Minimum Information for Conducting and Reporting In Vitro Intracellular Infection Assays.

Bacterial pathogens are constantly evolving to outsmart the host immune system and antibiotics developed to eradicate them. One key strategy involves the ability of bacteria to survive and replicate within host cells, thereby causing intracellular infections. To address this unmet clinical need, researchers are adopting new approaches, such as the development of novel molecules that can penetrate host cells, thus exerting their antimicrobial activity intracellularly, or repurposing existing antibiotics using nanocarriers (i.e., nanoantibiotics) for site-specific delivery. However, inconsistency in information reported across published studies makes it challenging for scientific comparison and judgment of experiments for future direction by researchers. Together with the lack of reproducibility of experiments, these inconsistencies limit the translation of experimental results beyond pre-clinical evaluation. Minimum information guidelines have been instrumental in addressing such challenges in other fields of biomedical research. Guidelines and recommendations provided herein have been designed for researchers as essential parameters to be disclosed when publishing their methodology and results, divided into four main categories: (i) experimental design, (ii) establishing an in vitro model, (iii) assessment of efficacy of novel therapeutics, and (iv) statistical assessment. These guidelines have been designed with the intention to improve the reproducibility and rigor of future studies while enabling quantitative comparisons of published studies, ultimately facilitating translation of emerging antimicrobial technologies into clinically viable therapies that safely and effectively treat intracellular infections.

Dynamics of IgG antibody response against Plasmodium antigens among Nigerian infants and young children.

Background: Plasmodium falciparum malaria is a leading cause of child mortality in Nigeria. Neonates are born with maternal antibodies from placental transfer which may protect against malaria infection in the first months of life. The IgG dynamics of the transition from passively transferred antimalarial antibodies to actively acquired IgG from natural exposure have not been well elucidated. Methods: Blood samples collected during a 2018 Nigeria nationwide HIV/AIDS household survey were available for 9,443 children under 5 years of age, with a subset of infants under 2 months of age having maternal samples available (n=41). Samples were assayed for the P. falciparum HRP2 antigen and anti-malarial IgG antibodies. LOESS regression examined the dynamics in IgG response in the first 5 years of life. Correlation with maternal IgG levels was assessed for mother/child pairs. Results: Consistent decreases were observed in median IgG levels against all Plasmodium spp. antigen targets for the first months of life. At a population level, P. falciparum apical membrane antigen-1 (AMA1) and merozoite surface protein-1 19kD (PfMSP1) IgG decreased during the first 12 months of life before reaching a nadir, whereas IgGs to other targets only declined for the first 4 months of life. Seropositivity showed a similar decline with the lowest seropositivity against AMA1 and PfMSP1 at 10-12 months, though remaining above 50% during the first 2 years of life in higher transmission areas. No protective association was observed between IgG positivity and P. falciparum infection in infants. Maternal antibody levels showed a strong positive correlation with infant antibody levels for all P. falciparum antigens from birth to 2 months of age, but this correlation was lost by 6 months of age. Discussion: Maternally transferred anti-malarial IgG antibodies rapidly decline during the first 6 months of life, with variations among specific antigens and malaria transmission intensity. From 3-23 months of age, there was a wide range in IgG levels for the blood-stage antigens indicating high individual variation in antibody production as children are infected with malaria. Non-falciparum species-specific antigens showed similar patterns in waning immunity and correlation with paired mother's IgG levels compared to P. falciparum antigens.

Oral administration of a 2-aminopyrimidine robenidine analogue (NCL195) significantly reduces Staphylococcus aureus infection and reduces Escherichia coli infection in combination with sub-inhibitory colistin concentrations in a bioluminescent mouse model.

