Interplay in the Selection of Fluoroquinolone Resistance and Bacterial Fitness

Fluoroquinolones are antibacterial drugs that inhibit DNA Gyrase and Topoisomerase IV. These essential enzymes facilitate chromosome replication and RNA transcription by regulating chromosome supercoiling. High-level resistance to fluoroquinolones in E. coli requires the accumulation of multiple mutations, including those that alter target genes and genes regulating drug efflux. Previous studies have shown some drug-resistance mutations reduce bacterial fitness, leading to the selection of fitness-compensatory mutations. The impact of fluoroquinolone-resistance on bacterial fitness was analyzed in constructed isogenic strains carrying up to 5 resistance mutations. Some mutations significantly decreased bacterial fitness both in vitro and in vivo. We identified low-fitness triple-mutants where the acquisition of a fourth resistance mutation significantly increased fitness in vitro and in vivo while at the same time dramatically decreasing drug susceptibility. The largest effect occurred with the addition of a parC mutation (Topoisomerase IV) to a low-fitness strain carrying resistance mutations in gyrA (DNA Gyrase) and marR (drug efflux regulation). Increased fitness was accompanied by a significant change in the level of gyrA promoter activity as measured in an assay of DNA supercoiling. In selection and competition experiments made in the absence of drug, parC mutants that improved fitness and reduced susceptibility were selected. These data suggest that natural selection for improved growth in bacteria with low-level resistance to fluoroquinolones could in some cases select for further reductions in drug susceptibility. Thus, increased resistance to fluoroquinolones could be selected even in the absence of further exposure to the drug

Differential Gene Expression by RamA in Ciprofloxacin-Resistant Salmonella Typhimurium

Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM). The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways

Genomic variation landscape of the human gut microbiome

While large-scale efforts have rapidly advanced the understanding and practical impact of human genomic variation, the latter is largely unexplored in the human microbiome. We therefore developed a framework for metagenomic variation analysis and applied it to 252 fecal metagenomes of 207 individuals from Europe and North America. Using 7.4 billion reads aligned to 101 reference species, we detected 10.3 million single nucleotide polymorphisms (SNPs), 107,991 short indels, and 1,051 structural variants. The average ratio of non-synonymous to synonymous polymorphism rates of 0.11 was more variable between gut microbial species than across human hosts. Subjects sampled at varying time intervals exhibited individuality and temporal stability of SNP variation patterns, despite considerable composition changes of their gut microbiota. This implies that individual-specific strains are not easily replaced and that an individual might have a unique metagenomic genotype, which may be exploitable for personalized diet or drug intake

Minimale Hemm-Konzentration (MHK) und minimale bakterizide Konzentration (MBK) von Polihexanid und Triclosan gegen Antibiotika-empfindliche und resistente Staphylococcus aureus- und Escherichia coli-Stämme

Background: An in-vitro study was conducted investigating the antim icrob ial efficacy of polihexanide and triclosan against clinical isola tes and reference laboratory strains of Staphylococcus aureus and E scherich ia coli .Methods: The minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined following DIN 58940-81 using a micro-dilution assay and a quantitative suspension test following EN 1040. Polihexanide was tested in polyethylene glycol 4000, triclosan in aqueous solutions.Results: Against all tested strains the MIC of polihexanide ranged between 1-2 µg/mL. For triclosan the MICs varied depending on strains ranging between 0.5 µg/mL for the reference strains and 64 µg/mL for two clinical isolates. A logRF >5 without and logRF >3 with 0.2% album in burden was achieved at 0.6 µg/mL triclosan. One exception was S. aureus strain H-5-24, where a triclosan concentration of 0.6 µg/mL required 1 minute without and 10 minutes with albumin burden to achieve the same logRFs. Polihexanide achieved a logRF >5 without and logRF >3 with albumin burden at a concentration of 0.6 µg/mL within 30 sec. The exception was the North-German epidemic MRSA strain, were an application time of 5 minutes was required. Conclusion: The clinical isolates of E. coli generally showed higher MICs against triclosan, both in the micro-dilution assay as well in the quantit ativ e suspension test than comparable reference laboratory strains. For polihexanide and triclosan strain dependant susceptibility was shown. However, both antimicrobial compounds are effective when used in concentrations common in practice.Hintergrund: In einer quantitativen in-vitro Studie wurde der antimikrobielle Effekt von Polihexanid und Triclosan unter identischen Verhältnissen gegen Referenzstämme sowie klinische Isolate von Staphylococcus aureus und Escherichia coli bestimmt.Methoden: Die minimale Hemmkonzentration (MHK) und die minimale mikrobiozide Konzentration (MMK) wurden gemäß DIN 58940-81 mittels Mikro-Verdünnungs-Essay und eines quantitativen Verdünnungstests gemäß EN 1040 bestimmt. Polihexanid wurde in einer Polyethylene-Glykol 4000 Lösung, Triclosan in wässriger Lösung angesetzt. Ergebnisse: Die MHK von Polihexanid gegen alle getesteten Stämme lag zwischen 1-2 µg/mL. Die MHK von Triclosan war stammabhängig und lag bei 0,5 µg/mL für die getesteten Referenzstämme und bei 64 µg/mL für zwei klinischen Isolate. Gegen alle Stämme erreichte Triclosan bei einer Konzentration von 0,6 µg/mL einen logRF >5 ohne und logRF >3 mit 0,2% Albuminbelastung. Als Ausnahme erwies sich S. aureus -Stamm H-5-24, für den eine Triclosan-Konzentration von 0,6 µg/mL bei einer Einwirkungszeit von 1 min ohne bzw. 10 min mit Albuminbelastung erforderlich war, um dieselben logRF zu erreichen. Polihexanid erreichte bei einer Konzentration von 0,6 µg/mL einen logRF >5 ohne und logRF >3 mit Belastung bei einer Einwirkungszeit von 30 s . Hier erwies sich als Ausnahme der Norddeutsche MRSA Epidemiestamm, gegen den bei selber Konzentration eine Einwirkungszeit von 5 min erforderlich war. Schlussfolgerungen: Die klinischen E. coli -Isolate erforderten höhere MHKs bei Triclosan als die untersuchten Referenzstämme. Für Polih exan id und Triclosan konnte eine stammabhängige Empfindlichkeit gezeigt werden. Beide Antiseptika werden im klinischen Einsatz jedoch regelhaft bei weit höheren Konzentrationen eingesetzt, womit die unterschiedlichen Empfindlichkeiten klinisch keine Rolle spielen sollten