Development of Daptomycin Susceptibility Breakpoints for Enterococcus faecium and Revision of the Breakpoints for Other Enterococcal Species by the Clinical and Laboratory Standards Institute.

Daptomycin is one of the few treatment options for infections caused by enterococci that are resistant to ampicillin and vancomycin, such as vancomycin-resistant Enterococcus faecium. The emergence and clinical significance of daptomycin-resistant enterococci and evolving microbiologic, pharmacokinetic-pharmacodynamic, and clinical data indicated that the pre-2019 Clinical and Laboratory Standards Institute (CLSI) susceptible-only breakpoint of ≤4 µg/mL for daptomycin and enterococci was no longer appropriate. After analyzing data that are outlined in this article, the CLSI Subcommittee on Antimicrobial Susceptibility Testing established new breakpoints for daptomycin and enterococci. For E. faecium, a susceptible dose-dependent (SDD) breakpoint of ≤4 µg/mL was established based on an increased dosage of 8-12 mg/kg/day (≥8 µg/mL-resistant). CLSI suggests infectious diseases consultation to guide daptomycin use for the SDD category. For Enterococcus faecalis and other enterococcal species, revised breakpoints of ≤2 µg/mL-susceptible, 4 µg/mL-intermediate, and ≥8 µg/mL-resistant were established based on a standard dosage of 6 mg/kg/day.

Evaluation of Ciprofloxacin and Levofloxacin Disk Diffusion and Etest Using the 2019 Enterobacteriaceae CLSI Breakpoints.

In 2019, the Clinical and Laboratory Standards Institute (CLSI) published revisions to the Enterobacteriaceae ciprofloxacin and levofloxacin breakpoints. We evaluated the performance of disk diffusion and Etest compared to that of reference broth microdilution by use of the revised breakpoints. Fifty-eight Enterobacteriaceae isolates with ciprofloxacin MICs of 0.5 µg/ml or 1.0 µg/ml on initial testing were specifically selected for evaluation. These MICs are susceptible by the 2018 breakpoints and not susceptible by the 2019 breakpoints. For ciprofloxacin disk diffusion, the categorical agreement (CA) was 46.6%, with 0 very major errors (VME), 4 major errors (ME) (21.1%), and 27 minor errors (mE) (46.6%) using the 2019 CLSI disk breakpoints. For levofloxacin, the CA was 72.4%, with 0 VME, 0 ME, and 16 mE (27.6%) using the 2019 CLSI disk breakpoints. Using an error rate-bound evaluation method, levofloxacin but not ciprofloxacin disk diffusion yielded an acceptable minor error rate of <40% for isolates with an MIC plus or minus 1 doubling dilution of the intermediate breakpoint. For Etest compared to the reference broth microdilution, the essential agreement was 100% for both ciprofloxacin and levofloxacin and the CA was 81.0% and 65.5%, respectively. No VME or ME were observed by Etest, and 11 minor errors for ciprofloxacin (19.0%) and 20 (34.5%) for levofloxacin were observed. By the error rate-bound method, the minor error rate for ciprofloxacin was acceptable, but minor error rates for levofloxacin remained outside the acceptance range (i.e., 42.6% for isolates with an MIC within 1 dilution of the breakpoint). In general, the disk diffusion and Etest methods performed well with this challenging collection of isolates, although laboratories must be aware of minor errors, particularly for isolates with results near the breakpoint.

Variability of Daptomycin MIC Values for Enterococcus faecium When Measured by Reference Broth Microdilution and Gradient Diffusion Tests.

