Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains.

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.

Impact of treatment modality on quality of life of head and neck cancer patients: Findings from an academic medical institution.

PURPOSE: The objective of this pilot study was to determine how different treatment modalities (surgery, radiation, and chemotherapy) impact quality of life (QOL) in a population of head and neck cancer (HNC) survivors. METHODS: Fifty-nine newly diagnosed, biopsy-confirmed HNC patients were recruited between 2007-2012. They completed the EORTC Quality of Life Questionnaire and Head & Neck Module at 5 intervals pre- and post-treatment. Participants were grouped into four categories based on modality: surgery only, surgery/radiation, chemoradiation, or surgery/chemoradiation. Repeated measures ANOVA examined effect of treatment modality on QOL over time. RESULTS: Xerostomia symptoms were significantly associated with chemoradiation (F(2.47, 59.27)=3.57, p=0.03), lowest at pretreatment and highest 6 months post-treatment. Time was significantly associated with head and neck pain, F(2.95,67.89)=3.39, p=0.02. CONCLUSIONS: HNC survivors exhibit different QOL related symptoms depending on combined treatment modalities, and time post-treatment. It is important to understand QOL differences based upon treatment modalities when developing treatment plans for HNC patients.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid detection of ß-lactam resistance in Enterobacteriaceae derived from blood cultures.

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 ß-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 ß-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the ß-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 ß-lactamases. Discrimination was more difficult in species expressing class A ß-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.

Liver pathology associated with increased mortality in turkey breeder and meat turkey flocks.

Between 2006 and 2011 a series of disease conditions characterized by raised mortality and liver disorders occurred in turkey breeder flocks and in meat turkey flocks in Germany. The flocks were between 12 and 23 wk of age, and mostly hens were affected. Clinical signs were nonspecific and accompanied by mortality varying between 1% and 7%. Affected birds displayed swollen livers that were marbled with black and red spots and yellowish areas. The pericardium was filled with an amber fluid, and the coronary groove was extensively filled with fat. Spleens were swollen, and a serous fluid that seemed to leak from the liver was present in the body cavity. Histopathological findings in all but one case included fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhages. Some animals showed cholangitis and hepatitis with intranuclear inclusion bodies. In three cases with breeders, electron microscopy detected virus particles that were between 23 and 30 nm and similar to parvo- or picornavirus. In addition, picornavirus RNA was detected in the livers of one meat turkey flock. Investigations by PCR for circovirus, polyomavirus parvovirus, and aviadenovirus yielded negative results in all cases, but an aviadenovirus was isolated from livers twice and a reovirus from the intestines once. Supplementation with vitamin E and selenium seemed to improve the situation. The most likely diagnosis is lipidosis, a metabolic disorder with complex etiology, which has rarely been described in turkeys.

Functional dissection of a Candida albicans zinc cluster transcription factor, the multidrug resistance regulator Mrr1.

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. The zinc cluster transcription factor Mrr1 controls MDR1 expression in response to inducing chemicals, and gain-of-function mutations in MRR1 are responsible for the constitutive MDR1 upregulation in fluconazole-resistant C. albicans strains. To understand how Mrr1 activity is regulated, we identified functional domains of this transcription factor. A hybrid protein consisting of the N-terminal 106 amino acids of Mrr1 and the transcriptional activation domain of Gal4 from Saccharomyces cerevisiae constitutively induced MDR1 expression, demonstrating that the DNA binding domain is sufficient to target Mrr1 to the MDR1 promoter. Using a series of C-terminal truncations and systematic internal deletions, we could show that Mrr1 contains multiple activation and inhibitory domains. One activation domain (AD1) is located in the C terminus of Mrr1. When fused to the tetracycline repressor TetR, this distal activation domain induced gene expression from a TetR-dependent promoter. The deletion of an inhibitory region (ID1) located near the distal activation domain resulted in constitutive activity of Mrr1. The additional removal of AD1 abolished the constitutive activity, but the truncated Mrr1 still could activate the MDR1 promoter in response to the inducer benomyl. These results demonstrate that the activity of Mrr1 is regulated in multiple ways and provide insights into the function of an important mediator of drug resistance in C. albicans.

Type C botulism in a commercial turkey farm: a case report.

Botulism is an intoxication caused by exotoxins of Clostridium botulinum. The case of botulism described here occurred on a commercial meat turkey farm with two houses. Toms and hens were maintained in two separate houses, toms in house A and hens in house B. At 10 wk of age, an increase in mortality was observed in the toms located in house A. Clinically the animals presented with paralysis of the legs, wings, and neck. Affected birds were sitting and reluctant to move. Necropsy failed to find any specific lesions. In liver, heart, muscles, crop, and gizzard as well as in intestinal contents, DNA of C. botulinum type C was detected by PCR. The result was confirmed by a mouse lethality neutralization test. During the 2 wk after the onset of the clinical signs the mortality was about 12%. The hens kept in house B did not show any symptoms and remained healthy. Investigations of environmental samples to detect the source of the toxin were not successful. After 2 wk clinical signs and mortality abated. At 16 wk of age, toms again showed the same clinical signs accompanied by raised mortality. Again C. botulinum toxin type C was detected. Within 2 wk the total mortality reached roughly 50%. Based on the "precautionary principle" and in agreement with the local authorities, the birds were euthanatized using CO2 in order to not compromise food safety.

Evolution of Fluconazole-Resistant Candida albicans Strains by Drug-Induced Mating Competence and Parasexual Recombination.

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.

An unusual outbreak of histomonosis in a commercial turkey flock.

In the past histomonosis was very well controlled with Dimetridazole as a treatment and/or Nifursol as feed additive. In the European Union both products were banned in 1995 and 2003, respectively. This was followed by the re-emergence of the disease in the recent years. In the present case a farm with two houses was affected by the disease. In each house 2620 hens and 2620 toms were kept, separated by wire mesh. At the 53rd day of age the toms in house 1 showed general clinical symptoms, accompanied by a slightly raised mortality, which sharply increased in the following days. At necropsy all dead birds showed lesions typical for histomonosis in caeca and liver. Histomonosis was diagnosed by histopathology and PCR. Within five days cumulative mortality was 25.1%.The hens kept at the same house didn't show any symptoms. At day 57 two toms, which were kept in house 2, died and showed similar symptoms and lesions. Within the next three days 48 more birds died. Again the hens in house 2 showed neither clinical signs nor mortality. Treatment trials using herbal products and a change of litter directly after the onset of clinical signs did not reduce the mortality. On day 62 the toms of both houses were euthanized by CO2 in closed containers. The hens were kept until they were slaughtered in week 16 and did not show any evidence of histomonosis.