We have previously reported promising in vivo activity of the first-generation 2-aminopyramidine robenidine analogue NCL195 against Gram-positive bacteria (GPB) when administered via the systemic route. In this study, we examined the efficacy of oral treatment with NCL195 (± low-dose colistin) in comparison to oral moxifloxacin in bioluminescent Staphylococcus aureus and Escherichia coli peritonitis-sepsis models. Four oral doses of 50 mg/kg NCL195, commencing immediately post-infection, were administered at 4 h intervals in the S. aureus peritonitis-sepsis model. We used a combination of four oral doses of 50 mg/kg NCL195 and four intraperitoneal doses of colistin at 0.125 mg/kg, 0.25 mg/kg, or 0.5 mg/kg in the E. coli peritonitis-sepsis model. Subsequently, the dose rates of four intraperitoneal doses of colistin were increased to 0.5 mg/kg, 1 mg/kg, or 2 mg/kg at 4 h intervals to treat a colistin-resistant E. coli infection. In the S. aureus infection model, oral treatment of mice with NCL195 resulted in significantly reduced S. aureus infection loads (P < 0.01) and longer survival times (P < 0.001) than vehicle-only treated mice. In the E. coli infection model, co-administration of NCL195 and graded doses of colistin resulted in a dose-dependent significant reduction in colistin-susceptible (P < 0.01) or colistin-resistant (P < 0.05) E. coli loads compared to treatment with colistin alone at similar concentrations. Our results confirm that NCL195 is a potential candidate for further preclinical development as a specific treatment for multidrug-resistant infections, either as a stand-alone antibiotic for GPB or in combination with sub-inhibitory concentrations of colistin for Gram-negative bacteria.

Impact of the new membrane-targeting lipoglycopeptide antibiotic MCC5145 on the treatment of bacteremic pneumococcal pneumonia in mice.

Bacteremic Streptococcus pneumoniae pneumonia is one of the most severe forms of invasive pneumococcal disease (IPD) and with particularly high case-fatality rates among the elderly and individuals with comorbidities, exacerbated by rising antibiotic resistance and time to initiation of therapy. Here, we examined the efficacy of the preclinical "vancapticin" glycopeptide MCC5145 against fulminant infection by S. pneumoniae serotype 2 strain D39 in a bioluminescent, neutropenic mouse model of bacteremic pneumonia. MCC5145 is a semisynthetic vancomycin derivative chemically modified at the C-terminus with a membrane-targeting motif designed to preferentially bind the anionic bacterial surface. We show that similar to vancomycin, subcutaneous administration of MCC5145 to mice 1 day after intranasal infection with a bioluminescent derivative of S. pneumoniae D39 elicited time and concentration-dependent reduction in total flux in the lungs and blood. Together, our finding supports the further development of MCC5145 as a potential new treatment option for pneumonia and/or bacteremic pneumonia in clinical settings, particularly for immunocompromised individuals. IMPORTANCE S. pneumoniae (the pneumococcus) causes severe community acquired lung and blood infection, especially among the elderly and people with underlying medical conditions and/or weakened immune systems. The rising incidence of antibiotic resistance and delays between diagnosis of infection and commencement of effective therapy make treatment difficult and result in high mortality rates. In this work, we show that a new derivative (MCC5145) of an existing antibiotic (vancomycin) rapidly eradicated lethal pneumococcal challenge from the lungs and blood of mice with a suppressed immune system. Our findings support that MCC5145 is a promising option for the treatment of lung and blood infections caused by the pneumococcus at point-of-care settings, particularly for the elderly and individuals with a weakened immune system.

Non-falciparum malaria infection and IgG seroprevalence among children under 15 years in Nigeria, 2018.

Plasmodium falciparum (Pf) is the dominant malaria parasite in Nigeria though P. vivax (Pv), P. ovale (Po), and P. malariae (Pm) are also endemic. Blood samples (n = 31,234) were collected from children aged 0-14 years during a 2018 nationwide HIV survey and assayed for Plasmodium antigenemia, Plasmodium DNA, and IgG against Plasmodium MSP1-19 antigens. Of all children, 6.6% were estimated to have Pm infection and 1.4% Po infection with no Pv infections detected. The highest household wealth quintile was strongly protective against infection with Pm (aOR: 0.11, 95% CI: 0.05-0.22) or Po (aOR= 0.01, 0.00-0.10). Overall Pm seroprevalence was 34.2% (95% CI: 33.3-35.2) with lower estimates for Po (12.1%, 11.6-12.5) and Pv (6.3%, 6.0-6.7). Pm seropositivity was detected throughout the country with several local government areas showing >50% seroprevalence. Serological and DNA indicators show widespread exposure of Nigerian children to Pm with lower rates to Po and Pv.

Plasmodium falciparum infection prevalence among children aged 6-59 months from independent DHS and HIV surveys: Nigeria, 2018.

