Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures.

The longstanding model is that most bloodstream infections (BSIs) are caused by a single organism. We perform whole genome sequencing of five-to-ten strains from blood culture (BC) bottles in each of ten patients with Candida glabrata BSI. We demonstrate that BCs contain mixed populations of clonal but genetically diverse strains. Genetically distinct strains from two patients exhibit phenotypes that are potentially important during BSIs, including differences in susceptibility to antifungal agents and phagocytosis. In both patients, the clinical microbiology lab recovered a fluconazole-susceptible index strain, but we identify mixed fluconazole-susceptible and -resistant populations. Diversity in drug susceptibility is likely clinically relevant, as fluconazole-resistant strains were subsequently recovered by the clinical laboratory during persistent or relapsing infections. In one patient, unrecognized respiration-deficient small colony variants are fluconazole-resistant and significantly attenuated for virulence during murine candidiasis. Our data suggest a population-based model of C. glabrata genotypic and phenotypic diversity during BSIs.

Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures.

The longstanding paradigm is that most bloodstream infections (BSIs) are caused by a single organism. We performed whole genome sequencing of five-to-ten strains from blood culture (BC) bottles in each of ten patients with Candida glabrata BSI. We demonstrated that BCs contained mixed populations of clonal but genetically diverse strains. Genetically distinct strains from two patients exhibited phenotypes that were potentially important during BSIs, including differences in susceptibility to antifungal agents and phagocytosis. In both patients, the clinical microbiology lab recovered a fluconazole-susceptible index strain, but we identified mixed fluconazole-susceptible and â€"resistant populations. Diversity in drug susceptibility was likely clinically relevant, as fluconazole-resistant strains were subsequently recovered by the clinical laboratory during persistent or relapsing infections. In one patient, unrecognized respiration-deficient small colony variants were fluconazole-resistant and significantly attenuated for virulence during murine candidiasis. Our data suggest a new population-based paradigm of C. glabrata genotypic and phenotypic diversity during BSIs.

Within-Host Genotypic and Phenotypic Diversity of Contemporaneous Carbapenem-Resistant Klebsiella pneumoniae from Blood Cultures of Patients with Bacteremia.

It is unknown whether bacterial bloodstream infections (BSIs) are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole-genome sequences of 10 sequence type 258 (ST258) CRKP strains from blood cultures in each of 6 patients (Illumina HiSeq). Strains clustered by patient by core genome and pan-genome phylogeny. In 5 patients, there was within-host strain diversity by gene mutations, presence/absence of antibiotic resistance or virulence genes, and/or plasmid content. Accessory gene phylogeny revealed strain diversity in all 6 patients. Strains from 3 patients underwent long-read sequencing for genome completion (Oxford Nanopore) and phenotypic testing. Genetically distinct strains within individuals exhibited significant differences in carbapenem and other antibiotic responses, capsular polysaccharide (CPS) production, mucoviscosity, and/or serum killing. In 2 patients, strains differed significantly in virulence during mouse BSIs. Genetic or phenotypic diversity was not observed among strains recovered from blood culture bottles seeded with index strains from the 3 patients and incubated in vitro at 37°C. In conclusion, we identified genotypic and phenotypic variant ST258 CRKP strains from blood cultures of individual patients with BSIs, which were not detected by the clinical laboratory or in seeded blood cultures. The data suggest a new paradigm of CRKP population diversity during BSIs, at least in some patients. If validated for BSIs caused by other bacteria, within-host microbial diversity may have implications for medical, microbiology, and infection prevention practices and for understanding antibiotic resistance and pathogenesis. IMPORTANCE The long-standing paradigm for pathogenesis of bacteremia is that, in most cases, a single organism passes through a bottleneck and establishes itself in the bloodstream (single-organism hypothesis). In keeping with this paradigm, standard practice in processing positive microbiologic cultures is to test single bacterial strains from morphologically distinct colonies. This study is the first genome-wide analysis of within-host diversity of Klebsiella pneumoniae strains recovered from individual patients with bloodstream infections (BSIs). Our finding that positive blood cultures comprised genetically and phenotypically heterogeneous carbapenem-resistant K. pneumoniae strains challenges the single-organism hypothesis and suggests that at least some BSIs are caused by mixed bacterial populations that are unrecognized by the clinical laboratory. The data support a model of pathogenesis in which pressures in vivo select for strain variants with particular antibiotic resistance or virulence attributes and raise questions about laboratory protocols and treatment decisions directed against single strains.

