Programmed Death Ligand 1: A Step Toward Immunoscore for Esophageal Cancer.

BACKGROUND: This study sought to evaluate the effect of tumor-infiltrating lymphocyte (TIL) density and programmed death ligand 1 (PD-L1) expression on the prognosis of esophageal cancer. METHODS: Banked tissue specimens from 53 patients who underwent esophagectomies for malignancy at a single institution over a 6-year period were stained for cluster of differentiation 3 (CD3), CD8, and PD-L1. Tumors were characterized as staining high or low density for CD3 and CD8, as well as positive or negative for PD-L1. TIL density and PD-L1 expression were analyzed in the context of survival, recurrence, and perioperative characteristics. RESULTS: Median follow-up was 823 days, with 92.5% survival and 26.8% recurrence rates. All tumors were adenocarcinomas. Neoadjuvant chemotherapy was given in 56.6% of cases, and neoadjuvant radiotherapy was given in 37.7%. High CD3 density was found in 83%, whereas high CD8 density was found in 56.6%. A total of 18.9% of the tumors stained positive for PD-L1. Survival was significantly shorter in Kaplan-Meier analysis for patients with primary tumors staining positive for PD-L1 (log rank: p = 0.05). Multivariable analysis controlling for neoadjuvant therapy, TIL markers, PD-L1, age, and sex found no significant difference in recurrence or survival. CONCLUSIONS: Positive staining for PD-L1 may be a prognostic marker for decreased survival in esophageal adenocarcinoma. Additional TIL cell types should be investigated for creation of an esophageal cancer Immunoscore. PD-L1 has potential as a therapeutic target.

An efflux pump (MexAB-OprM) of Pseudomonas aeruginosa is associated with antibacterial activity of Epigallocatechin-3-gallate (EGCG).

BACKGROUND: Pseudomonas aeruginosa is a notorious multidrug resistant nosocomial pathogen. An efflux pump (MexAB-OprM) is the main contributor to the multidrug resistance in clinical isolates of P. aeruginosa. Epigallocatechin-3-gallate (EGCG), a polyphenolic compound extracted from green tea, exhibits antibacterial activity. It is unclear that molecular details of the antibacterial activity of EGCG, EGCG-effect on antibiotic susceptibility, and clinical relevance of EGCG in bacteria. PURPOSE: This study aimed to determine the roles of the efflux pump and an efflux pump inhibitor (phenylalanine-arginine ß-naphthylamide; PAßN) in the antibacterial activity of EGCG and the EGCG-effect on antibiotic susceptibility. METHODS: Twenty-two multidrug resistant clinical isolates of P. aeruginosa and a wild type P. aeruginosa PAO1 were used to determine antibacterial activity of EGCG and EGCG-effect on antibiotic susceptibility. An efflux pump (MexAB-OPrM) mutant strain, its complemented strain carrying an intact mexAB-oprM, and their parental strain were used to determine roles of MexAB-OprM in the antibacterial activity of EGCG and EGCG-mediated antibiotic susceptibility. PAßN was also used to evaluate EGCG as a possible efflux pump inhibitor. RESULTS: EGCG inhibited cellular growth and killed 100% of cells at 64-512 µg/ml and at 256-1024 µg/ml, respectively, in all tested 22 clinical isolates including the wild type strain. A subinhibitory concentration of EGCG significantly enhanced susceptibility to antibiotics, unexceptionally to chloramphenicol and tetracyclines (≥4-fold) of the clinical isolates. Both the antibacterial activity of EGCG and the EGCG-mediated antibiotic susceptibility were enhanced more in the efflux pump mutant strain (mexB::Gm) than the parental strain, suggesting additionally accumulated-EGCG produced the more antibacterial activity in the mutant strain. EGCG was synergistically interacted with PAßN with enhancing susceptibility to all tested antibiotics (up to >500-fold) at higher levels than either EGCG alone or PAßN alone, suggesting EGCG may also inhibit the efflux pump with additional accumulation of the antibiotics. CONCLUSION: The results demonstrate that EGCG exhibits antibacterial activity and enhances antibiotic effects against clinical isolates of P. aeruginosa. EGCG may inhibit the efflux pump (MexAB-OprM) through which are associated with the antibacterial activity of EGCG and the EGCG-mediated antibiotic susceptibility in P. aeruginosa.

