MICs Of Amikacin Research Articles

New Bacteriophage Pseudomonas Phage Ka2 from a Tributary Stream of Lake Baikal

Pseudomonas aeruginosa, an opportunistic pathogen, causes various biofilm-associated infections like pneumonia, infections in cystic fibrosis patients, and urinary tract and burn infections with high morbidity and mortality, as well as low treatment efficacy due to the extremely wide spread of isolates with multidrug resistance. Here, we report the new bacteriophage Pseudomonas phage Ka2 isolated from a tributary stream of Lake Baikal and belonging to the Pbunavirus genus. Transmission electron microscopy resolved that Pseudomonas phage Ka2 has a capsid of 57 ± 9 nm and a contractile and inflexible tail of 115 ± 10 nm in the non-contracted state. The genome consists of 66,310 bp with a GC content of 55% and contains 96 coding sequences. Among them, 52 encode proteins have known functions, and none of them are potentially associated with lysogeny. The bacteriophage lyses 21 of 30 P. aeruginosa clinical isolates and decreases the MIC of amikacin, gentamicin, and cefepime up to 16-fold and the MIC of colistin up to 32-fold. When treating the biofilms with Ka2, the biomass was reduced by twice, and up to a 32-fold decrease in the antibiotics MBC against biofilm-embedded cells was achieved by the combination of Ka2 with cefepime for the PAO1 strain, along with a decrease of up to 16-fold with either amikacin or colistin for clinical isolates. Taken together, these data characterize the new Pseudomonas phage Ka2 as a promising tool for the combined treatment of infections associated with P. aeruginosa biofilms.

Amikacin treatment in patients with Enterobacterales bacteraemia: impact of MIC on mortality.

Recently, breakpoints of Enterobacterales to amikacin were changed from MIC ≤ 16 mg/L to MIC ≤ 4 mg/L based mainly on laboratory data with little supporting clinical evidence. Our aim was to investigate the relation between MIC of Enterobacterales to amikacin and mortality among patients with Enterobacterales bacteraemia from a urinary tract source treated with amikacin. This retrospective, single-centre study included patients with Enterobacterales urinary source bacteraemia treated with amikacin, with Low (MIC ≤ 4 mg/L) and High (MIC 8 or 16 mg/L) MICs. A cohort of patients treated with ertapenem was used to assess if amikacin MIC is a marker of severity independent of antimicrobial treatment. The primary outcome was 30-day mortality. Multivariate logistic regression analysis was done to assess risk factors for mortality. We included 85 patients, 46 (54.1%) were male, and mean age was 79.0 years (SD 11.7). Sixty-one patients (71.8%) had Low MIC and 24 (28.2%) had High MIC. Thirty-day mortality was 8.2% and 29.2% in the Low and High MIC groups, respectively (P = 0.031). Risk factors for 30-day mortality were age, infection by Enterobacterales other than Escherichia coli and high amikacin MIC. In a cohort of 88 patients treated with ertapenem, amikacin MIC was not associated with 30-day mortality. We demonstrated a relation between higher amikacin MIC levels (8 and 16 mg/L) and increased 30-day mortality in patients treated with amikacin for bacteraemia secondary to a urinary source. These findings support the new CLSI breakpoint change of Enterobacterales to amikacin.

