Amikacin Resistance Research Articles

Antimicrobial resistance and antibiotic consumption in a third level pediatric hospital in Mexico City

The increasing resistance to antibiotics is a public health problem and an imminent therapeutic challenge in hospitals. In this report we aimed to analyze the relationship between antimicrobial resistance and antibiotic consumption in a third-level pediatric hospital. A cross-sectional analysis was conducted using the information from the microbiology and pharmacy databases of the Pediatric Hospital "Doctor Silvestre Frenk Freund", during the period 2015-2018. Prevalence of antimicrobial resistance by microorganisms and dispensed grams of selected antibiotics were calculated annually. Antibiotic resistance trend over the time was evaluated using the Chi-square trends test and to assess the correlation between the dispensed grams of antibiotics with their antimicrobial resistance prevalence, we calculated the Pearson's coefficient (r). A total of 4,327 isolated bacterial samples were analyzed (56.5% Gram-positive and 44.5% Gram-negative). Most frequently isolated microorganisms were coagulase-negative staphylococci (CoNS), E. coli, K. pneumoniae, P. aeruginosa and S. aureus. We found a significant increase in resistance to clindamycin and oxacillin for CoNS and significant decrease in nitrofurantoin and amikacin resistance for E. coli and K. pneumoniae. We observed a strong positive and statistically significant correlation between amikacin resistance prevalence and amikacin dispensed grams for P. aeruginosa (r = 0.95, p = 0.05). The antibiotic resistance profile showed by our study highlights the need of an appropriate antibiotic control use in the Hospital setting.

Rapid macrolide and amikacin resistance testing for Mycobacterium abscessus in people with cystic fibrosis.

Introduction. Mycobacterium abscessus complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment. MABSC can acquire resistance against macrolides or amikacin via 23S or 16S rRNA gene mutations, respectively.Gap Statement. Current culture-based methods for MABSC detection and antibiotic resistance characterization are typically prolonged, limiting their utility to directly inform treatment or clinical trials. Culture-independent molecular methods may help address this limitation.Aim. To develop real-time PCR assays for characterization of key 23S or 16S rRNA gene mutations associated with constitutive resistance in MABSC.Methodology. We designed two real-time PCR assays to detect the key 23S and 16S rRNA gene mutations. The highly conserved nature of rRNA genes was a major design challenge. To reduce potential cross-reactivity, primers included non-template bases and targeted single-nucleotide polymorphisms unique to MABSC. We applied these assays, as well as a previously developed real-time PCR assay for MABSC detection, to 968 respiratory samples from people with cystic fibrosis. The results from the molecular methods were compared to those for gold standard culture methods and 23S and 16S rRNA gene sequencing.Results.The real-time PCR MABSC detection assay provided a sensitivity of 83.8 % and a specificity of 97.8 % compared to culture. The results from the real-time PCR resistance detection assays were mostly concordant (>77.4 %) with cultured isolate sequencing. The real-time PCR resistance detection assays identified several samples harbouring both resistant and susceptible MABSC, while culture-dependent methods only identified susceptible MABSC in these samples.Conclusion. Using the molecular methods described here, results for health care providers or researchers could be available days or weeks earlier than is currently possible via culture-based antibiotic susceptibility testing.

Phenotype-Genotype Characterization and Antibiotic-Resistance Correlations Among Colonizing and Infectious Methicillin-Resistant Staphylococcus aureus Recovered from Intensive Care Units.

