Isolation of Streptomyces inhibiting multiple-phytopathogenic fungi and characterization of lucensomycin biosynthetic gene cluster.

Soil microorganisms with diverse bioactive compounds such as Streptomyces are appreciated as valuable resources for the discovery of eco-friendly fungicides. This study isolated a novel Streptomyces from soil samples collected in the organic green tea fields in South Korea. The isolation process involved antifungal activity screening around 2400 culture extracts, revealing a strain designated as S. collinus Inha504 with remarkable antifungal activity against diverse phytopathogenic fungi. S. collinus Inha504 not only inhibited seven phytopathogenic fungi including Fusarium oxysporum and Aspergillus niger in bioassays and but also showed a control effect against F. oxysporum infected red pepper, strawberry, and tomato in the in vivo pot test. Genome mining of S. collinus Inha504 revealed the presence of the biosynthetic gene cluster (BGC) in the chromosome encoding a polyene macrolide which is highly homologous to the lucensomycin (LCM), a compound known for effective in crop disease control. Through genetic confirmation and bioassays, the antifungal activity of S. collinus Inha504 was attributed to the presence of LCM BGC in the chromosome. These results could serve as an effective strategy to select novel Streptomyces strains with valuable biological activity through bioassay-based screening and identify biosynthetic gene clusters responsible for the metabolites using genome mining approach.

Novel Variant and Known Mutation in 23S rRNA Gene of Mycoplasma pneumoniae, Northern Vietnam, 2023.

During a 2023 outbreak of Mycoplasma pneumoniae-associated community-acquired pneumonia among children in northern Vietnam, we analyzed M. pneumoniae isolated from nasopharyngeal samples. In almost half (6 of 13) of samples tested, we found known A2063G mutations (macrolide resistance) and a novel C2353T variant on the 23S rRNA gene.

Antimicrobial spiroketal macrolides and dichloro-diketopiperazine from Micromonospora sp. FIMYZ51.

Four compounds (1-4) featuring with an L-rhodinose and spiroketal, possess uncommon continuous hydroxy groups in the macrolide skeleton, and a dichloro-diketopiperazine (5) were isolated from a marine derived Micromonospora sp. FIMYZ51. The determination of the relative and absolute configurations of all isolates was achieved by extensive spectroscopic analyses, single-crystal X-ray diffraction analysis, and ECD calculations. According to structural characteristic and genomic sequences, a plausible biosynthetic pathway for compound 1-4 was proposed and a spirocyclase was inferred to be responsible for the formation of the rare spirocyclic moiety. Compounds 1-4 exhibited potent antifungal activities which is equal to itraconazole against Aspergillus niger. Compounds 1-5 exhibited different degree of inhibitory activities against opportunistic pathogenic bacteria of endocarditis (Micrococcus luteus) with MIC values ranging from 0.0625 µg/mL to 32 µg/mL. Compounds 2 and 3 showed moderate cytotoxicity against drug-resistant tumor cell lines (Namalwa and U266). The result not only provides active lead-compounds, but also reveal the potential of the spirocyclase gene resources from Micromonospora sp., which highlights the promising potential of the strain for biomedical applications.

Chronic toxic effects of erythromycin and its photodegradation products on microalgae Chlorella pyrenoidosa.

The photodegradation products (PDPs) of antibiotics in the aquatic environment received increasing concern, but their chronic effects on microalgae remain unclear. This study initially focused on examining the acute effects of erythromycin (ERY), then explored the chronic impacts of ERY PDPs on Chlorella pyrenoidosa. ERY of 4.0 - 32 mg/L ERY notably inhibited the cell growth and chlorophyll synthesis. The determined 96 h median effective concentration of ERY to C. pyrenoidosa was 11.78 mg/L. Higher concentrations of ERY induced more serious oxidative damage, antioxidant enzymes alleviated the oxidative stress. 6 PDPs (PDP749, PDP747, PDP719, PDP715, PDP701 and PDP557) were identified in the photodegradation process of ERY. The predicted combined toxicity of PDPs increased in the first 3 h, then decreased. Chronic exposure showed a gradual decreasing inhibition on microalgae growth and chlorophyll content. The acute effect of ERY PDPs manifested as growth stimulation, but the chronic effect manifested as growth inhibition. The malonaldehyde contents decreased with the degradation time of ERY at 7, 14 and 21 d. However, the malonaldehyde contents of ERY PDPs treatments were elevated compared to those in the control group after 21 d. Risk assessment still need to consider the potential toxicity of degradation products under long-term exposure.

