 and Ag have not pronounced antibacterial effect on Pseudomonas putida. A synergistic effect of Al2O3/Ag bilayer films in formation of oxidative species on the surface in contact with the bacterial suspension could be a reason for their antimicrobial effect on E. coli and P. putida.

Cerebrospinal fluid cytokines in the diagnosis of bacterial meningitis in infants.

Bacterial meningitis poses diagnostic challenges in infants. Antibiotic pretreatment and low bacterial density diminish cerebrospinal fluid (CSF) culture yield, while laboratory parameters do not reliably identify bacterial meningitis. Pro and anti-inflammatory cytokines are elevated in bacterial meningitis and may be useful diagnostic adjuncts when CSF cultures are negative. In a prospective cohort study of infants, we used cytometric bead arrays to measure tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), IL-6, IL-8, IL-10, and IL-12 in CSF. Receiver operating characteristic (ROC) analyses and Principal component analysis (PCA) were used to determine cytokine combinations that identified bacterial meningitis. Six hundred and eighty four infants < 6 mo were included; 11 had culture-proven bacterial meningitis. IL-6 and IL-10 were the individual cytokines possessing greatest accuracy in diagnosis of culture proven bacterial meningitis (ROC analyses; area under the concentration-time curve (AUC) 0.91; 0.9103 respectively), and performed as well as, or better than combinations identified using ROC and PCA. CSF cytokines were highly correlated with each other and with CSF white blood cell count (WBC) counts in infants with meningitis. A subset of antibiotic pretreated culture-negative subjects demonstrated cytokine patterns similar to culture positive subjects. CSF cytokine levels may aid diagnosis of bacterial meningitis, and facilitate decision-making regarding treatment for culture negative meningitis.

Stabilisation/solidification and bioaugmentation treatment of petroleum drill cuttings

Petroleum drill cuttings are usually treated by techniques suitable for particular contaminant groups. The significance of this study consists in the development of a treatment technology that can simultaneously handle the hydrocarbon and metal constituents of drill cuttings. Bioaugmentation is combined with stabilisation/solidification (S/S), within S/S monoliths and in granulated S/S monoliths. Portland cement was used for S/S treatment at 30% binder dosage. Bioaugmentation treatment involved two bacterial densities of a mixed culture bio-preparation. The effects of inclusion of compost, fertiliser and activated carbon were also evaluated. After 28 days, the combined S/S and bioaugmentation treatments recorded up to 15% higher total petroleum hydrocarbon (TPH) loss than control S/S treatment without bioaugmentation. Embedding fertiliser, activated carbon and higher bacterial density within S/S monoliths resulted in the highest (99%) TPH reduction but higher concentrations of metals. The addition of compost and lower bacterial density to granulated S/S monoliths led to similar (98%) TPH degradation and lower amounts of metals. The results suggest that with better mixture optimisation, combining S/S and bioaugmentation could engender more sustainable treatment of drill cuttings.

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat.

Biofilms formations of spoilage and pathogenic bacteria on food or food contact surfaces have attracted increasing attention. These events may lead to a higher risk of food spoilage and foodborne disease transmission. While Pseudomonas lundensis is one of the most important bacteria that cause spoilage in chilled meat, its capability for biofilm formation has been seldom reported. Here, we investigated biofilm formation characteristics of P. lundensis mainly by using crystal violet staining, and confocal laser scanning microscopy (CLSM). The swarming and swimming motility, biofilm formation in different temperatures (30, 10, and 4 °C) and the protease activity of the target strain were also assessed. The results showed that P. lundensis showed a typical surface-associated motility and was quite capable of forming biofilms in different temperatures (30, 10, and 4 °C). The strain began to adhere to the contact surfaces and form biofilms early in the 4 to 6 h. The biofilms began to be formed in massive amounts after 12 h at 30 °C, and the extracellular polysaccharides increased as the biofilm structure developed. Compared with at 30 °C, more biofilms were formed at 4 and 10 °C even by a low bacterial density. The protease activity in the biofilm was significantly correlated with the biofilm formation. Moreover, the protease activity in biofilm was significantly higher than that of the corresponding planktonic cultures after cultured 12 h at 30 °C.

Diagnostic methods for H. pylori infection: Choices, opportunities and pitfalls.

Diagnostic methods for H. pylori infection: Choices, opportunities and pitfalls.

Helicobacter Pylori Infection: Diagnostic Strategies in Primary Diagnosis and After Therapy.

Accurate diagnosis of Helicobacter pylori infection pre- and post-treatment is mandatory in the current era of decreasing prevalence and increasing antibiotic resistance. The diagnostic performance of most tests is poorer in clinical situations with low bacterial density which is seen in conditions such as atrophic gastritis or intake of antisecretory and antibiotic medications. Noninvasive tests require less cost and resource but provide excellent accuracy; however, endoscopy with testing of gastric biopsy specimens is indicated where alarming symptoms are present or antibiotic susceptibility testing by culture is desired. Newer modalities such as polymerase chain reaction testing provide additional virulence and antibiotic sensitivity profiling. This article outlines new developments and the key parameters of each test, as careful selection of test modality within the clinical context is required for adequate management of infected symptomatic patients.

Antibacterial Properties and Mechanism of Activity of a Novel Silver-Stabilized Hydrogen Peroxide.

Huwa-San peroxide (hydrogen peroxide; HSP) is a NSF Standard 60 (maximum 8mg/L-1) new generation peroxide stabilized with ionic silver suitable for continuous disinfection of potable water. Experiments were undertaken to examine the mechanism of HSP against planktonic and biofilm cultures of indicator bacterial strains. Contact/kill time (CT) relationships that achieve effective control were explored to determine the potential utility in primary disinfection. Inhibitory assays were conducted using both nutrient rich media and a medium based on synthetic wastewater. Assays were compared for exposures to three disinfectants (HSP, laboratory grade hydrogen peroxide (HP) and sodium hypochlorite) at concentrations of 20 ppm (therefore at 2.5 and 5 times the NSF limit for HP and sodium hypochlorite, respectively) and at pH 7.0 and 8.5 in dechlorinated tap water. HSP was found to be more or equally effective as hypochlorite or HP. Results from CT assays comparing HSP and HP at different bacterial concentrations with neutralization of residual peroxide with catalase suggested that at a high bacterial concentration HSP, but not HP, was protected from catalase degradation possibly through sequestration by bacterial cells. Consistent with this hypothesis, at a low bacterial cell density residual HSP was more effectively neutralized as less HSP was associated with bacteria and therefore accessible to catalase. Silver in HSP may facilitate this association through electrostatic interactions at the cell surface. This was supported by experiments where the addition of mono (K+) and divalent (Ca+2) cations (0.005-0.05M) reduced the killing efficacy of HSP but not HP. Experiments designed to distinguish any inhibitory effect of silver from that of peroxide in HSP were carried out by monitoring the metabolic activity of established P. aeruginosa PAO1 biofilms. Concentrations of 70-500 ppm HSP had a pronounced effect on metabolic activity while the equivalent concentrations of ionic silver (50- 375 ppb) had a negligible effect, demonstrating that the microbiocidal activity of HSP was due to peroxide rather than silver. Overall, it was found that the antimicrobial activity of HSP is enhanced over that of hydrogen peroxide; the presence of the ionic silver enhances interactions of HSP with the bacterial cell surface rather than acting directly as a biocide at the tested concentrations.

Pharmacodynamics of fosfomycin: insights into clinical use for antimicrobial resistance.

The aim of this study was to improve the understanding of the pharmacokinetic-pharmacodynamic relationships of fosfomycin against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains that have different fosfomycin MICs. Our methods included the use of a hollow fiber infection model with three clinical ESBL-producing E. coli strains. Human fosfomycin pharmacokinetic profiles were simulated over 4 days. Preliminary studies conducted to determine the dose ranges, including the dose ranges that suppressed the development of drug-resistant mutants, were conducted with regimens from 12 g/day to 36 g/day. The combination of fosfomycin at 4 g every 8 h (q8h) and meropenem at 1 g/q8h was selected for further assessment. The total bacterial population and the resistant subpopulations were determined. No efficacy was observed against the Ec42444 strain (fosfomycin MIC, 64 mg/liter) at doses of 12, 24, or 36 g/day. All dosages induced at least initial bacterial killing against Ec46 (fosfomycin MIC, 1 mg/liter). High-level drug-resistant mutants appeared in this strain in response to 12, 15, and 18 g/day. In the study arms that included 24 g/day, once or in a divided dose, a complete extinction of the bacterial inoculum was observed. The combination of meropenem with fosfomycin was synergistic for bacterial killing and also suppressed all fosfomycin-resistant clones of Ec2974 (fosfomycin MIC, 1 mg/liter). We conclude that fosfomycin susceptibility breakpoints (≤64 mg/liter according to CLSI [for E. coli urinary tract infections only]) should be revised for the treatment of serious systemic infections. Fosfomycin can be used to treat infections caused by organisms that demonstrate lower MICs and lower bacterial densities, although relatively high daily dosages (i.e., 24 g/day) are required to prevent the emergence of bacterial resistance. The ratio of the area under the concentration-time curve for the free, unbound fraction of fosfomycin versus the MIC (fAUC/MIC) appears to be the dynamically linked index of suppression of bacterial resistance. Fosfomycin with meropenem can act synergistically against E. coli strains in preventing the emergence of fosfomycin resistance.