Development of antibacterial drug + bacteriophage combination assays

Abstract Background The best methods to study the interactions between phages and antibacterials are unclear. As laboratory methodologies used to assess conventional antibacterials are established, we assessed their ability to evaluate phage plus antibacterial. Methods The efficacy of three characterized Escherichia coli phages (UP17, JK08, 113) were tested on a 100 MDR E coli. strains. The phages were assessed individually and in a 1:1:1 cocktail. In a phage microbial inhibitory concentration (PmIC) assay, a range of phage concentrations from 101 to 108 were inoculated with 5 × 105 bacteria/well in 96-well microtitre plates. The first fully lysed well was taken as the PmIC. Amikacin and meropenem MICs were determined by ISO 2776-1:2019 methods alone and in combination with a fixed phage concentration of 105/per well. Time–kill curves (TKCs) were conducted at fosfomycin concentrations of 133, 50 and 5 mg/L, with and without phage. Results The PmIC50/90 values, in plaque-forming units (pfu)/mL, for individual phage titre were >108/>108 for UP17, 107/>108 for JK08, 107/>108 for 113 and 106/>108 for the 1:1:1 phage cocktail, all with a standard deviation (SD) of <0.05. Amikacin and meropenem MIC50/90 (SD) values were 2/8 mg/L (<0.05–9.2) and 0.12/8 mg/L (<0.05–8.7), respectively. The addition of UP17 to amikacin increased amikacin MICs >2-fold in 78 strains, with equivalent trends in 39 strains with JK08, 54 strains with 113 and 45 strains with phage cocktail. Meropenem MICs in the presence of phage were reduced >2-fold in 24 strains with UP17. Equivalent decreases were seen with 34 strains with JK08, 26 strains with 113 and 29 strains with the cocktail. In TKCs, the addition of phage suppressed regrowth. Conclusions Microbroth methodologies based on ISO 2776-1:2019 lend themselves to be viable options for phage assessment, alone or in combination with antibiotics. The reproducibility of PmIC and familiarity of the methodology described allows for laboratory validation for phage and antibiotic combinations.

Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa.

Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum β-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate. PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other β-lactams.

Tigecycline Resistance-Associated Mutations in the MepA Efflux Pump in Staphylococcus aureus.

Tigecycline is an important antibacterial drug for treating infection by clinical multidrug-resistant bacteria, and tigecycline-resistant Staphylococcus aureus (TRSA) has been increasingly reported in recent years. Notably, only rpsJ and mepA are associated with the tigecycline resistance of S. aureus. The mepA gene encodes MepA efflux pumps, and the overexpression of mepA has been confirmed to be directly related to tigecycline resistance. Although the mutations of MepA widely occur, the associations between TRSA and mutations of MepA are still unclear. In this study, we explored mutations in the mepA genes from various sources. Then, tigecycline resistance-associated mutations T29I, E287G, and T29I+E287G in MepA were identified, and their effects were evaluated through mutant deletion and complementation, tigecycline accumulation assay, and molecular docking experiments. Results showed that the MICs of tigecycline, gentamicin, and amikacin increased in special complementary transformants and recovered after the addition of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The tigecycline accumulation assay of the mepA-deleted mutant strain and its complementary transformants showed that T29I, E287G, and T29I+E287G mutations promoted tigecycline efflux, and molecular docking showed that mutations T29I, E287G, and T29I+E287G decreased the binding energy and contributed to ligand binding. Moreover, we inferred the evolutionary trajectory of S. aureus under the selective pressure of tigecycline in vitro. Overall, our study indicated that mutations in MepA play important roles in tigecycline resistance in S. aureus. IMPORTANCE Previous analysis has shown that overexpression of MepA is an exact mechanism involved in tigecycline resistance apart from the rpsJ mutation and is usually dependent on the mutant mepR. However, no research has evaluated the effects of diverse mutations discovered in TRSA in MepA. This study demonstrates that the mutations in MepA confer resistance to tigecycline without overexpression and provides genotypic references for identifying TRSA. Although tigecycline resistance-associated mutations in MepA identified in this study have not been observed in clinical isolates, the mechanism should be explored given that S. aureus strains are prevalent in the environment. Measures should be implemented to contain TRSA within the time window before tigecycline resistance-associated mutations in MepA are prevalent.

148. Imipenem-Relebactam activity and genotypic characteristics of Carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa Isolates from Latin American Infections - Study for Monitoring Antimicrobial Resistance Trends (SMART) 2017 – 2020

