Theoretical considerations and empirical predictions of the pharmaco- and population dynamics of heteroresistance.

Antibiotics are considered one of the most important contributions to clinical medicine in the last century. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulation models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend that the definition of heteroresistance should include a consideration of the rate of return to susceptibility.

Dynamic within-host cefiderocol heteroresistance caused by blaSHV-12 amplification in pandrug-resistant and hypervirulent Klebsiella pneumoniae sequence type 11.

AIMS: Although cefiderocol (FDC) is not prescribed in China, FDC-resistant pandrug-resistant hypervirulent Klebsiella pneumoniae (PDR-hvKp) is emerging. In this study, we performed FDC susceptibility testing of clinical Kp isolates to explore the prevalence of FDC-resistant isolates and the mechanism of FDC-resistance. METHODS: We retrospectively selected 151 carbapenem-resistant Kp isolates to assess FDC susceptibility. Seven isolates harboring blaSHV-12 from two patients were enrolled for whole-genome sequencing. The antimicrobial resistance, virulence, blaSHV-12 expression, and fitness costs in different media were examined. The amplification of blaSHV-12 was further investigated by qPCR and long-read sequencing. RESULTS: The 151 isolates showed a low MIC50/MIC90 (1/4 mg/L) of FDC. The seven isolates were ST11 PDR-hvKp, and two represented FDC-resistance (MIC=32 mg/L). The IncR/IncFII plasmids of two FDC-resistant isolates harbored 6 and 15 copies of blaSHV-12, whereas four FDC-susceptible isolates carried one copy and one harbored three copies. These blaSHV-12 genes concatenated together and were located within the same 7.3 kb fragment flanked by IS26, which contributed to the increased expression and FDC resistance without fitness costs. The amplification of blaSHV-12 and FDC resistance could be induced by FDC in vitro and reversed during continuous passage. CONCLUSIONS: The amplification of blaSHV-12 and the consequent dynamic within-host heteroresistance are important concerns for the rational application of antibiotics. Long-read sequencing might be a superior way to detect resistance gene amplification rapidly and accurately.

Vancomycin heteroresistance caused by unstable tandem amplifications of the vanM gene cluster on linear conjugative plasmids in a clinical Enterococcus faecium.

Vancomycin heteroresistance is prone to missed detection and poses a risk of clinical treatment failure. We encountered one clinical Enterococcus faecium strain, SRR12, that carried a complete vanM gene cluster but was determined as susceptible to vancomycin using the broth microdilution method. However, distinct subcolonies appeared within the clear zone of inhibition in the E-test assay, one of which, named SRR12-v1, showed high-level resistance to vancomycin. SRR12 was confirmed as heteroresistant to vancomycin using population analysis profiling and displayed "revive" growth curves with a lengthy lag phase of over 13 hours when exposed to 2-32 mg/L vancomycin. The resistant subcolony SRR12-v1 was found to carry an identical vanM gene cluster to that of SRR12 but a significantly increased vanM copy number in the genome. Long-read whole genome sequencing revealed that a one-copy vanM gene cluster was located on a pELF1-like linear plasmid in SRR12. In comparison, tandem amplification of the vanM gene cluster jointed with IS1216E was seated on a linear plasmid in the genome of SRR12-v1. These amplifications of the vanM gene cluster were demonstrated as unstable and would decrease accompanied by fitness reversion after serial passaging for 50 generations under increasing vancomycin pressure or without antibiotic pressure but were relatively stable under constant vancomycin pressure. Further, vanM resistance in resistant variants was verified to be carried by conjugative plasmids with variable sizes using conjugation assays and S1-pulsed field gel electrophoresis blotting, suggesting the instability/flexibility of vanM cluster amplification in the genome and an increased risk of vanM resistance dissemination.

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin.

The siderophore-cephalosporin cefiderocol (FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux-mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA, or piuD from 498 unique isolates collected before the introduction of FDC from four clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n = 15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild-type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

Emergence of heteroresistance to carbapenems in Gram-negative clinical isolates from two Egyptian hospitals.