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) presents a profound hazard to public health. MRSA colonizing skin, mucous membranes, and the anterior nares without clinical symptoms is termed “colonizing MRSA”. Upon manifestation of clinical symptoms, it is termed “infectious MRSA”. Here, we characterize and differentiate colonizing and infectious MRSA, and analyze the phenotypic-genotypic and antibiotic susceptibility correlations.MethodologyClinical MRSA isolates were recovered from intensive care units (ICUs) of two major Egyptian hospitals and their biofilm formation ability was tested. Antibiograms against 16 antibiotics were determined, in addition to the minimum inhibitory concentrations (MICs) of vancomycin and linezolid. The entire collection was typed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as multi-locus sequence typing (MLST). Representative resistance and virulence genes were detected by PCR amplification.ResultsForty-nine isolates were confirmed as MRSA, of which 30 isolates were infectious and 19 were colonizing. Versatile resistance patterns were observed in both groups of isolates. We report a higher tendency for biofilm-formation and borderline minimum inhibitory concentrations among infectious isolates. A Positive antibiotic correlation was observed between susceptibility to protein synthesis inhibitors and cell wall inhibitors. Positive correlations were observed between isolation site and rifampicin resistance: nasal samples were enriched in rifampicin-resistant isolates, while urine and blood samples were enriched in susceptible ones. Furthermore, biofilm formation ability was slightly associated with amikacin resistance, and an association between teicoplanin resistance and the presence of the Panton-Valentine leukocidin gene was the only significant phenotype-genotype correlation observed. Finally, ERIC typing and MLST had congruent results.ConclusionLinezolid and vancomycin are still the most convenient choice for MRSA treatment. ERIC PCR and MLST show promising typing combination that could be easily used periodically for tracking the genotypic changes of MRSA, especially within the healthcare facilities. Several correlations were established between groups of antibiotics and the genotypes/phenotypes of the selected 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.

Assessment of version 2.5 of QMAC-dRAST for rapid antimicrobial susceptibility testing with reduced sample-to-answer turnaround time and an integrated expert system

ObjectiveTo assess the performance of the new rapid antimicrobial susceptibility testing (AST) QMAC-dRAST V2.5 system. MethodsASTs were performed using QMAC-dRAST-V2.5 and a disk diffusion method, directly from positive blood bottles with Gram-negative bacteria. Discrepancies between the results obtained using the two methods were categorized into very major errors (VME, S with dRAST vs. R with disk diffusion), major errors (ME, R vs. S, respectively), minor errors (mE, S vs. I or I vs. R, respectively), and very minor errors (Vme, I vs. S or R vs. I, respectively). For each AST, results were recorded after 4, 5, and 6h of incubation. ResultsFrom 106 bacteremia, 1416 individual AST results were obtained. Overall agreement between results using the two methods was 91%, ranging from 76.9% to 99.1% depending upon the antibiotic, with 128 errors, i.e. 14/1416 (1%) VME, 59/1416 (4.2%) ME, 25/1416 (1.8%) mE and 30/1416 (2.1%) Vme. VMEs were encountered for Klebsiellasp and Serratia marcescens isolates with low-level piperacillin and amikacin resistance, respectively. Using the integrated QMAC-dRAST-V2.5 expert system, all 14 VMEs and 3 mEs were eliminated, leading to 92.2% categorical agreement. After 45min of pre-incubation in the QMAC-dRAST-V2.5 device, 22.2% of the 1416 AST results were obtained after 4h, an additional 31.4% after 5h and a further 46.3% after 6h. ConclusionQMAC-dRAST-V2.5 is an optimized version of QMAC-dRAST V2.0, particularly with respect to utilization of an expert system and reduced TAT according to the antibiotic tested.

Comparing genotypes and antibiotic resistance profiles of Mycobacterium abscessus and Mycobacterium massiliense clinical isolates in China

Abstract This study aimed to investigate differences in the antimicrobial susceptibility of members of the Mycobacterium abscessus complex (MABC): subsp. massiliense and subsp. abscessus, and to identify associations between strain genotypes and antimicrobial resistance phenotypes. A total of 383 clinical MABC isolates (subsp. abscessus: n = 218, 56.9%; subsp. massiliense: n = 163, 42.6%; subsp. bolletii: n = 2, 0.5%) were characterised using multilocus variable number tandem repeat (VNTR) typing and drug susceptibility testing. Most isolates exhibited susceptibility to amikacin, clarithromycin and azithromycin but resistance to cefoxitin and minocycline was statistically more associated with isolates unclustered by VNTR type. The Simpson's diversity indexes of VNTR typing for M. abscessus and M. massiliense isolates were 0.999 and 0.997, respectively. Genotyping of M. abscessus and M. massiliense isolates by VNTR may provide valuable information for predicting resistance phenotype.