Abstract Background Imipenem/relebactam (IMI/REL) is a combination of Imipenem with Relebactam, an inhibitor of class A and C β-lactamases and has been approved in the US and EU, but not in Latin America. This report evaluates the in vitro activity of IMI/REL and comparators against Latin America (LATAM) Enterobacterales and P. aeruginosa (PSA) and the frequency of carbapenemase encoding genes (CEG) among gram-negative bacilli (GNB) isolated through the SMART Program (2017-2020). Methods 21606 nonconsecutive GNB isolates were collected in 10 LATAM countries. MICs for amikacin (AK), ceftazidime-avibactam (C-A), ceftolozane/tazobactam (C/T) and IMI/REL were determined by broth microdilution and interpreted by CLSI; a subset of Enterobacterales and P. aeruginosa carbapenem resistant was selected for characterization of carbapenemase encoding genes by PCR followed by DNA sequencing. Results Escherichia coli (N=9872; EC) tested susceptible to > 96% of all antibiotics analyzed; for P. aeruginosa (N=4528), C/T and C-A had the best susceptibility rates (85.7 and 86.6% respectively); for Enterobacter cloacae (N=1091; ECL) and Klebsiella pneumoniae (N=6115; KPN), we note that only IMI/REL and C-A were ≥ 95%. Profile of 2845 carbapenem resistant isolates was analysed and the main isolated agent in most countries was KPN, corresponding to 55% (1470), except in Mexico, Panama and Venezuela where PSA was the main carbapenemase producer. The blakpc-2, 3 were found in KPN 76.7, 79.2, 53.3, 52.1 and 85.3% in Argentina, Brazil, Colombia, Ecuador and Puerto Rico, respectively; Guatemala, Mexico, Venezuela presented blaNDM-1 in 74.3, 44.1 and 51.4%. Among ECL blakpc-2 (35.7%-Brazil, 48.2%-Colombia) and blaNDM-1(45.7%-Mexico) were most frequent, blaVIM-24 (16.6%) and blaIMP-18 (38.8%) were observed in Venezuela and Puerto Rico and in the latter, we observed first time reported blakpc-45 in 27.7 %. PSA expressed blakpc-2 (33.33%- Colombia), Chile and Venezuela blaVIM-2 (44.3%, 58.8%); blaspm-1 occurred only in Brazil (6.9%). Most common carbapenemase-producing bacteria in LATAM (n=2845) Antimicrobial susceptibility of Enterobacterales and P. aeruginosa (full data analysis=21606) Most Frequent CEG detected among carbapenemase producing isolates in Latin American countries (Genotypical sample analyzed n=2845) Conclusion The frequency of CEG is a threat in LATAM, mostly in Enterobacterales, whereas PSA as expected, has a lower frequency, but still a concern. In LATAM, IMI/REL has shown relevant activity against CEG producers, showing it is an option for treatment infections caused by MDR strains. Disclosures Gustavo Mizuno, PharmD, MSD Brazil: Employee Thales Polis, MD, MSD Brazil: Employee Jacqueline Ferrari, MD, MSD Brazil: Employee Jacqueline Pavia, MD, MSD COLOMBIA: EMPLOYEE Elisa Beirão, MD, MSD BRAZIL: Grant/Research Support Ana C. Gales, MD, MSD BRAZIL: Grant/Research Support Felipe Tuon, MD, MSD BRAZIL: Grant/Research Support Alexandre Alcantara, PharmD, MSD BRAZIL: EMPLOYEE Paula M. Batista, PharmD, MSD BRAZIL: EMPLOYEE.

In Vitro and In Vivo Effect of Amikacin and Imipenem Combinations against Multidrug-Resistant E. coli.

Introduction: The emergence of multidrug-resistant (MDR) E. coli has developed worldwide; therefore, the use of antibiotic combinations may be an effective strategy to target resistant bacteria and fight life-threatening infections. The current study was performed to evaluate the in vitro and in vivo efficacy of amikacin and imipenem alone and in combination against multidrug-resistant E. coli. Methods: The combination treatment was assessed in vitro using a checkerboard technique and time-killing curve and in vivo using a peritonitis mouse model. In resistant isolates, conventional PCR and quantitative real-time PCR techniques were used to detect the resistant genes of Metallo-β-lactamase gene Imipenemase (bla-IMP) and aminoglycoside 6′-N-acetyltransferase (aac (6′)-Ib). Scanning electron microscopy was used to detect the morphological changes in the resistant isolates after treatment with each drug alone and in combination. In vitro and in vivo studies showed a synergistic effect using the tested antibiotic combinations, showing fractional inhibitory concentration indices (FICIs) of ≤0.5. Regarding the in vivo study, combination therapy indicated a bactericidal effect after 24 h. E. coli isolates harboring the resistant genes Metallo-β-lactamase gene Imipenemase (bla-IMP) and aminoglycoside 6′-N-acetyltransferase (aac (6′)-Ib) represented 80% and 66.7%, respectively, which were mainly isolated from wound infections. The lowest effect on Metallo-β-lactamase gene Imipenemase (bla-IMP) and aminoglycoside 6′-N-acetyltransferase (aac (6′)-Ib) gene expression was shown in the presence of 0.25 × MIC of imipenem and 0.5 × MIC of amikacin. The scanning electron microscopy showed cell shrinkage and disruption in the outer membrane of E. coli in the presence of the antibiotic combination. Amikacin and imipenem combination can be expected to be effective in the treatment and control of serious infections caused by multidrug-resistant (MDR) E. coli and the reduction in bacterial resistance emergence.