BACKGROUND: Antimicrobial resistance is a global concern, linking bacterial genotype and phenotype. However, variability in antibiotic susceptibility within bacterial populations can lead to misclassification. Heteroresistance exemplifies this, where isolates have subpopulations less susceptible than the main population. This study explores heteroresistance in Gram-negative bacteria, distinguishing between carbapenem-sensitive isolates and stable heteroresistant isolates (SHIs). METHODS: A total of 151 Gram-negative clinical isolates including Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Proteus mirabilis from various sources were included. Heteroresistant isolates and their stability were detected by disc-diffusion technique while genotypic analysis was carried out by PCR and efflux activity was assessed by ethidium bromide (EtBr)-agar cartwheel method. RESULTS: A total of 51 heteroresistant subpopulations were detected, producing 16 SHIs upon stability-detection. Amplified resistance genes and EtBr-agar cartwheel method showed a significant difference between resistant subpopulations and their corresponding-sensitive main populations. CONCLUSION: Genotypic analysis confirmed that genetic mutation can lead to resistance development although the main populations were sensitive, thereby leading to treatment failure. This is a neglected issue which should be highly considered for better treatment outcomes.

The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran.

BACKGROUND: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates. METHODS: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP). RESULTS: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. CONCLUSIONS: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

Intrinsic clarithromycin heteroresistance in Mycobacterium avium.

BACKGROUND: Mycobacterium avium is associated with pulmonary disease in otherwise healthy adults. Several clarithromycin-refractory cases have been reported, including some cases caused by clarithromycin-susceptible strains. OBJECTIVES: To characterize the reason for the discrepancy between clinical response and antibiotic susceptibility results. METHODS: We conducted population analysis of clarithromycin-tolerant and heteroresistant subpopulations of M. avium cultured in vitro and in homogenates of infected lungs of mice. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for 28 M. avium and two M. kansasii strains. Mice were intranasally infected with M. avium and treated with or without clarithromycin (100 mg/kg) thrice weekly. They were sacrificed on day 35 and the bacteria in lung homogenates were tested for clarithromycin resistance. Population analysis assays were performed based on colony growth on plates containing two-fold dilutions of clarithromycin. RESULTS: The MBC/MIC ratios were ≥8 in all 28 strains of M. avium tested. In the population analysis assay, several colonies were observed on the plates containing clarithromycin concentrations above the MIC (2-64 mg/L). No growth of M. kansasii colonies was observed on the plates containing clarithromycin concentrations ≥2 mg/L. M. avium in the homogenates of infected lungs showed clearer clarithromycin-resistant subpopulations than in vitro, regardless of clarithromycin exposure. CONCLUSION: M. avium shows intrinsic heterogeneous resistance (heteroresistance) to clarithromycin. This may explain the observed discrepancies between clarithromycin susceptibility testing results and clinical response to clarithromycin treatment. Further studies are needed to confirm a link between heteroresistance and clinical outcomes.

Pyrazinamide-resistant Tuberculosis Obscured From Common Targeted Molecular Diagnostics.

Here, we describe a clinical case of pyrazinamide-resistant (PZA-R) tuberculosis (TB) reported as PZA-susceptible (PZA-S) by common molecular diagnostics. Phenotypic susceptibility testing (pDST) indicated PZA-R TB. Targeted Sanger sequencing reported wild-type PncA, indicating PZA-S TB. Whole Genome Sequencing (WGS) by PacBio and IonTorrent both detected deletion of a large portion of pncA, indicating PZA-R. Importantly, both WGS methods showed deletion of part of the primer region targeted by Sanger sequencing. Repeating Sanger sequencing from a culture in presence of PZA returned no result, revealing that 1) two minority susceptible subpopulations had vanished, 2) the PZA-R majority subpopulation harboring the pncA deletion could not be amplified by Sanger primers, and was thus obscured by amplification process. This case demonstrates how a small susceptible subpopulation can entirely obscure majority resistant populations from targeted molecular diagnostics and falsely imply homogenous susceptibility, leading to incorrect diagnosis. To our knowledge, this is the first report of a minority susceptible subpopulation masking a majority resistant population, causing targeted molecular diagnostics to call false susceptibility. The consequence of such genomic events is not limited to PZA. This phenomenon can impact molecular diagnostics' sensitivity whenever the resistance-conferring mutation is not fully within primer-targeted regions. This can be caused by structural changes of genomic context with phenotypic consequence as we report here, or by uncommon mechanisms of resistance. Such false susceptibility calls promote suboptimal treatment and spread of strains that challenge targeted molecular diagnostics. This motivates development of molecular diagnostics unreliant on primer conservation, and impels frequent WGS surveillance for variants that evade prevailing molecular diagnostics.

The Changing Paradigm of Drug-Resistant Tuberculosis Treatment: Successes, Pitfalls, and Future Perspectives.

