Bacterial Numbers Research Articles

Human airway epithelium controls Pseudomonas aeruginosa infection via inducible nitric oxide synthase

IntroductionAirway epithelial cells play a central role in the innate immune response to invading bacteria, yet adequate human infection models are lacking.MethodsWe utilized mucociliary-differentiated human airway organoids with direct access to the apical side of epithelial cells to model the initial phase of Pseudomonas aeruginosa respiratory tract infection.ResultsImmunofluorescence of infected organoids revealed that Pseudomonas aeruginosa invades the epithelial barrier and subsequently proliferates within the epithelial space. RNA sequencing analysis demonstrated that Pseudomonas infection stimulated innate antimicrobial immune responses, but specifically enhanced the expression of genes of the nitric oxide metabolic pathway. We demonstrated that activation of inducible nitric oxide synthase (iNOS) in airway organoids exposed bacteria to nitrosative stress, effectively inhibiting intra-epithelial pathogen proliferation. Pharmacological inhibition of iNOS resulted in expansion of bacterial proliferation whereas a NO producing drug reduced bacterial numbers. iNOS expression was mainly localized to ciliated epithelial cells of infected airway organoids, which was confirmed in primary human lung tissue during Pseudomonas pneumonia.DiscussionOur findings highlight the critical role of epithelial-derived iNOS in host defence against Pseudomonas aeruginosa infection. Furthermore, we describe a human tissue model that accurately mimics the airway epithelium, providing a valuable framework for systemically studying host-pathogen interactions in respiratory infections.

Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits.

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains a major health problem with 10.6 million cases of the disease and 1.6 million deaths in 2021. It is well understood that pulmonary TB is due to Mtb growth in the lung but quantitative estimates of rates of Mtb replication and death in lungs of patients or animals such as monkeys or rabbits remain largely unknown. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage, HN878, carrying an unstable plasmid pBP10. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted Mtb strain H37Rv (the segregation coefficient being s = 0.10 in HN878 vs. s = 0.18 in H37Rv). Interestingly, the kinetics of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of plasmid-bearing cells increased between 28 and 56 days post-infection and remained stable between 84 and 112 days post-infection despite a large increase in bacterial numbers in the lung at late time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication/death kinetics: one major population expanding early and being controlled/eliminated, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Importantly, a model with one kinetically homogeneous Mtb population could not explain the data including when the model was run stochastically. Given that in rabbits HN878 strain forms well circumscribed granulomas, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in future experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time. Taken together, our new data and mathematical modeling-based analyses illustrate differences in Mtb dynamics in mice and rabbits confirming a perhaps somewhat obvious observation that "rabbits are not mice".

Geographical distribution and driving force of microbial communities in the sediments of Diamantina and Kermadec trenches.

The distinctive geological characteristics of hadal trenches are recognized to affect the construction and ecological role of microbial communities; however, information on their population dynamics and assembly processes remains limited. In this study, bacteria and micro-eukaryotes in the sediments of the Diamantina and Kermadec trenches were explored utilizing high-throughput sequencing. Compared to the Diamantina Trench, significantly lower levels of bacterial and micro-eukaryotic biodiversity (p < 0.01), bacterial gene copy number (p < 0.05), and heterotrophic/parasitic micro-eukaryotic proportions (p < 0.05) were detected in the Kermadec Trench, which also exhibited a low community complexity based on the network analysis. Within each trench, no obvious population shifts were observed along the trench axis. Microbial communities in both trenches showed clear distance-decay distributions, mainly driven by stochastic processes. This study provided fresh perspectives on the microbial community assembly mechanism in deep-sea trenches. Studies of community complexity and diversified trophic states of microbes would contribute to an improved understanding of ecological functions and diversification in this extreme biosphere.

Modification of Antibacterial Copolymers on the Surface of PVA-Based Microfibers via Thermal Cross-Linking and Their Antibacterial Properties.

