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In the follow-up study of patients with pelvic fractures, rupture of the posterior urethra is registered in 3–25% of cases (Koraitim et al., 1996). The diagnostic gold standard for the assessment of hemodynamically stable trauma patients is contrast-enhanced CT scan, especially helical CT. Nevertheless, simultaneous suprapubic cystography and ascending urethrograms (the so-called up-and-downogram) are the investigation of choice in assessing the site, severity, and length of urethral injuries. (Carlin and Resnick, 1995) This paper discusses the evaluation and diagnosis of urethral injury in multiple-trauma patient.

Escape room’s popularity has raised over the past years among young adults. It creates a distended competitive environment, where participants collaborate to achieve a common objective through teamwork. We decided to apply this format as a teaching method for medical students at the University of Malaga, Spain. A peer-to-peer physiological cardiorespiratory escape room was designed by intern undergraduate students, collaborating within the Department of Human Physiology. This activity integrated the contents of the Human Physiology syllabus, which were organized into four stages that culminated in a final medical case. Intern students oversaw the design, promotion, preparation and execution of the activity, and were in charge of conducting the evaluation and follow up. The escape room was done in mid-December, after all theoretical and practical contents had been delivered, for four consecutive years, improving from each year’s experience. The target group for this activity were second year medical students, who were asked to team up freely in groups of four to six students before the start of the activity. The students in each group cooperated with each other while trying to solve the different puzzles and questions in each stage of the escape room. After the activity, the results of the final evaluation exam of these participants were compared against non-participants, who served as a control group. Qualitative feedback was also received from the participants via a special survey that was designed for this task. Results between 2020 and 2023 (three last activities) show that the final mark of the participants was significantly higher than in non-participants (6.39 ± 0.14 vs. 5.04 ± 0.2; p < 0.0007). The global exam mark also increased in the participants (5.43 ± 0.10 vs. 4.44 ± 0.15; p < 0.0007). A significant difference was observed in the performance in cardiovascular (p < 0.0007) and respiratory-related questions (p < 0.0007), which was substantial in the participants. The qualitative feedback received from the participants was mainly positive, indicating an overall acceptance of the format by the students. We conclude that escape room format with a peer-to-peer structure is an efficient teaching tool for medical students performed by medical students in the field of Human Physiology.

Linguistic anxiety (LA) is an abnormal stress response induced by situations that require the use of verbal behavior, and it is accentuated during language testing in persons with aphasia (PWA). The presence of LA in PWA may jeopardize the interpretation of cognitive evaluations, leading to biased conclusions about the severity of the language alteration and the effectiveness of the treatments. In the present study, we report the case of a woman (Mrs. A) with severe chronic mixed transcortical aphasia due to left frontal and parietal hemorrhages that partially spared the perisylvian area. Mrs. A was treated with the dopamine agonist Rotigotine alone and combined with Intensive Language-Action Therapy (ILAT). Complementary evaluations included autonomic reactivity during the performance of different language tasks, resting state functional magnetic resonance imaging (rs-fMRI) and [18F]-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). We found that formal language testing in a clinical setting triggered a dramatic increase of automatic echolalia, perseverations and frustration, making the task completion difficult. The treatment improved aphasia, but gains were more robust when evaluation was performed by Mrs. A’s husband at home than by clinicians. Autonomic evaluation under Rotigotine revealed higher reactivity during tasks tapping an impaired function in comparison with a task evaluating a preserved function (verbal repetition). Baseline 18F-FDG-PET analysis showed decreased metabolic activity in left limbic-paralimbic areas, whereas rs-fMRI revealed compensatory activity in the right hemisphere. We also analyzed the different factors (e.g., premorbid personality traits, task difficulty) that may have contributed to LA in Mrs. A during language testing. Our findings emphasize the usefulness of implicating adequately trained laypersons in the evaluation and treatment of PWA showing LA. Further studies using multidimensional evaluations are needed to disentangle the interplay between anxiety and abnormal language as well as the neural mechanisms underpinning LA in PWA.

