LB Medium Research Articles

Ex-vivo investigation of human salivary microbial growth with lysogeny broth for translational research–A pilot study

Background/purposeSalivary microbiome has become a surrogate indicator of oral disease due to its collective reservoirs and convenience in sampling. However, failed clinical trials often lead to wastes of resources, indicating a need for preclinical models. In this pilot study, we aimed to compare the salivary microbiome by metagenomics analysis before and after lysogeny broth culture for prospective translational studies. Materials and methodsThe study cohort included seven patients with severe periodontitis (Stage III/IV, Grade C), from whom unstimulated saliva was collected. The salivary microbiome was sequenced over the 16S rRNA gene V3–V4 hypervariable regions at baseline and after 6 hours of lysogeny broth culture. ResultsThe results revealed changes in salivary microbiome and reduced bacterial diversity after culture, mainly due to the expansion of genera Neisseria (Median (Mdn) 15.95% to 37.52%, P < 0.05), Rothia (Mdn 10.21% to 16.32%, P < 0.05), and Haemophilus (Mdn 5.88% to 13.25%, P < 0.05). Periodontitis-related pathogens such as phyla Bacteroidetes, Fusobacteria and Spirochaetes were identified, while genera Porphyromonas, Parvimonas, Peptostreptococcus, and Campylobacter showed a decrease after lysogeny broth culture. Caries-related pathogens, including genera Veillonella, Leptotrichia, and species Haemophilus parainfluenzae and Streptococcus salivarius, were also detected. ConclusionThis pilot study revealed that periodontitis- and caries-related bacteria could be identified in the saliva at baseline and after 6 hours ex-vivo culture with lysogeny broth. Our findings also suggested that lysogeny broth favored the growth of specific genera and may serve as a reference to monitor short-term modulation of these bacteria in salivary microbiome.

A novel chaperone-effector-immunity system identified in uropathogenic Escherichia coli UMN026.

Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75-95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi2 test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN+ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN-) and E. faecalis but not against isolate 4012 (ECUMN+). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

Application of UV LEDs to inactivate antibiotic resistant bacteria: Kinetics, efficiencies, and reactivations

Unregulated antibiotic use has led to the proliferation of antibiotic-resistant bacteria (ARB) in aquatic environments. Ultraviolet light-emitting diodes (UV LEDs) have evolved as an innovative technology for inactivating microorganisms offering several advantages over traditional mercury lamps. This research concentrated on utilizing UV LEDs with three distinct wavelengths (265 nm, 275 nm, and 285 nm) to inactivate E. coli DH10β encoding the ampicillin-resistant blaTEM-1 gene in its plasmid. Non-linear models, such as Geeraerd's and Weibull, provided more accurate characterization of the inactivation profiles than the traditional log-linear model due to the incorporation of both biological mechanisms and a deterministic approach within non-linear models. The inactivation rates of ARB were higher than antibiotic-sensitive bacteria (ASB) when subjected to UV LEDs. The highest inactivation rates were observed when all microorganisms were exposed to 265 nm. Photoreactivation emerged as the primary mechanism responsible for repairing DNA damage induced by UV LEDs. 285 nm showed the highest reactivation efficiencies for ARB under different fluences. At higher fluences, both 265 and 275 nm displayed similar effectiveness in suppressing reactivation, while at lower fluences, 275 nm exhibited better efficacies in controlling the reactivation. Therefore, the inhibition of reactivation was influenced by the extent of damage incurred to both DNA and enzymes. In nutrient-poor media (0.9 % NaCl), ASB did not exhibit any reactivation potential. However, the addition of Luria-Bertani (LB) broth promoted the reactivation of ASB. Lower fluence rate was more beneficial at 265 nm whereas higher fluence rates were more effective for longer wavelengths. The inactivation of ARB was enhanced by dissolved organic carbon (DOC) at low fluences. However, the removal of ARB was reduced due to the presence of DOC at higher fluences. The highest energy demand for ARB inactivation was reported at 285 nm. Environmental implicationThe excessive and unregulated utilization of antibiotics has emerged as a significant issue for public health. This paper presents a comprehensive analysis of the effectiveness of UV LEDs, an emerging technology, in the inactivation of antibiotic-resistant bacteria (ARB). This research paper explores the kinetics of UV LEDs with different wavelengths to inactivate ARB along with the reactivation efficiencies. This research work also explores the impact and relevant mechanisms of the impact of dissolved organic carbon (DOC) on the inactivation of ARB by UV LEDs.

