Methicillin-resistant Staphylococcus aureus (MRSA) is often resistant to multiple antibiotic classes and extremely difficult to treat, highlighting the urgent need for new drugs or alternative strategies. Hydroxyapatite (HAp) combined with silver (Ag) and zinc oxide (ZnO) to form an Ag/ZnO-HAp nanocomposite has been widely studied for its antibacterial properties. This nanocomposite is often assumed to produce additive or synergistic antibacterial effects. Four Ag/ZnO-HAp nanocomposites with different Ag-to-ZnO mole ratios: nanocomposite-A (0.005:0.005), nanocomposite-B (0.01:0.005), nanocomposite-C (0.005:0.01), and nanocomposite-D (0.01:0.01) were used to understand the roles of Ag and ZnO in antibacterial activity against MRSA ATCC700699. Agar diffusion tests of nanocomposites A, B, C, and D produced inhibition zones of 8, 9, 6, and 8 mm, respectively, whereas unmodified HAp showed no inhibition zone. Bactericidal assays revealed log reductions of 5.27, 7.16, 5.00, and 5.81 for nanocomposites A, B, C, and D at 0.0625 mg/mL over 18 h, respectively. Specific death rates (min-1) for nanocomposites A, B, C, and D over 240 min were 0.062, 0.075, 0.058, and 0.064; D-values (minutes) were 16.1, 13.3, 17.2, and 15.6, respectively. Minimum bactericidal concentrations for 9 MRSA and 1 methicillin-sensitive S. aureus clinical isolates ranged from ≤ 0.06 to > 2 mg/mL, regardless of antibiotic susceptibility profiles. Overall, the nanocomposites exhibit bactericidal activity in the order B > D> A > C. Ag content primarily drives bactericidal activity, whereas ZnO reduces Ag’s bactericidal effectiveness. These findings suggest that optimizing the Ag-to-ZnO ratio is crucial for maximizing bactericidal activity. Additionally, customizing the nanocomposites to clinical isolates may be essential to maximize biomedical applications.

The global spread of NDM-1-producing Klebsiella pneumoniae poses a serious therapeutic challenge because these strains are resistant to nearly all β-lactams and many other antibiotics. Glutathione (GSH) is essential for maintaining intracellular redox homeostasis, thereby protecting cells from oxidative damage. However, exogenous GSH may disrupt this homeostasis and increase bacterial vulnerability to oxidative stress, such as that induced by antibiotic exposure. This study investigates whether exogenous GSH exerts antibacterial activity and increases β-lactam susceptibility in NDM-1-producing K. pneumoniae ATCC BAA-2146, with potential implications for improved treatment strategies. Exogenous GSH inhibited the growth of NDM-1-producing K. pneumoniae at 5 and 10 mM. HCl-adjusted media matching the acidity of 10 mM GSH (pH 5.2 ± 0.1), but not 5 mM (pH 6.1 ± 0.1), similarly inhibited bacterial growth. Meropenem MICs decreased from 32 to 1 µg/mL with 5 mM GSH and to 8 µg/mL under matched acidity. Both 10 mM GSH and matched acidity decreased meropenem MICs from 32 to ≤ 0.25 µg/mL. For aztreonam, carbenicillin, and ceftazidime, 10 mM GSH decreased MICs 4-fold (> 256 to 64 µg/mL), whereas matched acidity decreased MICs 2-fold. In E. coli transformed with blaNDM−1, exogenous GSH and HCl-adjusted media matching the acidity of 5 and 10 mM GSH increased β-lactam susceptibility, comparable to the effects observed in NDM-1-producing K. pneumoniae. These results were confirmed by bacterial killing assays against NDM-1-producing K. pneumoniae and E. coli harboring blaNDM−1. Overall, GSH and GSH-induced acidity exhibited antibacterial activity and increased susceptibility to all β-lactam antibiotics, suggesting a potential therapeutic strategy against infections caused by NDM-1-producing K. pneumoniae.

Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterial pathogen that causes infections in the lungs, bloodstream, urinary tract, and wounds. This bacterium has become one of the most challenging pathogens for healthcare institutions worldwide. A big problem in treating these infections is that oxacillinase (OXA)-type carbapenemase-producing A. baumannii often resists many antibiotics that are not β-lactams, including almost all β-lactams. Glutathione is a tripeptide (l-glutathione, l-cysteine, and l-glycine) antioxidant synthesized in most Gram-negative bacteria and eukaryotic cells. Exogenous glutathione has been evaluated for antibacterial properties and various clinical implications. A previous study showed that exogenous glutathione significantly enhanced the susceptibility of β-lactam antibiotics in OXA-type carbapenemase-producing A. baumannii. However, the effect of exogenous glutathione on non-β-lactam antibiotics was unclear. In this study, the antibacterial activity of exogenous glutathione against A. baumannii was determined at various concentrations. Additionally, the effect of exogenous glutathione on several non-β-lactam antibiotics, including chloramphenicol, ciprofloxacin, erythromycin, novobiocin, kanamycin, and tetracycline, was evaluated on four OXA-type carbapenemase-producing strains of A. baumannii: blaOXA-23, blaOXA-24/40, blaOXA-51, and blaOXA-58. The results showed that exogenous glutathione significantly inhibited bacterial growth at concentrations greater than 10 mM, achieving complete growth inhibited at 16 mM. Interestingly, at the subinhibitory concentration of 10 mM, exogenous glutathione increased susceptibility (more sensitive) to chloramphenicol, novobiocin, and tetracycline by as much as 256-fold. Conversely, it decreased susceptibility (more resistant) to ciprofloxacin, erythromycin, and kanamycin by up to 32-fold at the same subinhibitory concentration. These findings highlight the need for caution when using glutathione in combination with antibiotics, especially for treating A. baumannii infections.

Pseudomonas aeruginosa is a human pathogen causing mild skin to life-threatening bloodstream infections. Antibiotic treatment of P. aeruginosa is uneasy because the bacterium possesses intrinsic resistance mechanisms to various antibiotics and can acquire resistance to nearly all available antibiotics. It was reported that some antibiotics can induce oxidative stress that contributes to cellular death, but bacterial cells can detoxify oxidative stress by the oxidative stress-scavenging systems. The relative amount of antibiotic-induced oxidative stress to oxidative stress-scavenging systems may determine the roles of antibiotic-induced oxidative stress in cellular death. Glutathione is one of the oxidative-scavenging systems and is synthesized by glutamyl-cysteine synthetase encoded by gshA and glutathione synthetase encoded by gshB. This study aims to determine the roles of glutathione in oxidative stress and antibiotic susceptibility in P. aeruginosa. Glutathione-encoding genes were knocked out in P. aeruginosa PAO1, and the mutant strains (gshA::Gm, gshB::Gm, and gshA::Gm/gshB::Tc) were used to determine susceptibility to hydrogen peroxide, superoxide-producing paraquat, and antibiotics. The mutant strains were 2- to eightfold more susceptible to hydrogen peroxide and superoxide and 2- to fourfold more susceptible to all tested antibiotics than their parental strain. The susceptibility of hydrogen peroxide, superoxide, and antibiotics was genetically complemented in P. aeruginosa PAO1. Overall results indicate that glutathione is crucial in detoxifying oxidative stress induced internally and by antibiotics in P. aeruginosa. This finding suggests that glutathione is one of the oxidative stress-scavenging systems and one of the intrinsic resistance mechanisms to antibiotics and, thus, a potential drug target for P. aeruginosa.

Quaternized cellulose nanocrystals (CNCs) were prepared by chloroacetylation and subsequent reaction with tertiary amines. The chloroacetylation of CNCs and quaternary ammonium-modified CNCs were characterized by the analysis of FTIR and solid-state NMR spectroscopies. Chloroacetylation of CNCs was found to be highly regioselective. CNCs can be chemically modified to tailor their properties to improve dispersion in the polymer matrix, which expands the application of CNCs as reinforcing materials. Biological evaluation of the ammonium-modified compounds was conducted using the disc diffusion test, minimum bactericidal concentration, and bacterial killing pattern for methicillin-associated multidrug-resistant clinical isolates of Staphylococcus aureus, one of the most problematic bacterial human pathogens. Results showed that the CNC with alkyl chains with 16 carbons had more antibacterial properties than that of 10 carbons against all tested clinical isolates of Staphylococcus aureus regardless of antibiotic resistance or sensitivity. This finding suggests that the ammonium-modified CNCs may be applicable to treat Staphylococcus aureus infections.

