Paraesophageal hernia repair in elderly patients: outcomes from a 10-year retrospective study.

BACKGROUND: Laparoscopic surgery has become the preferred management for paraesophageal hernias (PEH); however surgical management versus watchful waiting remains controversial in older patients. METHODS: This retrospective study analyzed the outcomes of PEH repair in elderly patients surgically managed at The Ottawa Hospital over a 10-year period. Patients older than 60 years who underwent PEH repair were examined with respect to presentation, technique and associated complications. RESULTS: Despite similar demographics, our study groups showed significantly different characteristics of surgical techniques. Most surgeries were performed laparoscopically; however, patients aged 70 years or older underwent more open and emergency surgeries than the younger group. Despite a 30-day postoperative complication rate of 45 % and 13 % in the older (≥ 70 yr) and younger (60-69 yr) groups, respectively, the rates during elective repair were similar. There were no deaths in the younger group, whereas the 30-day mortality rate was 5 % in patients aged 70 years and older, including a 2-fold increase with emergency repair (4 v. 2 patients). CONCLUSION: Management of PEH in older adults remains controversial in relation to a surgical versus watchful waiting approach. We found that in patients aged 70 years and older who undergo surgical management of PEH experience more open and emergency procedures, which are associated with higher complication rates. However, in the elective setting older patients had increased laparoscopic repairs and comparable complication rates to younger patients. We found the greatest outcomes with early, elective laparoscopic repair, irrespective of age.

Motor vehicle collision with seatbelt sign and traumatic abdominal wall hernia should raise suspicion for hollow viscus injury.

Diagnosing hollow viscus injury following motor vehicle collision (MVC) requires a high index of suspicion. Here we present two cases of high velocity MVC, with 3-point restrained occupants, who presented with a seatbelt sign and associated acute traumatic flank herniation. Both patients underwent a computer tomography (CT) scan which did not identify any hollow viscus injuries. Significant injuries were ultimately identified in the operating room (OR). The presence of a seatbelt sign and underlying acute traumatic hernia should prompt a heightened level of suspicion for intra-abdominal injury, particularly hollow viscus. A heightened level of suspision and a lower threshold for operative exploration is suggested to avoid the morbidity and mortality associated with a delayed diagnosis of hollow viscus injury.

Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to ß-lactams.

Rising drug resistance is limiting treatment options for infections by methicillin-resistant Staphylococcus aureus (MRSA). Herein we provide new evidence that wall teichoic acid (WTA) biogenesis is a remarkable antibacterial target with the capacity to destabilize the cooperative action of penicillin-binding proteins (PBPs) that underlie ß-lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted in the restoration of sensitivity of MRSA to a unique profile of ß-lactam antibiotics with a known selectivity for penicillin binding protein 2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously approved drugs with a cryptic capacity to potentiate its activity against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly potentiated cefuroxime, and this synergy was abolished in strains lacking tarO. The combination was also effective in a Galleria mellonella model of infection. Using both genetic and biochemical strategies, we determined the molecular target of ticlopidine as the N-acetylglucosamine-1-phosphate transferase encoded in gene tarO and provide evidence that WTA biogenesis represents an Achilles heel supporting the cooperative function of PBP2 and PBP4 in creating highly cross-linked muropeptides in the peptidoglycan of S. aureus. This approach represents a new paradigm to tackle MRSA infection.

Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168.

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.

Are essential genes really essential?

Gene essentiality has emerged as an often-asked question in the wake of bacterial genome sequencing and a renaissance in studies of prokaryotic physiology. Genome-scale efforts at describing essential gene sets have necessarily been carried out under standard and tractable growth conditions in a laboratory setting. In addition to reinforcing our understanding of core bacterial physiology, these studies have also uncovered large numbers of essential genes encoding proteins whose functions remain poorly described. Studies of these and other elements of core physiology have naturally followed and several paradoxes, relating to growth conditions and genetic context, have begun to challenge our understanding of the term "essential gene". Most recently genome-scale genetic interaction studies have revealed remarkable density and redundancy in biological systems with profound implications for dispensability phenotypes associated with single gene mutations. Consequently, the phenotype "essential" should be carefully viewed as contextual.

Probing teichoic acid genetics with bioactive molecules reveals new interactions among diverse processes in bacterial cell wall biogenesis.

