QSAR Reveals Decreased Lipophilicity of Polar Residues Determines the Selectivity of Antimicrobial Peptide Activity.

Antimicrobial resistance has increased rapidly, causing daunting morbidity and mortality rates worldwide. Antimicrobial peptides (AMPs) have emerged as promising alternatives to traditional antibiotics due to their broad range of targets and low tendency to elicit resistance. However, potent antimicrobial activity is often accompanied by excessive cytotoxicity toward host cells, leading to a halt in AMP therapeutic development. Here, we present multivariate analyses that correlate 28 peptide properties to the activity and toxicity of 46 diverse African-derived AMPs and identify the negative lipophilicity of polar residues as an essential physiochemical property for selective antimicrobial activity. Twenty-seven active AMPs are identified, of which the majority are of scorpion or frog origin. Of these, thirteen are novel with no previously reported activities. Principal component analysis and quantitative structure-activity relationships (QSAR) reveal that overall hydrophobicity, lipophilicity, and residue side chain surface area affect the antimicrobial and cytotoxic activity of an AMP. This has been well documented previously, but the present QSAR analysis additionally reveals that a decrease in the lipophilicity, contributed by those amino acids classified as polar, confers selectivity for a peptide to pathogen over mammalian cells. Furthermore, an increase in overall peptide charge aids selectivity toward Gram-negative bacteria and fungi, while selectivity toward Gram-positive bacteria is obtained through an increased number of small lipophilic residues. Finally, a conservative increase in peptide size in terms of sequence length and molecular weight also contributes to improved activity without affecting toxicity. Our findings suggest a novel approach for the rational design or modification of existing AMPs to increase pathogen selectivity and enhance therapeutic potential.

Development of Novel Membrane Disrupting Lipoguanidine Compounds Sensitizing Gram-Negative Bacteria to Antibiotics.

A new class of amphiphilic molecules, the lipoguanidines, designed as hybrids of guanidine and fatty acid compounds, has been synthesized and developed. The new molecules present both a guanidine polar head and a lipophilic tail that allow them to disrupt bacterial membranes and to sensitize Gram-negative bacteria to the action of the narrow-spectrum antibiotics rifampicin and novobiocin. The lipoguanidine 5g sensitizes Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to rifampicin, thereby reducing the antibiotic minimum inhibitory concentrations (MIC) up to 256-fold. Similarly, 5g is able to potentiate novobiocin up to 64-fold, thereby showing a broad spectrum of antibiotic potentiating activity. Toxicity and mechanism studies revealed the potential of 5g to work synergistically with rifampicin through the disruption of bacterial membranes without affecting eukaryotic cells.

Fibrous matrices facilitate pleurocidin killing of wound associated bacterial pathogens.

Conventional wound infection treatments neither actively promote wound healing nor address the growing problem of antibacterial resistance. Antimicrobial peptides (AMPs) are natural defense molecules, released from host cells, which may be rapidly bactericidal, modulate host-immune responses, and/or act as endogenous mediators for wound healing. However, their routine clinical use has hitherto been hindered due to their instability in the wound environment. Here we describe an electrospun carrier system for topical application of pleurocidin, demonstrating sufficient AMP release from matrices to kill wound-associated pathogens including Acinetobacter baumannii and Pseudomonas aeruginosa. Pleurocidin can be incorporated into polyvinyl alcohol (PVA) fiber matrices, using coaxial electrospinning, without major drug loss with a peptide content of 0.7% w/w predicted sufficient to kill most wound associated species. Pleurocidin retains its activity on release from the electrospun fiber matrix and completely inhibits growth of two strains of A. baumannii (AYE; ATCC 17978) and other ESKAPE pathogens. Inhibition of P. aeruginosa strains (PAO1; NCTC 13437) is, however, matrix weight per volume dependent, with only larger/thicker matrices maintaining complete inhibition. The resulting estimation of pleurocidin release from the matrix reveals high efficiency, facilitating a greater AMP potency. Wound matrices are often applied in parallel or sequentially with the use of standard wound care with biocides, therefore the presence and effect of biocides on pleurocidin potency was tested. It was revealed that combinations displayed additive or modestly synergistic effects depending on the biocide and pathogens which should be considered during the therapy. Taken together, we show that electrospun, pleurocidin-loaded wound matrices have potential to be investigated for wound infection treatment.

COVID-19 therapeutics: stewardship in England and considerations for antimicrobial resistance.

