Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics.

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

BACKGROUND: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. OBJECTIVES: To identify sources for the aac(6')-Im fragment found in A. baumannii. METHODS: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. RESULTS: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. CONCLUSIONS: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Possible impact of revisions in disc diffusion breakpoints for aminoglycosides and piperacillin/tazobactam in the 33rd edition of CLSI M100 document on clinical reporting and use in Indian settings with low susceptibility.

PURPOSE: The study explores the impact of significant interpretative breakpoint changes for aminoglycosides and piperacillin-tazobactam in Enterobacterales and Pseudomonas aeruginosa, considering PK/PD, clinical data, and susceptibility on clinical reporting and use. PROCEDURE: Between January 2021 and June 2023, a total of 189,583 samples were processed for bacterial pathogens and antimicrobial susceptibility testing was performed using disc diffusion method/VITEK® 2 Compact system/broth microdilution. WHONET software was utilised to capture and analyse the changes in the interpretation of disc diffusion method, following updates to CLSI M100 documents in comparison to previous editions. Antimicrobial consumption data was collected and interpreted as DDD/100 bed days using AMC tool software. Here, we present data for 13,615 members of Order Enterobacterales and 1793 Pseudomonas aeruginosa isolates. FINDING: Enterobacterales exhibited a significant susceptibility drop of 14.7% for gentamicin and 21.7% for amikacin. Pseudomonas aeruginosa showed an increase in isolates with intermediate tobramycin susceptibility, from 0.6% to 29.7%, with relatively minor changes in piperacillin-tazobactam interpretation. CONCLUSION: The changes indicate a shift toward increased 'resistance' and 'intermediate susceptibility' for these antibiotics, emphasizing the need for cautious use and leveraging PK/PD knowledge for improved antibiotic utilization, patient outcomes, and antimicrobial stewardship.

Proficiency of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China, 2008-2021.

To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency testing performed DST, and the sensitivity, specificity, reproducibility, and accordance rate were calculated from data of 13 rounds proficiency testing results for DST from 2008 to 2021. A total of 30 and 20 strains of Mycobacterium tuberculosis with known susceptibility results were sent to each laboratory in 2008 to 2019, 2020 and 2021, respectively. The number of participating laboratories ranged from 30 in 2009 to 546 in 2021. L-J DST was the predominant method. The specificity presented relatively higher than sensitivity. Improvement of specificity were observed for all drugs through the years, while sensitivity did not show improvement for amikacin and capreomycin. Accordance rate of pyrazinamide and kanamycin and reproducibility of capreomycin and pyrazinamide were not significantly improved through the years. Most of the participating laboratories significantly improved the quality of their DST through the consecutive rounds of proficiency testing except for second-line injectable drugs and pyrazinamide. The results highlight the importance of developing novel and/or improving existing methods for phenotypic DST for certain drugs.

Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Occurrence and characterization of rmtB-harbouring Salmonella and Escherichia coli isolates from a pig farm in the UK.

OBJECTIVES: To characterize and elucidate the spread of amikacin-resistant Enterobacteriaceae isolates from environmental samples on a pig farm in the UK, following the previous identification of index Salmonella isolates harbouring the rmtB gene, a 16S rRNA methylase. METHODS: Environmental samples were collected during two visits to a pig farm in the UK. Isolates were recovered using selective media (amikacin 128 mg/L) followed by real-time PCR and WGS to analyse rmtB-carrying Salmonella and Escherichia coli isolates. RESULTS: Salmonella and E. coli isolates harbouring the rmtB gene were detected at both farm visits. All Salmonella isolates were found to be monophasic S. enterica serovar Typhimurium variant Copenhagen of ST34. rmtB-harbouring E. coli isolates were found to be one of three STs: ST4089, ST1684 and ST34. Long-read sequencing identified the rmtB gene to be chromosomally located in Salmonella isolates and on IncFII-type plasmids in E. coli isolates. The results showed the rmtB gene to be flanked by IS26 elements and several resistance genes. CONCLUSIONS: We report on the occurrence of rmtB-harbouring Enterobacteriaceae on a pig farm in the UK. rmtB confers resistance to multiple aminoglycosides and this work highlights the need for surveillance to assess dissemination and risk.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

A pH/ROS-responsive antioxidative and antimicrobial GelMA hydrogel for on-demand drug delivery and enhanced osteogenic differentiation in vitro.

