Killing Curves Research Articles

Bacterial persistence to antibiotics activated by tRNA mutations.

Bacterial persistence is a significant cause of the intractability of chronic and relapsing infections. Despite its importance, many of the underlying mechanisms are still not well understood. Antibiotic-tolerant mutants of Burkholderia thailandensis were isolated through exposure to lethal doses of AMP or MEM, followed by whole-genome sequencing to identify mutations. Subsequently, these mutants underwent comprehensive characterization via killing curves, growth curves, and persistence-fraction plots. Northern blot analysis was employed to detect uncharged tRNA, while the generation of relA and spoT null mutations served to confirm the involvement of the stringent response in this persistence mechanism. Phenotypic reversion of the persistence mutation was demonstrated by incubating the mutants without antibiotics for 2 weeks. We have discovered a novel mechanism of persistence triggered by specific mutations at positions 32 or 38 within the anticodon loop of tRNAAsp. This leads to heightened persistence through a RelA-dependent stringent response. Notably, this persistence can be easily reverted to wild-type physiology by losing the mutant tRNA allele within the tRNA gene cluster when persistence is no longer essential for survival. This distinct form of persistence underscores the novel function of tRNA mutations at positions 32 or 38 within the anticodon loop, as well as the significance of the tRNA gene cluster in conferring adaptability to regulate persistence for enhanced survival.

Coevolutionary analysis of Pseudomonas syringae-phage interactions to help with rational design of phage treatments.

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges invitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.

Novel synergistic interactions between monolaurin, a mono-acyl glycerol and β lactam antibiotics against Staphylococcus aureus: an in vitro study

BackgroundA major worldwide health issue is the rising frequency of resistance of bacteria.Drug combinations are a winning strategy in fighting resistant bacteria and might help in protecting the existing drugs.Monolaurin is natural compound extracted from coconut oil and has a promising antimicrobial activity against Staphylococcus.aureus. This study aims to examine the efficacy of monolaurin both individually and in combination with β-lactam antibiotics against Staphylococcus aureus isolates.MethodsAgar dilution method was used for determination of minimum inhibitory concentration (MIC) of monolaurin against S.aureus isolates. Scanning electron microscope (SEM) was used to detect morphological changes in S.aureus after treatment with monolaurin. Conventional and Real-time Polymerase chain reaction (RT-PCR) were performed to detect of beta-lactamase (blaZ) gene and its expressional levels after monolaurin treatment. Combination therapy of monolaurin and antibiotics was assessed through fractional inhibitory concentration and time-kill method.ResultsThe antibacterial activity of monolaurin was assessed on 115 S.aureus isolates, the MIC of monolaurin were 250 to 2000 µg/ml. SEM showed cell elongation and swelling in the outer membrane of S.aureus in the prescence of 1xMIC of monolaurin. blaZ gene was found in 73.9% of S.aureus isolates. RT-PCR shows a significant decrease in of blaZ gene expression at 250 and 500 µg/ml of monolaurin. Synergistic effects were detected through FIC method and time killing curve. Combination therapy established a significant reduction on the MIC value. The collective findings from the antibiotic combinations with monolaurin indicated synergism rates ranging from 83.3% to 100%.In time-kill studies, combination of monolaurin and β-lactam antibiotics produced a synergistic effect.ConclusionThis study showed that monolaurin may be a natural antibacterial agent against S. aureus, and may be an outstanding modulator of β-lactam drugs. The concurrent application of monolaurin and β-lactam antibiotics, exhibiting synergistic effects against S. aureus in vitro, holds promise as potential candidates for the development of combination therapies that target particularly, patients with bacterial infections that are nearly incurable.