The COVID-19 pandemic saw unprecedented resources and funds driven into research for the development, and subsequent rapid distribution, of vaccines, diagnostics and directly acting antivirals (DAAs). DAAs have undeniably prevented progression and life-threatening conditions in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, there are concerns of antimicrobial resistance (AMR), antiviral resistance specifically, for DAAs. To preserve activity of DAAs for COVID-19 therapy, as well as detect possible mutations conferring resistance, antimicrobial stewardship and surveillance were rapidly implemented in England. This paper expands on the ubiquitous ongoing public health activities carried out in England, including epidemiologic, virologic and genomic surveillance, to support the stewardship of DAAs and assess the deployment, safety, effectiveness and resistance potential of these novel and repurposed therapeutics.

Phospholipid headgroup composition modulates the molecular interactions and antimicrobial effects of sulfobetaine zwitterionic detergents against the "ESKAPE" pathogen Pseudomonas aeruginosa.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa. Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

Generation of SARS-CoV-2 escape mutations by monoclonal antibody therapy.

COVID-19 patients at risk of severe disease may be treated with neutralising monoclonal antibodies (mAbs). To minimise virus escape from neutralisation these are administered as combinations e.g. casirivimab+imdevimab or, for antibodies targeting relatively conserved regions, individually e.g. sotrovimab. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to detect emerging drug resistance in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the antibody epitopes and for casirivimab+imdevimab multiple mutations are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.

Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Electrical Broth Micro-Dilution for Rapid Antibiotic Resistance Testing.

Rapid tests to assess the susceptibility of bacteria to antibiotics are required to inform antibiotic stewardship. We have developed a novel test, which measures changes in the impedance of a 100 nanoliter volume of bacterial suspension to determine an "electrical" minimum inhibitory concentration (eMIC). Two representative strains of Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were tested against a panel of frontline antibiotics with different modes of action (ciprofloxacin, doxycycline, colistin and imipenem, gentamicin, and ceftazidime). The eMIC measured at 1 h correlated strongly with a standard 24 h microbroth dilution MIC for all combinations of antibiotics and bacteria, allowing strains to be correctly assigned as sensitive or resistant measured in a fraction of the time.

Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens.

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.

Synergy between Winter Flounder antimicrobial peptides.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

Covalent DNA Binding Is Essential for Gram-Negative Antibacterial Activity of Broad Spectrum Pyrrolobenzodiazepines.

It is urgent to find new antibiotic classes against multidrug-resistant bacteria as the rate of discovery of new classes of antibiotics has been very slow in the last 50 years. Recently, pyrrolobenzodiazepines (PBDs) with a C8-linked aliphatic-heterocycle have been identified as a new broad-spectrum antibiotic class with activity against Gram-negative bacteria. The active imine moiety of the reported lead pyrrolobenzodiazepine compounds was replaced with amide to obtain the non-DNA binding and noncytotoxic dilactam analogues to understand the structure-activity relationship further and improve the safety potential of this class. The synthesised compounds were tested against panels of multidrug-resistant Gram-positive and Gram-negative bacteria, including WHO priority pathogens. Minimum inhibitory concentrations for the dilactam analogues ranged from 4 to 32 mg/L for MDR Gram-positive bacteria, compared to 0.03 to 2 mg/L for the corresponding imine analogues. At the same time, they were found to be inactive against MDR Gram-negative bacteria, with a MIC > 32 mg/L, compared to a MIC of 0.5 to 32 mg/L for imine analogues. A molecular modelling study suggests that the lack of imine functionality also affects the interaction of PBDs with DNA gyrase. This study suggests that the presence of N10-C11 imine moiety is crucial for the broad-spectrum activity of pyrrolobenzodiazepines.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

Establishing the selective phospholipid membrane coordination, permeation and lysis properties for a series of 'druggable' supramolecular self-associating antimicrobial amphiphiles.

The rise of antimicrobial resistance remains one of the greatest global health threats facing humanity. Furthermore, the development of novel antibiotics has all but ground to a halt due to a collision of intersectional pressures. Herein we determine the antimicrobial efficacy for 14 structurally related supramolecular self-associating amphiphiles against clinically relevant Gram-positive methicillin resistant Staphylococcus aureus and Gram-negative Escherichia coli. We establish the ability of these agents to selectively target phospholipid membranes of differing compositions, through a combination of computational host:guest complex formation simulations, synthetic vesicle lysis, adhesion and membrane fluidity experiments, alongside our novel 1H NMR CPMG nanodisc coordination assays, to verify a potential mode of action for this class of compounds and enable the production of evermore effective next-generation antimicrobial agents. Finally, we select a 7-compound subset, showing two lead compounds to exhibit 'druggable' profiles through completion of a variety of in vivo and in vitro DMPK studies.

Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms.

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.

Bactericidal Effect of Ultrasound-Responsive Microbubbles and Sub-inhibitory Gentamicin against Pseudomonas aeruginosa Biofilms on Substrates With Differing Acoustic Impedance.

The aim of this research was to explore the interaction between ultrasound-activated microbubbles (MBs) and Pseudomonas aeruginosa biofilms, specifically the effects of MB concentration, ultrasound exposure and substrate properties on bactericidal efficacy. Biofilms were grown using a Centre for Disease Control (CDC) bioreactor on polypropylene or stainless-steel coupons as acoustic analogues for soft and hard tissue, respectively. Biofilms were treated with different concentrations of phospholipid-shelled MBs (107-108 MB/mL), a sub-inhibitory concentration of gentamicin (4 µg/mL) and 1-MHz ultrasound with a continuous or pulsed (100-kHz pulse repetition frequency, 25% duty cycle, 0.5-MPa peak-to-peak pressure) wave. The effect of repeated ultrasound exposure with intervals of either 15- or 60-min was also investigated. With polypropylene coupons, the greatest bactericidal effect was achieved with 2 × 5 min of pulsed ultrasound separated by 60 min and a microbubble concentration of 5 × 107 MBs/mL. A 0.76 log (83%) additional reduction in the number of bacteria was achieved compared with the use of an antibiotic alone. With stainless-steel coupons, a 67% (0.46 log) reduction was obtained under the same exposure conditions, possibly due to enhancement of a standing wave field which inhibited MB penetration in the biofilm. These findings demonstrate the importance of treatment parameter selection in antimicrobial applications of MBs and ultrasound in different tissue environments.

Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile.

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defense. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A, but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

Nuclear Magnetic Resonance Metabolomics of Symbioses between Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome, characterized by low levels of lactobacilli and overgrowth of a diverse group of bacteria, associated with higher risk of a variety of infections, surgical complications, cancer, and preterm birth (PTB). Despite the lack of a consistently applicable etiology, Prevotella spp. are often associated with both BV and PTB, and Pr. bivia has known symbiotic relationships with both Peptostreptococcus anaerobius and Gardnerella vaginalis. Higher risk of PTB can also be predicted by a composite of metabolites linked to bacterial metabolism, but their specific bacterial source remains poorly understood. Here, we characterize diversity of metabolic strategies among BV-associated bacteria and lactobacilli and the symbiotic metabolic relationships between Pr. bivia and its partners and show how these influence the availability of metabolites associated with BV/PTB and/or pro- or anti-inflammatory immune responses. We confirm a commensal relationship between Pe. anaerobius and Pr. bivia, refining its mechanism, which sustains a substantial increase in acetate production. In contrast, the relationship between Pr. bivia and G. vaginalis strains, with sequence variant G2, is mutualistic, with outcome dependent on the metabolic strategy of the G. vaginalis strain. Taken together, our data show how knowledge of inter- and intraspecies metabolic diversity and the effects of symbiosis may refine our understanding of the mechanism and approach to risk prediction in BV and/or PTB. IMPORTANCE Bacterial vaginosis (BV) is the most common vaginal infection for women of childbearing age. Although 50% of women with BV do not have any symptoms, it approximately doubles the risk of catching a sexually transmitted infection and also increases the risk of preterm delivery in pregnant women. Recent studies of the vaginal microbiota have suggested that variation between species in the same genus or between strains of the same species explain better or poorer outcomes or at least some coexistence patterns for bacteria of concern. We tested whether such variation is manifested in how vaginal bacteria grow in the laboratory and whether and how they may share nutrients. We then showed that this affected the overall cocktail of chemicals they produce, including bacterially derived chemicals that we have previously shown are linked to a higher risk of preterm delivery.

Mutations in SilS and CusS/OmpC represent different routes to achieve high level silver ion tolerance in Klebsiella pneumoniae.

