Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice.

The high prevalence of multidrug-resistant Acinetobacter baumannii has emerged as a serious problem in the treatment of nosocomial infections in the past three decades. Recently, we developed a new small-molecule inhibitor belonging to a class of 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT, previously called CL-55). FT effectively suppressed the T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella sp. without affecting bacterial growth in vitro. In this study, we describe that prophylactic use of FT for 4 days prior to challenge with resistant clinical isolates of A. baumannii (ABT-897-17 and 52TS19) suppressed septic infection in mice, resulting in improved survival, limited bacteraemia and decreased bacterial load in the organs of the mice. We show that FT had an inhibitory effect on A. baumannii biofilm formation in vitro and, to a greater extent, on biofilm maturation. In addition, FT inhibited Acinetobacter isolate-induced death of HeLa cells, which morphologically manifested as apoptosis. The mechanism of FT action on A. baumannii is currently being studied. FT may be a promising candidate for the development of a broad-spectrum anti-virulence drug to use in the prevention of nosocomial infections.

Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro.

Obesity impairs host defense against Klebsiella pneumoniae, but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-wk-old male C57BL/6j mice a normal diet (ND) or high-fat diet (HFD) (13% vs. 60% fat, respectively) for 16 wk. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-d-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1ß, IL-6, IL-17, IFN-γ, CXCL2, and TNF-α were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, CXCL2, and IL-1ß (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intraalveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

In vivo antimicrobial activity of 0.6% povidone-iodine eye drops in patients undergoing intravitreal injections: a prospective study.

To investigate the antimicrobial activity of a preservative-free 0.6% povidone-iodine eye drop as an antiseptic procedure in decreasing the conjunctival bacterial load in eyes scheduled for intravitreal treatment and to compare its efficacy to the untreated fellow eye used as the control group. Prospective cohort analysis in which 208 patients received preservative-free 0.6% povidone-iodine eye drops three times a day for three days before intravitreal injection. Before and after the prophylactic treatment, a conjunctival swab was collected from both the study eye and the untreated contralateral eye, used as control. The swab was inoculated on different culture media and the colony-forming units were counted. Bacteria and fungi were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Treatment with 0.6% povidone-iodine eye drops significantly reduced the conjunctival bacterial load from baseline (p < 0.001 for blood agar and p < 0.001 for chocolate agar) with an eradication rate of 80%. The most commonly isolated pathogen at each time-point and in both groups was coagulase-negative Staphylococci, isolated in 84% of the positive cultures. The study provides evidence about the effectiveness of 0.6% povidone-iodine eye drops treatment in reducing the conjunctival bacterial load in eyes scheduled for intravitreal treatment.

EDTA and Taurolidine Affect Pseudomonas aeruginosa Virulence In Vitro-Impairment of Secretory Profile and Biofilm Production onto Peritoneal Dialysis Catheters.

Peritoneal catheter-associated biofilm infection is reported to be the main cause of refractory peritonitis in peritoneal dialysis patients. The application of antimicrobial lock therapy, based on results on central venous catheters, may be a promising option for treatment of biofilm-harboring peritoneal catheters. This study investigated the effects of two lock solutions, EDTA and taurolidine, on an in vitro model of Pseudomonas aeruginosa biofilm-related peritoneal catheter infection. Silicone peritoneal catheters were incubated for 24 h with a bioluminescent strain of P. aeruginosa. Then, serial dilutions of taurolidine and/or EDTA were applied (for 24 h) once or twice onto the contaminated catheters, and P. aeruginosa viability/persistence were evaluated in real time up to 120 h using a Fluoroskan reader. On selected supernatants, high-performance liquid chromatography mass spectrometry (HPLC-MS) analysis was performed to measure the production of autoinducers (AI), phenazines, and pyocyianines. Taurolidine alone or in combination with EDTA caused a significant decrease of bacterial load and biofilm persistence on the contaminated catheters. The treatment did not lead to the sterilization of the devices, yet it resulted in a substantial destructuration of the catheter-associated P. aeruginosa biofilm. HPLC-MS analysis showed that the treatment of biofilm-harboring catheters with taurolidine and EDTA also affected the secretory activity of the pathogen. EDTA and taurolidine affect P. aeruginosa biofilm produced on peritoneal catheters and profoundly compromise the microbial secretory profile. Future studies are needed to establish whether such lock solutions can be used to render peritoneal catheter-related infections more susceptible to antibiotic treatment. IMPORTANCE An in vitro model allows studies on the mechanisms by which the lock solutions exert their antimicrobial effects on catheter-associated biofilm, thus providing a better understanding of the management of devise-associated infections.

