Investigation of the Anti-Methicillin-Resistant Staphylococcus aureus Activity of (+)-Tanikolide- and (+)-Malyngolide-Based Analogues Prepared by Asymmetric Synthesis.

Herein, we report antibacterial and antifungal evaluation of a series of previously prepared (+)-tanikolide analogues. One analogue, (4S,6S)-4-methyltanikolide, displayed promising anti-methicillin-resistant Staphylococcus aureus activity with a MIC of 12.5 µg/mL. Based on the antimicrobial properties of the structurally related (-)-malyngolide, two further analogues (4S,6S)-4-methylmalyngolide and (4R,6S)-4-methylmalyngolide bearing a shortened n-nonyl alkyl side chain were prepared in the present study using a ZrCl4-catalysed deprotection/cyclisation as the key step in their asymmetric synthesis. When these were tested for activity against anti-methicillin-resistant Staphylococcus aureus, the MIC increased to 50 µg/mL.

Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae.

In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance.

Enriching antimicrobial peptides from milk hydrolysates using pectin/alginate food-gels.

Cationic antimicrobial peptides have raised interest as attractive alternatives to classical antibiotics, and also have utility in preventing food spoilage. We set out to enrich cationic antimicrobial peptides from milk hydrolysates using gels containing various ratios of anionic pectin/alginate. All processes were carried out with food-grade materials in order to suggest food-safe methods suited for producing food ingredients or supplements. Hydrolysed caseinate peptides retained in the gel fraction, identified by mass spectrometry, were enriched for potential antimicrobial peptides, as judged by a computational predictor of antimicrobial activity. Peptides retained in a 60:40 pectin:alginate gel fraction had a strong antimicrobial effect against 8 tested bacterial strains with a minimal inhibitory concentration of 1.5-5 mg/mL, while the unfractionated hydrolysate only had a detectable effect in one of the eight strains. Among 110 predicted antimicrobial peptides in the gel fraction, four are known antimicrobial peptides, HKEMPFPK, TTMPLW, YYQQKPVA and AVPYPQR. These results highlight the potential of pectin/alginate food-gels based processes as safe, fast, cost-effective methods to separate and enrich for antimicrobial peptides from complex food protein hydrolysates.

Klebsiella pneumoniae: Prevalence, Reservoirs, Antimicrobial Resistance, Pathogenicity, and Infection: A Hitherto Unrecognized Zoonotic Bacterium.

Klebsiella pneumoniae is considered an opportunistic pathogen, constituting an ongoing health concern for immunocompromised patients, the elderly, and neonates. Reports on the isolation of K. pneumoniae from other sources are increasing, many of which express multidrug-resistant (MDR) phenotypes. Three phylogroups were identified based on nucleotide differences. Niche environments, including plants, animals, and humans appear to be colonized by different phylogroups, among which KpI (K. pneumoniae) is commonly associated with human infection. Infections with K. pneumoniae can be transmitted through contaminated food or water and can be associated with community-acquired infections or between persons and animals involved in hospital-acquired infections. Increasing reports are describing detections along the food chain, suggesting the possibility exists that this could be a hitherto unexplored reservoir for this opportunistic bacterial pathogen. Expression of MDR phenotypes elaborated by these bacteria is due to the nature of various plasmids carrying antimicrobial resistance (AMR)-encoding genes, and is a challenge to animal, environmental, and human health alike. Raman spectroscopy has the potential to provide for the rapid identification and screening of antimicrobial susceptibility of Klebsiella isolates. Moreover, hypervirulent isolates linked with extraintestinal infections express phenotypes that may support their niche adaptation. In this review, the prevalence, reservoirs, AMR, Raman spectroscopy detection, and pathogenicity of K. pneumoniae are summarized and various extraintestinal infection pathways are further narrated to extend our understanding of its adaptation and survival ability in reservoirs, and associated disease risks.

Molecular characterisation of multi-drug resistant Escherichia coli of bovine origin.

Antimicrobial resistance reported in bacteria of animal origin is considered a major challenge to veterinary public health. In this study, the genotypic and phenotypic characterisation of twelve Escherichia coli isolates of bovine origin is reported. Twelve bacterial isolates of animal origin were selected from a previous study based on their multidrug resistant (MDR) profile. Efflux pump activity was measured using ethidium bromide (EtBr) and the biofilm forming ability of the individual strains was assessed using a number of phenotypic assays. All isolates were resistant to tetracyclines and a number of isolates expressed resistance to fluoroquinolones which was also confirmed in silico by the presence of these resistance markers. Amino acid substitutions in the quinolone resistance-determining regions were identified in all isolates and the presence of several siderophores were also noted. Whole genomesequence (WGS) data showed different STs that were not associated with epidemic STs or virulent clonal complexes. Seven isolates formed biofilms in minimal media with some isolates showing better adaptation at 25 °C while others at 37 °C. The capacity to efflux EtBr was found to be high in 4 isolates and impaired in 4 others. The pathogenicity of three selected isolates was assessed in zebrafish embryo infection models, revealing isolates CFS0355 and CFS0356 as highly pathogenic. These results highlight the application of NGS technologies combined with phenotypic assays in providing a better understanding of E. coli of bovine origin and their adaptation to this niche environment.