Bacterial infections on material surfaces are a serious public health concern worldwide. Although poly(vinyl alcohol) (PVA)-based materials have great potential as medical devices, they lack antibacterial properties on their surfaces and pose bacterial infection risks during implantation surgery. Copolymers containing antibacterial [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC) units were used to modify the surfaces of chemically cross-linked water-insoluble PVA-based microfibers. The copolymers also had carboxy units that were used to react with the hydroxy group of the PVA-based microfibers via a simple thermal treatment at 135 °C. PVA-based materials containing METAC units exhibit significant swelling due to electrostatic repulsions. Because the copolymers were modified on the extreme surface of the microfibers, no difference in the diameters between unmodified microfibers (PM-fiber) and copolymers with METAC unit-modified microfibers (PM-METAC-fiber), in both the dry and swollen states, was observed. The viable bacterial cell numbers, which were evaluated by colony counting, decreased by exposure to the poly(METAC-co-methacrylic acid (MAAc)) aqueous solution or PM-METAC-fibers. The value of CFU/mL decreased to 0.1% (against B. subtilis) and 3.9% (against E. coli) after contact with the PM-METAC-fibers compared to the PM-fibers. The percentage of hemolysis against rabbit red blood cells was equivalent to that of the negative control, suggesting that PM-METAC-fibers can selectively exhibit antibacterial properties. This modification method can be applied to various PVA-based materials if hydroxy groups are present on their surface. This study provides a facile, cost-effective, and promising strategy to impart antibacterial properties to the surface of PVA-based materials without significantly affecting their physicochemical properties.

Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs

BackgroundFlock-level prevalence and characterization of Mycoplasma ovipneumoniae is determined almost exclusively using nasal swabbing followed by molecular detection with either quantitative PCR or multi-locus sequence typing. However, the diagnostic performance and efficiency of swabbing the nasal passage compared to other anatomical locations has not been determined within sheep populations. The goal of this research was to assess the diagnostic capability of nasopharyngeal swabs in comparison to nasal swabs for the detection of Mycoplasma ovipneumoniae.ResultsNasal and nasopharyngeal swabs were collected during a controlled exposure study of domestic sheep with Mycoplasma ovipneumoniae. Both swab types were then analyzed via conventional and quantitative PCR. This dataset showed that the use of nasopharyngeal swabs in lieu of nasal swabs resulted in higher sensitivity, reduced inhibition during quantitative PCR, and higher bacterial copy numbers per swab. Moreover, it was demonstrated that diagnostic sensitivity could be further increased during quantitative PCR via ten-fold dilution of the extracted DNA. To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock.ConclusionsResults demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. This demonstrates that, in addition to anatomical location, the presence of inhibitory components in swab extracts also strongly influences diagnostic performance.

Façade eluates affect active and total soil microbiome

The application of biocides in building materials has become a prevalent practice to mitigate the growth of microorganisms such as algae, fungi, and bacteria on the façades. These can leach out from the material and reach the nearby soil environment. This study aimed to characterize the effect of façade eluates generated within different leaching experiments on total and metabolic active soil microbial community composition and functions. Façade eluates were produced by immersion testing DIN EN 16105 and a natural weathering experiment. Afterward, soil microcosms were treated with the respective façade eluate and incubated for 29 days. Subsequently, the active and total soil microbial community compositions were investigated.Fungal internal transcribed spacer region gene and bacterial 16S rRNA gene were sequenced for active (bromodeoxyuridine labeled DNA) microbial community and total community. Façade eluates reduced total bacterial and fungal gene copy numbers. Overall, active bacterial and fungal richness was reduced and altered in community composition in comparison to the total richness and composition, respectively. Façade eluates retrieved of façade samples without biocides did alter the soil microbial communities to the same extent as façade eluates with biocides. Additionally, members of the active microbiome that benefit from the presence of façade eluates and omitted ones could be identified. Our result demonstrated that façade eluates affect active and total soil microbial community composition and function regardless of the leaching procedure and biocides addition.

Laser NIR Irradiation Enhances Antimicrobial Photodynamic Inactivation of Biofilms of Staphylococcus aureus.