Antibiotic-chemoattractants enhance neutrophil clearance of Staphylococcus aureus.

The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term 'antibiotic-chemoattractants', consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development.

Responses of Escherichia coli and Listeria monocytogenes to ozone treatment on non-host tomato: Efficacy of intervention and evidence of induced acclimation.

Because of the continuous rise of foodborne illnesses caused by the consumption of raw fruits and vegetables, effective post-harvest anti-microbial strategies are necessary. The aim of this study was to evaluate the anti-microbial efficacy of ozone (O3) against two common causes of fresh produce contamination, the Gram-negative Escherichia coli O157:H7 and Gram-positive Listeria monocytogenes, and to relate its effects to potential mechanisms of xenobiosis by transcriptional network modeling. The study on non-host tomato environment correlated the dose × time aspects of xenobiosis by examining the correlation between bacterial survival in terms of log-reduction and defense responses at the level of gene expression. In E. coli, low (1 µg O3/g of fruit) and moderate (2 µg O3/g of fruit) doses caused insignificant reduction in survival, while high dose (3 µg/g of fruit) caused significant reduction in survival in a time-dependent manner. In L. monocytogenes, moderate dose caused significant reduction even with short-duration exposure. Distinct responses to O3 xenobiosis between E. coli and L. monocytogenes are likely related to differences in membrane and cytoplasmic structure and components. Transcriptome profiling by RNA-Seq showed that primary defenses in E. coli were attenuated after exposure to a low dose, while the responses at moderate dose were characterized by massive upregulation of pathogenesis and stress-related genes, which implied the activation of defense responses. More genes were downregulated during the first hour at high dose, with a large number of such genes getting significantly upregulated after 2 hr and 3 hr. This trend suggests that prolonged exposure led to potential adaptation. In contrast, massive downregulation of genes was observed in L. monocytogenes regardless of dose and exposure duration, implying a mechanism of defense distinct from that of E. coli. The nature of bacterial responses revealed by this study should guide the selection of xenobiotic agents for eliminating bacterial contamination on fresh produce without overlooking the potential risks of adaptation.

Chitosan propolis nanocomposite alone or in combination with apramycin: an alternative therapy for multidrug-resistant Salmonella Typhimurium in rabbits: in vitro and in vivo study.

Introduction. The emergence of multidrug-resistant Salmonella Typhimurium strains has increased the need for safe, alternative therapies from natural sources with antibacterial properties.Hypothesis/Gap Statement. There are no published data regarding the use of chitosan propolis nanocomposite (CPNP) either alone or in combination with antibiotics as antimicrobials against S. Typhimurium, especially in Egypt.Aim. This study evaluated the antibacterial activities of five antimicrobials [apramycin, propolis, chitosan nanoparticles (CNPs), chitosan propolis nanocomposite (CPNP) and CPNP +apramycin] against ten virulent and multidrug-resistant (MDR) S. Typhimurium field strains recovered from diarrheic rabbits through in vitro and in vivo study.Methodology. The expression levels of three virulence genes of S. Typhimurium strains were determined by quantitative reverse-transcription PCR (RT-qPCR) after exposure to sub-inhibitory concentrations of apramycin, propolis, CNPs, CPNP alone, and CPNP +apramycin. Additionally, 90 New Zealand rabbits were divided into control and experimentally S. Typhimurium-infected groups. The infected rabbits were orally administered saline solution (infected-untreated); 10 mg apramycin/kg (infected-apramycin-treated); 50 mg propolis/kg (infected-propolis-treated); 15 mg CPNP/kg (infected-CPNP-treated) and 15 mg CPNP +10 mg apramycin/kg (infected-CPNP +apramycin-treated) for 5 days.Results. The RT-qPCR analysis revealed different degrees of downregulation of all screened genes. Furthermore, the treatment of infected rabbits with CPNP or CPNP +apramycin significantly improved performance parameters, and total bacterial and Salmonella species counts, while also modulating both oxidative stress and altered liver and kidney parameters.Conclusion. This work demonstrates the use of CPNP alone or in combination with apramycin in the treatment of S. Typhimurium in rabbits.