Evaluation of hematological and blood biochemical indices in cultured Nile tilapia (Oreochromis niloticus) as affected by using phage therapy against Pseudomonas aeruginosa

Abstract Pseudomonas spp. causes significant losses in aquaculture, consecutive use of antibiotics, and reveals bacterial resistance; therefore, therapeutic bacteriophages, commonly called phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms, including cultured fish. The novelty of current work is represented in examining the lytic activity of four phages and their combination compared to the antibiotic streptomycin on Pseudomonas aeruginosa-infected Nile tilapia (Oreochromis niloticus) while measuring the hematological and blood biochemical parameters as a response for phage therapy. This study evaluated the in vitro killing curve for each phage using a growth curve that measured the OD600 after a single phage suspension was combined with the host P. aeruginosa, considered the best multiplicity of infection (MOI) for each phage. A144 healthy fish were acclimatized in the laboratory and divided into six groups: control, P. aeruginosa-infected fish, streptomycin, phage Ps1, Ps2, both (Ps1 and Ps2), were added to the T3, T4, T5, and T6 groups, respectively. Our findings demonstrated that P. aeruginosa infection caused surface body hemorrhages, tail and fin rot, irritated skin, superficial ulcers, and 100% mortality through 14 days. P. aeruginosa caused a reduction in hemoglobin (Hb), red blood cells (RBCs), platelet number (PLt), and platelet crit (PCT) count, protein, albumin, and A/G ratio; however, an increase in hematocrit (Hct), red cell distribution width (RDW), PDW, MPV compared to other groups after three days of infection and the effects increased after 12 days post-infection. The fish vaccinated with P1 (T4) and P1+P2 (T6) showed enhanced levels of Hb, RBCs, PLt, PCt, protein, albumin and decreased levels of RDW, PDW, MPV, and liver and kidney enzymes with enhanced contents more than streptomycin and closer to the control group. The biochemical markers recorded significant changes indicating liver and kidney impairments due to the infection with P. aeruginosa. It can be concluded that P1 and P1+P2 combinations could be used as therapy in Pseudomonas-infected fish to enhance their blood parameters and performance.

Phage P2-71 against multi-drug resistant Proteus mirabilis: isolation, characterization, and non-antibiotic antimicrobial potential.

Proteus mirabilis, a prevalent urinary tract pathogen and formidable biofilm producer, especially in Catheter-Associated Urinary Tract Infection, has seen a worrying rise in multidrug-resistant (MDR) strains. This upsurge calls for innovative approaches in infection control, beyond traditional antibiotics. Our research introduces bacteriophage (phage) therapy as a novel non-antibiotic strategy to combat these drug-resistant infections. We isolated P2-71, a lytic phage derived from canine feces, demonstrating potent activity against MDR P. mirabilis strains. P2-71 showcases a notably brief 10-minute latent period and a significant burst size of 228 particles per infected bacterium, ensuring rapid bacterial clearance. The phage maintains stability over a broad temperature range of 30-50°C and within a pH spectrum of 4-11, highlighting its resilience in various environmental conditions. Our host range assessment solidifies its potential against diverse MDR P. mirabilis strains. Through killing curve analysis, P2-71's effectiveness was validated at various MOI levels against P. mirabilis 37, highlighting its versatility. We extended our research to examine P2-71's stability and bactericidal kinetics in artificial urine, affirming its potential for clinical application. A detailed genomic analysis reveals P2-71's complex genetic makeup, including genes essential for morphogenesis, lysis, and DNA modification, which are crucial for its therapeutic action. This study not only furthers the understanding of phage therapy as a promising non-antibiotic antimicrobial but also underscores its critical role in combating emerging MDR infections in both veterinary and public health contexts.

Evaluation Of Antimicrobial Effects Of Orthodontic Band Cement Incorporated With Zirconia, Gold, Copper Biosynthesized Nanoparticles- An In-Vitro Study

OBJECTIVE: To evaluate the antimicrobial effects of orthodontic band cement incorporated with various biosynthesized nanoparticles. MATERIALS AND METHODS: Zirconia, gold, copper Nanoparticles were green synthesized using white tea extract and dry ginger which are incorporated into orthodontic band cement, characterized using FTIR analysis. Antimicrobial activity against Streptococcus mutans and Lactobacillus acidophilus was tested in vitro by disc diffusion and Time Kill Curve Assay. RESULTS: Zirconia nanoparticles incorporated GIC exhibited better antimicrobial activity against streptococcus mutans, and gold nanoparticles incorporated GIC against lactobacillus acidophilus at varying concentrations at different time period. CONCLUSION: The antibacterial effect against streptococcus mutans and lactobacillus acidophilus were distinctly noted in Zirconia nanoparticles, gold nanoparticles followed by copper nanoparticles. Zirconia and gold nanoparticles produced more efficient antibacterial property and eventually would be effective in curtailing White Spot Lesions.