Apramycin susceptibility of multidrug-resistant Gram-negative blood culture isolates in five countries in Southeast Asia

IntroductionBloodstream infections (BSIs) are a leading cause of sepsis, which is a life-threatening condition that significantly contributes to the mortality of bacterial infections. Aminoglycoside antibiotics such as gentamicin or amikacin are essential medicines in the treatment of BSIs, but their clinical efficacy is increasingly being compromised by antimicrobial resistance. The aminoglycoside apramycin has demonstrated preclinical efficacy against aminoglycoside-resistant and multidrug-resistant (MDR) Gram-negative bacilli (GNB) and is currently in clinical development for the treatment of critical systemic infections. MethodsThis study collected a panel of 470 MDR GNB isolates from healthcare facilities in Cambodia, Laos, Singapore, Thailand and Vietnam for a multicentre assessment of their antimicrobial susceptibility to apramycin in comparison with other aminoglycosides and colistin by broth microdilution assays. ResultsApramycin and amikacin MICs ≤ 16 µg/mL were found for 462 (98.3%) and 408 (86.8%) GNB isolates, respectively. Susceptibility to gentamicin and tobramycin (MIC ≤ 4 µg/mL) was significantly lower at 122 (26.0%) and 101 (21.5%) susceptible isolates, respectively. Of note, all carbapenem and third-generation cephalosporin-resistant Enterobacterales, all Acinetobacter baumannii and all Pseudomonas aeruginosa isolates tested in this study appeared to be susceptible to apramycin. Of the 65 colistin-resistant isolates tested, four (6.2%) had an apramycin MIC > 16 µg/mL. ConclusionApramycin demonstrated best-in-class activity against a panel of GNB isolates with resistances to other aminoglycosides, carbapenems, third-generation cephalosporins and colistin, warranting continued consideration of apramycin as a drug candidate for the treatment of MDR BSIs.

Detection and characterisation of 16S rRNA methyltransferase-producing Pseudomonas aeruginosa from the UK and Republic of Ireland from 2003–2015

16S rRNA methyltransferase (16S RMTase) genes confer high-level aminoglycoside resistance, reducing treatment options for multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa isolates (n=221) exhibiting high-level pan-aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) were screened for 16S RMTase genes to determine their occurrence among isolates submitted to a national reference laboratory from December 2003 to December 2015. 16S RMTase genes were identified using two multiplex PCRs, and whole-genome sequencing (WGS) was used to identify other antibiotic resistance genes, sequence types (STs) and the genetic environment of 16S RMTase genes. 16S RMTase genes were found in 8.6% (19/221) of isolates, with rmtB4 (47.4%; 9/19) being most common, followed by rmtD3 (21.1%; 4/19), rmtF2 (15.8%; 3/19) and single isolates harbouring rmtB1, rmtC and rmtD1. Carbapenemase genes were found in 89.5% (17/19) of 16S RMTase-positive isolates, with blaVIM (52.9%; 9/17) being most common. 16S RMTase genes were found in 'high-risk' clones known to harbour carbapenemase genes (ST233, ST277, ST357, ST654 and ST773). Analysis of the genetic environment of 16S RMTase genes identified that IS6100 was genetically linked to rmtB1; IS91 to rmtB4, rmtC or rmtD3; ISCR14 to rmtD1; and rmtF2 was linked to Tn3, IS91 or Tn1721. Although 16S RMTase genes explained only 8.6% of pan-aminoglycoside resistance in the P. aeruginosa isolates studied, the association of 16S RMTase genes with carbapenemase-producers and 'high-risk' clones highlights that continued surveillance is required to monitor spread as well as the importance of suppressing the emergence of dually-resistant clones in hospital settings.

