Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhône Valley.

Staphylococcus aureus is a major bovine mastitis pathogen. Although the reported antimicrobial resistance was generally low, the emergence of new genetic clusters in bovine mastitis requires examination of the link between antimicrobial resistance and genotypes. Here, amplified fragment length polymorphism (AFLP) profiles and standard antimicrobial resistance profiles were determined in order to characterize a total of 343 S. aureus cow mastitis isolates from two geographically close regions of Switzerland and France. AFLP profiles revealed similar population compositions in the two regions, with 4 major clusters (C8, C20, C97, and C151), but the proportions of isolates in each cluster significantly diverged between the two countries (P = 9.2 × 10⁻⁹). Antimicrobial resistance was overall low (< 5% resistance to all therapeutically relevant molecules), with the exception of penicillin resistance, which was detected in 26% of the isolates. Penicillin resistance proportions differed between clusters, with only 1 to 2% of resistance associated with C20 and C151 and up to 70% associated with bovine C97. The prevalence of C20 and C8 was unexpectedly high and requires further investigation into the mechanism of adaptation to the bovine host. The strong association of penicillin resistance with few clusters highlights the fact that the knowledge of local epidemiology is essential for rational choices of antimicrobial treatment in the absence of susceptibility testing. Taken together, these observations argue in favor of more routine scrutiny of antimicrobial resistance and antibiotic-resistant clones in cattle and the farm environment.

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis.

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

Human-to-bovine jump of Staphylococcus aureus CC8 is associated with the loss of a β-hemolysin converting prophage and the acquisition of a new staphylococcal cassette chromosome.

Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human.

Long-Term Trends of HIV Type 1 Drug Resistance Prevalence among Antiretroviral Treatment-Experienced Patients in Switzerland.

Background. Accurate quantification of the prevalence of human immunodeficiency virus type 1 (HIV-1) drug resistance in patients who are receiving antiretroviral therapy (ART) is difficult, and results from previous studies vary. We attempted to assess the prevalence and dynamics of resistance in a highly representative patient cohort from Switzerland. Methods. On the basis of genotypic resistance test results and clinical data, we grouped patients according to their risk of harboring resistant viruses. Estimates of resistance prevalence were calculated on the basis of either the proportion of individuals with a virologic failure or confirmed drug resistance (lower estimate) or the frequency-weighted average of risk group-specific probabilities for the presence of drug resistance mutations (upper estimate). Results. Lower and upper estimates of drug resistance prevalence in 8064 ART-exposed patients were 50% and 57% in 1999 and 37% and 45% in 2007, respectively. This decrease was driven by 2 mechanisms: loss to follow-up or death of high-risk patients exposed to mono- or dual-nucleoside reverse-transcriptase inhibitor therapy (lower estimates range from 72% to 75%) and continued enrollment of low-risk patients who were taking combination ART containing boosted protease inhibitors or nonnucleoside reverse-transcriptase inhibitors as first-line therapy (lower estimates range from 7% to 12%). A subset of 4184 participants (52%) had 1 study visit per year during 2002-2007. In this subset, lower and upper estimates increased from 45% to 49% and from 52% to 55%, respectively. Yearly increases in prevalence were becoming smaller in later years. Conclusions. Contrary to earlier predictions, in situations of free access to drugs, close monitoring, and rapid introduction of new potent therapies, the emergence of drug-resistant viruses can be minimized at the population level. Moreover, this study demonstrates the necessity of interpreting time trends in the context of evolving cohort populations.

Female plumage spottiness and parasite resistance in the barn owl (Tyto alba)

The hypothesis that extravagant ornaments signal parasite resistance has received support in several species for ornamented males but more rarely for ornamented females. However, recent theories have proposed that females should often be under sexual selection, and therefore females may signal the heritable capacity to resist parasites. We investigated this hypothesis in the socially monogamous barn owl, Tyto alba, in which females exhibit on average more and larger black spots on the plumage than males, and in which males were suggested to choose a mate with respect to female plumage spottiness. We hypothesized that the proportion of the plumage surface covered by black spots signals parasite resistance. In line with this hypothesis, we found that the ectoparasitic fly, Carnus hemapterus, was less abundant on young raised by more heavily spotted females and those flies were less fecund. In an experiment, where entire clutches were cross-fostered between nests, we found that the fecundity of the flies collected on nestlings was negatively correlated with the genetic mother's plumage spottiness. These results suggest that the ability to resist parasites covaries with the extent of female plumage spottiness. Among females collected dead along roads, those with a lot of black spots had a small bursa of Fabricius. Given that parasites bigger the development of this immune organ, this observation further suggests that more spotted females are usually less parasitized. The same analyses performed on male plumage spottiness all provided non-significant results. To our knowledge, this study is the first one showing that a heritable secondary sexual characteristics displayed by females reflects parasite resistance.

Clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer and primary or acquired resistance to aromatase inhibitors: final results of phase II Swiss Group for Clinical Cancer Research Trial (SAKK 21/00).

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of fulvestrant, an estrogen receptor antagonist, in postmenopausal women with hormone-responsive tumors progressing after aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: This is a phase II, open, multicenter, noncomparative study. Two patient groups were prospectively considered: group A (n=70) with AI-responsive disease and group B (n=20) with AI-resistant disease. Fulvestrant 250 mg was administered as intramuscular injection every 28 (+/-3) days. RESULTS: All patients were pretreated with AI and 84% also with tamoxifen or toremifene; 67% had bone metastases and 45% liver metastases. Fulvestrant administration was well tolerated and yielded a clinical benefit (CB; defined as objective response or stable disease [SD] for >or=24 weeks) in 28% (90% confidence interval [CI] 19% to 39%) of patients in group A and 37% (90% CI 19% to 58%) of patients in group B. Median time to progression (TTP) was 3.6 (95% CI 3.0 to 4.8) months in group A and 3.4 (95% CI 2.5 to 6.7) months in group B. CONCLUSIONS: Overall, 30% of patients who had progressed following prior AI treatment gained CB with fulvestrant, thereby delaying indication to start chemotherapy. Prior response to an AI did not appear to be predictive for benefit with fulvestrant.

Microcalorimetry Assay for Rapid Detection of Voriconazole Resistance in Aspergillus fumigatus.

We describe a calorimetric assay for detection of voriconazole-resistant Aspergillus fumigatus within 8 h. Among 27 genetically distinct strains, all 21 resistant and all 6 susceptible strains were correctly identified by measurement of fungal heat production in the presence of voriconazole. This proof-of-concept study demonstrates the potential of microcalorimetry for rapid detection of azole resistance in A. fumigatus.

HIV-1 drug resistance transmission networks in southwest Switzerland.

To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.

Consequences of smoking for body weight, body fat distribution, and insulin resistance

Our aim was to critically evaluate the relations among smoking, body weight, body fat distribution, and insulin resistance as reported in the literature. In the short term, nicotine increases energy expenditure and could reduce appetite, which may explain why smokers tend to have lower body weight than do nonsmokers and why smoking cessation is frequently followed by weight gain. In contrast, heavy smokers tend to have greater body weight than do light smokers or nonsmokers, which likely reflects a clustering of risky behaviors (eg, low degree of physical activity, poor diet, and smoking) that is conducive to weight gain. Other factors, such as weight cycling, could also be involved. In addition, smoking increases insulin resistance and is associated with central fat accumulation. As a result, smoking increases the risk of metabolic syndrome and diabetes, and these factors increase risk of cardiovascular disease. In the context of the worldwide obesity epidemic and a high prevalence of smoking, the greater risk of (central) obesity and insulin resistance among smokers is a matter of major concern

Evidence against a direct role of klotho in insulin resistance.

The klotho gene may be involved in the aging process. Klotho is a coactivator of FGF23, a regulator of phosphate and vitamin D metabolism. It has also been reported to be downregulated in insulin resistance syndromes and paradoxically to directly inhibit IGF-1 and insulin signaling. Our aim was to study klotho's regulation and effects on insulin and IGF-1 signaling to unravel this paradox. We studied klotho tissue distribution and expression by quantitative real-time polymerase chain reaction and Western blotting in obese Zucker rats and high-fat fed Wistar rats, two models of insulin resistance. Klotho was expressed in kidneys but at much lower levels (<1.5%) in liver, muscle, brain, and adipose tissue. There were no significant differences between insulin resistant and control animals. We next produced human recombinant soluble klotho protein (KLEC) and studied its effects on insulin and IGF-1 signaling in cultured cells. In HEK293 cells, FGF23 signaling (judged by FRS2-alpha and ERK1/2 phosphorylation) was activated by conditioned media from KLEC-producing cells (CM-KLEC); however, IGF-1 signaling was unaffected. CM-KLEC did not inhibit IGF-1 and insulin signaling in L6 and Hep G2 cells, as judged by Akt and ERK1/2 phosphorylation. We conclude that decreased klotho expression is not a general feature of rodent models of insulin resistance. Further, the soluble klotho protein does not inhibit IGF-1 and/or insulin signaling in HEK293, L6, and HepG2 cells, arguing against a direct role of klotho in insulin signaling. However, the hypothesis that klotho indirectly regulates insulin sensitivity via FGF23 activation remains to be investigated.