MODL-48. AUTOMATING ANALYSIS OF ORGANOTYPIC BRAIN SLICE CULTURE USING MACHINE LEARNING-POWERED COMPUTER VISION

Abstract The difficulty of image labeling and analysis poses a problem for many areas of scientific research, including the recently developed Organotypic Brain Slice Culture (OBSC) tumor characterization platform. An OBSC experiment generates dozens of images which, up to this point, had to be manually quantified. In order to increase the efficiency, consistency, and accuracy of the OBSC image analysis process, we have created a computer program which automates the analysis of OBSC images. This platform uses a number of computer vision (CV) techniques to identify objects of interest and measure their corresponding signals. For fluorescent images of single tumor spots, a straightforward thresholding algorithm divides the image into foreground and background. This automated approach substantially reduces analysis time, and the thresholding algorithm measures unusually shaped tumor spots up to 9% more accurately than previously used manual methods. Other images capture an entire plate of up to 12 OBSCs, complicating the image analysis task. A machine-learning algorithm trained on the web-based platform Biodock could identify individual OBSCs, even if they were touching each other or the sides of the wells. The ML-powered methods consistently reproduced killing curves for treatment effects on tumor tissue as well as the brain tissue substrate, and these results were obtained in much less time. By combining various CV approaches to image analysis, we have completely automated the image analysis component of our OBSC platform. This approach could be adapted to other tumor co-culture platforms.

Tenebrio molitor as a model system to study Staphylococcus spp virulence and horizontal gene transfer

Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6′)-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10−5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.

Evaluation of bioactivities of Pistacia vera L. hull extracts as a potential antimicrobial and antioxidant natural source.

Pistacia vera L. hull, a the major byproduct of pistachio processing, is a source of functional compounds with antioxidant and antimicrobial activities. The extraction of these natural compounds from pistachio hulls and their use instead of synthetic chemicals has gained great attention. In this work, the phytochemical contents and antioxidant and antimicrobial activities of pistachio hull ethanolic (PVE) and aqueous (PVD) extracts obtained by microwave-assisted extraction (MAE) were investigated. Gallic acid (1.9 and 1.5 mg/g dw), quercetin (0.025 and 0.009 mg/g dw), total phenolic (23.3 and 14.7 mg GAE/g dw) and flavonoid (5.0 and 2.9 mg QE/g dw) contents and antioxidant activities (SC50 0.63 and 0.56 mg/mL) of PVE and PVD extracts were determined, respectively. The extracts exhibited antimicrobial effects against Enterococcus faecalis, Staphylococcus aureus, Streptococcus uberis, Bacillus cereus, and Bacillus subtilis. Minimal inhibitory concentrations (MICs, 0.8-49.0 and 9.6-82.5 mg/mL) and the minimal bactericidal concentrations (MBCs, 1.3-99.1 and 15.5-150.0 mg/mL) of PVE and PVD extracts were determined, respectively. Kill curves revealed that PVE and PVD extracts could inhibit the growth of bacteria. It was shown that PVE and PVD extracts could represent a good economical source of functional and bioactive compounds.

Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure.