Draft Genome Sequences of Three Novel Acinetobacter Isolates from an Irish Commercial Pig Farm.

Acinetobacter species are important in the emergence and spread of antimicrobial resistance (AMR), which threatens human and animal health worldwide. Here, we present the draft genome sequences of three Acinetobacter species strains (RF14B, RF15A, and RF15B) isolated from pig feces and the floor of a pig hospital pen in Ireland.

Yersinia hibernica sp. nov., isolated from pig-production environments.

A Gram-stain-negative, rod-shaped strain isolated from pig-production environments was identified as a new species within the genus Yersinia using multifaceted genomic and biochemical approaches. The genome of this strain was closed using a hybrid assembly approach combining both high accuracy short read sequencing data with long read sequencing technology. Phylogenetic analysis of the 16S rRNA gene showed ~98 % similarity to Yersinia kristensenii and ~98 % similarity to Yersinia enterocolitica. Average nucleotide identity (OrthoANI) values were calculated as 85.79 % to Y. kristensenii ATCC 33638T and 85.73 % to Y. enterocolitica ATCC 9610T thereby providing evidence that this isolate should be considered as a novel species. The type strain is CFS1934T (=NCTC 14222T=LMG 31076T).

Susceptibility (re)-testing of a large collection of Listeria monocytogenes from foods in China from 2012 to 2015 and WGS characterization of resistant isolates.

OBJECTIVES: Our aim was to determine the antimicrobial susceptibilities of 2862 Listeria monocytogenes cultured from various foods in China and to use WGS to characterize the antimicrobial resistance and virulence genotypes of those expressing a resistance phenotype. METHODS: The susceptibilities of 2862 L. monocytogenes were determined by broth microdilution. Twenty-eight L. monocytogenes were found to be resistant to one to four antibiotics. All 28 resistant isolates were subsequently sequenced using short-read high accuracy protocols. The corresponding genomes were assembled and further analysis was carried out using appropriate bioinformatics pipelines. RESULTS: All 28 resistant L. monocytogenes were classified into five STs (ST3, ST8, ST9, ST155 and ST515). Both ST9 and ST155 were dominant and their genotypes correlated with their resistance phenotypes. All ST9 isolates were MDR and could be phylogenetically classified into two clusters. One was relatively close to clinical origins and one to food. Downstream analysis of the genetic contexts in which these resistance genotypes were found suggested that these may have been acquired from other bacteria by horizontal transfer or insertion into the chromosome. All isolates harboured Listeria pathogenicity island (LIPI)-1 and LIPI-2, and only two harboured LIPI-3. CONCLUSIONS: This study reported on the antimicrobial susceptibilities of 2862 foodborne L. monocytogenes along with the genomic characterization of 28 resistant isolates, 11 of which expressed an MDR phenotype. These data showed that this bacterium can acquire resistance by horizontal gene transfer in and between species. This study may necessitate a re-evaluation of risk to public health, associated with this bacterial species.

Exposure to Sub-inhibitory Concentrations of the Chemosensitizer 1-(1-Naphthylmethyl)-Piperazine Creates Membrane Destabilization in Multi-Drug Resistant Klebsiella pneumoniae.

Antimicrobial efflux is one of the important mechanisms causing multi-drug resistance (MDR) in bacteria. Chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) can inhibit an efflux pump and therefore can overcome MDR. However, secondary effects of NMP other than efflux pump inhibition are rarely investigated. Here, using phenotypic assays, phenotypic microarray and transcriptomic assays we show that NMP creates membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The NMP mediated membrane destabilization activity was measured using ß-lactamase activity, membrane potential alteration studies, and transmission electron microscopy assays. Results from both ß-lactamase and membrane potential alteration studies shows that both outer and inner membranes are destabilized in NMP exposed K. pneumoniae MGH 78578 cells. Phenotypic Microarray and RNA-seq were further used to elucidate the metabolic and transcriptional signals underpinning membrane destabilization. Membrane destabilization happens as early as 15 min post-NMP treatment. Our RNA-seq data shows that many genes involved in envelope stress response were differentially regulated in the NMP treated cells. Up-regulation of genes encoding the envelope stress response and repair systems show the distortion in membrane homeostasis during survival in an environment containing sub-inhibitory concentration of NMP. In addition, the lsr operon encoding the production of autoinducer-2 responsible for biofilm production was down-regulated resulting in reduced biofilm formation in NMP treated cells, a phenotype confirmed by crystal violet-based assays. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition.

Reversing Antimicrobial Resistance in Multidrug-Resistant Klebsiella pneumoniae of Clinical Origin Using 1-(1-Naphthylmethyl)-Piperazine.

