Establishment of a Fast Diagnostic Method for Sepsis Pathogens Based on M1 Bead Enrichment.

Blood culture-based sepsis diagnostic methods usually cannot obtain positive results in a timely manner. Molecular diagnostic methods, such as real-time PCR without blood culture, would be more time-saving and suitable for pathogenic diagnosis of sepsis, while their sensitivities have always been unsatisfactory for the usually low concentration of pathogens in the blood of sepsis patients. In this study, we established a fast diagnostic method using magnetic beads coated with human recombined mannose-binding lectin that makes it possible to concentrate pathogens from human plasma that have low concentrations of pathogens. With subsequent microculture (MC) and real-time PCR, this method allowed the detection of 1-10 CFUs/ml of Staphylococcus aureus, Group A Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Candida tropicalis, or C. albicans from human plasma within 9.5 h, which was 21-80 h earlier than blood culture. The combination of pathogen enrichment and MC made the detection of sepsis-causing pathogens more time-saving and more sensitive than blood culture or real-time PCR alone.

A narrative review of Clostridioides difficile infection in China.

Clostridioides difficile is the predominant pathogen responsible for antimicrobial associated diarrhea (AAD) and health care facility-associated infectious diarrhea. The role of C. difficile in China and its impact on public health have gained attention in recent years. Most clinical C. difficile isolates in China belong to multilocus sequence type clade 1 with sequence types (STs) 3, 35 and 54 predominating. Of note, the proportion of C. difficile isolates from clade 4, especially ST37 (PCR ribotype 17), is much higher in China than in other areas. In China, the antimicrobial-resistance profile of C. difficile is similar to that of other countries, demonstrating a higher resistance rate to erythromycin, clindamycin, and fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin). In general, susceptibility to vancomycin and metronidazole of clinical C. difficile in China is high, however, some resistance to metronidazole have recently been reported. Preclinical research on C. difficile in animals in China is limited, and different studies have reported varied isolation rates and antimicrobial resistance profiles. The diverse molecular types of C. difficile in China merit further epidemiological, genomic and evolutionary investigation. While the use of probiotics in preventing C. difficile infection (CDI) have received both support and opposition, the discovery of new probiotics and new formulations are showing promising results in combating the threat posed by CDI.

Direct Blood Culturing of Candida spp. on Solid Medium by a Rapid Enrichment Method with Magnetic Beads Coated with Recombinant Human Mannan-Binding Lectin.

A rapid and accurate method to identify the species and antibiotic resistance of Candida spp. in blood is vital to increase the survival rates of patients with bloodstream infections. However, the extremely low levels of Candida spp. in blood make rapid diagnosis by standard blood culture difficult. In this study, we constructed a direct blood culturing method (i.e., the M1 method) by a rapid enrichment method with magnetic beads coated with a recombined human mannan-binding lectin (rhMBL; i.e., M1 protein), which demonstrated much higher Candida sp.-binding capacity than that of full-length MBL expressed in vitro (i.e., M2). With the M1 method, individual colonies were obtained before the standard blood culture method for each species of Candida spp. tested at <1 CFU/ml (an average of 29 h earlier). Additionally, the clinical sensitivity of the M1 method was 90.5% compared with that of the standard blood culture method when detecting frozen plasma from patients. More significantly, the turnaround time of the M1 method for blood culture could be reduced by approximately 37 to 43 h compared with that of the standard blood culture method in clinical sample identification.

Response of the gut microbiota during the Clostridioides difficile infection in tree shrews mimics those in humans.

BACKGROUND: Clostridioides difficile is a major cause of antibiotic associated diarrhea. Several animal models are used to study C. difficile infection (CDI). The tree shrew has recently been developed as a model of primate processes. C. difficile infection has not been examined in tree shrews. We infected tree shrews with hyper-virulent C. difficile strains and examined the alterations in gut microbiota using 16S rRNA gene sequencing. RESULTS: C. difficile colonized the gastrointestinal tract of tree shrew and caused diarrhea and weight loss. Histopathologic examination indicated structures and mucosal cell destruction in ileal and colonic tissues. The gut microbial community was highly diversity before infection and was dominated by Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Antibiotic administration decreased the diversity of the gut microbiota and led to an outgrowth of Lactobacillus. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Lachnospiraceae, Enterobacteriaceae, Escherichia, Blautia, and Tyzzerella increased following C. difficile infection. These taxa could be biomarkers for C. difficile colonization. CONCLUSIONS: In general, the disease symptoms, histopathology, and gut microbiota changes following C. difficile infection in tree shrews were similar to those observed in humans.