Persister cells are drug-tolerant bacteria capable of surviving antibiotic treatment despite the absence of heritable resistance mechanisms. It is generally thought that persister cells survive antibiotic exposure through the implementation of stress responses and/or energy-sparing strategies. Exposure to DNA gyrase-targeting antibiotics could be particularly detrimental for bacteria that carry prophages integrated in their genomes. Gyrase inhibitors are known to induce prophages to switch from their dormant lysogenic state into the lytic cycle, causing the lysis of their bacterial host. However, the influence of resident prophages on the formation of persister cells has only been recently appreciated. Here, we evaluated the effect of endogenous prophage carriage on the generation of bacterial persistence during Salmonella enterica serovar Typhimurium exposure to both gyrase-targeting antibiotics and other classes of bactericidal antibiotics. Results from the analysis of strain variants harboring different prophage combinations revealed that prophages play a major role in limiting the formation of persister cells during exposure to DNA-damaging antibiotics. In particular, we present evidence that prophage Gifsy-1 (and its encoded lysis proteins) are major factors limiting persister cell formation upon ciprofloxacin exposure. Resident prophages also appear to have a significant impact on the initial drug susceptibility, resulting in an alteration of the characteristic biphasic killing curve of persister cells into a triphasic curve. In contrast, a prophage-free derivative of S. Typhimurium showed no difference in the killing kinetics for β-lactam or aminoglycoside antibiotics. Our study demonstrates that induction of prophages increased the susceptibility toward DNA gyrase inhibitors in S. Typhimurium, suggesting that prophages have the potential for enhancing antibiotic efficacy. IMPORTANCE Bacterial infections resulting from antibiotic treatment failure can often be traced to nonresistant persister cells. Moreover, intermittent or single treatment of persister cells with β-lactam antibiotics or fluoroquinolones can lead to the formation of drug-resistant bacteria and to the emergence of multiresistant strains. It is therefore important to have a better understanding of the mechanisms that impact persister formation. Our results indicate that prophage-associated bacterial killing significantly reduces persister cell formation in lysogenic cells exposed to DNA-gyrase-targeting drugs. This suggests that therapies based on gyrase inhibitors should be favored over alternative strategies when dealing with lysogenic pathogens.

In Vitro Killing Activities of Anidulafungin and Micafungin with and without Nikkomycin Z against Four Candida auris Clades.

Candida auris is a multidrug-resistant pathogen against which echinocandins are the drug of choice. However, information on how the chitin synthase inhibitor nikkomycin Z influences the killing activities of echinocandins against C. auris is currently lacking. We determined the killing activities of anidulafungin and micafungin (0.25, 1, 8, 16 and 32 mg/L each) with and without nikkomycin Z (8 mg/L) against 15 isolates representing four C. auris clades (South Asian n = 5; East Asian n = 3; South African n = 3; South American n = 4, two of which were of environmental origin). Two and one isolates from the South Asian clade harbored mutations in the hot-spot 1 (S639Y and S639P) and 2 (R1354H) regions of the FKS1 gene, respectively. The anidulafungin, micafungin and nikkomycin Z MIC ranges were 0.015-4, 0.03-4 and 2->16 mg/L, respectively. Anidulafungin and micafungin alone exerted weak fungistatic activity against wild-type isolates and the isolate with a mutation in the hot-spot 2 region of FKS1 but was ineffective against the isolates with a mutation in the hot-spot 1 region. The nikkomycin Z killing curves were always similar to their respective controls. Twenty-two of sixty (36.7%) anidulafungin plus nikkomycin Z and twenty-four of sixty (40%) micafungin plus nikkomycin Z combinations produced at least 100-fold decreases in the CFUs (synergy), with a 41.7% and 20% fungicidal effect, respectively, against wild-type isolates. Antagonism was never observed. Similar results were found with the isolate with a mutation in hot-spot 2 of FKS1, but the combinations were ineffective against the two isolates with prominent mutations in hot-spot 1 of FKS1. The simultaneous inhibition of β-1,3 glucan and chitin synthases in wild-type C. auris isolates produced significantly greater killing rates than either drug alone. Further studies are warranted to verify the clinical efficacy of echinocandin plus nikkomycin Z combinations against echinocandin susceptible C. auris isolates.

