Do intraoperative pyloric interventions predict the need for postoperative endoscopic interventions after minimally invasive esophagectomy?

Intraoperative pyloric procedures are often performed during esophagectomies to reduce the rates of gastric conduit dysfunction. They include pyloroplasty (PP), pyloromyotomy (PM), and pylorus botulinum toxin type-A injections (BI). Despite these procedures, patients frequently warrant further endoscopic interventions. The aim of this study is to compare intraoperative pyloric procedures and the rates of postoperative endoscopic interventions following minimally invasive esophagectomy (MIE). We identified patients who underwent MIE for esophageal carcinoma and grouped them as 'None' (no intervention), 'PP', 'PM', or 'BI' based on intraoperative pyloric procedure type. The rates of endoscopic interventions for the first six postoperative months were compared. To adjust for variability due to MIE type, the rates of >1 interventions were compared using a zero-inflated Poisson regression analysis. Significance was established at P < 0.05. There were 146 patients who underwent an MIE for esophageal cancer from 2008 to 2015; 77.4% were three-hole MIE, and 22.6% were Ivor- Lewis MIE. BI was most frequent in Ivor-Lewis patients (63.5%), while PP was most frequent (46.9%) in three-hole patients. Postoperative endoscopic interventions occurred in 38 patients (26.0%). The BI group had the highest percentage of patients requiring a postoperative intervention (n = 13, 31.7%). After adjusting for higher rates of interventions in three-hole MIE patients, the BI and None groups had the lowest rates of >1 postoperative interventions. Our data did not show superiority of any pyloric intervention in preventing endoscopic interventions. The patients who received BI to the pylorus demonstrated a trend toward a greater likelihood of having a postoperative intervention. However when adjusted for type of MIE, the BI and None groups had lower rates of subsequent multiple interventions. Further research is needed to determine if the choice of intraoperative pyloric procedure type significantly affects quality of life, morbidity, and overall prognosis in these patients.

Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus.

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible Staphylococcus aureus isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) isolates. The nucleotide sequences of the pbp4 structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two- to threefold decrease in vancomycin MICs. Inactivation of pbp4 by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.

American Society of Microbiology - 101st General Meeting. 20-24 May 2001, Orlando, FL, USA.

Sessions on prokaryotic genomics, bioinformatics, antibiotic resistance, intrinsic antibacterial resistance, and the identification of novel targets were the main highlights of this year's American Society Microbiology (ASM) meeting. In addition, updates on the status of antimicrobial developmental candidates and recently approved agents, were also discussed.

American Society of Microbiology 101st General Meeting. 20-24 May 2001, Orlando, FL, USA.

The application of sophisticated molecular biology, genetics and genomics has made possible the advanced analyses of microbial genes, the topology of DNA and chromosomes, and insight into the regulation of gene expression during all stages of the life cycle of microbes, both in vitro and in vivo. The struggle to control contagious pathogens continues world wide amidst resistance emergence to many classes of antimicrobial agents. Many hospital, research and community labs are applying themselves to a more thorough understanding of the molecular basis of this resistance. New drugs which improve on predecessor agents were presented. The following classes of antimicrobial agents were represented: quinolones, cephems, macrolides and natural products. New target opportunities against both lethal (essential) gene targets and virulence targets were presented throughout the conference. In addition, increasing attention to the involvement of microbial life forms in immune function and dysfunction were described in numerous presentations.

American Society of Microbiology - 100th General Meeting.

Symposium sessions on genomics, surveillance, and pharmaceutical intervention opportunities were highlights of this annual ASM meeting. Two-component signal transduction was highlighted by both academic and industrial representatives, as was prokaryotic genomics. Recurring themes throughout the meeting were the contribution of efflux mechanisms to worldwide resistance, target modifications responsible for fluoroquinolone resistance, and the role of structural biology in the discovery and exploitation of bacterial targets.

Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria.

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Escherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylococcus aureus were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain D-glutamate-adding activity and have Km and Vmax values similar to those of the regenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 microM for E. coli and H. influenzae MurD, respectively. Such substrate inhibition was not observed with the E. faecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO4(2-) had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.

39th Interscience Conference on Antimicrobial Agents and Chemotherapy of the American Society for Microbiology.