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 μg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 μg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 μg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 μg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Biodegradation of polyurethanes by Staphylococcus warneri and by microbial co-culture

With the increasing production and use of polyurethanes (PUs), it is necessary to develop sustainable techniques for the remediation of plastic pollution. The use of microorganisms capable of biodegrading PUs may be an environmentally desirable solution for controlling these plastic contaminants. To contribute to the discovery of alternatives for the mitigation of plastics in the environment, this study aimed to explore the potential of StaphylococcuswarneriUFV_01.21, isolated from the gut of Galleria mellonellalarvae, for biodegradation of PU in pure culture and microbial co-culture with Serratia liquefaciensL135. S. warneri grew using Impranil® PU as the sole carbon source in pure culture and co-culture. With six days of incubation, the biodegradation of Impranil® in Luria Bertani broth was 96, 88 and 76%, while in minimal medium, it was 58, 54 and 42% for S. warneri, S. liquefaciens, and co-culture, respectively. In addition, S. warneri in pure culture or co-culture was able to biodegrade, adhere and form biofilms on the surfaces of Impranil® disks and poly[4,4′-methylenebis (phenyl isocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone] (PCLMDI) films. Scanning electron microscopy also revealed biodegradation by detecting the formation of cracks, furrows, pores, and roughness on the surfaces of inoculated PU, both with pure culture and microbial co-culture. This study is the first to demonstrate the potential of S. warneriin PU biodegradation.

Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

The roles of extrinsic neurons in modulating neuroimmune crosstalk during acute enteric infection with Citrobacter rodentium

Background: The intestinal tract is one of the most densely innervated organs in our body, containing extrinsic sensory and sympathetic neurons. With their cell bodies located outside of the gut, these neurons directly detect enteric pathogens and release immunomodulatory neurotransmitters or neuropeptides to communicate with immune cells in the gut. Specifically, sympathetic neurons secrete neurotransmitter norepinephrine, which activate β2 adrenergic receptors (β2ΑR) on immune cells to regulate immune functions. Recent studies from our lab, as well as others, have begun to highlight the neural regulation of immune responses during enteric infections. However, the precise pathways responsible for orchestrating these effects remain elusive. Hypothesis: We sought to determine whether extrinsic sensory and sympathetic neurons are implicated in host protective responses against enteric pathogen Citrobacter rodentium (C. rodentium). Methods: Mice were inoculated by oral gavage with C. rodentium (108 colony-forming units) or sterile luria broth. Bacterial colonization was quantified by homogenization of distal colon tissues and plating serial dilutions onto MacConkey agar. Relevant tissues were collected for qPCR, flow cytometry or histology. Genetically engineered Arc Targeted Recombination in Active Population (TRAP) mice were used to identify neurons that were activated during infection. These ArcTRAP mice received single intraperitoneal (i.p.) injection of 4-hydroxytamoxifen post-infection to “trap” active neurons, marking them tdTomato+. 6-hydroxydopamine (6OHDA) was used to chemically ablate sympathetic innervations to the colon via 3 consecutive daily i.p. injections. Results: At day 4 post-infection, there were significantly more active tdTomato+ neurons in the rostral ventrolateral medulla (RVLM) of the infected mice compared to the non-infected controls. This increase was more pronounced as the infection peaked at day 10, indicating that more neurons were being activated in the RVLM in response to C. rodentium in the gut. 6OHDA treatment significantly increased bacterial burden in distal colon at day 10 post-infection. This was associated with decreased tyrosine hydroxylase expression compared to the vehicle controls. Subsequently, qPCR revealed that there was increased pro-inflammatory cytokines expression in the colon of sympathectomized infected mice compared to the infected controls. Similarly, β2AR knockout significantly increased bacterial burden in distal colon at day 3 and 10 post-infection. Increased pro-inflammatory cytokines expression suggests an immunomodulatory role for β2AR during C. rodentium infection. Conclusions: These studies provided novel insights into the neuroimmune crosstalk that is crucial for acute enteric infections with C. rodentium. Specifically, these experiments may reveal how extrinsic neurons shape local immune responses in the gut. NIH NIAID R01AI150647; Graduate Student Support Program (UC Davis: School of Veterinary Medicine). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In vitro analysis of various antibiotic and cement combinations against S. epidermidis and S. lugdunensis for treatment of periprosthetic shoulder infection