High cure rates of Mycoplasma genitalium following empiric treatment with azithromycin alongside frequent detection of macrolide resistance in Austria.

BACKGROUND: Mycoplasma genitalium (MG) is an emerging sexually transmitted infection, often harboring resistance-associated mutations to azithromycin (AZM). Global surveillance has been mandated to tackle the burden caused by MG, yet no data are available for Austria. Thus, we aimed to investigate the prevalence of MG, disease characteristics, and treatment outcomes at the largest Austrian HIV-and STI clinic. METHODS: All MG test results at the Medical University of Vienna from 02/2019 to 03/2022 were evaluated. Azithromycin resistance testing was implemented in 03/2021. RESULTS: Among 2671 MG tests, 199 distinct and mostly asymptomatic (68%; 135/199) MG infections were identified, affecting 10% (178/1775) of all individuals. This study included 83% (1479/1775) men, 53% (940/1775) men who have sex with men (MSM), 31% (540/1754) HIV+, and 15% (267/1775) who were using HIV pre-exposure prophylaxis (PrEP). In logistic regression analysis, 'MSM' (aOR 2.55 (95% CI 1.65-3.92)), 'use of PrEP' (aOR 2.29 (95% CI 1.58-3.32)), and 'history of syphilis' (aOR 1.57 (95% CI 1.01-2.24) were independent predictors for MG infections. Eighty-nine percent (178/199) received treatment: 11% (21/178) doxycycline (2 weeks), 52% (92/178) AZM (5 days), and 37% ( 65/178) moxifloxacin (7-10 days) and 60% (106/178) had follow-up data available showing negative tests in 63% (5/8), 76% (44/58) and 85% (34/40), respectively. AZM resistance analysis was available for 57% (114/199)) and detected in 68% (78/114). Resistance-guided therapy achieved a cure in 87% (53/61), yet, empiric AZM-treatment (prior to 03/2021) cleared 68% (26/38). CONCLUSIONS: Mycoplasma genitalium was readily detected in this Austrian observational study, affected predominantly MSM and often presented as asymptomatic disease. We observed a worryingly high prevalence of AZM resistance mutations; however, empiric AZM treatment cleared twice as many MG infections as expected.

Challenges in the treatment of pediatric Mycoplasma pneumoniae pneumonia.

