Evolutionary accumulation of FKS1 mutations from clinical echinocandin-resistant Candida auris.

INTRODUCTION: Drug resistance to echinocandins, first-line drugs used to treat Candida auris infection, is rapidly emerging. However, the accumulation of mutations in genes other than FKS1 (before an isolate develops to resistance via FKS1 mutations), remains poorly understood. Methods: Four clinical cases and 29 isolates associated with the incremental process of echinocandin resistance were collected and analyzed using antifungal drug susceptibility testing and genome sequencing to assess the evolution of echinocandin resistance. FINDINGS: Six echinocandin minimum inhibitory concentration (MIC)-elevated C. auris strains and seven resistant strains were isolated from the urinary system of patients receiving echinocandin treatment. Meanwhile, phylogenetic analyses illustrated that the echinocandin-resistant strains were closely related to other strains in the same patient. Genomic data revealed that the echinocandin-resistant strains had FKS1 mutations. Furthermore, three categories (ECN-S/E/R) of non-synonymous mutant SNP genes (such as RBR3, IFF6, MKC1, MPH1, RAD2, and MYO1) in C. auris appeared to be associated with the three-stage-evolutionary model of echinocandin resistance in C. glabrata: cell wall stress, drug adaptation, and genetic escape (FKS mutation). INTERPRETATION: Echinocandin-resistant C. auris undergoes spatial and temporal phase changes closely related to echinocandin exposure, particularly in the urinary system. These findings suggest that FKS1 mutations mediate an evolutionary accumulation of echinocandin resistance followed by modulation of chromosome remodelling and DNA repair processes that ultimately lead to FKS1 hot spot mutations and the development of drug resistance. This study provides an in-depth exploration of the molecular pathways involved in the evolution of Candida auris echinocandin resistance.

The first rare case of Candida palmioleophila infection reported in China and its genomic evolution in a human host environment.

Introduction: Candida palmioleophila is a rare human pathogenic fungus, which has been poorly characterized at the genome level. In this study, we reported the first fatal case of C. palmioleophila infection in China and investigate the microevolution of C. palmioleophila in the human host environment. Methods: A series of C. palmioleophila stains were collected from the patient at different time points for routine microbial and drug sensitivity testing. The first C. palmioleophila isolate 07202534 was identified by de novo whole genome sequencing. The in vitro and in vivo genetic evolutionary characteristics of C. palmioleophila were discussed based on the analysis of bioinformatics data. Results: The six C. palmioleophila isolates displayed dose-dependent sensitivity to fluconazole. The C. palmioleophila genome contained homologous genes such as CDR1 and MDR1, which were recognized to be related to azole resistance. In addition, amino acid variation was detected at F105L and other important sites of ERG11. In addition, the mean divergence time between C. palmioleophila and Scheffersomyces stipites CBS 6054 was 406.04 million years, indicating that C. palmioleophila originated earlier than its closest relative. In addition, the six strains of C. palmioleophila isolated form the patient had higher homology and fewer mutation sites, which indicated the stability in C. palmioleophila genome. We also found that C. palmioleophila had a wide natural niche and may evolve slowly. Discussion: We believe that this study will contribute to improve our understanding of the genetic evolution, pathogenicity, and drug resistance of C. palmioleophila and will aid in the prevention and control of its spread.

Risk factors and cerebrospinal fluid indexes analysis of intracranial infection by Acinetobacter baumannii after neurosurgery.

Background: Intracranial infection by Acinetobacter baumannii (A. baumannii) after neurosurgery has always been a difficult problem for neurosurgeons. This study analyzed risk factors that discriminated A. baumannii from other bacteria causing intracranial infection after neurosurgery. It also examined the differences in the cerebrospinal fluid (CSF) indexes to explore their value in the early diagnosis of intracranial infection by A. baumannii. Methods: We retrospectively reviewed ten years (January 2011 to May 2021) of postoperative central nervous system (CNS) infections in the First Hospital of China Medical University. According to the pathogen, CNS infections were divided into A. baumannii group and other species of bacteria group. We collected clinical and laboratory information of patients, and statistical analysis was performed with SPSS 26.0. Risk factors were screened by univariate analysis, and independent risk factors were screened by multiple logistic regression analysis. Finally, CSF-Pro, CSF-Glu, CSF-Cl, CSF-monocytes (%), CSF-multinucleated cells (%) levels, and CSF multinucleated cells%/monocytes% in the different groups were analyzed. Results: A total of 155 patients were included, 62 cases (40%) of intracranial infection by A. baumannii and 93 cases (60%) by other species of bacteria. The analysis showed that indwelling nasogastric tubes (P＜0.001, OR = 4.231), indwelling peripherally inserted central catheters (PICCs) (P = 0.041, OR = 2.765), and CSF drainage obstruction (P = 0.003, OR = 3.765) were independent risk factors for intracranial infection by A. baumannii after neurosurgery. Indwelling ventriculoperitoneal shunt (VPS) was a protective factor (P = 0.033, OR = 0.22). In addition, compared with other bacterial groups, the A. baumannii group had higher CSF-pro and CSF- multinucleated cells (%) levels and lower CSF-Glu and CSF- monocytes (%) levels, and the difference was statistically significant (P < 0.01). Conclusions: Our results elucidate risk factors and differences in CSF indexes for intracranial infection by A. baumannii after neurosurgery that could be detected and prevented early to reduce mortality.