Cajanin stilbene acid: A direct inhibitor of colistin resistance protein MCR-1 that restores the efficacy of polymyxin B against resistant Gram-negative bacteria

BackgroundThe resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli). PurposeIn this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery. Study design and methodsCheckerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments. ResultsHere, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 μg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18‑hydroxyl oxygens of CSA were the key sites binding to MCR-1. ConclusionCSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein.

The Lytic Activity of Bacteriophage ZCSE9 against Salmonella enterica and Its Synergistic Effects with Kanamycin.

Salmonella, the causative agent of several diseases in humans and animals, including salmonellosis, septicemia, typhoid fever, and fowl typhoid, poses a serious threat to global public health and food safety. Globally, reports of therapeutic failures are increasing because of the increase in bacterial antibiotic resistance. Thus, this work highlights the combined phage-antibiotic therapy as a promising approach to combating bacterial resistance. In this manner, the phage ZCSE9 was isolated, and the morphology, host infectivity, killing curve, combination with kanamycin, and genome analysis of this phage were all examined. Morphologically, phage ZCSE9 is a siphovirus with a relatively broad host range. In addition, the phage can tolerate high temperatures until 80 °C with one log reduction and a basic environment (pH 11) without a significant decline. Furthermore, the phage prevents bacterial growth in the planktonic state, according to the results of the time-killing curve. Moreover, using the phage at MOI 0.1 with kanamycin against five different Salmonella serotypes reduces the required antibiotics to inhibit the growth of the bacteria. Comparative genomics and phylogenetic analysis suggested that phage ZCSE9, along with its close relatives Salmonella phages vB_SenS_AG11 and wksl3, belongs to the genus Jerseyvirus. In conclusion, phage ZCSE9 and kanamycin form a robust heterologous antibacterial combination that enhances the effectiveness of a phage-only approach for combating Salmonella.

Effects of osmotic stress on Listeria monocytogenes ATCC 7644: persistent cells and heat resistance.

Persistent bacteria are a microbial subpopulation that, exposed to bactericidal treatment, is killed at a slower rate than the rest of the population they are part of. They can be triggered either following stress or stochastically without external signals. The hallmark of persistent bacteria is the biphasic killing curve, a sign that, within a microbial population, two subpopulations are inactivated at a different rate. Furthermore, when plated into a fresh medium and in the absence of stressors, persistent bacteria typically remain in the lag phase longer before resuming active replication. This study aims to evaluate in vitro whether the formation of persistent cells in a strain of Listeria monocytogenes can be triggered by exposure to osmotic stress and if this phenomenon can increase heat resistance in the bacterial population. In a first experiment, the lag time distribution of a L. monocytogenes strain grown in a 6% NaCl broth was evaluated using the software ScanLag. A stationary phase broth culture was inoculated on agar plates placed on an office scanner inside an incubator at 37°C. The plates were scanned every 20' for 4 days and the acquired images were automatically elaborated with the aid of MatLab software in order to evaluate the appearance times of every single colony. The experiment was also carried out on a control culture obtained by growing the strain in the broth without salt. In a second experiment, the same broth cultures, after proper dilutions to rebalance NaCl concentration, were subjected to a heat treatment at 51°C and the death curves obtained were parameterized using the GinaFit system. Results showed that the lag phase of 31.40% of the salt culture colonies was long enough to suppose the formation of persistent bacteria. Analyses of the thermal survival curves showed that the shoulder and tail model was the one that best represented the inactivation trend of the salt culture, unlike the control culture, whose trend was essentially linear. Results of the present study show how exposure to salt could induce the formation of persistent bacteria in a L. monocytogenes strain. The last raises concerns as persistent cells may not only be undetected with common analytical techniques but they even show a greater heat resistance.

