DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes.

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.

Mucin-induced surface dispersal of Staphylococcus aureus and Staphylococcus epidermidis via quorum-sensing dependent and independent mechanisms.

Nasopharyngeal carriage of staphylococci spreads potentially pathogenic strains into (peri)oral regions and increases the chance of cross-infections. Some laboratory strains can also move rapidly on hydrated agar surfaces, but the biological relevance of these observations is not clear. Using soft-agar [0.3% (wt/vol)] plate assays, we demonstrate the rapid surface dispersal of (peri)oral isolates of Staphylococcus aureus and Staphylococcus epidermidis and closely related laboratory strains in the presence of mucin glycoproteins. Mucin-induced dispersal was a stepwise process initiated by the passive spreading of the growing colonies followed by their rapid branching (dendrites) from the colony edge. Although most spreading strains used mucin as a growth substrate, dispersal was primarily dependent on the lubricating and hydrating properties of the mucins. Using S. aureus JE2 as a genetically tractable representative, we demonstrate that mucin-induced dendritic dispersal, but not colony spreading, is facilitated by the secretion of surfactant-active phenol-soluble modulins (PSMs) in a process regulated by the agr quorum-sensing system. Furthermore, the dendritic dispersal of S. aureus JE2 colonies was further stimulated in the presence of surfactant-active supernatants recovered from the most robust (peri)oral spreaders of S. aureus and S. epidermidis. These findings suggest complementary roles for lubricating mucins and staphylococcal PSMs in the active dispersal of potentially pathogenic strains from perioral to respiratory mucosae, where gel-forming, hydrating mucins abound. They also highlight the impact that interspecies interactions have on the co-dispersal of S. aureus with other perioral bacteria, heightening the risk of polymicrobial infections and the severity of the clinical outcomes. IMPORTANCE: Despite lacking classical motility machinery, nasopharyngeal staphylococci spread rapidly in (peri)oral and respiratory mucosa and cause cross-infections. We describe laboratory conditions for the reproducible study of staphylococcal dispersal on mucosa-like surfaces and the identification of two dispersal stages (colony spreading and dendritic expansion) stimulated by mucin glycoproteins. The mucin type mattered as dispersal required the surfactant activity and hydration provided by some mucin glycoproteins. While colony spreading was a passive mode of dispersal lubricated by the mucins, the more rapid and invasive form of dendritic expansion of Staphylococcus aureus and Staphylococcus epidermidis required additional lubrication by surfactant-active peptides (phenol-soluble modulins) secreted at high cell densities through quorum sensing. These results highlight a hitherto unknown role for gel-forming mucins in the dispersal of staphylococcal strains associated with cross-infections and point at perioral regions as overlooked sources of carriage and infection by staphylococci.

Competitive advantage of oral streptococci for colonization of the middle ear mucosa.

The identification of a diverse microbiome in otic secretions from healthy young adults challenged the entrenched dogma of middle ear sterility and underscored previously unknown roles for oral commensals in the seeding of otic biofilms. We gained insights into the selective forces that enrich for specific groups of oral migrants in the middle ear mucosa by investigating the phylogeny and physiology of 19 strains isolated previously from otic secretions and representing otic commensals (Streptococcus) or transient migrants (Staphylococcus, Neisseria and actinobacterial Micrococcus and Corynebacterium). Phylogenetic analyses of full length 16S rRNA sequences recovered from partially sequenced genomes resolved close relationships between the isolates and (peri)oral commensals. Physiological functions that facilitate mucosal colonization (swarming motility, surfactant production) and nutrition (mucin and protein degradation) were also widespread among the cultivars, as was their ability to grow in the presence or absence of oxygen. Yet, streptococci stood out for their enhanced biofilm-forming abilities under oxic and anoxic conditions and ability to ferment host-derived mucosal substrates into lactate, a key metabolic intermediate in the otic trophic webs. Additionally, the otic streptococci inhibited the growth of common otopathogens, an antagonistic interaction that could exclude competitors and protect the middle ear mucosa from infections. These adaptive traits allow streptococcal migrants to colonize the otic mucosa and grow microcolonies with syntrophic anaerobic partners, establishing trophic interactions with other commensals that mirror those formed by the oral ancestors in buccal biofilms.

Trauma Exposure Response: How Secondary Trauma Affects Personal and Professional Life.