Photodynamic inactivation (PDI) is a powerful technique for eradicating microorganisms, and our group previously demonstrated its effectiveness against planktonic cultures of Staphylococcus aureus bacteria using 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and visible light irradiation. However, biofilms exhibit a lower sensitivity to PDI, mainly due to limited penetration of the photosensitizer (PS). In the context of emerging antibacterial strategies, near-infrared treatments (NIRTs) have shown promise, especially for combating resistant strains. NIRT can act either through photon absorption by water, causing a thermal effect on bacteria, or by specific chromophores without a significant temperature increase. Our objective was to enhance biofilm sensitivity to TAPP-PDI by pretreatment with NIRT. This combined approach aims to disrupt biofilms and increase the efficacy of TAPP-PDI against bacterial biofilms. In vitro biofilm models of S. aureus RN6390 were utilized. NIRTs involved a 980 nm laser (continuous mode, 7.5 W/cm2, 30 s, totaling 225 J/cm2) post-TAPP exposure to enhance photosensitizer accumulation. Subsequent visible light irradiation at 180 J/cm2 was employed to perform PDI. Colony-forming unit counts evaluated the synergistic effect on bacterial viability. Scanning electron microscopy visualized the architectural changes in the biofilm structure. TAPP was extracted from bacteria to estimate the impact of NIRT on biofilm penetration. Using in vitro biofilm models, NIRT application following biofilm exposure to TAPP increased PS accumulation per bacteria. Under these conditions, NIRT induced a transient increase in the temperature of PBS to 46.0 ± 2.6°C (ΔT = 21.5°C). Following exposure to visible light, a synergistic effect emerged, yielding a substantial 4.4 ± 0.1-log CFU reduction. In contrast, the PDI and NIRT treatments individually caused a decrease in viability of 0.9 ± 0.1 and 0.8 ± 0.2-log respectively. Interestingly, preheating TAPP-PBS to 46°C had no significant impact on TAPP-PDI efficacy, suggesting the involvement of thermal and nonthermal effects of NIR action. In addition to the enhanced TAPP penetration, NIRT dispersed the biofilms and induced clefts in the biofilm matrix. Our findings suggest that NIR irradiation serves as a complementary treatment to PDI. This combined strategy reduces bacterial numbers at lower PS concentrations than standalone PDI treatment, highlighting its potential as an effective and resource-efficient antibacterial approach.

Optimization of antimicrobial properties of essential oils under rotating magnetic field

Essential oils (EOs) extracted from Thymus vulgaris L. (TEO) and Rosmarinus officinalis L. (REO) have aroused interest in their application to food preservation or in alternative medicine or aroma-therapy. In this study, the chemical compositions of essential oils were determined and analyzed. The purpose of the investigation was to investigate in detail the activity of essential oils extracted from herbs (thyme and rosemary) and exposed to rotating magnetic filed (RMF) against the model Gram-negative bacteria Escherichia coli The bacterial removal has been optimized by a surface response methodology (RSM). It was shown that TEO in a concentration of 10 µL/50 mL of water resulted in a total bacterial number reduction after 40 min under the RMF. Rosemary's antibacterial effect was much weaker. Thus, we summarized that the rotating magnetic fields at a frequency of 27 Hz can increase the antimicrobial efficiency but the effect depends on the type of essential oil. High-rate bacteria removal was obtain for thyme oil in concentration of 30 µL of thyme oil to 50 mL of bacterial suspension, exposed to RMF at a frequency of 27 Hz for 40–60 min.

