Rates of resistance and heteroresistance to newer ß-lactam/ß-lactamase inhibitors for carbapenem-resistant Enterobacterales.

Background: Heteroresistance (HR), the presence of antibiotic-resistant subpopulations within a primary isogenic population, may be a potentially overlooked contributor to newer ß-lactam/ß-lactamase inhibitor (BL/BLI) treatment failure in carbapenem-resistant Enterobacterales (CRE) infections. Objectives: To determine rates of susceptibility and HR to BL/BLIs ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in clinical CRE isolates. Methods: The first CRE isolate per patient per year from two >500 bed academic hospitals from 1 January 2016 to 31 December 2021, were included. Reference broth microdilution (BMD) was used to determine antibiotic susceptibility, and population analysis profiling (PAP) to determine HR. Carbapenemase production (CP) was determined using the Carba NP assay. Results: Among 327 CRE isolates, 46% were Enterobacter cloacae, 38% Klebsiella pneumoniae and 16% Escherichia coli. By BMD, 87% to 98% of CRE were susceptible to the three antibiotics tested. From 2016 to 2021, there were incremental decreases in the rates of susceptibility to each of the three BL/BLIs. HR was detected in each species-antibiotic combination, with the highest rates of HR (26%) found in K. pneumoniae isolates with imipenem/relebactam. HR or resistance to at least one BL/BLI by PAP was found in 24% of CRE isolates and 65% of these had detectable CP. Conclusion: Twenty-four percent of CRE isolates tested were either resistant or heteroresistant (HR) to newer BL/BLIs, with an overall decrease of ∼10% susceptibility over 6 years. While newer BL/BLIs remain active against most CRE, these findings support the need for ongoing antibiotic stewardship and a better understanding of the clinical implications of HR in CRE.

Development of a Broth Microdilution Method To Characterize Chlorhexidine MICs among Bacteria Collected from 2005 to 2019 at Three U.S. Sites.

Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms has been adopted by many U.S. hospitals, but increasing chlorhexidine use has raised concerns about possible emergence of resistance. We sought to establish a broth microdilution method for determining chlorhexidine MICs and then used the method to evaluate chlorhexidine MICs for bacteria that can cause health care-associated infections. We adapted a broth microdilution method for determining chlorhexidine MICs, poured panels, established quality control ranges, and tested Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex isolates collected at three U.S. sites. Chlorhexidine MICs were determined for 535 isolates including 129 S. aureus, 156 E. coli, 142 K. pneumoniae, and 108 E. cloacae complex isolates. The respective MIC distributions for each species ranged from 1 to 8 mg/L (MIC50 = 2 mg/L and MIC90 = 4 mg/L), 1 to 64 mg/L (MIC50 = 2 mg/L and MIC90 = 4 mg/L), 4 to 64 mg/L (MIC50 = 16 mg/L and MIC90 = 32 mg/L), and 1 to >64 mg/L (MIC50 = 16 mg/L and MIC90 = 64 mg/L). We successfully adapted a broth microdilution procedure that several laboratories were able to use to determine the chlorhexidine MICs of bacterial isolates. This method could be used to investigate whether chlorhexidine MICs are increasing. IMPORTANCE Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms and reduce health care-associated infections has been adopted by many hospitals. There is concern about the possible unintended consequences of using this agent widely. One possible unintended consequence is decreased susceptibility to chlorhexidine, but there are not readily available methods to perform this evaluation. We developed a method for chlorhexidine MIC testing that can be used to evaluate for possible unintended consequences.

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

Antibiotic Susceptibility Profile for the US Neisseria meningitidis Urethritis Clade.

The US Neisseria meningitidis urethritis clade (US_NmUC) harbors gonococcal deoxyribonucleic acid alleles and causes gonorrhea-like urogenital tract disease. A large convenience sample of US_NmUC isolates (N = 122) collected between January 2015 and December 2019 in Columbus, Ohio demonstrated uniform susceptibility to antibiotics recommended for gonorrhea treatment and meningococcal chemoprophylaxis.

Clinical and Genomic Epidemiology of mcr-9-Carrying Carbapenem-Resistant Enterobacterales Isolates in Metropolitan Atlanta, 2012 to 2017.

Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative infections. Recently, the ninth allele of the mobile colistin resistance (mcr) gene family, designated mcr-9, was reported. However, its clinical and public health significance remains unclear. We queried genomes of carbapenem-resistant Enterobacterales (CRE) for mcr-9 from a convenience sample of clinical isolates collected between 2012 and 2017 through the Georgia Emerging Infections Program, a population- and laboratory-based surveillance program. Isolates underwent phenotypic characterization and whole-genome sequencing. Phenotypic characteristics, genomic features, and clinical outcomes of mcr-9-positive and -negative CRE cases were then compared. Among 235 sequenced CRE genomes, 13 (6%) were found to harbor mcr-9, all of which were Enterobacter cloacae complex. The median MIC and rates of heteroresistance and inducible resistance to colistin were similar between mcr-9-positive and -negative isolates. However, rates of resistance were higher among mcr-9-positive isolates across most antibiotic classes. All cases had significant health care exposures. The 90-day mortality was similarly high in both mcr-9-positive (31%) and -negative (7%) CRE cases. Nucleotide identity and phylogenetic analysis did not reveal geotemporal clustering. mcr-9-positive isolates had a significantly higher number of median [range] antimicrobial resistance (AMR) genes (16 [4 to 22] versus 6 [2 to 15]; P < 0.001) than did mcr-9-negative isolates. Pangenome tests confirmed a significant association of mcr-9 detection with mobile genetic element and heavy metal resistance genes. Overall, the presence of mcr-9 was not associated with significant changes in colistin resistance or clinical outcomes, but continued genomic surveillance to monitor for emergence of AMR genes is warranted. IMPORTANCE Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative infections. A recently described allele of the mobile colistin resistance (mcr) gene family, designated mcr-9, has been widely reported among Enterobacterales species. However, its clinical and public health significance remains unclear. We compared characteristics and outcomes of mcr-9-positive and -negative CRE cases. All cases were acquired in the health care setting and associated with a high rate of mortality. The presence of mcr-9 was not associated with significant changes in colistin resistance, heteroresistance, or inducible resistance but was associated with resistance to other antimicrobials and antimicrobial resistance (AMR), virulence, and heavy metal resistance (HMR) genes. Overall, the presence of mcr-9 was not associated with significant phenotypic changes or clinical outcomes. However, given the increase in AMR and HMR gene content and potential clinical impact, continued genomic surveillance of multidrug-resistant organisms to monitor for emergence of AMR genes is warranted.

Biofilm Production by Uropathogens in Postmenopausal Women with Recurrent and Isolated Urinary Tract Infection.

OBJECTIVES: The aims of this study were to assess the in vitro biofilm-producing capabilities of uropathogens grown from a postmenopausal urogynecologic population with isolated and recurrent urinary tract infection (UTI) and to determine whether the biofilm-producing bacterial phenotype was associated with recurrent infection. METHODS: This was an institutional review board-approved cross-sectional analysis within a large academic referral center. Uropathogens were cultured from postmenopausal women with either isolated or recurrent acute UTI and then screened for in vitro biofilm formation using crystal violet microtiter assays. Demographic and clinical variables, including pelvic floor symptoms and surgical history were collected and analyzed. A multivariate model was developed to determine whether recurrent UTI was independently associated with biofilm production. RESULTS: Eighty-nine women were included: 67.4% White, 25.8% Black, 3.4% Asian, and 1.1% Hispanic with a mean age of 72 ± 10.5 years. Ninety-five uropathogen strains were isolated. Most uropathogens produced biofilm (n = 53, 55.8%). Uropathogens from women with recurrent UTI were significantly more likely to produce biofilm (70%) than uropathogens collected from women with isolated UTI (38.6%, P = 0.0033). Adjusting for age, prior pelvic reconstructive surgery, and body mass index, recurrent UTI bacteria were more likely to produce biofilm, compared with isolated UTI (odds ratio, 5.37; 95% confidence interval, 2.0-14.4; P = 0.001). CONCLUSIONS: In this cohort of postmenopausal urogynecology patients, in vitro biofilm formation was more frequently observed in uropathogens isolated from women with recurrent UTI compared with women with isolated UTI. Further study is needed to assess the role of biofilms in recurrent UTIs in postmenopausal women.

Prevalence of colistin heteroresistance in carbapenem-resistant Pseudomonas aeruginosa and association with clinical outcomes in patients: an observational study.

