The identification of pathogens directly from blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be a valuable tool for improving the treatment of patients with sepsis and bacteremia. However, the increasing incidence of multidrug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one ␤-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd-generation cephalosporins in a MALDI-TOF MS ␤-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the ␤-lactamase assay were compared with those of conventional methods. The assay permitted discrimination between E. coli strains that were resistant or susceptible to aminopenicillins with a sensitivity and a specificity of 100%. The same was true for resistance to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C ␤-lactamases. Discrimination was more difficult in species expressing class A ␤-lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. The test permitted the prediction of resistance within 2.5 h after the blood culture was flagged as positive.

SUMMARYLivestock-associated methicillin-resistantStaphylococcus aureus(LA-MRSA) have been isolated from a number of livestock species and persons involved in animal production. We investigated the prevalence of LA-MRSA in fattening turkeys and people living on farms that house fattening turkeys. Eighteen (90%) of 20 investigated flocks were positive for MRSA, and on 12 of the farms 22 (37·3%) of 59 persons sampled were positive for MRSA. People with frequent access to the stables were more likely to be positive for MRSA. In most flocks MRSA that could be assigned to clonal complex (CC) 398 were detected. In five flocks MRSA ofspa-type t002 that is not related to CC398 were identified. Moreover, other methicillin-resistantStaphylococcusspp. were detected on 11 farms and in eight people working on the farms.

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1 and Tac1, which result in constitutive overexpression of multidrug efflux pumps, and Upc2, which result in constitutive overexpression of ergosterol biosynthesis genes. However, the deregulated gene expression that is caused by hyperactive forms of these transcription factors also reduces the fitness of the cells in the absence of the drug. To investigate whether fluconazole-resistant clinical C. albicans isolates have overcome the fitness costs of drug resistance, we assessed the relative fitness of C. albicans isolates containing resistance mutations in these transcription factors in competition with matched drug-susceptible isolates from the same patients. Most of the fluconazole-resistant isolates were outcompeted by the corresponding drug-susceptible isolates when grown in rich medium without fluconazole. On the other hand, some resistant isolates with gain-of-function mutations in MRR1 did not exhibit reduced fitness under these conditions. In a mouse model of disseminated candidiasis, three out of four tested fluconazole-resistant clinical isolates did not exhibit a significant fitness defect. However, all four fluconazole-resistant isolates were outcompeted by the matched susceptible isolates in a mouse model of gastrointestinal colonization, demonstrating that the effects of drug resistance on in vivo fitness depend on the host niche. Collectively, our results indicate that the fitness costs of drug resistance in C. albicans are not easily remediated, especially when proper control of gene expression is required for successful adaptation to life within a mammalian host.

Pretreatment of skin with all-trans retinoic acid (tretinoin) has been shown to enhance wound healing. Previous studies have mainly used animal models to demonstrate this effect. We wanted to determine whether pretreatment could promote wound healing in severely photoaged dorsal forearm skin. Four elderly men with severely actinically damaged forearms were treated daily for 16 weeks. One arm was treated with 0.05-0.1% tretinoin cream (Retin A, Ortho), and the other with Purpose cream (Ortho) as a vehicle control. Four-millimetre punch biopsies were taken from both dorsal forearms prior to treatment. After 16 weeks, full-thickness 2-mm punch biopsies were taken from both sides. Serial photographs were taken, and healing of the wounds quantitatively assessed by image analysis. On the 11th day, the wounds were excised using a 4-mm biopsy punch. Biopsies were processed for light microscopy. After 16 weeks, the tretinoin-treated forearms showed moderate erythema and scaling. Polarized light photographs revealed multiple, red, vascularized foci and/or a diffuse network of small vessels. The histological effects were typical for tretinoin, i.e. compaction of the stratum corneum, epidermal acanthosis with correction of atypia, an increase in small vessels, and increased cellularity in the upper dermis. Purpose cream had no effect, either clinically or histologically. On the tretinoin-treated side, the wound areas were 35-37% smaller on days 1 and 4, and 47-50% smaller on days 6, 8, 11, compared with the controls. Clinically and histologically, reepithelialization occurred more rapidly. Thus tretinoin dramatically accelerated wound healing in photodamaged skin.

