Molecular mechanisms of azole resistance in Candida bloodstream isolates.

BACKGROUND: Antifungal resistance rates are increasing. We investigated the mechanisms of azole resistance of Candida spp. bloodstream isolates obtained from a surveillance study conducted between 2012 and 2015. METHODS: Twenty-six azole non-susceptible Candida spp. clinical isolates were investigated. Antifungal susceptibilities were determined using the Sensititre YeastOne® YO10 panel. The ERG11 gene was amplified and sequenced to identify amino acid polymorphisms, while real-time PCR was utilised to investigate the expression levels of ERG11, CDR1, CDR2 and MDR1. RESULTS: Azole cross-resistance was detected in all except two isolates. Amino acid substitutions (A114S, Y257H, E266D, and V488I) were observed in all four C. albicans tested. Of the 17 C. tropicalis isolates, eight (47%) had ERG11 substitutions, of which concurrent observation of Y132F and S154F was the most common. A novel substitution (I166S) was detected in two of the five C. glabrata isolates. Expression levels of the various genes differed between the species but CDR1 and CDR2 overexpression appeared to be more prominent in C. glabrata. CONCLUSIONS: There was interplay of various different mechanisms, including mechanisms which were not studied here, responsible for azole resistance in Candida spp in our study.

Genomic Surveillance of Carbapenem-Resistant Klebsiella pneumoniae from a Major Public Health Hospital in Singapore.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global public health threat. In this study, we employed whole-genome sequencing (WGS) to determine the genomic epidemiology of a longitudinal collection of clinical CRKP isolates recovered from a large public acute care hospital in Singapore. Phylogenetic analyses, a characterization of resistance and virulence determinants, and plasmid profiling were performed for 575 unique CRKP isolates collected between 2009 and 2020. The phylogenetic analyses identified the presence of global high-risk clones among the CRKP population (clonal group [CG] 14/15, CG17/20, CG147, CG258, and sequence type [ST] 231), and these clones constituted 50% of the isolates. Carbapenemase production was common (n = 497, 86.4%), and KPC was the predominant carbapenemase (n = 235, 40.9%), followed by OXA-48-like (n = 128, 22.3%) and NDM (n = 93, 16.2%). Hypervirulence was detected in 59 (10.3%) isolates and was most common in the ST231 carbapenemase-producing isolates (21/59, 35.6%). Carbapenemase genes were associated with diverse plasmid replicons; however, there was an association of blaOXA-181/232 with ColKP3 plasmids. This study presents the complex and diverse epidemiology of the CRKP strains circulating in Singapore. Our study highlights the utility of WGS-based genomic surveillance in tracking the population dynamics of CRKP. IMPORTANCE In this study, we characterized carbapenem-resistant Klebsiella pneumoniae clinical isolates collected over a 12-year period in the largest public acute-care hospital in Singapore using whole-genome sequencing. The results of this study demonstrate significant genomic diversity with the presence of well-known epidemic, multidrug-resistant clones amid a diverse pool of nonepidemic lineages. Genomic surveillance involving comprehensive resistance, virulence, and plasmid gene content profiling provided critical information for antimicrobial resistance monitoring and highlighted future surveillance priorities, such as the emergence of ST231 K. pneumoniae strains bearing multidrug resistance, virulence elements, and the potential plasmid-mediated transmission of the blaOXA-48-like gene. The findings here also reinforce the necessity of unique infection control and prevention strategies that take the genomic diversity of local circulating strains into consideration.

Ceftolozane/Tazobactam Resistance and Mechanisms in Carbapenem-Nonsusceptible Pseudomonas aeruginosa.

This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired ß-lactamases, especially metallo-ß-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by ß-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents.IMPORTANCEPseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel ß-lactam/ß-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future ß-lactams and ß-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

Genomic characterization of carbapenem-non-susceptible Pseudomonas aeruginosa in Singapore.

Pseudomonas aeruginosa is a clinically important pathogen implicated in many hospital-acquired infections. Its propensity to acquire broad-spectrum resistance has earned the organism its status as a severe public health threat requiring urgent control measures. While whole-genome sequencing-based genomic surveillance provides a means to track antimicrobial resistance, its use in molecular epidemiological surveys of P. aeruginosa remains limited, especially in the Southeast Asian region. We sequenced the whole genomes of 222 carbapenem-non-susceptible P. aeruginosa (CNPA) isolates collected in 2006-2020 at the largest public acute care hospital in Singapore. Antimicrobial susceptibilities were determined using broth microdilution. Clonal relatedness, multi-locus sequence types, and antimicrobial resistance determinants (acquired and chromosomal) were determined. In this study, CNPA exhibited broad-spectrum resistance (87.8% multi-drug resistance), retaining susceptibility only to polymyxin B (95.0%) and amikacin (55.0%). Carbapenemases were detected in 51.4% of the isolates, where IMP and NDM metallo-ß-lactamases were the most frequent. Carbapenem resistance was also likely associated with OprD alterations or efflux mechanisms (ArmZ/NalD mutations), which occurred in strains with or without carbapenemases. The population of CNPA in the hospital was diverse; the 222 isolates grouped into 68 sequence types (ST), which included various high-risk clones. We detected an emerging clone, the NDM-1-producing ST308, in addition to the global high-risk ST235 clone which was the predominant clone in our population. Our results thus provide a "snapshot" of the circulating lineages of CNPA locally and the prevailing genetic mechanisms contributing to carbapenem resistance. This database also serves as the baseline for future prospective surveillance studies.

