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.

DEL phenotype.

CONCLUSIONS: DEL red blood cells (RBCs) type as D- by routine serologic methods and are transfused routinely, without being identified as expressing a very weak D antigen, to D- recipients. DEL RBCs are detected only by adsorption and elution of anti-D or by molecular methods. Most DEL phenotypes have been reported in population studies conducted in East Asia, although DEL phenotypes have been detected also among Caucasian individuals. Approximately 98 percent of DEL phenotypes in East Asians are associated with the RHD*DEL1 or RHD*01EL.01 allele. The prevalence of DEL phenotypes has been reported among D- Han Chinese (30%), Japanese (28%), and Korean (17%) populations. The prevalence of DEL phenotypes is significantly lower among D- Caucasian populations (0.1%). Among the 3-5 percent of African individuals who are D-, there are no reports of the DEL phenotype. Case reports from East Asia indicate that transfusion of DEL RBCs to D- recipients has been associated with D alloimmunization. East Asian immigrants constitute 2.1 percent of the 318.9 million persons residing in the United States, and an estimated 2.8 percent are blood donors. Using these statistics, we estimate that 68-683 units of DEL RBCs from donors of East Asian ancestry are transfused as D- annually in the United States. Given the reports from East Asia of D alloimmunization attributed to transfusion of DEL RBCs, one would expect an occasional report of D alloimmunization in the United States following transfusion of DEL RBCs to a D- recipient. If such cases do occur, the most likely reason that they are not detected is the absence of active post-transfusion monitoring for formation of anti-D.

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.

Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with ß-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

BACKGROUND: Infections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. PURPOSE: We evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. MATERIALS AND METHODS: Seventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and ß-lactamase production were also examined in A. baumannii. RESULTS: Sixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512µg/ml and from 128 to ≥1024µg/ml, respectively among the clinical isolates. MIC90 and MBC86 levels were 256µg/ml and 512µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall≤1.0µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of < 0.5 and cell numbers' decrease per ml of >2log10 within 12h, respectively. EGCG significantly increased the effect of NMP but was unrelated to ß-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. CONCLUSION: Overall we suggest that EGCG-antibiotic combinations might provide an alternative approach to treat infections with A. baumannii regardless of antibiotic resistance.

Promoter deletions of Klebsiella pneumoniae carbapenemase (KPC)-encoding genes (blaKPC -2) and efflux pump (AcrAB) on ß-lactam susceptibility in KPC-producing Enterobacteriaceae.

Klebsiella pneumoniae carbapenemase (KPC)-encoding genes containing promoter-deletions (bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) have disseminated in Enterobacteriaceae. The minimal inhibitory concentrations (MICs) to ß-lactams in clinical KPC-producing Enterobacteriaceae range from susceptible to high-level resistant, resulting in diagnostic problems. To better understand the variability in ß-lactam MICs among KPC-producing Enterobacteriaceae, three isoforms of bla(KPC-2) gene were used to transform Escherichia coli W4573 and its deletion mutant of an efflux pump (AcrAB) to examine the effects on ß-lactam susceptibility. MICs to ß-lactams in E. coli W4573 and its acrAB mutant strain increased 1- to 500-fold (MIC from 0.125 to 64 µg mL(-1) of aztreonam) in the bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) transformants compared with the cloning vector alone. However, transformants of the acrAB mutant strain remained susceptible to all ß-lactams tested except for aztreonam and carbenicillin. Levels of the three promoters' length and carbapenemase activities in the transformants harboring the bla(KPC-2a), bla(KPC-2b), and bla(KPC-2c) were correlated to the levels of ß-lactam MICs in both E. coli W4573 and its mutant of an efflux pump (AcrAB). Overall, these results suggest that promoter-deletions of bla(KPC-2) gene and AcrAB may be associated with the variability in ß-lactam MICs in KPC-producing Enterobacteriaceae.

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.

Carbapenem-associated multidrug-resistant Acinetobacter baumannii are sensitised by aztreonam in combination with polyamines.