Drug-resistant tuberculosis (DR-TB) remains a global crisis due to the increasing incidence of drug-resistant forms of the disease, gaps in detection and prevention, models of care, and limited treatment options. The DR-TB treatment landscape has evolved over the last 10 years. Recent developments include the remarkable activity demonstrated by the newly approved anti-TB drugs bedaquiline and pretomanid against Mycobacterium tuberculosis. Hence, treatment of DR-TB has drastically evolved with the introduction of the short-course regimen for multidrug-resistant TB (MDR-TB), transitioning to injection-free regimens and the approval of the 6-month short regimens for rifampin-resistant TB and MDR-TB. Moreover, numerous clinical trials are under way with the aim to reduce pill burden and shorten the DR-TB treatment duration. While there have been apparent successes in the field, some challenges remain. These include the ongoing inclusion of high-dose isoniazid in DR-TB regimens despite a lack of evidence for its efficacy and the inclusion of ethambutol and pyrazinamide in the standard short regimen despite known high levels of background resistance to both drugs. Furthermore, antimicrobial heteroresistance, extensive cavitary disease and intracavitary gradients, the emergence of bedaquiline resistance, and the lack of biomarkers to monitor DR-TB treatment response remain serious challenges to the sustained successes. In this review, we outline the impact of the new drugs and regimens on patient treatment outcomes, explore evidence underpinning current practices on regimen selection and duration, reflect on the disappointments and pitfalls in the field, and highlight key areas that require continued efforts toward improving treatment approaches and rapid biomarkers for monitoring treatment response.

Inducible Mega-Mediated Macrolide Resistance Confers Heteroresistance in Streptococcus pneumoniae.

In Streptococcus pneumoniae (Spn), the 5.4 to 5.5 kb Macrolide Genetic Assembly (Mega) encodes an efflux pump (Mef[E]) and a ribosomal protection protein (Mel) conferring antibiotic resistance to commonly used macrolides in clinical isolates. We found the macrolide-inducible Mega operon provides heteroresistance (more than 8-fold range in MICs) to 14- and 15-membered ring macrolides. Heteroresistance is commonly missed during traditional clinical resistance screens but is highly concerning as resistant subpopulations can persist despite treatment. Spn strains containing the Mega element were screened via Etesting and population analysis profiling (PAP). All Mega-containing Spn strains screened displayed heteroresistance by PAP. The heteroresistance phenotype was linked to the mRNA expression of the mef(E)/mel operon of the Mega element. Macrolide induction uniformly increased Mega operon mRNA expression across the population, and heteroresistance was eliminated. A deletion of the 5' regulatory region of the Mega operon results in a mutant deficient in induction as well as in heteroresistance. The mef(E)L leader peptide sequence of the 5' regulatory region was required for induction and heteroresistance. Treatment with a noninducing 16-membered ring macrolide antibiotic did not induce the mef(E)/mel operon or eliminate the heteroresistance phenotype. Thus, inducibility of the Mega element by 14- and 15-membered macrolides and heteroresistance are linked in Spn. The stochastic variation in mef(E)/mel expression in a Spn population containing Mega provides the basis for heteroresistance.

Profiling daptomycin resistance among diverse methicillin-resistant Staphylococcus aureus lineages in China.

Daptomycin (DAP) is effective against methicillin-resistant Staphylococcus aureus (MRSA). However, reduced susceptibility to DAP in MRSA may lead to treatment failures. We aim to determine the distribution of DAP minimum inhibitory concentrations (MICs) and DAP heteroresistance (hDAP) among MRSA lineages in China. A total of 472 clinical MRSA isolates collected from 2015 to 2017 in China were examined for DAP susceptibility. All isolates (n = 472) were found to be DAP susceptible, but 35.17% (166/472) of them exhibited a high DAP MIC (MIC >0.5 µg/mL). The high DAP MIC group contained a larger proportion of isolates with a higher vancomycin or teicoplanin MIC (>1.5 µg/mL) than the low DAP MIC group (19.3% vs 7.8%, P < 0.001; 22.3% vs 8.2%, P < 0.001). We compared the clonal complex (CC) distributions and clinical characteristics in MRSA isolates stratified by DAP MIC. CC5 isolates were less susceptible to DAP (MIC50 = 1 µg/mL) than CC59 isolates (MIC50 = 0.5 µg/mL, P < 0.001). Population analysis profiling revealed that 5 of 10 ST5 and ST59 DAP-susceptible MRSA isolates investigated exhibited hDAP. The results also showed that CC5 MRSA with an agrA mutation (I238K) had a higher DAP MIC than those with a wild-type agrA (P < 0.001). The agrA-I238K mutation was found to be associated with agr dysfunction as indicated by the loss of δ-hemolysin production. In addition, agr/psmα defectiveness was associated with hDAP in MRSA. Whole-genome sequencing analysis revealed mutations in mprF and walR/walK in DAP-resistant subpopulations, and most DAP-resistant subpopulations (6/8, 75%) were stable. Our study suggests that the increased DAP resistance and hDAP in MRSA may threaten the effectiveness against MRSA infections.

