Isolates ofC Research Articles

Ploidy Variation and Spontaneous Haploid-Diploid Switching of Candida glabrata Clinical Isolates.

ABSTRACTThe human fungal pathogen Candida glabrata is phylogenetically closely related to Saccharomyces cerevisiae, a model eukaryotic organism. Unlike S. cerevisiae, which has both haploid and diploid forms and a complete sexual cycle, C. glabrata has long been considered a haploid and asexual species. In this study, we analyzed the ploidy states of 500 clinical isolates of C. glabrata from four Chinese hospitals and found that approximately 4% of the isolates were in or able to spontaneously switch to an aneuploid (genomic DNA, 1N-2N), diploid (2N), or hyperdiploid (>2N) form under in vivo or in vitro conditions. Stable diploid cells were identified in 3% of the isolates (15/500). Of particular interest, one clinical strain existed only in the diploid form. Multilocus sequence typing (MLST) assays revealed two major genetic clusters (A and B) of C. glabrata isolates. Most of the isolates (70%) from China belonged to the A cluster, whereas most of the isolates from other countries (such as Iran, Japan, United States, and European countries) belonged to the B cluster. Further investigation indicated that C. glabrata cells of different ploidy forms differed in a number of respects, including morphologies, antifungal susceptibility, virulence, and global gene expression profiles. Additionally, C. glabrata could undergo spontaneous switching between the diploid and haploid forms under both in vitro and in vivo conditions. Given the absence of an apparent sexual phase, one would expect that the ploidy shifts could function as an alternative strategy that promotes genetic diversity and benefits the ability of the fungus to rapidly adapt to the changing environment.IMPORTANCE The human fungal pathogen Candida glabrata has long been thought to be a haploid organism. Here, we report the population structure and ploidy states of 500 clinical isolates of C. glabrata from China. To our surprise, we found that the ploidy of a subset of clinical isolates varied dramatically. Some isolates were in or able to switch to an aneuploid, diploid, or hyperdiploid form. C. glabrata cells with different ploidy differed in a number of biological respects, including morphology, antifungal susceptibility, virulence, and global gene expression profile. Given the absence of an apparent sexual phase in this fungus, we propose that ploidy switching could be a strategy for rapid adaptation to environmental changes and could function as an alternative to sexual reproduction.

Antifungal Activity of Green and Red Brazilian Propolis Extracts

Background: Diseases associated to Candida spp. are recurrent and can be difficult to treat, mainly due to the new strains resistant to the limited number of available antifungals. Objective: The objective of this study is to evaluate the in vitro and in vivo activity of Brazilian green propolis (GrProp) and red propolis (RdProp) ethanolic extracts against standard strains of Candida albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis and 10 clinical isolates of C. albicans. Methods: Antifungal activity in vitro was tested using the M-27-A3/CLSI protocol. The in vivo antifungal activity was evaluated using Tenebrio molitor model and, the effect of extracts on adhesion of C. albicans in human buccal epithelial cells (BECs) was also studied. Results: GrProp and RdProp exhibited antifungal activity against at least one of the Candida strains tested. The adhesion inhibition of C. albicans in BECs was 45% (GrProp28), 60% (Gr- Prop50) and 82% (RdProp), in comparison to amphotericin B (82%). All propolis extracts showed synergistic activity with fluconazole and amphotericin B. GrProp50 (10 mg/kg) showed better protection of T. molitor, blocking the progression of C. albicans infection, increasing survival and delayed the larvae death. Conclusion: Brazilian GrProp and RdProp extracts inhibit the in vitro C. albicans growth and protect T. molitor against infection by this yeast. The physiochemical parameters found for the analyzed samples were in accordance to the standards established by the Brazilian Legislation for propolis and derivatives. GrProp and RdProp have potential to be used against Candida spp. infections, mainly in association with fluconazole or amphotericin B.

Resistance ofCorynespora cassiicolafrom soybean to QoI and MBC fungicides in Brazil