Mycoplasma pneumoniae (MP) is an important cause of community-acquired pneumonia in children and young adolescents. Despite macrolide antibiotics effectiveness as a first-line therapy, persistence of fever and/or clinical deterioration sometimes may complicate treatment and may even lead to severe systemic disease. To date, there is no consensus on alternative treatment options, optimal dosage, and duration for treating severe, progressive, and systemic MP pneumonia after macrolide treatment failure. Macrolide-resistant MP pneumonia and refractory MP pneumonia are the two major complex conditions that are clinically encountered. Currently, the vast majority of MP isolates are resistant to macrolides in East Asia, especially China, whereas in Europe and North America, whereas in Europe and North America prevalence is substantially lower than in Asia, varying across countries. The severity of pneumonia and extrapulmonary presentations may reflect the intensity of the host's immune reaction or the dissemination of bacterial infection. Children infected with macrolide-resistant MP strains who receive macrolide treatment experience persistent fever with extended antibiotic therapy and minimal decrease in MP-DNA load. Alternative second-line agents such as tetracyclines (doxycycline or minocycline) and fluoroquinolones (ciprofloxacin or levofloxacin) may lead to clinical improvement after macrolide treatment failure in children. Refractory MP pneumonia reflects a deterioration of clinical and radiological findings due to excessive immune response against the infection. Immunomodulators such as corticosteroids and intravenous immunoglobulin (IVIG) have shown promising results in treatment of refractory MP pneumonia, particularly when combined with appropriate antimicrobials. Corticosteroid-resistant hyperinflammatory MP pneumonia represents a persistent or recrudescent fever despite corticosteroid therapy with intravenous methylprednisolone at standard dosage. CONCLUSION:  This report summarizes the clinical significance of macrolide-resistant and refractory MP pneumonia and discusses the efficacy and safety of alternative drugs, with a stepwise approach to the management of MP pneumonia recommended from the viewpoint of clinical practice. WHAT IS KNOWN: • Although MP pneumonia is usually a benign self-limited infection with response macrolides as first line therapy, severe life-threatening cases may develop if additional treatment strategies are not effectively implemented. • Macrolide-resistant and refractory MP pneumonia are two conditions that may complicate the clinical course of MP pneumonia, increasing the risk for exacerbation and even death. WHAT IS NEW: • This report summarizes the clinical relevance of macrolide-resistant and refractory MP pneumonia and discusses the efficacy and safety of alternative drug therapies. • A practical stepwise approach to the management of MP pneumonia is developed based on a comprehensive analysis of existing evidence and expert opinion.

Isolation and structure determination of a new analog of polycavernosides from marine Okeania sp. cyanobacterium.

Polycavernoside E (1), a new polycavernoside analog, was isolated from a marine Okeania sp. cyanobacterium. The relative configuration was elucidated primarily by analyzing the two dimensional nuclear magnetism resonance (2D NMR) data. The absolute configuration was clarified by comparing the electronic circular dichroism (ECD) data of 1 with those of known analogs. Polycavernoside E (1) exhibited moderate antitrypanosomal activity against Trypanosoma brucei rhodesiense. Furthermore, the isolation of polycavernoside E (1) from marine cyanobacteria provides additional evidence that marine cyanobacteria, and not red algae, are responsible for the biosynthesis of polycavernosides.

Post-COVID-19 Pandemic Rebound of Macrolide-Resistant Mycoplasma pneumoniae Infection: A Descriptive Study.

The rebound characteristics of respiratory infections after lifting pandemic control measures were uncertain. From January to November 2023, patients presenting at a teaching hospital were tested for common respiratory viruses and Mycoplasma pneumoniae using a combination of antigen, nucleic acid amplification, and targeted next-generation sequencing (tNGS) tests. The number and rate of positive tests per month, clinical and microbiological characteristics were analyzed. A rapid rebound of SARS-CoV-2 was followed by a slower rebound of M. pneumoniae, with an interval of 5 months between their peaks. The hospitalization rate was higher, with infections caused by respiratory viruses compared to M. pneumoniae. Though the pediatric hospitalization rate of respiratory viruses (66.1%) was higher than that of M. pneumoniae (34.0%), the 4094 cases of M. pneumoniae within 6 months posed a huge burden on healthcare services. Multivariate analysis revealed that M. pneumoniae-infected adults had more fatigue, comorbidities, and higher serum C-reactive protein, whereas children had a higher incidence of other respiratory pathogens detected by tNGS or pathogen-specific PCR, fever, and were more likely to be female. A total of 85% of M. pneumoniae-positive specimens had mutations detected at the 23rRNA gene, with 99.7% showing A2063G mutation. Days to defervescence were longer in those not treated by effective antibiotics and those requiring a change in antibiotic treatment. A delayed but significant rebound of M. pneumoniae was observed after the complete relaxation of pandemic control measures. No unusual, unexplained, or unresponsive cases of respiratory infections which warrant further investigation were identified.