Prevalence estimates are critical for malaria programming efforts but generating these from non-malaria surveys is not standard practice. Malaria prevalence estimates for 6-59-month-old Nigerian children were compared between two national household surveys performed simultaneously in 2018: a Demographic and Health Survey (DHS) and the Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS). DHS tested via microscopy (n = 8298) and HRP2-based rapid diagnostic test (RDT, n = 11,351), and NAIIS collected dried blood spots (DBS) which were later tested for histidine-rich protein 2 (HRP2) antigen (n = 8029). National Plasmodium falciparum prevalence was 22.6% (95% CI 21.2- 24.1%) via microscopy and 36.2% (34.6- 37.8%) via RDT according to DHS, and HRP2 antigenemia was 38.3% (36.7-39.9%) by NAIIS DBS. Between the two surveys, significant rank-order correlation occurred for state-level malaria prevalence for RDT (Rho = 0.80, p < 0.001) and microscopy (Rho = 0.75, p < 0.001) versus HRP2. RDT versus HRP2 positivity showed 24 states (64.9%) with overlapping 95% confidence intervals from the two independent surveys. P. falciparum prevalence estimates among 6-59-month-olds in Nigeria were highly concordant from two simultaneous, independently conducted household surveys, regardless of malaria test utilized. This provides evidence for the value of post-hoc laboratory HRP2 detection to leverage non-malaria surveys with similar sampling designs to obtain accurate P. falciparum estimates.

In Vitro Activity of Robenidine Analogues NCL259 and NCL265 against Gram-Negative Pathogens.

Multidrug-resistant (MDR) Gram-negative pathogens, especially Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter spp., are recognized by the World Health Organization as the most critical priority pathogens in urgent need of drug development. In this study, the in vitro antimicrobial activity of robenidine analogues NCL259 and NCL265 was tested against key human and animal Gram-negative clinical isolates and reference strains. NCL259 and NCL265 demonstrated moderate antimicrobial activity against these Gram-negative priority pathogens with NCL265 consistently more active, achieving lower minimum inhibitory concentrations (MICs) in the range of 2−16 µg/mL. When used in combination with sub-inhibitory concentrations of polymyxin B to permeabilize the outer membrane, NCL259 and NCL265 elicited a synergistic or additive activity against the reference strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. A small minority of Klebsiella spp. isolates (three) were resistant to both NCL259 and NCL265 with MICs > 256 µg/mL. This resistance was completely reversed in the presence of the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PAßN) to yield MIC values of 8−16 µg/mL and 2−4 µg/mL for NCL259 and NCL256, respectively. When NCL259 and NCL265 were tested against wild-type E. coli isolate BW 25113 and its isogenic multidrug efflux pump subunit AcrB deletion mutant (∆AcrB), the MIC of both compounds against the mutant ∆AcrB isolate was reduced 16-fold compared to the wild-type parent, indicating a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. In vitro cytotoxicity testing revealed that NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to the parent robenidine, thus precluding their further development as novel antibiotics against Gram-negative pathogens.

Cinnamaldehyde derivatives act as antimicrobial agents against Acinetobacter baumannii through the inhibition of cell division.

Acinetobacter baumannii is a pathogen with high intrinsic antimicrobial resistance while multidrug resistant (MDR) and extensively drug resistant (XDR) strains of this pathogen are emerging. Treatment options for infections by these strains are very limited, hence new therapies are urgently needed. The bacterial cell division protein, FtsZ, is a promising drug target for the development of novel antimicrobial agents. We have previously reported limited activity of cinnamaldehyde analogs against Escherichia coli. In this study, we have determined the antimicrobial activity of six cinnamaldehyde analogs for antimicrobial activity against A. baumannii. Microscopic analysis was performed to determine if the compounds inhibit cell division. The on-target effect of the compounds was assessed by analyzing their effect on polymerization and on the GTPase activity of purified FtsZ from A. baumannii. In silico docking was used to assess the binding of cinnamaldehyde analogs. Finally, in vivo and in vitro safety assays were performed. All six compounds displayed antibacterial activity against the critical priority pathogen A. baumannii, with 4-bromophenyl-substituted 4 displaying the most potent antimicrobial activity (MIC 32 µg/mL). Bioactivity was significantly increased in the presence of an efflux pump inhibitor for A. baumannii ATCC 19606 (up to 32-fold) and significantly, for extensively drug resistant UW 5075 (greater than 4-fold), suggesting that efflux contributes to the intrinsic resistance of A. baumannii against these agents. The compounds inhibited cell division in A. baumannii as observed by the elongated phenotype and targeted the FtsZ protein as seen from the inhibition of polymerization and GTPase activity. In silico docking predicted that the compounds bind in the interdomain cleft adjacent to the H7 core helix. Di-chlorinated 6 was devoid of hemolytic activity and cytotoxicity against mammalian cells in vitro, as well as adverse activity in a Caenorhabditis elegans nematode model in vivo. Together, these findings present halogenated analogs 4 and 6 as promising candidates for further development as antimicrobial agents aimed at combating A. baumannii. This is also the first report of FtsZ-targeting compounds with activity against an XDR A. baumannii strain.