Effect of Imipenem and Amikacin Combination against Multi-Drug Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen associated with high morbidity and mortality rates. Combination of antibiotics has been found to combat multi-drug resistant or extensively drug resistance P. aeruginosa. In this study we investigate the in vitro and in vivo effect of amikacin and imipenem combination against resistant P. aeruginosa. The checkerboard technique and time-killing curve have been performed for in vitro studies showed synergistic effect for combination. A peritonitis mouse model has been used for evaluation of the therapeutic efficacy of this combination which confirmed this synergistic effect. The in vitro and in vivo techniques showed synergistic interaction between tested drugs with fractional inhibitory concentration indices (FICIs) of ≤0.5. Conventional PCR and quantitative real-time PCR techniques were used in molecular detection of bla IMP and aac(6′)-Ib as 35.5% and 42.2% of P. aeruginosa harbored bla IMP and aac(6′)-Ib respectively. Drug combination viewed statistically significant reduction in bacterial counts (p value < 0.5). The lowest bla IMP and aac(6′)-Ib expression was observed after treatment with 0.25 MIC of imipenem + 0.5 MIC of amikacin. Morphological changes in P. aeruginosa isolates were detected by scanning electron microscope (SEM) showing cell shrinkage and disruption in the outer membrane of P. aeruginosa that were more prominent with combination therapy than with monotherapy.

Aminoglycoside-resistance gene signatures are predictive of aminoglycoside MICs for carbapenem-resistant Klebsiella pneumoniae.

Aminoglycoside-containing regimens may be an effective treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp), but aminoglycoside-resistance genes are common in these strains. The relationship between the aminoglycoside-resistance genes and aminoglycoside MICs remains poorly defined. To identify genotypic signatures capable of predicting aminoglycoside MICs for CR-Kp. Clinical CR-Kp isolates (n = 158) underwent WGS to detect aminoglycoside-resistance genes. MICs of amikacin, gentamicin, plazomicin and tobramycin were determined by broth microdilution (BMD). Principal component analysis was used to initially separate isolates based on genotype. Multiple linear regression was then used to generate models that predict aminoglycoside MICs based on the aminoglycoside-resistance genes. Last, the performance of the predictive models was tested against a validation cohort of 29 CR-Kp isolates. Among the original 158 CR-Kp isolates, 91.77% (145/158) had at least one clinically relevant aminoglycoside-resistance gene. As a group, 99.37%, 84.81%, 82.28% and 10.76% of the CR-Kp isolates were susceptible to plazomicin, amikacin, gentamicin and tobramycin, respectively. The first two principal components explained 72.23% of the total variance in aminoglycoside MICs and separated isolates into four groups with aac(6')-Ib, aac(6')-Ib', aac(6')-Ib+aac(6')-Ib' or no clinically relevant aminoglycoside-resistance genes. Regression models predicted aminoglycoside MICs with adjusted R2 values of 56%-99%. Within the validation cohort, the categorical agreement when comparing the observed BMD MICs with the predicated MICs was 96.55%, 89.66%, 86.21% and 82.76% for plazomicin, gentamicin, amikacin and tobramycin, respectively. Susceptibility to each aminoglycoside varies in CR-Kp. Detection of aminoglycoside-resistance genes may be useful to predict aminoglycoside MICs for CR-Kp.

Comparison of in vitro activities of plazomicin and other aminoglycosides against clinical isolates of Klebsiella pneumoniae and Escherichia coli.