Identification of Novel Flavonoids and Ansa-Macrolides with Activities against Leishmania donovani through Natural Product Library Screening.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis (VL), a potentially fatal disease if left untreated. Given the limitations of current therapies, there is an urgent need for new, safe, and effective drugs. To discover novel antileishmanial compounds from previously unexplored chemical spaces, we conducted a high-throughput screening (HTS) of 2562 natural compounds, assessing their activity against L. donovani promastigotes and intracellular amastigotes. Utilizing the criteria of ≥70% parasite growth inhibition and ≥70% host cell (THP-1) viability, we selected 100 inhibitors for half-maximal inhibitory concentration (IC50) value determination. Twenty-six compounds showed activities in both forms of Leishmania with a selectivity index of over 3. Clustering analysis resulted in four chemical clusters with scaffolds of lycorine (cluster 1), 5-hydroxy-9,10-dihydro-4H,8H-pyrano[2,3-f]chromene-4,8-dione (cluster 2), and semi-synthetic derivatives of ansamycin macrolide (cluster 4). The enantiomer of lycorine, BMD-NP-00820, showed the highest anti-amastigote activity with an IC50 value of 1.74 ± 0.27 µM and a selectivity index (SI) > 29. In cluster 3, the most potent compound had an IC50 value of 2.20 ± 0.29 µM with an SI > 23, whereas in cluster 4, with compounds structurally similar to the tuberculosis drug rifapentine, BMD-NP-02085 had an IC50 value of 1.76 ± 0.28 µM, but the SI value was 7.5. Taken together, the natural products identified from this study are a potential source for the discovery of antileishmanial chemotypes for further development.

Dispersive Solid-Phase Extraction and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry-A Rapid and Accurate Method for Detecting 10 Macrolide Residues in Aquatic Products.

The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin (KIT), josamycin (JOS), spiramycin (SPI), tilmicosin (TIL), tylosin (TYL), and roxithromycin (ROX), was determined using solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The residues were extracted with 1% ammonia acetonitrile solution and purified by neutral alumina adsorption. Chromatographic separation was completed on an ACQUITY UPLC BEH C18 column with acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and mass spectrometry detection was performed by multiple reaction monitoring scanning with the positive mode in an electrospray ion source (ESI+). Five isotopically labeled compounds were used as internal standards for quality control purposes. The findings indicated that across the mass concentration span of 1.0-100 µg/L, there was a strong linear correlation (R2 > 0.99) between the concentration and instrumental response for the 10 MALs. The limit of detection of UPLC-MS/MS was 0.25-0.50 µg/kg, and the limit of quantitation was 0.5-1.0 µg/kg. The added recovery of blank matrix samples at standard gradient levels (1.0, 5.0, and 50.0 µg/kg) was 83.1-116.6%, and the intra-day precision and inter-day precisions were 3.7 and 13.8%, respectively. The method is simple and fast, with high accuracy and good repeatability, in line with the requirements for accurate qualitative and quantitative analysis of the residues for 10 MALs in aquatic products.

Identification of a 2-Aminobenzimidazole Scaffold that Potentiates Gram-Positive Selective Antibiotics Against Gram-Negative Bacteria.

The development of novel therapeutic approaches is crucial in the fight against multi-drug resistant (MDR) bacteria, particularly gram-negative species. Small molecule adjuvants that enhance the activity of otherwise gram-positive selective antibiotics against gram-negative bacteria have the potential to expand current treatment options. We have previously reported adjuvants based upon a 2-aminoimidazole (2-AI) scaffold that potentiate macrolide antibiotics against several gram-negative pathogens. Herein, we report the discovery and structure-activity relationship (SAR) investigation of an additional class of macrolide adjuvants based upon a 2-aminobenzimidazole (2-ABI) scaffold. The lead compound lowers the minimum inhibitory concentration (MIC) of clarithromycin (CLR) from 512 to 2 µg/mL at 30 µM against Klebsiella pneumoniae 2146, and from 32 to 2 µg/mL at 5 µM, against Acinetobacter baumannii 5075. Preliminary investigation into the mechanism of action suggests that the compounds are binding to lipopolysaccharide (LPS) in K. pneumoniae, and modulating lipooligosaccharide (LOS) biosynthesis, assembly, or transport in A. baumannii.