Cinnamomum cassia and Syzygium aromaticum Essential Oils Reduce the Colonization of Salmonella Typhimurium in an In Vivo Infection Model Using Caenorhabditis elegans.

The regulation of intestinal colonization in livestock by means of non-bactericidal additives is an important management lever for zoonotic bacteria such as Salmonella spp. Caenorhabditis elegans is proposed here as a model for the evaluation of five essential oils (EOs) as anti-colonization products against Salmonella Typhimurium. An evaluation of the toxicity of EOs for C. elegans showed LD50 values ranging from 74.5 ± 9.6 µg/mL for Cinnamomum cassia (CEO) to 271.6 ± 14.9 µg/mL for Syzygium aromaticum (SyEO). Both EOs significantly inhibited bacterial colonization in the digestive tract of C. elegans with reductions of 0.88 and 0.70 log CFU/nematode at nontoxic concentrations of 50 µg/mL and 150 µg/mL, respectively. With the minimal bactericidal concentrations of CEO and SyEO against S. Typhimurium being 312.5 µg/mL and 625 µg/mL, respectively, an antibacterial effect can be excluded to explain the inhibition of the bacterial load. The anti-colonizing activity of these two EOs could, however, be related to an inhibition of the swimming motility, which was significantly reduced by 23.47% for CEO at 50 µg/mL and 19.56% for SyEO at 150 µg/mL. This study shows the potential of C. elegans as a predictive in vivo model of anti-colonizing activities that is suitable for the evaluation of essential oils.

Pretreatment with Ranitidine Bismuth Citrate May Improve Success Rates of Helicobacter pylori Eradication: A Prospective, Randomized, Controlled and Open-Label Study.

Effective Helicobacter pylori (H. pylori) eradication is a major public health concern; however, eradication failure rates with the standard triple therapy remain high. We aimed to investigate the effectiveness and tolerability of ranitidine bismuth citrate (RBC) pretreatment before standard triple therapy for H. pylori eradication. A prospective, randomized, controlled, and open-label clinical trial was conducted from June to December 2019. H. pylori eradication rate, safety, and tolerability were compared between the standard treatment group (esomeprazole, amoxicillin, and clarithromycin for 7 days) and RBC pretreatment group (RBC for 2 weeks before standard triple therapy). This trial ended earlier than estimated owing to the N-nitrosodimethylamine concerns with ranitidine. Success rates of H. pylori eradication were 80.9% and 67.3% in the RBC pretreatment (n = 47) and standard treatment (n = 52) (p = 0.126) groups, respectively. Our trial was discontinued earlier than planned; however, a statistical significance would be achieved by expansion of our data (p = 0.031) if patient enrollment numbers reached those initially planned. Adverse event rates were comparable between groups (25.5% in the pretreatment group vs. 28.8% in the standard treatment group), without serious event. Tolerability was excellent in both groups, recorded as 97.9% and 100% in the pretreatment and standard treatment groups, respectively. Compared with the standard triple regimen, RBC pretreatment for 2 weeks may achieve higher H. pylori eradication rates, with excellent safety and tolerability. However, this study necessitates further validation as it was discontinued early owing to the N-nitrosodimethylamine issues of ranitidine.

Novel Small Molecule Growth Inhibitor Affecting Bacterial Outer Membrane Reduces Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection in Avian Model.