Long-term inflammation, including those induced by bacterial infections, contributes to the superfluous accumulation of reactive oxygen species (ROS), further aggravating this condition, decreasing the local pH, and adversely affecting bone defect healing. Conventional drug delivery scaffold materials struggle to meet the demands of this complex and dynamic microenvironment. In this work, a smart gelatin methacryloyl (GelMA) hydrogel was synthesized for the dual delivery of proanthocyanidin and amikacin based on the unique pH and ROS responsiveness of boronate complexes. Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) demonstrated the co-crosslinking of two boronate complexes with GelMA. The addition of the boronate complexes improved the mechanical properties, swelling ratio, degradation kinetics and antioxidative properties of the hydrogel. The hydrogel exhibited pH and ROS responses and a synergistic control over the drug release. Proanthocyanidin was responsively released to protect mouse osteoblast precursor cells from oxidative stress and promote their osteogenic differentiation. The hydrogel responded to pH changes and released sufficient amikacin in a timely manner, thereby exerting an efficient antimicrobial effect. Overall, the hydrogel delivery system exhibited a promising strategy for solving infectious and inflammatory problems in bone defects and promoting early-stage bone healing.

D-histidine combated biofilm formation and enhanced the effect of amikacin against Pseudomonas aeruginosa in vitro.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against Pseudomonas aeruginosa biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on Pseudomonas aeruginosa. Therefore, this study was undertaken to explore the effect of D-histidine on Pseudomonas aeruginosa in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in Pseudomonas aeruginosa PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating Pseudomonas aeruginosa by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against Pseudomonas aeruginosa biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by Pseudomonas aeruginosa.

Murepavadin promotes the killing efficacies of aminoglycoside antibiotics against Pseudomonas aeruginosa by enhancing membrane potential.

Murepavadin is a peptidomimetic that specifically targets the lipopolysaccharide transport protein LptD of Pseudomonas aeruginosa. Here, we found that murepavadin enhances the bactericidal efficacies of tobramycin and amikacin. We further demonstrated that murepavadin enhances bacterial respiration activity and subsequent membrane potential, which promotes intracellular uptake of aminoglycoside antibiotics. In addition, the murepavadin-amikacin combination displayed a synergistic bactericidal effect in a murine pneumonia model.

Antibiotic Resistance, Biofilm Formation, and Persistent Phenotype of Klebsiella pneumoniae in a Vietnamese Tertiary Hospital: A Focus on Amikacin.

Klebsiella pneumoniae stands out as a major opportunistic pathogen responsible for both hospital- and community-acquired bacterial infections. This study comprehensively assesses the antibiotic resistance, amikacin persistent patterns, and biofilm-forming ability of 247 isolates of K. pneumoniae obtained from an intensive care unit of a tertiary hospital in Vietnam. Microdilution assays, conducted on a 96-well plate, determined the minimum inhibitory concentrations (MICs) of amikacin. Susceptibility data for other antibiotics were gathered from the antibiogram profile. Stationary-phase bacteria were exposed to 50 × MIC, and viable bacteria counts were measured to determine amikacin persistence. Biofilm forming capacity on 96-well polystyrene surfaces was assessed by biomass and viable bacteria. The prevalence of resistance was notably high across most antibiotics, with 64.8% classified as carbapenem-resistant K. pneumoniae and 81.4% as multidrug resistant. Amikacin, however, exhibited a relatively low rate of resistance. Of the isolates, 58.2% demonstrated a moderate to strong biofilm formation capacity, and these were found to be poorly responsive to amikacin. K. pneumoniae reveals a significant inclination for amikacin persistence, with ∼45% of isolates displaying an antibiotic antibiotic-survival ratio exceeding 10%. The study sheds light on challenges in treating of K. pneumoniae infection in Vietnam, encompassing a high prevalence of antibiotic resistance, a substantial ability to form biofilm, and a notable rate of antibiotic persistence.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.IMPORTANCEAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6'-N-acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance.

Multifunctional gelatin nanoparticle stabilized-Pickering emulsion hydrogel based on dextran and amikacin with controlled drug release and enhanced antibacterial capability for promoting infected wound healing.