Efficacy of doxycycline therapy for macrolide-resistant Mycoplasma pneumoniae pneumonia in children at different periods.

BACKGROUND: The prevalence of macrolide-resistant Mycoplasma pneumoniae has increased considerably. Treatment in children has become challenging. This study aimed to evaluate the efficacy of doxycycline therapy for macrolide-resistant Mycoplasma pneumoniae pneumonia in children at different periods. METHODS: We retrospectively analyzed the data of patients with macrolide-resistant Mycoplasma pneumoniae pneumonia hospitalized between May 2019 to August 2022. According to treatment, patients were divided into three groups: oral doxycycline treatment alone (DOX group), changed from intravenous azithromycin to oral doxycycline (ATD group), and intravenous azithromycin treatment alone (AZI group). ATD group cases were separated into two sub-groups: intravenous azithromycin treatment<3 days (ATD1 group) and ≥ 3 days (ATD2 group). Clinical symptoms were compared in each group and adjusted by Propensity score matching (PSM) analysis. RESULTS: A total of 106 were recruited in this study. 17 (16%) were in DOX group, 58 (55%) in ATD group, and 31(29%) in AZI group. Compared with ATD group and AZI group, the DOX group showed shorter hospitalization duration and fever duration after treatment, while higher rate of chest radiographic improvement. After using PSM analysis, shorter days to hospitalization duration (P = 0.037) and to fever duration after treatment (P = 0.027) in DOX + ATD1 group than in ATD2 group was observed. A higher number of patients in the DOX + ATD1 group achieved defervescence within 72 h (P = 0.031), and fewer children received glucocorticoid adjuvant therapy (P = 0.002). No adverse reactions associated with doxycycline was observed during treatment. CONCLUSIONS: Children receiving early oral doxycycline had a shorter duration of fever and hospitalization in macrolide-resistant Mycoplasma pneumoniae patients.

Macrolide sesquiterpene pyridine alkaloids from Celastrus monospermus and evaluation of their immunosuppressive and anti-osteoclastogenesis activities.

Phytochemical investigation of the stems of Celastrus monospermus Roxb enabled isolation and identification of fifteen new macrolide sesquiterpene pyridine alkaloids (1-15) along with five known analogues. Their structures were elucidated by comprehensive spectroscopic analysis (NMR, HRESIMS, IR, UV), chemical hydrolysis, and single crystal X-ray diffraction analysis. Bioassay of the abundant isolates revealed that seven compounds inhibited the proliferation of B lymphocytes with IC50 values ranging between 1.4 and 19.9 µM. Among them, celasmondine C (3) could significantly promote the apoptosis of activated B lymphocyte, especially late-stage apoptosis. Besides, compounds 3, 16, and 20 exhibited potent suppression of osteoclast formation at a concentration of 1.0 µM. This investigation enriched the chemical diversity of macrolide sesquiterpene pyridine alkaloids, and supported evidence for the development of new immunosuppressive and anti-osteoclastogenesis agents.