INTRODUCTION: Well-being, both psychological and emotional, is crucial to the development of the competent, caring physician. The ACGME calls on sponsoring institutions to educate learners about topics related to well-being. Trauma exposure response, also known as secondary trauma, is a common phenomenon experienced by physicians. It is important to recognize and mitigate the effects of trauma exposure response, as it can have profound effects on personal and professional lives. We found no MedEdPORTAL resources on trauma exposure response or secondary trauma that include physicians as the audience. METHODS: This 1-hour, interactive session was developed to embed a wellness program into protected time for residents and fellows across the institution. The session was led by a faculty member and consisted of an interactive presentation and a small-group discussion. RESULTS: Twenty-eight of 32 programs at our institution participated in the sessions. This included a total of 292 residents and fellows to whom this session was offered. The session was successful in meeting the educational objectives and was rated as valuable or extremely valuable by most residents and fellows. Trainees appreciated protected time for this discussion and valued the opportunity to have open, honest conversations with their colleagues. DISCUSSION: This effective session delivered meaningful content about trauma exposure response and reviewed coping strategies. Institutional support of protected time was a success factor. The sessions were well received by residents and fellows and can be used across disciplines.

Oral seeding and niche-adaptation of middle ear biofilms in health.

The entrenched dogma of a sterile middle ear mucosa in health is incongruent with its periodic aeration and seeding with saliva aerosols. To test this, we sequenced 16S rRNA-V4 amplicons from otic secretions collected at the nasopharyngeal orifice of the tympanic tube and, as controls, oropharyngeal and buccal samples. The otic samples harbored a rich diversity of oral keystone genera and similar functional traits but were enriched in anaerobic genera in the Bacteroidetes (Prevotella and Alloprevotella), Fusobacteria (Fusobacterium and Leptotrichia) and Firmicutes (Veillonella) phyla. Facultative anaerobes in the Streptococcus genus were also abundant in the otic and oral samples but corresponded to distinct, and sometimes novel, cultivars, consistent with the ecological diversification of the oral migrants once in the middle ear microenvironment. Neutral community models also predicted a large contribution of oral dispersal to the otic communities and the positive selection of taxa better adapted to growth and reproduction under limited aeration. These results challenge the traditional view of a sterile middle ear in health and highlight hitherto unknown roles for oral dispersal and episodic ventilation in seeding and diversifying otic biofilms.

Successful balloon angioplasty of pulmonary artery stenosis in two cats and associated complications.

Two cats (2.5 months and 8 months old) were each evaluated due to a loud systolic murmur, and each was diagnosed with severe pulmonary artery stenosis at the bifurcation of the main pulmonary artery. Echocardiograms confirmed significant right atrial dilation and right ventricular dilation and/or hypertrophy that was progressive in one cat. Atenolol was initiated and the cats were referred for interventional therapy. Balloon angioplasty was performed via the jugular vein. In case 1, the pressure gradient across the stenosis was reduced from 169.7 mmHg to 23.6 mmHg and 52.4 mmHg across the left and right branch pulmonary arteries, respectively. In case 2, the stenotic echocardiographic gradient was reduced from 64 mmHg to 38.0 mmHg and 35.3 mmHg across the left branch and right-branched pulmonary arteries respectively. Both patients developed moderate to severe dynamic right ventricular outflow tract obstruction post angioplasty. Case 2 developed hypotension, desaturation, and ventricular arrhythmias intra-operatively. Case 1 was discharged but appeared to develop acute lung perfusion injury approximately 36 h after procedure that was manifested by radiographic pulmonary congestion and pulmonary infiltrate of the left lung fields. The congestion was successfully managed medically. Serial echocardiograms over the following 4 years in case 1 showed near complete resolution of the stenosis and associated right heart enlargement.

Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens.

The CrbS/R two-component signal transduction system is a conserved regulatory mechanism through which specific Gram-negative bacteria control acetate flux into primary metabolic pathways. CrbS/R governs expression of acetyl-CoA synthase (acsA), an enzyme that converts acetate to acetyl-CoA, a metabolite at the nexus of the cell's most important energy-harvesting and biosynthetic reactions. During infection, bacteria can utilize this system to hijack host acetate metabolism and alter the course of colonization and pathogenesis. In toxigenic strains of Vibrio cholerae, CrbS/R-dependent expression of acsA is required for virulence in an arthropod model. Here, we investigate the function of the CrbS/R system in Pseudomonas aeruginosa, Pseudomonas entomophila, and non-toxigenic V. cholerae strains. We demonstrate that its role in acetate metabolism is conserved; this system regulates expression of the acsA gene and is required for growth on acetate as a sole carbon source. As a first step towards describing the mechanism of signaling through this pathway, we identify residues and domains that may be critical for phosphotransfer. We further demonstrate that although CrbS, the putative hybrid sensor kinase, carries both a histidine kinase domain and a receiver domain, the latter is not required for acsA transcription. In order to determine whether our findings are relevant to pathogenesis, we tested our strains in a Drosophila model of oral infection previously employed for the study of acetate-dependent virulence by V. cholerae. We show that non-toxigenic V. cholerae strains lacking CrbS or CrbR are significantly less virulent than are wild-type strains, while P. aeruginosa and P. entomophila lacking CrbS or CrbR are fully pathogenic. Together, the data suggest that the CrbS/R system plays a central role in acetate metabolism in V. cholerae, P. aeruginosa, and P. entomophila. However, each microbe's unique environmental adaptations and pathogenesis strategies may dictate conditions under which CrbS/R-mediated acs expression is most critical.