Avian pathogenic Escherichia coli (APEC), a subgroup of extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly implicated in urinary tract infections and meningitis in humans. A major limitation for the current ExPEC antibiotic therapy is the development of resistance, and antibacterial drugs that can circumvent this problem are critically needed. Here, we evaluated eight novel membrane-affecting anti-APEC small molecule growth inhibitors (GIs), identified in our previous study, against APEC infection in chickens. Among the GIs tested, GI-7 (the most effective), when administered orally (1 mg/kg of body weight), reduced the mortality (41.7%), severity of lesions (62.9%), and APEC load (2.6 log) in chickens. Furthermore, GI-7 administration at an optimized dose (60 mg/liter) in drinking water also reduced the mortality (14.7%), severity of lesions (29.5%), and APEC load (2.2 log) in chickens. The abundances of Lactobacillus and oleate were increased in the cecum and serum, respectively, of GI-7-treated chickens. Pharmacokinetic analysis revealed that GI-7 was readily absorbed with minimal accumulation in the tissues. Earlier, we showed that GI-7 induced membrane blebbing and increased membrane permeability in APEC, suggesting an effect on the APEC membrane. Consistent with this finding, the expression of genes essential for maintaining outer membrane (OM) integrity was downregulated in GI-7-treated APEC. Furthermore, decreased levels of lipopolysaccharide (LPS) transport (Lpt) proteins and LPS were observed in GI-7-treated APEC. However, the mechanism of action of GI-7 currently remains unknown and needs further investigation. Our studies suggest that GI-7 represents a promising novel lead compound that can be developed to treat APEC infection in chickens and related human ExPEC infections. IMPORTANCE APEC is a subgroup of ExPEC, and genetic similarities of APEC with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), have been reported. Our study identified a novel small molecule growth inhibitor, GI-7, effective in reducing APEC infection in chickens with an efficacy similar to that of the currently used antibiotic sulfadimethoxine, notably with an 8-times-lower dose. GI-7 affects the OM integrity and decreases the Lpt protein and LPS levels in APEC, an antibacterial mechanism that can overcome the antibiotic resistance problem. Overall, GI-7 represents a promising lead molecule/scaffold for the development of novel antibacterial therapies that could have profound implications for treating APEC infections in chickens, as well as human infections caused by ExPECs and other related Gram-negative bacteria. Further elucidation of the mechanism of action of GI-7 and identification of its target(s) in APEC will benefit future novel antibacterial development efforts.

Synergistic anti-oxidative and antimicrobial effects of oat phenolic compounds and ascorbate palmitoyl on fish balls during cold storage.

This study investigated the effect of antioxidants on lipid stability of mackerel (Scomber japonicus) fish balls. Oat phenolic acid compounds (OPC) and ascorbate palmitoyl (AP) were used to prolong the shelf life of steamed mackerel fish balls. Fish balls were stored at 4°C for 21 days, and the total bacterial count, hardness, whiteness, water holding capacity (WHC), pH, total volatile basic nitrogen (TVB-N), and thiobarbituric acid reactive substances (TBARS) value were monitored regularly. The results indicated that OPC+AP composite as a biological preservative could significantly inhibit the increase of the total bacterial count. Meanwhile, OPC and AP could maintain better hardness, whiteness, and WHC of fish balls during refrigerated storage. Furthermore, OPC and AP slowed down the increase of TVB-N and TBARS values. The results showed that OPC+AP had a synergistic effect on the storage time and could prolong the shelf life within the storage time. Adding OPC and AP was a promising strategy to improve the quality and shelf life of fish balls. PRACTICAL APPLICATION: The research provided a new application of OPC and AP for improving fish balls quality and shelf life during cold storage (4°C). OPC is a natural plant secondary metabolite from oat which exhibits excellent anti-oxidation. The research showed that OPC and AP combined with synergistic effect as biological preservatives can effectively inhibit the total bacterial count and reduce TBARS and TVB-N value of fish balls during the shelf life and maintain the hardness, which improved the quality and shelf life of fish balls.

Potential Role of CXCL10 in Monitoring Response to Treatment in Leprosy Patients.

The treatment of multibacillary cases of leprosy with multidrug therapy (MDT) comprises 12 doses of a combination of rifampicin, dapsone and clofazimine. Previous studies have described the immunological phenotypic pattern in skin lesions in multibacillary patients. Here, we evaluated the effect of MDT on skin cell phenotype and on the Mycobacterium leprae-specific immune response. An analysis of skin cell phenotype demonstrated a significant decrease in MRS1 (SR-A), CXCL10 (IP-10) and IFNG (IFN-γ) gene and protein expression after MDT release. Patients were randomized according to whether they experienced a reduction in bacillary load after MDT. A reduction in CXCL10 (IP-10) in sera was associated with the absence of a reduction in the bacillary load at release. Although IFN-γ production in response to M. leprae was not affected by MDT, CXCL10 (IP-10) levels in response to M. leprae increased in cells from patients who experienced a reduction in bacillary load after treatment. Together, our results suggest that CXCL10 (IP-10) may be a good marker for monitoring treatment efficacy in multibacillary patients.

Interleukin-4 protects mice against lethal influenza and Streptococcus pneumoniae co-infected pneumonia.