High Prevalence of Multidrug Resistant <i>Klebsiella</i> Species Isolated from the Yaounde University Teaching Hospital, Cameroon

Background and Purpose: Klebsiella species are amongst the most common causes of a variety of community-acquired and hospital-acquired infections (HAI), characterized by high morbidity and mortality rates. Most infections caused by Klebsiella species are usually treated using antibiotics. The aim of this study was to determine the antimicrobial resistance profile of Klebsiella species isolated from in-patients and out-patients at the Yaounde University Teaching Hospital. The data generated will go a long way to improve on the choice of an adequate empiric antibiotic treatment for infections caused by Klebsiella species. Methodology: A cross-sectional descriptive study was carried out over a period of 6 months, spanning from February 2019 to July 2019 with a sample size of 37 isolates, obtained from 6 different clinical specimens. Identification of isolates was done using API 20E identification system (Biomerieux SA, Lyon, France). Susceptibility to antibiotics was tested as described by Kirby-Bauer in 1956. Inhibition diameters were interpreted according to recommendations from the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2019). Results and Conclusion: Among the 37 Klebsiella isolates identified, Klebsiella pneumoniae was the most prevalent species isolated with a percentage of 54.1%, followed by Klebsiella rhinoscleromatis 18.9%, Klebsiella ozaenae 16.2% and Klebsiella oxytoca, 10.8%. The resistance pattern of Klebsiella to amoxicillin, amoxicillin/clavulanate, tircacillin, tircacillin + clavulanic acid, piperacillin, piperacillin + tazobactam, cefalotin, cefuroxim, ceftazidime, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, aztreonam, amikacin, gentamicin, tobramycin, trimethoprim/ sulfamethoxazole, nalidixic acid, pipemidic acid, norfloxacin, ciprofloxacin, levofloxacin, ofloxacin, and moxifoxacin was as follows; 100%, 86.5%, 97.3%, 83.6%, 86.5%, 16.2%, 86.5%, 83.8%, 78.4%, 32.4%, 78.4%, 76.7%, 2.7%, 2.7%, 76.7%, 13.5%, 75.7%, 73.0%, 91.9%, 51.4%, 48.6%, 64.9%, 48.6%, 48.6%, 73.0% and 62.2% respectively. Multidrug resistance was observed in 94.6% of the Klebsiella isolates. Conclusion: This study shows that the level of multidrug resistance is high. The isolates expressed good sensitivity to carbapenems, piperacillin + tazobactam, amikacin and high resistance to all other antimicrobials tested. Therefore, antimicrobial susceptibility testing prior to prescriptions should be encouraged and sensitization of the population about consequences of inappropriate antibiotic treatment and auto medication should be enforced as a means to curb antimicrobial resistance.

1309. Imipenem/Cilastatin/Relebactam (I/R) Alone and in Combination against Pseudomonas aeruginosa (PSA) in the In Vitro Pharmacodynamic Model

BackgroundI/R, a carbapenem-beta-lactamase inhibitor antibiotic, is active against most imipenem-resistant PSA, including MDR isolates. Combination therapy may enhance activity against MDR pathogens and suppress resistance. This study’s objective was to assess the efficacy of I/R compared with combinations including colistin (CST) or amikacin (AMK) against PSA in an IVPD model.MethodsHuman-simulated concentrations of I/R 500/250 mg every six hours, a total daily dose of CST 360 mg, and AMK 25 mg/kg daily were reproduced alone and in combination against 6 imipenem-non-susceptible PSA with I/R MICs of 1/4 to 8/4 mg/L in an IVPD over 24h. The primary endpoint was the difference in 24h colony forming units (CFU) between each combination regimen and its components alone. The log ratio differences of the area under the CFU curve were also calculated to compare the overall bacterial burden resulting from exposure to I/R alone with those treated by combination regimens. Emergence of resistance was tested at 24h using drug-containing plates at 3xMIC.ResultsI/R, CST, and AMK alone produced 24hCFU changes consistent with isolate MICs. One isolate (already I/R non-susceptible) developed I/R resistance, and 4 and 3 developed CST and AMK resistance, respectively. I/R plus CST suppressed all resistance and resulted in synergistic or additive interactions against three of six isolates with 24h CFU reductions ranging from -2.62 to -4.67 log10CFU/mL. This combination further reduced overall bacterial burden by 79-81% compared with I/R alone against two I/R-non-susceptible strains. I/R plus AMK also prevented resistance emergence but exhibited indifferent interactions against all isolates at 24h with the combined drugs achieving -0.51 to -3.33 log10CFU/mL reductions. Minor overall reductions in bacterial burden were observed relative to I/R alone.ConclusionI/R plus CST resulted in additivity or synergy against three of six PSA and prevented I/R and CST resistance, whereas the addition of AMK only suppressed resistance. The greatest overall reductions in bacterial burden, however, were observed with I/R plus CST against I/R-non-susceptible isolates, supporting targeted use of this combination against this phenotype when alternatives are unavailable.Disclosures David P. Nicolau, PharmD, Cepheid (Other Financial or Material Support, Consultant, speaker bureau member or has received research support.)Merck & Co., Inc. (Consultant, Grant/Research Support, Speaker’s Bureau)Wockhardt (Grant/Research Support) Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)bioMérieux (Research Grant or Support, Other Financial or Material Support, Speaker Honorarium)Melinta (Research Grant or Support)Merck & Co., Inc. (Research Grant or Support)Paratek (Speaker’s Bureau)Summit (Other Financial or Material Support, Research funding (clinical trials))

