Isolation, genome analysis and tissue localization of Ceratobasidium theobromae, a new encounter pathogen of cassava in Southeast Asia.

In Southeast Asia (SEA) fastidious fungi of the Ceratobasidium genus are associated with proliferation of sprouts and vascular necrosis in cacao and cassava, crops that were introduced from the tropical Americas to this region. Here, we report the isolation and in vitro culture of a Ceratobasidium sp. isolated from cassava with symptoms of witches' broom disease (CWBD), a devastating disease of this crop in SEA. The genome characterization using a hybrid assembly strategy identifies the fungus as an isolate of the species C. theobromae, the causal agent of vascular streak dieback of cacao in SEA. Both fungi have a genome size > 31 Mb (G+C content 49%), share > 98% nucleotide identity of the Internal Transcribed Spacer (ITS) and > 94% in genes used for species-level identification. Using RNAscope® we traced the pathogen and confirmed its irregular distribution in the xylem and epidermis along the cassava stem, which explains the obtention of healthy planting material from symptom-free parts of a diseased plant. These results are essential for understanding the epidemiology of CWBD, as a basis for disease management including measures to prevent further spread and minimize the risk of introducing C. theobromae via long-distance movement of cassava materials to Africa and the Americas.

Bacillus coagulans-associated severe necrotizing scleral infection.

Purpose: We describe a case of severe scleritis possibly caused by Bacillus coagulans. Observations: Conventional laboratory evaluation was inconclusive. The associated organism was identified with metagenomic RNA deep sequencing (MDS). The infection resolved with trimethoprim-sulfamethoxazole treatment. Conclusions: This case demonstrates the utility of unbiased, high-throughput sequencing for infectious scleritis.

Hollow-fibre infection model: adaptations for the culture and assessment of fastidious organisms.

The hollow-fibre infection model (HFIM) is a valuable in vitro platform for emulating antimicrobial drug pharmacokinetic profiles. Despite its potential, standardized protocols for HFIM operation, especially concerning fastidious organisms, are lacking. This study addresses this gap by examining challenges in culturing Pasteurella multocida and Actinobacillus pleuropneumoniae, two fastidious organisms, in the HFIM. Our findings reveal effective strategies to prevent system clogging, involving multiple freeze-thaw cycles of horse blood, centrifugation and cell straining to enhance the clarity of the Mueller-Hinton fastidious medium defined by the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute. Additionally, we propose that the provision of a CO2 atmosphere, along with the utilization of gas-permeable tubing and gas vent filters, significantly facilitates the growth of fastidious organisms. Remarkably, both P. multocida and A. pleuropneumoniae were sustained for a period of up to 10 days under these optimized conditions. This study provides crucial insights into the modifications necessary to successfully culture fastidious organisms in the HFIM, paving the way for more accurate and representative in vitro models for antimicrobial drug testing. These advancements hold promise for advancing research in the field of antimicrobial pharmacokinetics and efficacy against challenging pathogens.

Assessing the Prevalence and Dynamics of Emerging Campylobacterales in Human Stool Samples in Brussels by Filtration Culture.

Thermophilic C. jejuni/coli is reported to be the first bacterial cause of gastroenteritis worldwide and the most common zoonosis in Europe. Although non-jejuni/coli Campylobacter sp. are increasingly suspected to be responsible for diarrhoea or to be involved in inflammatory bowel disease, they remain poorly isolated due to their fastidious and non-thermophilic nature. Additionally, they are not targeted by commercial syndromic PCR assays. In this study, we present routine diagnostic results over 6 years (2017-2019 and 2021-2023) of Campylobacter sp. and related species, obtained by optimised culture from 51,065 stools by both 0.65 µm pore filtration on antibiotic-free agar, incubated in an H2-enriched atmosphere at 37 °C (also known as the Cape Town protocol), and the use of selective inhibitory Butzler medium incubated at 42 °C. This allowed the isolation of 16 Campylobacter species, 2 Aliarcobacter species, and 2 Helicobacter species, providing a completely different view of the epidemiology of Campylobacterales, in which C. jejuni/coli represents only 30.0% of all isolates, while C. concisus represents 44.4%. C. ureolyticus, representing only 5.5% of all Campylobacterales pre-COVID-19, represented 20.6% of all strains post-COVID-19 (218% increase; p < 0.05). At the same time, the proportions of C. jejuni, C. coli, and C. concisus decreased by 37, 53, and 28%, respectively (p < 0.05).

Aspergillus terreus panophthalmitis with orbital cellulitis.