Different Conformations Revealed by NMR Underlie Resistance to Ceftazidime/Avibactam and Susceptibility to Meropenem and Imipenem among D179Y Variants of KPC ß-Lactamase.

ß-Lactamase-mediated resistance to ceftazidime-avibactam (CZA) is a serious limitation in the treatment of Gram-negative bacteria harboring Klebsiella pneumoniae carbapenemase (KPC). Herein, the basis of susceptibility to carbapenems and resistance to ceftazidime (CAZ) and CZA of the D179Y variant of KPC-2 and -3 was explored. First, we determined that resistance to CZA in a laboratory strain of Escherichia coli DH10B was not due to increased expression levels of the variant enzymes, as demonstrated by reverse transcription PCR (RT-PCR). Using timed mass spectrometry, the D179Y variant formed prolonged acyl-enzyme complexes with imipenem (IMI) and meropenem (MEM) in KPC-2 and KPC-3, which could be detected up to 24 h, suggesting that IMI and MEM act as covalent ß-lactamase inhibitors more than as substrates for D179Y KPC-2 and -3. This prolonged acyl-enzyme complex of IMI and MEM by D179Y variants was not observed with wild-type (WT) KPCs. CAZ was studied and the D179Y variants also formed acyl-enzyme complexes (1 to 2 h). Thermal denaturation and differential scanning fluorimetry showed that the tyrosine substitution at position 179 destabilized the KPC ß-lactamases (KPC-2/3 melting temperature [Tm] of 54 to 55°C versus D179Y Tm of 47.5 to 51°C), and the D179Y protein was 3% disordered compared to KPC-2 at 318 K. Heteronuclear 1H/15N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy also revealed that the D179Y variant, compared to KPC-2, is partially disordered. Based upon these observations, we discuss the impact of disordering of the Ω loop as a consequence of the D179Y substitution. These conformational changes and disorder in the overall structure as a result of D179Y contribute to this unanticipated phenotype.

Molecular Epidemiology, Natural History, and Long-Term Outcomes of Multidrug-Resistant Enterobacterales Colonization and Infections Among Solid Organ Transplant Recipients.

BACKGROUND: Multidrug-resistant Enterobacterales (MDR-E), including carbapenem-resistant and third-generation cephalosporin-resistant Enterobacterales (CRE, CefR-E), are major pathogens following solid organ transplantation (SOT). METHODS: We prospectively studied patients who underwent lung, liver, and small bowel transplant from February 2015 through March 2017. Weekly perirectal swabs (up to 100 days post-transplant) were cultured for MDR-E. Whole-genome sequencing (WGS) was performed on gastrointestinal (GI) tract-colonizing and disease-causing isolates. RESULTS: Twenty-five percent (40 of 162) of patients were MDR-E GI-colonized. Klebsiella pneumoniae was the most common CRE and CefR-E. Klebsiella pneumoniae carbapenemases and CTX-M were leading causes of CR and CefR, respectively. Thirty-five percent of GI colonizers developed MDR-E infection vs 2% of noncolonizers (P < .0001). The attack rate was higher among CRE colonizers than CefR-E colonizers (53% vs 21%, P = .049). GI colonization and high body mass index were independent risk factors for MDR-E infection (P ≤ .004). Thirty-day mortality among infected patients was 6%. However, 44% of survivors developed recurrent infections; 43% of recurrences were late (285 days to 3.9 years after the initial infection). Long-term survival (median, 4.3 years post-transplant) did not differ significantly between MDR-E-infected and MDR-E-noninfected patients (71% vs 77%, P = .56). WGS phylogenetic analyses revealed that infections were caused by GI-colonizing strains and suggested unrecognized transmission of novel clonal group-258 sublineage CR-K. pneumoniae and horizontal transfer of resistance genes. CONCLUSIONS: MDR-E GI colonization was common following SOT and predisposed patients to infections by colonizing strains. MDR-E infections were associated with low short- and long-term mortality, but recurrences were frequent and often occurred years after initial infections. Findings provide support for MDR-E surveillance in our SOT program.