Anti-biogram pattern of uro-pathogens isolated from patients in a Tertiary Care Hospital in Karnataka, India

Introduction: Urinary tract infection (UTI) is one of the important causes of morbidity in the community.Development of antibiotic resistance among uro-pathogensposeproblem in the treatment of UTI.Hence it is essential to examine the prevalence of such uro-pathogens and study theiranti-biogram. Materials and Methods: Bacteria isolated from midstream urine samples collected aseptically from 266 patients over a period of 6 months from November 2016 to April 2017 were identifiedbystandard microbiological procedures. Anti-biogram was done by Kirby–Bauer disc diffusion method and interpreted as per the CLSI guidelines. Result: Out of 266 urine samples, 136 (51.12%) were found to be positive for microbial isolates, of which 96 (71.3%) samples were from females and 40 (29.4%) samples were from males. Escherichia coli was the predominant organism (47.05%) followed by Pseudomonas species (13.2%), Enterococcus species (11.7%), Klebsiella species (7.3%), Citrobacter koseri (4.4%) and Proteus species (2.9%). Enterobacteriaceae shows highest sensitivity towards nitrofurantoin (86%), amikacin (68%) and gentamicin (59%) and resistance towards norfloxacin (74%). Sensitivity of Pseudomonas spp was 72.2% for piperacillin/tazobactam, 44.4% for amikacin and 66% resistance to ceftazidime. Sensitivity pattern of Enterococci spp to nitrofurantoin 96.6%, aminoglycosides 78%, and fluroquinolones 50%. An attempt was made to study ESBL production among Enterobacteriaceae members. The isolates were 54.4% multidrug resistant (MDR) and 38.8% were ESBL producers. Conclusion: In our study E.coli was the most common uropathogen isolated. Patterns of antibiotic resistance in a wide variety of pathogenic organism were noted. To prevent the development of resistance, periodic evaluation of antibiotic susceptibility patterns is necessary. Keywords: Enterobacteriaceae, Extended spectrum beta-lactamase, Uro-pathogens.

Multi-drug resistant and extended-spectrum lactamase producing bacteria in pediatric urinary tract infection

Background: Urinary tract infection (UTI) is one of most common pediatric infections. The study was conducted with an aim to assess the etiological agents, antimicrobial resistance, their risk factors and clinical significance in pediatric UTI cases. Methods and materials: This is a cross-sectional study conducted at Department of Microbiology and Infectious Diseases for 1 year (January to December 2018). A total of 4020 pediatric patients <14 years old, suspected to have UTI, were included in the study. Clinical data was obtained from software and enquiry with patients whenever feasible; standard microbiological guidelines were followed for laboratory testing. Chi-square test was applied for comparison of categorical variables. P value less than 0.05 was considered as statistically significant. Results: Of the 4020 children with suspected UTI, 724 (18%) were culture positive. Escherichia coli was the most common etiological agent (55% n = 402), followed by Enterococcus faecalis (17%, n = 120), and Klebsiella pneumoniae (9%, n = 65). Among total isolates, 32% (n = 231) were multi-drug resistant (MDR) while 35% (192) of gram-negative bacilli were extended-spectrum b lactamase (ESBL) producers. Multidrug resistance and ESBL production occurred more frequently in previously hospitalized patients (p value 0.0001 and 0.02), patients with longer duration of fever (p value 0.03 and 0.01) and higher recurrence rate (p value 0.001 and 0.001). ESBL production was observed most commonly in neonates (40%), followed by infants (32%). Degree of antimicrobial resistance was significantly higher in ESBL producer isolates as compared to non-producers. Amikacin resistance was observed in 22% of ESBL producer isolates as compared to 14% in non-producers (p value 0.01). Similar pattern of resistance were observed for other antimicrobials. Conclusion: High-level antimicrobial resistance was observed in pediatric UTI with alarming incidence superbugs MDR and ESBL. Previous hospitalization, longer duration of fever and recurrence is significantly associated with increase in MDR and ESBL production. Regular antimicrobial surveillance should be carried out and judicial use of antimicrobials based on susceptibility reports when available and local antimicrobial resistance data should be encouraged for the optimal management of pediatric UTI.