An eleven year old male reported a ten-day history of unilateral pain, redness, and sudden loss of vision. Ophthalmic examination revealed panophthalmitis that did not respond to conventional intravenous antibiotics, and systemic deterioration raised suspicion of a fungal aetiology. However, the worsening of the ocular condition from panophthalmitis to orbital cellulitis upon commencement of amphotericin B suggests the presence of a fastidious microorganism. Aspergillus terreus was isolated from a vitreous tap sample and responded well to intravenous voriconazole, exhibiting a distinct antimicrobial susceptibility spectrum and emphasising its possible involvement in relatively healthy early adolescence. To the author's knowledge, panophthalmitis with orbital cellulitis in early adolescence, without prior ocular insult, paranasal sinus involvement, or immunocompromised status, has not been reported previously.

Practice of standardization of CLSI M45 A3 antimicrobial susceptibility testing of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in Guangdong Province 2017-2021.

The concentration of antimicrobial agents in environments like water and food has increased rapidly, which led to a rapid increase in antimicrobial resistance levels in the environment. Monitoring of bacterial resistance levels is considered as a necessary means to control the bacterial resistance. Reference standards are critical for antimicrobial susceptibility testing. CLSI M45 A3 standard defines pathogenic microorganisms that cause infections less frequently than those covered by CLSI M02, M07, and M100 as Infrequently Isolated or Fastidious Bacteria and specifies antimicrobial susceptibility testing methods. Our study investigated the epidemiology and antimicrobial susceptibility testing data of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in 70 hospitals in Guangdong Province between 2017 and 2021. We defined testing methods other than those specified in CLSI M45 A3 as "Non-Standardized." The proportion of standardized antimicrobial susceptibility testing for penicillin increased significantly (Corynebacterium spp. 17.4% vs. 50.0% p < 0.05; Micrococcus spp. 50.0% vs. 77.8% p < 0.05; Abiotrophia spp. and Granulicatella spp. 21.4% vs. 90.9% p < 0.001), while for cefotaxime (Corynebacterium spp. 0.0% vs. 45.2% p < 0.05; Abiotrophia spp. and Granulicatella spp. 0.0% vs. 14.3% p = 0.515) and vancomycin increased finitely. Non-standardized methods were used for all other antimicrobials. Due to limitations in the economic and medical environment, some clinical laboratories are unable to fully comply with CLSI M45 A3 standard. We recommend that CLSI should add breakpoints for disk diffusion method to improve the standardization of antimicrobial susceptibility testing.

Microbroth dilution method for antibiotic susceptibility testing of fastidious and anaerobic bacteria of the urinary microbiome.

Bacterial isolates from the human urinary microbiome have been extensively studied for their antibiotic resistance; however, little work has been done on those isolates that are difficult to grow in vitro. This study was designed to qualify a serum-based medium, New York City Broth III (NYCIII), and a broth microdilution method to determine the antibiotic susceptibility of previously underreported or undescribed microbes that have a difficult time growing in standard Mueller-Hinton broth. Here, we demonstrate that NYCIII microbroth dilution can be an effective method for the determination of antibiotic susceptibility of species found in the human urinary microbiome. We show that this method serves well to characterize fastidious and anaerobic urinary microbes that have no Clinical and Laboratory Standards Institute (CLSI) guidelines, including several in the families Aerococcaceae, Lactobacillaceae, or Actinomycetaceae. Previous studies using expanded quantitative urine culture reveal that urine samples from clinical patients are commonly polymicrobial in composition. Thus, we test whether NYCIII can serve as a viable harmonized medium, capable of supporting antibiotic susceptibility testing in a range of fastidious, non-fastidious, and anaerobic urinary microbes. We propose this methodology to be standardized comparable to CLSI standards to allow for resistance testing in uncharacterized urinary bacteria. IMPORTANCE: Antibiotic susceptibilities of fastidious and anaerobic bacteria of the human urinary microbiome are largely underreported due to difficulty in growing them in the lab environment. The current standard medium, Muller-Hinton broth, has difficulty supporting the growth of many of these species, leaving microbiologists without a standardized method. To address this need, this study offers a methodology to survey susceptibilities in a high-throughput manner of these understudied microbes with a proposed harmonized medium, NYCIII, which is capable of supporting the growth of both fastidious and non-fastidious urinary microbes. Broader standardization of this method can allow for the development of antibiotic-resistant breakpoints of the many uncharacterized urinary microbes.