Bacterial cells growing under aerobic conditions produce reactive oxygen species that damage macromolecules, which leads to cellular death. However, bacterial cells possess oxidative stress-scavenging systems that detoxify these reactive oxygen species. Antibiotics can induce oxidative stress, which contributes to cellular death alongside their specific killing mechanisms. An imbalance between the oxidative stress-scavenging systems and antibiotic-induced oxidative stress may affect bacterial survival or death. Catalases are one of the oxidative stress-scavenging systems and detoxify hydrogen peroxide, thereby protecting cells from oxidative stress. Pseudomonas aeruginosa is a Gram-negative human pathogen and has multiple genes encoding catalase. This study explores the roles of catalase-encoding genes (katA and katB) in response to oxidative stress and antibiotics in P. aeruginosa. The catalase-encoding genes were knocked out in P. aeruginosa PAO1 and clinical isolates. The resulting mutant strains (katA::Tc, katB::Gm, katA::Tc/katB::Gm) were tested for their susceptibility to hydrogen peroxide, superoxide, and antibiotics. The results showed that the katA::Tc was more susceptible to hydrogen peroxide, while the katB::Gm was more susceptible to superoxide. MIC (minimum inhibitory concentration) levels for katB::Gm with chloramphenicol were decreased by 2- to 4-fold compared to the parental strain. However, MIC levels for katA::Tc remained unchanged for all antibiotics tested. These results indicate that katA and katB detoxify both hydrogen peroxide and superoxide, with katA being more effective against hydrogen peroxide and katB being more effective against superoxide than vice versa. Furthermore, katB appears to confer resistance to oxidative stress induced by chloramphenicol in P. aeruginosa.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and Pseudomonas aeruginosa, associated with systemic and hospital-acquired infections, have spread globally and pose a significant public health concern. Glutathione is a multifunctional thiol-antioxidant compound synthesized in most Gram-negative bacteria and crucial in maintaining intracellular redox homeostasis. Exogenous glutathione exhibits antibiotic properties and has differential effects on conventional antibiotics. Therefore, its effect on specific antibiotics needs to be clarified in bacterial species. In this study, we investigated the antibacterial activity of glutathione and its effect on meropenem susceptibility in KPC-producing bacteria. Two major KPC-encoding genes cloned from two different clinical KPC-producing K. pneumoniae were introduced into E. coli and P. aeruginosa. Then, the KPC-producing K. pneumoniae, E. coli, and P. aeruginosa were used for minimum inhibitory concentration (MIC), population analysis, checkerboard, and time-killing assays. The results showed that glutathione exhibited antibacterial activity at >10 mM in K. pneumoniae, E. coli, and P. aeruginosa. MIC levels of meropenem combined with 10 mM of glutathione were synergistically decreased by 8- to ≥ 256-fold in KPC-producing bacteria. Furthermore, this combination killed 100

Gram-negative bacterial pathogens are responsible for various infections. Over the past decade, these pathogens have acquired resistance to multiple antibiotics, and the multidrug-resistant (MDR) bacteria have rapidly spread globally, creating significant treatment challenges. Quaternized cellulose nanocrystals (CNCs) have promising antibacterial properties. We previously reported quaternized CNCs with ten-carbon (CNC-3) and sixteen-carbon (CNC-4) alkyl chains and an unmodified CNC (CNC-1). We found that CNC-4 exhibited a significant bactericidal effect against Staphylococcus aureus. In this study, we aim to evaluate the antibacterial properties of the quaternized CNCs against Gram-negative MDR clinical isolates of Acinetobacter baumannii (21 isolates), Klebsiella pneumoniae (18 isolates), and Escherichia coli (7 isolates), including each of their reference species. Agar diffusion, minimum bactericidal concentration (MBC), and bacterial killing pattern were conducted. The results showed that CNC-3 exhibited an MBC of 50 μg ml−1 for 28

