Efflux-Related Carbapenem Resistance in Acinetobacter baumannii Is Associated with Two-Component Regulatory Efflux Systems' Alteration and Insertion of ΔAbaR25-Type Island Fragment.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

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.

Efflux-mediated tolerance to cationic biocides, a cause for concern?

AbstractWith an increase in the number of isolates resistant to multiple antibiotics, infection control has become increasingly important to help combat the spread of multi-drug-resistant pathogens. An important component of this is through the use of disinfectants and antiseptics (biocides). Antibiotic resistance has been well studied in bacteria, but little is known about potential biocide resistance genes and there have been few reported outbreaks in hospitals resulting from a breakdown in biocide effectiveness. Development of increased tolerance to biocides has been thought to be more difficult due to the mode of action of biocides which affect multiple cellular targets compared with antibiotics. Very few genes which contribute towards increased biocide tolerance have been identified. However, the majority of those that have are components or regulators of different efflux pumps or genes which modulate membrane function/modification. This review will examine the role of efflux in increased tolerance towards biocides, focusing on cationic biocides and heavy metals against Gram-negative bacteria. As many efflux pumps which are upregulated by biocide presence also contribute towards an antimicrobial resistance phenotype, the role of these efflux pumps in cross-resistance to both other biocides and antibiotics will be explored.

Pseudomonas aeruginosa adapts to octenidine via a combination of efflux and membrane remodelling.

Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.

Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland.

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D ß-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.

Long-Term Exposure to Octenidine in a Simulated Sink Trap Environment Results in Selection of Pseudomonas aeruginosa, Citrobacter, and Enterobacter Isolates with Mutations in Efflux Pump Regulators.

Octenidine-based disinfection products are becoming increasingly popular for infection control of multidrug-resistant (MDR) Gram-negative isolates. When a waste trap was removed from a hospital and allowed to acclimatize in a standard tap rig in our laboratory, it was shown that Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter and Enterobacter spp. were readily isolated. This study aimed to understand the potential impact of prolonged exposure to low doses of a commercial product containing octenidine on these bacteria. Phenotypic and genotypic analyses showed that P. aeruginosa strains had increased tolerance to octenidine, which was characterized by mutations in the Tet repressor SmvR. Enterobacter species demonstrated increased tolerance to many other cationic biocides, although not octenidine, as well as the antibiotics ciprofloxacin, chloramphenicol, and ceftazidime, through mutations in another Tet repressor, RamR. Citrobacter species with mutations in RamR and MarR were identified following octenidine exposure, and this is linked to development of resistance to ampicillin, piperacillin, and chloramphenicol, as well as an increased MIC for ciprofloxacin. Isolates were able to retain fitness, as characterized by growth, biofilm formation, and virulence in Galleria mellonella, after prolonged contact with octenidine, although there were strain-to-strain differences. These results demonstrate that continued low-level octenidine exposure in a simulated sink trap environment selects for mutations that affect smvR It may also promote microbial adaptation to other cationic biocides and cross-resistance to antibiotics, while not incurring a fitness cost. This suggests that hospital sink traps may act as a reservoir for more biocide-tolerant organisms.IMPORTANCE Multidrug-resistant (MDR) strains of bacteria are a major clinical problem, and several reports have linked outbreaks of MDR bacteria with bacterial populations in hospital sinks. Biocides such as octenidine are used clinically in body washes and other products, such as wound dressings for infection control. Therefore, increased tolerance to these biocides would be detrimental to infection control processes. Here, we exposed bacterial populations originally from hospital sink traps to repeated dosing with an octenidine-containing product over several weeks and observed how particular species adapted. We found mutations in genes related to biocide and antibiotic susceptibility, which resulted in increased tolerance, although this was species dependent. Bacteria that became more tolerant to octenidine also showed no loss of fitness. This shows that prolonged octenidine exposure has the potential to promote microbial adaptation in the environment and that hospital sink traps may act as a reservoir for increased biocide- and antibiotic-tolerant organisms.

Profiling protein expression in Klebsiella pneumoniae with a carbohydrate-based covalent probe.

We report the application of a covalent probe based on a d-glucosamine scaffold for the profiling of the bacterial pathogen Klebsiella pneumoniae. Incubation of K. pneumoniae lysates with the probe followed by electrophoretic separation and in-gel fluorescence detection allowed the generation of strain-specific signatures and the differentiation of a carbapenem-resistant strain. The labelling profile of the probe was independent of its anomeric configuration and included several low-abundance proteins not readily detectable by conventional protein staining. Initial target identification experiments by mass spectrometry suggest that target proteins include several carbohydrate-recognising proteins, which indicates that the sugar scaffold may have a role for target recognition.

