Accuracy of Sensititre YeastOne echinocandins epidemiological cut-off values for identification of FKS mutant Candida albicans and Candida glabrata: a ten year national survey of the Fungal Infection Network of Switzerland (FUNGINOS).

OBJECTIVES: Echinocandins represent the first-line treatment of candidaemia. Acquired echinocandin resistance is mainly observed among Candida albicans and Candida glabrata and is associated with FKS hotspot mutations. The commercial Sensititre YeastOne™ (SYO) kit is widely used for antifungal susceptibility testing, but interpretive clinical breakpoints are not well defined. We determined echinocandins epidemiological cut-off values (ECV) for C. albicans/glabrata tested by SYO and assessed their ability to identify FKS mutants in a national survey of candidaemia. METHODS: Bloodstream isolates of C. albicans and C. glabrata were collected in 25 Swiss hospitals from 2004 to 2013 and tested by SYO. FKS hotspot sequencing was performed for isolates with an MIC≥ECV for any echinocandin. RESULTS: In all, 1277 C. albicans and 347 C. glabrata were included. ECV 97.5% of caspofungin, anidulafungin and micafungin were 0.12, 0.06 and 0.03 µg/mL for C. albicans, and 0.25, 0.12 and 0.03 µg/mL for C. glabrata, respectively. FKS hotspot sequencing was performed for 70 isolates. No mutation was found in the 52 'limit wild-type' isolates (MIC=ECV for at least one echinocandin). Among the 18 'non-wild-type' isolates (MIC>ECV for at least one echinocandin), FKS mutations were recovered in the only two isolates with MIC>ECV for all three echinocandins, but not in those exhibiting a 'non-wild-type' phenotype for only one or two echinocandins. CONCLUSION: This 10-year nationwide survey showed that the rate of echinocandin resistance among C. albicans and C. glabrata remains low in Switzerland despite increased echinocandin use. SYO-ECV could discriminate FKS mutants from wild-type isolates tested by SYO in this population.

Meningitis and Bacteremia Due to Neisseria cinerea following a Percutaneous Rhizotomy of the Trigeminal Ganglion.

Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed.

First documented outbreak of KPC-2-producing Klebsiella pneumoniae in Switzerland: infection control measures and clinical management.

We report the epidemiological and clinical features of the first outbreak of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) type 2 in Switzerland. The outbreak took place in the medical intensive care unit (MICU) of our tertiary care hospital and affected three severely ill patients. After the implementation of strict infection control measures, no further patients colonised with KPC-KP could be detected by the screening of exposed patients. Successful treatment of patients infected with KPC-KP consisted of a combination therapy of meropenem, colistin and tigecycline.

Transvaginal access for NOTES: a cohort study of microbiological colonization and contamination.

BACKGROUND AND STUDY AIMS: Animal data and limited clinical evidence suggest a low incidence of infection following transvaginal natural orifice transluminal endoscopic surgery (NOTES). However, a systematic microbiological evaluation has not yet been carried out. The aim of this prospective cohort study was to evaluate the extent of microbiological contamination of the peritoneal cavity caused by the transvaginal access for NOTES and the impact of preoperative vaginal disinfection on vaginal colonization. PATIENTS AND METHODS: Consecutive female patients with symptomatic cholecystolithiasis were offered either transvaginal rigid-hybrid cholecystectomy (tvCCE) or conventional laparoscopic cholecystectomy. Patients who opted for tvCCE were prospectively evaluated between February and June 2010. Disinfection in patients undergoing tvCCE included hexetidine tablets and octenidine applied vaginally. All patients received a single dose of perioperative cefuroxime. Swabs were obtained from the posterior fornix and the peritoneal cavity at different intervals. RESULTS: Of 32 patients, 27 (84 %) opted to undergo tvCCE. One patient (4 %; 95 % confidence interval [CI] 0.7 % - 18.3 %) had a positive bacterial culture in the Douglas pouch prior to transvaginal access compared with two patients (7 %; 95 %CI 2.1 % - 23.4 %) following colpotomy closure (P = 1.000). Vaginal disinfection significantly decreased vaginal bacterial load (P = 0.001) and bacterial growth in routine cultures (P < 0.001); in 16 patients (59 %; 95 %CI 40.7 % - 75.5 %) vaginal swabs were sterile after disinfection. No postoperative surgical site infections occurred (95 %CI 0 % - 12.5 %). CONCLUSIONS: In selected patients and following vaginal antisepsis, transvaginal access for NOTES is associated with microbiological contamination of the peritoneal cavity in a minority of patients, indicating a low risk of peritoneal contamination caused by the transvaginal access.