BACKGROUND: Silver ions have potent broad-spectrum antimicrobial activity and are widely incorporated into a variety of products to limit bacterial growth. In Enterobacteriaceae, decreased silver susceptibility has been mapped to two homologous operons; the chromosomally located cus operon and the plasmid based sil operon. Here we characterised the mechanisms and clinical impact of induced silver tolerance in Klebsiella pneumoniae. RESULTS: In K. pneumoniae carriage of the sil operon alone does not give elevated silver tolerance. However, when exposed to increasing concentrations of silver nitrate (AgNO3), K. pneumoniae strains which contain the sil operon, will preferentially mutate SilS, resulting in overexpression of the genes encoding the RND efflux pump silCBA. Those strains which do not carry the sil operon also adapt upon exposure to increasing silver concentrations through mutations in another two-component regulator CusS. Secondary mutations leading to disruption of the outer membrane porin OmpC were also detected. Both routes result in a high level of silver tolerance with MIC's of >512 mg/L. When exposed to a high concentration of AgNO3 (400 mg/L), only strains that contained the sil operon were able to survive, again through mutations in SilS. The AgNO3 adapted strains were also resistant to killing by challenge with several clinical and commercial silver containing dressings. CONCLUSIONS: This study shows that K. pneumoniae has two possible pathways for development of increased silver tolerance but that the sil operon is preferentially mutated. This operon is essential when K. pneumoniae is exposed to high concentrations of silver. The potential clinical impact on wound management is shown by the increased survivability of these adapted strains when exposed to several silver impregnated dressings. This would make infections with these strains more difficult to treat and further limits our therapeutic options.

Contribution of the efflux pump AcrAB-TolC to the tolerance of chlorhexidine and other biocides in Klebsiella spp.

Introduction. We are becoming increasingly reliant on the effectiveness of biocides to combat the spread of Gram-negative multi-drug-resistant (MDR) pathogens, including Klebsiella pneumoniae. It has been shown that chlorhexidine exposure can lead to mutations in the efflux pump repressor regulators SmvR and RamR, but the contribution of each individual efflux pump to biocide tolerance is unknown.Hypothesis. Multiple efflux pumps, including SmvA and AcrAB-TolC, are involved in increased tolerance to biocides. However, strains with upregulated AcrAB-TolC caused by biocide exposure are more problematic due to their increased MDR phenotype.Aim. To investigate the role of AcrAB-TolC in the tolerance to several biocides, including chlorhexidine, and the potential threat of cross-resistance to antibiotics through increased expression of this efflux pump.Methodology. Antimicrobial susceptibility testing was performed on K. pneumoniae isolates with ramR mutations selected for after exposure to chlorhexidine, as well as transposon mutants in components and regulators of AcrAB-TolC. RTPCR was used to detect the expression levels of this pump after biocide exposure. Strains from the globally important ST258 clade were compared for genetic differences in acrAB-TolC and its regulators and for phenotypic differences in antimicrobial susceptibility.Results. Cross-resistance to antimicrobials was observed following mutations in ramR. Exposure to chlorhexidine led to increased expression of acrA and its activator ramA, and transposon mutants in AcrAB-TolC have increased susceptibility to several biocides, including chlorhexidine. Variations in ramR within the ST258 clade led to an increase in tolerance to certain biocides, although this was strain dependent. One strain, MKP103, that had increased levels of biocide tolerance showed a unique mutation in ramR that was reflected in enhanced expression of acrA and ramA. MKP103 transposon variants were able to further enhance their tolerance to specific biocides with mutations affecting SmvA.Conclusions. Biocide tolerance in K. pneumoniae is dependent upon several components, with increased efflux through AcrAB-TolC being an important one.

Genomic Diversity of Bacteriophages Infecting the Genus Acinetobacter.

The number of sequenced Acinetobacter phage genomes in the International Nucleotide Sequence Database Collaboration has increased significantly in recent years, from 37 in 2017 to a total of 139 as of January 2021 with genome sizes ranging from 31 to 378 kb. Here, we explored the genetic diversity of the Acinetobacter phages using comparative genomics approaches that included assessment of nucleotide similarity, shared gene content, single gene phylogeny, and the network-based classification tool vConTACT2. Phages infecting Acinetobacter sp. are genetically diverse and can be grouped into 8 clusters (subfamilies) and 46 sub-clusters (genera), of which 8 represent genomic singletons (additional genera). We propose the creation of five new subfamilies and suggest a reorganisation of the genus Obolenskvirus. These results provide an updated view of the viruses infecting Acinetobacter species, providing insights into their diversity.