To compare the in vitro activity of plazomicin and two older aminoglycosides (gentamicin and amikacin) against 180 isolates of Escherichia coli and Klebsiella pneumoniae, including subsets of 60 non-ESBL-producing, 60 ESBL-producing and 60 carbapenem-resistant (46 carrying blaOXA-48, 11 carrying blaNDM and 3 carrying blaOXA-48 and blaNDM) strains. MICs of plazomicin, gentamicin and amikacin were determined by a gradient diffusion method. Gentamicin and amikacin MICs were interpreted according to CLSI criteria and EUCAST breakpoint tables. Plazomicin MICs were interpreted using FDA-defined breakpoints. All non-ESBL-producing and ESBL-producing isolates were susceptible to plazomicin. The plazomicin susceptibility rate (71.7%) in carbapenem-resistant isolates was significantly higher than those observed for gentamicin (45%) and amikacin (56.7% and 51.7% according to CLSI and EUCAST breakpoints, respectively). Gentamicin, amikacin and plazomicin susceptibility rates (35.6% for gentamicin; 44.4% and 37.8% for amikacin according to CLSI and EUCAST breakpoints, respectively; 64.4% for plazomicin) in carbapenem-resistant K. pneumoniae were significantly lower than those observed for carbapenem-resistant E. coli isolates (73.3% for gentamicin; 93.3% for amikacin and plazomicin). Gentamicin, amikacin and plazomicin susceptibility rates for blaNDM-positive isolates were lower than those observed for blaOXA-48-positive isolates, but differences were not statistically significant. Among the isolates that were non-susceptible to both gentamicin and amikacin, the plazomicin susceptibility rate was less than 30%. Although plazomicin showed excellent in vitro activity against carbapenem-susceptible isolates, the plazomicin resistance rate increased to 35.6% among carbapenem-resistant K. pneumoniae and further increased to 45.5% among blaNDM-positive isolates.

Association between 16S rRNA gene mutations and susceptibility to amikacin in Mycobacterium avium Complex and Mycobacterium abscessus clinical isolates

We evaluated the association between 16S rRNA gene (rrs) mutations and susceptibility in clinical isolates of amikacin-resistant nontuberculous mycobacteria (NTM) in NTM-pulmonary disease (PD) patients. Susceptibility was retested for 134 amikacin-resistant isolates (minimum inhibitory concentration [MIC] ≥ 64 µg/ml) from 86 patients. Amikacin resistance was reconfirmed in 102 NTM isolates from 62 patients with either Mycobacterium avium complex-PD (MAC-PD) (n = 54) or M. abscessus-PD (n = 8). MICs and rrs mutations were evaluated for 318 single colonies from these isolates. For the 54 MAC-PD patients, rrs mutations were present in 34 isolates (63%), comprising all 31 isolates with amikacin MICs ≥ 128 µg/ml, but only three of 23 isolates with an MIC = 64 µg/ml. For the eight M. abscessus-PD patients, all amikacin-resistant (MIC ≥ 64 µg/ml) isolates had rrs mutations. In amikacin-resistant isolates, the A1408G mutation (n = 29) was most common. Two novel mutations, C1496T and T1498A, were also identified. The culture conversion rate did not differ by amikacin MIC. Overall, all high-level and 13% (3/23) of low-level amikacin-resistant MAC isolates had rrs mutations whereas mutations were present in all amikacin-resistant M. abscessus isolates. These findings are valuable for managing MAC- and M. abscessus-PD and suggest the importance of phenotypic and genotypic susceptibility testing.

Pharmacodynamics of once- versus twice-daily dosing of nebulized amikacin in an in vitro Hollow-Fiber Infection Model against 3 clinical isolates of Pseudomonas aeruginosa

This study aims to compare the bacterial killing of once- versus twice-daily nebulized amikacin against Pseudomonas aeruginosa and to determine the optimal duration of therapy. Three clinical P. aeruginosa isolates (amikacin MICs 2, 8, and 64 mg/L) were exposed to simulated epithelial lining fluid exposures of nebulized amikacin with dosing regimens of 400 mg and 800 mg once- or twice-daily up to 7-days using the in vitro hollow-fiber infection model. Quantitative cultures were performed. Simulated amikacin dosing regimens of 400 mg twice-daily and 800 mg once-daily achieved ≥2-log reduction in the bacterial burden within the first 24-hours of therapy for all isolates tested. No dosing regimen suppressed the emergence of amikacin resistance. No difference in bacterial killing or regrowth was observed between 3- and 7-days of amikacin. Amikacin doses of 800 mg once-daily for up to 3-days may be considered for future clinical trials.