The 39th ASM ICAAC Meeting, attended by over 16,000 delegates, highlighted numerous late-stage development antibacterials. The presentation of over 600 reports (posters, symposia and oral presentations) regarding resistance emergence underscores the evolving landscape of antibiotic susceptibility worldwide. Although bacterial genomic efforts are slowly expanding the potential sources of novel targets, the established antibacterial classes, including quinolones, beta-lactams, protein synthesis inhibitors and carbapenems, were most prevalent among the presentations. Quinolones dominated this conference, with many presentations of moxifloxacin (> 72 presentations), gatifloxacin (> 66 presentations) and gemifloxacin (> 59 presentations). The new class of antibacterials, the oxazolidinones, represented by Linezolid, was also highlighted.

38th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), September 24-27, 1998, San Diego, CA, USA.

This year's ICAAC meeting was dominated by exciting late-stage development antibiotics that represent breakthroughs in covering pathogens of unmet medical need, including drug-resistant pathogens. There are several exciting antibiotics in late-stage development, among them, linezolid, gatifloxacin, moxifloxacin, SB-265805, HMR 3647, SCH27899 and the resurrected daptomycin. The development of new technology for screening and the cross-application into genomics have led to numerous breakthroughs in techniques and strategies for the identification of novel prokaryotic targets.

Why are there no new antibiotics?

Renewed efforts are underway in the pharmaceutical industry to address the growing problem of antibiotic resistant pathogens. With this rededication to novel antibiotic discovery, new tools and technologies are being deployed to understand critical therapeutic intervention points in bacterial metabolism.

98th General Meeting of the American Society for Microbiology. May 17-21, 1998; Atlanta, GA.

The 98th General Meeting of the American Society for Microbiology was held from May 17-21, 1998, in Atlanta, Georgia and was attended by well over 10,000 scientists. The theme of antibiotic resistance dominated the meeting with numerous presentations on resistance mechanisms, new targets and potential antimicrobial agents. Many new insights into the understanding of microbial physiology were provided. Microbial genomics was shown to be revolutionizing the way in which scientists can probe and explore bacteria and fungi.

Identification and characterization of cell wall-cell division gene clusters in pathogenic gram-positive cocci.

Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.

The 97th Annual Meeting of the American Society for Microbiology.

The Annual Meeting of the American Society for Microbiology took place in Miami Beach, Florida, from May 4-8, 1997. Over 9000 scientists attended this meeting, which covers all major aspects of prokaryotic research (basic, applied, medical, and diagnostic). Genomics discussions were a major part of the meeting agenda, with scientists detailing both basic and applied research effort using genomics and bioinformatics. New ideas for potential novel antimicrobials have also surfaced as the tools to pursue Drug Discovery have fallen into place and pharmaceutical companies have ;rediscovered' anti-infectives.

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.

Role of mecA transcriptional regulation in the phenotypic expression of methicillin resistance in Staphylococcus aureus.

The gene required for methicillin resistance in staphylococci, mecA, encodes the low-affinity penicillin-binding protein 2a (PBP2a). Transcriptional regulation of mecA is accomplished in some isolates by mecR1 and mecI, cotranscribed chromosomal genes that encode a putative signal transducer and a transcriptional repressor, respectively. Two Staphylococcus aureus strains that have identical mecR1-mecI nucleotide sequences, BMS1 and N315P, both exhibit low-level, heterotypic expression of methicillin resistance and contain no beta-lactamase coregulatory sequences. mecR1-mecI was amplified from BMS1 by PCR and was shown to be functional on a high-copy-number plasmid when introduced into an S. aureus strain with a deleted mecR1-mecI locus. Cloned mecR1-mecI repressed phenotypic expression of methicillin resistance, mecA transcription and PBP2a production and mediated PBP2a induction in response to certain beta-lactam antibiotics. However, mecR1-mecI had different regulatory activities in its native chromosomal location in N315P compared with those in BMS1. Uninduced mecA transcription was markedly repressed in N315P, and mecI inactivation increased mecA transcription and PBP2a production 5- and 40-fold, respectively. Furthermore, the N315P phenotype changed from low-level, heterotypic resistance with intact mecI to high-level, homotypic resistance in strains with disrupted mecI. In contrast, uninduced BMS1 produced abundant mecA transcript and PBP2a, while the disruption of mecI had no effect on phenotype and little effect on mecA transcription or PBP2a production. Thus, mecI-mediated repression of mecA appears to be dysfunctional in BMS1 because of the presence or absence of additional regulatory cofactors. Furthermore, heterotypic resistance expression in this strain is independent of mecA transcriptional regulation.

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid.

A striking feature of recent outbreaks of vancomycin-resistant (VmR) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of VmR. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative VmR transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E.faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which VmR was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.

Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.