A32 PHAGE TREATMENT DELAYS ONSET OF CROHN’S-ASSOCIATED E. COLI DRIVEN COLITIS IN MICE COLONIZED WITH A DEFINED MICROBIOTA

Abstract Background Opportunistic pathogens have been postulated to drive dysregulated inflammation in inflammatory bowel disease (IBD). Indeed, adherent-invasive Escherichia coli (AIEC) isolated from IBD patients have pathobiont and pro-inflammatory characteristics. Current treatments for IBD suppress the immune response and do not target key microbial drivers, therefore novel strategies are required. Purpose Our aim was to determine whether bacteriophage therapy targeted against AIEC could reduce the severity of E. coli-driven colitis in gnotobiotic mice. Method Adult germ-free C57BL/6 mice were colonized with altered Schaedler-like flora (ASF) and E. coli NRG857c, a Crohn’s disease-associated bacterial isolate. Three weeks later, mice were treated with daily phage (selected by killing curves bioassays against E. coli NRG857c) or PBS for 2 weeks (n=6/group). Mice were then exposed to low-dose dextran sulfate sodium (2%; DSS) in drinking water for 5 days, followed by 2 days of water. PBS-treated mice (n=6) that received no DSS were used as additional negative controls. Mice were monitored daily for weight, stool consistency, and occult blood. At sacrifice, colon tissue was collected for histological analysis and fecal contents were cultured to determine bacterial load. In separate experiments, C57BL/6NTac-Il10em8Tac (IL-10-/-) mice were colonized with ASF-like microbiota and E. coli NRG857c. Three weeks later, mice (n=5) were treated with weekly phage or PBS (n=5) for 7 weeks. Mice were monitored weekly as described above. Result(s) Daily phage treatment reduced the severity of clinical symptoms induced by acute DSS administration (p < 0.001 vs. DSS-PBS treated mice). At endpoint, phage treatment was associated with lower histological scores as compared with DSS-PBS controls (p < 0.0001). A 1-log reduction in AIEC bacterial load was observed in phage treated mice as compared with DSS-PBS controls (p < 0.001). In IL-10-/- mice, weekly phage treatment delayed the spontaneous onset of colitis (p < 0.0001 vs. PBS-treated mice). At endpoint, mice treated with phage had lower colitis scores. Reduced weekly AIEC bacterial load was observed in phage-treated mice. Conclusion(s) Lytic phages, targeting a known AIEC pathobiont isolated from Crohn’s disease patients, ameliorate acute intestinal injury and delay onset of spontaneous colitis. Future work will investigate the mechanisms by which phage therapy prevents and treats colitis, to better inform clinical trial design. Disclosure of Interest None Declared

Artemisia afra and Artemisia annua Extracts Have Bactericidal Activity against Mycobacterium tuberculosis in Physiologically Relevant Carbon Sources and Hypoxia.

Mycobacterium tuberculosis (Mtb) is a deadly pathogen and causative agent of human tuberculosis, causing ~1.5 million deaths every year. The increasing drug resistance of this pathogen necessitates novel and improved treatment strategies. A crucial aspect of the host-pathogen interaction is bacterial nutrition. In this study, Artemisia annua and Artemisia afra dichloromethane extracts were tested for bactericidal activity against Mtb strain mc26230 under hypoxia and various infection-associated carbon sources (glycerol, glucose, and cholesterol). Both extracts showed significant bactericidal activity against Mtb, regardless of carbon source. Based on killing curves, A. afra showed the most consistent bactericidal activity against Mtb for all tested carbon sources, whereas A. annua showed the highest bactericidal activity in 7H9 minimal media with glycerol. Both extracts retained their bactericidal activity against Mtb under hypoxic conditions. Further investigations are required to determine the mechanism of action of these extracts and identify their active constituent compounds.

Impact of surfactants and other body fluids on in vitro activity of a novel β-lactamase inhibitor enmetazobactam in combination with cefepime against clinical isolates of Klebsiella pneumoniae.

