The performance and costs of XTT, resazurin, MTS and luciferin as viability dyes in in vitro susceptibility testing of Madurella mycetomatis.

BACKGROUND: in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. METHODS: To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. RESULTS: Each of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC. CONCLUSION: All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. LAY SUMMARY: To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them.

In vitro determination of the susceptibility of Malassezia furfur biofilm to different commercially used antimicrobials.

Malassezia furfur is a yeast known as the etiological agent of seborrheic dermatitis. We evaluated the action of five different antimicrobials (amphotericin B, chloramphenicol, ketoconazole, fluconazole, and nystatin) on inhibiting biofilm formation and removing biofilm already formed by M. furfur. The assays were carried out using the microdilution method, and scanning electron microscopy images were used to analyze the biofilm structure. According to the results obtained, the percentage of inhibition was higher for chloramphenicol, followed by ketoconazole, nystatin, and amphotericin B. Regarding the eradication of the biofilm formed, the highest percentage was chloramphenicol, followed by ketoconazole and nystatin. Amphotericin B did not affect biofilm eradication, whereas fluconazole did not cause significant changes inhibiting or removing M. furfur biofilm. Therefore, except for fluconazole, all evaluated antimicrobials had inhibiting effects on the biofilm of M. furfur, either in its formation and/or eradication. Although the results achieved with chloramphenicol have been highlighted, further in vitro and in vivo studies are still needed in order to include this antimicrobial in the therapy of seborrheic dermatitis due to its toxicity, especially to the bone marrow.

Absence of association between Pfnfs1 mutation and in vitro susceptibility to lumefantrine in Plasmodium falciparum.

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

Antifungal activity of azoles, allylamines, and 8-hidroxiquinolines, alone and in combination, against Malassezia pachydermatis in vitro and in vivo.

Malassezia pachydermatis is often reported as the causative agent of dermatitis in dogs. This study aims to evaluate the in vitro and in vivo antifungal activity of azoles and terbinafine (TRB), alone and in combination with the 8-hydroxyquinoline derivatives (8-HQs) clioquinol (CQL), 8-hydroxyquinoline-5-(n-4-chlorophenyl)sulfonamide (PH151), and 8-hydroxyquinoline-5-(n-4-methoxyphenyl)sulfonamide (PH153), against 16 M. pachydermatis isolates. Susceptibility to the drugs was evaluated by in vitro broth microdilution and time-kill assays. The Toll-deficient Drosophila melanogaster fly model was used to assess the efficacy of drugs in vivo. In vitro tests showed that ketoconazole (KTZ) was the most active drug, followed by TRB and CQL. The combinations itraconazole (ITZ)+CQL and ITZ+PH151 resulted in the highest percentages of synergism and none of the combinations resulted in antagonism. TRB showed the highest survival rates after seven days of treatment of the flies, followed by CQL and ITZ, whereas the evaluation of fungal burden of dead flies showed a greater fungicidal effect of azoles when compared to the other drugs. Here we showed for the first time that CQL is effective against M. pachydermatis and potentially interesting for the treatment of malasseziosis.

Lodderomyces elongisporus as causative organism of oropharyngeal infections - Two case reports.

We report two cases of patients with oropharyngeal infection by Lodderomyces elongisporus. The identification of the two isolates was confirmed after sequencing the ITS1 and ITS4 regions. The antifungal susceptibility test revealed low MIC values for the different antifungals tested. This is the first reported case of L. elongisporus present during an oropharyngeal infection and describes the laboratory methodology employed in the diagnosis.

The combination of manogepix and itraconazole is synergistic and inhibits the growth of Madurella mycetomatis in vitro but not in vivo.

Mycetoma is a neglected tropical disease commonly caused by the fungus Madurella mycetomatis. Standard treatment consists of extensive treatment with itraconazole in combination with surgical excision of the infected tissue, but has a low success rate. To improve treatment outcomes, novel treatment strategies are needed. Here, we determined the potential of manogepix, a novel antifungal agent that targets the GPI-anchor biosynthesis pathway by inhibition of the GWT1 enzyme. Manogepix was evaluated by determining the minimal inhibitory concentrations (MICs) according to the CLSI-based in vitro susceptibility assay for 22 M. mycetomatis strains and by in silico protein comparison of the target protein. The synergy between manogepix and itraconazole was determined using a checkerboard assay. The efficacy of clinically relevant dosages was assessed in an in vivo grain model in Galleria mellonella larvae. MICs for manogepix ranged from <0.008 to >8 mg/l and 16/22 M. mycetomatis strains had an MIC ≥4 mg/ml. Differences in MICs were not related to differences observed in the GWT1 protein sequence. For 70% of the tested isolates, synergism was found between manogepix and itraconazole in vitro. In vivo, enhanced survival was not observed upon admission of 8.6 mg/kg manogepix, nor in combination treatment with 5.7 mg/kg itraconazole. MICs of manogepix were high, but the in vitro antifungal activity of itraconazole was enhanced in combination therapy. However, no efficacy of manogepix was found in an in vivo grain model using clinically relevant dosages. Therefore, the therapeutic potential of manogepix in mycetoma caused by M. mycetomatis seems limited.