Ten Challenges to Understanding and Managing the Insect-Transmitted, Xylem-Limited Bacterial Pathogen Xylella fastidiosa.

An unprecedented plant health emergency in olives has been registered over the last decade in Italy, arguably more severe than what occurred repeatedly in grapes in the United States in the last 140 years. These emergencies are epidemics caused by a stealthy pathogen, the xylem-limited, insect-transmitted bacterium Xylella fastidiosa. Although these epidemics spurred research that answered many questions about the biology and management of this pathogen, many gaps in knowledge remain. For this review, we set out to represent both the U.S. and European perspectives on the most pressing challenges that need to be addressed. These are presented in 10 sections that we hope will stimulate discussion and interdisciplinary research. We reviewed intrinsic problems that arise from the fastidious growth of X. fastidiosa, the lack of specificity for insect transmission, and the economic and social importance of perennial mature woody plant hosts. Epidemiological models and predictions of pathogen establishment and disease expansion, vital for preparedness, are based on very limited data. Most of the current knowledge has been gathered from a few pathosystems, whereas several hundred remain to be studied, probably including those that will become the center of the next epidemic. Unfortunately, aspects of a particular pathosystem are not always transferable to others. We recommend diversification of research topics of both fundamental and applied nature addressing multiple pathosystems. Increasing preparedness through knowledge acquisition is the best strategy to anticipate and manage diseases caused by this pathogen, described as "the most dangerous plant bacterium known worldwide."

The performance of a Fully Automated Urine Particle Analyzer, Sysmex UF-5000, in detecting fastidious bacteria in urine samples.

Several types of fastidious bacteria can cause tract infections. We evaluated the performance of counting fastidious bacteria using a Fully Automated Urine Particle Analyzer UF-5000. The results showed that UF-5000 counts fastidious bacteria in urine without the need for culture using measurement principles based on flow cytometry.

Microbiome of limb-threatening diabetic foot ulcers indicates the association of fastidious Stenotrophomonas and major amputation.

BACKGROUND: Proper identification of the polymicrobial microorganisms in patients with limb-threatening diabetic foot ulcers (LTDFUs) using conventional culture is insufficient. This prospective study evaluates the potential value of adjuvant molecular testing assisting in identify fastidious micro-organisms in LTDFUs compared to standard treatment alone. METHODS: Ninety patients with LTDFUs received interdisciplinary and standard antibiotic treatment in a referral diabetic foot center. A simultaneous 16S amplicon sequencing (16S AS) specimen along with conventional culture collected at admission was used to retrospectively evaluate the microbiological findings and its association with amputation outcomes. RESULTS: The microorganism count revealed by 16S AS overwhelmed that of conventional culturing (17 vs. 3 bacteria/ulcer respectively). The Stenotrophomonas spp. revealed in 29 patients were highly correlated with major (above ankle) amputation (OR: 4.76, 95% CI 1.01-22.56), while only one had been concomitantly identified by conventional culturing. Thus, there were 27 cases without proper antibiotics coverage during treatment. CONCLUSIONS: Adjuvant molecular testing assisted identification of fastidious pathogens such as Stenotrophomonas infection and might be associated with major amputation in patients with LTDFUs.

Buffer-free CAS assay using a diluted growth medium efficiently detects siderophore production and microbial growth.

Siderophores are iron chelators and low-molecular-weight compounds secreted by various microorganisms under low-iron conditions. Many microorganisms produce siderophores in the natural environment as iron is an essential element for many of them. CAS assays are widely used to detect siderophores in cultures of various microorganisms; however, it is necessary to improve their sensitivity for the efficient application to fastidious microorganisms. We developed a simple, high-throughput CAS assay employing a buffer-free CAS reagent and diluted growth medium (10% dR2A) in a 96-well microplate. Using a diluted growth medium in agar plates suitable for iron-restricted conditions supported siderophore production by microorganisms from activated sludge. A buffer-free CAS reagent combined with a diluted growth medium revealed that these microorganisms tended to produce more siderophores or iron chelators than microorganisms under iron-rich conditions. Moreover, this buffer-free CAS assay easily and efficiently detected not only siderophore production but also the growth of fastidious microorganisms.

The Essential Role of PCR and PCR Panel Size in Comparison with Urine Culture in Identification of Polymicrobial and Fastidious Organisms in Patients with Complicated Urinary Tract Infections.