OBJECTIVES: To describe the prevalence of colistin heteroresistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) and evaluate the association with clinical outcomes. METHODS: Colistin heteroresistance was evaluated in CRPA isolates collected from patients without cystic fibrosis in Atlanta, Georgia, USA using two definitions: HR1, growth at 4 and 8 mg/L of colistin at a frequency ≥1 × 10-6 the main population; and HR2, growth at a colistin concentration ≥8× the MIC of the main population at a frequency ≥1 × 10-7. A modified population analysis profile (mPAP) technique was compared with reference PAP for detecting heteroresistance. For adults hospitalized at the time of or within 1 week of CRPA culture, multivariable logistic regression estimated the association between heteroresistance and 90 day mortality. RESULTS: Of 143 colistin-susceptible CRPA isolates, 8 (6%) met the HR1 definition and 37 (26%) met the HR2 definition. Compared with the reference PAP, mPAP had a sensitivity and specificity of 50% and 100% for HR1 and 32% and 99% for HR2. Of 82 hospitalized patients, 45 (56%) were male and the median age was 63 years (IQR 49-73). Heteroresistance was not associated with 90 day mortality using HR1 (0% in heteroresistant versus 22% in non-heteroresistant group; P = 0.6) or HR2 (12% in heteroresistant versus 24% in non-heteroresistant group; P = 0.4; adjusted OR 0.8; 95% CI 0.2-3.4). CONCLUSIONS: Colistin heteroresistance was identified in up to 26% of patients with CRPA in our sample, although the prevalence varied depending on the definition. We did not observe an apparent association between colistin heteroresistance and 90 day mortality.

Mortality in patients with carbapenem-resistant Pseudomonas aeruginosa with and without susceptibility to traditional antipseudomonal ß-lactams.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates can frequently retain susceptibility to traditional antipseudomonal ß-lactams including cefepime, ceftazidime and piperacillin/tazobactam. OBJECTIVES: This observational study aimed to determine the proportion of CRPA isolates that were susceptible to all tested other traditional antipseudomonal ß-lactams (S-CRPA) and assess whether patients with S-CRPA had improved 30 day mortality compared with patients with NS-CRPA (non-susceptible to cefepime, ceftazidime or piperacillin/tazobactam). METHODS: Patients with CRPA isolated from normally sterile sites, urine, lower respiratory tracts and wounds were identified using active population- and laboratory-based surveillance through the Georgia Emerging Infections Program from August 2016 to July 2018 in Atlanta, GA, USA. Only unique patients who were hospitalized at the time of, or within 1 week of, culture were included. We excluded patients with cystic fibrosis. Multivariable logistic regression estimated the association between S-CRPA and 30 day mortality. RESULTS: Among 635 adults hospitalized with CRPA, 219 (34%) had S-CRPA. Patients with S-CRPA were more likely to be white (50% versus 38%, P = 0.01) and live in a private residence prior to culture (44% versus 28%, P < 0.01), and less likely to have required ICU care within the prior week (23% versus 36%, P < 0.01) compared with patients with NS-CRPA. Compared with those with NS-CRPA, patients with S-CRPA had an increased 30 day mortality (18% versus 15%, adjusted OR 1.9; 95% CI 1.2-3.1). CONCLUSIONS: S-CRPA was associated with higher 30 day mortality than NS-CRPA in hospitalized patients. The reason for this observed increase in mortality deserves further investigation.

Effect of genetic background on the evolution of Vancomycin-Intermediate Staphylococcus aureus (VISA).

Vancomycin-intermediate Staphylococcus aureus (VISA) typically arises through accumulation of chromosomal mutations that alter cell-wall thickness and global regulatory pathways. Genome-based prediction of VISA requires understanding whether strain background influences patterns of mutation that lead to resistance. We used an iterative method to experimentally evolve three important methicillin-resistant S. aureus (MRSA) strain backgrounds-(CC1, CC5 and CC8 (USA300)) to generate a library of 120 laboratory selected VISA isolates. At the endpoint, isolates had vancomycin MICs ranging from 4 to 10 µg/mL. We detected mutations in more than 150 genes, but only six genes (already known to be associated with VISA from prior studies) were mutated in all three background strains (walK, prs, rpoB, rpoC, vraS, yvqF). We found evidence of interactions between loci (e.g., vraS and yvqF mutants were significantly negatively correlated) and rpoB, rpoC, vraS and yvqF were more frequently mutated in one of the backgrounds. Increasing vancomycin resistance was correlated with lower maximal growth rates (a proxy for fitness) regardless of background. However, CC5 VISA isolates had higher MICs with fewer rounds of selection and had lower fitness costs than the CC8 VISA isolates. Using multivariable regression, we found that genes differed in their contribution to overall MIC depending on the background. Overall, these results demonstrated that VISA evolved through mutations in a similar set of loci in all backgrounds, but the effect of mutation in common genes differed with regard to fitness and contribution to resistance in different strains.

Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States.

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States.IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

Carbapenem-resistant Enterobacterales bacteriuria and subsequent bacteremia: A population-based study.

OBJECTIVE: To describe the epidemiology of carbapenem-resistant Enterobacterales (CRE) bacteriuria and to determine whether urinary catheters increase the risk of subsequent CRE bacteremia. DESIGN: Using active population- and laboratory-based surveillance we described a cohort of patients with incident CRE bacteriuria and identified risk factors for developing CRE bacteremia within 1 year. SETTING: The study was conducted among the 8 counties of Georgia Health District 3 (HD3) in Atlanta, Georgia. PATIENTS: Residents of HD3 with CRE first identified in urine between 2012 and 2017. RESULTS: We identified 464 patients with CRE bacteriuria (mean yearly incidence, 1.96 cases per 100,000 population). Of 425 with chart review, most had a urinary catheter (56%), and many resided in long-term care facilities (48%), had a Charlson comorbidity index >3 (38%) or a decubitus ulcer (37%). 21 patients (5%) developed CRE bacteremia with the same organism within 1 year. Risk factors for subsequent bacteremia included presence of a urinary catheter (odds ratio [OR], 8.0; 95% confidence interval [CI], 1.8-34.9), central venous catheter (OR, 4.3; 95% CI, 1.7-10.6) or another indwelling device (OR, 4.3; 95% CI, 1.6-11.4), urine culture obtained as an inpatient (OR, 5.7; 95% CI, 1.3-25.9), and being in the ICU in the week prior to urine culture (OR, 2.9; 95% CI, 1.1-7.8). In a multivariable analysis, urinary catheter increased the risk of CRE bacteremia (OR, 5.3; 95% CI, 1.2-23.6). CONCLUSIONS: In patients with CRE bacteriuria, urinary catheters increase the risk of CRE bacteremia. Future interventions should aim to reduce inappropriate insertion and early removal of urinary catheters.

Absence of mgrB Alleviates Negative Growth Effects of Colistin Resistance in Enterobacter cloacae.

Colistin is an important last-line antibiotic to treat highly resistant Enterobacter infections. Resistance to colistin has emerged among clinical isolates but has been associated with a significant growth defect. Here, we describe a clinical Enterobacter isolate with a deletion of mgrB, a regulator of colistin resistance, leading to high-level resistance in the absence of a growth defect. The identification of a path to resistance unrestrained by growth defects suggests colistin resistance could become more common in Enterobacter.

High Rates of New Delhi Metallo-ß-Lactamase Carbapenemase Genes in Multi-Drug Resistant Gram-Negative Bacteria in Nicaragua.

Antimicrobial resistance (AMR) is a global public health crisis. Much of the burden of AMR in resource-limited settings remains unknown. This pilot study characterized clinical isolates of multidrug-resistant Gram-negative rods (MDR-GNRs) from Nicaragua. New Delhi metallo-ß-lactamase (NDM) carbapenemase genes were detected in 60% of isolates. Enterobacteriaceae had the highest rates of NDM detection, with 92% (50/54 isolates) positive by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) analysis revealed patterns of clustering among isolates by two factors: plasmid profiles and year of culture. These findings of very high rates of NDM-carbapenemase genes in MDR-GNRs from hospitals throughout Nicaragua are alarming. Further research is needed to determine clinical and epidemiologic factors associated with multidrug-resistant isolates and to guide interventions to limit further spread.

Invasive Nontypeable Haemophilus influenzae Infection Among Adults With HIV in Metropolitan Atlanta, Georgia, 2008-2018.