AbstractFungicide sprays on soybean in Brazil have contributed to the selection of less sensitive isolates ofCorynespora cassiicola. We collected 59 isolates ofC. cassiicolafrom three Brazilian states and two isolates from Paraguay. We investigated their EC50to quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC), any cross‐resistance to compounds within QoI and MBC groups, and characterized the polymorphisms in theircytbandβ‐tubulingenes. Local associations of polymorphisms identified in each gene were statistically correlated with assays results. In total, 79% and 74% of the isolates were classified as resistant to QoI and MBC fungicides, respectively. There was positive cross‐resistance to active ingredients within QoI and MBC groups. For QoI, all isolates presented heteroplasmy in G143A ofcytbgene; the mutations F129L and G137R were not found. For MBC, 63% of isolates possessed E198A and 21% possessed F200Y mutations, associated with reduced control by MBC fungicides. Heteroplasmy was identified in two and one isolates from Brazil with E198A and F200Y mutations, respectively. The resistance factor for isolates with E198A (10.9) was statistically similar to the isolate with F200Y (8.8) mutation. Genic association analysis of the in vitro assays using discriminatory doses proved them to be accurate. Reduced sensitivity ofC. cassiicolato QoI and MBC was also identified in isolates from Paraguay and resistance to QoI and MBC was widely present inC. cassiicolaisolates from the main soybean‐producing states in Brazil. Thus, integrated management measures should be adopted to manage soybean target spot in these countries.

Mangrove horseshoe crab (Carcinoscorpius rotundicaudaLatreille, 1802) populations reveal genetic break in Strait of Malacca, with connectivity along southern coasts of Peninsular Malaysia

AbstractThe mangrove horseshoe crab,Carcinoscorpius rotundicauda, has divergent populations between the east and west coasts of Peninsular Malaysia, with the southern coast acting as a land barrier. The actual position of such a genetic break along Peninsular Malaysia as well as the connectivity status of the southernmostC. rotundicaudapopulations with east and west coast populations remain unexplored, however.The aim was to investigate the genetic diversity and structure ofC. rotundicaudapopulations from the west (Kuala Sepetang in State Perak), east (Balok in State Pahang), and southern (Pendas in State Johor) coasts of Peninsular Malaysia. Haemolymph samples from adultC. rotundicaudaspecimens (n = 152) and eggs from their freshly deposited nests (n = 190) were collected monthly (from January 2016 to January 2017) for the sequencing of mitochondrial cytochrome coxidase subunit I (COI).Gene isolates ofC. rotundicaudafrom the present study were compared with National Center for Biotechnology Information (NCBI) GenBank sequences to cover most of the range of the species in Asia. A neighbour‐joining tree strongly supported two clades, separating the west‐coast populations from the south‐ and east‐coast populations, with further substructure patterns.Both haplotype network and barrier analyses revealed a genetic break within the Strait of Malacca instead of the southern tip of Peninsular Malaysia. The southernmost samples from the Strait of Johor formed a haplotypic diverse gene pool that appeared only as a subclade of the eastern populations. In a detailed haplotype network of 347 individuals, individuals with similarCOIsequences indicate connectivity betweenC. rotundicaudaon the east andC. rotundicaudaon south and south west of Peninsular Malaysia.Overall, the genetic break betweenC. rotundicaudapopulations is better explained by the convergent ocean currents and available mangrove habitats on the west coast (i.e. Strait of Malacca), rather than the point of the Malay Peninsula acting as a land barrier alone.

Vaginal Isolates of Candida glabrata Are Uniquely Susceptible to Ionophoric Killer Toxins Produced by Saccharomyces cerevisiae

ABSTRACTCompared to other species of Candida yeasts, the growth of Candida glabrata is inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins are broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

Environmental Candida auris and the Global Warming Emergence Hypothesis.

Global warming was proposed to be a contributing cause for the nearly simultaneous emergence of different clades of Candida auris as a nosocomial pathogen in different continents. The global warming emergence hypothesis posits that C. auris existed in the environment prior to its clinical recognition and became pathogenic for humans because of thermal adaptation in response to climate change. The isolation of C. auris from two sites in the remote Andaman Islands establishes it as an environmental organism, a necessary condition for the hypothesis. The observation that one environmental isolate grew slower at mammalian temperatures than clinical strains is consistent with the notion that their ancestor recently adapted to higher temperatures. The knowledge that C. auris can be recovered from the environment should prompt additional searches to define its ecological niches, and the analysis of future environmental isolates will provide evidence for validating or refuting the global warming emergence hypothesis.

Clinical relevance and antimicrobial susceptibility profile of the unknown human pathogen Corynebacterium aurimucosum.