Vancomycin-resistant enterococci (VRE) are important pathogens in hospitalized patients; however, the factors involved in VRE colonization of hospitalized patients are not well characterized. Bacteriocins provide a competitive advantage to enterococci in experimental models of colonization, but little is known about bacteriocin content in samples derived from humans and even less is known about their dynamics in the clinical setting. To identify bacteriocins which may be relevant in the transmission of VRE, we present a systematic analysis of bacteriocin content in the genomes of 2,248 patient-derived E. faecium isolates collected over a 6-year period from a single hospital system. We used computational methods to broadly search for bacteriocin structural genes and a functional assay to look for phenotypes consistent with bacteriocin expression. We identified homology to 15 different bacteriocins, with 2 having a high presence in this clinical cohort. Bacteriocin 43 (bac43) was found in a total of 58% of isolates, increasing from 8% to 91% presence over the 6-year collection period. There was little genetic variation in the bac43 structural or immunity genes across isolates. The enterocin A structural gene was found in 98% of isolates, but only 0.3% of isolates had an intact enterocin A gene cluster and displayed a bacteriocin-producing phenotype. This study presents a wide survey of bacteriocins from hospital isolates and identified bac43 as highly conserved, increasing in prevalence, and phenotypically functional. This makes bac43 an interesting target for future investigation for a potential role in E. faecium transmission.IMPORTANCEWhile enterococci are a normal inhabitant of the human gut, vancomycin-resistant E. faecalis and E. faecium are urgent public health threats responsible for hospital-associated infections. Bacteriocins are ribosomally synthesized antimicrobial proteins and are commonly used by bacteria to provide a competitive advantage in polymicrobial environments. Bacteriocins have the potential to be used by E. faecium to invade and dominate the human gut leading to a greater propensity for transmission. In this work, we explore bacteriocin content in a defined clinically derived population of E. faecium using both genetic and phenotypic studies. We show that one highly active bacteriocin is increasing in prevalence over time and demonstrates great potential relevance to E. faecium transmission., Competing Interests: The authors declare no conflict of interest.

Streptococcus pneumoniae is an important human pathogen that normally resides in the human nasopharynx. Competence-mediated bacteriocin expression by S. pneumoniae plays a major role in both the establishment and persistence of colonization on this polymicrobial surface. Over 20 distinct bacteriocin loci have been identified in pneumococcal genomes, but only a small number have been characterized phenotypically. In this work, we demonstrate that three-fourths of S. pneumoniae strains contain a highly conserved scb locus that encodes an active lactococcin 972-like bacteriocin called streptococcin B. In these backgrounds, the scbABC locus is part of the early competence cascade due to a ComE binding site in the promoter region. Streptococcin B producing strains target both members of the population that have failed to activate competence and the 25% of the population that carry a naturally occurring deletion of the ComE binding site and the functional bacteriocin gene. The ComR-type regulator found directly upstream of the scb locus in S. pneumoniae strains can activate scb expression independent of the presence of the ComE binding site but only when stimulated by a peptide that is encoded in the scb locus of Streptococcus pseudopneumoniae , a closely related bacterium that also inhabits the human nasopharynx. Given the co-regulation with competence and the phenotypic confirmation of activity, streptococcin B represents a previously unrecognized fratricide effector that gives producing strains an additional advantage over the naturally occurring deleted strains during colonization., Importance: Streptococcus pneumoniae is a common cause of pneumonia, meningitis, sinusitis, and otitis media. In order to successfully colonize humans, a prerequisite to the development of invasive disease, S. pneumoniae must compete with other bacterial inhabitants of the nasal surface for space and nutrients. Bacteriocins are small antimicrobial peptides produced by bacteria that typically target neighboring bacteria by disruption of the cell surface. S. pnuemoniae encodes a large number of potential bacteriocin, but, for most, their role in competitive interactions has not been defined. This work demonstrates that isolates that produce the bacteriocin streptococcin B have an advantage over non-producers. These observations contribute to our understanding of the competitive interactions that precede the development of S. pneumoniae disease.