Complicated urinary tract infections (cUTIs) are difficult to treat, consume substantial resources, and cause increased patient morbidity. Data suggest that cUTI may be caused by polymicrobial and fastidious organisms (PMOs and FOs, respectively); as such, urine culture (UC) may be an unreliable diagnostic tool for detecting cUTIs. We sought to determine the utility of PCR testing for patients presumed to have a cUTI and determine the impact of PCR panel size on organism detection. We reviewed 36,586 specimens from patients with presumptive cUTIs who received both UC and PCR testing. Overall positivity rate for PCR and UC was 52.3% and 33.9%, respectively (p < 0.01). PCR detected more PMO and FO than UC (PMO: 46.2% vs. 3.6%; FO: 31.3% vs. 0.7%, respectively, both p < 0.01). Line-item concordance showed that PCR detected 90.2% of organisms identified by UC whereas UC discovered 31.9% of organisms detected by PCR (p < 0.01). Organism detection increased with expansion in PCR panel size from 5-25 organisms (p < 0.01). Our data show that overall positivity rate and the detection of individual organisms, PMO and FO are significantly with PCR testing and that these advantages are ideally realized with a PCR panel size of 25 or greater.

Granulicatella adiacens Endocarditis of a Bioprosthetic Aortic Valve.

Infective endocarditis (IE) is relatively uncommon; however, when it is diagnosed, it is usually among those with known cardiac valvular abnormalities. The most common pathogens that cause endocarditis are streptococci (mainly viridans), enterococci, and other streptococci species. An extremely rare pathogen that could cause IE is Granulicatella. This gram-positive coccus classically inhabits human mucosal surfaces and only rarely causes disease. We present an incredibly rare case of a 74-year-old female with a bioprosthetic aortic valve replacement, who presented with headache and weakness and was subsequently found to have recurrent Granulicatella adiacens infective endocarditis.

Direct 16S/18S rRNA Gene PCR Followed by Sanger Sequencing as a Clinical Diagnostic Tool for Detection of Bacterial and Fungal Infections: a Systematic Review and Meta-Analysis.

rRNA gene Sanger sequencing is being used for the identification of cultured pathogens. A new diagnostic approach is sequencing of uncultured samples by using the commercial DNA extraction and sequencing platform SepsiTest (ST). The goal was to analyze the clinical performance of ST with a focus on nongrowing pathogens and the impact on antibiotic therapy. A literature search used PubMed/Medline, Cochrane, Science Direct, and Google Scholar. Eligibility followed PRISMA-P criteria. Quality and risk of bias were assessed drawing on QUADAS-2 (quality assessment of diagnostic accuracy studies, revised) criteria. Meta-analyses were performed regarding accuracy metrics compared to standard references and the added value of ST in terms of extra found pathogens. We identified 25 studies on sepsis, infectious endocarditis, bacterial meningitis, joint infections, pyomyositis, and various diseases from routine diagnosis. Patients with suspected infections of purportedly sterile body sites originated from various hospital wards. The overall sensitivity (79%; 95% confidence interval [CI], 73 to 84%) and specificity (83%; 95% CI, 72 to 90%) were accompanied by large effect sizes. ST-related positivity was 32% (95% CI, 30 to 34%), which was significantly higher than the culture positivity (20%; 95% CI, 18 to 22%). The overall added value of ST was 14% (95% CI, 10 to 20%) for all samples. With 130 relevant taxa, ST uncovered high microbial richness. Four studies demonstrated changes of antibiotic treatment at 12% (95% CI, 9 to 15%) of all patients upon availability of ST results. ST appears to be an approach for the diagnosis of nongrowing pathogens. The potential clinical role of this agnostic molecular diagnostic tool is discussed regarding changes of antibiotic treatment in cases where culture stays negative.

Bartholin's gland cyst caused by Sneathia amnii: a case report.

BACKGROUND: Sneathia amnii is a conditional pathogen of the female genital tract that is involved in bacterial vaginosis and poor reproductive and perinatal outcomes. Few studies have reported subcutaneous cysts following invasive infection caused by S amnii. CASE PRESENTATION: Here we report the case of a 27-year-old woman who presented with Bartholin's gland cyst due to S amnii infection, and was successfully treated with surgical neostomy and antibiotic agents. The isolate was gram-negative, bacillary, anaerobic, and was identified by polymerase chain reaction (PCR) amplification of the 16 S rRNA. CONCLUSIONS: S amni is an important but underappreciated pathogen that needs further investigation. This report describes the microbial and pathogenic characteristics of S amnii and is expected to provide a valuable reference in obstetric and gynecologic clinical practice.