Plant essential oils possess broad-spectral antimicrobial property, but the applications are impeded by their insolubility in water, extreme volatility, and strong irritation. Nanoparticle-stabilized emulsion (Pickering emulsion) gels are colloidal systems with ability to accommodate two immiscible phases in one system. The thick adsorption nanoparticle layers and the cross-linked networks in continuous phase could provide protective barriers for antibacterial oil and achieve on-demand controlled release. An emulsion hydrogel templated from gelatin nanoparticle-stabilized emulsion is one-pot constructed by conducting a tunable cross-linking process between oxidized dextran (Odex) and amikacin in the continuous phase and concomitantly trapping tea tree essential oil (TO) droplets in the three-dimensional network. The resulted emulsion hydrogel presents tunable gelation time, adequate mechanical strength, fascinating injectability, and self-healing capability. It is pH-responsiveness and presents controlled release of amikacin and TO, exhibiting a long-term bacteriostasis of 144 h. The emulsion hydrogel facilitates the outstanding wound healing efficiency in 14 days (95.2 ± 0.8 % of wound closure), accompanied with enhanced collagen deposition and angiogenic activities. The incorporation of TO into emulsion hydrogel system reduced its irritation and improved its biosafety, showing potential application in bacteria inhibition even as implants in vivo.

Amikacin sulphate loaded chitosan-diopside nanoparticles composite scaffold for infectious wound healing.

A wound dressing material should inhibit infections that may occur at the wound site, and at the same time, it should enhance the healing process. In this study, we developed an amikacin sulphate (AK) incorporated chitosan (Ch) and Diopside nanoparticles composite dressing (Ch-nDE-AK) for controlling wound infection and healing. The diopside nanoparticles (nDE) were prepared using sol-gel synthesis and characterized using XRD, FT-IR, and FESEM. nDE shows a size range of 142 ± 31 nm through FESEM analysis. Later, the developed composite dressing was characterized using SEM, EDS, and FT-IR analysis. Ch-nDE-AK dressing possesses a porous nature that will aid in easy cell infiltration and proliferation. The swelling studies indicated the expansion capability of the scaffold when applied to the injured site. Ch-nDE-AK scaffold showed a 69.6 ± 8.2 % amikacin sulphate release up to 7 days, which indicates the sustained release of the drug from Ch-nDE-AK scaffold. The drug release data was subjected to various kinetics models and was observed to follow the Higuchi model. The scaffold showed antibacterial activity against ATCC strains of S. aureus and E. coli for 7 days by in vitro. Ch-nDE-AK scaffold also showed antibacterial activity against S. aureus and E. coli clinical strains in vitro. The ex vivo antibacterial study confirmed the antibacterial ability of Ch-nDE-AK scaffold against S. aureus and E. coli. Ch-nDE-AK scaffold also exhibits anti-biofilm activity against S. aureus and E. coli. The Ch-nDE-AK scaffold showed cytocompatibility and cell attachment to fibroblast cells. Additionally, the scratch assay using fibroblast cells confirmed the role of the nDE in the scaffold, helping in cell migration. Thus, the developed Ch-nDE-AK dressing can potentially be used to treat infectious wound healing.

Wound Infection in Surgical Ward of a Tertiary Care Hospital in Dhaka City: The Identification of Organisms and Their Sensitivity Pattern.

Wound infection is one of the most important causes of morbidity and mortality worldwide. The aim of this study was to identify the organisms and their sensitivity pattern from wound infection patients attending in a tertiary care hospital in Dhaka city. This cross-sectional study was carried out in a total of 240 aseptically collected wound swab samples from wound infection suspected patients visiting Bangladesh Medical College Hospital, Dhaka, Bangladesh were analyzed from July 2017 to June 2019. Bacteriological culture of the samples, colony morphology, Gram's staining, and biochemical tests were done following standard microbiological techniques. The antimicrobial susceptibility testing was performed by modified Kirby-Bauer disc diffusion technique following clinical and laboratory standards institute guidelines. Out of 240 wound swab samples from suspected patients of wound infection, 126(52.5%) showed bacterial growth whereas 114(47.5%) were culture negative. No sample yielded more than one organism. Among 126 culture positive cases 75(59.52%) were male and 51(40.48%) were female. The higher rate of bacterial infections 26.19% was noted in the age group of 21-30 years, followed by the age group of 31-40 years, 41-50 years, 51-60 years. Among 126 culture positive cases, 74.6% were Gram negative and 25.4% were Gram positive bacteria. Out of total 126 isolates, E. coli was the most prevalent pathogen 31(24.60%) followed by Staphylococcus aureus 29(23.01%); Pseudomonas 27(21.43%); Klebsiella 18(14.29%); Enterobacter 12(9.52%); Acinetobacter 4(3.17%), while Coagulase negative Staphylococcus 3(2.38%) and Proteus 2(1.59%) were least detected isolates in wound swab. Highly effective antibiotics against Staph aureus were vancomycin 100.0%; imipenem 100.0%; linezolid 100.0% and meropenem 89.65%. Amikacin; gentamicin; netilmicin; imipenem and meropenem showed higher sensitivity in E coli, Klebsiella and Enterobacter species. Colistin was 88.88% effective against Pseudominas spp. followed by imipenem 81.48%, piperacillin-tazobactam 77.78%, meropenem 70.37% and amikacin 51.85%. Acinetobacter spp. showed 75.0% and 50.0% sensitivity to netilmicin and colistin respectively. Injectable and reserve drugs were sensitive to bacterial populations among patients of wound infections in our hospital. It is a wake-up call for clinician to treat wound infections. To prevent the increase resistance to antibiotics, it is necessary to avoid the administration of uncontrolled and unnecessary antibiotics available.