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most problematic human pathogens.Antibiotic treatment of MRSA often associated with resistance to multiple classes of antibiotics is extremely challenging and urgently demands action to treat MRSA.Glutathione (GSH) is a biogenic thiol-compound that maintains an optimal intracellular redox-potential required for various normal cellular processes.Antibacterial activity of exogenous GSH has been reported in some bacterial pathogens but is largely unknown in MRSA.Aim: This study aimed to understand antibacterial activity of GSH, its role in antibiotic susceptibility, and a potential antibacterial mechanism in clinical isolates of S. aureus.Materials and Methods: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), checkerboard, time-killing, and bacterial killing assays were performed for 14 clinical isolates of S. aureus including 10 MRSA and two type strains (ATCC 700699 and 35556).Results: MIC and MBC levels for the clinical and type strains were 15 -20 mM and 25 -40 mM of GSH, respectively.Subinhibitory concentrations of GSH synergistically enhanced susceptibility of all tested-antibiotics, resulting in sensitizing all-tested S. aureus.Bacterial-killing produced by GSH-mediated acidity was significantly higher than that by hydrochloric acid-mediated acidity.Conclusion: Overall results concluded that GSH exhibited antibacterial activity on S. aureus regardless of antibiotic susceptibility and synergistically enhanced antibiotic susceptibility.Additionally, GSH-mediated acidity was one of the antibacterial mechanisms.These findings suggest that GSH may be a potential antimicrobial agent or adjuvant for the conventional anti-MRSA regimens.

Pseudomonas aeruginosa is a major causative agent of the hospital- and community-acquired infections. These infections are often antibiotic resistant and difficult to treat. Several intrinsic and acquired resistance mechanisms to antibiotics have reported in P. aeruginosa. Recently, oxidative- stress-scavenging-systems have suggested as a possible intrinsic resistance mechanism to antibiotics because oxidative stresses induced by bactericidal antibiotics contribute to bacterial killing effects. However, this remains controversial such that further clarification is required. Glutathione reductase is a key enzyme in the maintenance of the optimum level of intracellular glutathione-redox potential to ensure normal functioning of cellular processes including the detoxification of oxidative stress. In this study, the role of a glutathione-reductase-encoding gene (gor) in oxidative stress and antibiotic susceptibility was determined in P. aeruginosa. Results showed that a gor-mutant strain was more susceptible to hydrogen peroxide (but not superoxide) than the parental strain and 100% of cells were killed with 0.01% hydrogen peroxide while the parental strain survived at the same concentration of hydrogen peroxide. The gor-mutant strain was also more susceptible to carbenicillin, chloramphenicol, ciprofloxacin, and tetracycline than the parental strain, which was confirmed by bacterial killing-kinetics. These results suggest that the gor gene is associated with oxidative stress and susceptibility to bactericidal as well as bacteriostatic antibiotics and that the oxidative-stress-scavenging-systems may be a possible drug-target for multidrug resistant P. aeruginosa.

ABSTRACT A novel and simple surface modification of cellulose nanocrystals (CNC) was performed by chloroacetylation and subsequent reaction with tertiary amines to form quaternary ammonium modified CNCs. The acetylation of CNC and quaternary ammonium modified CNCs was confirmed using IR spectroscopy and solid state NMR spectroscopy. The 13 C NMR spectrum of quaternary ammonium modified CNC showed several additional resonances ranging from 14.5 ppm to 58.0 ppm compared to 13 C NMR spectrum of pure CNC, suggesting that alkyl chains have been added to the pure CNC. The disc diffusion method was used to evaluate the antimicrobial properties of quaternary ammonium modified CNCs. It was found that modified CNCs with alkyl chain longer than ten carbons are effective antimicrobial agents against Staphylococcus aureus and E. coli bacteria. These CNCs can be chemically modified to tailor the properties to improve dispersion in the polymer matrix. This will expand the application of CNC as a reinforcing material. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 44789.

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 β-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 β-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.

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 β-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 β-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.

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 beta-naphthylamide; PA beta N) 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. PA beta N 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 mu g/ml and at 256-1024 mu g/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 PA beta N with enhancing susceptibility to all tested antibiotics (up to >500-fold) at higher levels than either EGCG alone or PA beta N 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.

Metronidazole resistance is a key factor associated with Helicobacter pylori treatment failure. Although this resistance is mainly associated with mutations in the rdxA and frxA genes, the question of whether metronidazole resistance is caused by the inactivation of frxA alone is still debated. Furthermore, it is unclear whether there are other mutations involved in addition to the two genes that are associated with resistance. A metronidazole-resistant strain was cultured from the metronidazole-susceptible H. pylori strain 26695-1 by exposure to low concentrations of metronidazole. The genome sequences of both susceptible and resistant H. pylori strains were determined by Illumina next-generation sequencing, from which putative candidate resistance mutations were identified. Natural transformation was used to introduce PCR products containing candidate mutations into the susceptible parent strain 26695-1, and the metronidazole MIC was determined for each strain. Mutations in frxA (hp0642), rdxA (hp0954), and rpsU (hp0562) were confirmed by the Sanger method. The mutated sequence in rdxA was successfully transformed into strain 26695-1, and the transformants showed resistance to metronidazole. The transformants containing a single mutation in rdxA showed a low MIC (16 mg/liter), while those containing mutations in both rdxA and frxA showed a higher MIC (48 mg/liter). No transformants containing a single mutation in frxA or rpsU were obtained. Next-generation sequencing was used to identify mutations related to drug resistance. We confirmed that the mutations in rdxA are mainly associated with metronidazole resistance, and mutations in frxA are able to enhance H. pylori resistance only in the presence of rdxA mutations. Moreover, mutations in rpsU may play a role in metronidazole resistance.