Carbapenem-associated multidrug-resistant Acinetobacter baumannii (MDR-Ab) are common among clinical isolates worldwide and are a major therapeutic challenge. Previously it was shown that exogenous polyamines (spermine and spermidine) enhanced susceptibility to ß-lactams but induced resistance to polymyxins in Pseudomonas aeruginosa. This study aimed to explore the possible availability of exogenous polyamines in treating carbapenem-associated MDR-Ab. The effects of polyamines on the growth rate of A. baumannii, minimum inhibitory concentrations (MICs) of antibiotics, and time-kill and chequerboard assays were determined. Roles of lipopolysaccharide (LPS) and ß-lactamase activity of A. baumannii were also assessed for the polyamine effects. Growth of A. baumannii was unaffected at 4mM spermine and 16 mM spermidine alone, but was significantly inhibited by a subinhibitory concentration of aztreonam (AZT) (8 µg/mL) and those concentrations of the polyamines. MICs to AZT alone (≥128 µg/mL) were reduced to the range 0.25-8 µg/mL in combination with polyamines in all carbapenem-associated MDR-Ab. MICs to penicillins, but not to ceftazidime and meropenem, were also significantly reduced, whilst MICs to other antibiotics, including polymyxin B, were unaffected in combination with polyamines for all tested A. baumannii. Polyamine effects on AZT were strongly synergistic with bactericidal activity and were retained at concentrations of 5mM MgCl(2) (or CaCl(2)) or 200 mM NaCl. Roles of LPS and ß-lactamase in the polyamine effects were excluded. Overall results suggest that AZT in combination with polyamines may be useful for the treatment of carbapenem-associated MDR-Ab.

Differential Role of Two-Component Regulatory Systems (phoPQ and pmrAB) in Polymyxin B Susceptibility of Pseudomonas aeruginosa.

Polymyxins are often considered as a last resort to treat multidrug resistant P. aeruginosa but polymyxin resistance has been increasingly reported worldwide in clinical isolates. Polymyxin resistance in P. aeruginosa is known to be associated with alterations in either PhoQ or PmrB. In this study, mutant strains of P. aeruginosa carrying amino acid substitution, a single and/or dual inactivation of PhoQ and PmrB were constructed to further understand the roles of PhoQ and PmrB in polymyxin susceptibility. Polymyxin B resistance was caused by both inactivation and/or amino acid substitutions in PhoQ but by only amino acid substitutions of PmrB. Alterations of both PhoQ and PmrB resulted in higher levels of polymyxin B resistance than alteration of either PhoQ or PmrB alone. These results were confirmed by time-killing assays suggesting that high-level polymyxin resistance in P. aeruginosa is caused by alterations of both PhoQ and PmrB.

Co-existence of multidrug-resistant and -susceptible strains of Pseudomonas aeruginosa from a single clinical isolate.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and difficult to treat due to acquired-resistance to multiple antibiotics. A pair of strains, M38100A and M38100B, previously identified from a single clinical isolate of P. aeruginosa was investigated to understand phenotypic and genotypic characteristics. Results revealed that the pair of strains was very similar for serum susceptibility, growth rate in a complex medium (Luria-Bertani), RAPD-genotype profiles, status of genes encoding type III secretion toxins, and no extra-chromosomal DNA. However, antibiotic susceptibility of the strain M38100B showed resistant to all tested-antibiotics while the strain M38100A showed susceptible to the same tested-antibiotics as similar levels of P. aeruginosa PAO1. The strain M38100B exhibited no growth in a minimal medium as a sole carbon and nitrogen source of glutamate while the strain M38100A grew well in the same minimal medium. These results suggest that multidrug resistance of the strain M38100B may be caused by multiple mutations on its genomic DNA and a precursor stage for a homogeneous multidrug resistant population.

Alterations in two-component regulatory systems of phoPQ and pmrAB are associated with polymyxin B resistance in clinical isolates of Pseudomonas aeruginosa.

Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 microg/ml polymyxin B (MIC, 8 microg/ml), whereas P. aeruginosa PAO1 grew with 0.25 mug/ml polymyxin B (MIC, 0.5 microg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 microg/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 microg/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.

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.