Complete Genome Sequence of emm4 Streptococcus pyogenes MEW427, a Throat Isolate from a Child Meeting Clinical Criteria for Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).

We report the complete genome assembly of the Streptococcus pyogenes type emm4 strain MEW427 (also referred to as strain UM001 in the Pediatric Acute-Onset Neuropsychiatric Syndrome [PANS] Research Consortium), a throat isolate from a child with acute-onset neuropsychiatric symptoms meeting clinical criteria for PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus). The genome length is 1,814,455 bp with 38.51% G+C%.

Complete Genome Sequence of emm28 Type Streptococcus pyogenes MEW123, a Streptomycin-Resistant Derivative of a Clinical Throat Isolate Suitable for Investigation of Pathogenesis.

We present here the complete genome sequence of Streptococcus pyogenes type emm28 strain MEW123, a streptomycin-resistant derivative of a pediatric throat isolate. The genome length is 1,878,699 bp, with 38.29% G+C% content. The genome sequence adds value to this virulent emm28 representative strain and will aid in the investigation of streptococcal pathogenesis.

Promotion of Wellness and Mental Health Awareness Among Physicians in Training: Perspective of a National, Multispecialty Panel of Residents and Fellows.

BACKGROUND: Physicians in training are at high risk for depression, and physicians in practice have a substantially elevated risk of suicide compared to the general population. The graduate medical education community is currently mobilizing efforts to improve resident wellness. OBJECTIVE: We sought to provide a trainee perspective on current resources to support resident wellness and resources that need to be developed to ensure an optimal learning environment. METHODS: The ACGME Council of Review Committee Residents, a 29-member multispecialty group of residents and fellows, conducted an appreciative inquiry exercise to (1) identify existing resources to address resident wellness; (2) envision the ideal learning environment to promote wellness; and (3) determine how the existing infrastructure could be modified to approach the ideal. The information was aggregated to identify consensus themes from group discussion. RESULTS: National policy on resident wellness should (1) increase awareness of the stress of residency and destigmatize depression in trainees; (2) develop systems to identify and treat depression in trainees in a confidential way to reduce barriers to accessing help; (3) enhance mentoring by senior peers and faculty; (4) promote a supportive culture; and (5) encourage additional study of the problem to deepen our understanding of the issue. CONCLUSIONS: A multispecialty, national panel of trainees identified actionable goals to broaden efforts in programs and sponsoring institutions to promote resident wellness and mental health awareness. Engagement of all stakeholders within the graduate medical education community will be critical to developing a comprehensive solution to this important issue.

Secondary prevention of cardiogenic arterial thromboembolism in the cat: The double-blind, randomized, positive-controlled feline arterial thromboembolism; clopidogrel vs. aspirin trial (FAT CAT).

OBJECTIVES: To determine if clopidogrel administration is associated with a reduced likelihood of recurrent cardiogenic arterial thromboembolism (CATE) in cats compared to aspirin administration. Secondary aims were to determine if clopidogrel administration had an effect on the composite endpoint of recurrent CATE and cardiac death and to identify adverse effects of chronic clopidogrel or aspirin therapy. ANIMALS: Seventy-five cats that survived a CATE event. METHODS: Multicenter, double-blind, randomized, positive-controlled study. Cats were assigned to clopidogrel (18.75 mg/cat PO q 24 h) or aspirin (81 mg/cat PO q 72 h). Kaplan-Meier survival curves were created for each endpoint and the log rank test performed to compare treatment groups with respect to time to event and the likelihood of the event occurring. RESULTS: The mean age of all cats was 8.0 ± 3.5 yr and 57/75 (76%) were male (p < 0.001); 62/75 (83%) were mixed breed with the remainder including Persian, Abyssinian, American Shorthair, Bengal, Birman, Himalayan, Maine Coon, Ragdoll, Snowshoe, and Sphynx breeds. Only 15% (11/75) of cats had a history of heart disease recorded prior to the CATE event. Clopidogrel administration was associated with significantly reduced likelihood of recurrent CATE compared to aspirin (p = 0.024) and had a longer median time to recurrence [443 (95% CI 185-990) days vs. 192 (95% CI 62-364) days, respectively]. Clopidogrel was also associated with a significantly reduced likelihood of the composite endpoint of recurrent CATE or cardiac death (p = 0.033) with a longer median time to event [346 (95% CI 146-495) days vs. 128 (95% CI 58-243) days]. CONCLUSIONS: Clopidogrel administration significantly reduces the likelihood of recurrent CATE compared with aspirin in cats; both drugs were well tolerated.