The molecular characters and antibiotic resistance of Clostridioides difficile from economic animals in China.

BACKGROUND: It has been performed worldwidely to explore the potential of animals that might be a reservoir for community associated human infections of Clostridioides difficile. Several genetically undistinguished PCR ribotypes of C. difficile from animals and human have been reported, illustrating potential transmission of C. difficile between them. Pig and calf were considered as the main origins of C. difficile with predominant RT078 and RT033, respectively. As more investigations involved, great diversity of molecular types from pig and calf were reported in Europe, North American and Australia. However, there were quite limited research on C. difficile isolates from meat animals in China, leading to non-comprehensive understanding of molecular epidemiology of C. difficile in China. RESULTS: A total of 55 C. difficile were isolated from 953 animal stool samples, within which 51 strains were from newborn dairy calf less than 7 days in Shandong Province. These isolates were divided into 3 STs and 6 RTs, of which ST11/RT126 was predominant type, and responsible for majority antibiotic resistance isolates. All the isolates were resistant to at least one tested antibiotics, however, only two multidrug resistant (MDR) isolates were identified. Furthermore, erythromycin (ERY) and clindamycin (CLI) were the two main resistant antibiotics. None of the isolates were resistant to vancomycin (VAN), metronidazole (MTZ), tetracycline (TET), and rifampin (RIF). CONCLUSIONS: In this study, we analyzed the prevalence, molecular characters and antibiotic resistance of C. difficile from calf, sheep, chicken, and pig in China. Some unique features were found here: first, RT126 not RT078 were the dominant type from baby calf, and none isolates were got from pig; second, on the whole, isolates from animals display relative lower resistant rate to these 11 tested antibiotics, compared with isolates from human in China in our previous report. Our study helps to deep understanding the situation of C. difficile from economic animals in China, and to further study the potential transmission of C. difficile between meat animals and human.

Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing.

BACKGROUND: Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates. RESULTS: Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes. CONCLUSIONS: We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

Bacteria-induced susceptibility to Candida albicans super-infection in mice via monocyte methyltransferase Setdb2.

Systemic bacterial infections are prone to secondary Candida albicans super-infection. However, the molecular mechanisms involved remain poorly understood. In this study, a model comprising sublethal cecal ligation and puncture plus C. albicans intravenous injection was applied to mimic the situation in super-infection. Compared with mice without systemic bacterial infection, mice with systemic bacterial infection had lower antifungal gene expression (including Il1b, Tnf, Il6, Ifnb, Ifng, Cxcl1, and Ccr2) in monocytes and less inflammatory monocytes and neutrophils infiltrating into the kidney when challenged with C. albicans. Further, lentivirus-mediated Setdb2-knockout and overexpression experiments verified that Setdb2 levels in monocytes correlated negatively with antifungal gene expression and survival rates. Transcriptional repression was probably achieved by Setdb2 through H3 methylation at lysine 9 in promoter regions of these antifungal genes.

Antibiotic resistance of clinical isolates of Clostridioides difficile in China and its association with geographical regions and patient age.

It is known that antibiotic usage is associated with the development of Clostridioides difficile infection (CDI), especially clindamycin, third-generation cephalosporins, and fuoroquinolones. Antibiotic resistance rates to many antibiotics varies a lot by study. We performed a study focused on antibiotic resistance in clinical isolates of C. difficile from more widespread geographic regions across China. Of 319 C. difficile isolates tested against 11 antibiotics, 313 (98.1%) were resistant to at least one antibiotic. The highest rate of resistance was to ciprofloxacin, clindamycin, and erythromycin across all age groups, similar to previous studies. However, all isolates were susceptible to metronidazole and vancomycin. Overall the resistance rate to tested antibiotics was lower than other reports in China except for chloramphenicol and meropenem. Genotype ST37/RT017 in clade 4 was resistant to more antibiotics than other types. Unexpectedly, RT078 isolates in this study were susceptible to almost all tested antibiotics. In addition, the proportion of multi-drug resistant (MDR) isolates observed (17%) in this study was much lower than several European studies (up to 55%) and a previous study in China (78%). Although isolates from patients aged between 65 and 85 were more resistant to antibiotics in comparison to other age groups, MDR isolates were still detected in children below 2-years of age. The highest percentage of MDR isolates was determined in South China, an area that is most developed economically. The clade 4, RT017 (ST37) has been associated with outbreaks in Europe and North America and is responsible for most C. difficile infections (CDIs) in Asia. In addition, RT017 is often clindamycin and fluoroquinolone resistant. This study provided a relatively comprehensive description of antibiotic resistance of C. difficile in China, and further elucidates the epidemiology and antibiotic resistance of clinical isolates of C. difficile in China at a national level.