Polyamines induce resistance to cationic peptide, aminoglycoside, and quinolone antibiotics in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa, a gram-negative bacterium of human pathogens, is noted for its environmental versatility, enormous metabolic capacity, and resistance to antibiotics. Overexpression of the outer membrane protein OprH and increased resistance to polycationic peptide antibiotics (e.g., polymyxin B) mediated by the PhoPQ two-component system on induction of a putative lipopolysaccharide (LPS) modification operon (PA3552-PA3559) have been reported as part of the adaptive responses to magnesium limitation in P. aeruginosa. Induction of the oprH-phoPQ operon and the LPS modification operon by exogenous spermidine was revealed from GeneChip analysis during studies of polyamine metabolism and was confirmed by the lacZ fusions of affected promoters. From the results of MIC measurements, it was found that addition of spermidine or other polyamines to the growth medium increased the MIC values of multiple antibiotics, including polycationic antibiotics, aminoglycosides, quinolones, and fluorescent dyes. MIC values of these compounds in the transposon insertion mutants of oprH, phoP, phoQ, and pmrB were also determined in the presence and absence of spermidine. The results showed that the spermidine effect on cationic peptide antibiotic and quinolone resistance was diminished in the phoP mutant only. The spermidine effect on antibiotics was not influenced by magnesium concentrations, as demonstrated by MICs and oprH::lacZ fusion studies in the presence of 20 muM or 2 mM magnesium. Furthermore, in spermidine uptake mutants, MICs of cationic peptide antibiotics and fluorescent dyes, but not of aminoglycosides and quinolones, were increased by spermidine. These results suggested the presence of a complicated molecular mechanism for polyamine-mediated resistance to multiple antibiotics in P. aeruginosa.

Polyamines increase antibiotic susceptibility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen. Treatment is complicated by frequent acquired resistance to antipseudomonal therapies. Polyamines (cadaverine, putrescine, spermidine, and spermine) are ubiquitous polycationic compounds essential for all living organisms. In a dose-dependent manner, polyamines increased the susceptibility of P. aeruginosa to 14 beta-lactam antibiotics, chloramphenicol, nalidixic acid, and trimethoprim as demonstrated by a reduction in MIC of up to 64-fold. This effect was partially antagonized (25 to 50%) by the presence of 10 mM of Mg(2+) or Ca(2+). In contrast, the effects of the outer membrane permeabilizers, polymyxin B nonapeptide and EDTA, were completely abolished by 3 mM Mg(2+) or Ca(2+). Changes on the outer membrane barrier by these compounds were assessed by activity measurements of periplasmic beta-lactamase. The results showed that while EDTA and polymyxin B serve as outer membrane disorganizing agents as expected, exogenous spermidine and spermine did not exhibit any apparent effect on outer membrane permeability or rupture. In summary, these results strongly suggest that the increased antibiotic susceptibility by polyamines is exerted by a mechanism that differs from that of EDTA and polymyxin B. Polyamines might be potentially useful in antipseudomonal therapies by increasing the effectiveness of certain beta-lactam antibiotics.

Isogenic variation of Helicobacter pylori strain resulting in heteroresistant antibacterial phenotypes in a single host in vivo.

BACKGROUND: Antibiotic-susceptible and -resistant Helicobacter pylori can be present simultaneously in the same host. The aim of this study was to evaluate the genomic diversity of H. pylori strains resulting in heteroresistant antibacterial phenotypes. MATERIALS AND METHODS: Twenty-one pairs of H. pylori strains isolated from the antrum and body displaying heteroresistant antibacterial phenotypes were included. We compared the genotypes of paired-isolates by random arbitrarily primed polymerase chain reaction (PCR), flagella gene PCR-based restriction fragment length polymorphism, and flaA gene sequencing. In metronidazole-heteroresistant isolates, the sequence variation of rdxA and frxA genes was analyzed using phylogenetic analysis. RESULTS: The DNA fingerprinting patterns of the paired isolates revealed that 12 pairs (57.1%) were identical, whereas one pair (3.8%) was different. The remaining eight pairs (38.1%) of isolates showed minor heterogenecity in fingerprinting patterns. In flaA gene sequencing, these identical and similar isolates showed close sequence similarity between the antrum and body, whereas different isolate showed 31 points of different nucleotide sequences. Phylogenetic analysis of the metronidazole-heteroresistant pairs showed consistent genetic relatedness of each paired isolates despite the sequence variation of the rdxA or frxA genes in five pairs (71.4%). CONCLUSIONS: These results suggest that continuing genomic diversities in the same strain may play an important role in modulating the antibiotic-heteroresistant H. pylori in vivo.

In vitro induction of resistance to metronidazole, and analysis of mutations in rdxA and frxA genes from Helicobacter pylori isolates.