New Broad-Spectrum Antibiotics Containing a Pyrrolobenzodiazepine Ring with Activity against Multidrug-Resistant Gram-Negative Bacteria.

It is urgent to find new antibiotic classes with activity against multidrug-resistant (MDR) Gram-negative pathogens as the pipeline of antibiotics is essentially empty. Modified pyrrolobenzodiazepines with a C8-linked aliphatic heterocycle provide a new class of broad-spectrum antibacterial agents with activity against MDR Gram-negative bacteria, including WHO priority pathogens. The structure-activity relationship established that the third ring was particularly important for Gram-negative activity. Minimum inhibitory concentrations for the lead compounds ranged from 0.125 to 2 mg/L for MDR Gram-negative, excluding Pseudomonas aeruginosa, and between 0.03 and 1 mg/L for MDR Gram-positive species. The lead compounds were rapidly bactericidal with >5 log reduction in viable count within 4 h for Acinetobacter baumannii and Klebsiella pneumoniae. The lead compound inhibited DNA gyrase in gel-based assays, with an IC50 of 3.16 ± 1.36 mg/L. This study provides a new chemical scaffold for developing novel broad-spectrum antibiotics which can help replenish the pipeline of antibiotics.

Mutations in the two component regulator systems PmrAB and PhoPQ give rise to increased colistin resistance in Citrobacter and Enterobacter spp.

Introduction. Colistin is a last resort antibiotic for treating infections caused by carbapenem-resistant isolates. Mechanisms of resistance to colistin have been widely described in Klebsiella pneumoniae and Escherichia coli but have yet to be characterized in Citrobacter and Enterobacter species.Aim. To identify the causative mutations leading to generation of colistin resistance in Citrobacter and Enterobacter spp.Methodology. Colistin resistance was generated by culturing in increasing concentrations of colistin or by direct culture in a lethal (above MIC) concentration. Whole-genome sequencing was used to identify mutations. Fitness of resistant strains was determined by changes in growth rate, and virulence in Galleria mellonella.Results. We were able to generate colistin resistance upon exposure to sub-MIC levels of colistin, in several but not all strains of Citrobacter and Enterobacter resulting in a 16-fold increase in colistin MIC values for both species. The same individual strains also developed resistance to colistin after a single exposure at 10× MIC, with a similar increase in MIC. Genetic analysis revealed that this increased resistance was attributed to mutations in PmrB for Citrobacter and PhoP in Enterobacter, although we were not able to identify causative mutations in all strains. Colistin-resistant mutants showed little difference in growth rate, and virulence in G. mellonella, although there were strain-to-strain differences.Conclusions. Stable colistin resistance may be acquired with no loss of fitness in these species. However, only select strains were able to adapt suggesting that acquisition of colistin resistance is dependent upon individual strain characteristics.

Evaluating the level of nitroreductase activity in clinical Klebsiella pneumoniae isolates to support strategies for nitro drug and prodrug development.

To understand the potential utility of novel nitroreductase (NR)-activated prodrugs, NR enzyme activity was assessed in clinical Klebsiella pneumoniae isolates using a NR-activated fluorescent probe. NR activity was constant throughout the bacterial growth cycle, but individual K. pneumoniae isolates exhibited a wide range of NR activity levels. The genes of major NR enzymes (nfsA and nfnB) showed a number of sequence variants. Aside from a C-terminal extension of NfnB, which may be responsible for lower NR activity in specific isolates, the genetic differences did not explain the variation in activity. Analysis of important clinical strains (ST11, ST258, ST14 and ST101) showed significant variation in NR activity between isolates within the same sequence type despite conservation of nfsA/nfnB sequences. Addition of methyl viologen (MV), a known activator of soxRS, caused a significant increase in NR activity for all strains, with proportionally larger increases in activity seen for strains with low uninduced NR levels. Real-time PCR on selected strains following exposure to MV showed upregulation of soxS (15-32-fold) and nfsA (5-22-fold) in all strains tested. Expression of nfnB was upregulated 2-5-fold in 4/6 strains tested. High levels of NR activity in the absence of MV activation correlated with nitrofurantoin susceptibility. These data provide evidence that NR gene mutations and regulatory pathways influence NR activity in K. pneumoniae isolates and this is likely to impact treatment efficacy with novel nitro-containing drugs or prodrugs.