Remediation of Mucorales-contaminated Healthcare Linens at a Laundry Facility Following an Investigation of a Case Cluster of Hospital-acquired Mucormycosis.

BACKGROUND: In an investigation of hospital-acquired mucormycosis cases among transplant recipients, healthcare linens (HCLs) delivered to our center were found to be contaminated with Mucorales. We describe an investigation and remediation of Mucorales contamination at the laundry supplying our center. METHODS: We performed monthly RODAC cultures of HCLs upon hospital arrival, and conducted site inspections and surveillance cultures at the laundry facility. Remediation was designed and implemented by infection prevention and facility leadership teams. RESULTS: Prior to remediation, 20% of HCLs were culture-positive for Mucorales upon hospital arrival. Laundry facility layout and processes were consistent with industry standards. Significant step-ups in Mucorales and mold culture-positivity of HCLs were detected at the post-dryer step (0% to 12% [P = .04] and 5% to 29% [P = .01], respectively). Further increases to 17% and 40% culture-positivity, respectively, were noted during pre-transport holding. Site inspection revealed heavy Mucorales-positive lint accumulation in rooftop air intake and exhaust vents that cooled driers; intake and exhaust vents that were facing each other; rooftop and plant-wide lint accumulation, including in the pre-transport clean room; uncovered carts with freshly-laundered HCLs. Following environmental remediation, quality assurance measures and education directed toward these sources, Mucorales culture-positivity of newly-delivered HCLs was reduced to 0.3% (P = .0001); area of lint-contaminated rooftop decreased from 918 m2 to 0 m2 on satellite images. CONCLUSIONS: Targeted laundry facility interventions guided by site inspections and step-wise culturing significantly reduced Mucorales-contaminated HCLs delivered to our hospital. Collaboration between infection prevention and laundry facility teams was crucial to successful remediation.

Evaluation of a high-throughput, cost-effective Illumina library preparation kit.

Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.

Genetic diversity of clinical and environmental Mucorales isolates obtained from an investigation of mucormycosis cases among solid organ transplant recipients.

Mucormycoses are invasive infections by Rhizopus species and other Mucorales. Over 10 months, four solid organ transplant (SOT) recipients at our centre developed mucormycosis due to Rhizopus microsporus (n=2), R. arrhizus (n=1) or Lichtheimia corymbifera (n=1), at a median 31.5 days (range: 13-34) post-admission. We performed whole genome sequencing (WGS) on 72 Mucorales isolates (45 R. arrhizus, 19 R. delemar, six R. microsporus, two Lichtheimia species) from these patients, from five patients with community-acquired mucormycosis, and from hospital and regional environments. Isolates were compared by core protein phylogeny and global genomic features, including genome size, guanine-cytosine percentages, shared protein families and paralogue expansions. Patient isolates fell into six core phylogenetic lineages (clades). Phylogenetic and genomic similarities of R. microsporus isolates recovered 7 months apart from two SOT recipients in adjoining hospitals suggested a potential common source exposure. However, isolates from other patients and environmental sites had unique genomes. Many isolates that were indistinguishable by core phylogeny were distinct by one or more global genomic comparisons. Certain clades were recovered throughout the study period, whereas others were found at particular time points. In conclusion, mucormycosis cases could not be genetically linked to a definitive environmental source. Comprehensive genomic analyses eliminated false associations between Mucorales isolates that would have been assigned using core phylogenetic or less extensive genomic comparisons. The genomic diversity of Mucorales mandates that multiple isolates from individual patients and environmental sites undergo WGS during epidemiological investigations. However, exhaustive surveillance of fungal populations in a hospital and surrounding community is probably infeasible.

Ceftolozane-Tazobactam for the Treatment of Multidrug-Resistant Pseudomonas aeruginosa Infections: Clinical Effectiveness and Evolution of Resistance.