BackgroundPeriprosthetic joint infections (PJIs) are devastating potential complications after arthroplasty surgery with significant therapeutic challenges. Many authors advocate for two-stage revision arthroplasty for PJI of the shoulder which includes explanation with antibiotic spacer placement. The optimal antibiotic/cement combination for PJI in the shoulder is not known. The purpose of this study is to analyze various cement and antibiotic concentrations against Coagulase negative staphylococcus species, S. epidermidis and S. lugdunensis. MethodsFive strains of coagulase negative Staph were taken from clinically documented orthopedic infections Both Simplex and Palacos cement were used to create antibiotic laden beads and mixed with either ertapenem, vancomycin, gentamicin, or a combination of ertapenem + vancomycin or ertapenem + gentamicin. The bacteria were plated on lysogeny broth agar plates and 3 beads were added per plate. Samples were analyzed for zone of inhibition at 24 hours, 72 hours, and 1 week. After 1 week, beads were transferred to new plates with bacteria and the process was repeated for 6 weeks. ResultsResults showed that ertapenem beads with both Simplex and Palacos cement showed the largest zones of inhibition for all samples. Vancomycin in Palacos cement and vancomycin in combination with ertapenem in Simplex and Palacos cement showed consistent zones of inhibition for the duration of the experiments. Ertapenem in combination with either vancomycin or gentamicin may allow for a powerful initial burst of killing followed by consistent antibiotic elution as opposed to gentamicin alone. ConclusionWhile many premade spacers on the market are infused with gentamicin, our in vitro models demonstrate more efficacious bacterial eradication for antibiotics such as ertapenem and vancomycin specifically for certain low virulence organisms that are commonly found in PJI around the shoulder.

ANTIMICROBIAL ACTIVITY OF OILS FROM PUMKIN SEEDS, GRAPE SEEDS AND SEA-BUCKTHORN FRUITs

Currently, the search for new alternative effective sources for obtaining compounds with antimicrobial activity does not lose its relevance. Aim. To investigate the antimicrobial activity of oils obtained by cold pressing from pumpkin seeds, grape seeds and sea-buckthorn fruits. Methods. Antimicrobial activity of oils from pumpkin seeds, grape seeds and sea-buckthorn fruits against strains of indicator microorganisms Staphylococcus aureus АТСС 25923, Micrococcus luteus АТСС 4698, Enterococcus faecalis АТСС 29212, Bacillus subtilis АТСС 6633, Kocuria rhizophila DSM 3480, Escherichia coli АТСС 25922, Proteus vulgaris АТСС 6896, Salmonella enterica NCTC 6017, Klebsiella pneumoniae АТСС 10031, Pseudomonas aeruginosa АТСС 27853, Pseudomonas putida KT 2440 and Candida albicans АТСС 18804 was determined on semi-liquid LB medium by the well method. Results. All tested plant oils showed antimicrobial activity against certain strains of Gram-positive and Gram-negative bacteria, but were ineffective against B. subtilis, P. aeruginosa, P. putida and the eukaryotic microorganism C. albicans. Strains of Gram-positive bacteria were more sensitive to the action of all three types of plant oils. The most sensitive strain was M. luteus ATCC 4698, the size of which zones of no growth exceeded 16 mm under the influence of all tested oils. Pumpkin seeds oil was more effective against sensitive test strains of indicator microorganisms. Conclusions. Plant oils obtained by cold pressing from pumpkin seeds, grape seeds and sea-buckthorn fruits showed variable antimicrobial activity, which was better in pumpkin seeds oil. K. rhizophila DSM 3480 and M. luteus ATCC 4698 strains were the most sensitive to its influence, the sizes of the zones of no growth were 16.5 ± 0.1 mm and 16.3 ± 0.2 mm, respectively. Further researches are necessary to determine the chemical composition and quantitative assessment of the bioactive compounds of these oils, as well as the mechanisms of their action on the cells of microorganisms.

