Biological Impact of a Large-Scale Genomic Inversion That Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of the Streptococcus pyogenes Chromosome.

A large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-long Streptococcus pyogenes serotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces the ori site relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, both in vitro and in vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place near oriC always happen to secure the linkage of oriC to DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed.IMPORTANCE Based on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of the S. pyogenes genome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growth in vitro and in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of the oriC and the chromosome partition cluster is preserved.

Low incidence of complications after cephalic vein cutdown for pacemaker lead implantation in children weighing less than 10 kilograms: A single-center experience with long-term follow-up.

BACKGROUND: Only a few studies on the cephalic vein cutdown technique for pacemaker lead implantation in children weighing ≤10 kg have been reported even though the procedure is widely accepted in adults. OBJECTIVE: The purpose of this study was to prove that cephalic vein cutdown for pacemaker lead implantation is a reliable technique with a low incidence of complications in children weighing ≤10 kg. METHODS: The study included 44 children weighing ≤10 kg with an endocardial pacemaker. Cephalic, subclavian, and axillary vein diameters were measured by ultrasound before implantation. The measured diameters were used to select either an endocardial or epicardial surgical technique. Regular 6-month follow-up visits included pacemaker interrogation and clinical and ultrasound examinations. RESULTS: Two dual-chamber and 42 single-chamber pacemakers were implanted. Mean weight at implantation was 6.24 kg (range 2.25-10.40 kg), and mean age was 11.4 months (range 1 day-47 months). In 40 children (90.1%), the ventricular leads were implanted using the cephalic vein cutdown technique, and implantation was accomplished via the prepared right external jugular vein in 4 of the children (9.9%). The atrial leads were implanted using axillary vein puncture and external jugular vein preparations. Mean follow-up was 8.9 years (range 0-20.9 years). Only 1 pacemaker-related complication was detected (a lead fracture near the connector that was successfully resolved using a lead repair kit). CONCLUSION: The cephalic vein cutdown technique is feasible and reliable in children weighing ≤10 kg, which justifies the application of additional surgical effort in the treatment of these small patients.

Right ventricular myxoma - a case report.

INTRODUCTION: Myxomas arising from the right ventricle are extremely rare. CASE REPORT: We presented a 71-year-old patient with worsening symptoms of the exertional dyspnea and atypical chest pains lasting 6 months. A transthoracic and transesophageal echocardiogram revealed a large, 2.6 x 2.2 cm, ovoid, well-circumscribed, echogenic mass in the right ventricle outflow tract attached by small pedicle, partly obstructing the right ventricular outflow tract and protruding through the pulmonic valve during systole. The tumor was completely removed with the stalk and 5 mm of the surrounding tissue. The histopathological findings confirmed the diagnosis of myxoma. CONCLUSION: This case illustrates the usefulness of echocardiography both in diagnosis of patients with atypical symptoms without family history and associated syndromes (like Carney's complex), and in surgical approach planning. It also stresses the importance of surgical excision of tumor as soon as possible following the diagnosis to prevent the complications such are: valvular obstruction, pulmonary embolization and syncopes.

Penetrating wound of the heart manifested with peripheral embolism--case report.

INTRODUCTION: Heart injuries can be classified as penetrating and non-penetrating (blunt). Penetrating wounds are usually caused by stabbing with a piercing object, weapon or projectiles--missiles. The right atrium is damaged in most cases, because of its anatomical position--making the most of the anterior side of the heart. Morbidity caused by stabbing injuries to the heart is 20%-30%, while piercing wounds cause 30%-60% of deaths. CASE REPORT: . A 28-year-old patient was admitted to our clinic with acute ischemia of the extremities. Angiography revealed a bullet in the right common femoral artery, occluding it. The patient denied having any piercing or shooting wound to his leg, but he said that four years before he had been shot to his chest. Echocardiography revealed an atrial septal defect of secondary type. An event reconstruction revealed that, four years after shooting, the bullet was displaced from the heart to the right common femoral artery. CONCLUSION: This case report is unique because of the rare type of injury, time that passed from the injury, the way bullet entered the artery (via atrial septal defect) and especially the success of both surgical procedures (embolectomy and repair of atrial septal defect).

Long-term survival of Streptococcus pyogenes in rich media is pH-dependent.

The mechanisms that allow Streptococcus pyogenes to survive and persist in the human host, often in spite of antibiotic therapy, remain poorly characterized. Therefore, the determination of culture conditions for long-term studies is crucial to advancement in this field. Stationary cultures of S. pyogenes strain NZ131 and its spontaneous small-colony variant OK171 were found to survive in rich medium for less than 2 weeks, and this inability to survive resulted from the acidification of the medium to below pH 5.5, which the cells did not tolerate for longer than 6-7 days. The growth of NZ131 resulted in acidification of the culture to below pH 5.5 by the onset of stationary phase, and the loss of viability occurred in a linear fashion. These results were also found to be true for M49 strain CS101 and for M1 strain SF370. The S. pyogenes strains could be protected from killing by the addition of a buffer that stabilized the pH of the medium at pH 6.5, ensuring bacterial survival to at least 70 days. By contrast, increasing the glucose added to the medium accelerated the loss of culture viability in strain NZ131 but not OK171, suggesting that the small-colony variant is altered in glucose uptake or metabolism. Similarly, acidification of the medium prior to inoculation or at the middle of exponential phase resulted in growth inhibition of all strains. These results suggest that control of the pH is crucial for establishing long-term cultures of S. pyogenes.

