Whole genome sequencing analysis demonstrates therapy-induced echinocandin resistance in Candida auris isolates.

Candida auris is an emerging, multidrug-resistant yeast, causing outbreaks in healthcare facilities. Echinocandins are the antifungal drugs of choice to treat candidiasis, as they cause few side effects and resistance is rarely found. Previously, immunocompromised patients from Kuwait with C. auris colonisation or infection were treated with echinocandins, and within days to months, resistance was reported in urine isolates. To determine whether the development of echinocandin resistance was due to independent introductions of resistant strains or resulted from intra-patient resistance development, whole genome sequencing (WGS) single-nucleotide polymorphism (SNP) analysis was performed on susceptible (n = 26) and echinocandin-resistant (n = 6) isolates from seven patients. WGS SNP analysis identified three distinct clusters differing 17-127 SNPs from two patients, and the remaining isolates from five patients, respectively. Sequential isolates within patients had a maximum of 11 SNP differences over a time period of 1-10 months. The majority of isolates with reduced susceptibility displayed unique FKS1 substitutions including a novel FKS1M690V substitution, and nearly all were genetically related, ranging from only three to six SNP differences compared to susceptible isolates from the same patient. Resistant isolates from three patients shared the common FKS1S639F substitution; however, WGS analysis did not suggest a common source. These findings strongly indicate that echinocandin resistance is induced during antifungal treatment. Future studies should determine whether such echinocandin-resistant strains are capable of long-term colonisation, cause subsequent breakthrough candidiasis, have a propensity to cross-infect other patients, or remain viable for longer time periods in the hospital environment.

In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris.

OBJECTIVE: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance. METHODS: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling. RESULTS: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation. CONCLUSIONS: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat.

Invasive Candida auris infections in Kuwait hospitals: epidemiology, antifungal treatment and outcome.

PURPOSE: Candida auris is a recently recognized yeast pathogen, which has attracted worldwide attention due to its multidrug-resistant nature and associated high mortality rates. Its persistence in hospital environment and propensity of nosocomial transmission underscores the need of continuous monitoring to prevent outbreaks. Since the first case of C. auris candidemia in May, 2014, we have identified 17 additional invasive cases, which are described here. METHODS: Identity of 17 isolates originating from proven or possible cases of invasive C. auris infection and identified as Candida haemulonii by Vitek 2 yeast identification system was confirmed by PCR-sequencing of rDNA. Information about risk factors, treatment and outcomes were retrospectively retrieved from case files. Antifungal susceptibility testing was performed by Etest. RESULTS: Thirteen cases of candidemia and 4 cases of other invasive infections were detected in 6 hospitals across Kuwait. Major risk factors included adult patients with cancer, diabetes, gastrointestinal/liver diseases and extended (> 25 days) hospital stay. All isolates were resistant to fluconazole. Additionally, 5 and 4 isolates were also resistant to voriconazole and amphotericin B, respectively. Despite antifungal treatment, 9 of 15 patients died. Most patients (n = 12) were hospitalized in 2 hospitals that are in close proximity, whereas 5 other patients were from 3 hospitals that are situated > 10 km apart. CONCLUSIONS: Occurrence of successive cases of invasive C. auris infections with resulting mortality in nine patients suggests persistence of this multidrug-resistant yeast in major hospitals in Kuwait. Early detection by continuous surveillance and enforcement of infection control measures are recommended.

Development of Echinocandin Resistance in Candida tropicalis following Short-Term Exposure to Caspofungin for Empiric Therapy.

Isolation of two echinocandin-resistant Candida tropicalis strains from endotracheal secretions of a patient following short-term exposure to caspofungin is described. Both strains exhibited resistance to echinocandins by Etest and reference broth microdilution, showing a homozygous S645P mutation within the hot spot 1 (HS-1) region of FKS1 and belonging to a unique multilocus sequence type. Other C. tropicalis isolates collected from patients in the same intensive care unit within a 60-day period were susceptible to echinocandins and contained wild-type FKS1 sequences.

Implications of Streptococcus pneumoniae penicillin resistance and serotype distribution in Kuwait for disease treatment and prevention.

Streptococcus pneumoniae causes serious infections. Treatment is difficult because of the emergence of penicillin resistance in S. pneumoniae. Pneumococcal vaccines offer the promise of control and prevention of pneumococcal infections. Serotype prevalence and penicillin susceptibility data for a country will predict the usefulness of the vaccines in that country. In Kuwait, the 23-valent polysaccharide and the 7-valent conjugate vaccines are being used without knowledge of the prevalent serotypes in the country. To obtain the necessary background information, data on penicillin susceptibility and serogroups were obtained from 397 consecutive clinical isolates collected during 2004 and 2005. Two hundred fifty-three isolates (64%) were penicillin resistant, and resistance was significantly higher in patients < or =15 years old and among the upper respiratory tract and eye isolates. The most common serotypes were 23F, 19F, 6A, 6B, 14, and 19A. Among the penicillin-resistant strains, the most common serotypes were 23F, 19F, 6B, 14, and 9A. Among the invasive strains, the most common serotypes were 14, 23F, 19A, and 9V. The polysaccharide vaccine gave 82% coverage against invasive infections in all age groups >2 years. The coverage of the 7-valent conjugate vaccine against invasive serotypes in children < or =2 years old was 55%. This moderate coverage by the conjugate vaccine against invasive infections in children necessitates a revised strategy on the use of the present conjugate vaccine and shows the need for formulation of an improved vaccine for superior coverage for Kuwait and possibly other countries of the Arabian Gulf.

Increasing prevalence of antimicrobial resistance in Streptococcus pneumoniae in Kuwait: implications for therapy.

From 2001 to 2004, 824 consecutive Streptococcus pneumoniae isolates obtained from all the teaching hospitals and primary-care centers serving all of Kuwait were studied for their susceptibility to a number of antibiotics. Of these, 514 (63%) were resistant to penicillin, 55% of which were of intermediate resistance, with a minimum inhibitory concentration (MIC) of 0.16-1 microg/ml, and 8% were of full resistance (MIC > or =2 microg/ml). The prevalence of penicillin resistance was significantly higher among the eye isolates than the rest of the isolates combined. Resistance across all classes of antibiotics was more common with the isolates from blood and cerebrospinal fluid (CSF). Decreased susceptibility to ceftriaxone, cefotaxime, imipenem, meropenem, erythromycin, and cotrimoxazole was observed in 15%, 12.8%, 10.2%, 8.6%, 42%, and 69% of isolates, respectively. Multidrug-resistant S. pneumoniae was isolated mostly from invasive diseases. There has been a remarkable increase in the prevalence of drug-resistant S. pneumoniae isolates in Kuwait over the past 20 years, and Kuwait has thus joined the league of hyperendemic countries for penicillin-resistant S. pneumoniae (PRSP). It is also conceivable that Kuwait, with a large expatriate population of workers, may serve as a focal point for further dissemination of resistant clones to the rest of the world. These data are unique because they are representative of the whole Kuwaiti population.