Black hairy tongue associated with linezolid therapy in a patient with mycobacterial abscesses infection- Report from rural india

Background: Black Hairy tongue consists of a benign condition related to abnormal keratin build-up in filiform papillae, giving them an elongated hair-like appearance. We report a case of a 35 y/m presented who developed black discoloration of tongue while on linezolid therapy. Case Description: A 35 y/m, presented to gastroenterology OPD with chief complaints of on and off chromic discharging sinuses from port site right hypochondrium since past 1 y. Pus swabs taken from discharging site were taken and sent for gram stain, ZN stain and culture. Gram stain showed few pus cells,gram positive cocci in clusters. ZN stain showed few acid fast bacilli. Discussion: Bacterial culture grew MSSA and LJ media showed growth of rapidly growing mycobacteria(RGM) after 6 d of incubation. RGM was further identified by HAINS genotypic assay as Mycobacterium abscesses subspecies abscesses. Antibiotic susceptibility was performed on Middlebrook 7H11 with OADC and isolate was found to be sensitive to amikacin, gentamycin,imipenem and linezolid and resistance to macrolides, fluroquinolones and doxycycline. Diagnosis of MSSA and Mycobacterium abscesses infection was made and patient was started on amikacin and linezolid 600 mg OD. On 16thday of therapy patient developed dark discoloration of dorsum of tongue, teeth and oral mucosa. Other antibiotics, drugs, poor oral hygiene, smoking, and consumption of coffee or other coloured beverages were rule out as risk factors. Patient was switched on to meropenem and aminoglycoside. Discoloration improved after three weeks of discontinuing Linezolid. Conclusion: The diagnosis of BHT primarily relies on a visual intra- oral examination. Described adverse reaction is uncommonly described with prolonged Linezolid therapy.Careful review of known precipitating factors and recent medication changes is also fundamental in the diagnosis of BHT. Overall clinical prognosis of BHT is excellent.

Emergence of Imipenem Resistance in a CpxA-H208P-Variant-Producing Proteus mirabilis Clinical Isolate

The Proteus mirabilis PmirS clinical isolate, which was susceptible to imipenem (0.5 μg/mL) and amikacin (1 μg/mL), was recovered from a bronchial aspirate of a patient who recently underwent lung transplantation. The P. mirabilis PmirR clinical isolate, which exhibited resistance to imipenem (16 μg/mL) and amikacin (24 μg/mL), was isolated 3 weeks later from the same patient and the same specimen type. Using short-read sequencing technology, these isolates appeared to be genetically identical except the cpxA gene of the PmirR isolate that was mutated leading to the His-208-Pro substitution. The structural alteration was localized in the histidine kinase, adenylate cyclase, methyl accepting protein, phosphatase (HAMP) domain, which is involved in the signal transduction between the sensor kinase and the regulator response of the CpxA/CpxR two-component system (TCS). No significant defect in the growth rate was found between the PmirS and PmirR isolates. This study suggests that alteration in CpxA might confer imipenem and amikacin resistance in P. mirabilis. This study brings new evidence that the TCS alteration could provide an adaptive capacity in a clinical context by conferring antibiotic resistance without fitness cost.