Surfactants might impact treatment of lower respiratory tract infections. Moreover, other body fluids, such as urine or serum, could impact antibacterial activity as well. Therefore, the impact of surfactants, urine, and serum on the antibacterial activity of the novel β-lactam/β-lactamase inhibitor combination of cefepime-enmetazobactam (FPE) was determined. Ten clinical isolates of Klebsiella pneumoniae, and the quality control strains K. pneumoniae ATCC 700603 and Escherichia coli NCTC 13353, were tested. Minimal Inhibitory Concentration (MIC) determinations (all strains) and Time Kill Curves (TKC) (one clinical isolate) were determined for FPE and piperacillin-tazobactam (TZP) with and without surfactant formulations Survanta® (SUR; 1%v/v) and Curosurf® (CUR; 1 mg ml-1). Determination of daptomycin MIC against Staphylococcus aureus ATCC 29213 in the presence and absence of surfactants was used as a positive control. Additionally, the impact of growth media supplemented with pooled human urine or serum were also evaluated by MIC testing. Expectedly, media supplemented with SUR increased the daptomycin MIC against S. aureus ATCC 29213. In contrast, the surfactants had no impact on the antibacterial activity of FPE against the tested Enterobacterales isolates. TKC experiments also revealed no impact of CUR on the antibacterial activity of FPE. These results demonstrate that the antibacterial activity of FPE is unaffected in the presence of lung surfactant. Moreover, FPE was not impacted by media supplemented with urine or serum.

Epifriedelanol is the key compound to antibacterial effects of extracts of Synadenium glaucescens (Pax) against medically important bacteria

IntroductionSynadenium glaucescens has been used for the treatment of bacterial infections in many parts of the world. We investigated the antibacterial and cytotoxicity activities of secondary metabolites of this plant.MethodsHexane, dichloromethane, methanol, and water were used as extraction solvents. The extract of the root bark was fractionated with ethyl acetate and methanol. The isolation of compounds from root barks, leaves and stem wood extracts were carried out using column chromatography. Antibacterial activities were characterized based on growth curves, killing curves and MIC determinations. Haemolytic effect towards sheep red blood cells (RBCs) was analysed with spectrophotometer at the wavelength of 540nm. Results and DiscussionExtracts from whole root and root bark showed strong activity against Staphylococcus aureus, and Streptococci and Enterococci species, and moderate to weak activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella species, Shigella sonnei and Yersinia enterocolitica. Staphylococcus aureus was the most susceptible, and E. coli and Klebsiella pneumonia were the least susceptible ones. Likewise, extracts, fractions, sub-fractions and epifriedelanol demonstrated bacteriostatic activity against S. aureus. The haemolytic activity of the extracts, fractions, sub-fractions and epifriedelanol was significantly low compared to the positive control, hydrogen peroxide. But extract from leaves showed high haemolytic effects at the concentrations of 500 μg/mL and 1000 μg/mL. Thus, extracts of S. glaucescens have antibacterial activity against several Gram-positive bacteria including Methicillin Resistant S. aureus with low haemolytic activity. At high concentrations, the extracts from leaves have toxicity risk. More studies for the active compounds are required for biological testing.

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.

Application of the lytic bacteriophage Rostam to control Salmonella enteritidis in eggs

Foodborne Salmonella enteritidis infections place human health at risk, driven by regular outbreaks and individual cases by different contaminated food materials. This study was conducted to characterize and employ a single bacteriophage as a potential biocontrol agent. Phage Rostam was isolated, characterized and then applied as biocontrol agent against S. enteritidis in liquid whole eggs and eggshell. Rostam is a novel myovirus belonging to the Rosemountvirus genus and active against Escherichia coli and Salmonella spp. Rostam is stable in a pH range from 4 to 10, a salt concentration of 1–9 %, whereas UV radiation gradually reduces phage stability, and its 53 kb genome sequence indicates this phage does not contain known toxins or lysogeny-associated genes. Its latent period is short with a burst size of 151 PFU/cell, under standard growth conditions. Killing curves indicate that at higher multiplicities of infection (MOI), the reduction in S. enteritidis count is more pronounced. Phage Rostam (MOI 10,000) reduces S. enteritidis growth to below the detection limit at 4 °C in both liquid whole eggs and on the eggshell within 24 h. Due to its high lytic activity and stability in relevant conditions, Rostam has the potential to be an efficient biopreservative for egg and egg products.