Jinxing Lu

Sequence Variation in tcdA and tcdB of Clostridium difficile: ST37 with Truncated tcdA Is a Potential Epidemic Strain in China

ABSTRACT 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.

Pichia fabianii blood infection in a premature infant in China: case report

BackgroundInvasive fungal infections caused by uncommon fungi have increased in recent years. Hospitalized low-birth-weight infants are at high risk for neonatal fungal infections. Pichia fabianii is a rare pathogen causing blood infection, which has reportedly caused only 4 cases of fungemia and 1 case of endocarditis worldwide. Here, we describe the first case of a P. fabianii blood infection in a premature infant in China.Case presentationOn July 28th, a low-birth-weight (LBW, 1760xa0g) female infant born at 33+4xa0weeks of gestation was admitted to the pediatric intensive care unit with mild neonatal asphyxia. Until August 2nd, a mechanical respirator was used to assist respiration under the Continuous Positive Airway Pressure (CPAP) model. The baby had an increased body temperature and a fever. To prevent infection, Ceftriaxone Sodium (CS) was administered intravenously for three days, after which Cefepime was administered until August 13th. Chest X-rays showed suspected plaque-like shadows in the right lung. Blood cultures twice tested positive for fungal infection caused by Candida pelliculosa (recognized as Pichia fabianii later), which is first mis-identified by commercial kit. Hence, intravenous fluconazole was administered. However, cultures of other body fluids (e.g., urine, feces and sputum) tested negative for fungal infection. Routine tests and biochemistry of cerebrospinal fluid (CSF) were normal. Latex agglutination of Cryptococcus neoformans and fungi cultures in the CSF were also negative. After 14xa0days of intravenous fluconazole, blood was re-cultured, the result of which was negative. On August 30th, intravenous fluconazole was suspended. On Sep 3rd, the infant left the hospital in good health.ConclusionsThis is the first case of a blood infection caused by P. fabianii in a LBW premature female infant in China. Risk factors for fungal infection include premature birth, as well as mechanical invasive operation and antibacterial drug usage. Whether such risk factors necessitate prophylactic use of antifungal drugs is an important question that has yet to be fully addressed. Additionally, the pathogen P. fabianii collected in this study was resistant to amphotericin B (AMB) and itraconazole (ITR). With the exception of the azole-resistant endocarditis case, all other cases have not demonstrated such a resistance. Finally, commercial biochemical methods used in routine practice are limited in their ability to identify P. fabianii. Molecular genetic based methods are imperative for identification of uncommon fungal species from disseminated infections.

Analysis of the Clonality of Candida tropicalis Strains from a General Hospital in Beijing Using Multilocus Sequence Typing

Multilocus sequence typing (MLST) based on six loci was used to analyze the relationship of 58 Candida tropicalis isolates from individual patients in a general hospital in Beijing, China. A total of 52 diploid sequence types (DSTs) were generated by the MLST, all of which were new to the central database. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrograms were constructed, which showed that the 58 isolates were distributed robustly and 6 main groups were clustered regardless of the specimen source and medical department. The minimum spanning tree (MST) of the 58 isolates (52 DSTs) and all 401 isolates (268 DSTs) in the C. tropicalis central database (http://pubmlst.org/ctropicalis/) indicated that the isolates in this study clustered in three relative pure clonal complexes, and 2 clustered with isolates from Taiwan, Belgium, Brazil, and the US. This study presents the first MLST analysis of C. tropicalis isolates from Mainland China, which may be useful for further studies on the similarity, genetic relationship, and molecular epidemiology of C. tropicalis strains worldwide.

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 90xa0%.

Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates.

BackgroundCandida 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.ResultsDNA 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.ConclusionsThis 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.

Distinct Expression Levels of ALS, LIP, and SAP Genes in Candida tropicalis with Diverse Virulent Activities

Candia tropicalis is an increasingly important human pathogen, causing nosocomial fungemia among patients with neutropenia or malignancy. However, limited research has been published concerning its pathogenicity. Based on the phenotypes of C. tropicalis in our previous study, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases, and hemolysins), and one reference strain, ATCC750. The present study aimed to investigate the filamentation ability, the expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) and the cell damage of C. tropicalis strains with diverse virulences. C. tropicalis exhibited strain-dependent filamentation ability, which was positively correlated with biofilm formation. Reverse transcriptase PCR analysis showed that the ALST3 and SAPT3 genes had the highest expression in their corresponding genes for most C. tropicalis. The expressions of virulence genes, except ALST3 on polystyrene, were upregulated compared with growth in the planktonic and on human urinary bladder epithelial cell line (TCC-SUP) surface. Clustering analysis of virulence genes showed that isolates had a high biofilm forming ability on polystyrene formed a group. Lactate dehydrogenase assays showed that the cell damage induced by C. tropicalis markedly increased with longer infection time (24 and 48 h). Strain FXCT01, isolated from blood, caused the most serious cell damage; while ZRCT52, which had no filamentation ability, caused the least cell damage. Correlation analysis demonstrated significant correlation existed between adhesion on epithelial cells or the expression of ALST2-3 and cell damage. Overall, our results supported the view that adhesion and filamentation may play significant roles in the cell damage caused by C. tropicalis.