Genomic Modification of TonB and Emergence of Small-Colony Phenotype in VIM- and NDM-Producing Escherichia coli following Cefiderocol Exposure In Vitro.

Knowledge on resistance mechanisms toward cefiderocol, a novel siderophore-conjugated cephalosporin antibiotic, is still limited. Although the presence of New-Delhi metallo-ß-lactamase has been demonstrated to facilitate the resistance development toward cefiderocol via siderophore receptor mutations in Enterobacter cloacae and Klebsiella pneumoniae, the impact of metallo-ß-lactamases on facilitating such mutations in Escherichia coli is not yet elucidated. Our study aimed to study the effect of the presence of various ß-lactamases, such as NDM-5, VIM-1, KPC-2, and OXA-48, on the development of cefiderocol resistance in E. coli. To this end, we performed liquid mating to transfer these ß-lactamases onto a defined K-12 E. coli background (J53) and exposed these transconjugants to increasing cefiderocol concentrations in a serial passage experiment. Cefiderocol-resistant isolates were genotyped by whole-genome sequencing to investigate the underlying resistance mechanism. Cefiderocol-resistant isolates emerged only in isolates producing VIM-1 and NDM-5 metallo-ß-lactamase, but not in those producing the serine ß-lactamases KPC-2 and OXA-48. We observed two distinct morphological changes of the J53 E. coli strain exhibiting reduced colony size after insertions of transposable elements in the tonB gene leading to alterations in the TonB binding site and morphological changes consistent with the small-colony variant (SCV) phenotype due to mutations in the hemB and hemH genes. Passaging experiments suggested that these phenotypes were highly plastic. The SCV phenotype is attributed to immune evasion and decreased susceptibility toward antibiotics. The emergence of SCV following cefiderocol exposure may have clinical implications for bacterial clearance and warrants further investigation.

Multiple heteroresistance to tigecycline and colistin in Acinetobacter baumannii isolates and its implications for combined antibiotic treatment.

BACKGROUND: We investigated the presence of heteroresistance against both tigecycline and colistin in Acinetobacter baumannii and then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations. METHODS: We performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance in A. baumannii isolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels of pmrB. Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays. RESULTS: All tigecycline-heteroresistant A. baumannii isolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression of pmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells. CONCLUSION: Our results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent among A. baumannii clinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections.

Antibiotic resistance, heteroresistance, and eradication success of Helicobacter pylori infection in children.

BACKGROUND: Antibiotic resistance is a well-known factor of Helicobacter pylori eradication failure. Heteroresistance indicates the coexistence of resistant and susceptible strains and might lead to underestimating antimicrobial resistance. This study aims to evaluate the susceptibility profile, the frequency of heteroresistance of H. pylori strains, and their effect on eradication success in a pediatric population. MATERIALS AND METHODS: Children aged 2-17 years who underwent an upper gastrointestinal endoscopy from 2011 to 2019 with positive H. pylori status were included. Susceptibility was measured by disk diffusion and E-test. The difference in susceptibility profiles between isolates from the antrum and the corpus was used to detect heteroresistance. For those who received eradication treatment, we evaluated eradication rate and factors affecting treatment success. RESULTS: Inclusion criteria were met by 565 children. Strains susceptible to all antibiotics were detected in 64.2%. Primary resistance rates for clarithromycin (CLA), metronidazole (MET), levofloxacin (LEV), tetracyclin (TET), and amoxicillin (AMO) were 11%, 22.9%, 6.9%, 0.4%, and 0% and secondary resistance rates were 20.4%, 29.4%, 9.3%, 0%, and 0%. Heteroresistance was present in untreated children in 2%, 7.1%, 0.7%, 0.7%, and 0% for CLA, MET, LEV, TET, and AMO. First-line eradication rates were 78.5% in intention-to-treat (ITT), 88.3% in full-analysis-set (FAS), and 94.1% in per-protocol (PP). Factors affecting eradication success were the duration of treatment when the triple-tailored treatment was used, the number of daily doses of amoxicillin administered, and the patient's adherence to treatment. CONCLUSIONS: This study shows the presence of relatively low primary resistance rates for H. pylori isolates but demonstrates the presence of heteroresistance in our population. Routine biopsies from the antrum and corpus must be considered for susceptibility testing to allow tailored treatments and increase eradication rates. Treatment success is affected by treatment choice, correct dosing of medications, and adherence. All these factors should be considered when evaluating the efficacy of an eradication regimen.

