Sensitive Isolates Research Articles

Significance of genotypes and resistance status of Mycobacterium tuberculosis strains in gene expression of apoptosis cell death and inflammatory pathways in A549 lung epithelial cell line.

Tuberculosis (TB) has been a major health issue throughout history. As part of TB infection, host-Mycobacterium tuberculosis (Mtb) interactions are important. Through immune pathology and cell death control processes, Mtb infection facilitates intracellular growth. The relationship between apoptosis and inflammation in Mtb infection remains unclear. In this study, the levels of related apoptosis and inflammatory genes were assessed in A549 cells infected with a variety of Mtb strains. Mtb isolates with different phenotypes (sensitive, INHR, RifR, MDR, and XDR) were collected from the Pasteur Institute of Iran, during this study. Whole genome sequencing was previously performed on all strains, and the Beijing genotype was selected as sensitive. Also, for other resistant strains, the New-1 genotype was available and isolated for genotype comparison. A549 lung carcinoma cells were also grown and infected with selected Mtb strains. Genes involved in inflammation and apoptosis were detected using reverse transcription-PCR (RT-PCR). All sensitive strains and resistant strains were found to significantly up-regulate anti-apoptotic (bcl2 and rb1), chemokine (IL-8 and MCP-1), and pro-inflammatory cytokine (TNF-α and IFN-γ) expression, while significant down-regulation was observed after 24 and 48 hr of infection in anti-inflammatory genes (IL-10) and pro-apoptotic genes (bad and bax). Besides resistance strains, Mtb genotypes also affected gene expression. The Beijing genotype (sensitive isolate) influences inflammatory and apoptotic genes more sharply than the New-1 genotype (INHR, RifR, MDR, and XDR). Gene expression differences related to apoptosis and inflammation examined in the current study may be attributed to genotypes rather than resistance status since the expression of most genes has been observed to be lower in resistant strains (INHR, RifR, MDR, and XDR belonging to the New-1 genotype) compared to sensitive strains (Beijing genotype).

Distribution of lineages and type II toxin-antitoxin systems among rifampin-resistant Mycobacterium Tuberculosis Isolates.

Type II toxin-antitoxin systems such as mazEF3, vapBC3, and relJK play a role in antibiotic resistance and tolerance. Among the different known TA systems, mazEF3, vapBC3, and relJK, which are type II systems, have specific roles in drug resistance. Therefore, the aim of this study was to investigate the mutations in these genes in sensitive and resistant isolates of Mycobacterium tuberculosis. Thirty-two rifampin-resistant and 121 rifampin-sensitive M. tuberculosis isolates were collected from various regions of Iran. Lineage typing was performed using the ASO-PCR method. Mutations in the rpoB gene were analyzed in all isolates by MAS-PCR. Furthermore, mutations in the mazEF3, relJK, and vapBC3 genes of the type II toxin system were assessed through PCR sequencing. These sequences were analyzed using COBALT and SnapGene 2017, and submitted to the GenBank database. Among the 153 M. tuberculosis samples, lineages 4, 3 and 2 were the most common. Lineage 2 had the highest rate of rifampin resistance. Mutations in rpoB531 were the most frequent in resistant isolates. Examination of the toxin-antitoxin system showed that rifampin-resistant isolates belonging to lineage 3 had mutations in either the toxin or antitoxin parts of all three TA systems. A mutation in nucleotide 195 (codon 65) of mazF3 leading to an amino acid change from threonine to isoleucine was detected in all rifampin-resistant isolates. M. tuberculosis isolates belonging to lineage 2 exhibited the highest rifampin resistance in our study. Identifying the mutation in mazF3 in all rifampin-resistant isolates can highlight the significance of this mutation in the development of drug resistance in M. tuberculosis. Expanding the sample size in future studies can help develop a new method for identifying resistant isolates.

Exposure of Helicobacter pylori to clarithromycin in vitro resulting in the development of resistance and triggers metabolic reprogramming associated with virulence and pathogenicity.