Eleven clinical Klebsiella pneumoniae fluoroquinolone-resistant isolates were tested to access the potential of adjuvant therapies to reduce antimicrobial resistance using fixed concentrations of the chemosensitizers chlorpromazine (CPZ), thioridazine (TZ), phenylalanine-arginine-ß-naphthylamide (PAßN), and 1-(1-naphthylmethyl)-piperazine-(NMP) with varying concentrations of antimicrobial agents nalidixic acid (NAL), ciprofloxacin (CIP), moxifloxacin (MXF), tetracycline (TET), and chloramphenicol (CHL). Ethidium bromide dye was used together with the chemosensitizers to investigate permeabilization effects. NMP was assessed for its capacity to reduce the mass of biofilm alone and in combination with CIP and MXF. Of the selected chemosensitizers, NMP exhibited the greatest capacity to reverse resistance and inhibit efflux, based on the concentrations tested. Susceptibility to antimicrobial agents including (fluoro)quinolones, TET, and CHL were found to be increased in the presence of NMP, in a concentration-dependent manner. PAßN also demonstrated similar effects when combined with the chemosensitizers tested. In the case of half of the isolates studied, NMP alone reduced preformed biofilm biomass. Combinations of latter along with CIP or MXF were also found to reduce the mass of preformed biofilm, in the case of only some of the bacterial isolates. The capacity of NMP to reduce antimicrobial resistance could be of relevance as a strategy to limit bacterial colonization on abiotic surfaces.

Exploring the Genome and Phenotype of Multi-Drug Resistant Klebsiella pneumoniae of Clinical Origin.

Klebsiella pneumoniae is an important nosocomial pathogen with an extraordinary resistant phenotype due to a combination of acquired resistant-elements and efflux mechanisms. In this study a detailed molecular characterization of 11 K. pneumoniae isolates of clinical origin was carried out. Eleven clinical isolates were tested for their susceptibilities, by disk diffusion and broth microdilution and interpreted according to CLSI guidelines. Efflux activity was determined by measuring the extrusion of ethidium bromide and biofilm formation was assessed following static growth in Müeller-Hinton and minimal media M9 broths at two temperatures and time points. Template DNA from all 11 isolates was extracted and sequenced. The study collection was found to be resistant to several (extended-spectrum beta-lactam) ESBL-type compounds along with several (fluoro)quinolones (FQ). Resistance to tetracycline accounted for 55% of the study collection (n = 6) and three of the 11 isolates were resistance to carbapenems. Genotyping identified blaCTX-M-15 (82%), blaSHV-12 (55%), and blaTEM-1B (45%) ESBL encoding genes and FQ resistance was associated the presence of the oqxAB operon, identified in 10 of the 11 isolates and qnrB gene in one isolate. The polymorphisms detected in the quinolone resistance-determining regions (QRDRs) were associated with isolates of the clonal group CG15. Sequence types (ST) identified were representative of previously described clonal groups including CG258 (n = 7), CG15 (n = 3), and CG147 (n = 1). Plasmid replicon type databases were queried indicating the presence of IncFII and IncFIB replicon types in the majority of the isolates (91%), followed by IncFIA (45%), and IncR (45%). Two of the 11 isolates were found positive for yersiniabactin siderophore-encoding genes. No differences in the ability to efflux ethidium bromide were identified. Biofilm formation was stronger when the isolates were grown under stressed conditions at 37°C for a period up to 96 h. These data confirm the fact that well-recognized clonal groups of K. pneumoniae of importance to human health carries a diverse repertoire of antimicrobial resistance determinants, particularly related to critically important drugs in the ESBL and FQ classes. The capacity of most isolates to form strong biofilms, when stressed under laboratory-simulated conditions, supports the risk to human health associated with nosocomial infections deriving from indwelling medical devices.

The ins and outs of RND efflux pumps in Escherichia coli.

Infectious diseases remain one of the principal causes of morbidity and mortality in the world. Relevant authorities including the WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. They have also reaffirmed the urgent need for investment in the discovery and development of new antibiotics and therapeutic approaches to treat multidrug resistant (MDR) bacteria. The extensive use of antimicrobial compounds in diverse environments, including farming and healthcare, has been identified as one of the main causes for the emergence of MDR bacteria. Induced selective pressure has led bacteria to develop new strategies of defense against these chemicals. Bacteria can accomplish this by several mechanisms, including enzymatic inactivation of the target compound; decreased cell permeability; target protection and/or overproduction; altered target site/enzyme and increased efflux due to over-expression of efflux pumps. Efflux pumps can be specific for a single substrate or can confer resistance to multiple antimicrobials by facilitating the extrusion of a broad range of compounds including antibiotics, heavy metals, biocides and others, from the bacterial cell. To overcome antimicrobial resistance caused by active efflux, efforts are required to better understand the fundamentals of drug efflux mechanisms. There is also a need to elucidate how these mechanisms are regulated and how they respond upon exposure to antimicrobials. Understanding these will allow the development of combined therapies using efflux inhibitors together with antibiotics to act on Gram-negative bacteria, such as the emerging globally disseminated MDR pathogen Escherichia coli ST131 (O25:H4). This review will summarize the current knowledge on resistance-nodulation-cell division efflux mechanisms in E. coli, a bacteria responsible for community and hospital-acquired infections, as well as foodborne outbreaks worldwide.