Antibacterial activity of exogenous glutathione and its synergism on antibiotics sensitize carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

A major clinical impact of A. baumannii is hospital-acquired infections including ventilator-associated pneumonia. The treatment of this pathogen is often difficult due to its innate and acquired resistance to almost all commercially available antibiotics. Infections with carbapenem-associated multidrug resistant A. baumannii is the most problematic. Glutathione is a tripeptide thiol-antioxidant and antibacterial activity of exogenous glutathione was reported in some bacteria. However, clinical relevance and molecular details of the antibacterial activity of glutathione are currently unclear. Seventy clinical isolates of A. baumannii including 63 carbapenem-associated multidrug resistant isolates and a type strain A. baumannii ATCC 19606 were used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Fractional inhibitory concentration (FIC) and time-killing activity with meropenem and/or glutathione were also determined in the carbapenem-associated multidrug resistant isolates. In addition, the roles of exogenous glutathione in multidrug efflux pumps and ß-lactamase production were examined. Levels of MIC and MBC were ranged from 10 to 15mM of exogenous glutathione. All tested carbapenem-associated multidrug resistant isolates were sensitized by all tested antibiotics in combination with subinhibitory concentrations of glutathione. FIC levels of glutathione with carbapenem (meropenem) were all<0.5 and the carbapenem-associated multidrug resistant isolates were killed by subinhibitory concentrations of both glutathione and meropenem at>2log10 within 12h, suggesting glutathione synergistically interacts with meropenem. The roles of multidrug efflux pumps and ß-lactamase production were excluded for the glutathione-mediated antibiotic susceptibility. Overall results demonstrate that the antibacterial activity of glutathione is clinically relevant and its synergism on antibiotics sensitizes clinical isolates of A. baumannii regardless of their resistance or susceptibility to antibiotics. This finding suggests that exogenous glutathione alone and/or in combination with existing antibiotics may be applicable to treat infections with carbapenem-associated multidrug resistant A. baumannii.

Homeostasis of glutathione is associated with polyamine-mediated ß-lactam susceptibility in Acinetobacter baumannii ATCC 19606.

Glutathione is a tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) thiol compound existing in many bacteria and maintains a proper cellular redox state, thus protecting cells against toxic substances such as reactive oxygen species. Polyamines (spermine and spermidine) are low-molecular-weight aliphatic polycations ubiquitously presenting in all living cells and modulate many cellular functions. We previously reported that exogenous polyamines significantly enhanced ß-lactam susceptibility of ß-lactam-associated multidrug-resistant Acinetobacter baumannii. In this study, three genes differentially associated with the polyamine effects on ß-lactam susceptibility were identified by transposon mutagenesis of A. baumannii ATCC 19606. All three genes encoded components of membrane transport systems. Inactivation of one of the genes encoding a putative glutathione transport ATP-binding protein increased the accumulation of intracellular glutathione (∼150 to ∼200%) and significantly decreased the polyamine effects on ß-lactam susceptibility in A. baumannii ATCC 19606. When the cells were grown with polyamines, the levels of intracellular glutathione in A. baumannii ATCC 19606 significantly decreased from ∼0.5 to ∼0.2 nmol, while the levels of extracellular glutathione were correspondingly increased. However, the levels of total glutathione (intra- plus extracellular) were unchanged when the cells were grown with or without polyamines. Overall, these results suggest that exogenous polyamines induce glutathione export, resulting in decreased levels of intracellular glutathione, which may produce an improper cellular redox state that is associated with the polyamine-mediated ß-lactam susceptibility of A. baumannii. This finding may provide a clue for development of new antimicrobial agents and/or novel strategies to treat multidrug-resistant A. baumannii.