Antibiotic resistance and heteroresistance in Helicobacter pylori isolates from symptomatic Vietnamese children: A prospective multicenter study.

BACKGROUND: Antibiotic resistance of Helicobacter pylori (H. pylori) is increasing worldwide, with geographical variations, impacting the treatment outcomes. This study assessed the antibiotic resistance patterns of H. pylori in Vietnamese children. MATERIALS AND METHODS: Symptomatic children undergoing gastroduodenoscopy at two tertiary Children's Hospitals in Ho Chi Minh City were recruited. Antral and corpus biopsies were obtained and cultured separately. Susceptibility to amoxicillin (AMO), clarithromycin (CLA), metronidazole (MET), levofloxacin (LEV), and tetracycline (TET) was determined using E-test. Polymerase chain reaction was performed on another antral biopsy to detect the urease gene, cytotoxin-associated gene A (cagA), vacuolating cytotoxin A (vacA) genotypes, and 23S rRNA mutations conferring CLA resistance. RESULTS: Among 123 enrolled children, a high primary resistance rate was found for CLA (68.5%, 61/89), followed by LEV (55.1%), MET (31.5%), AMO (25.8%), and TET (1.1%). Secondary resistance rates were 82.1% (7/28), 71.4%, 53.6%, and 3.6% for CLA, LEV, MET, and TET, respectively. Multidrug resistance was frequent (67.7%), with common patterns including CLA + LEV (20.3%) and CLA + MTZ + LEV (15.2%). Heteroresistance was detected in eight children (6.5%). The A2143G mutation was detected in 97.5% (119/122) of children. 86.1% of children had positive cagA strains and 27.9% had multiple vacA genotypes. No factor was significantly associated with antibiotic resistance. CONCLUSIONS: The alarming rate of antibiotic resistance for H. pylori, especially for CLA, with emerging multi- and hetero-resistant strains, pose a major treatment challenge that precludes CLA use as empirical therapy. Biopsies from both antrum and corpus can improve H. pylori culture, allowing tailored treatment based on antimicrobial susceptibility.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.

BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

Why the Vonoprazan Helicobacter pylori Therapies in the US-European Trial Produced Unacceptable Cure Rates.

INTRODUCTION: Helicobacter pylori infects a large percentage of the world's population and is etiologically related to gastric cancer. The U.S. Food and Drug Administration recently approved two 14-day vonoprazan-containing regimens (vonoprazan-amoxicillin with or without clarithromycin) for H. pylori infections in the United States/Europe. METHODS: We critically reviewed the trial methods to discover why the results were unacceptable low [i.e., no regimen achieved clinically acceptable (≥ 90%) or even conditionally acceptable cure rates (≥ 85%)]. Cure rates with antibiotic susceptible strains were 84.7 for vonoprazan triple therapy, 78.5 for vonoprazan-amoxicillin, and 78.7 for lansoprazole triple therapy, respectively. As was previously shown in Japan, the benefit from adding clarithromycin to vonoprazan-amoxicillin was minimal and the majority of the clarithromycin administered was unnecessary. RESULTS: The possible reasons for failure to achieve high cure rates discussed include (a) reduced intragastric antibiotic concentrations, (b) an increase in heteroresistance, and (c) failure to achieve an intragastric pH conducive for amoxicillin to eradicate the infection. In addition, there was no pilot study or other attempt to optimize any regimen. CONCLUSION: The most likely reason for failure was failure to achieve high intragastric concentrations of antibiotics or to achieve an intragastric pH conducive for amoxicillin to be active. Importantly, vonoprazan triple therapy resulted in > 10 tons of unneeded clarithromycin/million courses of vonoprazan triple therapy. Antibiotic misuse combined with low cure rates suggest that vonoprazan-clarithromycin triple therapies should not be prescribed for H. pylori infection. Dual vonoprazan-amoxicillin therapy has proven effective elsewhere and after optimization may eventually prove useful in the U.S./Europe.

Heteroresistance to rifampicin & isoniazid in clinical samples of patients with presumptive drug-resistant tuberculosis in Central India.