A lipoglycopeptide antibiotic for Gram-positive biofilm-related infections.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria (Staphylococcus aureus and Streptococcus pneumoniae) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC90 ≤ 0.06 µg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae, as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

An assessment of infection prevention and control preparedness of healthcare facilities in Nigeria in the early phase of the COVID-19 pandemic (February-May 2020).

Background: Infection prevention and control (IPC) activities play a large role in preventing the transmission of SARS-CoV-2 in healthcare settings. This study describes the state of IPC preparedness within health facilities in Nigeria during the early phase of coronavirus disease (COVID-19) pandemic. Methods: We carried out a cross sectional study of health facilities across Nigeria using a COVID-19 IPC checklist adapted from the U.S Centers for Disease Control and Prevention. The IPC aspects assessed were the existence of IPC committee and teams with terms of reference and workplans, IPC training, availability of personal protective equipment and having systems in place for screening, isolation and notification of COVID-19 patients. Existence of the assessed aspects was regarded as preparedness in that aspect. Results: In total, 461 health facilities comprising, 350 (75.9%) private and 111 (24.1%) public health facilities participated. Only 19 (4.1%) health facilities were COVID-19 treatment centres with 68% of these being public health facilities. Public health facilities were better prepared in the areas of IPC programme with 69.7% of them having an IPC focal point versus 32.3% of private facilities. More public facilities (59.6%) had an IPC workplan versus 26.8% of private facilities. Neither the public nor the private facilities were adequately prepared for triaging, screening, and notifying suspected cases, as well as having trained staff and equipment to implement triaging. Conclusions: The results highlight the need for government, organisations and policymakers to establish conducive IPC structures to reduce the risk of COVID-19 transmission in healthcare settings.

Impact of a Novel Anticoccidial Analogue on Systemic Staphylococcus aureus Infection in a Bioluminescent Mouse Model.

In this study, we investigated the potential of an analogue of robenidine (NCL179) to expand its chemical diversity for the treatment of multidrug-resistant (MDR) bacterial infections. We show that NCL179 exhibits potent bactericidal activity, returning minimum inhibitory concentration/minimum bactericidal concentrations (MICs/MBCs) of 1-2 µg/mL against methicillin-resistant Staphylococcus aureus, MICs/MBCs of 1-2 µg/mL against methicillin-resistant S. pseudintermedius and MICs/MBCs of 2-4 µg/mL against vancomycin-resistant enterococci. NCL179 showed synergistic activity against clinical isolates and reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in the presence of sub-inhibitory concentrations of colistin, whereas NCL179 alone had no activity. Mice given oral NCL179 at 10 mg/kg and 50 mg/kg (4 × doses, 4 h apart) showed no adverse clinical effects and no observable histological effects in any of the organs examined. In a bioluminescent S. aureus sepsis challenge model, mice that received four oral doses of NCL179 at 50 mg/kg at 4 h intervals exhibited significantly reduced bacterial loads, longer survival times and higher overall survival rates than the vehicle-only treated mice. These results support NCL179 as a valid candidate for further development to treat MDR bacterial infections as a stand-alone antibiotic or in combination with existing antibiotic classes.

Repurposing of the Fasciolicide Triclabendazole to Treat Infections Caused by Staphylococcus spp. and Vancomycin-Resistant Enterococci.