In vitro Bactericidal Activities of Combination Antibiotic Therapies Against Carbapenem-Resistant Klebsiella pneumoniae With Different Carbapenemases and Sequence Types.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is becoming increasingly problematic due to the limited effectiveness of new antimicrobials or other factors such as treatment cost. Thus, combination therapy remains a suitable treatment option. We aimed to evaluate the in vitro bactericidal activity of various antibiotic combinations against CRKP with different carbapenemase genotypes and sequence types (STs). Thirty-seven CRKP with various STs and carbapenemases were exposed to 11 antibiotic combinations (polymyxin B or tigecycline in combination with ß-lactams including aztreonam, cefepime, piperacillin/tazobactam, doripenem, meropenem, and polymyxin B with tigecycline) in static time-kill studies (TKS) using clinically achievable concentrations. Out of the 407 isolate-combination pairs, only 146 (35.8%) were bactericidal (≥3 log10CFU/mL decrease from initial inoculum). Polymyxin B in combination with doripenem, meropenem, or cefepime was the most active, each demonstrating bactericidal activity in 27, 24, and 24 out of 37 isolates, respectively. Tigecycline in combination with ß-lactams was rarely bactericidal. Aside from the lower frequency of bactericidal activity in the dual-carbapenemase producers, there was no apparent difference in combination activity among the strains with other carbapenemase types. In addition, bactericidal combinations were varied even in strains with similar STs, carbapenemases, and other genomic characteristics. Our findings demonstrate that the bactericidal activity of antibiotic combinations is highly strain-specific likely owing to the complex interplay of carbapenem-resistance mechanisms, i.e., carbapenemase genotype alone cannot predict in vitro bactericidal activity. The availability of WGS information can help rationalize the activity of certain combinations. Further studies should explore the use of genomic markers with phenotypic information to predict combination activity.

Risk factors and outcomes associated with the isolation of polymyxin B and carbapenem-resistant Enterobacteriaceae spp.: A case-control study.

Increasing resistance to polymyxin, a last-line antibiotic, is a growing public health concern worldwide. The primary objective of this study was to identify predictors for the isolation of polymyxin-resistant (PR) carbapenem-resistant Enterobacteriaceae (CRE) among hospitalized patients. The secondary objective was to describe the clinical outcomes of patients with PR-CRE infections. A retrospective case-control study including patients admitted to Singapore General Hospital between June 2012 and June 2016 was conducted. Cases were defined as patients who had clinical cultures from which a PR-CRE was isolated. Controls were randomly selected from patients with polymyxin-susceptible (PS) CRE admitted during the same period, and frequency-matched to site of isolation. We included 37 PR cases and 111 PS controls. Polymyxin resistance was detected predominantly in Enterobacter spp. (54.1%) and Klebsiella pneumoniae (43.2%). Multilocus sequence typing showed little clonal relatedness among the isolates. mcr-1 was detected in two PR-CRE isolates. Multivariable analyses showed that PR-CRE isolation was associated with prior polymyxins (adjusted odds ratio (OR), 21.31; 95% confidence interval (CI), 3.04-150.96) and carbapenem exposures (OR 3.74; CI 1.13-12.44), when adjusted for time at risk and bacteria species. In PR-CRE patients with infections, the 30-day all-cause in-hospital mortality was 50.0% as compared to 38.1% in patients with PS-CRE (P = 0.346). Prior polymyxin and carbapenem exposures were independent risk factors for isolation of PR-CRE. Outcomes of PR-CRE and PS-CRE infections were similar in this study.

Correction to: Candidemia in a major regional tertiary referral hospital - epidemiology, practice patterns and outcomes.

[This corrects the article DOI: 10.1186/s13756-017-0184-1.].

Candidemia in a major regional tertiary referral hospital - epidemiology, practice patterns and outcomes.

BACKGROUND: Candidemia is a common cause of nosocomial bloodstream infections, resulting in high morbidity and mortality. This study was conducted to describe the epidemiology, species distribution, antifungal susceptibility patterns and outcomes of candidemia in a large regional tertiary referral hospital. METHODS: A retrospective surveillance study of patients with candidemia was conducted at Singapore General Hospital between July 2012 and December 2015. In addition, incidence densities and species distribution of candidemia episodes were analysed from 2008 to 2015. RESULTS: In the period of 2012 to 2015, 261 candidemia episodes were identified. The overall incidence was 0.14/1000 inpatient-days. C. glabrata (31.4%), C. tropicalis (29.9%), and C. albicans (23.8%) were most commonly isolated. The incidence of C. glabrata significantly increased from 2008 to 2015 (Coefficient 0.004, confidence interval 0-0.007, p = 0.04). Fluconazole resistance was detected primarily in C. tropicalis (16.7%) and C. glabrata (7.2%). fks mutations were identified in one C. albicans and one C. tropicalis. Candidemia episodes caused by C. tropicalis were more commonly encountered in patients with haematological malignancies (p = 0.01), neutropenia (p < 0.001) and higher SAPS II scores (p = 0.02), while prior exposure to echinocandins was associated with isolation of C. parapsilosis (p = 0.001). Echinocandins (73.3%) were most commonly prescribed as initial treatment. The median (range) time to initial treatment was 1 (0-9) days. The 30-day in-hospital mortality rate was 49.8%. High SAPS II score (Odds ratio, OR 1.08; 95% confidence interval, CI 1.05-1.11) and renal replacement therapy (OR 5.54; CI 2.80-10.97) were independent predictors of mortality, while drain placement (OR 0.44; CI 0.19-0.99) was protective. CONCLUSIONS: Decreasing azole susceptibilities to C. tropicalis and the emergence of echinocandin resistance suggest that susceptibility patterns may no longer be sufficiently predicted by speciation in our institution. Candidemia is associated with poor outcomes. Strategies optimising antifungal therapy, especially in the critically-ill population, should be explored.