Ceftolozane-tazobactam and ceftazidime-avibactam activity against ß-lactam-resistant Pseudomonas aeruginosa and extended-spectrum ß-lactamase-producing Enterobacterales clinical isolates from U.S. medical centres.

BACKGROUND: Despite availability of ceftolozane-tazobactam (C/T) and ceftazidime-avibactam (CZA) for several years, the individual spectrum of activity of each agent may not be widely known. We compared the activity of C/T and CZA against convenience samples of 119 extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales and 60 ß-lactam-resistant Pseudomonas aeruginosa clinical isolates collected from three U.S. institutions. METHODS: Minimal inhibitory concentrations (MICs) for C/T and CZA were determined by broth microdilution. Molecular identification of nine ß-lactamase gene targets was conducted for Enterobacterales and P. aeruginosa isolates with increased MICs to C/T or CZA. RESULTS: More than 90% of Enterobacterales isolates demonstrated susceptibility to both C/T and CZA, in contrast to the other traditional ß-lactam agents tested, which were much less active. The MIC50/90 values were nearly equivalent between agents. The most common ß-lactamase genes identified in Enterobacterales isolates with MIC values ≥2 mg/L were the CTX-M-1 group (85%) and CMY-2-like (23%) ß-lactamases. Both agents were active against >80% of ß-lactam-resistant P. aeruginosa isolates tested, most of which had oprD mutations identified. One P. aeruginosa isolate was positive for a Klebsiella pneumoniae carbapenemase-type gene but remained meropenem-susceptible. The MIC50 values were four-fold lower in favour of C/T (1 mg/L vs. 4 mg/L) against P. aeruginosa. CONCLUSIONS: Our data suggest that either agent may be a reasonable choice for centres with a high proportion of ESBL producers; however, C/T may have improved activity against P. aeruginosa and may be preferred in institutions with a higher frequency of resistant pseudomonal isolates.

Detection of high-risk carbapenem-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates using volatile molecular profiles.

Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are alarming in the clinical setting, as CRE isolates often exhibit resistance to most clinically-available antibiotics. Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase carried by CRE in North America and Europe, frequently detected in isolates of K. pneumoniae, Escherichia coli, and Enterobacter cloacae. Notably, KPC-expressing strains often arise from clonal lineages, with sequence type 258 (ST258) representing the dominant lineage in K. pneumoniae, ST131 in E. coli, and ST78 and ST171 in E. cloacae. Prior studies have demonstrated that carbapenem-resistant K. pneumoniae differs from carbapenem-susceptible K. pneumoniae at both the transcriptomic and soluble metabolomic levels. In the present study, we sought to determine whether carbapenem-resistant and carbapenem-susceptible isolates of K. pneumoniae, E. coli, and E. cloacae produce distinct volatile metabolic profiles. We were able to identify a volatile metabolic fingerprint that could discriminate between CRE and non-CRE with an area under the receiver operating characteristic curve (AUROC) as high as 0.912. Species-specific AUROCs were as high as 0.988 for K. pneumoniae and 1.000 for E. cloacae. Paradoxically, curing of KPC-expressing plasmids from a subset of K. pneumoniae isolates further accentuated the metabolic differences observed between ST258 and non-ST258.

The two transmembrane regions of Candida albicans Dfi1 contribute to its biogenesis.