Vancomycin resistant enterococci (VRE) are important pathogens in hospitalized patients, however, the factors involved in VRE colonization of hospitalized patients are not well characterized. Bacteriocins provide a competitive advantage to enterococci in experimental models of colonization, but little is known about bacteriocin content in samples derived from humans and even less is known about their dynamics in the clinical setting. To identify bacteriocins which may be relevant in the transmission of VRE, we present a systematic analysis of bacteriocin content in the genomes of 2,428 patient derived E. faecium isolates collected over a six-year period from a single hospital system. We used computational methods to broadly search for bacteriocin structural genes and a functional assay to look for phenotypes consistent with bacteriocin expression. We identified homology to 15 different bacteriocins with two having high presence in this clinical cohort. Bacteriocin 43 (bac43) was found in a total of 58% of isolates, increasing from 8% to 91% presence over the six-year collection period. There was little genetic variation in the bac43 structural or immunity genes across isolates. The enterocin A structural gene was found in 98% of isolates but only 0.3% of isolates had an intact enterocin A gene cluster and displayed a bacteriocin producing phenotype. This study presents a wide survey of bacteriocins from hospital isolates and identified bac43 as highly conserved, increasing in prevalence, and phenotypically functional. This makes bac43 an interesting target for future investigation for a potential role in E. faecium transmission., Importance: While enterococci are a normal inhabitant of the human gut, vancomycin-resistant E. faecalis and E. faecium are urgent public health threats responsible for hospital associated infections. Bacteriocins are ribosomally synthesized antimicrobial proteins and are commonly used by bacteria to provide a competitive advantage in polymicrobial environments. Bacteriocins have the potential be used by E. faecium to invade and dominate the human gut leading to a greater propensity for transmission. In this work, we explore bacteriocin content in a defined clinically derived population of E. faecium using both genetic and phenotypic studies. We show that one highly active bacteriocin is increasing in prevalence over time and demonstrates great potential relevance to E. faecium transmission.

BACKGROUND Osteogensis imperfecta (OI) is a rare disorder associated with brittle bones, skeletal deformities, short stature, and conductive hearing loss. It is caused by mutations in genes encoding collagen type I production and is associated with multiple fractures occurring during a patient's lifetime. Atypical fractures can occur without a history of previous injury or diagnosis. CASE REPORT A 52-year old man sustained a fall from his own height. He experienced pelvic pain but could bear weight. He was referred to the hospital by his primary care physician and was admitted to the Orthopedic Department with a pelvic ring fracture. We performed open reduction and internal fixation with a reconstruction plate via an intrapelvic approach. At 12 weeks after discharge, he sustained a peritrochanteric fracture. It was surgically treated with a dynamic hip screw. The patient commenced weight-bearing 20 weeks after the initial procedure. Bone union was achieved clinically and radiographically. He did not have any revision procedures. At the time of this report, 5 years after the described period, he felt no discomfort around the hips, sacroiliac joints, and pubic symphysis. CONCLUSIONS OI poses a difficult treatment challenge, but, achieving a good clinical and functional result is possible with a precise surgical technique and meticulous preoperative planning. Clinicians should always consider the possibility of a simultaneous fracture occurrence in different parts of the body. Some patients present without a previous diagnosis, and we should always have some suspicion of OI in cases of young patients presenting with low-energy fractures.