Phenotypic and genetic features of a novel clinically isolated rough morphotype Candida auris.

Introduction: Candida auris is a newly emerging pathogenic fungus of global concern and has been defined by the World Health Organization (WHO) as a member of the critical group of the most health-threatening fungi. Methods: This study reveals and reports for the first time that a rough morphotype C. auris strain causes urinary tract infections in non-intensive care unit (ICU) inpatients. Furthermore, the morphology, the scanning electronmicroscopy (SEM), Whole-genome resequencing and RNA sequencing of C. auris possessing rough morphotype colonies compared to their smooth morphotype counterparts. Results: The newly identified phenotypic variation of C. auris appears round, convex, dry, and burr-like with a rough texture. SEM shows that rough type C. auris has a rough and uneven colony surface with radial wrinkles and irregular spore arrangement. Cells of the rough morphotype C. auris naturally aggregate into clusters with tight connections in the liquid, and it seems that the cell division is incomplete. A genome-wide analysis of the rough type C. auris confirmed its genetic association with the smooth type of C. auris prevalent in China (Shenyang) two years ago; however, single nucleotide polymorphism (SNP) mutations of five genes (ACE2, IFF6, RER2, UTP20, and CaO19.5847) were identified more recently. RNA-seq revealed IFF2/HYR3, DAL5, PSA31, and SIT1 were notably up-regulated, while multiple cell wall-associated genes (ALS1, MNN1, PUL1, DSE1, SCW11, PGA38, RBE1, FGR41, BGLI, GIT3, CEP3, and SAP2) were consistently down-regulated in rough morphotype C. auris. Discussion: The rough phenotypic variation of C. auris is likely to be related to the structural and functional changes in cell wall proteins. This novel rough morphotype C. auris will provide a basis for further studies concerning the evolutionary characteristics of C. auris.

Carbapenem-resistant Enterobacter cloacae complex in a tertiary Hospital in Northeast China, 2010-2019.

BACKGROUND: Carbapenem-resistant Enterobacter cloacae complex (CREC) is a new emerging threat to global public health. The objective of the study was to investigate the clinical characteristics and molecular epidemiology of CREC infections in the medical center of northeast China. METHODS: Twenty-nine patients were infected/colonized with CREC during a ten-year period (2010-2019) by WHONET analysis. Antibiotic susceptibilities were tested with VITEK 2 and micro broth dilution method (for polymyxin B and tigecycline). Carbapenemase encoding genes, ß-lactamase genes, and seven housekeeping genes for MLST were amplified and sequenced for 18 cryopreserved CREC isolates. Maximum likelihood phylogenetic tree was built with the concentrated sequences to show the relatedness between the 18 isolates. RESULTS: There was a rapid increase in CREC detection rate during the ten-year period, reaching 8.11% in 2018 and 6.48% in 2019. The resistance rate of CREC isolates to imipenem and meropenem were 100.0 and 77.8%, however, they showed high sensitivity to tigecycline, polymyxin B and amikacin. The 30-day crude mortality of CREC infection was 17.4%, indicating that it may be a low-virulence bacterium. Furthermore, molecular epidemiology revealed that ST93 was the predominant sequence type followed by ST171 and ST145, with NDM-1 and NDM-5 as the main carbapenemase-encoding genes. Moreover, E. hormaechei subsp. steigerwaltii and E. hormaechei subsp. oharae were the main species, which showed different resistance patterns. CONCLUSION: Rising detection rate of CREC was observed in a tertiary hospital, which showed heterogeneity in drug resistance patterns, resistance genes, and MLST types. Effective infection prevention and control measures should be taken to reduce the spread of CREC.

Genomic epidemiology of Candida auris in a general hospital in Shenyang, China: a three-year surveillance study.