Evaluation of Effectiveness of Antibiotic Combination Therapy in Multi Drug Resistant Escherichia Coli in Vitro and in Vivo

The antimicrobial resistance to commonly used antibiotics including broad spectrum antibiotics like carbapenem and colistin and aminoglycosides are increasing among Escherichia coli which has resulted in emergence of MDR strains as well as limiting therapeutic options to treat infection by them. The use of combination therapy has been found to be an effective strategy to overcome this situation. The aim of the present study was to see the efficacy of combination of imipenem-colistin, imipenem-amikacin and colistin-amikacin both in vitro and in vivo in MDR Escherichia coli resistant to all of them. Total 65 Escherichia coli were included in this study isolated from urine, wound swab and blood samples of infected patients. Escherichia coli were identified by biochemical test followed by PCR. Antibiotic susceptibility pattern was performed by disc-diffusion method. MIC of imipenem, colistin and amikacin were done by agar dilution method. Antibiotic combinations (imipenem+colistin, imipenem+amikacin and colistin+amikacin) effectiveness in treating resistant Escherichia coli were evaluated in vitro by agar dilution method and in vivo by using a mouse model. Among 65 Escherichia coli 11 were MDR among which 2 were resistant to imipenem& colistin; 4 were resistant to imipenem & amikacin; 4 were resistant to amikacin & colistin; one was resistant to all the three drugs. Combination of two antibiotics of these three in vitro against resistant Escherichia coli showed synergistic effect when 1/4 or 1/8 MIC for each antimicrobial were used. The best in vivo efficacy appeared with imipenem-colistin and imipenemamikacin combination which showed 100% blood culture negative results after 72 hours of antibiotic combination therapy.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures

ObjectiveThe aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. Material and methodsGram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. ResultsMALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n=68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5h. ConclusionsA simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients’ clinical outcomes.

Antibiotic resistance mechanisms in Acinetobacter spp. strains isolated from patients in a paediatric hospital in Mexico

ObjectivesThe aim of this study was to identify Acinetobacter spp. strains from paediatric patients, to determine their genetic relationship, to detect antibiotic resistance genes and to evaluate the role of efflux pumps in antibiotic resistance. MethodsA total of 54 non-duplicate, non-consecutive Acinetobacter spp. isolates were collected from paediatric patients. Their genetic relationship, antibiotic resistance profile, efflux pump activity, antibiotic resistance genes and plasmid profile were determined. ResultsThe isolates were identified as 24 Acinetobacter haemolyticus, 24 Acinetobacter calcoaceticus–baumannii (Acb) complex and 1 strain each of Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter indicus, Acinetobacter lwoffii, Acinetobacter ursingii and Acinetobacter venetianus. The 24 A. haemolyticus were considered genetically unrelated. One strain was resistant to carbapenems, two to cephalosporins, two to ciprofloxacin and sixteen to aminoglycosides. The antibiotic resistance genes blaOXA-214 (29%), blaOXA-215 (4%), blaOXA-264 (8%), blaOXA-265 (29%), blaNDM-1 (4%), aac(6ʹ)-Ig (38%) and the novel variants blaOXA-575 (13%), blaTEM-229 (75%), aac(6ʹ)-Iga (4%), aac(6ʹ)-Igb (13%) and aac(6ʹ)-Igc (42%) were detected. Among 24 Acb complex, 5 were multidrug-resistant, carbapenem-resistant strains carrying blaOXA-51 and blaOXA-23; they were genetically related and had the same plasmid profile. Other species were susceptible. In some strains of A. haemolyticus and Acb complex, the role of RND efflux pumps was evidenced by a decrease in the MICs for cefotaxime, amikacin and ciprofloxacin in the presence of an efflux pump inhibitor. ConclusionsThis study identified isolates of A. haemolyticus carrying new β-lactamase variants and shows for the first time the contribution of efflux pumps to antibiotic resistance in this species.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures

ObjectiveThe aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. Material and methodsGram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. ResultsMALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n=68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5h. ConclusionsA simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients’ clinical outcomes.