Assessment of amoxicillin biodegradation using a fluidized bed reactor

In recent decades, emerging pharmaceutical contaminants, such as personal care items, pharmaceuticals, and disinfectants, have increased alarmingly in aquatic ecosystems. The efficacy of wastewater treatment plants in eliminating these contaminants has been demonstrated. This study aims to find an alternative method, such as using the fluidized bed reactor (FBR) with bacteria to remove amoxicillin from wastewater. The active bacteria Acinetobacter baumannii and Klebsiella pneumoniae were isolated from one of the pharmaceutical factories in Baghdad, Iraq, from Al-Jazeera wastewater, which was used in FBR technology to decompose the antibiotic amoxicillin. The study examined several operational parameters, including pH, total organic carbon (TOC), dissolved carbon dioxide (CO2) concentration, airflow rate, amoxicillin concentration, and bacterial number, while maintaining a constant temperature. High-performance liquid chromatography (HPLC) was used to find the reaction route. The maximum amoxicillin removal efficiency was 93% and 91% for Acinetobacter baumannii and Klebsiella pneumoniae, respectively. For both bacterial species, the pH decreased due to the formation of amino acids and CO2. The suitable operation condition was 5 mg/L of amoxicillin concentration, flow rate of 30 m/s, and the number of bacteria 47 x 105 cfm/mL and 40 x 105 cfm/mL for Acinetobacter baumannii and Klebsiella pneumoniae, respectively. After treatment, the number of bacteria increased due to the degradation of amoxicillin. The steady-state time is found at 64 hours. The organic matter reaction pathway ends at the aromatic amino acid lysine, which kills gram-negative bacteria. This method was found to be successful in removing amoxicillin in low concentrations by using Acinetobacter baumannii. In the future, treating amoxicillin in wastewater using FBR technology with gram-positive bacteria is recommended.

Calcium sorption and isotope fractionation in Bacillus subtilis and Pseudomonas aeruginosa

Bacteria are a key component of the critical zone, because of their role in the nutrient availability for the vegetation. There is still little knowledge on the direct role of bacteria on Ca storage/leaching in soils while it is an essential macronutrient for vegetation growth. In recent years, Ca stable isotopes have shown their potential in understanding the Ca biogeochemical cycle. Preliminary studies highlighted that in the presence of soil bacteria, the plant uptake of nutrients is increased due to the mineral bioweathering. Moreover, Ca isotope signatures of nutrient media also showed differences between growth experiments in batch in the presence and absence of bacteria. In this study, the focus is to verify if Ca adsorption and incorporation into/onto bacterial strains induce such isotopic fractionation. Batch experiments were carried out on Pseudomonas aeruginosa (a Gram-negative bacterium) and on the vegetative and sporulated forms of Bacillus subtilis (a Gram-positive bacterium). These experimentations showed that: (i) no observable isotopic fractionations were induced during calcium/bacteria contact for all experimental parameters (pH, kinetic, bacterial cell number, interaction time, dead/alive bacteria); (ii) Ca was mainly stored in the bacterial cell wall compartments. On the other hand, significant Ca isotopic differences between the spores and the sporulation medium (Δ44/40Caspores–sporulation medium ranging from − 0.53 to − 1.15‰), suggest isotopic fractionation during the sporulation process, likely occurring during the attachment of Ca to carboxyl acid groups as calcium chelates with dipicolinic acid. The absence of Ca isotope fractionation during Ca sorption on vegetative and sporulated bacteria via passive channels indicates that the tested bacteria’s contribution to the Ca biogeochemical cycle is indirect primary enhancing bioweathering and Ca bioavailability for vegetation. If confirmed by further studies, only the sporulation mechanisms itself may directly impact the Ca biogeochemical cycle.

Diffusive Phyllosphere Microbiome Potentially Regulates Harm and Defence Interactions Between Stephanitis nashi and Its Crabapple Host.

Pear lace bug (Stephanitis nashi) is a significant herbivorous pest, harbouring a diverse microbiome crucial for crabapple (Malus sp.) host adaptation. However, the mutual influence of S. nashi- and plant-associated microbiomes on plant responses to pest damage remains unclear. This study found that S. nashi damage significantly altered bacterial community structure and reduced bacterial evenness in the crabapple phyllosphere. Notably, bacterial diversity within S. nashi was significantly lower than that in the environment, potentially influenced by insect developmental stage, bacterial diffusion stage and endosymbiont species number and abundance. Extensive bacterial correlation and diffusion effect between S. nashi and adjacent plant environments were observed, evident in a gradual decrease in bacterial diversity and an increase in bacterial acquisition ratio from soil to phyllosphere to S. nashi. Correspondingly, S. nashi significantly impacted the metabolic response of crabapple leaves, altering pathways involved in vitamin, amino acid and lipid metabolism and so forth. Furthermore, association analysis linked these metabolic changes to phyllosphere bacterial alterations, emphasizing the important role of diffusive phyllosphere microbiome in regulating S. nashi-crabapple interactions. This study highlights bacterial diffusion effect between insect and plants and their potential role in regulating insect adaptability and plant defence responses, providing new insights into plant-insect-microbiome interactions.