Introduction. Even though Corynebacterium aurimucosum has been described in 2002, this species has long been underestimated due to the unreliability of conventional identification methods and only a few cases of infections have been reported.Hypothesis/Gap Statement. Little is known about clinical significance and antimicrobial susceptibility profile of this uncommon species.Aim. To evaluate the clinical relevance of C. aurimucosum and its antimicrobial susceptibility profile.Methodology. All C. aurimucosum isolates, collected from 2010 to 2019 in 10 French university hospitals, were retrospectively included. Demographic, clinical and microbiological data were collected for all cases. Antimicrobial susceptibility testing was performed according to the 2019 EUCAST guidelines.Results. Fifty-seven clinical isolates of C. aurimucosum were collected in 57 patients (median age, 65.8 years; male/female sex ratio, 1.1), mostly from urine (28 %), blood culture (28 %) and bone/synovial fluid (19 %) samples. Of them, 14 cases of infection were confirmed, mainly bone and joint infections (50 %) followed by urinary tract infections (UTIs) (21 %), bacteremia (14 %), skin and soft-tissue infections (14 %). C. aurimucosum was recovered in pure culture in 36 % of cases (UTIs and bacteremia) while mixed cultures were observed for other infections. By testing 52 clinical isolates in vitro, this species appeared to be fully susceptible to linezolid and vancomycin while most isolates (>80 %) were susceptible to amoxicillin (MIC90, 2 µg ml-1), gentamicin, tetracycline and rifampicin. Both cefotaxime and ciprofloxacin seemed to have a limited activity (ca. 50 % of susceptible strains). The MIC distribution for ciprofloxacin showed a bimodal profile with a population of highly-resistant strains with MICs >2 µg ml-1. Most isolates (>90 %) were categorized as resistant to penicillin G and clindamycin.Conclusion. C. aurimucosum should be considered as an actual opportunistic pathogen, and treatment with amoxicillin, vancomycin or linezolid should be preferred.

Identification of Genomewide Alternative Splicing Events in Sequential, Isogenic Clinical Isolates of Candida albicans Reveals a Novel Mechanism of Drug Resistance and Tolerance to Cellular Stresses.

Alternative splicing (AS)-a process by which a single gene gives rise to different protein isoforms in eukaryotes-has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans, has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3, which contains a single intron. The sod3Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicansIMPORTANCE The emergence of resistance in Candida albicans, an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans, here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress.

Chitosan Ameliorates Candida auris Virulence in a Galleria mellonella Infection Model.

Candida auris has emerged as a multidrug-resistant nosocomial pathogen over the last decade. Outbreaks of the organism in health care facilities have resulted in life-threatening invasive candidiasis in over 40 countries worldwide. Resistance by C. auris to conventional antifungal drugs such as fluconazole and amphotericin B means that alternative therapeutics must be explored. As such, this study served to investigate the efficacy of a naturally derived polysaccharide called chitosan against aggregative (Agg) and nonaggregative (non-Agg) isolates of C. aurisin vitro and in vivo. In vitro results indicated that chitosan was effective against planktonic and sessile forms of Agg and non-Agg C. auris In a Galleria mellonella model to assess C. auris virulence, chitosan treatment was shown to ameliorate killing effects of both C. auris phenotypes (NCPF 8973 and NCPF 8978, respectively) in vivo Specifically, chitosan reduced the fungal load and increased survival rates of infected Galleria, while treatment alone was nontoxic to the larvae. Finally, chitosan treatment appeared to induce a stress-like gene expression response in NCPF 8973 in the larvae likely arising from a protective response by the organism to resist antifungal activity of the compound. Taken together, results from this study demonstrate that naturally derived compounds such as chitosan may be useful alternatives to conventional antifungals against C. auris.

Tracing the Evolutionary History and Global Expansion of Candida auris Using Population Genomic Analyses.

Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. aurisIMPORTANCE In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology.

Mechanisms of Acquired In Vivo and In Vitro Resistance to Voriconazole by Candida krusei following Exposure to Suboptimal Drug Concentration.

Five Candida krusei isolates (susceptible and resistant) recovered from the urine of a kidney transplant patient treated with voriconazole (VRC) 200 mg twice daily for 20 days were studied. Eight unrelated clinical isolates of C. krusei were exposed in vitro to VRC 0.001 μg/ml for 30 days. Development of VRC transient resistance occurred in vivo, and induction of permanent resistance occurred in vitro Mostly, ABC1 and ERG11 genes were overexpressed, and a homozygous T418C mutation in the ERG11 gene was found.

Candida auris Clinical Isolates from South Korea: Identification, Antifungal Susceptibility, and Genotyping.

Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.

In Vitro Activity of Omadacycline against Chlamydia pneumoniae.

The in vitro activities of omadacycline, azithromycin, doxycycline, moxifloxacin, and levofloxacin were tested against 15 isolates of Chlamydia pneumoniae The minimum inhibitory concentration at which 90% of the isolates of C. pneumoniae were inhibited by omadacycline was 0.25 μg/ml (range, 0.03 to 0.5 μg/ml).