Inhibition of bacterial fouling by soft coral natural products

Six common soft coral species (Sarcophyton sp., Sinularia sp.1 and sp.2, Cladiella sp., Scleronephthya sp. and Dendronephthya sp.) from Bandar Al-Khayran (Sultanate of Oman) had significantly lower bacterial density in comparison with surfaces of empty shells. Methanol: chloroform (1:1) extracts of these species were tested against Gram positive (Micrococcus luteus, Staphlococcus aureus, Bacillus subtilis, Bacillus sp.) and Gram negative (Salmonella enterica, Pseudomonas aeruginosa, Escherichia coli, Cytophaga sp., Pseudomonas sp., Shewanella sp.) marine biofouling and pathogenic bacterial strains. All tested extracts had some activity against human pathogens and the highest antimicrobial activity was observed for extracts of Sinularia sp.1 and Cladiella sp. (inhibited 50% and 60% of the strains, respectively). Only ethyl acetate extracts of Cladiella sp. inhibited growth of biofouling bacteria. The active fraction was purified and identified as a mixture of hexadecyl palmitate and hexadecyl stearate. Pure hexadecyl palmitate inhibited growth (Bacillus sp. and Psychrobacter sp.) and attachment (Bacillus sp., Cytophaga sp., Pseudomonas sp., Psychrobacter sp., Shewanella sp.) of the marine biofouling bacteria. The results of this study suggest that soft corals have developed mechanisms to combat microbial infections and inhibit bacterial fouling.

Hydrogen production by Escherichia coli without nitrogen sparging and subsequent use of the waste culture for fast mass scale one-pot green synthesis of silver nanoparticles

Abstract In view of the transition to hydrogen as a major energy carrier in the future, new routes for bringing down the cost of biological hydrogen production need to be explored. The current study was devoted to optimizing the dark fermentation by Escherichia coli HD701 for hydrogen production from an acid-hydrolyzed potato starch residue stream without nitrogen sparging to reduce the cost. To further increase the economic feasibility of hydrogen production by E. coli, this study explores the use of the waste culture after hydrogen production in mass scale one-pot green synthesis of silver nanoparticles. Hydrogen gas production by E. coli was conducted from an optimum 10 g/L reducing sugars of acid-hydrolyzed potato starch residue stream. At low initial bacterial optical density OD600 of 0.02, the optimum air space was 3% of the fermentor for hydrogen production without nitrogen sparging. However, the cumulative hydrogen gas produced was 17% lower than that of the hydrogen produced after nitrogen sparging. Increasing the initial bacterial optical density OD600 increased the cumulative hydrogen produced up to that of the nitrogen sparged control. The initial bacterial optical density of 0.04 was optimum for hydrogen production without nitrogen sparging producing 0.444 mol/mole glucose compared to 0.448 mol/mole glucose obtained after nitrogen sparging. Eliminating the nitrogen sparging step extended the lag phase of the hydrogen production profile, but the slight increase in the initial bacterial optical density enhanced the rate of hydrogen formation at the log phase. The waste culture after hydrogen production could be efficiently used in one-pot mass scale green synthesis of silver nanoparticles. The waste culture after hydrogen production was autoclaved and centrifuged. The supernatant containing the bacterial cell extracts after autoclaving and the fermentation residual reducing sugars was used for bioreduction of silver ions into nanoparticles at 15 psi and 121 °C for only 5 min. Silver nanoparticles with a size range of 5–25 nm and an average nanoparticles size of 15.6 ± 2.46 nm, as analyzed using transmission electron microscopy, were obtained at an optimum of pH 7 and 10 mM silver nitrate. The synthesized nanoparticles had a reddish-brown color in aqueous solution and showed the typical wavelength spectrum of silver nanoparticles with a maximum wavelength of 430 nm. The use of the waste culture after hydrogen production for fast one-pot mass scale green synthesis of silver nanoparticles suggests interlinking the two biotechnologies in future applications to increase the economic feasibility of both biotechnologies.

DNA sequencing diagnosis of off-season spirochetemia with low bacterial density in Borrelia burgdorferi and Borrelia miyamotoi infections.

A highly conserved 357-bp segment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.

Biodegradation of the Antituberculosis Drug Isoniazid in the Aquatic Environment

As part of our study on pharmaceutical waste management in Ghana, we investigated the biodegradability of the most widely used antituberculosis drug isoniazid in the aquatic environment. The experiment was conducted using three tests from the Organization for Economic Co‐operation and Development guidelines (close bottle test (CBT) 301D, low bacterial density i.e. &lt;1000 colony forming units (CFU) mL−1, manometric respiratory test (MRT) 301 F, i.e. &lt;1000 CFU mL−1 and the Zahn‐Wellens test (ZWT) 302B, i.e. &lt;1000 CFU mL−1) employing different concentrations. At the end of the study, the results showed that isoniazid at concentrations of 4.29 mg L−1 for the CBT and 25.7 mg L−1 for the MRT were found not to be readily biodegradable in accordance with the test guidelines. In the ZWT, at a concentration of 142.92 mg L−1 which allows evaluation of aerobic degradation at high bacterial density, the results provided evidence for biological degradation of isoniazid. Finally, the test samples after 28 days analyzed by high performance liquid chromatography coupled to ultraviolet detector showed a complete primary elimination of the test substance in MRT and ZWT. The difference of the results obtained by HPLC and oxygen consumption in the MRT indicated the formation of stable transformation products, i.e. incomplete biodegradation, whilst there was no biodegradation at all in CBT.

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 105, 107 and 109 CFU ml−1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.