[Ascending aorta false aneurysm as a late complication of aortic valve surgery].

INTRODUCTION: False aneurysms of the ascending aorta represent a rare but potentially fatal complication of cardiac surgical procedures. Predisposing factors are aortic dissection, infection, connective tissue disorders, chronic hypertension, aortic calcifications and aortotomy dehiscence. At the beginning they are usually asymptomatic, but later various symptoms arise as a consequence of vital structures compression. Potential risk of rupture rises with time and pseudoaneurysm enlargement. From surgical point of view treatment of such cases represents a unique challenge because of the great danger of inadvertent opening of the aneurysm during resternotomy. CASE OUTLINE: A 58-year-old female patient underwent aortic valve replacement due to severe aortic stenosis in 2004. Operation and postoperative recovery were uneventful.Three years later she started complaining about chest pain. On chest X-ray there was upper mediastinal widening. CT scan showed a pseudoaneurysm of the ascending aorta located in front of the right atrium and right ventricle, which was subsequently verified by angiography. During redo operation the pseudoaneurysm was successfully resected and aorta closed with separate ethybond sutures with pledgets. CONCLUSION: Postoperative pseudoaneurysms of the ascending aorta mostly arise from the suture lines. The most useful diagnostic procedures are contrast CT scan, echocardiography, angiography and MRI. Surgical intervention is absolutely indicated. The institution of cardiopulmonary bypass by alternative ways before chest opening is strongly recommended.

Pregnancy-related aortic aneurysm and dissection in patients with Marfan's syndrome: medical and surgical management during pregnancy and after delivery.

In the current article, 3 cases of aortic aneurysm and dissection in pregnant patients with Marfan's syndrome are reported. It is well known that pregnancy is a risk factor for the development of aortic aneurysm and dissection in women with Marfan's syndrome since it is shown that dissection can develop both before and after labor. Marfan patients with an aortic root diameter greater than 4 cm should undergo preconceptual counseling for surgical aortic repair before pregnancy. Pregnant Marfan patients with an aortic aneurysm should be closely and continuously evaluated by multidisciplinary specialists in order to prevent possible aortic dissection that could be fatal for both the mother and the fetus.

Permanent endovenous pediatric pacing: absence of lead failure--20 years follow-up study.

BACKGROUND: The aim of the study was to analyze endovenous pacing lead survival in pediatric population implanted by cephalic cut down, or by axillary vein puncture. METHODS: All implantations were performed in total endotracheal anesthesia, by the same surgeon. Implantations of ventricular leads were performed by cephalic vein cut down or by external jugular vein preparation. In dual-chamber pacing, atrial leads were implanted via cephalic vein (along with ventricular lead), by axillary vein puncture or via external jugular vein. All implanted leads were secured by resorbable suture. RESULTS: Over the 20-year follow-up period, 105 children of 5.7 years average age (range 1 day-15 years) were implanted with a permanent endovenous pacing system for congenital or postsurgical complete atrioventricular block or sinus node disease. Within the group, 27 patients (25.7%) weighed less than 10 kg on implantation. A total of 121 endovenous leads were implanted. All ventricular leads were with a passive fixation mechanism, and most of them unipolar (87.6%) and steroid eluting (94.2%). Leads implanted in atrial position were 82% bipolar, predominantly with active fixation (94%), and all steroid eluting. The most frequently used mode of stimulation was VVIR (66.6%). No acute or chronic lead displacement, exit block, sensing problem, lead conductor fracture, insulation defect or infections were observed during the total follow-up of 709 pacing years (average 6.9, range 0-20 years). CONCLUSION: Implantation of the endovenous leads by preparation of the cephalic or puncture of the axillary vein, with lead fixation by resorbable suture represents a method of choice.

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes.

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.

Fracture of the subcutaneous patch electrode in a patient with an implanted cardioverter-defibrillator.

There are more than 20 years of experience with implantation of defibrillator devices in humans and the procedure is an important therapeutic option for patients at high risk of life-threatening ventricular arrhythmias. The incidence of new defibrillator implantation has gradually increased, being used even in children, (1) although pediatric use is associated with several complications, especially with epicardial systems,(2) including fracture of the subcutaneous patch,(3-5) mainly because of growth.(3) We present a case of subcutaneous patch electrode fracture in the left axillary pectoral region of a patient who needed the patch for effective defibrillation, and we discuss the methods of treatment.

[Implantable "loop recorder"--a new diagnostic tool for syncope of unknown cause].