A Genome-Wide Transcriptional Analysis of Yeast-Hyphal Transition in Candida tropicalis by RNA-Seq.

Candida tropicalis is considered as the leading pathogen in nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly in leukemia. The yeast-filament transition is required for virulent infection by Candida. Several studies have explored the genome-wide transcription profile of Candida, however, no report on the transcriptional profile of C. tropicalis under yeast-filament transition has been published. In this study, the transcriptomes of three C. tropicalis isolates with different adhesion and biofilm formation abilities, identified in our previous studies, were analyzed in both the yeast and filament states using RNA-Seq. Differentially expressed genes were found for each isolate during the transition. A total of 115 genes were up- or down- regulated in the two hyphal-producing isolates (ZRCT 4 and ZRCT 45). Among these differentially expressed genes, only two were down-regulated during the yeast-filament transition. Furthermore, six filament-associated genes were up-regulated in the hyphae-producing isolates. According to Candida Hypha Growth Database established in this study, 331 hyphae- related genes were discovered in C. tropicalis. ALS1 and ALS3 were down-regulated and up-regulated, respectively, during filamentous growth of C. tropicalis. These findings proved a better understanding of gene expression dynamics during the yeast-filament transition in C. tropicalis.

A retrospective study of community-acquired Clostridium difficile infection in southwest China

To identify the prevalence and characteristics of community-acquired Clostridium difficile infection (CA-CDI) in southwest China, we conducted a cross-sectional study. 978 diarrhea patients were enrolled and stool specimens’ DNA was screened for virulence genes. Bacterial culture was performed and isolates were characterized by PCR ribotyping and multilocus sequence typing. Toxin genes tcdA and/or tcdB were found in 138/978 (14.11%) cases for fecal samples. A total of 55u2009C. difficile strains were isolated (5.62%). The positive rate of toxin genes and isolation results had no statistical significance between children and adults groups. However, some clinical features, such as fecal property, diarrhea times before hospital treatment shown difference between two groups. The watery stool was more likely found in children, while the blood stool for adults; most of children cases diarrhea ≤3 times before hospital treatment, and adults diarrhea >3 times. Independent risk factor associated with CA-CDI was patients with fever. ST35/RT046 (18.18%), ST54/RT012 (14.55%), ST3/RT001 (14.55%) and ST3/RT009 (12.73%) were the most distributed genotype profiles. ST35/RT046, ST3/RT001 and ST3/RT009 were the commonly found in children patients but ST54/RT012 for adults. The prevalence of CA-CDI in Yunnan province was relatively high, and isolates displayed heterogeneity between children and adults groups.

Molecular Characterization of Clostridium difficile Isolates in China From 2010 to 2015

Clostridium difficile infection (CDI) has become a worldwide public health problem causing high mortality and a large disease burden. Molecular typing and analysis is important for surveillance and infection control of CDI. However, molecular characterization of C. difficile across China is extremely rare. Here, we report on the toxin profiles, molecular subtyping with multilocus sequence typing (MLST) and PCR ribotyping, and epidemiological characteristics of 199 C. difficile isolates collected between 2010 through 2015 from 13 participating centers across China. We identified 35 STs and 27 ribotypes (RTs) among the 199 C. difficile isolates: ST35 (15.58%), ST3 (15.08%), ST37 (12.06%), and RT017 (14.07%), RT001 (12.06%), RT012 (11.56%) are the most prevalent. One isolate with ST1 and 8 isolates with ST 11 were identified. We identified a new ST in this study, denoted ST332. The toxin profile tcdA+tcdB+tcdC+tcdR+tcdE+CDT- (65.83%) was the predominant profile. Furthermore, 11 isolates with positive binary toxin genes were discovered. According to the PCR ribotyping, one isolate with RT 027, and 6 isolates with RT 078 were confirmed. The epidemiological characteristics of C. difficile in China shows geographical differences, and both the toxin profile and molecular types exhibit great diversity across the different areas.

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.