SmvA is an important efflux pump for cationic biocides in Klebsiella pneumoniae and other Enterobacteriaceae.

The multidrug resistant (MDR) opportunistic pathogen Klebsiella pneumoniae has previously been shown to adapt to chlorhexidine by increasing expression of the MFS efflux pump smvA. Here we show that loss of the regulator SmvR, through adaptation to chlorhexidine, results in increased resistance to a number of cationic biocides in K. pneumoniae and other members of the Enterobacteriaceae. Clinical Enterobacteriaceae isolates which lack smvA and smvR also have an increased susceptibility to chlorhexidine. When smvA from Salmonella and K. pneumoniae are expressed in Escherichia coli, which lacks a homologue to SmvAR, resistance to chlorhexidine increased (4-fold) but plasmid carriage of smvA alone was detrimental to the cell. Challenge of K. pneumoniae with chlorhexidine and another cationic biocide, octenidine, resulted in increased expression of smvA (approx. 70 fold). Adaptation to octenidine was achieved through mutating key residues in SmvA (A363V; Y391N) rather than abolishing the function of SmvR, as with chlorhexidine adaptation. Molecular modelling was able to predict that octenidine interacted more strongly with these mutated SmvA forms. These results show that SmvA is a major efflux pump for cationic biocides in several bacterial species and that increased efflux through SmvA can lead to increased chlorhexidine and octenidine tolerance.

Intra Strain Variation of the Effects of Gram-Negative ESKAPE Pathogens on Intestinal Colonization, Host Viability, and Host Response in the Model Organism Caenorhabditis elegans.

In its native environment of rotting vegetation, the soil nematode Caenorhabditis elegans encounters a range of bacteria. This includes species from the ESKAPE group of pathogens that pose a clinical problem in acquired hospital infections. Here, we investigated three Gram-negative members of the ESKAPE group, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Pathogenicity profiles as measured by time to kill adult C. elegans showed that P. aeruginosa was the most pathogenic, followed by K. pneumoniae, while C. elegans cultured on A. baumannii exhibited the same survival as those on the standard laboratory food source for C. elegans, Escherichia coli OP50. The pathogenicity was paralleled by a reduction in time that C. elegans resided on the bacterial lawn with the most pathogenic strains triggering an increase in the frequency of food-leaving. Previous reports indicate that gut colonization is a feature of pathogenicity, but we found that the most pathogenic strains were not associated with the highest level of colonization. Indeed, clearance of P. aeruginosa strains from the C. elegans gut was independent of bacterial pathogenicity. We show that this clearance is regulated by neuromodulation as C. elegans mutants in unc-31 and egl-3 have enhanced clearance of P. aeruginosa. Intriguingly this is also not linked to their pathogenicity. It is likely that there is a dynamic balance occurring in the C. elegans intestinal environment between maintaining a healthy, beneficial microbiota and removal of pathogenic bacteria.

Clinically Relevant Plasmid-Host Interactions Indicate that Transcriptional and Not Genomic Modifications Ameliorate Fitness Costs of Klebsiella pneumoniae Carbapenemase-Carrying Plasmids.

The rapid dissemination of antimicrobial resistance (AMR) around the globe is largely due to mobile genetic elements, such as plasmids. They confer resistance to critically important drugs, including extended-spectrum beta-lactams, carbapenems, and colistin. Large, complex resistance plasmids have evolved alongside their host bacteria. However, much of the research on plasmid-host evolution has focused on small, simple laboratory plasmids in laboratory-adapted bacterial hosts. These and other studies have documented mutations in both host and plasmid genes which occur after plasmid introduction to ameliorate fitness costs of plasmid carriage. We describe here the impact of two naturally occurring variants of a large AMR plasmid (pKpQIL) on a globally successful pathogen. In our study, after pKpQIL plasmid introduction, no changes in coding domain sequences were observed in their natural host, Klebsiella pneumoniae However, significant changes in chromosomal and plasmid gene expression may have allowed the bacterium to adapt to the acquisition of the AMR plasmid. We hypothesize that this was sufficient to ameliorate the associated fitness costs of plasmid carriage, as pKpQIL plasmids were maintained without selection pressure. The dogma that removal of selection pressure (e.g., antimicrobial exposure) results in plasmid loss due to bacterial fitness costs is not true for all plasmid/host combinations. We also show that pKpQIL impacted the ability of K. pneumoniae to form a biofilm, an important aspect of virulence. This study used highly relevant models to study the interaction between AMR plasmids and pathogens and revealed striking differences from results of studies done on laboratory-adapted plasmids and strains.IMPORTANCE Antimicrobial resistance is a serious problem facing society. Many of the genes that confer resistance can be shared between bacteria through mobile genetic elements, such as plasmids. Our work shows that when two clinically relevant AMR plasmids enter their natural host bacteria, there are changes in gene expression, rather than changes to gene coding sequences. These changes in gene expression ameliorate the potential fitness costs of carriage of these AMR plasmids. In line with this, the plasmids were stable within their natural host and were not lost in the absence of selective pressure. We also show that better understanding of the impact of resistance plasmids on fundamental pathogen biology, including biofilm formation, is crucial for fighting drug-resistant infections.