Deletion of yghZ in Escherichia coli promotes growth in presence of furfural with xylose as carbon source.

Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain-SSK201-was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96h and in presence of 1.0g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

Volatile Fatty Acids Effective as Antibacterial Agents against Three Enteric Bacteria during Mesophilic Anaerobic Incubation.

The antibacterial effects of a selection of volatile fatty acids (acetic, propionic, butyric, valeric, and caproic acids) relevant to anaerobic digestion were investigated at 1, 2 and 4 g/L. The antibacterial effects were characterised by the dynamics of Enterococcus faecalis NCTC 00775, Escherichia coli JCM 1649 and Klebsiella pneumoniae A17. Mesophilic anaerobic incubation to determine the minimum bactericidal concentration (MBC) and median lethal concentration of the VFAs was carried out in Luria Bertani broth at 37 °C for 48 h. Samples collected at times 0, 3, 6, 24 and 48 h were used to monitor bacterial kinetics and pH. VFAs at 4 g/L demonstrated the highest bactericidal effect (p < 0.05), while 1 g/L supported bacterial growth. The VFA cocktail was the most effective, while propionic acid was the least effective. Enterococcus faecalis NCTC 00775 was the most resistant strain with the VFAs MBC of 4 g/L, while Klebsiella pneumoniae A17 was the least resistant with the VFAs MBC of 2 g/L. Allowing a 48 h incubation period led to more log decline in the bacterial numbers compared to earlier times. The VFA cocktail, valeric, and caproic acids at 4 g/L achieved elimination of the three bacteria strains, with over 7 log10 decrease within 48 h.

Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium.

Cysteine and its derivatives, including H2S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H2S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H2S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H2S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Complete genome sequence and genome-wide transposon mutagenesis enable the determination of genes required for sodium hypochlorite tolerance and drug resistance in pathogen Aeromonas veronii GD2019

Aeromonas veronii, a significant pathogen in aquatic environments, poses a substantial threat to both human and animal health, particularly in aquaculture. In this study, we isolated A. veronii strain GD2019 from diseased largemouth bass (Micropterus salmoides) during a severe outbreak of aeromonad septicemia in Guangdong Province, China. The complete genome sequence of A. veronii GD2019 revealed that GD2019 contains a single chromosome of 4703,168 bp with an average G+C content of 58.3%. Phylogenetic analyses indicated that GD2019 forms a separate sub-branch in A. veronii and comparative genomic analyses identified the existence of an intact Type III secretion system. Moreover, to investigate the genes that are required for the conditional fitness of A. veronii under various stresses, a high-density transposon insertion library in GD2019 was generated by a Tn5-based transposon and covers 6311 genomic loci including 4155 genes and 2156 intergenic regions. Leveraging this library, 630 genes were classified as essential genes for growth in rich-nutrient LB medium. Furthermore, the genes GE001863/NtrC and GE002550 were found to confer tolerance to sodium hypochlorite in A. veronii. GE002562 and GE002614 were associated with the resistance to carbenicillin. Collectively, our results provide abundant genetic information on A. veronii, shedding light on the pathogenetic mechanisms of Aeromonas.

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

Bioprospecting of extremophilic perchlorate-reducing bacteria: report of promising Bacillus spp. isolated from sediments of the bay of Cartagena, Colombia