Comparison of gradient diffusion and molecular methods using Allplex™ NG&DR assay (Seegene®) for macrolide and fluoroquinolone screening resistance in Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is increasing worldwide. Second-line treatments with macrolides or fluoroquinolones are an option for NG infections in some cases following the STI guideline recommendations. In our study, we compared the gradient diffusion test using EUCAST 2024 breakpoints with a new molecular method using the Allplex™ NG&DR assay (Seegene®) including A2059G/C2611 mutations (23S rRNA) associated with high/moderate-level macrolide resistance and S91F mutation (gyrA) relationship with fluoroquinolone resistance in NG isolates (n = 100). We calculated the sensitivity, specificity, and correlation of the molecular test for fluoroquinolone using the gradient diffusion as the reference method. In twenty-three strains was not detected any mutation associated with macrolides or fluoroquinolone resistance. No A2059G/C2611T mutations were detected, and the S91F mutations were detected in 77 out of the 100 isolates screened. Twenty-three NG isolates were reported to be resistant to azithromycin (ECOFF: >1 mg/L), and 78 NG isolates were resistant to ciprofloxacin (MIC: >0.06 mg/L). The molecular method showed a sensitivity of 96.1% and, a specificity of 90.9% for fluoroquinolone susceptibility, but the statistical analysis between the molecular test and gradient diffusion test was not statistically significant for fluoroquinolone resistance (p = 1). Statistical analysis was not performed for macrolides because of the absence of positive RT-PCR results. According to our data, Allplex™ assay cannot replace the gradient diffusion test for macrolide resistance. However, the assay could be used to test fluoroquinolone resistance in NG isolates as a replacement for phenotypic methods.

Estimation of antimicrobial resistance of Mycoplasma genitalium, Belgium, 2022.

BackgroundAntimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking.AimWe aimed to estimate the occurrence of resistant MG in Belgium.MethodsBetween July and November 2022, frozen remnants of MG-positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance-associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioural variables via Fisher's exact test and logistic regression analysis.ResultsOf the 244 MG-positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics.ConclusionAlthough limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies.

Mycoplasma genitalium: Key Information for the Primary Care Clinician.

Mycoplasma genitalium (MG) is an emerging sexually transmitted infection, which appears to be a cause of urethritis and cervicitis and has been associated with pelvic inflammatory disease (PID), epididymitis, proctitis, infertility, complications during pregnancy, and human immunodeficiency virus (HIV) transmission. Three Food and Drug Administration (FDA) approved tests are available. Testing should be focused to avoid inappropriate antibiotic use. The Center of Disease Control and Prevention (CDC) guidelines recommend testing for persistent male urethritis, cervicitis, and proctitis and state that testing should be considered in cases of PID. Testing is also recommended for sexual contacts of patients with MG. Testing is not recommended in asymptomatic patients, including pregnant patients, who do not have a history of MG exposure. Although resistance-guided therapy is recommended, there are currently no FDA approved tests for MG macrolide resistance, and tests are not widely available in the United States. The CDC recommends 2-step treatment with doxycycline followed by azithromycin or moxifloxacin. Moxifloxacin is recommended if resistance testing is unavailable or testing demonstrates macrolide resistance..

Macrolide and lincosamide resistance of Streptococcus agalactiae in pregnant women in Poland.

Knowing about the antibiotic resistance, serotypes, and virulence-associated genes of Group B Streptococcus for epidemiological and vaccine development is very important. We have determined antimicrobial susceptibility patterns, serotype, and virulence profiles. The antibiotic susceptibility was assessed for a total of 421 Streptococcus agalactiae strains, isolated from pregnant women and neonates. Then, 89 erythromycin and/or clindamycin-resistant strains (82 isolates obtained from pregnant women and seven isolates derived from neonates) were assessed in detail. PCR techniques were used to identify the studied strains, perform serotyping, and assess genes encoding selected virulence factors. Phenotypic and genotypic methods determined the mechanisms of resistance. All tested strains were sensitive to penicillin and levofloxacin. The constitutive MLSB mechanism (78.2%), inducible MLSB mechanism (14.9%), and M phenotype (6.9%) were identified in the macrolide-resistant strains. It was found that macrolide resistance is strongly associated with the presence of the ermB gene and serotype V. FbsA, fbsB, fbsC, scpB, and lmb formed the most recurring pattern of genes among the nine surface proteins whose genes were analysed. A minority (7.9%) of the GBS isolates exhibited resistance to lincosamides and macrolides, or either, including those that comprised the hypervirulent clone ST-17. The representative antibiotic resistance pattern consisted of erythromycin, clindamycin, and tetracycline resistance (71.9%). An increase in the fraction of strains resistant to macrolides and lincosamides indicates the need for monitoring both the susceptibility of these strains and the presence of the ST-17 clone.