Background: Data on the use of ceftolozane-tazobactam and emergence of ceftolozane-tazobactam resistance during multidrug resistant (MDR)-Pseudomonas aeruginosa infections are limited. Methods: We performed a retrospective study of 21 patients treated with ceftolozane-tazobactam for MDR-P. aeruginosa infections. Whole genome sequencing and quantitative real-time polymerase chain reaction were performed on longitudinal isolates. Results: Median age was 58 years; 9 patients (43%) were transplant recipients. Median simplified acute physiology score-II (SAPS-II) was 26. Eighteen (86%) patients were treated for respiratory tract infections; others were treated for bloodstream, complicated intraabdominal infections, or complicated urinary tract infections. Ceftolozane-tazobactam was discontinued in 1 patient (rash). Thirty-day all-cause and attributable mortality rates were 10% (2/21) and 5% (1/21), respectively; corresponding 90-day mortality rates were 48% (10/21) and 19% (4/21). The ceftolozane-tazobactam failure rate was 29% (6/21). SAPS-II score was the sole predictor of failure. Ceftolozane-tazobactam resistance emerged in 3 (14%) patients. Resistance was associated with de novo mutations, rather than acquisition of resistant nosocomial isolates. ampC overexpression and mutations were identified as potential resistance determinants. Conclusions: In this small study, ceftolozane-tazobactam was successful in treating 71% of patients with MDR-P. aeruginosa infections, most of whom had pneumonia. The emergence of ceftolozane-tazobactam resistance in 3 patients is worrisome and may be mediated in part by AmpC-related mechanisms. More research on treatment responses and resistance during various types of MDR-P. aeruginosa infections is needed to define ceftolozane-tazobactam's place in the armamentarium.

Mutations in blaKPC-3 That Confer Ceftazidime-Avibactam Resistance Encode Novel KPC-3 Variants That Function as Extended-Spectrum ß-Lactamases.

We identified four blaKPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant blaKPC-3 encoded enzymes that functioned as extended-spectrum ß-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other ß-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

Emergence of Ceftazidime-Avibactam Resistance Due to Plasmid-Borne blaKPC-3 Mutations during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

Ceftazidime-avibactam is a novel ß-lactam/ß-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne blaKPC-3, which were not present in baseline isolates. blaKPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and blaKPC cloning into competent Escherichia coli In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ≥4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced ≥4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of blaKPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to blaKPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring blaKPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.

Candida albicans Transcriptional Profiling Within Biliary Fluid From a Patient With Cholangitis, Before and After Antifungal Treatment and Surgical Drainage.

We used ribonucleic acid sequencing to profile Candida albicans transcription within biliary fluid from a patient with cholangitis; samples were collected before and after treatment with fluconazole and drainage. Candida albicans transcriptomes at the infection site distinguished treated from untreated cholangitis. After treatment, 1131 C. albicans genes were differentially expressed in biliary fluid. Up-regulated genes were enriched in hyphal growth, cell wall organization, adhesion, oxidation reduction, biofilm, and fatty acid and ergosterol biosynthesis. This is the first study to define Candida global gene expression during deep-seated human infection. Successful treatment of cholangitis induced C. albicans genes involved in fluconazole responses and pathogenesis.

Association between the Presence of Aminoglycoside-Modifying Enzymes and In Vitro Activity of Gentamicin, Tobramycin, Amikacin, and Plazomicin against Klebsiella pneumoniae Carbapenemase- and Extended-Spectrum-ß-Lactamase-Producing Enterobacter Species.

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum ß-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL-, 6 KPC-/ESBL+, and 38 KPC-/ESBL- isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL- (25% and 38%, respectively), KPC-/ESBL+ (50% and 17%, respectively), and KPC-/ESBL- (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and <0.0001, respectively) or multiple AMEs (100% versus 13%, 33%, and 0%, respectively; P < 0.01 for all). KPC+/ESBL+ isolates also had a greater number of AMEs (mean of 4.6 versus 1.5, 0.9, and 0.05, respectively; P < 0.01 for all). GEN and TOB MICs were higher against isolates with >1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 µg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority.

Coordination of Candida albicans Invasion and Infection Functions by Phosphoglycerol Phosphatase Rhr2.