Three extremophile bacterial strains (BBCOL-009, BBCOL-014 and BBCOL-015), capable of degrading high concentrations of perchlorate at a range of pH (6.5 to 10.0), were isolated from Colombian Caribbean Coast sediments. Morphological features included Gram negative strain bacilli with sizes averaged of 1.75 × 0.95, 2.32 × 0.65 and 3.08 × 0.70 μm, respectively. The reported strains tolerate a wide range of pH (6.5 to 10.0); concentrations of NaCl (3.5 to 7.5% w/v) and KClO4− (250 to 10000 mg/L), reduction of KClO4− from 10 to 25%. LB broth with NaCl (3.5–30% w/v) and KClO4ˉ (250-10000 mg/L) were used in independent trials to evaluate susceptibility to salinity and perchlorate, respectively. Isolates increased their biomass at 7.5 % (w/v) NaCl with optimal development at 3.5 % NaCl. Subsequently, ClO4ˉ reduction was assessed using LB medium with 3.5% NaCl and 10000 mg/L ClO4ˉ. BBCOL-009, BBCOL-014 and BBCOL-015 achieved 10%, 17%, and 25% reduction of ClO4ˉ, respectively. The 16 S rRNA gene sequence grouped them as Bacillus flexus T6186-2, Bacillus marisflavi TF-11 (T), and Bacillus vietnamensis 15 − 1 (T) respectively, with < 97.5% homology. In addition, antimicrobial resistance to ertapenem, vancomycine, amoxicillin clavulanate, penicillin, and erythromycin was present in all the isolates, indicating their high adaptability to stressful environments. The isolated strains from marine sediments in Cartagena Bay, Colombia are suitable candidates to reduce perchlorate contamination in different environments. Although the primary focus of the study of perchlorate-reducing and resistant bacteria is in the ecological and agricultural realms, from an astrobiological perspective, perchlorate-resistant bacteria serve as models for astrobiological investigations.

Brilliant green decolorization by dye decolorizing peroxidase producing bacterium, Achromobacter insolitus isolated from Forest soils of Similipal Biosphere Reserve, Odisha

Dye-decolorizing peroxidases (DyPs) are recently identified superfamily of heme-containing peroxidases which has received much attention for its potential application in dye decolourization. In the current research, six bacterial colonies (SDB1-6) showing lignolytic activity were isolated from forest soils of SBR using a minimal salt medium supplemented with alkali lignin and screened for DyP activity using brilliant green dye supplemented luria bertani agar medium. The highest DyP activity showing bacterium was identified using biochemical and 16SrRNA sequencing and was identified as Achromobacter insolitus. A. insolitus was found to be an efficient strain showing the highest DyP activity of 2.7 U/mL/ml at un-optimized condition. The RSM optimization was conducted to increase the DyP enzyme activity. A. insolitus demonstrated DyP activity of 6.20 U/mL/min with an increase of 2.29 times of enzyme activity in optimal condition of pH 7.0, temperature 25 °C, H2O2 concentration 0.1 mM, and time 24 h. The bacterium was further screened for dye decolorization taking crystal violet, methyl orange, methylene blue, brilliant green and methyl red dyes supplemented in LB medium. The bacterium showed maximum brilliant green degradation ability was analyzed using UV-Vis and FTIR analyses to characterize the degraded product. The highest brilliant green degradation activity of the A. insolitus in the present study could be exploited for bioremediation of brilliant green contaminated industrial effluents in a ecofriendly and cost effective way.

Increased susceptibility to azithromycin of Pseudomonas aeruginosa biofilms using RPMI 1640 testing media.

Azithromycin (AZM) is efficient for treatment of chronic Pseudomonas aeruginosa biofilm lung infections, despite of resistance in conventional susceptibility testing. It has been shown that planktonic P. aeruginosa are more susceptible to AZM when tested in RPMI 1640 medium. The aim of the study was to test the susceptibility to AZM of P. aeruginosa biofilms in LB vs RPMI 1640 media. We investigated the effect of AZM on planktonic and biofilms of (WT) P. aeruginosa (PAO1), the hypermutable (ΔmutS) and the antibiotic-resistant phenotype(ΔnfxB) mutants. The effect of AZM on young and mature biofilms was investigated in the modified Calgary Biofilm Device by estimation of the minimal biofilm inhibitory concentration (MBIC). The AZM MBIC90 in LB/RPMI1640 on young biofilms treated for 24 h was 16/4 μg/mL for PAO1, 32/8 μg/mL for ΔmutS, and 256/16 μg/mL for ΔnfxB, while in mature biofilms was 256/2 μg/mL for PAO1 and ΔmutS and 16/1 μg/mL for ΔnfxB. The effect of AZM was improved when the treatment was prolonged to 72 h, supporting the intracellular accumulation of AZM. An increased susceptibility of P. aeruginosa biofilms to AZM was observed in RPMI 1640 than in LB medium. Our results might improve susceptibility testing and dosing of AZM for treatment of biofilm infections.

Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases.

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.

Abstract 239: Establishing a murine intratumoral bacterial model

Abstract Background: Long thought to be sterile spaces, current evidence now suggests that host microbiota can be found within cancerous tissue. Emerging data has illuminated that microbes are integral components of tumor tissue itself, which may correlate with tumor progression, treatment responsiveness, and disease prognosis. A recent Cell article from Fu et al. demonstrated that tumor-resident intracellular microbiota can promote metastatic colonization in breast cancer. Others within this research landscape see therapeutic opportunity - harnessing the tropism of these microbes to deliver TME modifying payloads directly to cancerous lesions. Purpose: Before studying the mechanism underlying their tropism to solid tumors, it is necessary to first establish a murine model that would allow for an oncotropic bacteria to be detected and quantified within tissues of interest. My research entailed the design and execution of pilot experiments developing xenograft melanoma and genetic colorectal models for future research projects. Methods: In all mouse studies, an E. coli Nissle 1917 strain called “NisLux” was used, who’s integration with a luxCDABE cassette allowed it to be selectively identified via luminescence using traditional exposure photography or endpoint luminescence detection using a 96-well plate reader. A xenograft melanoma model was developed using C57BL/6J mice and a B16-F10 murine melanoma line. A genetics model was developed using C57BL/6J-Apcmin mice which spontaneously develop colorectal adenomas as they age. Tumors, spleens, and healthy tissue were harvested and processed following tail vein injections of either NisLux or PBS solution. Harvested tissue was normalized by weight and concentration of resident NisLux was calculated from serially diluted samples plated on LB agar. Results: In both the xenograft and genetics murine models, NisLux exhibited tropism to tumor sites, enriching at a concentration above that of healthy background tissue. NisLux dosing correlated with splenomegaly without changes to the mass of the primary lesion compared to control. Conclusions: NisLux exhibits tropism to tumor sites in our xenograft and genetics murine models. Both models should undergo further procedural and control optimization. Two possible modifications to future melanoma xenograft experiments include the collection of non-cancerous skin negative control samples and a further delayed takedown day for tissue processing. In future C57BL/6J-Apcmin mice experiments, healthy colon sections from NisLux dosed C57BL/6J mice should be used as negative controls. Overall, the results from both models were promising and encourage future exploration of the mechanism underlying NisLux’s tropism to solid tumors. Subsequent experiments could involve comparing the tropism of engineered KO strains to WT NisLux or performing comparative sequencing of isolated tumor resident NisLux to the initial dosing population. Citation Format: Ryan A. Hannon, Dongqing Xu, Fengyi Wan. Establishing a murine intratumoral bacterial model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 239.

The metabolic pathway of clodinafop-propargyl degradation by consortium WP and its bacterial diversity analysis

Clodinafop-propargyl (CP) is a widely used post-emergence aryloxyphenoxy propionate (AOPP) herbicide and causes serious environmental problems. The elimination of CP from the environment is urgently required, but there are few reports on microbial degradation of CP. In this study, a bacterial consortium WP was enriched from the activated sludge of CP containment, which could completely degrade 50 mg L−1 of CP within 7 days. UPLC-MS analysis showed that the main intermediate metabolites of CP degradation were clodinafop acid (CA), 4-(5-chloro-3-fluoro-2-pyridyloxy) phenol, and 5-chloro-3-fluoropyridin-2-ol. And the metabolic pathway of CP degradation by consortium WP has been proposed. High-throughput sequencing revealed that the consortium WP had rich bacterial diversity, including 4 phyla, 14 classes, and 148 genera. Ten bacteria capable of converting CP to CA were isolated from the consortium WP using LB medium containing CP, and the strain Pseudoxanthomonas sp. WP66 had the strongest conversion ability, which could utilize CP as the sole source for growth and degrade 91.20% of 100 mg L−1 CP within 15 h. This study presented the main degradation pathways of CP by microorganisms, and provided resource of bacterial strain for the removal of CP from the environment.