Streptococcus pneumoniae (pneumococcus) is an important human pathogen that primarily resides in the nasopharynx. To persist in this polymicrobial environment, pneumococcus must compete with other members of the bacterial community. Competition is mediated in part by the action of the blp locus, which encodes a variable array of bacteriocins and their associated immunity proteins. The locus is controlled by a two-component regulatory system that senses the extracellular concentration of the peptide pheromone, BlpC. There are four major pherotypes of BlpC that can be found in most pneumococcal genomes. Here, we show that the protease SepM is required for activation of three of the four major pherotypes. The only SepM-independent BlpC type is 9 amino acids shorter than the SepM-dependent peptides, consistent with a cleavage event at the C-terminal end. The processing event occurs following secretion, and removal of the C-terminal region is required for binding to the histidine kinase receptor. Synthetic truncated peptides or full-length peptides preincubated with SepM-expressing bacteria can upregulate the blp locus independent of SepM. We show that naturally secreted SepM-independent peptides accumulate in the supernatant of secreting cells at low levels, suggesting a role for the tail in peptide secretion, stability, or solubility and demonstrating a significant trade-off for SepM-independence. IMPORTANCE Streptococcus pneumoniae is an important cause of disease in humans that occurs when the bacteria in the nasopharynx bypasses host defenses to invade deeper tissues. Colonization fitness thus represents an important initial step in pathogenesis. S. pneumoniae produces antimicrobial peptides called bacteriocins that provide a competitive advantage over neighboring bacteria in the nasopharynx. The blp locus encodes a variable array of bacteriocins that participate in competition. Here, we demonstrate that activation of the blp locus requires a surface protease that activates the blp signal peptide. There are naturally occurring signal peptides that do not require cleavage, but these are characterized by poor secretion. We describe an additional, previously unappreciated activation step in the control of bacteriocin production in S. pneumoniae.

Abstract: Presentation and mortality of coronary artery disease (CAD) substantially differs in both sexes. Most of the existing data analyzing sex differences is older than 10 years and mostly was retrieved in clinical trials, which are potentially structured with a bias against the inclusion of women, leading to a potential selection-bias. Meanwhile, with better diagnostic and therapeutic options, actual data analyzing sex differences in emergency CAD patients is rare.Data on all emergency case numbers with CAD diagnosis in Germany 2017 was retrieved from the German Institute for Medical Documentation and Information. DRG, OPS, and ICD codes were used to determine comorbidities, in-hospital course, and outcome. Competing risk regression analysis for in-hospital mortality was performed analyzing age, European System for Cardiac Operative Risk Evaluation (EuroSCORE), severity of CAD, clinical presentation type and sex.264,742 patients were included. Female patients were older and had more comorbidities. Three-vessel CAD was significantly less present in female patients (36.5% vs 47.5%; P < .001). After adjusting for age, EuroSCORE and severity of CAD, female sex was an independent predictor of lower in-hospital mortality (subdistribution hazard ratio [sHR] 0.94, 95% CI: 0.90-0.98, P = .002) in the whole cohort and in non-ST-segment elevation myocardial infarction (NSTEMI) patients (sHR 0.85, 95% CI: 0.79-0.92, P < .001), whereas in ST-segment elevation myocardial infarction (STEMI) patients, female sex was associated with a higher in-hospital mortality (sHR 1.07, 95% CI: 1.01-1.14, P = .029).In all patients admitted as emergency with CAD diagnosis and in all NSTEMI patients, female sex is protective, whereas in STEMI patients, females show a higher in-hospital mortality risk., Competing Interests: The authors have no conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Gram-positive bacteria employ an array of secreted peptides to control population-level behaviors in response to environmental cues. We review mechanistic and functional features of secreted peptides produced by the human pathogen Streptococcus pneumoniae. We discuss sequence features, mechanisms of transport, and receptors for 3 major categories of small peptides: the double-glycine peptides, the Rap, Rgg, NprR, PlcR, and PrgX (RRNPP)-binding peptides, and the lanthionine-containing peptides. We highlight the impact of factors that contribute to carriage and pathogenesis, specifically genetic diversity, microbial competition, biofilm development, and environmental adaptation. A recent expansion in pneumococcal peptide studies reveals a complex network of interacting signaling systems where multiple peptides are integrated into the same signaling pathway, allowing multiple points of entry into the pathway and extending information content in new directions. In addition, since peptides are present in the extracellular milieu, there are opportunities for crosstalk, quorum sensing (QS), as well as intra- and interstrain and species interactions. Knowledge on the manner that population-level behaviors contribute to disease provides an avenue for the design and development of anti-infective strategies., Competing Interests: The authors have declared that no competing interests exist.