In H. pylori infection, antibiotic-resistance is one of the most common causes of treatment failure. Bacterial metabolic activities, such as energy production, bacterial growth, cell wall construction, and cell-cell communication, all play important roles in antimicrobial resistance mechanisms. Identification of microbial metabolites may result in the discovery of novel antimicrobial therapeutic targets and treatments. The purpose of this work is to assess H. pylori metabolomic reprogramming in order to reveal the underlying mechanisms associated with the development of clarithromycin resistance. Previously, four H. pylori isolates were induced to become resistant to clarithromycin in vitro by incrementally increasing the concentrations of clarithromycin. Bacterial metabolites were extracted using the Bligh and Dyer technique and analyzed using metabolomic fingerprinting based on Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). The data was processed and analyzed using the MassHunter Qualitative Analysis and Mass Profiler Professional software. In parental sensitivity (S), breakpoint isolates (B), and induced resistance isolates (R) H. pylori isolates, 982 metabolites were found. Furthermore, based on accurate mass, isotope ratios, abundances, and spacing, 292 metabolites matched the metabolites in the Agilent METLIN precise Mass-Personal Metabolite Database and Library (AM-PCDL). Several metabolites associated with bacterial virulence, pathogenicity, survival, and proliferation (L-leucine, Pyridoxone [Vitamine B6], D-Mannitol, Sphingolipids, Indoleacrylic acid, Dulcitol, and D-Proline) were found to be elevated in generated resistant H. pylori isolates when compared to parental sensitive isolates. The elevated metabolites could be part of antibiotics resistance mechanisms. Understanding the fundamental metabolome changes in the course of progressing from clarithromycin-sensitive to breakpoint to resistant in H. pylori clinical isolates may be a promising strategy for discovering novel alternatives therapeutic targets.

The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC50 < 0.83 μg/mL), moderately resistant isolates (MR, 1.18 < EC50 < 55.67 μg/mL), and highly resistant isolates (HR, EC50 > 56.98 μg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola.

Effects of Silybum marianum L. Seed Extracts on Multi Drug Resistant (MDR) Bacteria.

Wound infections became a great challenge, especially after the emergence of bacterial resistance to commonly used antibiotics. Medicinal plants can be the source of alternative antibacterial agents effective against multi drug resistant (MDR) bacteria. This research aimed to evaluate the effectiveness of different Silybum marianum seed extracts in fighting MDR bacteria that infect wounds. First, thirty purified bacterial cultures obtained from superficial, infected wounds were subjected to antibiotic sensitivity tests. The selected MDR isolates were then used to test the antimicrobial effects of different S. marianum seed extracts. The most potent extract was evaluated for its impact on the ultrastructure of the cells of sensitive bacterial isolates using transmission electron microscopy (TEM). The bioactive ingredients of this extract were analyzed by means of gas chromatography-mass spectroscopy (GC-MS). Then, in-silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties were predicted for the main components. The results indicated that four out of 30 bacterial isolates were considered MDR bacteria. Primary morphological features of colonies, secondary (automatic) identification using the Biomerieux Vitek 2 System, and 16S rRNA sequencing of the four isolates confirmed that they represent Staphylococcus aureus, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Escherichia coli. Among different extracts of S. marianum seeds, ethanol extract showed the strongest inhibitory effect on both Gram-positive and Gram-negative bacteria, with minimum inhibitory concentration (MIC) values between 9.375 and 1.172 mg/mL. However, at concentrations four times higher, this extract was unable to kill bacterial cells, indicating that it had a bacteriostatic effect on the tested MDR strains. TEM revealed denaturation and distorted cell ultrastructure in S. aureus and S. maltophilia after exposure to ethanol extract. In addition, GC-MS analysis of the ethanol extract identified nine compounds known to have important biological activities, and ADMET analysis showed good drug-likeness for two of these compounds. Consequently, S. marianum seeds could be a good source of alternative bacteriostatic agents effective against MDR bacterial strains that cause wound infections.