Candida auris is an emerging pathogenic fungal species found worldwide. Since April 2016, C. auris colonization/infection cases have been found in a general hospital in Shenyang, China. The genome-based phylogenetic studies of these isolates remain undefined. In the current study, the microbiological characteristics and antifungal susceptibility of these C. auris isolates, which were collected in Shenyang during the three-year period (2016-2018), were investigated. Whole-genome sequencing was applied to investigate the genetic variation and molecular epidemiological characteristics. A total of 93 C. auris isolates, including 92 clinical isolates and 1 environmental screening isolate were identified. Among the investigated wards, the C. auris cases were the most prevalent (97.4%, 37/38) in four intensive care units (ICUs). The Shenyang isolates carrying the VF125AL mutation in the key drug-resistance gene ERG11 were mainly fluconazole resistant and formed a distinct subclade under the South African clade according to the phylogenetic and population structural analyses. In addition, the Shenyang subclade was found to be closely related to the British subclade in the aspect of genetic distance. As a conclusion, this study provides an important clue for revealing the origin of C. auris found in Shenyang and could also contribute to improve the understanding of the epidemiological characteristics of C. auris worldwide.

Filamentous growth is a general feature of Candida auris clinical isolates.

A striking feature of pathogenic Candida species is morphological plasticity that facilitates environmental adaptation and host infection. Candida auris is an emerging multidrug-resistant fungal pathogen first described in Japan in 2009. In this study, we demonstrate that clinical isolates of C. auris have multiple colony and cellular morphologies including the yeast, filamentous, aggregated, and elongated forms. This phenotypic diversity has been observed in eight clinical isolates of C. auris representing four major genetic clades, suggesting that it could be a general characteristic. We further demonstrate that different cell types of C. auris exhibit distinct antifungal resistance and virulence properties in a Galleria mellonella infection model. Our findings imply that morphological diversity is an important biological feature of C. auris and could be a contributor to its emergence and rapid prevalence worldwide. LAY SUMMARY: Candida auris is an emerging multidrug-resistant fungal pathogen. Morphological analyses indicate that filamentation is a general feature of clinical isolates of C. auris. This ability is associated with antifungal resistance and virulence.

Is the superbug fungus really so scary? A systematic review and meta-analysis of global epidemiology and mortality of Candida auris.

BACKGROUND: Candida auris is a new pathogen called "superbug fungus" which caused panic worldwide. There are no large-scale epidemiology studies by now, therefore a systematic review and meta-analysis was undertaken to determine the epidemic situation, drug resistance patterns and mortality of C. auris. METHODS: We systematically searched studies on the clinical report of Candida auris in Pubmed, Embase and Cochrane databases until October 6, 2019. A standardized form was used for data collection, and then statics was performed with STATA11.0. RESULTS: It showed that more than 4733 cases of C. auris were reported in over 33 countries, with more cases in South Africa, United States of America, India, Spain, United Kingdom, South Korea, Colombia and Pakistan. C. auirs exhibited a decrease in case count after 2016. Clade I and III were the most prevalent clades with more cases reported and wider geographical distribution. Blood stream infection was observed in 32% of the cases, which varied depending on the clades. Resistance to fluconazole, amphotericin B, caspofungin, micafungin and anidulafungin in C. auris were 91, 12, 12.1, 0.8 and 1.1%. The overall mortality of C. auris infection was 39%. Furthermore, subgroup analyses showed that mortality was higher in bloodstream infections (45%), and lower in Europe (20%). CONCLUSIONS: Over 4000 cases of C. auris were reported in at least 33 countries, which showed high resistance to fluconazole, moderate resistance to amphotericin B and caspofungin, high sensitivity to micafungin and anidulafungin. The crude mortality for BSI of C. auris was 45% which was similar to some drug-resistant bacteria previously reported. In conclusion, C. auris displayed similar characteristics to some drug resistance organisms. This study depicts several issues of C. auris that are most concerned, and is of great significance for the clinical management.

Clinical Characteristics and Outcomes of Candidemia Caused by Meyerozyma guilliermondii Complex in Cancer Patients Undergoing Surgery.

Although Meyerozyma guilliermondii complex is an uncommon cause of invasive candidiasis worldwide, reported cases, mainly regarding bloodstream infections, increased over years, and patients with cancer who have undergone recent surgery are most commonly affected. However, the clinical characteristics and outcomes of candidemia caused by M. guilliermondii complex remain poorly understood. A retrospective case-control study was conducted to evaluate the clinical characteristics and mortality of candidemia caused by M. guilliermondii complex in cancer patients undergoing surgery. Demographic and clinical data were collected from the hospital medical records system with a standardized data collection form and were analyzed with SPSS 20.0. Sixty-six cancer patients who have undergone recent surgery and were diagnosed with candidemia caused by M. guilliermondii complex were included in the study. Regarding the clinical manifestations, most patients' body temperatures ranged from 38 to 40 °C, with a median fever duration of 4 (IQR: 3-6) days. Multivariate analysis indicated that the presence of central venous catheter (OR: 6.68; 95% CI 2.80-15.94) and gastric tube (OR: 3.55; 95% CI 1.22-10.34) were independent risk factors for M. guilliermondii complex fungemia. The 30-day crude mortality of candidemia caused by M. guilliermondii complex was 12.1%, twice that of the control group. Moreover, increased WBC count, age ≥ 60 years, septic shock, and ICU admission were identified as predictors of mortality through univariate analysis. These findings will provide a foundation for the clinical management of candidemia caused by M. guilliermondii complex in post-surgical cancer patients.