First Report of Klebsiella oxytoca Strain Simultaneously Producing NDM-1, IMP-4, and KPC-2 Carbapenemases.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that blaNDM-1 was carried on an IncX3 plasmid. The blaIMP-4 and blaKPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate.

Molecularly Imprinted Membrane Electrospray Ionization for Direct Sample Analyses.

Typically dealing with practical samples with very complex matrices, ambient ionization mass spectrometry suffers from low detection sensitivity. In this study, molecular imprinting technology was explored and integrated with the membrane electrospray ionization (MESI) method for direct sample analyses. By enriching targeted analytes on molecularly imprinted membranes (MIMs), improvement (by 10- to 50-fold) in the limit of quantitation could be achieved, compared to conventional nanoelectrospray ionization methods or other ambient ionization methods. MIMs were prepared by cross-linking a synthesized molecularly imprinted polymer layer onto a polyvinylidene difluoride (PVDF) membrane. The characteristics of MIM in recognizing target analytes were investigated and verified. Experiments showed that MIM-ESI could provide satisfactory performances for direct quantification of targeted analytes in complex samples using mass spectroscopy (MS), and the quantitative performance of this methodology was validated. With the capability of target enrichment, the uses of MIM-ESI MS in different application fields were also demonstrated, including food safety, quantification of drug concentrations in blood, pesticide residues in soil, and antibiotic residues in milk.

Genomic study of the Type IVC secretion system in Clostridium difficile: understanding C. difficile evolution via horizontal gene transfer.

Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.

Extreme diversity and multiple SCCmec elements in coagulase-negative Staphylococcus found in the Clinic and Community in Beijing, China.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are recognized as a large reservoir of staphylococcal cassette chromosome mec (SCCmec) harboured by Staphylococcus aureus. However, data of SCCmec in CoNS are relatively absent particularly in China. METHODS: Seventy-eight CoNS clinical and 47 community isolates were collected in Beijing. PCR was performed to classify SCCmec types. Under oxacillin treatment, quantitative real-time reverse transcription PCR (qRT-PCR) was performed to compare mecA mRNA levels and mRNA half-life between isolates with single SCCmec element and those with multiple one. Their growth curves were analysed. Their bacterial cell wall integrity was also compared by performing a Gram stain. All ccr complex segments were sequenced and obtained ccr segments were analysed by phylogenetic analyses. RESULTS: All 78 clinical isolates had mecA segments compared with 38% in community isolates (total 47). Only 29% clinical isolates and 33% community isolates (among mecA positive isolates) harboured a single previously identified SCCmec type; notably, 17% clinical isolates and 28% community isolates had multiple SCCmec types. Further studies indicated that isolates with multiple SCCmec elements had more stable mecA mRNA expression compared with isolates with single SCCmec elements. CoNS with multiple SCCmec elements demonstrated superior cell wall integrity. Interestingly, phylogenetic analyses of obtained 70 ccr segments indicated that horizontal gene transfer of the ccr complex might exist among various species of clinical CoNS, community CoNS and S. aureus. CONCLUSIONS: CoNS recovered from patients carried extremely diverse but distinctive SCCmec elements compared with isolates from the community. More attention should be given to CoNS with multiple SCCmec not only because they had superior cell wall integrity, but also because CoNS and S. aureus might acquire multiple SCCmec through the ccr complex.

Rapid determination of bacterial aminoglycoside resistance in environmental samples using membrane electrospray ionization mass spectrometry.