Repurposing drugs for treatment of Mycobacterium abscessus: a view to a kill.

The current treatment regimens recommended for Mycobacterium abscessus subspecies abscessus (Mab) pulmonary disease are not effective. We identified 16 drugs with potential to build new regimens, translating to 560 possible three-drug combination regimens. To determine MICs and efficacy of drugs from different antibiotic classes for treatment against Mab, in order to winnow down the potential drugs for combination therapy to tractable numbers, for future use in hollow-fibre studies. The MICs of levofloxacin, minocycline, meropenem, imipenem, tedizolid, bedaquiline, azithromycin, clarithromycin, amikacin, vancomycin, delafloxacin, tebipenem/avibactam and omadacycline were determined for 20 Mab isolates. In addition, concentration-response studies with tedizolid, bedaquiline, clarithromycin, amikacin, tebipenem/avibactam, cefdinir, faropenem, omadacycline and daunorubicin were performed and data were fitted to the inhibitory sigmoid Emax model. Efficacy was defined as maximal kill, expressed as cfu/mL kill below day 0 burden. The lowest MICs among the 13 antibiotics were of bedaquiline, tebipenem/avibactam and omadacycline. The antibiotics that killed Mab below the day 0 burden were the anticancer agent daunorubicin (3.36 log10 cfu/mL), cefdinir (1.85 log10 cfu/mL), faropenem (2.48 log10 cfu/mL) and tebipenem/avibactam (1.71 log10 cfu/mL kill). The EC50 values of these drugs were 11.67, 9.52, 48.2 and 0.33 mg/L, respectively, below peak concentrations of these drugs. The low MICs and efficacy at clinically achievable concentrations mean that tebipenem/avibactam, daunorubicin, omadacycline and bedaquiline give a view of components of a three-drug regimen likely to effectively kill Mab. We propose pharmacokinetic/pharmacodynamic studies to identify such a regimen and the doses to be combined.

In-vitro activity of cefiderocol against multidrug-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii isolates from the UK

Background. Cefiderocol is a parenteral siderophore cephalosporin, with a catechol-containing 3’ side chain. We evaluated its activity against MDR Gram-negative bacteria Methods. MICs of cefiderocol, meropenem, ceftolozane-tazobactam, cefepime, ceftazidime, aztreonam, ciprofloxacin, ceftazidime-avibactam, amikacin and colistin were determined in cation-adjusted Mueller-Hinton broth; the medium was iron depleted for cefiderocol only. The panel comprised 305 Enterobacterales, 111 P. aeruginosa and 99 A. baumannii selected for carbapenemases, ESBL production or carbapenem resistance via combinations of porin-loss with AmpC or ESBL. Results. The activity of cefiderocol was unrelated to Enterobacterales species. At 4mg/L cefiderocol inhibited 92.1% of all Enterobacterales, with rates of 95-100% for isolates with AmpC+porin-loss, VIM, IMP, OXA-48-like, KPC, GES, SME or IMI. Only isolates with NDM (72.1%) or ESBL+porin-loss (88.5%) had lower rates. No comparator agent inhibited &gt;90% of isolates at EUCAST breakpoint. Cefiderocol 4mg/L inhibited 86.5% of all P. aeruginosa, with rates of 80-100% for those with VIM, IMP, GES or VEB beta-lactamases. Lower rates were seen for those with NDM (72.7%) and PER (73.3%) enzymes. No comparator inhibited &gt;85% of isolates at breakpoint. Cefiderocol 4mg/L also inhibited 88.9% of A. baumannii isolates with rates &gt;85% for those with OXA-51, -23, -24, or -58. A lower rate (80%) was seen for those with NDM carbapenemases. A concentration of 16mg/L was needed to inhibit ≥90% of A. baumannii. Conclusions. Cefiderocol was widely active at low concentrations against MDR Enterobacterales, P. aeruginosa and A. baumannii. MICs for isolates with NDM enzymes nonetheless were higher than for those with other carbapenemases.