A new point mutation (D1158N) in histidine kinase Bos1 confers high-level resistance to fludioxonil in field gray mold disease

Gray mold, caused by the fungus Botrytis cinerea, is one of the most important plant diseases worldwide that is prone to developing resistance to fungicides. Currently, the phenylpyrrole fungicide fludioxonil exhibits excellent efficacy in the control of gray mold in China. In this study, we detected the fludioxonil resistance of gray mold disease in Shouguang City of Shandong Province, where we first found fludioxonil-resistant isolates of B. cinerea in 2014. A total of 87 single spore isolates of B. cinerea were obtained from cucumbers in greenhouse, and 3 of which could grow on PDA plates amended with 50 μg/mL fludioxonil that was defined as high-level resistance, with a resistance frequency of 3.4%. Furthermore, the 3 fludioxonil-resistant isolates also showed high-level resistance to the dicarboximide fungicides iprodione and procymidone. Sequencing comparison revealed that all the 3 fludioxonil-resistant isolates had a point mutation at codon 1158, GAC (Asp) → AAC (Asn) in the histidine kinase Bos1, which was proved to be the reason for fludioxonil resistance. In addition, the fludioxonil-resistant isolates possessed an impaired biological fitness compared to the sensitive isolates based on the results of mycelial growth, conidiation, virulence, and osmotic stress tolerance determination. Taken together, our results indicate that the high-level resistance to fludioxonil caused by the Bos1 point mutation (D1158N) has emerged in the field gray mold disease, and the resistance risk is relatively high, and fludioxonil should be used sparingly.

Antibacterial and Antifungal Activities of Apis mellifera L. Honey, Propolis, Royal Jelly in Iraqi Kurdistan Region

This study was conducted at a laboratory in the Biology, College of Education, University of Salahaddin, from February to May 2022 to investigate propolis, honey and royal jelly’s chemical composition, antioxidant and antibacterial activities. The honeybee production extract showed that Gram (+) bacteria were more resistant to the antibacterial compounds of honey and propolis than Gram (-) bacteria and fungi. E. coli was a more sensitive isolate than all the other bacteria examined against the honey types tested. At the same time, it revealed more resistance against all types of propolis. Royal jelly with honey displayed more antimicrobial activity than other bee products and exhibited superior activity; the minimum inhibitory concentration of honey and propolis samples ranged from 32 to 512µg/mL. The MIC value of the most effective honey (Honey 1, Honey 2 and Royal jelly) was 32µg/mL. The lowest concentration of Qaladze propolis was (32µg/mL) for E. coli ATCC 25922, followed by 128µg/mL in some other propolis types. Keywords: Apis mellifera; antimicrobial activities; honey; propolis; royal jelly.

Characteristics of famoxadone-resistant mutants of Phytophthora litchii and their effect on lychee fruit quality

Lychee downy blight (LDB), a common disease caused by the oomycete Phytophthora litchii, poses a significant threat to both pre- and post-harvest stages, leading to substantial economic losses. Famoxadone, a quinone outside inhibitor fungicide, was registered for controlling LDB in China in 2002. However, limited information is available regarding the risk, mechanism, and impact on lychee fruit quality associated with famoxadone resistance. In this study, we determined the sensitivity of 133 P. litchii isolates to famoxadone, yielding a mean EC50 value of 0.46 ± 0.21 μg/mL. Through fungicide adaption, we derived resistant mutants with M124I and Y131C substitutions in PlCyt b (Cytochrome b in P. litchii) from wild-type isolates. In vitro assessments revealed that the fitness of the resistant mutants was significantly lower compared to the parental isolates. These laboratory findings demonstrate a moderate resistance risk of P. litchii to famoxadone. Molecular docking analyses indicated that the M124I and Y131C alterations disrupted hydrogen bonds and weakened the binding energy between famoxadone and PlCyt b. This indicates that the M124I and Y131C changes do indeed confer famoxadone resistance in P. litchii. Infection caused by famoxadone-resistant mutants exhibited a decreased or comparable impact on the characteristic traits of lychee fruit compared to the sensitive isolate. For future detection of famoxadone-resistant strains, AS-PCR primers were designed based on the M124I substitution.

Ametoctradin resistance risk and its resistance-related point mutation in PsCytb of Phytophthora sojae confirmed using ectopic overexpression

Ametoctradin is mainly used to treat plant oomycetes diseases, but the mechanism and resistance risk of ametoctradin in Phytophthora sojae remain unknown. This study determined the ametoctradin sensitivity of 106 P. sojae isolates and found that the frequency distribution of the median effective concentration (EC50) of ametoctradin was unimodal with a mean value of 0.1743 ± 0.0901 μg/mL. Furthermore, ametoctradin-resistant mutants had a substantially lower fitness index compared with that of wild-type isolates. Although ametoctradin did not show cross-resistance to other fungicides, negative cross-resistance to amisulbrom was found. In comparison to sensitive isolates, the control efficacy of ametoctradin to resistant mutants was lower, implying a low to moderate ametoctradin resistance risk in P. sojae. All ametoctradin-resistant mutants contained a S33L point mutation in PsCytb. A system with overexpression of PsCytb in the nucleus was established. When we ectopically overexpressed S33L-harboring PsCytb, P. sojae developed ametoctradin resistance. We hypothesized that the observed negative resistance between ametoctradin and amisulbrom could be attributed to conformational changes in the binding cavity of PsCytb at residues 33 and 220.

Self-Isolated Dual-Mode High-Pass Birdcage RF Coil for Proton and Sodium MR Imaging at 7 T MRI

This study presents the feasibility of a dual-mode high-pass birdcage RF coil to acquire MR images at both 1H and 23Na frequencies at ultra-high-field MR scanner, 7 T. A dual-mode circuit (DMC) in the dual-mode birdcage (DMBC) RF coil operates at two frequencies, addressing the limitations of sensitivity reduction and isolation between two frequencies as in traditional dual-tuned RF coil. Finite-difference time-domain (FDTD) based electromagnetic (EM) simulations were performed to verify the RF coil at each frequency on the three-dimensional human head model. The DMBC RF coil resonated at proton (1H) and sodium (23Na) frequencies, and also single-tuned high-pass birdcage RF coils were constructed for both 1H and 23Na frequencies. The bench test performance of the RF coils was evaluated using network analysis parameters, including the measurement of scattering parameters (S-parameters) and quality factors (Q-factors). Q-factor of the DMBC coil at 1H port was 10.2% lower than that of 1H single-tuned birdcage (STBC) coil, with a modest SNR reduction of 6.5%. Similarly, the Q-factor for the DMBC coil at 23Na port was 12.3% less than that of 23Na STBC coil, and the SNR showed a minimal reduction of 5.4%. Utilizing the DMBC coil, promising 1H and 23Na MR images were acquired compared to those by using STBC coils. In conclusion, deploying a DMBC 1H/23Na coil has been demonstrated to overcome traditional constraints associated with dual-tuned RF coils, achieving this with only nominal signal attenuation across both nuclei operational frequencies.

Sensitivity of 240 Listeria monocytogenes isolates to common industrial biocides is more dependent on the presence of residual organic matter or biofilm than on genetic determinants

Listeria monocytogenes continues to be a problem in ready-to-eat foods. Tolerance and adaptation to biocides have been proposed to contribute to the persistence of L. monocytogenes in the food processing industry. This study aimed to determine if the sensitivity to common biocides varies among 240 L. monocytogenes isolates. The impact of residual organic matter (soils), biofilm, and ability to adapt to biocides was also evaluated. Food and food processing related isolates of L. monocytogenes (n = 240) were whole genome sequenced and assigned to sequence types (n = 53) and clonal complexes (n = 32). Isolates were screened for genetic elements associated with biocide tolerance and tested for their minimum inhibitory concentration (MIC) to benzalkonium chloride (BC), peracetic acid (PAA), sodium hypochlorite (SH) and ethanol (ET). Biocide tolerance under soiled conditions, in biofilm and in a broth suspension test (BST) was further tested for 19 representative isolates. Adaptation to sub-MICs of BC, PAA or SH (18 days, 15 °C) was tested for 15 isolates. MIC for PAA (62 mg/L) showed no variation, while for SH (47–94 mg/L) and ET (4.7–9.4% v/v) values differed 2 × among isolates. For BC, an 8 × difference (0.3–2.5 mg/L) was observed. Eighty-seven of 88 BC tolerant isolates (MIC of 2.5 mg/L) harbored known BC tolerance genes. Soiled conditions increased MIC-values 8–33 × for BC and 8 × for SH but not for PAA and ET. Biofilm increased minimum bactericidal concentrations 4–8 × for BC but not for PAA and SH. Survival of isolates with BC tolerance genes was not significantly (p > 0.05) improved in BSTs with PAA, SH or BC. Lineage I isolates (n = 7 of 19) showed significantly (p < 0.05) better survival than lineage II isolates (n = 12) when exposed to PAA (100 mg/L) in BSTs. Adaptation to BC (8 × MIC increase) occurred exclusively for originally sensitive isolates, while no adaptation to SH and PAA were observed. In conclusion, L. monocytogenes isolates showed limited variation in sensitivity to biocides in MIC assays. However, lineage I isolates were less sensitive to PAA than lineage II isolates in BSTs. The efficacy of SH and BC were affected by residual organic matter and/or biofilm, demonstrating the need for proper cleaning prior to biocide use with PAA being less perturbed.

In vitro susceptibility test of Cladosporium cladosporioides isolates from Argentinian tomato crops against commercial fungicides

Tomato cultivation is an important agricultural activity in northeast of Argentina, and disease control is indispensable for its production. The purpose of the current study was to identify two fungi isolated from tomato plants cultivated in greenhouses, with symptoms of leaf mould disease and a strain of the genus Cladosporium from a culture collection, and evaluate their in vitro susceptibility to four commercial fungicides. Macro and microscopic examination, molecular characterisation and sequence analysis were applied for identification. Broth dilution and spread plate methods were used to determine the minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC). The active ingredients of the products were azoxystrobin+difenoconazole, trifloxystrobin+tebuconazole, chlorothalonil and metalaxyl-M+mancozeb. The results were processed using the Kruskal-Wallis method. The isolates were identified as Cladosporium cladosporioidesa-c; consequently, lesions found on tomato plants did not corrspond to Cladosporium fulvum. There was a significant statistical difference between the obtained values. Qualitatively, the three strains had a similar behaviour for chlorothalonil (MIC values: 0.25 - 0.5 μg/ml, MFC values: 4 μg/ml). In all cases, tests with metalaxyl-M+mancozeb yielded higher values than those achieved for chlorothalonil (MIC values: 8 μg/ml, MFC values: 8- 32 μg/ml). trobilurin-formulated fungicides were less effective against C. cladosporioidesa-b (MIC values: 16-256 μg/ml, MFC values: &gt;64 μg/ml). C. cladosporioidesc was the most sensitive isolate. The information about the presence of a non-frequent fungus and its fungicide susceptibility, would be useful for establishing control strategies and enhance production.

Molecular Characteristics and the Effect of Mutations in Different Sites of the rplD Gene Among Clinical Isolates of Azithromycin Resistance Neisseria gonorrhoeae in Eastern China

Background: The emergence of resistance to azithromycin complicates the treatment of Neisseria gonorrhoeae, the causative agent of gonorrhea. Objectives: The main objective of this study was to investigate the impact of mutations in different sites of the rplD gene on azithromycin resistance (AZM-R) and the molecular characteristics of N. gonorrhoeae. This study aimed to provide evidence for drug resistance and transmission. Methods: A total of 37 isolates of N. gonorrhoeae were collected within January 2018 to December 2020. These isolates were obtained from urine, semen, or vaginal secretions of different patients. Azithromycin resistance was assessed, and genes associated with AZM-R, namely rplD, and rplV, were analyzed using polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencing. All clinical isolates were characterized through multi-locus sequence typing (MLST). Results: The study comprised 21 azithromycin-resistant N. gonorrhoeae isolates, with two of them demonstrating high resistance, indicated by a minimum inhibitory concentration (MIC) greater than 256 µg/mL. Additionally, 16 sensitive isolates were included in the study. Ten isolates were observed to have rplD point mutations, including mutations such as G70D, G70S, G68D, and A43T. No mutations were detected in rplV. The rate of point mutations in rplD was significantly different between the azithromycin-sensitive (AZM-S) group and the AZM-R group (P &lt; 0.05). Among the 37 isolates studied, 12 distinct MLST types were identified and further grouped into four different MLST goeBURST groups. The two isolates with high-level AZM-R were ST1901 and ST1588, respectively. Conclusions: The clinical isolates of N. gonorrhoeae from Wenzhou, Eastern China, exhibit significant genetic diversity and a relatively high prevalence of AZM-R. Mutations in the rplD gene were identified, which reduced susceptibility to macrolides and were significantly associated with increased AZM-R.

A growing threat: Investigating the high incidence of benzimidazole fungicides resistance in Iranian Botrytis cinerea isolates.

Effective management of fungicide application programs requires monitoring the profile of resistant populations of Botrytis cinerea, given its high-risk nature. This research aimed to examine the sensitivity of 200 B. cinerea isolates collected from different plant species and regions across Iran towards thiophanate-methyl and carbendazim fungicides. To distinguish between susceptible and resistant isolates, the discriminatory dose assay was employed, followed by the selection of representative isolates from each group for EC50 analysis. To identify potential modifications in codon 198 of the β-tubulin gene in B. cinerea resistant isolates, the researchers employed the PCR-RFLP diagnostic method. More than two-thirds of the isolates exhibited a varying degree of resistance to MBC fungicides, even in farms where the application of these fungicides had not taken place in recent years. After treatment with the BsaI enzyme, the PCR product of sensitive isolates displayed two bands measuring 98 and 371 bp, while only one band of 469 bp was identified in resistant isolates. The study also evaluated whether resistance to fungicides could affect the pathogenicity and mycelial growth of the isolates. The findings showed no significant difference between the resistant and sensitive groups in terms of these factors, indicating that resistance does not come at a cost to the pathogen's fitness. Considering the high incidence of resistance and the absence of negative consequences on fitness, it is recommended to exercise caution in the employment of benzimidazole fungicides as part of B. cinerea management strategies.

CRISPR-enabled investigation of fitness costs associated with the E198A mutation in β-tubulin of Colletotrichum siamense.

Understanding fitness costs associated with fungicide resistance is critical to improve resistance management strategies. E198A in b-tubulin confers resistance to the fungicide thiophanate-methyl and has been widely reported in several plant pathogens including Colletotrichum siamense. To better understand potential fitness costs associated with the resistance, a ribonucleoprotein (RNP) complex mediated CRISPR/Cas9 system was used to create a point mutation (E198A) through homology directed repair (HDR) in each of the sensitive (E198) C. siamense isolates collected from strawberries, raspberries, and peaches. The RNP complex was delivered into fungal protoplasts using polyethylene glycol-mediated (PEG) transfection. The transformation efficiency, the proportion of transformants of sensitive parental isolates containing the E198A mutation, averaged 72%. No off-target mutations were observed when sequences similar to the b-tubulin target region with a maximum of four mismatch sites were analyzed, suggesting that the CRISPR/Cas9 system used in this study was highly specific for genome editing in C. siamense. Of the 41 comparisons of fitness between mutant and wild type isolates through in vitro and detached fruit assays, mutant isolates appeared to be as fit (24 of 41 comparisons), if not more fit than wild-type isolates (10 of 41 comparisons). The use of CRISPR/Cas9 to evaluate fitness costs associated with point mutations in this study represents a novel and useful method, since wild-type and mutant isolates were genetically identical except for the target mutation.

Sensitivity of tomato leaf mould–causing Fulvia fulva to seven succinate dehydrogenase inhibitor (SDHI) fungicides in Nara Prefecture, Japan and high efficacy of isofetamid in controlling SDHI‐resistant isolates

AbstractLeaf mould caused by Fulvia fulva is a major disease of tomato in Japan. Resistance to several types of fungicides, including succinate dehydrogenase inhibitors (SDHIs), has been reported in isolates from Japan. In this study, we evaluated the sensitivity of F. fulva to SDHI fungicides, SDHI fungicide efficacy in controlling sensitive and resistant isolates, and the presence of mutations in the Sdh B gene, the target site of SDHI fungicides. The minimum inhibitory concentration (MIC) of six SDHI fungicides, excluding isofetamid, ranged from 0.1 to &gt;100 μg/mL against mycelial growth of 45 F. fulva isolates collected in Nara prefecture, Japan, whereas that of isofetamid ranged from 0.1 to only 10 μg/mL. The peak MIC distribution of fluopyram and isofetamid was 10 μg/mL, that of inpyrfluxam was 100 μg/mL and was &gt;100 μg/mL for the remaining SDHI fungicides. In a greenhouse inoculation experiment, only isofetamid controlled the highly resistant isolates with an MIC of 10 μg/mL and the remaining fungicides had MIC &gt;100 μg/mL. In addition, moderately and highly resistant isolates were controlled by the application of isofetamid 6 h or 7 days before inoculation or 3 days after inoculation. Mutations in the Sdh B gene were not observed among sensitive, moderately resistant and highly resistant isolates, suggesting that mutations in other Sdh genes and/or changes in efflux pump expression were involved. Our results indicate the presence of cross‐resistance and different levels of sensitivity reduction among SDHI fungicides. Isofetamid showed the lowest resistance development among the tested SDHI fungicides and is considered effective in controlling tomato leaf mould.