Importance: Invasive nontypeable Haemophilus influenzae (NTHi) infection among adults is typically associated with bacteremic pneumonia. Nontypeable H influenzae is genetically diverse and clusters of infection are uncommon. Objective: To evaluate an increase in invasive NTHi infection from 2017-2018 among HIV-infected men who have sex with men in metropolitan Atlanta, Georgia. Design, Setting, and Participants: A population-based surveillance study with a cohort substudy and descriptive epidemiological analysis identified adults aged 18 years or older with invasive NTHi infection (isolation of NTHi from a normally sterile site) between January 1, 2008, and December 31, 2018 (final date of follow-up). Exposures: Time period, HIV status, and genetic relatedness (ie, cluster status) of available NTHi isolates. Main Outcomes and Measures: The primary outcome was incidence of invasive NTHi infection (from 2008-2016 and 2017-2018) among persons with HIV and compared with NTHi infection from 2008-2018 among those without HIV. The secondary outcomes were assessed among those aged 18 to 55 years with invasive NTHi infection and included epidemiological, clinical, and geographic comparisons by cluster status. Results: Among 553 adults with invasive NTHi infection (median age, 66 years [Q1-Q3, 48-78 years]; 52% male; and 38% black), 60 cases occurred among persons with HIV. Incidence of invasive NTHi infection from 2017-2018 among persons with HIV (41.7 cases per 100 000) was significantly greater than from 2008-2016 among those with HIV (9.6 per 100 000; P < .001) and from 2008-2018 among those without HIV (1.1 per 100 000; P < .001). Among adults aged 18 to 55 years with invasive NTHi infections from 2017-2018 (n = 179), persons with HIV (n = 31) were significantly more likely than those from 2008-2018 without HIV (n = 124) to be male (94% vs 49%, respectively; P < .001), black (100% vs 53%; P < .001), and have septic arthritis (35% vs 1%; P < .001). Persons with HIV who had invasive NTHi infection from 2017-2018 (n = 31) were more likely than persons with HIV who had invasive NTHi infection from 2008-2016 (n = 24) to have septic arthritis (35% vs 4%, respectively; P = .01). Pulsed-field gel electrophoresis of 174 of 179 NTHi isolates from 18- to 55-year-olds identified 2 genetically distinct clonal groups: cluster 1 (C1; n = 24) and cluster 2 (C2; n = 23). Whole-genome sequencing confirmed 2 clonal lineages of NTHi infection and revealed all C1 isolates (but none of the C2 isolates) carried IS1016 (an insertion sequence associated with H influenzae capsule genes). Persons with HIV were significantly more likely to have C1 or C2 invasive NTHi infection from 2017-2018 (28/31 [90%]) compared with from 2008-2016 among persons with HIV (10/24 [42%]; P < .001) and compared with from 2008-2018 among those without HIV (9/119 [8%]; P < .001). Among persons with C1 or C2 invasive NTHi infection who had HIV (n = 38) (median age, 34.5 years; 100% male; 100% black; 82% men who have sex with men), 32 (84%) lived in 2 urban counties and an area of significant spatial aggregation was identified compared with those without C1 or C2 invasive NTHi infection. Conclusions and Relevance: Among persons with HIV in Atlanta, the incidence of invasive nontypeable H influenzae infection increased significantly from 2017-2018 compared with 2008-2016. Two unique but genetically related clonal strains were identified and were associated with septic arthritis among black men who have sex with men and who lived in geographic proximity.

Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection.

Antibiotic-resistant bacteria are a significant threat to human health, with one estimate suggesting they will cause 10 million worldwide deaths per year by 2050, surpassing deaths due to cancer1. Because new antibiotic development can take a decade or longer, it is imperative to effectively use currently available drugs. Antibiotic combination therapy offers promise for treating highly resistant bacterial infections, but the factors governing the sporadic efficacy of such regimens have remained unclear. Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination2. Here, using carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates, we reveal the underlying basis for the majority of effective combinations to be heteroresistance. Heteroresistance is a poorly understood mechanism of resistance reported for different classes of antibiotics3-6 in which only a subset of cells are phenotypically resistant7. Within an isolate, the subpopulations resistant to different antibiotics were distinct, and over 88% of CRE isolates exhibited heteroresistance to multiple antibiotics ('multiple heteroresistance'). Combinations targeting multiple heteroresistance were efficacious, whereas those targeting homogenous resistance were ineffective. Two pan-resistant Klebsiella isolates were eradicated by combinations targeting multiple heteroresistance, highlighting a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.