The Candida albicans RHR2 gene, which specifies a glycerol biosynthetic enzyme, is required for biofilm formation in vitro and in vivo. Prior studies indicate that RHR2 is ultimately required for expression of adhesin genes, such as ALS1. In fact, RHR2 is unnecessary for biofilm formation when ALS1 is overexpressed from an RHR2-independent promoter. Here, we describe two additional biological processes that depend upon RHR2: invasion into an abiotic substrate and pathogenicity in an abdominal infection model. We report here that abiotic substrate invasion occurs concomitantly with biofilm formation, and a screen of transcription factor mutants indicates that biofilm and hyphal formation ability correlates with invasion ability. However, analysis presented here of the rhr2Δ/Δ mutant separates biofilm formation and invasion. We found that an rhr2Δ/Δ mutant forms a biofilm upon overexpression of the adhesin gene ALS1 or the transcription factor genes BRG1 or UME6. However, the biofilm-forming strains do not invade the substrate. These results indicate that RHR2 has an adhesin-independent role in substrate invasion, and mathematical modeling argues that RHR2 is required to generate turgor. Previous studies have shown that abdominal infection by C. albicans has two aspects: infection of abdominal organs and persistence in abscesses. We report here that an rhr2Δ/Δ mutant is defective in both of these infection phenotypes. We find here that overexpression of ALS1 in the mutant restores infection of organs, but does not improve persistence in abscesses. Therefore, RHR2 has an adhesin-independent role in abdominal infection, just as it does in substrate invasion. This report suggests that RHR2, through glycerol synthesis, coordinates adherence with host- or substrate-interaction activities that enable proliferation of the C. albicans population.

Mutational analysis of essential septins reveals a role for septin-mediated signaling in filamentation.

Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful analysis in the model yeast Saccharomyces cerevisiae has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic fungus Candida albicans has shown that septins also have vital roles in cell wall regulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins, which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.

Candida glabrata intra-abdominal candidiasis is characterized by persistence within the peritoneal cavity and abscesses.

The pathogenesis of Candida glabrata infections is poorly understood. We studied the pathogenesis of intra-abdominal candidiasis (IAC) in mice that were infected intraperitoneally with C. glabrata and sterile feces. C. glabrata BG2 (5 × 10(8) CFU) caused a 100% mortality rate. Sublethal inocula of BG2 (1 × 10(8) or 1 × 10(7) CFU) caused peritonitis that progressed to abscesses. Three clinical C. glabrata strains (5 × 10(8) CFU) caused 80 to 100% mortality rates, while a fourth (strain 346) caused a 29% mortality rate. Following sublethal inocula (1 × 10(7) CFU), the intra-abscess burdens of virulent strain 356 were ∼1 log greater than those of strain 346. A C. glabrata Δplb1-2 mutant (phospholipase B genes disrupted) killed mice as well as BG2 did. When sublethal inocula were used, however, the Δplb1-2 mutant was associated with more rapid abscess resolution and lower intra-abscess burdens; these findings were reversed by PLB1 and PLB2 reinsertion. The Δplb1-2 mutant was also more susceptible than BG2 to killing by human neutrophils in vitro. BG2 and the Δplb1-2 mutant were indistinguishable during hematogenously disseminated candidiasis. C. albicans SC5314 was more virulent than C. glabrata BG2 during IAC, causing a 100% mortality rate following a challenge with 5 × 10(7) CFU. In contrast, a sublethal inoculum (1 × 10(7) CFU) of BG2 caused less neutrophil infiltration and greater burdens in peritoneal fluid than SC5314 did and abscesses that persisted longer and contained greater burdens. In conclusion, a mouse model of C. glabrata IAC mimics disease in humans and distinguishes the relative virulence of clinical and gene disruption strains. C. glabrata differed from C. albicans during IAC by being less lethal and eliciting dampened neutrophil responses but resulting in more persistent peritonitis and abscesses.

Profiling of Candida albicans gene expression during intra-abdominal candidiasis identifies biologic processes involved in pathogenesis.