Prediction of presence of fastidious bacteria by the Fully Automated Urine Particle Analyzer UF-1000i in the case of ineffective antimicrobial therapy for urinary tract infection.

INTRODUCTION: Recent studies have reported associations between fastidious bacteria that are difficult to grow and isolate in conventional urine culture conditions and urinary tract infections (UTIs). Because the Fully Automated Urine Particle Analyzer UF-1000i (hereinafter referred to as "UF-1000i") detects fastidious bacteria without being affected by culture conditions, owing to its flow cytometry-based principle, we evaluated the robustness of UF-1000i detection using clinical urine samples from patients with UTIs following ineffective antimicrobial therapy. METHODS: A total of 150 patients diagnosed with UTIs were enrolled, and their laboratory findings were analyzed, focusing on the discrepancy in bacterial numbers between UF-1000i and conventional culture at each antimicrobial therapy effectiveness classification. In addition, gene identification was conducted by molecular analysis using 16S ribosomal RNA gene sequencing and next-generation sequencing (NGS) to elucidate the reason for the presence of fastidious bacteria in these samples. RESULTS: The ineffective therapy cases showed more than 100-fold discrepancy in bacterial counts, with a higher proportion (30.8%) than effective therapy cases without secondary administration (5.7%) between the bacterial counts in UF-1000i and conventional culture methods. The presence rates of fastidious bacteria were 100% and 66.7% in discrepant cases of ineffective and effective without secondary administrations, respectively. CONCLUSION: This study suggests that discrepancies in bacterial numbers between the conventional culture method and UF-1000i measurement at the primary visit can predict the presence of fastidious bacteria, especially in cases of ineffective antimicrobial therapy.

Development of a long-read next generation sequencing workflow for improved characterization of fastidious respiratory mycoplasmas.

Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick's broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4-5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.

Identification and Characterization of Potato Zebra Chip Resistance Among Wild Solanum Species.

Potato zebra chip (ZC) disease, associated with the uncultured phloem-limited bacterium, Candidatus Liberibacter solanacearum (CLso), is transmitted by the potato psyllid Bactericera cockerelli. Potato ZC disease poses a significant threat to potato production worldwide. Current management practices mainly rely on the control of the psyllid to limit the spread of CLso. The present study investigated new sources of ZC resistance among wild Solanum species. A taxonomically diverse collection of tuber-bearing Solanum species was screened; one ZC-resistant accession and three ZC-tolerant accessions were identified among the 52 screened accessions. Further characterization of the resistant accession showed that the resistance was primarily associated with antibiosis effects due to differences in leaf trichome density and morphology of the wild accession, which could limit the psyllid feeding and oviposition. This germplasm offers a good resource for further understanding ZC and psyllid resistance mechanisms, contributing to potato breeding efforts to develop ZC resistance cultivars. Alternatively, it could be used as a potential trap crop to manage psyllid and control ZC disease.

A Rare Presentation of Infective Endocarditis Due to Serratia marcescens.

Serratia marcescens is an opportunistic organism that can commonly cause respiratory tract infections in immunocompromised individuals. It has also been shown to cause urinary tract infections and soft tissue infections. It has several virulence factors including fimbriae-like adhesions that allow for surface attachment and biofilm formation to increase the likelihood of infections in humans. However, it has rarely been shown to cause infective endocarditis but has an increased mortality compared to the usual microbial agents associated with it (Staphylococcus and Streptococcus). Therefore, a high index of suspicion is necessary to accurately diagnose and treat patients at risk. Most published cases of S. marcescens endocarditis show that almost all described patients had chronic medical conditions or cardiovascular abnormalities. Furthermore, treatment has become difficult as S. marcescens has been shown to exhibit antibacterial resistance with beta-lactamase production. Here, we present a complicated case of S. marcescens pneumonia and infective endocarditis with a good prognosis. Our patient had a rapid onset of complications (i.e. including joint infections, splenic abscesses, myositis, and septic arthritis), despite the initial benign presentation concerning for pneumonia. However, the patient had a favorable outcome due to the prompt work-up and treatment that was initiated. Therefore, S. marcescens bacteremia in a patient with risk factors should prompt further investigation with a thorough evaluation of source followed by immediate management. This case highlights the fastidious nature of S. marcescens. Further investigation needs to be done to elucidate the pathogenesis of the organism that can serve as a target for future therapeutic intervention.

Multicenter Evaluation of a Gradient Diffusion Method for Antimicrobial Susceptibility Testing of Helicobacter pylori.

Helicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization. IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.