Metabolic origin of insulin resistance in obesity with and without type 2 (non-insulin-dependent) diabetes mellitus.

A metabolic hypothesis is presented for insulin resistance in obesity, in the presence or absence of Type 2 (non-insulin-dependent) diabetes mellitus. It is based on physiological mechanisms including a series of negative feed-back mechanisms, with the inhibition of the function of the glycogen cycle in skeletal muscle as a consequence of decreased glucose utilization resulting from increased lipid oxidation in the obese. It considers the inhibition of glycogen synthase activity together with inhibition of glucose storage and impaired glucose tolerance. The prolonged duration of increased lipid oxidation, considered as the initial cause, may lead to Type 2 diabetes. This hypothesis is compatible with others based on the inhibition of insulin receptor kinase and of glucose transporter activities.

Shifts in species richness, herbivore specialization, and plant resistance along elevation gradients.

Environmental gradients have been postulated to generate patterns of diversity and diet specialization, in which more stable environments, such as tropical regions, should promote higher diversity and specialization. Using field sampling and phylogenetic analyses of butterfly fauna over an entire alpine region, we show that butterfly specialization (measured as the mean phylogenetic distance between utilized host plants) decreases at higher elevations, alongside a decreasing gradient of plant diversity. Consistent with current hypotheses on the relationship between biodiversity and the strength of species interactions, we experimentally show that a higher level of generalization at high elevations is associated with lower levels of plant resistance: across 16 pairs of plant species, low-elevation plants were more resistant vis-à-vis their congeneric alpine relatives. Thus, the links between diversity, herbivore diet specialization, and plant resistance along an elevation gradient suggest a causal relationship analogous to that hypothesized along latitudinal gradients.

CYP3A activity measured by the midazolam test is not related to 3435 C >T polymorphism in the multiple drug resistance transporter gene.

A recent study with 69 Japanese liver transplants treated with tacrolimus found that the MDR13435 C >T polymorphism, but not the MDR12677 G >T polymorphism, was associated with differences in the intestinal expression level of CYP3A4 mRNA. In the present study, over 6 h, we measured the kinetics of a 75 microg oral dose of midazolam, a CYP3A substrate, in 21 healthy subjects genotyped for the MDR13435 C >T and 2677 G >T polymorphism. No statistically significant differences were found in the calculated pharmacokinetic parameters between the three 3435 C >T genotypes (TT, CT and CC group, respectively: Cmax (mean +/- SD: 0.30 +/- 0.08 ng/ml, 0.31 +/- 0.09 ng/ml and 0.31 +/- 0.11 ng/ml; Apparent clearance: 122 +/- 29 l/h, 156 +/- 92 l/h and 111 +/- 35 l/h; t1/2: 1.9 +/- 1.1 h, 1.6 +/- 0.90 h and 1.7 +/- 0.7 h). In addition, the 30-min 1'OH midazolam to midazolam ratio, a marker of CYP3A activity, determined in 74 HIV-positive patients before the introduction of antiretroviral treatment, was not significantly different between the three 3435 C >T genotypes (mean ratio +/- SD: 3.65 +/- 2.24, 4.22 +/- 3.49 and 4.24 +/- 2.03, in the TT, CT and CC groups, respectively). Similarly, no association was found between the MDR12677 G >T polymorphism and CYP3A activity in the healthy subjects or in the HIV-positive patients. The existence of a strong association between the activity of CYP3A and MDR13435 C >T and 2677 G >T polymorphisms appears unlikely, at least in Caucasian populations and/or in the absence of specific environmental factors.

Molecular mechanisms of drug resistance in clinical Candida species isolated from tunisian hospitals.

Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 μg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 μg/ml), voriconazole (MIC > 16 μg/ml), and amphotericin B (MIC > 32 μg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6β-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14α-demethylase and sterol Δ(5,6)-desaturase leading to azole-polyene cross-resistance.