Use of pericardial fluid pH to distinguish between idiopathic and neoplastic effusions.

Pericardial effusion (PE) resulting from neoplasia usually is associated with a poor prognosis, whereas idiopathic PE frequently has a good prognosis. This study examined the utility of pH measurement to distinguish between these 2 etiologies. Dogs were classified as having idiopathic PE (n = 12) if pericarditis was diagnosed on histopathology (n = 4) or if no historical, physical, or echocardiographic evidence of recurrent PE was present for at least 6 months after pericardiocentesis (n = 8). Dogs were classified as having neoplastic PE (n = 25) if pericardial or myocardial neoplasia was detected on histopathology (n = 11) or a discrete mass associated with the right atrium, right ventricle, or the aorta was visualized on echocardiography (n = 14). Samples of PE were centrifuged and the supernatant pH was measured with a portable pH meter. The lowest pH (6.40) was found in a dog with idiopathic PE and the highest pH (7.85) was found in a dog with neoplastic PE. However, data from the 2 groups overlapped in 33 out of 37 (89%) instances, and median pH from the idiopathic and neoplastic groups was not significantly different (7.40 and 7.47, respectively; P = 0.28; difference in medians = -0.7; 95% CI, -0.26-0.06). Because of the degree of overlap, our data provide little justification for the use of pH measurement as a diagnostic test in cases of PE.

Arterial thromboembolism in cats: acute crisis in 127 cases (1992-2001) and long-term management with low-dose aspirin in 24 cases.

Records of 127 cats with arterial thromboembolism (ATE) were reviewed. Abyssinian, Birman, Ragdoll, and male cats were overrepresented. Tachypnea (91%), hypothermia (66%), and absent limb motor function (66%) were common. Of 90 cats with diagnostics performed, underlying diseases were hyperthyroidism (12), cardiomyopathy (dilated [8], unclassified [33], hypertrophic obstructive [5], hypertrophic [19]), neoplasia (6), other (4), and none (3). Common abnormalities were left atrial enlargement (93%), congestive heart failure (CHF, 44%), and arrhythmias (44%). Of cats without CHF, 89% were tachypneic. Common biochemical abnormalities were hyperglycemia, azotemia, and abnormally high serum concentrations of muscle enzymes. Of 87 cats treated for acute limb ATE, 39 (45%) survived to be discharged. Significant differences were found between survivors and nonsurvivors for temperature (P < .00001), heart rate (P = .038), serum phosphorus concentration (P = .024), motor function (P = .008), and number of limbs affected (P = .001). No significant difference was found between survivors and nonsurvivors when compared by age, respiratory rate, other biochemical analytes, or concurrent CHE A logistic regression model based on rectal temperature predicted a 50% probability of survival at 98.9 degrees F (37.2 degrees C). Median survival time (MST) for discharged cats was 117 days. Eleven cats had ATE recurrences, and 5 cats developed limb problems. Cats with CHF (MST: 77 days) had significantly shorter survival than cats without CHF (MST: 223 days; P = .016). No significant difference was found in survival or recurrence rate between cats receiving high-dose aspirin (> or = 40 mg/cat q72h) and cats receiving low-dose aspirin (5 mg/cat q72h). Adverse effects were less frequent and milder for the lower dosage.

Supravalvular mitral stenosis in a cat.

A 3-year-old, 4-kg, castrated male domestic shorthair cat presented with signs of progressive respiratory distress. Thoracic radiographs showed pulmonary edema and pleural effusion. Echocardiography revealed a perforate membrane immediately above the mitral valve that divided the left atrium into proximal and distal chambers. The left auricle was proximal to the dividing membrane and connected to the markedly enlarged proximal left atrial chamber, consistent with the diagnosis of supravalvular mitral stenosis (SMS). Position of the obstructing membrane relative to the left auricle distinguishes SMS from cor triatriatum sinister (CTS). In CTS, the left auricle is distal to the dividing membrane and connects to the distal left atrial chamber.