Characterization of RaeE-RaeF-RopN, a putative RND efflux pump system in Riemerella anatipestifer

Resistance-nodulation-division (RND) efflux systems are ubiquitous in Gram-negative bacteria and play a predominant role in antimicrobial resistance and other diverse phenotypes, but the knowledges of RND efflux systems are poorly understood so far in Riemerella anatipestifer. According to the sequence annotation, RIA_1117-RIA_1118-RIA_1119 operon in RA-GD strain encodes a putative tripartite RND efflux system. RIA_1117, RIA_1118 and RIA_1119 genes encode an outer member protein (OMP), an inner membrane pump protein (pump transporter), and a periplasmic membrane fusion protein (MFP), respectively. Furthermore, RIA_1119 protein is annotated as a MexE component. In this work, the biological functions of RIA_1117-RIA_1118-RIA_1119 proteins were studied. The antibiotic susceptibility testing showed that the inactivation of RIA_1117, RIA_1118 and RIA_1119 genes all raised susceptibility to amikacin, streptomycin and SDS. By induction with the above antimicrobial agents, the transcription levels of RIA_1117 and RIA_1118 genes were up-regulated significantly using qRT-PCR detection, but no significance difference was observed for the transcription level of RIA_1119 gene. CCCP inhibitor assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins mediated amikacin, streptomycin and SDS resistance depending on proton motive force (PMF). Spot assay and streptomycin accumulation assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins contributed to export streptomycin, and CCCP increased the accumulation of streptomycin. Furthermore, RIA_1117, RIA_1118 and RIA_1119 proteins also were involved in the fitness and virulence of RA-GD strain. These results showed that RIA_1117-RIA_1118-RIA_1119 operon encoded a RND efflux system, which has the substrate specificity for streptomycin, amikacin and SDS and contributed to the growth and virulence of RA-GD. RIA_1117-RIA_1118-RIA_1119 was designated RaeE-RaeF-RopN efflux system. Based on the above results and structural analysis, RIA_1117, RIA_1118 and RIA_1119 proteins corresponded to RopN (OMP), RaeF (pump transporter) and RaeE (MFP), respectively.

Pharmacodynamic Evaluation of Plasma and Epithelial Lining Fluid Exposures of Amikacin against Pseudomonas aeruginosa in a Dynamic In Vitro Hollow-Fiber Infection Model.

Given that aminoglycosides, such as amikacin, may be used for multidrug-resistant Pseudomonas aeruginosa infections, optimization of therapy is paramount for improved treatment outcomes. This study aims to investigate the pharmacodynamics of different simulated intravenous amikacin doses on susceptible P. aeruginosa to inform ventilator-associated pneumonia (VAP) and sepsis treatment choices. A hollow-fiber infection model with two P. aeruginosa isolates (MICs of 2 and 8 mg/liter) with an initial inoculum of ∼108 CFU/ml was used to test different amikacin dosing regimens. Three regimens (15, 25, and 50 mg/kg) were tested to simulate a blood exposure, while a 30 mg/kg regimen simulated the epithelial lining fluid (ELF) for potential respiratory tract infection. Data were described using a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model. Whole-genome sequencing was used to identify mutations associated with resistance emergence. While bacterial density was reduced by >6 logs within the first 12 h in simulated blood exposures following this initial bacterial kill, there was amplification of a resistant subpopulation with ribosomal mutations that were likely mediating amikacin resistance. No appreciable bacterial killing occurred with subsequent doses. There was less (<5 log) bacterial killing in the simulated ELF exposure for either isolate tested. Simulation studies suggested that a dose of 30 and 50 mg/kg may provide maximal bacterial killing for bloodstream and VAP infections, respectively. Our results suggest that amikacin efficacy may be improved with the use of high-dose therapy to rapidly eliminate susceptible bacteria. Subsequent doses may have reduced efficacy given the rapid amplification of less-susceptible bacterial subpopulations with amikacin monotherapy.

Evidence for Expanding the Role of Streptomycin in the Management of Drug-Resistant Mycobacterium tuberculosis.