First cases and risk factors of super yeast Candida auris infection or colonization from Shenyang, China.

For the first time, we identified 15 cases of Candida auris in Shenyang, China, and then performed a risk factor assessment for these patients compared with 30 control subjects who were hospitalized in the same ward during the same period of time as the infected patients. We found that diarrhea, gastrointestinal decompression, infection, or colonization with other Candida isolates (especially Candida albicans) and tetracycline antibiotics were all risk factors for C. auris infection or colonization. Diarrhea and tetracycline antibiotics were independent risk factors. We suggest clinicians pay special attention to the emergence of multidrug-resistant C. auris infections or colonization.

Antimicrobial resistance, genotypic characterization and pulsed-field gel electrophoresis typing of extended spectrum ß-lactamases-producing clinical Escherichia coli strains in Macao, China.

BACKGROUND: The rise of the production of CTX-M class extended spectrum ß-lactamases (ESBLs) has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. METHODS: Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis (PFGE). RESULTS: Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracycline, ciprofloxacin, sulfamethoxazole trimethoprim and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs (76.2%). Two strains showed indistinguishable patterns by PFGE. CONCLUSIONS: The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.

[Molecular epidemiological study of multi-drug resistant Acinetobacter baumannii].

OBJECTIVE: To determine the possible genetic background and the source of our hospital's 43 clinical isolates of multidrug-resistant Acinetobacter baumannii, and the category of gene cassettes in type 1 integrons of all strains. METHODS: Restriction enzyme Apa I was chosen for all strains in pulsed-field gel electrophoresis (PFGE) methods. Multilocus sequence typing (MLST) was used to compare the allelic profiles of all the strains. PCR method was used for amplify the integrons of all strains. RESULTS: PFGE results showed that 43 strains were divided into four types. A-type and B-type were divided into 4 and 2 subtypes, respectively. The MLST results showed the existing of three allelic profiles: 1-3-3-2-2-7-3, 1-3-3-2-2-11-3, and 1-3-3-2-2-14-3. B-type and D-type of PFGE have the same allelic profile (1-3-3-2-2-11-3). A-type strains were detected mainly in ICU, and in burn unit only found B- and D-type. The same integron was detected in 62.8% of the strains. The constituent ratio of A1, A2, A3, A4, B1, B2, C and D-type was 40.7%, 18.5%, 7.4%, 3.7%, 14.8%, 3.7%, 3.7% and 7.4%, respectively. CONCLUSIONS: The coexistence of multiple cloning system in this region was proved by the PFGE and MLST, and the same clone can evolve to different subtypes when stimulated by different environmental conditions; and the different carrying-situation of the same integron in strains prove the possibility of the change during the evolution of resistance mechanisms.

[Novel gene sequence variants of pbp2b in penicillin-nonsusceptible Streptococcus pneumonia isolates].

OBJECTIVE: To investigate the alternations in gene/amino acid sequence of penicillin-binding protein (PBP)2b from clinical isolates of penicillin-nonsusceptible Streptococcus pneumonia (PNSP) in this region. METHODS: 24 strains of Streptococcus pneumonia were collected from January to December 2006. The antibiotics susceptibility of these strains was detected. PCR amplification and direct sequencing of pbp2b genes were performed. The sequence variations of PBP genes of the PNSP in this region were studied with sequence BLAST analysis. RESULTS: Three prominent substitutions were common to 13 PNSP isolates with minimal inhibitory concentration (MIC) at least 0.1 mg/L. These included the replacement of Thr(445)--> Ala following the conservative motif SSN, Glu(475)-->Gly and Thr(488)-->Ala/Ser. The exchange of Glu(332)-->Gly was identified in 12 PNSP isolates of which the MIC was at least 0. 25 mg/L. Seven penicillin resistant Streptococcus pneumonia (PRSP) isolates (MIC > or = 3 mg/L) shared the amino acid substitution Ala(618)-->Gly adjacent to third conserved (KTG) motif and the PBP2b sequences of seven PRSP isolates were classified within Baek's group II and were very similar to those of the Korean J77 isolate. Novel gene and amino acid sequence variants in isolate 14, 15, 8, 11 and 24 was identified in this study and these gene sequences have been deposited in the GenBank database and assigned accession no. EU035970, EU056919, EU056920, EU056921 and EU106886. CONCLUSION: Analysis of pbp2b genes revealed highly similar patterns of nucleotide and amino acid sequence variation among most resistant isolates, while penicillin intermediate Streptococcus pneumonia might be associated with novel gene sequence variants.