INTRODUCTION: The implantable loop recorder (ILR) is a new diagnostic tool in cardiology for establishing the causes of unexplained syncope in patients where standard conventional tests, invasive tests included, have failed. The device is a diagnostic "pacemaker," surgically implanted underneath the skin of the chest, with leads attached to the case of the device, not requiring endovenous lead implantation. Heart rhythm is monitored continuously on the basis of an endless loop, up to a maximum period of 14 months. Recording is carried out either by applying an outside activator whenever symptoms occur, or automatically, according to a pre-set algorithm for bradycardia, tachycardia, and/or asystolic detection. OBJECTIVE: The aim of this study was to present this new diagnostic method as well as our first experiences with its implementation. METHOD: We followed 5 patients (3 male, 2 female, mean age: 46.4 +/- 19) who had ILRs ("Reveal Plus," Medtronic Inc., USA) implanted at our centre, over a period of 14 months (7.6 +/- 5.5), concentrating on their clinical course, symptom occurrence, and electronically monitored heart rhythm at the time of ILR auto activation and/or recordings triggered by outside activation whenever a patient's symptoms were discernible. RESULTS: In three patients, the ILR revealed syncope aetiology by documenting heart rhythm at the time of its occurrence. In one patient, involving a lethal outcome, the ILR was not explanted, so that the rhythm at the time of the fatal syncope, although assumed, remained undocumented. In one, most recently implanted patient, follow-up is still in progress. CONCLUSION: The implantable loop recorder represents an important innovation and a step forward in establishing the causes of recurrent syncope, which cannot be determined by standard invasive and non-invasive testing.

[Resynchronisation therapy in patients with heart failure: our results].

INTRODUCTION: Resynchronisation therapy with biventricular permanent pacing stimulation is one method of treating patients with systolic heart failure, with echocardiograph signs of ventricular asynchrony and a prolonged QRS of longer than 120 milliseconds. This method has been accepted in most medical centres around the world and was instigated in our Pacemaker Centre in December 2001, 3 months after FDA approval for human use. OBJECTIVE: The aim of the study was to present this new procedure and the results obtained from our own group of patients. METHOD: A multi-site, biventricular pacemaker, with a special electrode for left-half heart stimulation was implanted in the coronary sinus of 17 patients who had suffered systolic heart failure (12 male and 5 female, average age 59.9 years). For all of them, the duration of the QRS interval was longer than 120 ms, with left bundle branch morphology, and an ejection fraction below 30%. All the patients were NYHA class II or III. Prior to and after the implantation, a 12-channel ECG and ECHO were carried out, a 6-minute hall walk test was performed, additionally, the total walked distance on a flat surface was measured, the general condition of the patient was evaluated, the number of medications being taken was noted, as was the number of days of hospitalisation. RESULTS: The average time from diagnosis to implantation was 22 months, and the average post-operative follow-up was 14 months. Two of the patients died 10 and 7 months after the implantation, due to a new myocardial infarction and refractory heart failure. In addition, one patient did not show any improvement after the implantation of the multi-site pacemaker (there were three "non-responder" patients). All the other patients felt much better: decreased NYHA class for I - II class, increased left ventricle ejection fraction, reduced use of diuretics, increased 6-minute hall walk distance and general walk distance on a flat surface, and decreased number of days of hospitalisation. CONCLUSION: Resynchronisation heart failure therapy in the majority of patients with systolic left ventricular dysfunction and a prolonged QRS interval considerably improves cardiac function, in addition to reducing symptoms and hospital stays.

The LysR-type transcriptional regulator CysB controls the repression of hslJ transcription in Escherichia coli.

The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor in Escherichia coli cells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. The hslJ gene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium. cysB mutants showed upregulation of the hslJ : : lacZ gene fusion and exhibited increased novobiocin resistance. In this study the hslJ transcription start point and the corresponding putative sigma(70) promoter were determined. The hslJ promoter region was defined by employing different hslJ-lacZ operon fusions, and transcription of the hslJ gene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor of hslJ transcription and binds the hslJ promoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligand N-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression of hslJ transcription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.

Identification of the CysB-regulated gene, hslJ, related to the Escherichia coli novobiocin resistance phenotype.

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.

Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene.

A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed.

Autonomous expression of the slo gene of the bicistronic nga-slo operon of Streptococcus pyogenes.

A recent model for cytolysin-mediated translocation in Streptococcus pyogenes proposes that NAD-glycohydrolase is translocated through streptolysin O-generated pores into a host cell (J. Madden, N. Ruiz, and M. Caparon, Cell 104:143-152, 2001). This model also assumes that the NAD-glycohydrolase (nga) and streptolysin O (slo) genes that code for these products are organized in an operon-like structure expressed from a single promoter only (nga). We expand this model by showing that slo possesses its own autonomous promoter, which is located 155 bp upstream of the slo gene. Under experimental conditions in which S. pyogenes is grown in THY medium, the strength of the slo promoter, as measured by the activity of a lacZ reporter gene, resulted in low but highly reproducible values. Finally, we demonstrated that sloR, a S. pyogenes gene that closely resembles the Clostridium perfringens pfoR gene, exerts a negative effect on the expression of the slo gene.