Streptococcus pneumoniae co-infection post-influenza is a major cause of mortality characterized by uncontrolled bacteria burden and excessive immune response during influenza pandemics. Interleukin (IL)-4 is a canonical type II immune cytokine known for its wide range of biological activities on different cell types. It displays protective roles in numerous infectious diseases and immune-related diseases, but its role in influenza and S. pneumoniae (influenza/S. pneumoniae) co-infected pneumonia has not been reported. In our study, we used C57BL/6 wild-type (WT) and IL-4-deficient (IL-4-/- ) mice to establish co-infection model with S. pneumoniae after influenza virus infection. Co-infected IL-4-/- mice showed increased mortality and weight loss compared with WT mice. IL-4 deficiency led to increased bacterial loads in lungs without altering influenza virus replication, suggesting a role of IL-4 in decreasing post-influenza susceptibility to S. pneumoniae co-infection. Loss of IL-4 also resulted in aggravated lung damage together with massive proinflammatory cytokine production and immune cell infiltration during co-infection. Administration of recombinant IL-4 rescued the survival and weight loss of IL-4-/- mice in lethal co-infection. Additionally, IL-4 deficiency led to more immune cell death in co-infection. Gasdermin D (GSDMD) during co-infection was induced in IL-4-/- mice that subsequently activated cell pyroptosis. Treatment of recombinant IL-4 or inhibition of GSDMD activity by disulfiram decreased immune cell death and bacterial loads in lungs of IL-4-/- co-infected mice. These results suggest that IL-4 decreases post-influenza susceptibility to S. pneumoniae co-infection via suppressing GSDMD-induced pyroptosis. Collectively, this study demonstrates the protective role of IL-4 in influenza/S. pneumoniae co-infected pneumonia.

Selection of ESBL-producing Escherichia coli in the gut of calves experimentally fed with milk containing antibiotic residues.

In the bovine sector, the spread of Enterobacterales producing extended-spectrum and AmpC ß-lactamases (ESBL/AmpC) mostly concerns veal calves, and the use of waste milk containing antibiotic residues has been recurrently incriminated. In this study, calves were experimentally fed with milk containing either 2,000 µg/L or 20,000 µg/L of the critically important antibiotic cefquinome. The total counts of enterobacterales and ESBL-producing E. coli were monitored using non-selective and selective media. Our data highlighted the important combination of two main factors (cefquinome exposure and initial ESBL colonization level) in the ESBL selection and amplification process in the gut of calves. Results also proved the dose-independent effect of cefquinome administration on the selection and amplification of ESBL-producing E. coli. Finally, the blaCTX-M-1/IncI1 ST3 plasmid was systematically recovered after cefquinome exposure, highlighting its epidemic success. Altogether, this work is one of the rare experimental studies providing quantitative information on the impact of waste milk containing antimicrobials on the ESBL load in calves' microbiota, and the first one using cefquinome. These data emphasise the need for global guidelines on the use of waste milk on dairy farms in order to decrease the antimicrobial resistance burden in this sector.

Gut microbiome alterations in high-fat-diet-fed mice are associated with antibiotic tolerance.

Antibiotic tolerance, the ability of a typically susceptible microorganism to survive extended periods of exposure to antibiotics, has a critical role in chronic and recurrent bacterial infections, and facilitates the evolution of antibiotic resistance. However, the physiological factors that contribute to the development of antibiotic tolerance, particularly in vivo, are not fully known. Despite the fact that a high-fat diet (HFD) is implicated in several human diseases, the relationship between HFD and antibiotic efficacy is still poorly understood. Here, we evaluated the efficacy of multiple clinically relevant bactericidal antibiotics in HFD-fed mice infected with methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli. We found that HFD-fed mice had higher bacterial burdens and these bacteria displayed lower susceptibility to bactericidal antibiotic treatment compared with mice that were fed a standard diet, while microbiota-depleted standard-diet- or HFD-fed mice showed similar susceptibility. Faecal microbiota transplantation from HFD-fed mice impaired antibiotic activity in mice fed a standard diet, indicating that alteration of the gut microbiota and related metabolites in HFD-fed mice may account for the decreased antibiotic activity. 16S rRNA sequencing and metabolomics analysis of faecal samples revealed decreased microbial diversity and differential metabolite profiles in HFD-fed mice. Notably, the tryptophan metabolite indole-3-acetic acid (IAA) was significantly decreased in HFD-fed mice. Further in vitro studies showed that IAA supplementation inhibited the formation of bacterial persisters and promoted the elimination of persisters in combination with antibiotic treatment, potentially through the activation of bacterial metabolic pathways. In vivo, the combination of IAA and ciprofloxacin increased the survival rate of HFD-fed mice infected with MRSA persisters. Overall, our data reveal that a HFD has an antagonistic effect on antibiotic treatment in a mouse model, and this is associated with the alteration of the gut microbiota and IAA production.