Effectiveness of a new decolonisation regimen for eradication of extended-spectrum ß-lactamase-producing Enterobacteriaceae.

Gram-negative bacteria expressing extended-spectrum ß-lactamases (ESBL) have emerged worldwide. ESBL colonisation can persist for years and may favour ESBL transmission. Interventions include contact isolation precautions and restriction of antibiotic use, but decolonisation (DC) for ESBL is not established. We performed a prospective controlled open-label cohort-study from 1/2000 to 1/2008 to determine the effectiveness of a standardised DC programme. ESBL-positive patients routinely underwent screening from rectum, throat, and urine. DC included: chlorhexidine 0.2% mouth rinse three times daily (throat colonisation), paromomycin 4 × 1 g daily (intestinal colonisation), and oral antibiotics for urinary tract colonisation. ESBL elimination was defined as ≥ 1 set of negative follow-up screenings (throat, rectal, urine). Of 100 enrolled patients, 83% of patients were infected and 17% colonised with ESBL. Escherichia coli (71%) and Klebsiella pneumoniae (25%) were the most frequent pathogens. Overall, 76% (76/100) of patients became negative for ESBL at follow-up. Fifty-five percent (42/76) of the successfully treated patients received systemic treatment for infection. Of those who completed DC, 83% (15/18) were free of ESBL at follow-up. DC success correlated with the number of risk factors and colonised sites. DC may be beneficial in a selected group of patients, potentially shortening duration of ESBL colonisation and subsequently reducing the risk for transmission.

Vertebral osteomyelitis caused by Actinobaculum schaalii: a difficult-to-diagnose and potentially invasive uropathogen.

We report the first case of vertebral osteomyelitis caused by Actinobaculum schaalii and review all cases of A. schaalii identified at our institution between 2002 and 2005. A. schaalii causes urinary tract infections - especially in elderly people - occasionally with septic complications.

Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites.

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

Subtle mutagenesis by ends-in recombination in malaria parasites.

The recent advent of gene-targeting techniques in malaria (Plasmodium) parasites provides the means for introducing subtle mutations into their genome. Here, we used the TRAP gene of Plasmodium berghei as a target to test whether an ends-in strategy, i.e., targeting plasmids of the insertion type, may be suitable for subtle mutagenesis. We analyzed the recombinant loci generated by insertion of linear plasmids containing either base-pair substitutions, insertions, or deletions in their targeting sequence. We show that plasmid integration occurs via a double-strand gap repair mechanism. Although sequence heterologies located close (less than 450 bp) to the initial double-strand break (DSB) were often lost during plasmid integration, mutations located 600 bp and farther from the DSB were frequently maintained in the recombinant loci. The short lengths of gene conversion tracts associated with plasmid integration into TRAP suggests that an ends-in strategy may be widely applicable to modify plasmodial genes and perform structure-function analyses of their important products.

Primary structure and biochemical properties of a variant-specific surface protein of Giardia.

Trophozoites of Giardia duodenalis express at their cell surface variant-specific proteins (VSPs) that are believed to contribute to the protection of the parasite from immunological and other host defense mechanisms. In the present study, we have cloned and characterized the gene encoding a VSP (VSP4A1, originally designated CRISP-90) that is expressed by the sheep-derived Giardia variant clone O2-4A1. The gene was isolated by probing a genomic library with a near-full-length gene-specific polymerase chain reaction (PCR) product. The VSP4A1 gene specifies a 70729 Da protein with features common to all previously reported VSPs, including a high cysteine and threonine content, a highly conserved hydrophobic carboxy-terminal domain and little similarity in the remaining polypeptide sequence. Comparison of the predicted sequence with the amino-terminal sequence of purified VSP4A1 revealed the absence of an amino-terminal hydrophobic extension from the mature protein. VSP4A1 purified from the O2-4A1 variant clone was found to undergo conformational changes resulting in the formation of two additional electrophoretic species. Free thiol groups were not detected in purified VSP4A1, indicating that all cysteine residues may be involved in disulphide crosslinking. Possibly as a consequence of this VSP4A1 was found to be fairly resistant to proteolytic digestion. Although VSP4A1 is able to bind zinc following blotting to a nitrocellulose membrane, other analyses with both the purified and cell associated VSP have failed to confirm significant zinc ion binding to this protein. The latter result questions the assumption previously made by other authors that zinc binding to VSPs constitutes an important structural and functional aspect of these proteins.

Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis.

This paper reports the cloning and molecular characterization of the gene encoding pyruvate phosphate dikinase (PPDK) from Giardia. The ORF is 2652 nucleotide residues in length and not interrupted by introns. The gene appears to exist as a single copy in the genome and predicts a 97629 Da protein containing 884 amino acid residues. Comparison of the deduced Giardia PPDK sequence with those of homologous enzymes from other organisms revealed high sequence similarities and the presence of various conserved domains known to be essential for substrate binding and catalysis. Analysis of the ppdk gene and 19 other protein-coding genes from the protist revealed no typical TATA boxes, positioned at around -30, but the presence of two novel consensus sequence motifs in the 5' flanking regions. One is an AT-rich element immediately preceding the translation initiation codon and the other a 14-bp box centered at -30. These shared consensus sequence patterns present in the 5' flanking region of Giardia genes are suggested to play a role in the control of transcription initiation.

Comparison of genetic groups determined by molecular and immunological analyses of Giardia isolated from animals and humans in Switzerland and Australia.

Nine axenic isolates of Giardia originating from four different host species in Switzerland were subjected to genetic analysis using the polymerase chain reaction (PCR) to amplify segments of genes encoding different trophozoite variant surface proteins (VSPs). Three genotypes were identified on the basis of product yield, size, and restriction-fragment-length polymorphisms (RFLPs). Five G. duodenalis isolates (O1, B1, B2, B3-1A1 and C1--from a sheep, three calves and a dog, respectively) were classified as belonging to genetic group I of Andrews et al. (1989). DNA amplified from the VSP genes tsp11, tsa417 and vsp1267 of these isolates was indistinguishable in size and restriction characteristics from that amplified from group-I Giardia isolated from humans in Australia. One human-derived Swiss isolate (H2-17A1), typed as belonging to genetic group II, yielded a vsp1267-specific PCR product that was indistinguishable by size or restriction sites from the equivalent 1.6-kb product amplified from human-derived Australian group-II organisms. This isolate also yielded 1.8-kb tsp11 and 0.52-kb tsa417/tsp11-like PCR products possessing RFLPs typical of group-II organisms. Three isolates (O2-4A1, O3 and H3-15K2--originating from two sheep and a human, respectively) represent a novel genotype that is closely related to genetic groups I and II. These three isolates exhibited identical RFLPs in their tsp11 PCR products and failed to yield a vsp1267 PCR product. An antiserum specific for the 90-kDa VSP of the sheep-derived clone O2-4A1 reacted strongly by immunofluorescence and on Western blots with surface proteins from the O2, O3 and H3 isolates only--consistent with the genetic classification determined above. The data provide no evidence for the occurrence of host-specific genotypes.

Separation of a cysteine-rich surface antigen-expressing variant from a cloned Giardia isolate by fluorescence-activated cell sorting.

Clonally derived trophozoites (clone O2-4A1, from a sheep) of the morphologically defined group of Giardia duodenalis change their cysteine-rich surface proteins (CRISPs) spontaneously during in vitro culture. This phenomenon constitutes a serious obstacle for studies that rely on a pure population of cells bearing a particular variant CRISP. We describe herein the successful separation and quantitation of a trophozoite subpopulation expressing a 90-kDa major surface antigen (CRISP-90) from a heterologous population using fluorescence-activated cell sorting (FACS) on the basis of fluorescein-tagged antibodies directed specifically against O2-4A1 CRISP-90. During subsequent in vitro culture, the purified cell population exhibited a progressive decline in the proportion of cells labeled by CRISP-90 specific antibodies. After 46 generations, only one-third of the total trophozoite population reacted with the anti-CRISP-90 antibodies.