The opportunistic pathogen Candida albicans forms invasive filaments that grow into host tissues during disease. The glycosylated, integral plasma membrane protein Dfi1 is important for invasive filamentation in a laboratory model, and for lethality in murine disseminated candidiasis. However, Dfi1 topology and essential domains for Dfi1 biogenesis were undefined. Sequence analysis predicted that Dfi1 contains two transmembrane regions, located near the N- and C-termini. In this communication, we show that Dfi1 remains an integral membrane protein despite deletion of either predicted transmembrane region, whereas deletion of both regions results in a soluble protein. Additionally, Dfi1 that was properly oriented in the membrane, as indicated by N-linked glycosylation, was observed when either transmembrane region was deleted, but was absent when both transmembrane regions were deleted. Interestingly, deletion of the N-terminal transmembrane region resulted in production of two forms of Dfi1. Most of the protein molecules acquired normal N-linked glycosylation and a smaller population failed to become normally N-linked glycosylated. This defect was reversed by replacement of the N-terminal hydrophobic sequence with one synthetic transmembrane sequence but not another. Finally, microscopy studies revealed that Dfi1 lacking the N-terminal transmembrane region was observed at the cell periphery, where full-length Dfi1 normally localizes, whereas the double-truncation mutant was diffusely intracellular. Therefore, mature Dfi1 protein contains two transmembrane domains which contribute to its biogenesis.

Activity of minocycline against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae clinical isolates, with comparison to doxycycline and tigecycline.

We examined the in vitro activity of minocycline against 103 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae isolates and found approximately half had susceptible (26%) or intermediate (26%) MICs. For a subset of 35 isolates, susceptibility was highest to tigecycline (71% FDA vs. 20% EUCAST) followed by minocycline (14%) and then doxycycline (6%).

The Activity of Antimicrobial Surfaces Varies by Testing Protocol Utilized.

BACKGROUND: Contaminated hospital surfaces are an important source of nosocomial infections. A major obstacle in marketing antimicrobial surfaces is a lack of efficacy data based on standardized testing protocols. AIM: We compared the efficacy of multiple testing protocols against several "antimicrobial" film surfaces. METHODS: Four clinical isolates were used: one Escherichia coli, one Klebsiella pneumoniae, and two Staphylococcus aureus strains. Two industry methods (modified ISO 22196 and ASTM E2149), a "dried droplet", and a "transfer" method were tested against two commercially available antimicrobial films, one film in development, an untreated control, and a positive (silver) control film. At 2 (only ISO) and 24 hours following inoculation, bacteria were collected from film surfaces and enumerated. RESULTS: Compared to untreated films in all protocols, there were no significant differences in recovery on either commercial brand at 2 or 24 hours after inoculation. The silver surface demonstrated significant microbicidal activity (mean loss 4.9 Log10 CFU/ml) in all methods and time points with the exception of 2 hours in the ISO protocol and the transfer method. Using our novel droplet method, no differences between placebo and active surfaces were detected. The surface in development demonstrated variable activity depending on method, organism, and time point. The ISO demonstrated minimal activity at 2 hours but significant activity at 24 hours (mean 4.5 Log10 CFU/ml difference versus placebo). The ASTEM protocol exhibited significant differences in recovery of staphylococci (mean 5 Log10 CFU/ml) but not Gram-negative isolates (10 fold decrease). Minimal activity was observed with this film in the transfer method. CONCLUSIONS: Varying results between protocols suggested that efficacy of antimicrobial surfaces cannot be easily and reproducibly compared. Clinical use should be considered and further development of representative methods is needed.

Susceptibility of Multidrug-Resistant Gram-Negative Urine Isolates to Oral Antibiotics.

Increasing resistance among Gram-negative uropathogens limits treatment options, and susceptibility data for multidrug-resistant isolates are limited. We assessed the activity of five oral agents against 91 multidrug-resistant Gram-negative urine isolates that were collected from emergency department/hospitalized patients. Fosfomycin and nitrofurantoin were most active (>75% susceptibility). Susceptibilities to sulfamethoxazole-trimethoprim, ciprofloxacin, and ampicillin were ≤40%; empirical use of these agents likely provides inadequate coverage in areas with a high prevalence of multidrug-resistant uropathogens.

Activity of fosfomycin and comparison of several susceptibility testing methods against contemporary urine isolates.