Treatment of Madurella mycetomatis-caused mycetoma consists of extensive exposure to antifungals and surgery. To improve therapy, we evaluated manogepix, a novel antifungal agent, as a therapeutic option against M. mycetomatis. Our findings suggest limited therapeutic potential for manogepix.

The In Vitro Antimicrobial Susceptibility of Borrelia burgdorferi sensu lato: Shedding Light on the Known Unknowns.

Human Lyme borreliosis (LB) represents a multisystem disorder that can progress in stages. The causative agents are transmitted by hard ticks of the Ixodes ricinus complex that have been infected with the spirochete Borrelia burgdorferi sensu lato. Today, LB is considered the most important human tick-borne illness in the Northern Hemisphere. The causative agent was identified and successfully isolated in 1982 and, shortly thereafter, antibiotic treatment was found to be safe and efficacious. Since then, various in vitro studies have been conducted in order to improve our knowledge of the activity of antimicrobial agents against B. burgdorferi s. l. The full spectrum of in vitro antibiotic susceptibility has still not been defined for some of the more recently developed compounds. Moreover, our current understanding of the in vitro interactions between B. burgdorferi s. l. and antimicrobial agents, and their possible mechanisms of resistance remains very limited and is largely based on in vitro susceptibility experiments on only a few isolates of Borrelia. Even less is known about the possible mechanisms of the in vitro persistence of spirochetes exposed to antimicrobial agents in the presence of human and animal cell lines. Only a relatively small number of laboratory studies and cell culture experiments have been conducted. This review summarizes what is and what is not known about the in vitro susceptibility of B. burgdorferi s. l. It aims to shed light on the known unknowns that continue to fuel current debates on possible treatment resistance and mechanisms of persistence of Lyme disease spirochetes in the presence of antimicrobial agents.

Sensibilidad de Escherichia coli frente a fosfomicina, en infecciones urinarias provenientes de personas con discapacidad que acudieron a la Secretaría Nacional por los Derechos Humanos de las Personas con Discapacidad-SENADIS / Susceptibility of Escherichia coli against fosfomycin in urinary tract infections from people with disabilities who attended SENADIS

INTRODUCCIÓN: Fosfomicina es un antimicrobiano de amplio espectro utilizado para el tratamiento de las infecciones urinarias bajas; tiene actividad sobre bacilos gramnegativos y cocos grampositivos, así también sobre microorganismos multirresistentes, además de ofrecer una alternativa terapéutica de administración vía oral en dosis única, alcanzando una efectividad de 90%. OBJETIVO: Conocer la sensibilidad in vitro de Escherichia coli frente a fosfomicina, en infecciones urinarias provenientes de personas con discapacidad. MATERIAL Y MÉTODO: Estudio observacional, descriptivo, prospectivo, en el que se incluyó un total de 273 muestras de urocultivo, de pacientes de ambos sexos que acudieron a SENADIS, y que en el momento de la consulta presentaban síntomas de infección del tracto urinario, por lo que se les solicitó el análisis de orina simple y cultivo. De las muestras procesadas en el laboratorio de microbiología, que fueron positivas con crecimiento bacteriano significativo, se procedió a la identificación bacteriana y a la realización del antibiograma según las recomendaciones de CLSI. RESULTADOS: De estas 273 muestras, 91 fueron positivas para diferentes uropatógenos, 62/91 (68%) resultaron ser E. coli. De estas cepas de E. coli, 59/62 (95%) mostraron sensibilidad in vitro a fosfomicina. Comentario: Aunque el número de muestra obtenido es pequeño y no extrapolable ampliamente, pretendemos extender el trabajo por un tiempo más para compararlo más adelante. CONCLUSIONES: Se observa que fosfomicina presenta buena actividad in vitro frente a cepas de E. coli aisladas de urocultivo, pudiendo representar una buena alternativa terapéutica a ser utilizada en la población en estudio.