The Decontamination Effect of an Oscillating Chitosan Brush Compared With an Ultrasonic PEEK-Tip: An InVitro Study Using a Dynamic Biofilm Model.

This study aimed to assess the effect of an oscillating chitosan brush (OCB) compared with an ultrasonic device with PEEK tip (US-PEEK) for mechanical implant surface decontamination using an invitro model combining 3D models and a validated dynamic multispecies biofilm. A multispecies biofilm using six bacterial strains (Streptococcus oralis, Veillonella parvula, Actinomyces naeslundii, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans) was seeded on dental implants with machined and sandblasted, large-grit and acid-etched (SLA) surfaces. These were installed in 3D models depicting peri-implant defect. Mechanical decontamination was performed for 120 s using either an OCB or a US-PEEK. A negative control group received no treatment. Scanning electron microscopy (SEM) was used to evaluate the bacterial composition and quantitative PCR (qPCR) analyzed the number of each bacterial species [colony-forming units per milliliter (CFU/mL)]. Well-structured biofilms with a dense microbial distribution were observed on the negative control implants after 72 h. qPCR following mechanical decontamination showed a scarce bacterial reduction in the OCB group. The US-PEEK group exhibited a significant decrease in bacterial species compared to both OCB and control groups (p < 0.05). A biofilm removal effect was also observed in the OCB group for the machined implant surfaces. In vitro assessment using an anatomical 3D model showed that mechanical decontamination effectively reduced biofilm. The US-PEEK group demonstrated biofilm reduction on the SLA surface, while the OCB group showed a reduction on the machined implant surface. Additionally, the US-PEEK group demonstrated greater efficacy in reducing bacterial numbers.

A double ttrA and pduA knock-out mutant of Salmonella Typhimurium is not attenuated for mice (Mus musculus).

Despite numerous studies on Salmonella enterica subsp. enterica serovar Typhimurium, the underlying mechanisms of several aspects of its virulence are still under investigation, including the role of the pdu and ttrA genes, associated with the metabolism of 1,2-propanediol using tetrathionate as an electron acceptor respectively. Our objective was to contribute to an understanding of the role of these genes inbacterial virulence for mice (Mus musculus) using an S. Typhumirum ΔttrApduA mutant. The experiment was conducted with a group infected by the S. Typhimurium mutant and a control group infected with a wild-type strain. The mutant was not attenuated compared with the parent strain. There were no differences in the bacterial numbers recovered from the mesenteric lymph nodes and Peyer's patches but at 8-day after oral infection higher numbers were recovered from the spleen, liver, and cecum. Unlike the single pduA and ttrA mutants, the double ΔttrApduA mutation did not affect invasion and survival in mice, which highlights the need for further studies to clarify the role of these important metabolism genes under reduced redox conditions linked to Salmonella virulence.

Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits.

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains a major health problem with 10.6 million cases of the disease and 1.6 million deaths in 2021. It is well understood that pulmonary TB is due to Mtb growth in the lung but quantitative estimates of rates of Mtb replication and death in lungs of patients or animals such as monkeys or rabbits remain largely unknown. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage, HN878, carrying an unstable plasmid pBP10. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted Mtb strain H37Rv (the segregation coefficient being s = 0.10 in HN878 vs. s = 0.18 in H37Rv). Interestingly, the kinetics of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of plasmid-bearing cells increased between 28 and 56 days post-infection and remained stable between 84 and 112 days post-infection despite a large increase in bacterial numbers in the lung at late time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication/death kinetics: one major population expanding early and being controlled/eliminated, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Importantly, a model with one kinetically homogeneous Mtb population could not explain the data including when the model was run stochastically. Given that in rabbits HN878 strain forms well circumscribed granulomas, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in future experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time. Taken together, our new data and mathematical modeling-based analyses illustrate differences in Mtb dynamics in mice and rabbits confirming a perhaps somewhat obvious observation that "rabbits are not mice".

