Otolaryngologic Community-Acquired MRSA Infections in a Suburban Private Practice

A retrospective review was conducted of all cases of head and neck community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections seen in the senior author\u27s suburban private practice during a 12-month period. An office and hospital chart review was performed for all patients identified with culture-positive CA-MRSA infections. Several parameters were evaluated, including site of infection, prior therapy, imaging, comorbid disease, culture and sensitivities, surgical and medical management, and long-term results. Of the 10 patients identified, 3 had infections of nasal soft tissues, 3 had infections of the lips, 2 had infections of the paranasal sinus, 1 had an infection of the chin, and 1 had a diffuse, hemifacial infection. All patients were managed successfully with a combination of surgical drainage, wound care, and antibiotics. CA-MRSA is a burgeoning problem in otolaryngology. Appropriate management-including early recognition, appropriate empirical therapy, prompt and thorough surgical drainage with culture and sensitivities, correct antibiotic choice, and meticulous postoperative care-appears to offer excellent results

Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.Dr. Kasthuri Venkateswaran, Dr. Irene Ouoba, Dr. Joseph W. Kloepper, Dr. Cecilie From and Dr. Maria Morea are gratefully acknowledged for providing isolates FO-36bT, SAFN-027, SAFN-037, KL-052, 51-3C and 82-2C; Bs31; SE 49 (AP3) and SE 52 (AP7); FEL 55, UNG22 and MIL46, respectively. Raquel Branquinho was supported by a PhD fellowship (Ref. SFRH/BD/61410/2009) and Clara Sousa by a post-doctoral fellowship (Ref. SFRH/BPD/70548/2010), from FCT (Fundacao para a Ciencia e Tecnologia, Portugal). Hugo Osorio acknowledges the funding from QREN-FEDER through the Operational Program ON. 2 - O Novo Norte. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by the Portuguese Foundation for Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competin

Early events of Bacillus anthracis germination identified by time-course quantitative proteomics

Germination of Bacillus anthracis spores involves rehydration of the spore interior and rapid degradation of several of the protective layers, including the spore coat. Here, we examine the temporal changes that occur during B. anthracis spore germination using an isobaric tagging system. Over the course of 17 min from the onset of germination, the levels of at least 19 spore proteins significantly decrease. Included are acid-soluble proteins, several known and predicted coat proteins, and proteins of unknown function. Over half of these proteins are small (less than 100 amino acids) and would have been undetectable by conventional gel-based analysis. We also identified 20 proteins, whose levels modestly increased at the later time points when metabolism has likely resumed. Taken together, our data show that isobaric labeling of complex mixtures is particularly effective for temporal studies. Furthermore, we describe a rigorous statistical approach to define relevant changes that takes into account the nature of data obtained from multidimensional protein identification technology coupled with the use of isobaric tags. This study provides an expanded list of the proteins that may be involved in germination of the B. anthracis spore and their relative levels during germination.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55849/1/5199_ftp.pd

Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology

Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis , the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called CotΒ, which is encoded only in the genomes of the Bacillus cereus group. We found that CotΒ is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotΒ null mutant in the Sterne strain reveals that CotΒ has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotΒ null mutation has no effect on virulence in a murine model of B. anthracis infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72138/1/j.1574-6968.2008.01380.x.pd

Constructing Fluorogenic Bacillus Spores (F-Spores) via Hydrophobic Decoration of Coat Proteins

Background: Bacterial spores are protected by a coat consisting of about 60 different proteins assembled as a biochemically complex structure with intriguing morphological and mechanical properties. Historically, the coat has been considered a static structure providing rigidity and mainly acting as a sieve to exclude exogenous large toxic molecules, such as lytic enzymes. Over recent years, however, new information about the coat’s architecture and function have emerged from experiments using innovative tools such as automated scanning microscopy, and high resolution atomic force microscopy. Principal Findings: Using thin-section electron microscopy, we found that the coat of Bacillus spores has topologically specific proteins forming a layer that is identifiable because it spontaneously becomes decorated with hydrophobic fluorogenic probes from the milieu. Moreover, spores with decorated coat proteins (termed F-spores) have the unexpected attribute of responding to external germination signals by generating intense fluorescence. Fluorescence data from diverse experimental designs, including F-spores constructed from five different Bacilli species, indicated that the fluorogenic ability of F-spores is under control of a putative germination-dependent mechanism. Conclusions: This work uncovers a novel attribute of spore-coat proteins that we exploited to decorate a specific layer imparting germination-dependent fluorogenicity to F-spores. We expect that F-spores will provide a model system to gai

YwdL in Bacillus cereus: Its Role in Germination and Exosporium Structure

In members of the Bacillus cereus group the outermost layer of the spore is the exosporium, which interacts with hosts and the environment. Efforts have been made to identify proteins of the exosporium but only a few have so far been characterised and their role in determining spore architecture and spore function is still poorly understood. We have characterised the exosporium protein, YwdL. ΔywdL spores have a more fragile exosporium, subject to damage on repeated freeze-thawing, although there is no evidence of altered resistance properties, and coats appear intact. Immunogold labelling and Western blotting with anti-YwdL antibodies identified YwdL to be located exclusively on the inner surface of the exosporium of B. cereus and B. thuringiensis. We conclude that YwdL is important for formation of a robust exosporium but is not required to maintain the crystalline assembly within the basal layer or for attachment of the hairy nap structure. ΔywdL spores are unable to germinate in response to CaDPA, and have altered germination properties, a phenotype that confirms the expected defect in localization of the cortex lytic enzyme CwlJ in the coat

Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor

Hypervirulent Clostridium difficile PCR-Ribotypes Exhibit Resistance to Widely Used Disinfectants

The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings