G143A Mutation Research Articles

First Detection of Frogeye Leaf Spot in Soybean Fields in North Dakota and the G143A Mutation in the Cytochrome b Gene of Cercospora sojina

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from North Dakota soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in North Dakota. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in North Dakota will need to use an integrated approach of cultural practices, genetic resistance, and fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

The G143A Mutation in the Cytochrome b Gene Is Associated with Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina from Soybean Fields in Wisconsin

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important management tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from Wisconsin soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in Wisconsin. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in Wisconsin will need to use an integrated approach of cultural practices, genetic resistance, and using fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Prevalence of the A2058G mutation in 23S rRNA gene, which determines Treponema pallidum macrolide resistance in Russian population

Objective. To investigate prevalence of the A2058G mutation in the Russian population of T. pallidum and its association with molecular subtypes. Materials and Methods. We analyzed DNA isolated from 325 samples of clinical material obtained from patients of dermatovenereological treatment and prophylactic institutions in 6 federal districts of the Russia in the period from 2014 to 2021. Patients were diagnosed with primary syphilis of the genital organs, primary syphilis of other sites, or secondary syphilis of the skin and mucous membranes. DNA was isolated using the Proba-NK reagent kit (DNA-technology, Russia) according to the manufacturer’s instructions. The presence of T. pallidum genetic material was confirmed by PCR with primers for the species-specific polA gene. Molecular typing was performed based on the analysis of polymorphic regions of species-specific T. pallidum genes. The primary decoding of the nucleotide sequences of the 23S rRNA gene fragment was carried out using the Sequencing Analysis 5.3.1 program. The analyzed fragments were aligned using the Mega 5 program. Results. Eight molecular subtypes of T. pallidum – 14d/f, 14d/g, 14b/f, 14c/f, 14i/f, 9d/f, 14b/g and 14e/f with stable dominance of subtype 14d/f – were identified in the Russian Federation in the interval 2014–2021. Three subtypes, 14d/g, 14b/g and 14b/f, carrying the A2058G mutation associated with azithromycin resistance, were identified. Conclusions. Studies on molecular typing of T. pallidum strains in the Russia have shown significant population heterogeneity. Three sublines containing the A2058G mutation were shown to exist, one of which – 14b/f – is described as rare. The obtained data confirm the relevance of continuous monitoring of the emergence of resistant strains and the development of new mutations.

Distribution of G143A Mutations Conferring Fungicide Resistance in Virginia Populations of Parastagonospora nodorum Infecting Wheat

Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum is an important leaf spot disease in the mid-Atlantic United States. Disease management approaches include use of resistant varieties, cultural control, and foliar fungicides. Frequent use of foliar fungicides can select for fungicide resistance within pathogen populations. Recently, the first report of quinone outside inhibitor (QoI) fungicide resistance in the United States was made based on a relatively small collection of P. nodorum isolates from Virginia. The objective of this study was to conduct a statewide, 2-year survey of P. nodorum populations in Virginia wheat and quantify frequencies of the target-site mutation that confers QoI resistance. A total of 318 isolates of P. nodorum were obtained from wheat collected at seven locations distributed throughout the wheat-growing regions of Virginia in 2018 and 2019. A previously designed pyrosequencing assay that detects the G143A substitution in the cytochrome b gene of P. nodorum was used to screen isolates for the presence or absence of the target site mutation. The G143A substitution was detected in all sampled fields. Among locations and years, frequencies of the mutation in P. nodorum populations ranged from 5 to 32% (mean = 19%). Thus, the QoI-resistance-conferring G143A mutation was widespread in P. nodorum populations in Virginia, and it occurred at a relatively high frequency. Results suggest that fungicides containing QoI active ingredients may not be effective for controlling SNB in Virginia and the surrounding region, and application of standalone QoI fungicides for disease control in wheat is not recommended.

Analysis of Mycoplasma pneumoniae antibiotic resistance based on data obtained in the Tula region

Objective. To study the prevalence of mutations causing resistance to macrolides in Mycoplasma pneumoniae in the Tula region and compare the data with similar studies from European and Asian countries. Material and methods. A retrospective study of 76 samples of biological materials (nasal and pharynx swabs) was conducted. The materials were collected from the patients with an established diagnosis: the lower respiratory tract infection (pneumonia, bronchitis). All the patients included in the study were treated in specialized infections units and clinics of the Tula region for the period 2017–2018. Antibiotic therapy was not conducted before the initial survey. All the samples contained M. pneumoniae DNA. A real-time polymerase chain reaction (real-time PCR) technology was used to detect the DNA of M. pneumoniae in the materials. The analysis of mutations in the 23S rRNA gene was performed at the Scientific Research Institute of Antimicrobial Chemotherapy in Smolensk. During the study, a modified real-time PCR technique with the effect of the fluorescence quenching of the probe with a primer (patent No. 2646123) was used. The statistical analysis was carried out with the MS Excel software package. Results. It was identified that 9 samples (11.84%) had mutations associated with resistance to macrolides. Two samples had the A2059G mutation, while seven samples had the A2058G mutation (numbering of nucleotide sequences in the M. pneumoniae rRNA gene was compiled following E. coli numbering). The data obtained by us are slightly higher than the mean values of European countries and are significantly lower according to the studies of Asian countries. Conclusions. Provided unique data on the prevalence and identification of mutations of macrolide-resistant strains of M. pneumoniae in one of the regions of the Central Federal District is a contribution to the study of antibiotic-resistant strains of M. pneumoniae in Russia and all over the world. There is a necessity to monitor antibiotic resistance in other regions and increase diligence on atypical forms of community-acquired pneumonia since the co-infection of M. pneumoniae patients with COVID-19 leads to a deterioration in the condition and an increase in the duration of treatment. Keywords: community-acquired pneumonia, macrolides mycoplasmas, mutations of antibiotic resistance

Determination of In Vitro Antimicrobial Susceptibility for Lefamulin (Pleuromutilin) for Ureaplasma Spp. and Mycoplasma hominis.

Lefamulin is the first of the pleuromutilin class of antimicrobials to be available for therapeutic use in humans. Minimum inhibitory concentrations of lefamulin were determined by microbroth dilution for 90 characterised clinical isolates (25 Ureaplasma parvum, 25 Ureaplasma urealyticum, and 40 Mycoplasma hominis). All Mycoplasma hominis isolates possessed lefamulin MICs of ≤0.25 mg/L after 48 h (MIC50/90 of 0.06/0.12 mg/L), despite an inherent resistance to macrolides; while Ureaplasma isolates had MICs of ≤2 mg/L after 24 h (MIC50/90 of 0.25/1 mg/L), despite inherent resistance to clindamycin. Two U. urealyticum isolates with additional A2058G mutations of 23S rRNA, and one U. parvum isolate with a R66Q67 deletion (all of which had a combined resistance to macrolides and clindamycin) only showed a 2-fold increase in lefamulin MIC (1–2 mg/L) relative to macrolide-susceptible strains. Lefamulin could be an effective alternative antimicrobial for treating Ureaplasma spp. and Mycoplasma hominis infections irrespective of intrinsic or acquired resistance to macrolides, lincosamides, and ketolides. Based on this potent in vitro activity and the known good, rapid, and homogenous tissue penetration of female and male urogenital tissues and glands, further exploration of clinical efficacy of lefamulin for the treatment of Mycoplasma and Ureaplasma urogenital infections is warranted.

Correlation between clarithromycin resistance, virulence factors and clinical characteristics of the disease in Helicobacter pylori infected patients in Shahrekord, Southwest Iran

The purpose of this study was to determine the mutations associated with clarithromycin resistance in Helicobacter pylori strains isolated from biopsy samples that were collected from the endoscopic ward of Shahrekord Hajar teaching Hospital and also to study the frequency of virulence factor and their correlation and pathological findings with clarithromycin resistance during the years 2019–2020. In this cross-sectional descriptive study, 152 patients with Helicobacter pylori infection were considered, and then, two common A2142G and A2143G mutations in the 23SrRNA gene associated with resistance were analyzed by Real-time PCR (Taq man). The presence of vacA, iceA1, iceA2, cagA, babA2, and oipA virulence genes was investigated by PCR and electrophoresis in 8% polyacrylamide gel. Then, data were analyzed using the relevant statistical tests. In this study, the frequency of Helicobacter pylori was 76% and the frequency of mutant isolates was 57.2%. The frequencies of A2142G and A2143G point mutations were 42.1% and 28.3%. There was a significant correlation among oipA, vacA, and iceA1 virulence factors, type of disease, chronic inflammatory score, and glandular atrophy with the antibiotic resistance to clarithromycin. There was no significant correlation between the age and sex of the patients with antibiotic resistance. According to the results of this study, it seems that the use of clarithromycin to combat this bacterium should be limited.

Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy.

Aims and ObjectivesMore than half of the world’s population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies.Materials and MethodsSpecific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay’s sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.ResultsIn this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and −LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively.ConclusionThe effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.

Method for QoI sensitivity monitoring in Colletotrichum lentis by a species-specific pyrosequencing assay

Colletotrichum lentis is an important pathogen of lentils in Canada causing the disease anthracnose. Fungicide applications are an important tool for the control of this disease and, historically, QoI fungicides have been used intensively. Reduced field efficacy of QoI fungicides on C. lentis has been reported recently and could be linked to the presence of QoI resistant isolates. In this study, the cytochrome b gene of C. lentis was sequenced, and the mutation G143A was identified as the causal resistance mechanism. Furthermore, a sensitive and reliable molecular biological assay was developed for the quantitative detection of the G143A mutation by pyrosequencing in field samples.

Detection of clarithromycin resistance and 23SrRNA point mutations in clinical isolates of Helicobacter pylori isolates: Phenotypic and molecular methods

Background and objectivesPeptic ulcer disease, chronic gastritis, and stomach cancer are all caused by H. pylori. The most notable drug for the treatment is the antibiotic clarithromycin, which is currently the drug of choice. H. pylori clarithromycin resistance has been associated with point mutations in 23srRNA, the most prominent of which are A2143 and A2144G. In H. pylori bacteria, methylase synthesis, macrolide-inactivating enzyme activity, and active efflux have all been found to be resistance mechanisms. The goal of the study is to determine how resistant H. pylori is to clarithromycin and what the minimum inhibitory concentration is for various antimicrobials. Furthermore, gastro-endoscopy will be performed on Iraqi patients to detect the presence of A2143G and A2144G point mutations in Helicobacter pylori infections, as diagnosed from the pyloric region and other anatomical regions. MethodsOne hundred fifteen samples were collected from patients strongly suspected of H. pylori infection presented for upper gastrointestinal endoscopy at Ramadi Teaching Hospitals and Private Clinics for the period from January 2020 until February 2021. Specimens were cultured on brain heart infusion agar containing various antibiotics and were incubated at 37 °C under microaerophilic conditions. For identification of H. pylori, isolates of the biochemical tests and RT-PCR assay were applied. The Epsilometer test was used in the antibiotic susceptibility testing as dependent on the CLSI standard. The Restriction Fragment Length Polymorphism technique was used to determine point mutations. ResultsIn total, 55 (47.8%) Helicobacter pylori isolates were cultured from the 115 biopsy specimens, among which 16 (29.1%), 38 (69.1%), 20 (36.4%), and 40 (72.7%) revealed some degree of resistance to levofloxacin, clarithromycin, ciprofloxacin, and metronidazole, respectively. The frequency of A2144G and A2143 point mutations were 23 (60.5%) and 19 (50%), respectively. ConclusionsAccording to our results, Helicobacter pylori showed high resistance to clarithromycin. Our results demonstrate the requirement for antibiotic susceptibility testing and molecular methods in selecting drug regimens.

Efficacy of Loop-Mediated Isothermal Amplification for H. pylori Detection as Point-of-Care Testing by Noninvasive Sampling.

For targeted eradication of Helicobacter pylori (H. pylori) to reduce gastric cancer burden, a convenient approach is definitely needed. The purpose of this study was to evaluate the LAMP assay for H. pylori detection using samples collected by noninvasive and self-sampling methods. The available LAMP assay for H. pylori detection was appraised and verified using reference and clinically isolated H. pylori strains. In addition, a clinical study was conducted to assess the LAMP assay on 51 patients, from whom saliva, oral brushing samples, feces, corpus, and antrum specimens were available. Clarithromycin resistance was also analysed through detection of A2143G mutation using the LAMP-RFLP method. The validation and verification analysis demonstrated that the LAMP assay had an acceptable result in terms of specificity, sensitivity, reproducibility, and accuracy for clinical settings. The LAMP assay showed a detection limit for H. pylori down to 0.25 fg/µL of genomic DNA. An acceptable consensus was observed using saliva samples (sensitivity 58.1%, specificity 84.2%, PPV 85.7%, NPV 55.2%, accuracy 68%) in comparison to biopsy sampling as the gold standard. The performance testing of different combinations of noninvasive sampling methods demonstrated that a combination of saliva and oral brushing could achieve a sensitivity of 74.2% and a specificity of 57.9%. A2143G mutation detection by LAMP-RFLP showed perfect consensus with Sanger sequencing results. It appears that the LAMP assay in combination with noninvasive and self-sampling as a point-of-care testing (POCT) approach has potential usefulness to detect H. pylori infection in clinic settings and screening programs.

Tailored eradication strategy vs concomitant therapy for Helicobacter pylori eradication treatment in Korean patients.

BACKGROUNDAntibiotic resistance to Helicobacter pylori (H. pylori) infection, which ultimately results in eradication failure, has been an emerging issue in the clinical field. Recently, to overcome this problem, an antibiotic sensitivity-based tailored therapy (TT) for H. pylori infection has received attention. AIMTo investigate the efficacy and safety profiles of TT for H. pylori infection treatment compared to a non-bismuth quadruple therapy, concomitant therapy (CT) regimen. METHODSWe included patients (> 18 years) with an H. pylori infection and without a history of Helicobacter eradication who visited the Gil Medical Center between March 2016 and October 2020. After being randomly assigned to either the TT or CT treatment group in 1 to 1 manner, patient compliance, eradication success rate (ESR), and patient-reported side effects profiles were assessed and compared between the two groups. H. pylori infection was diagnosed using a rapid urease test, Giemsa stain, or dual priming oligonucleotide polymerase chain reaction (DPO-PCR). Tailored eradication strategy based through the presence of a 23S ribosomal RNA point mutation. For the TT group, a DPO-PCR test, which detected A2142G and/or A2143G point mutations, and a clarithromycin resistance test were performed. Patients in the clarithromycin-resistant group were treated with a bismuth-containing quadruple combination therapy, while those with sensitive results were treated with the standard triple regimen. RESULTSOf the 217 patients with a treatment naive H. pylori infection, 110 patients [mean age: 58.66 ± 13.03, men, n = 55 (50%)] were treated with TT, and 107 patients [mean age: 56.67 ± 10.88, men, n = 52 (48.60%)] were treated with CT. The compliance (TT vs CT, 100% vs 98.13%, P = 0.30), and follow-up loss rates (8.18% vs 9.35%, P = 0.95) were not significantly different between the groups. The ESR after treatment was also not statistically different between the groups (TT vs CT, 82.73% vs 82.24%, P = 0.95). However, the treatment-related and patient-reported side effects were significantly lower in the TT group than in the CT group (22.77% vs 50.52%, P < 0.001).CONCLUSIONThe DPO-based TT regimen shows promising results in efficacy and safety profiles as a first-line Helicobacter eradication regimen in Korea, especially when physicians are confronted with increased antibiotic resistance rates.

Multilocus Sequence Typing of Treponema Pallidum Subsp. Pallidum in Barcelona

Aim: To implement the multilocus sequence typing (MLST) methodology in syphilis samples previously characterized by enhanced CDC typing (ECDCT) and macrolide resistance. Materials & methods: MLST was performed on genital ulcer and blood samples by analyzing a region of the tp0136, tp0548 and tp0705 loci using Sanger sequencing. Results: Up to 59/85 (69.4%) of genital ulcer and 4/39 (10.3%) of whole blood samples were fully typed. The most frequent profiles were 1.3.1 (56%) and 1.1.1 (11%). All the 1.3.1 samples typed carried the A2058G mutation, responsible for macrolide resistance. MLST and ECDCT showed similar overall typing yields. Conclusion: Several allelic profiles of T. pallidum subsp. pallidum were identified and classified into two major genetic clades in Barcelona. Our results were similar to that described in Europe.

Molecular epidemiology of Bordetella pertussis and analysis of vaccine antigen genes from clinical isolates from Shenzhen, China

BackgroundAlthough pertussis cases globally have been controlled through the Expanded Programme on Immunization (EPI), the incidence of pertussis has increased significantly in recent years, with a “resurgence” of pertussis occurring in developed countries with high immunization coverage. Attracted by its fast-developing economy, the population of Shenzhen has reached 14 million and has become one of the top five largest cities by population size in China. The incidence of pertussis here was about 2.02/100,000, far exceeding that of the whole province and the whole country (both < 1/100,000). There are increasing numbers of reports demonstrating variation in Bordetella pertussis antigens and genes, which may be associated with the increased incidence. Fifty strains of Bordetella pertussis isolated from 387 suspected cases were collected in Shenzhen in 2018 for genotypic and molecular epidemiological analysis.MethodsThere were 387 suspected cases of pertussis enrolled at surveillance sites in Shenzhen from June to August 2018. Nasopharyngeal swabs from suspected pertussis cases were collected for bacterial culture and the identity of putative Bordetella pertussis isolates was confirmed by real-time PCR. The immunization history of each patient was taken. The acellular pertussis vaccine (APV) antigen genes for pertussis toxin (ptxA, ptxC), pertactin (prn) and fimbriae (fim2 and fim3) together with the pertussis toxin promoter region (ptxP) were analyzed by second-generation sequencing. Genetic and phylogenetic analysis was performed using sequences publicly available from GenBank, National Institutes of Health, Bethesda, MD, USA (https://www.ncbi.nlm.nih.gov/genbank/). The antimicrobial susceptibility was test by Kirby-Bauer disk diffusion.ResultsFifty strains of Bordetella pertussis were successfully isolated from nasopharyngeal swabs of 387 suspected cases, with a positivity rate of 16.79%, including 28 males and 22 females, accounting for 56.0% and 44.0% respectively. Thirty-eight of the 50 (76%) patients were found to be positive for B. pertussis by culture. Among the positive cases with a history of vaccination, 30 of 42 (71.4%) cases had an incomplete pertussis vaccination history according to the national recommendation. Three phylogenetic groups (PG1-PG3) were identified each containing a predominant genotype. The two vaccines strains, CS and Tohama I, were distantly related to these three groups. Thirty-one out of fifty (62%) isolates belonged to genotype PG1, with the allelic profile prn2/ptxC2/ptxP3/ptxA1/fim3-1/fim2-1. Eighteen out of fifty (36%) isolates contained the A2047G mutation and were highly resistant to erythromycin, and all belonged to genotype PG3 (prn1/ptxA1/ptxP1/ptxC1/fim3-1/fim2-1), which is closely related to the recent epidemic strains found in northern China.ConclusionsThe positive rate of cases under one-year-old was significantly higher than that of other age groups and should be monitored. The dominant antigen genotypes of 50 Shenzhen isolates are closely related to the epidemic strains in the United States, Australia and many countries in Europe. Despite high rates of immunization with APV, epidemics of pertussis have recently occurred in these countries. Therefore, genomic analysis of circulating isolates of B. pertussis should be continued, for it will benefit the control of whooping cough and development of improved vaccines and therapeutic strategies.

Macrolide Resistance, Clinical Features, and Cytokine Profiles in Taiwanese Children With Mycoplasma pneumoniae Infection.

BackgroundThe factors that predict the progression of Mycoplasma pneumoniae infection remain inconclusive. Therefore, we investigated macrolide resistance prevalence, M pneumoniae genotype, and clinical characteristics of childhood M pneumoniae respiratory tract infections in Taiwan.MethodsA total of 295 children hospitalized with respiratory tract infections with positive serological M pneumoniae immunoglobulin M test results were enrolled in this 3-year prospective study. Oropharyngeal swabs were obtained for M pneumoniae cultures and polymerase chain reaction tests. All M pneumoniae specimens were further characterized by P1 typing, multilocus variable-number tandem-repeat analysis (MLVA), and macrolide resistance genotyping. The clinical characteristics and blood cytokine profiles were analyzed accordingly.ResultsOf 138 M pneumoniae specimens, type I P1 was the predominant (136 of 138, 98.6%). The MLVA type P (4-4-5-7-2) was the leading strain (42 of 138, 30.4%), followed by type J, U, A, and X. The overall macrolide-resistant rate was 38.4% (53 of 138); the resistance rate increased dramatically yearly: 10.6% in 2017, 47.5% in 2018, and 62.5% in 2019 (P < .001). All macrolide-resistant M pneumoniae (MRMP) harbored the A2063G mutation and were MLVA type 4-5-7-2 (49 of 53, 92.5%), especially type U and X. No significant differences in clinical symptoms, duration of hospital stay, and radiographic findings were identified among patients between MRMP and macrolide-sensitive M pneumoniae (MSMP) groups. Patients with MRMP infection had more febrile days before and during hospitalization and higher interleukin (IL)-13 and IL-33 levels than patients with MSMP infection (P < .05).ConclusionsMacrolide-resistant M pneumoniae surged in Taiwan throughout the study period, but macrolide resistance was not a determinant factor of clinical severity.

Characterization of Domain V Mutations in Clinical Isolates of Helicobacter pylori in Pakistan and Their Effect on Clarithromycin MIC.

PurposeClarithromycin is commonly prescribed for H. pylori infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of H. pylori in Pakistan.Materials and MethodsInfection was diagnosed in 93 patients’ biopsies using culture, rapid urease test, 16S rRNA, and vacA gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new MboII RFLP pattern.ResultsA total of 48 (83%) strains were obtained from 58 (62.3%) PCR H. pylori-positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new MboII RFLP pattern in an intermediated resistant strains was due to A1761G mutation.ConclusionH. pylori domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to H. pylori against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in H. pylori seem crucial at this stage.

Detection and characterization of QoI resistance in Pyrenophora tritici‐repentis populations causing tan spot of wheat in Argentina

AbstractTan spot, caused by the fungus Pyrenophora tritici‐repentis (Ptr), is a disease that has become more prevalent and intense in wheat crops in Argentina in recent years. Failure to control the disease with strobilurin fungicides, which were once effective, has been observed in different zones where wheat is grown. However, whether or not true resistance is present in the pathogen population in the region is not scientifically confirmed. This study evaluated the sensitivity of numerous Ptr isolates to representative QoI fungicides used in Argentina through in vitro and in planta assays, as well as through molecular analysis. Eighty‐two monosporic isolates obtained in different locations in the north and south of Buenos Aires province in 2014, 2016, and 2018 were tested to determine sensitivity to selected QoI fungicides in conidial germination and mycelial inhibition assays, as well as in molecular analysis. Conidial germination was not inhibited at 1 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin. On the other hand, mycelial growth was inhibited by 59%, 56%, and 86% at 100 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin, respectively. The molecular analysis detected the G143A mutation in the cytb gene of all the 82 Ptr isolates, but the F129L and G137R substitutions were not present. This study documents the G143A mutation conferring QoI resistance in Ptr in South America. The findings of this study are key for future decisions regarding use of fungicide and rotation in the region.

Occurrence and antimicrobial resistance of Campylobacter isolates from broiler chicken and slaughter house environment in India

Campylobacteriosis is among the most frequently reported foodborne zoonoses. A total of 848 samples were screened for Campylobacter spp. and occurrence was found to be 8.7%, 2.3% and 1.65% in broiler cecum samples, chicken meat samples and slaughter house environment swabs, respectively. High level of antimicrobial resistance was found against tetracycline (64.1%), doxycycline (54.4%), ampicillin (46.6%), nalidixic acid (42.7%), kanamycin (35.9%), and ciprofloxacin (33.33%). Resistance to co-amoxiclav (19.4%) and erythromycin (21.4%) was less common. The MAR index of the isolates was in the range of 0.11–0.78. Multi-drug resistance was observed in 54.4% of the isolates, with 53.2% C. jejuni and 55.3% C. coli isolates found resistant against three or more classes of antimicrobials. Presence of mutations in gyrA and 23S rRNA genes was investigated, which revealed that all the fluoroquinolone resistant isolates possessed Thr-86-Ile point mutation, whereas only 68% of erythromycin resistant isolates had A2075G mutation. The tetO gene was present in 91.7% tetracycline resistant isolates and bla OXA-61 gene was detected in 97.9% of the ampicillin resistant isolates. The occurrence of antimicrobial resistant Campylobacter spp. in broiler chicken samples and slaughter house settings is a public health risk and calls for judicial use of antimicrobials.

Fungicide Resistance Evolving in Ramularia collo-cygni Population in Estonia.

Ramularia leaf spot caused by the fungus Ramularia collo-cygni, has recently become widespread in Estonian barley fields. Currently, disease control in barley fields relies on SDHI and DMI fungicides, which might be threatened by R. collo-cygni isolates that are well-adapted to fungicide pressure. In a two-year study, 353 R. collo-cygni isolates were collected from spring barley fields in Estonia. A total of 153 R. collo-cygni isolates were examined for sensitivity to azoles (DMIs; prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs; boscalid, fluxapyroxad). Epoxiconazole was the least effective and a new fungicide mefentrifluconazole was the most effective DMI. Among SDHIs, fluxapyroxad was more effective than boscalid. Also, single R. collo-cygni isolates with high resistance to tested fungicides occurred, which could affect fungicide control of the pathogen. The entire collection of R. collo-cygni was analysed for mutations in fungicide target proteins. Six mutations were identified in CYP51 gene, the most dominant being I381T, I384T, and S459C. Also, numerous point mutations in the SdhC gene were present. The mutation G143A in strobilurin target protein CytB dominates in over 80% of the R. collo-cygni population, confirming the low efficacy of strobilurin fungicides in barley disease control.

High prevalence of clarithromycin resistant Helicobacter pylori in Turkish children with gastric disorders

ObjectivesHelicobacter pylori (H. pylori) is one of the leading cause of most gastroduodenal diseases. Commonly, clarithromycin is prescribed to treat H. pylori infections. Increasing resistance to clarithromycin is now one of the main problems of health worldwide. This study is aimed to survey clarithromycin resistant H. pylori infection in children with gastric disorders in Turkey. Patients and methodsA total of 110 biopsy samples were collected from the gastric antral of children referred to Balcali Hospital in Adana city, Turkey from October 2015 to November 2016. After the DNA extraction, a PCR reaction was performed for the detection of glmM gene as a diagnostic marker for Helicobacter. To examine resistance to clarithromycin, RFLP-PCR was performed to detect A2142G and A2143G point mutations in the 23SrRNA amplification product. ResultsBased on histopathological and RUT tests and PCR, H. pylori was detected in 26.4% (29/110), 23.6% (26/110), and 33.6% (37/110) of the pediatric patients respectively. The frequency of H. pylori infection was dependent on age but independent of gender. The A2143G and A2142G point mutations were found in (n: 13, 61.90%) and (n: 8, 38.09%) of H. pylori isolates. ConclusionOur findings highlight a high rate of resistance to clarithromycin in Turkish children with H. pylori infection. Hence, it is essential to note other classes of antibiotics except clarithromycin for the effective treatment of H. pylori infection in children younger than 18 years in the study region.