RATIONALE: Antibiotic resistance in pathogenic bacteria is becoming a global public health problem, such as aminoglycoside resistance encoded by the armA gene. Although many methods have been reported, rapid analysis of environmental samples is still challenging. METHOD: A rapid analytical method was developed in this study to determine bacterial aminoglycoside resistance using membrane electrospray ionization mass spectrometry (MESI-MS). Precursor/product-ion pairs of ArmA unique peptides were detected with minimal sample preparation. Standard peptides were synthesized and used for developing and validating the methodology, and then the method was verified by both ArmA positive and ArmA negative simulated environmental samples. RESULTS: A rapid method for determination of bacterial aminoglycoside resistance was developed using MESI-MS/MS. The bacterial cultural time was optimized to 2 hours, and the precision, accuracy and recovery of this method were investigated. The peptide IHSSTNER (IR-8) unique to ArmA in simulated environmental samples can be successfully identified within 3 hours. CONCLUSIONS: The novel assay offered a rapid method to determine bacterial aminoglycoside resistance with high sensitivity, accuracy and precision in simulated environmental samples. This method could also be applied to identify other drug-resistance proteins in clinical/environmental samples. Copyright © 2016 John Wiley & Sons, Ltd.

Saccharicrinis marinus sp. nov., isolated from marine sediment.

A novel bacterial strain, designated Y11T, was isolated from marine sediment at Weihai in China. Comparative analysis of 16S rRNA gene sequences demonstrated that the novel isolate showed highest similarity to Saccharicrinis fermentans DSM 9555T (94.0 %) and Saccharicrinis carchari SS12T (92.7 %). Strain Y11T was a Gram-stain-negative, rod-shaped, non-endospore-forming, yellow-pigmented bacterium and was able to hydrolyse agar weakly. It was catalase-negative, oxidase-positive, facultatively anaerobic and motile by gliding. Optimal growth occurred at 28-30 °C, at pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. The DNA G+C content was 34.4 mol%. The strain contained MK-7 as the prevalent menaquinone. The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and C15 : 1ω6c. The predominant polar lipids were phosphatidylethanolamine and two unknown lipids. Data from the present polyphasic taxonomic study clearly place the strain as representing a novel species within the genus Saccharicrinis, for which the name Saccharicrinis marinus sp. nov. is proposed. The type strain is Y11T ( = CICC10837T = KCTC42400T).

Sequence variation in tcdA and tcdB of Clostridium difficile: ST37 with truncated tcdA is a potential epidemic strain in China.

Clostridium difficile is a well-known nosocomial infectious pathogen. Research on C. difficile infection has primarily focused on strains such as the hypervirulent PCR ribotype 027 (sequence type 1 [ST1]) emerging in Europe and North America. However, other new emerging ribotypes in some countries have attracted attention, such as PCR ribotype 17 (ST37) in Asia and Latin America. We collected 70 strains and sequenced their toxin genes, tcdA and tcdB. Multilocus sequence typing (MLST) was used to study their population structure. In addition, tcdA and/or tcdB sequences of 25 other isolates were obtained from GenBank. Single nucleotide polymorphisms (SNPs) were identified and analyzed. Phylogenetic analyses were performed to study toxin gene evolution. All tcdA and tcdB sequences were divided into 1 of 16 types (denoted A01 to -16 and B01 to -16, respectively). Hypervirulent strain RT027 is A13B12, and RT078 is A14B10, whereas the newly epidemic strain RT017 is A15B13. SNP analysis suggests the possibility of recombination in tcdB, perhaps through horizontal gene transfer. SNPs were also found in the sequences corresponding to the PCR primers widely used for toxin detection. Our study shows that ST037 shares a few genotypic features in its tcdA and tcdB genes with some known hypervirulent strains, indicating that they fall into a unique clade. Our findings can be used to map the relationships among C. difficile strains more finely than can be done with less sensitive methods, such as toxinotyping or even MLST, to reveal their inherent epidemiological characteristics.

Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates.