WarA, a remote homolog of NpmA and KamB from Nocardia wallacei, confers broad spectrum aminoglycoside resistance in Nocardia and Mycobacteria.

OBJECTIVES: Aminoglycoside resistance in bacteria is typically conferred by specific drug-modifying enzymes. Infrequently, such resistance is achieved through 16S ribosomal RNA methyltransferases, such as NpmA and KamB encoded by Escherichia coli and Streptoalloteichus tenebrarius, respectively. These enzymes are not widespread and have not been described in Nocardia species to date. METHODS: We report the genomic mining of 18 Nocardia wallacei isolates that were found to be specifically and substantially resistant to amikacin. RESULTS: We identified a gene coding for a protein with very distant homology to NpmA and KamB. However, 3-D modeling revealed that the tertiary structure of these three proteins was highly similar. Cloning and expressing this gene in two susceptible bacteria Nocardia asteroides, and Mycobacterium smegmatis (another Actinobacterium) led to high-level, pan-aminoglycoside resistance in both cases. We named this gene warA (Wallacei Amikacin Resistance A). CONCLUSIONS: This is the first description and experimental characterization of a gene of this family in Nocardia, and the first demonstration that such activity could lead to pan-aminoglycoside resistance in Mycobacteria as well. The discovery of this novel gene has important biotechnology and clinical implications.

Molecular pharmacodynamics of meropenem for nosocomial pneumonia caused by Pseudomonas aeruginosa.

Hospital-acquired pneumonia (HAP) is a leading cause of morbidity and mortality, commonly caused by Pseudomonas aeruginosa. Meropenem is a commonly used therapeutic agent, although emergent resistance occurs during treatment. We used a rabbit HAP infection model to assess the bacterial kill and resistance pharmacodynamics of meropenem. Meropenem 5 mg/kg administered subcutaneously (s.c.) q8h (±amikacin 3.33-5 mg/kg q8h administered intravenously[i.v.]) or meropenem 30 mg/kg s.c. q8h regimens were assessed in a rabbit lung infection model infected with P. aeruginosa, with bacterial quantification and phenotypic/genotypic characterization of emergent resistant isolates. The pharmacokinetic/pharmacodynamic output was fitted to a mathematical model, and human-like regimens were simulated to predict outcomes in a clinical context. Increasing meropenem monotherapy demonstrated a dose-response effect to bacterial kill and an inverted U relationship with emergent resistance. The addition of amikacin to meropenem suppressed the emergence of resistance. A network of porin loss, efflux upregulation, and increased expression of AmpC was identified as the mechanism of this emergent resistance. A bridging simulation using human pharmacokinetics identified meropenem 2 g i.v. q8h as the licensed clinical regimen most likely to suppress resistance. We demonstrate an innovative experimental platform to phenotypically and genotypically characterize bacterial emergent resistance pharmacodynamics in HAP. For meropenem, we have demonstrated the risk of resistance emergence during therapy and identified two mitigating strategies: (i) regimen intensification and (ii) use of combination therapy. This platform will allow pre-clinical assessment of emergent resistance risk during treatment of HAP for other antimicrobials, to allow construction of clinical regimens that mitigate this risk.IMPORTANCEThe emergence of antimicrobial resistance (AMR) during antimicrobial treatment for hospital-acquired pneumonia (HAP) is a well-documented problem (particularly in pneumonia caused by Pseudomonas aeruginosa) that contributes to the wider global antimicrobial resistance crisis. During drug development, regimens are typically determined by their sufficiency to achieve bactericidal effect. Prevention of the emergence of resistance pharmacodynamics is usually not characterized or used to determine the regimen. The innovative experimental platform described here allows characterization of the emergence of AMR during the treatment of HAP and the development of strategies to mitigate this. We have demonstrated this specifically for meropenem-a broad-spectrum antibiotic commonly used to treat HAP. We have characterized the antimicrobial resistance pharmacodynamics of meropenem when used to treat HAP, caused by initially meropenem-susceptible P. aeruginosa, phenotypically and genotypically. We have also shown that intensifying the regimen and using combination therapy are both strategies that can both treat HAP and suppress the emergence of resistance.