Iron Restriction to Clinical Isolates of Candida albicans by the Novel Chelator DIBI Inhibits Growth and Increases Sensitivity to Azoles In Vitro and In Vivo in a Murine Model of Experimental Vaginitis.

Candida albicans is an important opportunistic pathogen causing various human infections that are often treated with azole antifungals. The U.S. CDC now regards developing candidal antifungal resistance as a threat, creating a need for new and more effective antifungal treatments. Iron is an essential nutrient for all living cells, and there is growing evidence that interference with iron homeostasis of C. albicans can improve its response to antifungals. This study was aimed at establishing whether withholding iron by currently used medical iron chelators and the novel chelator DIBI could restrict growth and also enhance the activity of azoles against clinical isolates of C. albicans DIBI, but not deferoxamine or deferiprone, inhibited the growth of C. albicans at relatively low concentrations in vitro, and this inhibition was reversed by iron addition. DIBI in combination with various azoles demonstrated stronger growth inhibition than the azoles alone and greatly prolonged the inhibition of cell multiplication. In addition, the administration of DIBI along with fluconazole (FLC) to mice inoculated with an FLC-sensitive isolate in a model of experimental C. albicans vaginitis showed a markedly improved clearance of infection. These results suggest that iron chelation by DIBI has the potential to enhance azole efficacy for the treatment of candidiasis.

High Prevalence of Toxigenic and Nontoxigenic Clostridium difficile Strains in Malaysia.

Accumulating evidence shows a high prevalence of Clostridium difficile in Southeast Asia associated with a range of clinical presentations. However, severe infections are rarely reported. We investigated C. difficile infection (CDI) across four hospitals in Kuala Lumpur and Kota Bharu, Malaysia. Enzyme immunoassays for glutamate dehydrogenase (GDH) and toxin A or B were performed on diarrheal stool specimens collected from patients in 2015 and 2016. Specimens were also cultured and isolates of C. difficile characterized by PCR ribotyping and detection of toxin genes. In total, 437 specimens were collected and fecal toxin was detected in 3.0%. A further 16.2% of specimens were GDH positive and toxin negative. After culture, toxigenic strains were isolated from 10.3% and nontoxigenic strains from 12.4% of specimens. The most prevalent PCR ribotypes (RTs) were RT 017 (20.0%) and RT 043 (10.0%). The high prevalence of RT 017 and nontoxigenic strains in Malaysia and in neighboring Thailand and Indonesia suggests that they localize to the region of Southeast Asia, with an implication that they may mediate the burden of CDI in the region.

Role of the Inducible Adhesin CpAls7 in Binding of Candida parapsilosis to the Extracellular Matrix under Fluid Shear.

The yeast Candida parapsilosis is an increasingly common cause of systemic fungal infections among immunocompromised individuals, including premature infants. Adhesion to host surfaces is an important step in pathogenesis, but this process has not been extensively studied in this organism. A microfluidics assay was developed to test the ability of C. parapsilosis to adhere to immobilized host extracellular matrix proteins under physiological fluid shear conditions. Growth in mammalian tissue culture medium at 37°C for 3 to 6 h led to the induction of an adhesive phenotype at shear forces of 1 to 5 dynes/cm2 in some isolates of C. parapsilosis Glutamic acid, proline, and calcium appeared to be the minimally necessary requirements for increased adhesion in these assays. To determine whether genes homologous to the ALS gene family of C. albicans were important for the adhesive phenotype, the expression levels of 5 homologous C. parapsilosis genes were quantified by using quantitative PCR (qPCR) under conditions leading to increased adhesion. CPAR2_404800 (CpALS7) and CPAR2_404780 showed increased expression levels compared to those in control yeast. The extent of adhesion was variable among different isolates, and linear regression identified the expression of CpALS7 but not CPAR2_404780 as having a strong positive correlation with adhesion. A homozygous CpALS7 deletion strain was deficient in adhesion, whereas the expression of CpALS7 in Saccharomyces cerevisiae resulted in increased adhesion. Together, these data provide strong evidence that CpAls7 aids in the adherence of C. parapsilosis to the extracellular matrix under shear forces and support its previously reported role in virulence.

Antimicrobial Octapeptin C4 Analogues Active against Cryptococcus Species.