Metabolomic Response of Thalassiosira weissflogii to Erythromycin Stress: Detoxification Systems, Steroidal Metabolites, and Energy Metabolism.

Erythromycin, a macrolide antibiotic, is a prioritized pollutant that poses a high risk to environmental health. It has been detected in different environmental matrices and can cause undesired effects in aquatic organisms, particularly freshwater algae, which are primary producers. However, the impact of erythromycin on marine algae remains largely unexplored. Erythromycin has been reported to induce hormetic effects in the marine diatom Thalassiosira weissflogii (T. weissflogii). These effects are associated with the molecular pathways and biological processes of ribosome assembly, protein translation, photosynthesis, and oxidative stress. However, the alterations in the global gene expression have yet to be validated at the metabolic level. The present study used non-targeted metabolomic analysis to reveal the altered metabolic profiles of T. weissflogii under erythromycin stress. The results showed that the increased cell density was possibly attributed to the accumulation of steroidal compounds with potential hormonic action at the metabolic level. Additionally, slight increases in the mitochondrial membrane potential (MMP) and viable cells were observed in the treatment of 0.001 mg/L of erythromycin (an environmentally realistic level). Contrarily, the 0.75 and 2.5 mg/L erythromycin treatments (corresponding to EC20 and EC50, respectively) showed decreases in the MMP, cell density, and viable algal cells, which were associated with modified metabolic pathways involving ATP-binding cassette (ABC) transporters, the metabolism of hydrocarbons and lipids, thiamine metabolism, and the metabolism of porphyrin and chlorophyll. These findings suggest that metabolomic analysis, as a complement to the measurement of apical endpoints, could provide novel insights into the molecular mechanisms of hormesis induced by antibiotic agents in algae.

Detection of resistance to macrolides and fluoroquinolones in Mycoplasma genitalium by targeted next-generation sequencing.

Mycoplasma genitalium is fastidious to culture, and its detection in human clinical specimens relies mainly on molecular methods. Phenotypic determination of antibiotic susceptibility for this bacterium is not a timely or feasible option for most clinical laboratories. This study sought to determine whether next-generation sequencing technologies can effectively be employed in determining genetic mutations associated with drug resistance in M. genitalium samples collected in Aptima Hologic tubes and possibly integrating them into viable workflows in public health laboratories. Following analysis by a custom-designed bioinformatics pipeline, at least one mutation/sample has been identified in 94/98 specimens in at least one of seven loci (macrolides: rrl, rplD, rplV; fluoroquinolones: parC, parE, gyrA, gyrB) described previously to be connected to antibiotic resistance. This method identified a total of 469 single nucleotide polymorphisms (SNPs) (452 mutations): 134 of 23S rRNA SNPs and 318 amino acid mutations: 114 substitutions and 204 synonymous; the turnaround time (sample to analyzed sequence) was typically 3 days. The assays and workflows described in this work demonstrated that the determination of a drug resistance profile for macrolides and fluoroquinolones of M. genitalium samples by using next-generation sequencing in clinical samples is a feasible approach that can be implemented in clinical laboratories, following thorough and extensive validation studies.IMPORTANCEThe mechanisms of drug resistance in Mycoplasma genitalium are complex and involve several genetic loci. The molecular methods for accurately characterizing resistance to fluoroquinolones and macrolides in this organism are often not available or approved for patient use and do not cover all genetic determinants. To this end, we propose a next-generation sequencing-based method with a turnaround time of 3 days that includes the investigation of all drug resistance loci of M. genitalium. Following adaptation, validation, and verification for routine clinical use, assays based on this method may yield molecular results that can be used to guide proper treatment regimens and for surveillance of drug resistance in the general population.