Daptomycin has become a mainstay therapy for the treatment of serious vancomycin-resistant Enterococcus faecium infections. However, concern exists that current testing methods do not accurately predict the clinical success of daptomycin therapy. We evaluated a collection of 40 isolates of E. faecium across three centers by reference broth microdilution (BMD), and two gradient strips, to determine the precision of daptomycin MICs by these methods and the correlation of daptomycin MIC testing with mutations in the liaFSR system, one of the primary daptomycin resistance mechanisms among the enterococci. Daptomycin MICs spanned 3-log2 dilutions by BMD for 60.0% of isolates, 17.5% spanned 4 dilutions, 2.5% spanned 5 dilutions, and 20.0% spanned 6 or more dilutions. Fifteen isolates had MICs interpreted as susceptible by some tests and nonsusceptible by others. Neither BMD nor gradient diffusion tests could reliably differentiate isolates with or without mutations in liaFSR, resulting in a 59.8% very major error rate compared to determination of genotype by BMD, 63.5% by Etest, and 68.5% by MIC test strip. Imprecision in daptomycin MIC determination for E. faecium make establishment of a revised breakpoint challenging. Clinicians should be aware of this testing variability when making treatment decisions for patients with serious infections caused by this organism.

Alp, an arthropod-associated outer membrane protein of Borrelia species that cause relapsing fever.

Borrelia hermsii and other relapsing fever (RF) species are noted for their highly polymorphic surface antigens, the variable major proteins (VMP). Less is known about other surface proteins of these pathogens in either their vertebrate reservoirs or arthropod vectors. To further characterize these proteins, we elicited antibodies against VMP-less cells, noted antibody reactions against whole cells and cell components, and then subjected selected antigens to mass spectroscopy for amino acid sequencing for comparison against a B. hermsii genome database. One of the derived monoclonal antibodies, H0120, agglutinated spirochetes, and in Western blot analyses, it bound to a 14-kDa protein of whole cells and their membrane fractions but not after protease treatment. A search of open reading frames of the B. hermsii genome with extracted peptides identified the 14-kDa protein with bha128, a 453-nucleotide gene of the 175-kb linear plasmid. The bha128 gene was synthesized and expressed in Escherichia coli. The protein product was bound by antibody H0120. Genes homologous to bha128 occur in the RF species Borrelia turicatae, B. duttonii, and B. recurrentis but not in Lyme disease Borrelia species or other organisms. The following findings indicated an association of BHA128, renamed Alp, with the tick environment: (i) Alp was produced at higher levels at 23°C than at 34 °C; (ii) almost all spirochetes in tick salivary glands were bound by the H0120 antibody, but only ~1% of spirochetes in the blood of infected mice were bound; and (iii) infected mice produced antibodies to several B. hermsii antigens but not detectably to native or recombinant Alp.

Tfap2 transcription factors in zebrafish neural crest development and ectodermal evolution.

Transcription factor AP2 (Tfap2) genes play essential roles in development of the epidermis and migratory cells of the neural crest (NC) in vertebrate embryos. These transcriptional activators are among the earliest genes expressed in the ectoderm and specify fates within the epidermis/crest through both direct and indirect mechanisms. The Tfap2 family arose from a single ancestral gene in a chordate ancestor that underwent gene duplication to give up to five family members in living vertebrates. This coincided with the acquisition of important roles in NC development by Tfap2 genes suggesting that this gene family was important in ectodermal evolution and possibly in the origin of NC. Here, we show that a zebrafish tfap2c is expressed in the nonneural ectoderm during early development and functions redundantly with tfap2a in NC specification. In zebrafish embryos depleted of both tfap2a and tfap2c, NC cells are virtually eliminated. Cell transplantation experiments indicate that tfap2c functions cell-autonomously in NC specification. Cells of the enveloping layer, which forms a temporary skin layer surrounding the ectoderm, also fail to differentiate or to express appropriate keratins in tfap2c deficient embryos. The role of Tfap2 genes in epidermal and NC development is considered here in the broader context of ectodermal evolution. Distinct, tissue-specific functions for Tfap2 genes in different vertebrates may reflect subfunctionalisation of an ancestral gene that consequently led to the gain of novel roles for different subfamily members in patterning the epidermis and NC.