One approach to combat the increasing incidence of multidrug-resistant (MDR) bacterial pathogens involves repurposing existing compounds with known safety and development pathways as new antibacterial classes with potentially novel mechanisms of action. Here, triclabendazole (TCBZ), a drug originally developed to treat Fasciola hepatica (liver fluke) in sheep and cattle, and later in humans, was evaluated as an antibacterial alone or in combination with sub-inhibitory concentrations of polymyxin B (PMB) against clinical isolates and reference strains of key Gram-positive and Gram-negative bacteria. We show for the first time that in vitro, TCBZ selectively kills methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius at a minimum inhibitory concentration (MIC) range of 2-4 µg/mL, and vancomycin-resistant enterococci at a MIC range of 4-8 µg/mL. TCBZ also inhibited key Gram-negative bacteria in the presence of sub-inhibitory concentrations of PMB, returning MIC90 values of 1 µg/mL for Escherichia coli, 8 µg/mL for Klebsiella pneumoniae, 2 µg/mL for Acinetobacter baumannii and 4 µg/mL for Pseudomonasaeruginosa. Interestingly, TCBZ was found to be bacteriostatic against intracellular S. aureus but bactericidal against intracellular S. pseudintermedius. Additionally, TCBZ's favourable pharmacokinetic (PK) and pharmacodynamic (PD) profile was further explored by in vivo safety and efficacy studies using a bioluminescent mouse model of S. aureus sepsis. We show that repeated four-hourly oral treatment of mice with 50 mg/kg TCBZ after systemic S. aureus challenge resulted in a significant reduction in S. aureus populations in the blood to 18 h post-infection (compared to untreated mice) but did not clear the bacterial infection from the bloodstream, consistent with in vivo bacteriostatic activity. These results indicate that additional pharmaceutical development of TCBZ may enhance its PK/PD, allowing it to be an appropriate candidate for the treatment of serious MDR bacterial pathogens.

Evaluation of Benzguinols as Next-Generation Antibiotics for the Treatment of Multidrug-Resistant Bacterial Infections.

Our recent focus on the "lost antibiotic" unguinol and related nidulin-family fungal natural products identified two semisynthetic derivatives, benzguinols A and B, with unexpected in vitro activity against Staphylococcus aureus isolates either susceptible or resistant to methicillin. Here, we show further activity of the benzguinols against methicillin-resistant isolates of the animal pathogen Staphylococcus pseudintermedius, with minimum inhibitory concentration (MIC) ranging 0.5-1 µg/mL. When combined with sub-inhibitory concentrations of colistin, the benzguinols demonstrated synergy against Gram-negative reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MICs of 1-2 µg/mL in the presence of colistin), whereas the benzguinols alone had no activity. Administration of three intraperitoneal (IP) doses of 20 mg/kg benzguinol A or B to mice did not result in any obvious adverse clinical or pathological evidence of acute toxicity. Importantly, mice that received three 20 mg/kg IP doses of benzguinol A or B at 4 h intervals exhibited significantly reduced bacterial loads and longer survival times than vehicle-only treated mice in a bioluminescent S. aureus murine sepsis challenge model. We conclude that the benzguinols are potential candidates for further development for specific treatment of serious bacterial infections as both stand-alone antibiotics and in combination with existing antibiotic classes.

Considerations for quality assurance of multiplex malaria antigen detection assays with large sample sets.

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of < 5%, with the exception of HRP2 in month 5 (CV of 11%). Regression fittings for inter-plate control signals provided mean and standard deviations (SDs), and of 504 assay plates, 6 (1.2%) violated the acceptable deviation limits and were repeated. For the 40,272 human blood samples assayed in this study, of 161,088 potential data points (each sample × 4 antigens), 160,641 (99.7%) successfully passed quality checks. The QA framework presented here can be utilized to ensure quality of laboratory antigen detection for large sample sets.

Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis.

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.

Comparison of Two Transmission Electron Microscopy Methods to Visualize Drug-Induced Alterations of Gram-Negative Bacterial Morphology.

In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.

In vitro synergistic activity of NCL195 in combination with colistin against Gram-negative bacterial pathogens.

In this study, the potential of using the novel antibiotic NCL195 combined with subinhibitory concentrations of colistin against infections caused by Gram-negative bacteria (GNB) was investigated. We showed synergistic activity of the combination NCL195 + colistin against clinical multidrug-resistant GNB pathogens with minimum inhibitory concentrations (MICs) for NCL195 ranging from 0.5-4 µg/mL for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas NCL195 alone had no activity. Transmission electron microscopy of the membrane morphology of E. coli and P. aeruginosa after single colistin or combination drug treatment showed marked ultrastructural changes most frequently in the cell envelope. Exposure to NCL195 alone did not show any change compared with untreated control cells, whereas treatment with the NCL195 + colistin combination caused more damage than colistin alone. Direct evidence for this interaction was demonstrated by fluorescence-based membrane potential measurements. We conclude that the synergistic antimicrobial activity of the combination NCL195 + colistin against GNB pathogens warrants further exploration for specific treatment of acute GNB infections.