Acinetobacter baumannii is one of the most important human pathogens causing a variety of nosocomial infections. Carbapenem antibiotics have been primarily used to treat the A. baumannii infections. However, carbapenem resistant A. baumannii producing carbapenemases causes serious treatment problems worldwide. Outbreaks of carbapenem resistant isolates have reported in some area of the United States, but their dissemination and genetic structure of the carbapenemase encoding genes are currently little known. To understand outbreaks, dissemination, and genetic structure of the carbapenemase encoding genes in Southern Texas, 32 clinical isolates collected from Austin and Houston, TX were characterized. Twenty-eight of 32 isolates were resistant to all tested β-lactam antibiotics including carbapenem (imipenem and meropenem). Three of them carried blaOXA-23 as a part of Tn2008 integrated into a known plasmid (pACICU2) and all others carried blaOXA-24 flanked by XerC/XerD-like recombinase binding sites that were adjoined by DNA sequences originated from multiple plasmids. Genotype analysis revealed that the 25 isolates carrying blaOXA-24 were all identical genotypes same as a representative isolate carrying blaOXA-24 from Chicago, IL but the 3 isolates carrying blaOXA-23 was a distinct genotype as compared with isolates carrying blaOXA-23 from Chicago, IL and Washington, D.C. Each of the blaOXA-23 and blaOXA-24 was transferred to carbapenem susceptible A. baumannii and E. coli with similar minimal inhibitory concentration (MIC) of carbapenem as that of their parental isolates but significantly lower levels of MIC in E. coli. Overall results suggest that a unique strain carrying blaOXA-23 and a similar strain carrying blaOXA-24 as seen in other geographic areas are currently disseminated in Southern Texas.

ABSTRACT We report here the complete genome sequence of a metronidazole-resistant Helicobacter pylori strain (MET r ). The MET r strain was obtained under exposure of H. pylori 26695 on agar plates with low metronidazole concentrations. The genome data provide insight into the genomic changes of H. pylori under selection by metronidazole in vitro .

Resistance to clarithromycin is the most important factor causing failure of Helicobacter pylori eradication. Although clarithromycin resistance is mainly associated with three point mutations in the 23S rRNA genes, it is unclear whether other mutations are associated with this resistance.Two types of clarithromycin-resistant strains (low- and high-resistance strains) were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations. The genome sequences were determined with a next-generation sequencer. Natural transformation was used to introduce the candidate mutations into strain 26695. Etest and an agar dilution method were used to determine the MICs.High-resistance strains contained the mutation A2143G in the 23S rRNA genes, whereas low-resistance strains did not. There were seven candidate mutations in six genes outside of the 23S rRNA genes. The mutated sequences in hp1048 (infB), hp1314 (rpl22) and the 23S rRNA gene were successfully transformed into strain 26695 and the transformants showed an increased MIC of and low resistance to clarithromycin. The transformants containing a single mutation in infB or rpl22 (either a 9 bp insertion or a 3 bp deletion) or the 23S rRNA gene showed low MICs (0.5, 2.0, 4.0 and 32 mg/L, respectively) while the transformants containing double mutations (mutation in the 23S rRNA genes and mutation in infB or rpl22) showed higher MICs (> 256 mg/L).Next-generation sequencing can be a useful tool for screening mutations related to drug resistance. We discovered novel mutations related to clarithromycin resistance in H. pylori (infB and rpl22), which have synergic effects with 23S rRNA resulting in higher MICs.

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 β-lactams in clinical KPC-producing Enterobacteriaceae range from susceptible to high-level resistant, resulting in diagnostic problems. To better understand the variability in β-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 β-lactam susceptibility. MICs to β-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 β-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 β-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 β-lactam MICs in KPC-producing Enterobacteriaceae.