Peptidase-containing ABC transporters (PCATs) are a widely distributed family of transporters which secrete double-glycine (GG) peptides. In the opportunistic pathogen Streptococcus pneumoniae (pneumococcus), the PCATs ComAB and BlpAB have been shown to secrete quorum-sensing pheromones and bacteriocins related to the competence and pneumocin pathways. Here, we describe another pneumococcal PCAT, RtgAB, encoded by the rtg locus and found intact in 17% of strains. The Rgg/SHP-like quorum-sensing system RtgR/S, which uses a peptide pheromone with a distinctive Trp-X-Trp motif, regulates expression of the rtg locus and provides a competitive fitness advantage in a mouse model of nasopharyngeal colonization. RtgAB secretes a set of coregulated rtg GG peptides. ComAB and BlpAB, which share a substrate pool, do not secrete the rtg GG peptides. Similarly, RtgAB does not efficiently secrete ComAB/BlpAB substrates. We examined the molecular determinants of substrate selectivity between ComAB, BlpAB, and RtgAB and found that the GG peptide signal sequences contain all the information necessary to direct secretion through specific transporters. Secretion through ComAB and BlpAB depends largely on the identity of four conserved hydrophobic signal sequence residues previously implicated in substrate recognition by PCATs. In contrast, a motif situated at the N-terminal end of the signal sequence, found only in rtg GG peptides, directs secretion through RtgAB. These findings illustrate the complexity in predicting substrate-PCAT pairings by demonstrating specificity that is not dictated solely by signal sequence residues previously implicated in substrate recognition. IMPORTANCE The export of peptides from the cell is a fundamental process carried out by all bacteria. One method of bacterial peptide export relies on a family of transporters called peptidase-containing ABC transporters (PCATs). PCATs export so-called GG peptides which carry out diverse functions, including cell-to-cell communication and interbacterial competition. In this work, we describe a PCAT-encoding genetic locus, rtg , in the pathogen Streptococcus pneumoniae (pneumococcus). The rtg locus is linked to increased competitive fitness advantage in a mouse model of nasopharyngeal colonization. We also describe how the rtg PCAT preferentially secretes a set of coregulated GG peptides but not GG peptides secreted by other pneumococcal PCATs. These findings illuminate a relatively understudied part of PCAT biology: how these transporters discriminate between different subsets of GG peptides. Ultimately, expanding our knowledge of PCATs will advance our understanding of the many microbial processes dependent on these transporters., (Copyright © 2020 Wang et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

In the polymicrobial environment of the human nasopharynx, Streptococcus pneumoniae (pneumococcus) competes with other members of the microbial community for limited nutrients in part by secreting small peptide bacteriocins called pneumocins. Pneumocin production is controlled by a quorum sensing system encoded by the blp locus. Although the locus is found in all pneumococci, there is significant variability in the repertoire of pneumocins and associated immunity proteins encoded in the Bacteriocin Immunity Region (BIR) and in the presence or absence of a functional Blp transporter. Strains without an active Blp transporter are inactive in plate overlay assays and rely on a homologous transporter that is only produced during brief periods of competence to stimulate the blp locus and secrete pneumocins. The variability of the locus suggests that selective pressure is influencing the content to promote the optimal competitive environment. Much of the variability in the blp locus has been described at the genome level; the phenotypic activity attributable to the various BIR genes has not been fully described. To examine the role of the predicted pneumocin peptides in competition, 454 isolates were screened for competence independent blp pheromone secretion using plate assays. Active strains were characterized for inhibition, BIR content, BlpC pherotype and serotype. Deletion analysis on inhibitory strains demonstrated that BlpI and BlpJ peptides function as a two-peptide bacteriocin and that BlpIJ immunity is encoded by the co-transcribed blpU4/5 genes. BlpIJ secretion promotes inhibitory activity against the majority of pneumococcal isolates when expressed in a Blp transporter intact background. Intermediate levels of competition in biofilms were noted when BlpIJ containing strains carried the non-functional Blp transporter. Based on genome data, the combination of BlpIJ in a Blp transporter intact strain is surprisingly rare, despite clear advantages during colonization and biofilm growth. In contrast, we show that the blpK/pncF operon encoding the single-peptide pneumocin BlpK and its immunity protein is found in the majority of isolates. Unlike, BlpIJ and BlpK were shown to promote a limited spectrum of inhibition due in part to immunity that is independent of activation of the blp locus.