Revisiting unexploited antibiotics in search of new antibacterial drug candidates: the case of γ-actinorhodin.

Of the thousands of natural product antibiotics discovered to date, only a handful have been developed for the treatment of bacterial infection. The clinically unexploited majority likely include compounds with untapped potential as antibacterial drugs, and in view of the ever-growing unmet medical need for such agents, warrant systematic re-evaluation. Here we revisit the actinorhodins, a class that was first reported 70 years ago, but which remains poorly characterized. We show that γ-actinorhodin possesses many of the requisite properties of an antibacterial drug, displaying potent and selective bactericidal activity against key Gram-positive pathogens (including Staphylococcus aureus and enterococci), a mode of action distinct from that of other agents in clinical use, an extremely low potential for the development of resistance, and a degree of in vivo efficacy in an invertebrate model of infection. Our findings underscore the utility of revisiting unexploited antibiotics as a source of novel antibacterial drug candidates.

Retention of virulence following colistin adaptation in Klebsiella pneumoniae is strain-dependent rather than associated with specific mutations.

This study aimed to understand the impact on virulence and fitness of mutations in specific genes found after adaptation of Klebsiella pneumoniae to colistin. Isolates with an increase in their inhibitory concentration (MIC) to colistin of 32- to >128-fold were shown to have mutations in mgrB, phoPQ and pmrAB, all known regulators of pathways affecting membrane lipid content. When these strains were used in studies in Galleria mellonella there was no clear correlation between mutations in specific genes per se and loss of virulence. Strains which showed sequence duplication in the HAMP-domain of PmrB showed reduced virulence but strains with point mutations in pmrAB showed no decrease in virulence. Similarly, specific mutations in mgrB in individual strains showed either loss of virulence or no effect/increased virulence. This study suggests that the impact on virulence may be independent of the colistin resistance mechanism and reflects differences in individual strain backgrounds.

Novel pyridyl nitrofuranyl isoxazolines show antibacterial activity against multiple drug resistant Staphylococcus species.

A novel series of pyridyl nitrofuranyl isoxazolines were synthesized and evaluated for their antibacterial activity against multiple drug resistant (MDR) Staphylococcus strains. Compounds with piperazine linker between the pyridyl group and isoxazoline ring showed better activity when compared to compounds without the piperazine linker. 3-Pyridyl nitrofuranyl isoxazoline with a piperazine linker was found to be more active than corresponding 2-and 4-pyridyl analogues with MICs in the range of 4-32µg/mL against MDR Staphylococcus strains. The eukaryotic toxicity of the compounds was tested by MTT assay and were found to be non-toxic against both non-tumour lung fibroblast WI-38 and cervical cancer cell line HeLa. The most active pyridyl nitrofuranyl isoxazoline compound showed improved activity against a panel of Staphylococcus strains compared to nitrofuran group containing antibiotic nitrofurantoin.

Triaryl Benzimidazoles as a New Class of Antibacterial Agents against Resistant Pathogenic Microorganisms.

A new class of nontoxic triaryl benzimidazole compounds, derived from existing classes of DNA minor groove binders, were designed, synthesized, and evaluated for their antibacterial activity against multidrug resistant (MDR) Gram-positive and Gram-negative species. Molecular modeling experiments suggest that the newly synthesized class cannot be accommodated within the minor groove of DNA due to a change in the shape of the molecules. Compounds 8, 13, and 14 were found to be the most active of the series, with MICs in the range of 0.5-4 µg/mL against the MDR Staphylococci and Enterococci species. Compound 13 showed moderate activity against the MDR Gram-negative strains, with MICs in the range of 16-32 µg/mL. Active compounds showed a bactericidal mode of action, and a mechanistic study suggested the inhibition of bacterial gyrase as the mechanism of action (MOA) of this chemical class. The MOA was further supported by the molecular modeling study.