Randomized Pilot Study of a Keratin-based Topical Cream for Radiation Dermatitis in Breast Cancer Patients.

Purpose: Radiotherapy (RT) is commonly used in the treatment of breast cancer and often, despite advances in fractionated dosing schedules, produces undesirable skin toxicity. The purpose of this study was to evaluate the feasibility of using a keratin-based topical cream, KeraStat® Cream (KC; KeraNetics, Inc., Winston Salem, NC, USA) to manage the symptoms of radiation dermatitis (RD) in breast cancer patients undergoing RT. Materials and Methods: A total of 24 subjects were enrolled on this single-center, randomized, open-label study. Participants were randomly assigned to KC or standard of care (SOC, patient's choice of a variety of readily available creams or moisturizers). Patients were asked to apply the assigned treatment to the irradiated area twice daily, beginning with day 1 of RT, through 30 days post-RT. The primary outcome was compliance of use. Secondary outcomes included safety and tolerability of KC, as well as RD severity assessed using the Radiation Therapy Oncology Group (RTOG) scale and the patient-reported Dermatology Life Quality Index (DLQI). Results: All subjects in the KC group were assessed as compliant with no adverse events. The rate of RTOG Grade 2 RD was lower in the KC group (30.8%) compared to the SOC group (54.5%, P = .408). At the final RT visit, the mean RTOG RD score was lower in the KC group (1.0) versus the SOC group (1.4). Similarly, patient-reported quality of life measured by the DLQI at the end of RT was improved in the KC group (mean 4.25, small effect) versus the SOC group (mean 6.18, moderate effect, P = .412). Conclusions: KC was safe and well tolerated with no adverse events. Though efficacy measures were not powered to draw definitive conclusions, trends and clinical assessments suggest that there is a benefit of using KC compared to SOC for breast cancer patients treated with RT, and a larger powered study for efficacy is warranted. Trial Registry: This clinical trial is registered as NCT03374995 titled KeraStat(R) Cream for Radiation Dermatitis.

One third of middle ear effusions from children undergoing tympanostomy tube placement had multiple bacterial pathogens.

BACKGROUND: Because previous studies have indicated that otitis media may be a polymicrobial disease, we prospectively analyzed middle ear effusions of children undergoing tympanostomy tube placement with multiplex polymerase chain reaction for four otopathogens. METHODS: Middle ear effusions from 207 children undergoing routine tympanostomy tube placement were collected and were classified by the surgeon as acute otitis media (AOM) for purulent effusions and as otitis media with effusion (OME) for non-purulent effusions. DNA was isolated from these samples and analyzed with multiplex polymerase chain reaction for Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis. RESULTS: 119 (57%) of 207 patients were PCR positive for at least one of these four organisms. 36 (30%) of the positive samples indicated the presence of more than one bacterial species. Patient samples were further separated into 2 groups based on clinical presentation at the time of surgery. Samples were categorized as acute otitis media (AOM) if pus was observed behind the tympanic membrane. If no pus was present, samples were categorized as otitis media with effusion (OME). Bacteria were identified in most of the children with AOM (87%) and half the children with OME (51%, p < 0.001). A single bacterial organism was detected in middle ear effusions from children with AOM more often than those with OME (74% versus 33%, p < 0.001). Haemophilus influenzae was the predominant single organism and caused 58% of all AOM in this study. Alloiococcus otitidis and Moraxella catarrhalis were more frequently identified in middle ear effusions than Streptococcus pneumoniae. CONCLUSIONS: Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis were identified in the middle ear effusions of some patients with otitis media. Overall, we found AOM is predominantly a single organism infection and most commonly from Haemophilus influenzae. In contrast, OME infections had a more equal distribution of single organisms, polymicrobial entities, and non-bacterial agents.

Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains.

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.

Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model.

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.

Activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant.

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of naïve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity.

Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation.

BACKGROUND: Group A Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro. FINDINGS: Allelic replacement of speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation. CONCLUSIONS: The constitutive production of SpeB by the srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

Loss of the group A Streptococcus regulator Srv decreases biofilm formation in vivo in an otitis media model of infection.

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.

Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.