In 2019, the WHO tuberculosis (TB) treatment guidelines were updated to recommend only limited use of streptomycin, in favor of newer agents or amikacin as the preferred aminoglycoside for drug-resistant Mycobacterium tuberculosis However, the emergence of resistance to newer drugs, such as bedaquiline, has prompted a reanalysis of antitubercular drugs in search of untapped potential. Using 211 clinical isolates of M. tuberculosis from South Africa, we performed phenotypic drug susceptibility testing (DST) to aminoglycosides by both critical concentration and MIC determination in parallel with whole-genome sequencing to identify known genotypic resistance elements. Isolates with low-level streptomycin resistance mediated by gidB were frequently misclassified with respect to streptomycin resistance when using the WHO-recommended critical concentration of 2 μg/ml. We identified 29 M. tuberculosis isolates from South Africa with low-level streptomycin resistance concomitant with high-level amikacin resistance, conferred by gidB and rrs 1400, respectively. Using a large global data set of M. tuberculosis genomes, we observed 95 examples of this corresponding resistance genotype (gidB-rrs 1400), including identification in 81/257 (31.5%) of extensively drug resistant (XDR) isolates. In a phylogenetic analysis, we observed repeated evolution of low-level streptomycin and high-level amikacin resistance in multiple countries. Our findings suggest that current critical concentration methods and the design of molecular diagnostics need to be revisited to provide more accurate assessments of streptomycin resistance for gidB-containing isolates. For patients harboring isolates of M. tuberculosis with high-level amikacin resistance conferred by rrs 1400, and for whom newer agents are not available, treatment with streptomycin may still prove useful, even in the face of low-level resistance conferred by gidB.

Underutilization of nontuberculous mycobacterial drug susceptibility testing in Ontario, Canada, 2010-2015.

Drug susceptibility testing (DST) in nontuberculous mycobacterial pulmonary disease (NTM-PD) is useful for some Mycobacterium species. International guidelines recommend routine use of DST for clinically relevant mycobacteria. DST use and results are poorly studied at the population level. We sought to identify the frequency of DST utilization for nontuberculous mycobacteria (NTMs) and describe the potential relevance of these results in Ontario. Using public health laboratory data, we performed a population-based retrospective analysis of NTM DST utilization in Ontario from May 2010 to June 2015. We determined the proportion of incident NTM-PD infections for which DST was performed and analyzed minimum inhibitory concentration (MIC) distributions from NTM testing overall, using thresholds recommended by the Clinical and Laboratory Standards Institute. The proportion of incident cases of NTM-PD tested for DST was 6.3% (240/3,806) for Mycobacterium avium complex (MAC), 36.2% (67/185) for M. abscessus, and 1.8% (19/1,057) for M. xenopi. Among specimens from all body sites, MAC resistance to clarithromycin occurred in 8.0% of specimens (21/262) and MAC resistance to amikacin (intravenous, MIC > 64 µg/mL) occurred in 22.6% (19/84). M.abscessus resistance occurred as follows: to amikacin, 3.8% (3/79); cefoxitin, 14.0% (11/79); imipenem, 30.4% (14/46); linezolid, 39.2% (31/79); clarithromycin, 54.2% (13/24); ciprofloxacin, 92.4% (73/79); and moxifloxacin, 91.1% (51/56). M. xenopi analysis was limited by few DST requests and a lack of DST clinical correlation. We found that NTM DST is underutilized in Ontario and observed a very high frequency of amikacin resistance among MAC isolates.

Characterization of carbapenem‐resistant Klebsiella pneumoniae in a tertiary hospital in Fuzhou, China

AimsThe emergence of carbapenem‐resistant Klebsiella pneumoniae (CRKP) strains has led to increased mortality and morbidity rates. Tigecycline, a new class of broad‐spectrum glycyl‐tetracycline antibiotics, has been used to target multi‐ and pan‐drug‐resistant bacterial infections. This study aimed to assess the molecular characteristics of CRKP in a tertiary hospital, and its susceptibility to tigecycline, to create a reference for hospital infection control and clinical drug use.Methods and ResultsWe retrieved patient clinical information and CRKP characterization from medical records and detected the MIC of tigecycline using the micro‐broth dilution method. Multi‐locus sequence typing was performed, and antibiotic resistance genes associated with CRKP were detected by qPCR. A total of 166 CRKP strains were detected in the sputum, urine and blood among intensive care unit patients (average age, 69·6 years). The most infrequently observed resistance genes were amikacin resistance genes, followed by tobramycin resistance genes. KPC ‐2, CTX‐M9 and CTX‐M1 were the most frequently detected resistance genes.ConclusionsNo strain was resistant to tigecycline (MIC ≥ 8 µg ml−1). Twenty‐four sequence types were identified, with ST11 being the most common type.Significance and Impact of the StudyClinicians and infection control experts should be aware of CRKP prevalence to facilitate clinical treatment and improve nosocomial infection control.