The bacterial cell wall has been a celebrated target for antibiotics and holds real promise for the discovery of new antibacterial chemical matter. In addition to peptidoglycan, the walls of Gram-positive bacteria contain large amounts of the polymer teichoic acid, covalently attached to peptidoglycan. Recently, wall teichoic acid was shown to be essential to the proper morphology of Bacillus subtilis and an important virulence factor for Staphylococcus aureus. Additionally, recent studies have shown that the dispensability of genes encoding teichoic acid biosynthetic enzymes is paradoxical and complex. Here, we report on the discovery of a promoter (P(ywaC)), which is sensitive to lesions in teichoic acid synthesis. Exploiting this promoter through a chemical-genetic approach, we revealed surprising interactions among undecaprenol, peptidoglycan, and teichoic acid biosynthesis that help explain the complexity of teichoic acid gene dispensability. Furthermore, the new reporter assay represents an exciting avenue for the discovery of antibacterial molecules.

The N-acetylmannosamine transferase catalyzes the first committed step of teichoic acid assembly in Bacillus subtilis and Staphylococcus aureus.

There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the N-acetylmannosamine transferase (tagA in Bacillus subtilis; tarA in Staphylococcus aureus), which adds N-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the N-acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable gene in the pathway, suggesting that tagA (or tarA) encodes the first committed step in wall teichoic acid synthesis.

Duplication of teichoic acid biosynthetic genes in Staphylococcus aureus leads to functionally redundant poly(ribitol phosphate) polymerases.

Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI'J' and tarIJ) as well as poly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI'J') could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI'J' is due to poor translation of the TarJ' enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.

The Amino terminus of Bacillus subtilis TagB possesses separable localization and functional properties.

The function(s) of gram-positive wall teichoic acid is emerging with recent findings that it is an important virulence factor in the pathogen Staphylococcus aureus and that it is crucial to proper rod-shaped cell morphology of Bacillus subtilis. Despite its importance, our understanding of teichoic acid biosynthesis remains incomplete. The TagB protein has been implicated in the priming step of poly(glycerol phosphate) wall teichoic acid synthesis in B. subtilis. Work to date indicates that the TagB protein is localized to the membrane, where it adds a single glycerol phosphate residue to the nonreducing end of the undecaprenol-phosphate-linked N-acetylmannosamine-beta(1,4)-N-acetylglucosamine-1-phosphate. Thus, membrane association is critical to TagB function. In this work we elucidate the mechanism of TagB membrane localization. We report the identification of a membrane targeting determinant at the amino terminus of TagB that is necessary and sufficient for membrane localization. The putative amphipathicity of this membrane targeting determinant was characterized and shown to be required for TagB function but not localization. This work shows for the first time that the amino terminus of TagB mediates membrane targeting and protein function.

Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis.

An extensive literature has established that the synthesis of wall teichoic acid in Bacillus subtilis is essential for cell viability. Paradoxically, we have recently shown that wall teichoic acid biogenesis is dispensable in Staphylococcus aureus (M. A. D'Elia, M. P. Pereira, Y. S. Chung, W. Zhao, A. Chau, T. J. Kenney, M. C. Sulavik, T. A. Black, and E. D. Brown, J. Bacteriol. 188:4183-4189, 2006). A complex pattern of teichoic acid gene dispensability was seen in S. aureus where the first gene (tarO) was dispensable and later acting genes showed an indispensable phenotype. Here we show, for the first time, that wall teichoic acid synthesis is also dispensable in B. subtilis and that a similar gene dispensability pattern is seen where later acting enzymes display an essential phenotype, while the gene tagO, whose product catalyzes the first step in the pathway, could be deleted to yield viable mutants devoid of teichoic acid in the cell wall.

Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway.

An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.

Crystal structure of CTP:glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus: examination of structural basis for kinetic mechanism.

Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD(Sa) in Staphylococcus aureus and TagD(Bs) in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD(Sa) and TagD(Bs) (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD(Sa) in the apo state to 3 A resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD(Bs) crystal structures reveals that the overall structure of TarD(Sa) is essentially the same as that of TagD(Bs), except in the C-terminus, where it forms a helix in TagD(Bs) but is disordered in the apo TarD(Sa) structure. In addition, TarD(Sa) can exist both as a tetramer and as a dimer, unlike TagD(Bs), which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD(Sa) and TagD(Bs).