Polymorphism of virulence genes and biofilm associated with in vitro induced resistance to clarithromycin in Helicobacter pylori

BackgroundClarithromycin-containing triple therapy is commonly used to treat Helicobacter pylori infections. Clarithromycin resistance is the leading cause of H. pylori treatment failure. Understanding the specific mutations that occur in H. pylori strains that have evolved antibiotic resistance can help create a more effective and individualised antibiotic treatment plan. However, little is understood about the genetic reprogramming linked to clarithromycin exposure and the emergence of antibiotic resistance in H. pylori. Therefore, this study aims to identify compensatory mutations and biofilm formation associated with the development of clarithromycin resistance in H. pylori. Clarithromycin-sensitive H. pylori clinical isolates were induced to develop clarithromycin resistance through in vitro exposure to incrementally increasing concentration of the antibiotic. The genomes of the origin sensitive isolates (S), isogenic breakpoint (B), and resistant isolates (R) were sequenced. Single nucleotide variations (SNVs), and insertions or deletions (InDels) associated with the development of clarithromycin resistance were identified. Growth and biofilm production were also assessed.ResultsThe S isolates with A2143G mutation in the 23S rRNA gene were successfully induced to be resistant. According to the data, antibiotic exposure may alter the expression of certain genes, including those that code for the Cag4/Cag protein, the vacuolating cytotoxin domain-containing protein, the sel1 repeat family protein, and the rsmh gene, which may increase the risk of developing and enhances virulence in H. pylori. Enhanced biofilm formation was detected among R isolates compared to B and S isolates. Furthermore, high polymorphism was also detected among the genes associated with biofilm production.ConclusionsTherefore, this study suggests that H. pylori may acquire virulence factors while also developing antibiotic resistance due to clarithromycin exposure.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern

IntroductionThe development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. Aim.This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. MethodologyBacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. ResultsAmong A. baumannii isolates, 55% isolates (n ​= ​22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3′)-I gene (50%, 11/22), aac(6′)-Ib gene (45.4%, 10/22), aph(3′)-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. ConclusionThe presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. RepositoriesGenbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3′)-I], MT903334, MT903335 [aph(3′)-I)] and MT903336, MT940242 [ aac(6’)-Ib].

Iminoctadine tris (albesilate) resistance in Geotrichum citri-aurantii and its management

The bis-guanidine fungicide iminoctadine tris (albesilate) (ITA) is widely used to control citrus sour rot pathogen Geotrichum citri-aurantii but its resistance status in the orchards is still unclear. In the present study, ITA resistance in G. citri-aurantii was assessed and resistance mechanisms and its alternative fungicides were explored. ITA resistance frequency in field isolates of G. citri-aurantii collected between 2018 and 2020 from orchards in China was 6.2%. Consecutively culturing on potato dextrose agar (PDA) for 10 generations showed that ITA resistance for field resistant isolates was relatively stable, whereas resistance for the laboratory induced resistant isolates declined by 53.9%. The competitive ability on citrus fruit for resistant isolates was substantially lower than that of the sensitive isolates. Resistance mechanism studies showed that an ATP-binding cassette (ABC) gene (EVM0003677) and two major facilitator superfamily (MFS) genes (EVM0000087 and EVM0003101) might be involved in ITA resistance only in the induced resistant isolates but not in the field resistant isolates. Bioassay of 26 fungicides found that the demethylation inhibitor fungicides metconazole and propiconazole, and the dinitro-aniline fungicide fluazinam were highly effective to ITA-sensitive isolates of G. citri-aurantii and their EC50 values were 0.33, 0.56, and 0.54 mg/L, respectively. The protective control efficacies of ITA, propiconazole, metconazole, and fluazinam all at 400 mg/L were 100%, 60.4%, 33.9%, and 18.5%, respectively. No positive cross resistance was detected between ITA and metconazole, propiconazole or fluazinam. The mixture of ITA and propiconazole at the mass ratio of 9:8 had a synergistic co-toxicity factor of 26. These results have significant implications for our awareness of ITA resistance in orchards and its management.

Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 μg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 μg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the β-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.