The efficacy of antimicrobial solutions against multispecies bacterial biofilm with or without negative pressure wound therapy in an in vitro wound model.

Biofilm is the major challenge in chronic wound management. Instilling a wound cleansing solution aids in wound bed cleaning and infectious pathogen elimination. Negative pressure wound therapy (NPWT) improves the wound-healing process. This study investigated the efficacy of two antimicrobials (Vashe Wound Cleanser and Prontosan Wound Irrigation Solution) against a multispecies bacterial biofilm with or without NPWT in an in vitro wound model. A mixed multispecies biofilm containing Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, and Acinetobacter baumannii was developed and verified by scanning electron microscopy and fluorescent in situ hybridization. The efficacy of Vashe and Prontosan against multispecies biofilm with or without NPWT was evaluated by colony-forming unit (cfu) of each species and total bacterial number, and visually confirmed by live/dead stain and confocal microscopy. Prontosan reduced biofilm cell numbers significantly: 6 instils over 24 h resulting in 3.86 ± 0.14 cfu log10 reduction without NPWT and 4.75 ± 0.13 cfu log10 reduction combined with NPWT (P < 0.01) and 12 instils over 48 h resulting in 5.24 ± 0.11 cfu log10 reduction without NPWT and biofilm eradication with NPWT (P < 0.001). NPWT alone or combined with Vashe failed to reduce multispecies biofilm numbers significantly over 24 or 48 h. Prontosan significantly reduced biofilm cell numbers, with better efficacy over 48 than 24 h, emphasizing the necessity for persistent and robust treatment. NPWT enhanced the effectiveness of Prontosan instillation. However, NPWT alone or combined with Vashe showed limited efficacy and difficulty when combating the multispecies biofilm in vitro.

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

The infectious capacity of Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus saprophyticus in a porcine model of urinary tract infection.

The purpose of this study was to establish a porcine model of urinary tract infection (UTI) with gram-positive uropathogens. Ten female domestic pigs were experimentally inoculated with human UTI isolates of Enterococcus faecalis (n = 3), Staphylococcus saprophyticus (n = 3), or Staphylococcus aureus (n = 4) and followed with regular urine samples. Bladders and kidneys were aseptically removed at termination (5-7 days post infection) and assessed by gross pathology and bacterial enumeration. Enterococcus faecalis (n = 3 of 3) and S. aureus (n = 2 of 4) successfully colonized the pig bladders. Inoculation with S. saprophyticus never resulted in detectable bacteriuria. All infected pigs had cleared the infection spontaneously before termination. Surprisingly, three (of four) pigs inoculated with S. aureus led to spontaneous infection with opportunistic pathogens. Also, one pig colonized with E. faecalis resulted in spontaneous infection with E. coli. In conlusion, the pig supports experimental UTI with E. faecalis for up to 24 h but not prolonged infection. S. aureus and S. saprophyticus fails to cause UTI in pigs and other animals should be considered for studying these pathogens.

PSV-12 Evaluating the effect of lyoprotectants in preserving community structure and bacterial viability in the lyophilized fecal community of pigs