A single amino acid substitution in PmrB is associated with polymyxin B resistance in clinical isolate of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major causative agent of hospital-acquired infections and infections in cystic fibrosis patients. Treatment of P. aeruginosa is complicated by the presence of intrinsic and acquired multidrug-resistant isolates. Polymyxin B has often been used as the last option to treat the multidrug-resistant isolates. However, polymyxin B-resistant clinical isolates have been increasingly reported worldwide. To understand molecular details of polymyxin resistance we characterized polymyxin B-resistant clinical isolate of P. aeruginosa. The clinical isolate grew with 4 microg mL(-1) of polymyxin B while a reference P. aeruginosa PAO1 grew with 0.25 microg mL(-1). Polymyxin B susceptibility was restored (minimal inhibitory concentration from 8 to 0.5 microg mL(-1)) by an intact clone of pmrAB, but not by an intact clone of phoPQ or the cloning vector. DNA sequence analysis of pmrB from the resistant isolate revealed a single nucleotide substitution (T to C) substituted methionine to threonine at position 292 of PmrB. Involvement of this amino acid substitution in polymyxin B resistance was confirmed by complementation of a pmrAB null-mutant strain with the pmrAB containing the amino acid substitution. These results suggest that amino acid substitution in PmrB is one mechanism of polymyxin B resistance in clinical isolates of P. aeruginosa.

Fur-independent induction of Helicobacter pylori flavodoxin-encoding gene (fldA) under iron starvation.

BACKGROUND AND AIMS: Helicobacter pylori infection is associated with a variety of diseases including gastric cancer. Flavodoxin is an electron transfer protein containing a flavin mononucleotide prosthetic group and substituted an iron-containing electron transfer protein under iron-limiting conditions. H. pylori flavodoxin has been reported but its pathogenic role is unclear. The aim of this study is to understand a pathogenic role of H. pylori flavodoxin under iron-limiting condition. METHODS: The flavodoxin-encoding gene (fldA) was cloned from one of clinical H. pylori isolates (DU17) and its transcript was quantified by primer extension, Northern hybridization, and real-time polymerase chain reaction in different concentrations of an iron chelator. The fldA transcript was also quantified in H. pylori ATCC 700392, lacking a ferric uptake regulatory (fur) protein. RESULT: Nucleotide sequence of the fldA from H. pylori DU17 revealed a 492-bp (164 amino acids) open reading frame with a deduced amino acid sequence having 97% identity to that from the complete genomic sequence of H. pylori 26695. The deduced promoter [-35, -10, and +1] of the fldA was 56-bp upstream from the first codon of FldA. The fldA transcript (approximately 0.55-kb) was induced up to 14-fold in both wild-type and fur-knocked-out strains under iron-limiting conditions, suggesting that the fldA induction is independent to the Fur protein. CONCLUSION: The fldA gene may play an important role in iron starvation conditions.

Mixed-infection of antibiotic susceptible and resistant Helicobacter pylori isolates in a single patient and underestimation of antimicrobial susceptibility testing.

Antibiotic resistance among Helicobacter pylori has been increasing worldwide and has begun to affect the overall efficacy of current antibiotic regimens adversely. We examined 220 pairs of H. pylori isolates obtained from both the antrum and corpus of separate patients; 109 (50%) harbored antibiotic-resistant H. pylori: amoxicillin (0.5%), clarithromycin (5.9%), furazolidone (1.4%), metronidazole (45.5%), nitrofurantoin (1.4%), and tetracycline (6.8%). Heteroresistance among the two biopsy sites from each patient was present in 41 of the 109 patients (38%) with antibiotic resistant H. pylori (e.g. 34% with resistant strains would be misclassified as susceptible if a biopsy of the antrum alone used for antimicrobial susceptibility testing). DNA fingerprinting genotype analysis was carried out on the 41 pairs of isolates with heteroresistance. While different patients had different fingerprinting patterns, each pair of isolates showed identical or similar fingerprinting patterns. These results suggest that antibiotic-resistant H. pylori typically develop from pre-existing susceptible strain rather than coinfection with a different strain. The minor differences in genotype (degeneration of genotype) seen reflect one of the processes for development of genetic diversity in H. pylori. No biopsy single site can be considered representative for antimicrobial susceptibility testing.