In clinical Helicobacter pylori isolates, metronidazole resistance has been associated with mutations in the rdxA and frxA genes. The aim of this study was to examine the role of the rdxA and frxA genes after the in vitro induction of metronidazole resistance. A total of five suscep-tible H. pylori isolates were initially exposed to different subinhibitory metronidazole concentrations to induce in vitro resistance to metronidazole. Susceptible and resistant strains after the in vitro induction of resistance were examined to evaluate mutations of the rdxA and frxA genes by sequence analysis. After the in vitro induction of resistance, analysis revealed that two and four susceptible strains developed resistance when cultured with 0.3 microg/ml and 0.6 microg/ml of metronidazole, respectively. Before and after the induction of resistance, none of the susceptible strains that developed low and moderate levels of resistance presented any mutation in either of the evaluated genes, whereas strains with high-level metronidazole resistance contained a simple mutation of the frxA gene, but no specific changes in the rdxA gene. Strains with moderate-level resistance contained both single and multiple mutations of rdxA and frxA, respectively, and the low-level-metronidazole-resistant strain contained a single mutation in the frxA gene, without any significant change in the rdxA gene. In this study, the strains that developed resistance were mainly associated with mutations of the frxA gene, suggesting the possibility that inactivation of this gene could originate metronidazole resistance. The results after the in vitro induction of resistance to metronidazole suggested the presence of additional metronidazole resistance mechanisms, other than mutations of the rdxA and/or frxA genes.

Tetracycline-resistant clinical Helicobacter pylori isolates with and without mutations in 16S rRNA-encoding genes.

Tetracycline-resistant Helicobacter pylori strains have been increasingly reported worldwide. However, only a small number of tetracycline-resistant strains have been studied with regard to possible mechanisms of resistance and those studies have focused on mutations in the tetracycline binding sites of 16S rRNA-encoding genes. We here report studies of 41 tetracycline-resistant H. pylori strains (tetracycline MICs, 4 to 32 microg/ml) from North America (n = 12) and from East Asia (n = 29). DNA sequence analyses of 16S rRNA-encoding genes revealed that 22 (54%) of the resistant isolates carried one of five different single-nucleotide substitutions (CGA, GGA, TGA, AGC, or AGT) at the putative tetracycline binding site (AGA(965-967)). Single-nucleotide substitutions were associated with reduced ribosomal binding and with slightly increased tetracycline MICs (1 to 2 microg/ml). The 19 tetracycline-resistant isolates with no detectable mutations in the tetracycline binding site had normal tetracycline-ribosome binding. All tetracycline-resistant isolates, including those with and those without mutations in the tetracycline binding site, showed decreased accumulation of tetracycline. These results suggest that tetracycline resistance is multifactorial, involving alterations both in ribosomal binding and in membrane permeability.

High-level beta-lactam resistance associated with acquired multidrug resistance in Helicobacter pylori.

Four clinical Helicobacter pylori isolates with high-level resistance to beta-lactams exhibited low- to moderate-level resistance to the structurally and functionally unrelated antibiotics ciprofloxacin, chloramphenicol, metronidazole, rifampin, and tetracycline. This pattern of multidrug resistance was transferable to susceptible H. pylori by natural transformation using naked genomic DNA from a clinical multidrug-resistant isolate. Acquisition of the multidrug resistance was also associated with a change in the genotype of the transformed multidrug-resistant H. pylori. DNA sequence analyses of the gene encoding penicillin binding protein 1A (PBP 1A) showed 36 nucleotide substitutions resulting in 10 amino acid changes in the C-terminal portion (the putative penicillin binding domain). Acquisition of beta-lactam resistance was consistently associated with transfer of a mosaic block containing the C-terminal portion of PBP 1A. No changes of genes gyrA, rpoB, rrn16S, rdxA, and frxA, and nine other genes (ftsI, hcpA, llm, lytB, mreB, mreC, pbp2, pbp4, and rodA1) encoding putative PBPs or involved in cell wall synthesis were found among the transformed resistant H. pylori. Antibiotic accumulations of chloramphenicol, penicillin, and tetracycline were all significantly decreased in the natural and transformed resistant H. pylori compared to what was seen with susceptible H. pylori. Natural transformation also resulted in the outer membrane protein profiles of the transformed resistant H. pylori becoming similar to that of the clinical resistant H. pylori isolates. Overall, these results demonstrate that high-level beta-lactam resistance associated with acquired multidrug resistance in clinical H. pylori is mediated by combination strategies including alterations of PBP 1A and decreased membrane permeability.

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.