The clonal population structure of Candida albicans suggests that (para)sexual recombination does not play an important role in the lifestyle of this opportunistic fungal pathogen, an assumption that is strengthened by the fact that most C. albicans strains are heterozygous at the mating type locus (MTL) and therefore mating-incompetent. On the other hand, mating might occur within clonal populations and allow the combination of advantageous traits that were acquired by individual cells to adapt to adverse conditions. We have investigated if parasexual recombination may be involved in the evolution of highly drug-resistant strains exhibiting multiple resistance mechanisms against fluconazole, an antifungal drug that is commonly used to treat infections by C. albicans. Growth of strains that were heterozygous for MTL and different fluconazole resistance mutations in the presence of the drug resulted in the emergence of derivatives that had become homozygous for the mutated allele and the mating type locus and exhibited increased drug resistance. When MTLa/a and MTLα/α cells of these strains were mixed in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Therefore, fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence, which allows cells in an originally clonal population to exchange individually acquired resistance mechanisms and generate highly drug-resistant progeny. IMPORTANCE Sexual reproduction is an important mechanism in the evolution of species, since it allows the combination of advantageous traits of individual members in a population. The pathogenic yeast Candida albicans is a diploid organism that normally propagates in a clonal fashion, because heterozygosity at the mating type locus (MTL) inhibits mating between cells. Here we show that C. albicans cells that have acquired drug resistance mutations during treatment with the commonly used antifungal agent fluconazole rapidly develop further increased resistance by genome rearrangements that result in simultaneous loss of heterozygosity for the mutated allele and the mating type locus. This enables the drug-resistant cells of a population to switch to the mating-competent opaque morphology and mate with each other to combine different individually acquired resistance mechanisms. The tetraploid mating products reassort their merged genomes and, under selective pressure by the drug, generate highly resistant progeny that have retained the advantageous mutated alleles. Parasexual propagation, promoted by stress-induced genome rearrangements that result in the acquisition of mating competence in cells with adaptive mutations, may therefore be an important mechanism in the evolution of C. albicans populations.

No abstract

The overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the widely used antimycotic agent fluconazole and other toxic compounds in the pathogenic yeast Candida albicans. Infections by the pathogenic yeast Candida albicans are commonly treated with the antifungal agent fluconazole, which inhibits ergosterol biosynthesis. C. albicans can develop resistance to fluconazole by different mechanisms, which often are combined to result in clinically relevant, high-level resistance (20). Besides mutations in the target enzyme that decrease its affinity for the drug, alterations in gene expression are a frequent cause of fluconazole resistance. The constitutive upregulation of ergosterol biosynthesis genes and the overexpression of multidrug efflux pumps of the ABC transporter and major facilitator superfamilies all result in increased azole resistance. Zinc cluster proteins, a family of transcription factors that is unique to the fungal kingdom (17), play a central role in the regulation of genes involved in drug resistance. In C. albicans, Upc2 controls the expression of ergosterol biosynthesis genes (16, 34), Tac1 regulates the expression of the ABC transporters CDR1 and CDR2 (4), and Mrr1 controls the expression of the major facilitator MDR1 (22). These transcription factors mediate the upregulation of their respective target genes in response to inducing stimuli. In addition, gain-of-function mutations in Upc2, Tac1, and Mrr1 result in the constitutive activation of the transcription factors and overexpression of their target genes in fluconazole-resistant strains (1-4, 6, 7, 11, 12, 22, 33, 37). However, it is currently not understood how these transcriptional regulators achieve an activated state under inducing conditions or after the acquisition of gain-offunction mutations.The zinc cluster proteins are defined by a conserved DNA binding motif, which consists of six cysteine residues that coordinate two zinc atoms (Zn 2 Cys 6 ). Most zinc cluster transcription factors have their DNA binding domain at the N terminus, a large negative regulatory domain in the middle of the protein, and an activation domain at the C terminus (17). The transcriptional activity of zinc cluster proteins can be regulated in different ways, but many of them are activated by the binding of inducing molecules (17,23). These can be metabolites, as for transcription factors regulating genes involved in nutrient utilization and biosyntheses (e.g., Put3 and Leu3), or xenobiotics, which activate the pleiotropic drug resistance regulators Pdr1 and Pdr3 in Saccharomyces cerevisiae and Candida glabrata. Pdr1 and Pdr3 are functionally related to Tac1 and Mrr1 of C. albicans, because they also control the expression of multidrug efflux pumps. It has been shown that the binding of drugs and other toxic chemicals to the negative regulatory domain of Pdr1/Pdr3 results in a conformational change that enables the C-terminal activation domain to interact with the...

On the dorsal hands, MAL-IPL reduced AK more efficaciously than placebo-IPL; both treatment modalities significantly improved photoaged skin.