Background & objectives: A combination of resistant and susceptible Mycobacterium tuberculosis (MTB) isolated from clinical specimens is referred to as heteroresistance. Heteroresistance leads to difficulties in drug resistance testing and may adversely affect treatment outcomes. The present study estimated the proportion of heteroresistance among MTB in clinical samples of presumptive drug-resistant tuberculosis (TB) patients in Central India. Methods: A retrospective analysis of data generated from line probe assay (LPA) at a tertiary care hospital in Central India between January 2013 and December 2018 was carried out. A heteroresistant MTB in a sample was indicated by the presence of both wild-type and mutant-type patterns on an LPA strip. Results: Data analysis was carried out on interpretable 11,788 LPA results. Heteroresistance in MTB was detected in 637 (5.4%) samples. Of these, heteroresistance in MTB was detected in 413 (64.8%), 163 (25.5%) and 61 (9.5%) samples with respect to rpoB, katG and inhA genes, respectively. Interpretation & conclusions: Heteroresistance is considered a preliminary step in the development of drug resistance. Delayed or suboptimal anti-tubercular therapy in patients with heteroresistance of MTB may elicit full clinical resistance and negatively impact the National TB Elimination Programme. Further studies are, however, needed to determine the impact of heteroresistance on treatment outcomes in individual patients.

Quantitative Pharmacodynamic Characterization of Resistance versus Heteroresistance of Colistin in E. coli Using a Semimechanistic Modeling of Killing Curves.

Heteroresistance corresponds to the presence, in a bacterial isolate, of an initial small subpopulation of bacteria characterized by a significant reduction in their sensitivity to a given antibiotic. Mechanisms of heteroresistance versus resistance are poorly understood. The aim of this study was to explore heteroresistance in mcr-positive and mcr-negative Escherichia coli strains exposed to colistin by use of modeling killing curves with a semimechanistic model. We quantify, for a range of phenotypically (susceptibility based on MIC) and genotypically (carriage of mcr-1 or mcr-3 or mcr-negative) different bacteria, a maximum killing rate (Emax) of colistin and the corresponding potency (EC50), i.e., the colistin concentrations corresponding to Emax/2. Heteroresistant subpopulations were identified in both mcr-negative and mcr-positive E. coli as around 0.06% of the starting population. Minority heteroresistant bacteria, both for mcr-negative and mcr-positive strains, differed from the corresponding dominant populations only by the maximum killing rate of colistin (differences for Emax by a factor of 12.66 and 3.76 for mcr-negative and mcr-positive strains, respectively) and without alteration of their EC50s. On the other hand, the resistant mcr-positive strains are distinguished from the mcr-negative strains by differences in their EC50, which can reach a factor of 44 for their dominant population and 22 for their heteroresistant subpopulations. It is suggested that the underlying physiological mechanisms differ between resistance and heteroresistance, with resistance being linked to a decrease in the affinity of colistin for its site of action, whereas heteroresistance would, rather, be linked to an alteration of the target, which will be more difficult to be further changed or destroyed.

Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species.

The Enterobacter cloacae complex (ECC) is a group of diverse environmental and clinically relevant bacterial species associated with a variety of infections in humans. ECC have emerged as one of the leading causes of nosocomial infections worldwide. The purpose of this paper is to evaluate the activity of NOSO-502 and colistin (CST) against a panel of ECC clinical isolates, including different Hoffmann's clusters strains, and to investigate the associated resistance mechanisms. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). MIC50 and MIC90 of NOSO-502 against ECC are 1 µg/mL and 2 µg/mL, respectively, with a MIC range from 0.5 µg/mL to 32 µg/mL. Only strains belonging to clusters XI and XII showed decreased susceptibility to both NOSO-502 and CST while isolates from clusters I, II, IV, and IX were only resistant to CST. To understand this phenomenon, E. cloacae ATCC 13047 from cluster XI was chosen for further study. Results revealed that the two-component system ECL_01761-ECL_01762 (ortholog of CrrAB from Klebsiella pneumoniae) induces NOSO-502 hetero-resistance by expression regulation of the ECL_01758 efflux pump component (ortholog of KexD from K. pneumoniae) which could compete with AcrB to work with the multidrug efflux pump proteins AcrA and TolC. In E. cloacae ATCC 13047, CST-hetero-resistance is conferred via modification of the lipid A by addition of 4-amino-4-deoxy-l-arabinose controlled by PhoPQ. We identified that the response regulator ECL_01761 is also involved in this resistance pathway by regulating the expression of the ECL_01760 membrane transporter.