Comparing minimum inhibitory concentrations of amikacin for pulmonary Mycobacterium avium complex disease: An analysis of culture media differences.

Mycobacterium avium complex (MAC) is considered a paramount microbe, especially in East Asia, including Japan. The commonly used commercial Minimum Inhibitory Concentrations (MIC) assay using Middlebrook 7H9 (7H9) medium deviates from the latest Clinical and Laboratory Standards Institute (CLSI) guidelines. Alternatively, measurement with cation-adjusted Mueller-Hinton broth (CAMHB) that conforms to CLSI standards is not yet widely available. Following the approval and commercialization of amikacin liposome inhalation suspension (ALIS) in 2021, a more precise evaluation of amikacin (AMK) susceptibility in MAC is necessary for treatment decisions. In the present study, 33 sputum samples were extracted from 27 patients, and MICs of AMK were compared between the frequently used 7H9 and the recommended CAMHB of the isolated MAC strains. The history of exposure to aminoglycosides for each sample was also added as clinical information. The findings indicated that there was only an 18% concordance rate in MIC between the two media, with 19 samples (58%) indicating lower MICs in 7H9 relative to CAMHB. The 17 samples had a history of exposure to aminoglycosides for periods ranging from 1.5 to 28 months. Specifically, 10 samples were exposed to amikacin by inhalation and intravenous injection, and the remaining seven samples had a history of ALIS inhalation. Samples with a prior utilization of aminoglycosides were significantly predisposed to developing resistance to ALIS compared to those without such a history (P = 0.046). Physicians are encouraged to scrutinize the findings of susceptibility testing utilizing CLSI-endorsed MIC assay using CAMHB medium to ascertain the optimal therapeutic approach.

GlnR activated transcription of nitrogen metabolic pathway genes facilitates biofilm formation by mycobacterium abscessus.

OBJECTIVES: Nitrogen is indispensable for the synthesis of biomacromolecules. The correlation between nitrogen metabolism and Mycobacterium abscessus (M. abscessus) biofilm formation is unclear. This study constructed global nitrogen regulator gene GlnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains. METHODS: Global nitrogen regulator gene glnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains were constructed. Sauton's medium was used to culture M. abscessus pellicle biofilm. To test the antibiotic susceptibility of pellicle biofilm, clarithromycin, amikacin, cefoxitin or imipenem was added to the medium under biofilms after 14 days of incubation. RT-qPCR and ChIP-qPCR were performed to analyse the transcriptional regulatory function of GlnR. RESULTS: GlnR knockout decreased the growth rate of planktonic cells, reduced biofilm mass and wrinkle formation, and diminished the resistance of biofilms to antibiotics. However, the susceptibility of planktonic cells to antibiotics was not changed by glnR knockout. The growth rate of planktonic ΔglnR cells was accelerated by adding nitrogen sources to the medium; the addition of glutamine or sodium glutamate rescued ΔglnR biofilm morphology and resistance to amikacin, cefoxitin, clarithromycin and imipenem. GlnR bound the promoter region and activated the transcription of eight nitrogen metabolic pathway genes (i.e. glnA, amt, ansP, nirB, nirD, glnD, glnK and narK3), which are closely related to glutamine/glutamate biosynthesis and, thus, regulate biofilm formation. CONCLUSION: This study provides insights into the mechanisms of M. abscessus biofilm formation and its resistance to antibiotics.