ABSTRACTResistance to antimicrobials is a growing problem in both developed and developing countries. In nations where AIDS is most prevalent, the human fungal pathogen Cryptococcus neoformans is a significant contributor to mortality, and its growing resistance to current antifungals is an ever-expanding threat. We investigated octapeptin C4, from the cationic cyclic lipopeptide class of antimicrobials, as a potential new antifungal. Octapeptin C4 was a potent, selective inhibitor of this fungal pathogen with an MIC of 1.56 μg/ml. Further testing of octapeptin C4 against 40 clinical isolates of C. neoformans var. grubii or neoformans showed an MIC of 1.56 to 3.13 μg/ml, while 20 clinical isolates of C. neoformans var. gattii had an MIC of 0.78 to 12.5 μg/ml. In each case, the MIC values for octapeptin C4 were equivalent to, or better than, current antifungal drugs fluconazole and amphotericin B. The negatively charged polysaccharide capsule of C. neoformans influences the pathogen's sensitivity to octapeptin C4, whereas the degree of melanization had little effect. Testing synthetic octapeptin C4 derivatives provided insight into the structure activity relationships, revealing that the lipophilic amino acid moieties are more important to the activity than the cationic diaminobutyric acid groups. Octapeptins have promising potential for development as anticryptococcal therapeutic agents.

High-throughput sorting of eggs for synchronization of C. elegans in a microfluidic spiral chip.

In this study, we report the use of a high-throughput microfluidic spiral chip to screen out eggs from a mixed age nematode population, which can subsequently be cultured to a desired developmental stage. For the sorting of a mixture containing three different developmental stages, eggs, L1 and L4, we utilized a microfluidic spiral chip with a trapezoidal channel to obtain a sorting efficiency of above 97% and a sample purity (SP) of above 80% for eggs at different flow rates up to 10 mL min-1. The result demonstrated a cost-effective, simple, and highly efficient method for synchronizing C. elegans at a high throughput (∼4200 organisms per min at 6 mL min-1), while eliminating challenges such as clogging and non-reusability of membrane-based filtration. Due to its simplicity, our method can be easily adopted in the C. elegans research community.

Contribution to the History of Serbian Medicine: Ten Years from the Introduction of Clostridium difficile into Microbiological Diagnostics in Serbia

SummaryIn Serbia, the first isolates ofC. difficilewere isolated in the Public Health Institute (PHI), Center for Microbiology in Niš, at the end of 2005.The National Reference Laboratory for Anaerobic Infections (NRLA) in PHI Niš confirmed the toxigenic strains that caused the first three registered hospital epidemics in Serbia, in 2006 at the Clinic of Neurology, Clinical Center Niš, in 2007 at the Clinical Center of Vojvodina in Novi Sad, and in 2009 in the General Hospital in Požarevac.In 2014,C. difficilespecies were isolated for the first time from 175 environment samples in the research studies which were conducted in NRLA of PHI Niš. In the samples of soil taken from the ground within the Clinical Center Niš, those taken from the parks at the territory of the Municipality of Niš, samples of mud and sand around the illegal sewage systems at the territory of the Municipality of Niška Banja, a small number of bacteriaC. difficileproducing the toxins (A+B+) as well as non-toxigenic isolates (A−B−) were found.Results of the first epidemiological investigations of cases of diarrhea associated with prior antibiotic treatment applied in hospitalized patients in a number of health centers in our country, microbiological investigations done in the Public Health Institute, valuable discussions at professional and scientific meetings influenced the general attitude that isolation and identification ofC. difficileand/or detection of toxin produced by this bacteria should be part of the routine work in the Serbian microbiological laboratories.

Multilocus sequence typing and clonal population genetic structure ofCyclospora cayetanensisin humans

To investigate the prevalence of Cyclospora cayetanensis in a longitudinal study and to conduct a population genetic analysis, fecal specimens from 6579 patients were collected during the cyclosporiasis - prevalent seasons in two urban areas of central China in 2011-2015. The overall incidence of C. cayetanensis infection was 1·2% (76/6579): 1·6% (50/3173) in Zhengzhou and 0·8% (26/3406) in Kaifeng (P 0·05). All the isolates clustered in the C. cayetanensis clade based on the small subunit ribosomal RNA gene sequence phylogenetic analysis. There were 45 specimens positive for all the five C. cayetanensis microsatellite loci, and formed 29 multilocus genotypes (MLGs). The phylogenetic relationships of 54 distinct MLGs (including 25 known reference MLGs), based on the concatenated multilocus sequences, formed three main clusters. A population structure analysis showed that the 79 isolates (including 34 known reference isolates) of C. cayetanensis produced three distinct subpopulations based on allelic profile data. In conclusion, we determined the frequency of C. cayetanensis infection in humans in Henan Province. The clonal population structure of the human C. cayetanensis isolates showed linkage disequilibrium and three distinct subpopulations.