The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) uses natural genetic competence to increase its adaptability through horizontal gene transfer. One method of acquiring DNA is through predation of neighboring strains with antimicrobial peptides called "bacteriocins." Competence and production of the major family of pneumococcal bacteriocins, pneumocins, are regulated by the quorum-sensing systems com and blp , respectively. In the classical paradigm, the ABC transporters ComAB and BlpAB each secretes its own system's signaling pheromone and in the case of BlpAB also secretes the pneumocins. While ComAB is found in all pneumococci, only 25% of strains encode an intact version of BlpAB [BlpAB(+)] while the rest do not [BlpAB(-)]. Contrary to the classical paradigm, it was previously shown that BlpAB(-) strains can activate blp through ComAB-mediated secretion of the blp pheromone during brief periods of competence. To better understand the full extent of com - blp crosstalk, we examined the contribution of each transporter to competence development and pneumocin secretion. We found that BlpAB(+) strains have a greater capacity for competence activation through BlpAB-mediated secretion of the com pheromone. Similarly, we show that ComAB and BlpAB are promiscuous and both can secrete pneumocins. Consequently, differences in pneumocin secretion between BlpAB(+) and BlpAB(-) strains derive from the regulation and kinetics of transporter expression rather than substrate specificity. We speculate that BlpAB(-) strains (opportunists) use pneumocins mainly in a narrowly tailored role for DNA acquisition and defense during competence while BlpAB(+) strains (aggressors) expand their use for the general inhibition of rival strains., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Many streptococci have evolved the ability for natural genetic competence. Recent studies have uncovered regulatory links between competence and the production of antimicrobial peptides called bacteriocins in multiple streptococcal species. This reveals a broadly distributed strategy among streptococci to exploit bacteriocin-mediated killing during competence for adaptive gain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Campylobacter jejuni , a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni -derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase ( ctsM ), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM + (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni , whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria., Competing Interests: The authors declare no conflict of interest.

Pneumonia due to either Streptococcus pneumoniae (Sp) or Staphylococcus aureus (Sa) accounts for most mortality after influenza and acute respiratory illness (ARI). Because carriage precedes infection, we estimated Sp and Sa carriage to examine the co-colonization dynamics between Sp, Sa and respiratory viruses in the presence of ARI in the oropharynx. We tested oropharyngeal specimens of community subjects (aged ⩾2 years) with ARI for the presence of influenza A and B, 11 other common respiratory viruses, Sp and Sa, using real-time PCR. A total of 338 participants reported 519 ARI episodes of which 119 (35%) carried Sp, 52 (13%) carried Sa and 25 (7%) carried both. Thirty-five subjects tested positive for influenza, of which 14 (40%) carried Sp and six (17%) carried Sa, significantly more than in the influenza-negative group (P = 0·03 and P = 0·04, respectively). In subjects infected by any virus compared to those with no virus, Sp carriage (39·2% vs. 27·9%, P = 0·03) but not Sa carriage (11·6% vs. 14%, P = 0·6) was more frequent. For children, when Sa was present, Sp carriage tended to be less frequent than expected given the presence of viral infection, but not significantly [observed relative risk 1·14, 95% confidence interval (CI) 0·4-3·1; with a relative excess risk due to interaction of -0·11]. Independent of age, Sp carriers were more likely to return that season with subsequent ARI (odds ratio 2·14, 95% CI 1·1-4·3, P = 0·03). Both Sp and Sa carriage rates in the oropharynx increase during influenza infection in children. However, no negative interaction between Sp and Sa was observed. Sp carriers are more likely to suffer subsequent ARI episodes than non-carriers.