BACKGROUND: The pathogenesis of intra-abdominal candidiasis is poorly understood. METHODS: Mice were intraperitoneally infected with Candida albicans (1 × 10(6) colony-forming units) and sterile stool. nanoString assays were used to quantitate messenger RNA for 145 C. albicans genes within the peritoneal cavity at 48 hours. RESULTS: Within 6 hours after infection, mice developed peritonitis, characterized by high yeast burdens, neutrophil influx, and a pH of 7.9 within peritoneal fluid. Organ invasion by hyphae and early abscess formation were evident 6 and 24 hours after infection, respectively; abscesses resolved by day 14. nanoString assays revealed adhesion and responses to alkaline pH, osmolarity, and stress as biologic processes activated in the peritoneal cavity. Disruption of the highly-expressed gene RIM101, which encodes an alkaline-regulated transcription factor, did not impact cellular morphology but reduced both C. albicans burden during early peritonitis and C. albicans persistence within abscesses. RIM101 influenced expression of 49 genes during intra-abdominal candidiasis, including previously unidentified Rim101 targets. Overexpression of the RIM101-dependent gene SAP5, which encodes a secreted protease, restored the ability of a rim101 mutant to persist within abscesses. CONCLUSIONS: A mouse model of intra-abdominal candidiasis is valuable for studying pathogenesis and C. albicans gene expression. RIM101 contributes to persistence within intra-abdominal abscesses, at least in part through activation of SAP5.

Mutations of the ompK36 porin gene and promoter impact responses of sequence type 258, KPC-2-producing Klebsiella pneumoniae strains to doripenem and doripenem-colistin.

Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 µg/ml) and colistin (2 µg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 µg/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS5, and wild-type/other mutants, respectively (P = 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8 µg/ml (P = 0.0007 and 0.09, respectively), but not if the MIC was >8 µg/ml (P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5 mutants (P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin.

Characterization of porin expression in Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae identifies isolates most susceptible to the combination of colistin and carbapenems.

We characterized carbapenem resistance mechanisms among 12 Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (referred to here as KPC K. pneumoniae) clinical isolates and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem, and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to the ST258 clonal group and harbored blaKPC-2, blaSHV-12, and blaTEM-1. As determined by time-kill assays, doripenem (8 µg/ml) and ertapenem (2 µg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 µg/ml) exerted some activity (range, 0.39 to 2.5 log10) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem, and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12), and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other (R(2) = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than those with low expression (100% [8/8] versus 0% [0/4]; P = 0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem (P ≤ 0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC K. pneumoniae, including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC K. pneumoniae isolates may be a practical tool for identifying effective combination regimens.

A competitive infection model of hematogenously disseminated candidiasis in mice redefines the role of Candida albicans IRS4 in pathogenesis.

Candida albicans IRS4 encodes a protein that regulates phosphatidylinositol-(4,5)-bisphosphate, which was shown to contribute to hematogenously disseminated candidiasis (DC) after several days in the standard mouse model. Our objective was to more accurately define the temporal contributions of IRS4 to pathogenesis. During competition assays in vitro, an irs4-null (Δirs4) mutant exhibited wild-type fitness. In DC experiments, mice were infected intravenously with the Δirs4 mutant, strain CAI-12 (1 × 10(5) CFU), or a mixture of the strains (0.5 × 10(5) CFU each). In single-strain infections, quantitative PCR revealed reduced Δirs4 mutant burdens within kidneys at days 1, 4, and 7 but not 6 h. In competitive infections, the Δirs4 mutant was outcompeted by CAI-12 in each mouse at ≥6 h (competitive indices, P ≤ 0.0001). At 4 and 7 days, the Δirs4 mutant burdens during competitive infections were significantly lower than those during single-strain infections (P = 0.01 and P < 0.001, respectively), suggesting increased susceptibility to inflammatory responses. Phagocytic infiltration of kidneys in response to CAI-12 or competitive infections was significantly greater than that in response to Δirs4 mutant infection at days 1 and 4 (P < 0.001), and the Δirs4 mutant was more susceptible to phagocytosis and killing by human polymorphonuclear cells (P = 0.01 and P = 0.006, respectively) and mouse macrophages in vitro (P = 0.04 and P = 0.01, respectively). Therefore, IRS4 contributes to tissue invasion at early stages of DC and mediates resistance to phagocytosis as DC progresses. Microarray analysis revealed remarkably similar gene expression by the Δirs4 mutant and reference strain CAI-12 within blood, suggesting that IRS4 is not significantly involved in the hematogenous stage of disease. A competitive DC model detects attenuated virulence that is not evident with the standard model.