Fosfomycin is recommended as first-line treatment for acute uncomplicated cystitis in women. It has demonstrated in vitro activity against a variety of pathogens; however, a paucity of data are available from the USA. We determined the susceptibility of a collection of urine isolates to fosfomycin and compared multiple methods of susceptibility testing. Consecutive non-duplicate Enterobacteriaceae, enterococci and Pseudomonas aeruginosa isolates were collected from the clinical microbiology laboratory between August 2013 and January 2014. Isolates represented hospitalised or emergency department patients with monomicrobial bacteriuria. Fosfomycin MICs were determined in duplicate, on separate days, by Etest and disk diffusion and results were compared with agar dilution. Nitrofurantoin and ciprofloxacin were used as comparators. MIC results were categorised using Clinical and Laboratory Standards Institute interpretive criteria for Escherichia coli and Enterococcus faecalis. Correlation between the three testing methods was evaluated. Overall susceptibility to fosfomycin was 94.4%, 93.5% and 87.9% by agar dilution, disk diffusion and Etest, respectively. Five fosfomycin-resistant isolates were identified, including two Morganella morganii, one P. aeruginosa, one Proteus mirabilis and one Enterobacter aerogenes. Across all organisms, rates of essential agreement, categorical agreement, minor errors, major errors and very major errors for Etest/disk diffusion compared with agar dilution were 77.3%/NA, 89.5/93.8%, 7.1/5.0%, 3.6/1.3% and 0/0%, respectively. Fosfomycin displayed fairly consistent activity against a majority of isolates collected when using the susceptibility breakpoint of 64 µg/mL. MICs for E. coli were particularly low (≤2 µg/mL). These data lend support to current guidelines that recommend fosfomycin as empirical first-line therapy for uncomplicated UTI.

An evaluation of multiple phenotypic screening methods for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae may display MICs to carbapenems within susceptible or intermediate ranges, prompting confirmatory testing. Four phenotypic methods to detect KPC producers were evaluated against a collection of clinical Enterobacteriaceae isolates. Meropenem-phenylboronic acid double disk synergy testing demonstrated the best performance with 100% sensitivity and specificity.

Calmodulin binding to Dfi1p promotes invasiveness of Candida albicans.

Candida albicans, a dimorphic fungus, undergoes hyphal development in response to many different environmental cues, including growth in contact with a semi-solid matrix. C. albicans forms hyphae that invade agar when cells are embedded in or grown on the surface of agar, and the integral membrane protein Dfi1p is required for this activity. In addition, Dfi1p is required for full activation of mitogen activated protein kinase Cek1p during growth on agar. In this study, we identified a putative calmodulin binding motif in the C-terminal tail of Dfi1p. This region of Dfi1p bound to calmodulin in vitro, and mutations that affected this region affected both calmodulin binding in vitro and invasive filamentation when incorporated into the full length Dfi1p protein. Moreover, increasing intracellular calcium levels led to calcium-dependent, Dfi1p-dependent Cek1p activation. We propose that conformational changes in Dfi1p in response to environmental conditions encountered during growth allow the protein to bind calmodulin and initiate a signaling cascade that activates Cek1p.

Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis.

The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-D-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis.

A Candida albicans cell wall-linked protein promotes invasive filamentation into semi-solid medium.

Growth of cells in contact with an abiotic or biological surface profoundly affects cellular physiology. In the opportunistic human pathogen, Candida albicans, growth on a semi-solid matrix such as agar results in invasive filamentation, a process in which cells change their morphology to highly elongated filamentous hyphae that grow into the matrix. We hypothesized that a plasma membrane receptor-type protein would sense the presence of matrix and activate a signal transduction cascade, thus promoting invasive filamentation. In this communication, we demonstrate that during growth in contact with a semi-solid surface, activation of a MAP kinase, Cek1p, is promoted, in part, by a plasma membrane protein termed Dfi1p and results in invasive filamentation. A C. albicans mutant lacking Dfi1p showed reduced virulence in a murine model of disseminated candidiasis. Dfi1p is a relatively small, integral membrane protein that localizes to the plasma membrane. Some Dfi1p molecules become cross-linked to the carbohydrate polymers of the cell wall. Thus, Dfi1p is capable of linking the cell wall to the plasma membrane and cytoplasm.