Unlabelled: Nasopharyngeal colonization is important for Streptococcus pneumoniae evolution, providing the opportunity for horizontal gene transfer when multiple strains co-occur. Although colonization with more than one strain of pneumococcus is common, the factors that influence the ability of strains to coexist are not known. A highly variable blp (bacteriocin-like peptide) locus has been identified in all sequenced strains of S. pneumoniae This locus controls the regulation and secretion of bacteriocins, small peptides that target other bacteria. In this study, we analyzed a series of cocolonizing isolates to evaluate the impact of the blp locus on human colonization to determine whether competitive phenotypes of bacteriocin secretion restrict cocolonization. We identified a collection of 135 nasopharyngeal samples cocolonized with two or more strains, totaling 285 isolates. The blp locus of all strains was characterized genetically with regard to pheromone type, bacteriocin/immunity content, and potential for locus functionality. Inhibitory phenotypes of bacteriocin secretion and locus activity were assessed through overlay assays. Isolates from single colonizations (n = 298) were characterized for comparison. Cocolonizing strains had a high diversity of blp cassettes; approximately one-third displayed an inhibitory phenotype in vitro Despite in vitro evidence of competition, pneumococci cocolonized the subjects independently of blp pheromone type (P = 0.577), bacteriocin/immunity content, blp locus activity (P = 0.798), and inhibitory phenotype (P = 0.716). In addition, no significant differences were observed when single and cocolonizing strains were compared. Despite clear evidence of blp-mediated competition in experimental models, the results of our study suggest that the blp locus plays a limited role in restricting pneumococcal cocolonization in humans., Importance: Nasopharyngeal colonization with Streptococcus pneumoniae (pneumococcus) is important for pneumococcal evolution, as the nasopharynx represents the major site for horizontal gene transfer when multiple strains co-occur, a phenomenon known as cocolonization. Understanding how pneumococcal strains interact within the competitive environment of the nasopharynx is of chief importance in the context of pneumococcal ecology. In this study, we used an unbiased collection of naturally co-occurring pneumococcal strains and showed that a biological process frequently used by bacteria for competition-bacteriocin production-is not decisive in the coexistence of pneumococci in the host, in contrast to what has been shown in experimental models., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Streptococcus pneumoniae (pneumococcus) has remained a persistent cause of invasive and mucosal disease in humans despite the widespread use of antibiotics and vaccines. The resilience of this organism is due to its capacity for adaptation through the uptake and incorporation of new genetic material from the surrounding microbial community. DNA uptake and recombination is controlled by a tightly regulated quorum sensing system that is triggered by the extracellular accumulation of competence stimulating peptide (CSP). In this study, we demonstrate that CSP can stimulate the production of a diverse array of blp bacteriocins. This cross stimulation occurs through increased production and secretion of the bacteriocin pheromone, BlpC, and requires a functional competence regulatory system. We show that a highly conserved motif in the promoter of the operon encoding BlpC and its transporter mediates the upregulation by CSP. The accumulation of BlpC following CSP stimulation results in augmented activation of the entire blp locus. Using biofilm-grown organisms as a model for competition and genetic exchange on the mucosal surface, we demonstrate that DNA exchange is enhanced by bacteriocin secretion suggesting that co-stimulation of bacteriocins with competence provides an adaptive advantage. The blp and com regulatory pathways are believed to have diverged and specialized in a remote ancestor of pneumococcus. Despite this, the two systems have maintained a regulatory connection that promotes competition and adaptation by targeting for lysis a wide array of potential competitors while simultaneously providing the means for incorporation of their DNA.