BACKGROUND: Candida tropicalis is considered to be the leading pathogen causing nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly those with leukemia. Microsatellite-based typing methods using sets of genetic markers have been developed and reported for population structure analysis of C. albicans, C. glabrata, and C. parapsilosis, but no studies have been published for genetic analysis of C. tropicalis. The objective of this study was to develop new microsatellite loci that have the ability to distinguish among C. tropicalis isolates. RESULTS: DNA sequences containing over 10 bi- or tri-nucleotide repeats were selected from the C. tropicalis genome database. Thirty PCR primers sets specific for the microsatellite loci were designed and tested using eight clinically independent isolates. According to the amplification efficiency, specificity, and observed polymorphisms, eight markers were selected for further population structure analysis and molecular typing. Sixty-five independent C. tropicalis isolates were genotyped using these 8 markers. Based on these analyses, six microsatellite loci were confirmed, although two loci were found to be with unstable flanking areas. The six polymorphic loci displayed 4-22 alleles and 7-27 genotypes. The discriminatory power of the six loci ranged from 0.70 to 0.95. Genotyping results obtained by microsatellite analysis were compared to PCR-fingerprinting and multi-locus sequence typing (MLST). The comparisons showed that microsatellite analysis and MLST had the similar discriminatory power for C. tropicalis, which were more powerful than PCR-fingerprinting. CONCLUSIONS: This is the first attempt to develop new microsatellite loci for C. tropicalis. These newly developed markers will be a valuable resource for the differentiation of C. tropicalis isolates. More C. tropicalis isolates will need to be sequenced and analyzed in order to fully show the potential of these newly developed microsatellite markers.

Detection of human herpesviruses (HHVs) in semen of human male infertile patients.

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21-49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.

A rapid multiplex real-time PCR detection of toxigenic Clostridioides difficile directly from fecal samples.

This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/µL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03434-6.

Identification and molecular analysis of pathogenic yeasts in droppings of domestic pigeons in Beijing, China.

Feral pigeons are known as reservoirs of pathogenic yeasts that cause opportunistic infections in human. In the outskirts of Beijing, China, pigeons are more frequently raised at homes than are encountered in public areas. Many studies have focused on the presence of pathogenic yeasts in the excreta (fresh or withered) of a variety kinds of birds, pigeon crop and cloacae. One hundred and forty-three samples of fresh droppings were collected from three suburban pigeon-raising homes in an area of northern Beijing, China. The internal transcribed sequences (ITS) of all strains (except for 8 strains of Rhodotorula sp. ) were sequenced and compared with those of the databases of the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov ) using the Basic Local Alignment Search Tool (BLAST). Yeasts representing 8 genera, Cryptococcus, Filobasidium, Rhodotorula, Candida, Debaryomyces, Saccaromyces, Trichosporon and Sporidiobolus, were identified from 120 isolates. Cryptococcus was the most prolific genera represented by eight species. The populations of yeast species isolated from fresh pigeon droppings were different among homes. Although it is well established that Cryptococcus neoformans exists mainly in old pigeon guano, several C. neoformans strains were still isolated from fresh pigeon excreta, providing a clue that live cryptococcal cells could move through the gastrointestinal tract of the pigeons. Eight genera identified from fresh droppings of domestic pigeons further confirm that pigeons serve as reservoirs, carriers and even spreaders of Cryptococcus species and other medically significant yeasts. The proportion of pathogenic yeasts in all isolates is more than 90 %.

Application of EMA-qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems.

Legionella are prevalent in human-made water systems and cause legionellosis in humans. Conventional culturing and polymerase chain reaction (PCR) techniques are not sufficiently accurate for the quantitative analysis of live Legionella bacteria in water samples because of the presence of viable but nonculturable cells and dead cells. Here, we report a rapid detection method for viable Legionella that combines ethidium monoazide (EMA) with quantitative real-time PCR (qPCR) and apply this method to detect Legionella in a large number of water samples from different sources. Results yielded that samples treated with 5 µg/ml EMA for 10 min and subsequently exposed to light irradiation for 5 min were optimal for detecting Legionella. EMA treatment before qPCR could block the signal from approximately 4 log(10) of dead cells. When investigating environmental water samples, the percent-positive rate obtained by EMA-qPCR was significantly higher than conventional PCR and culture methods, and slightly lower than qPCR. The bacterial count of Legionella determined by EMA-qPCR were mostly greater than those determined by culture assays and lower than those determined by qPCR. Acceptable correlations were found between the EMA-qPCR and qPCR results for cooling towers, piped water and hot spring water samples (r = 0.849, P < 0.001) and also found between the EMA-qPCR and culture results for hot spring water samples (r = 0.698, P < 0.001). The results indicate that EMA-qPCR could be used as a complementary tool for the detection and monitoring of Legionella in water systems, especially in hot spring water samples.