Abstract Fecal microbiota transplantation (FMT) stands as a potential method for enhancing pig health. FMT has progressed from the basic delivery of fresh stool, to lyophilized preparations which is beneficial for sample storage, but can damage bacterial cells. This has necessitated the need for lyoprotectants to preserve bacterial viability. However, the ideal lyoprotectant for retaining community structure and viability remains unclear. Furthermore, DNA-based microbial profiling methods such as 16S rRNA and metagenomics sequencing do not distinguish live and dead bacteria, prompting the need for other methods of distinguishing live and dead members of the bacterial community after lyophilization. PMA-seq (propidium monoazide treatment followed by 16S rRNA gene amplicon sequencing) has been shown to differentiate between live and dead bacteria in microbial communities. To refine FMT in pigs, we sought to ascertain the impact of four lyoprotectants on bacterial viability and fecal microbiome structure. Fecal samples from six pigs were treated with mannitol, maltodextrin, trehalose, maltodextrin-trehalose mix (2.5% each) as lyoprotectants or left untreated. Lyophilized fecal samples underwent PMA treatment prior to 16S rRNA sequencing to capture the active community, and total and viable bacterial cells number were quantified. One-way ANOVA was used to analyze total and viable bacterial cells, and richness, while PERMANOVA was used to compare beta diversity. Results revealed no initial differences in total, viable bacteria, or community structure (P &amp;gt; 0.05). However, untreated, and mannitol-treated groups showed significantly reduced total and viable bacterial cells post-lyophilization (P &amp;lt; 0.01), unlike the other lyoprotectants (P &amp;gt; 0.05). Mannitol and the untreated group also displayed decreased richness and beta diversity shifts (P &amp;lt; 0.05). Overall, lyophilization with mannitol or no lyoprotectant notably impacted pig fecal microbiota community structure, while maltodextrin, trehalose, and maltodextrin-trehalose effectively preserved bacterial viability and structure.

Impact of different illegal Artisanal Small-Scale Mining techniques on soil properties in a major mining landscape in Ghana

The widespread prevalence of illegal Artisanal Small-Scale Mining has raised significant concerns about its environmental and health impacts. This study investigates the effects of two mining methods (alluvial and chamfi) on soil physical, chemical, and microbial properties four years after mining in three municipalities of the Western Region of Ghana. The study identified four species of Aspergillus fungi (niger, flavus, ochraceous and fumigatus) and Trichoderma species in both mined and control soils. The quantity of A. fumigatus was significantly higher in control plots compared to mined plots. In alluvial sites, bacterial numbers were significantly higher in control plots than in mined plots, while in chamfi sites, bacterial numbers did not differ significantly between mined and control plots. Soil texture in both mined sites was predominantly sandy loam and loamy sand, with a generally high silt to clay ratio. Mined plots at both sites were significant and more acidic (p < 0.05) than the control plots. Additionally, cation exchange capacity (CEC), electrical conductivity (EC), organic carbon (OC), total nitrogen (TN), phosphorus (P), potassium (K+), calcium (Ca2+), and magnesium (Mg2+) levels were significantly higher in control plots compared to mined plots. The control plots also exhibited higher base saturation and lower exchangeable acidity than mined plots. Bacteria count at Chamfi sites was generally higher at mined areas than the control sites, with the reverse being the case for Alluvial sites. Soil chemical parameters were higher at control areas than at mined sites, irrespective of the mining approach used. However, there was no clear pattern of differences in soil chemical properties at Chamfi and Alluvial mined sites. This study provides insights and additional perspectives for improving decision-making and guiding the potential restoration of these areas.

Role of the JAK2/STAT3 pathway on infection of Francisella novicida.

Francisella tularensis is a causative agent of the zoonotic disease tularemia, and is highly pathogenic to humans. The pathogenicity of this bacterium is largely attributed to intracellular growth in host cells. Although several bacterial factors important for the intracellular growth have been elucidated, including the type VI secretion system, the host factors involved in the intracellular growth of F. tularensis are largely unknown. To identify the host factors important for F. tularensis infection, 368 compounds were screened for the negative regulation of F. tularensis subsp. novicida (F. novicida) infection. Consequently, 56 inhibitors were isolated that decreased F. novicida infection. Among those inhibitors, we focused on cucurbitacin I, an inhibitor of the JAK2/ STAT3 pathway. Cucurbitacin I and another JAK2/STAT3 inhibitor, Stattic, decreased the intracellular bacterial number of F. novicida. However, these inhibitors failed to affect the cell attachment or the intrasaccular proliferation of F. novicida. In addition, treatment with these inhibitors destabilized actin filaments. These results suggest that the JAK2/STAT3 pathway plays an important role in internalization of F. novicida into host cells through mechanisms involving actin dynamics, such as phagocytosis.