Variant cysteine-rich surface proteins of Giardia isolates from human and animal sources.

Cloned Giardia isolates obtained from a sheep, a calf, and a human possessed a major membrane protein that showed marked intraspecific variations in size as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following surface biotinylation and radioiodination. Metabolic labeling with [35S] cysteine and electrophoretic analysis also revealed for each cloned isolate a predominant protein that corresponded in size to the major surface protein demonstrated by surface labeling techniques. Immunoprecipitation studies with a polyclonal antiserum specifically directed against the 90-kDa major cysteine-rich protein purified from a subclone of the sheep isolate (O2-4A1) showed that the cysteine-rich protein and the major surface protein are identical. The surface location of the antigen was further corroborated by the reaction of fluorescence-labeled antibodies raised against the 90-kDa O2-4A1 cysteine-rich protein with the entire surface of live trophozoites of the homologous clone. The ability of the cloned Giardia isolates to undergo variations of their cysteine-rich surface protein (CRISP) was demonstrated by the spontaneous appearance of new CRISPs in clonally derived populations during prolonged in vitro culturing and in cultures of the O2-4A1 clone that had survived treatment with the cytotoxic anti-90-kDa CRISP antiserum specific for the surface antigen of this clone. The surviving progeny were devoid of the original CRISP, as judged by resistance to the immune serum. Subsequent cysteine metabolic labeling of the recloned surviving trophozoites demonstrated a large number of new variants, each expressing a single CRISP that varied significantly in molecular weight from those in the different cloned lines. These studies suggest that the presence of CRISPs and their variations are not restricted to Giardia isolates obtained from humans but are universal phenomena among the Giardia duodenalis types of organisms.

Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding.

The amino acid sequence of the Bradyrhizobium japonicum nitrogen fixation regulatory protein NifA, as derived from the nucleotide sequence of the nifA gene, was aligned to the corresponding protein sequences from Klebsiella pneumoniae, Rhizobium meliloti and Rhizobium leguminosarum biovar viciae. High conservation was found in the central domain and in the COOH-terminal, putative DNA binding domain, whereas very little homology was present within the first 250 amino acids from the NH2-terminus. Upon deletion of the first 218 amino acids (37% of the protein) and expression of the remainder as a Cat'-'NifA hybrid protein, a fully active, nif-specific transcriptional activator protein was obtained which also retained oxygen sensitivity, a characteristic property of the wild-type B. japonicum NifA protein. In contrast, an unaltered COOH-terminal domain was required for an active NifA protein. Between the central and the DNA binding domains, a so-called interdomain linker region was identified which was conserved in all rhizobial species but missing in the K.pneumoniae NifA protein. Two conserved cysteine residues in this region were changed to serine residues, by oligonucleotide-directed mutagenesis. This resulted in absolutely inactive NifA mutant proteins. Similar null phenotypes were obtained by altering two closely adjacent cysteine residues in the central domain to serine residues. Nif gene activation in vivo by the B.japonicum NifA protein, but not by the K.pneumoniae NifA protein, was sensitive to treatment with chelating agents, and this inhibition could be overcome by the addition of divalent metal ions. On the basis of these observations and previous data on oxygen sensitivity we raise the hypothesis that at least some, if not all, of the four essential cysteine residues may be involved in oxygen reactivity or metal binding or both.

The symbiotic nitrogen fixation regulatory operon (fixRnifA) of Bradyrhizobium japonicum is expressed aerobically and is subject to a novel, nifA-independent type of activation.

The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and its transcription start site determined. Between the start of transcription and the nifA gene an open reading frame of 278 codons was found and named fixR. A deletion in fixR which allowed transcription into nifA resulted in a 50% reduced Fix activity. The fixRnifA operon was expressed in soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and in aerobic cultures. The transcription start site (+1) was preceded by a characteristic nif(-24/-12)-type promoter consensus sequence. Double base-pair exchanges in the -12 but not in the -24 region resulted in a 'promoter-down' phenotype. A promoter-upstream DNA region between -50 and -148 was essential for maximal promoter activity. Expression from the promoter was not dependent on nifA. We conclude that the fixRnifA promoter is positively controlled, and that it requires a newly postulated transcriptional factor in order to become activated.