Inoculum effect of antimicrobial peptides.

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 105 cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.

Comparing rates of mycobacterial clearance in sputum smear-negative and smear-positive adults living with HIV.

BACKGROUND: Pulmonary tuberculosis (TB) in people living with HIV (PLH) frequently presents as sputum smear-negative. However, clinical trials of TB in adults often use smear-positive individuals to ensure measurable bacterial responses following initiation of treatment, thereby excluding HIV-infected patients from trials. METHODS: In this prospective case cohort study, 118 HIV-seropositive TB patients were assessed prior to initiation of standard four-drug TB therapy and at several time points through 35 days. Sputum bacillary load, as a marker of treatment response, was determined serially by: smear microscopy, Xpert MTB/RIF, liquid culture, and colony counts on agar medium. RESULTS: By all four measures, patients who were baseline smear-positive had higher bacterial loads than those presenting as smear-negative, until day 35. However, most smear-negative PLH had significant bacillary load at enrolment and their mycobacteria were cleared more rapidly than smear-positive patients. Smear-negative patients' decline in bacillary load, determined by colony counts, was linear to day 7 suggesting measurable bactericidal activity. Moreover, the decrease in bacterial counts was comparable to smear-positive individuals. Increasing cycle threshold values (Ct) on the Xpert assay in smear-positive patients to day 14 implied decreasing bacterial load. CONCLUSION: Our data suggest that smear-negative PLH can be included in clinical trials of novel treatment regimens as they contain sufficient viable bacteria, but allowances for late exclusions would have to be made in sample size estimations. We also show that increases in Ct in smear-positive patients to day 14 reflect treatment responses and the Xpert MTB/RIF assay could be used as biomarker for early treatment response.

Randomized Placebo-Controlled Trial of Topical Mupirocin to Reduce Staphylococcus aureus Colonization in Infants in the Neonatal Intensive Care Unit.

OBJECTIVE: To evaluate the efficacy of topical mupirocin in reducing Staphylococcus aureus colonization in infants in the neonatal intensive care unit (NICU). STUDY DESIGN: A prospective double-blind randomized controlled trial of mupirocin vs placebo in S aureus-colonized infants was conducted in a tertiary care NICU between October 2016 and December 2019. Weekly universal active surveillance with polymerase chain reaction screening identified colonized infants. Colonized infants received a 5-day course of mupirocin (mupirocin group) or petroleum jelly (control group). Repeat courses were given for additional positive screens. RESULTS: A total of 216 infants were enrolled; 205 were included in data analyses. Primary decolonization was more successful for mupirocin-treated infants (86 of 104 [83%]) than for controls (20 of 101; 20%) (P < .001). Although recurrent S aureus colonization occurred frequently (59 of 81 [73%] mupirocin-treated and 26 of 33 [79%] controls), subsequent decolonization remained more successful for mupirocin-treated infants than for controls (38 of 49 [78%] vs 2 of 21 [10%]; P < .001). Subgroup analyses of infants of ≤30 weeks' gestational age yielded similar results; decolonization occurred more often in mupirocin-treated infants compared with control infants (63 of 76 [83%] vs 13 of 74 [18%]; P < .001). Bacterial sterile site infections tended to be less frequent in mupirocin-treated infants compared with controls (2 of 104 [2%] vs 8 of 101 [8%]; P = .057). No invasive S aureus infections occurred in mupirocin-treated infants, but 50% of infections in controls were from S aureus, and 1 resulted in death. CONCLUSIONS: Universal active surveillance and targeted treatment with topical mupirocin is a successful decolonization strategy for NICU infants and may prevent S aureus infection. However, S aureus colonization frequently recurs, necessitating repeat treatment. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02967432.

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG.

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.