Semisynthesis and biological evaluation of a focused library of unguinol derivatives as next-generation antibiotics.

In this study, we report the semisynthesis and in vitro biological evaluation of thirty-four derivatives of the fungal depsidone antibiotic, unguinol. Initially, the semisynthetic modifications were focused on the two free hydroxy groups (3-OH and 8-OH), the three free aromatic positions (C-2, C-4 and C-7), the butenyl side chain and the depsidone ester linkage. Fifteen first-generation unguinol analogues were synthesised and screened against a panel of bacteria, fungi and mammalian cells to formulate a basic structure activity relationship (SAR) for the unguinol pharmacophore. Based on the SAR studies, we synthesised a further nineteen second-generation analogues, specifically aimed at improving the antibacterial potency of the pharmacophore. In vitro antibacterial activity testing of these compounds revealed that 3-O-(2-fluorobenzyl)unguinol and 3-O-(2,4-difluorobenzyl)unguinol showed potent activity against both methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MIC 0.25-1 µg mL-1) and are promising candidates for further development in vivo.

Neutral electrolyzed oxidizing water is effective for pre-harvest decontamination of fresh produce.

Pre-harvest sanitization of irrigation water has potential for reducing pathogen contamination of fresh produce. We compared the sanitizing effects of irrigation water containing neutral electrolyzed oxidizing water (EOW) or sodium hypochlorite (NaClO) on pre-harvest lettuce and baby spinach leaves artificially contaminated with a mixture of Escherichia coli, Salmonella Enteritidis and Listeria innocua (~1 × 108 colony-forming units/mL each resuspended in water containing 100 mg/L dissolved organic carbon, simulating a splash-back scenario from contaminated soil/manure). The microbial load and leaf quality were assessed over 7 days, and post-harvest shelf life evaluated for 10 days. Irrigation with water containing EOW or NaClO at 50 mg/L free chlorine significantly reduced the inoculated bacterial load by ≥ 1.5 log10, whereas tap water irrigation reduced the inoculated bacterial load by an average of 0.5 log10, when compared with untreated leaves. There were no visual effects of EOW or tap water irrigation on baby spinach or lettuce leaf surfaces pre- or post-harvest, whereas there were obvious negative effects of NaClO irrigation on leaf appearance for both plants, including severe necrotic zones and yellowing/browning of leaves. Therefore, EOW could serve as a viable alternative to chemical-based sanitizers for pre-harvest disinfection of minimally processed vegetables.

A pH-neutral electrolyzed oxidizing water significantly reduces microbial contamination of fresh spinach leaves.

There are growing demands globally to use safe, efficacious and environmentally friendly sanitizers for post-harvest treatment of fresh produce to reduce or eliminate spoilage and foodborne pathogens. Here, we compared the efficacy of a pH-neutral electrolyzed oxidizing water (Ecas4 Anolyte; ECAS) with that of an approved peroxyacetic acid-based sanitizer (Ecolab Tsunami® 100) in reducing the total microbial load and inoculated Escherichia coli, Salmonella Enteritidis and Listeria innocua populations on post-harvest baby spinach leaves over 10 days. The impact of both sanitizers on the overall quality of the spinach leaves during storage was also assessed by shelf life and vitamin C content measurements. ECAS at 50 ppm and 85 ppm significantly reduced the bacterial load compared to tap water-treated or untreated (control) leaves, and at similar levels (approx. 10-fold reduction) to those achieved using 50 ppm of Ecolab Tsunami® 100. While there were no obvious deleterious effects of treatment with 50 ppm Tsunami® 100 or ECAS at 50 ppm and 85 ppm on plant leaf appearance, tap water-treated and untreated leaves showed some yellowing, bruising and sliming. Given its safety, efficacy and environmentally-friendly characteristics, ECAS could be a viable alternative to chemical-based sanitizers for post-harvest treatment of fresh produce.