Analysis on drug sensitivity spectrum of 167 multidrug-resistant Mycobacterium tuberculosis in China

Objective: To investigate the drugs-sensitivity spectrum of multidrug-resistant tuberculosis (MDR-TB) in China and provide a scientific evidence for the drug selection in clinical therapy and the control of MDR-TB. Methods: A total of 167 strains of MDR-TB were included in this study. Every strain was genotyped by lysX gene sequencing and their sensitivity to 13 different anti-TB drugs was tested by using MicroDST(TM) and BACTEC(TM) MGIT 960(TM) liquid-culturing method. The association between drug resistance and genotypes as well as cross drug resistance was also analyzed. The results were analyzed by means of the comparison of enumeration data between two groups with χ(2) test. Results: The overall resistance rate of 167 MDR-TB strains to 11 anti-TB drugs, except isoniazide and rifampicin, was 95.81%, the rates of pre-extensive drug-resistance (pre-XDR) and extensive drug-resistance were 31.14%(52/167) and 6.59% (11/167), respectively. The streptomycin resistance rate of Beijing genotypes was significantly higher than that of the non-Beijing genotypes ( χ(2)=30.682, P<0.05), while the pre-XDR proportion in Beijing genotypes was lower than that in non-Beijing genotypes (χ(2)=5.332, P<0.05). The resistance rates of Ofloxacin and Pyrazinamide in the modern Beijing genotype were significantly higher than those in classical ones (χ(2)=4.105 and χ(2)=3.912, P<0.05). In addition, the cross-resistance rate to rifampicin and rifabutin was 86.23%. A significant difference in drug-resistance rate to rifabutin was seen among groups with different levels of rifampicin resistance (χ(2)=45.912, P<0.05). There was positive correlation not only between ofloxac resistance and moxifloxac resistance, but also between amikacin resistance and kanamycin resistance, with the coefficient of 0.87 and 0.91, respectively. Conclusions: In this study, we observed that there were high incidences of the resistance to 11 anti-TB drugs in 167 clinical MDR-TB strains and the cross resistance phenomena between drugs of the same type were quite serious. The majority of MDR-TB strains belonged to Beijing genotype, which was highly associated with streptomycin resistance.

RanB, a putative ABC-type multidrug efflux transporter contributes to aminoglycosides resistance and organic solvents tolerance in Riemerella anatipestifer

Riemerella anatipestifer is a Gram-negative bacterium, which is an important pathogen infecting ducks and resistant to various antibiotics. The efflux pump is an important resistance mechanism of Gram-negative bacteria, but little research has been done in R. anatipestifer. In this study, the drug resistance mediated by RIA_1614 gene of R. anatipestifer RA-GD strain was studied, because the gene was presumed to be an efflux pump component of ABC. Firstly, the deletion strain RA-GD△RIA_1614 and complemented strain RA-GD△RIA_1614 pCPRA::RIA_1614 were constructed. Then, MICs of various antimicrobial agents to parent and deletion strains and the tolerance of the strains to organic solvents were detected to screen the substrates for RIA_1614 gene. Moreover, the transcription levels of RIA_1614 gene in the parent and the complemented strains exposed to the substrates were detected by quantitative real-time RT-PCR. Furthermore, the efflux abilities of parent, deletion and complemented strains to substrates were determined by antibiotic accumulation test. In addition, in vitro competition ability and virulence of the strains were also detected. The results showed that the deletion strain was more sensitive to aminoglycosides and organic solvents than parental strain RA-GD. When RA-GD and complemented strain were exposed to sub-repression levels of aminoglycosides and organic solvents, the transcription levels of RIA_1614 gene were significantly up-regulated. Sodium o-vanadate inhibitor assay confirmed that RIA_1614 protein contributed to amikacin and streptomycin resistance and organic solvent tolerance. Streptomycin accumulation test showed that the RIA_1614 protein was able to export streptomycin, and the addition of ATPase inhibitor sodium o-vanadate increased the accumulation of streptomycin, indicating that RIA_1614 protein was an ATP-dependent efflux transporter. Growth and competition experiments revealed that RIA_1614 protein had no significant effect on growth of RA-GD, but decreased in vitro competition ability of the strain. Furthermore, pathogenicity tests showed that RIA_1614 protein involved in the virulence of the strain. Based on the results and amino acid sequence analysis, it was determined that RIA_1614 protein was a member of ABC efflux pumps, and the protein was named RanB.