BACKGROUND: Fosfomycin is a broad-spectrum antibiotic used for the treatment of lower urinary tract infections, it is active against gramnegative bacilli and grampositive cocci, as well as against multi-resistant microorganism, in addition to offering a therapeutic alternative for oral administration in a single dose, reaching an effectiveness of 90%. AIM: To study the susceptibility of Escherichia coli to fosfomycin in urinary tract infections, of isolated strains obtained from patients with disabilities. METHODS: It is an observational, descriptive, prospective study in which a total of 273 urine culture samples of patients of both sexes who attended the SENADIS were included, and who at the time of the consultation presented symptoms of urinary tract infection. The urine positive cultures with significant bacterial growth were performed to determine its bacterial identification and the antibiogram according to CLSI recommendations. RESULTS: Of these 273 samples, 91 samples were positive for different uropathogens, with 62/91 (68%) being positive for E. coli. Of these E. coli strains, 59/62 (95%) showed in vitro susceptibility to fosfomycin. Comment: Although the number of samples obtained is small and it cannot be extrapolated, we pretend to extend the work for a while longer to be able to compare it later. CONCLUSION: Fosfomycin has good activity in vitro against E. coli isolated from urine culture in our institution, representing a good alternative to be used in our study population

In Vitro Activity of Two Novel Antimicrobial Compounds on MDR-Resistant Clinical Isolates.

The development of novel antibiotics is mandatory to curb the growing antibiotic resistance problem resulting in difficult-to-treat bacterial infections. Here, we have determined the spectrum of activity of cystobactamids and chelocardins, two novel and promising classes of molecules with different modes of action. A panel of 297 clinically relevant Gram-negative and Gram-positive isolates with different antibiotic susceptibility profiles, going from wild type to multi- or even extremely drug resistant (MDR, XDR) and including carbapenem-resistant isolates, were tested using broth microdilution assays to determine the minimal inhibitory concentrations (MICs), MIC50s and MIC90s of two cystobactamids derivatives (CN-861-2 and CN-DM-861) and two chelocardin derivatives (CHD and CDCHD). Cystobactamids revealed potent activities on the majority of tested Enterobacterales (MIC50s ranging from 0.25 to 4 µg/mL), except for Klebsiella pneumoniae isolates (MIC50s is 128 µg/mL). Pseudomonas aeruginosa and Acinetobacter baumannii showed slightly higher MIC50s (4 µg/mL and 8 µg/mL, respectively) for cystobactamids. Chelocardins inhibited the growth of Enterobacterales and Stenotrophomas maltophilia at low to moderate MICs (0.25-16 µg/mL) and the chemically modified CDCHD was active at lower MICs. A. baumannii and P. aeruginosa were less susceptible to these molecules with MICs ranging from 0.5 to 32 µg/mL. These molecules show also interesting in vitro efficacies on clinically relevant Gram-positive bacteria with MICs of 0.125-8 µg/mL for cystobactamids and 0.5-8 µg/mL for chelocardins. Taken together, the cystobactamid CN-DM-861 and chelocardin CDCHD showed interesting antibiotic activities on MDR or XDR bacteria, without cross-resistance to clinically relevant antibiotics such as carbapenems, fluoroquinolones, and colistin.

In Vitro Susceptibility of Achromobacter Species Isolated from Cystic Fibrosis Patients: a 6-Year Survey.

We conducted in vitro antimicrobial susceptibility testing of 267 Achromobacter isolates for 16 antibiotics from 2017 to 2022. The highest susceptibility was found for piperacillin-tazobactam (70%) and ceftazidime-avibactam (62%). Between 30% and 49% of strains were susceptible to tigecycline, ceftazidime, and meropenem. We applied species-specific Achromobacter xylosoxidans breakpoints for piperacillin-tazobactam, meropenem, and trimethoprim-sulfamethoxazole and EUCAST pharmacokinetic/pharmacodynamic (PK/PD) breakpoints for the others. A. xylosoxidans was the most frequently isolated species, followed by Achromobacter insuavis and Achromobacter ruhlandii.

Antifungal Susceptibility of Oral Candida Isolates from Mother-Infant Dyads to Nystatin, Fluconazole, and Caspofungin.

The carriage of Candida albicans in children's oral cavities is associated with a higher risk for early childhood caries, so controlling this fungus in early life is essential for preventing caries. In a prospective cohort of 41 mothers and their children from 0 to 2 years of age, this study addressed four main objectives: (1) Evaluate in vitro the antifungal agent susceptibility of oral Candida isolates from the mother-child cohort; (2) compare Candida susceptibility between isolates from the mothers and children; (3) assess longitudinal changes in the susceptibility of the isolates collected between 0 and 2 years; and (4) detect mutations in C. albicans antifungal resistance genes. Susceptibility to antifungal medications was tested by in vitro broth microdilution and expressed as the minimal inhibitory concentration (MIC). C. albicans clinical isolates were sequenced by whole genome sequencing, and the genes related to antifungal resistance, ERG3, ERG11, CDR1, CDR2, MDR1, and FKS1, were assessed. Four Candida spp. (n = 126) were isolated: C. albicans, C. parapsilosis, C. dubliniensis, and C. lusitaniae. Caspofungin was the most active drug for oral Candida, followed by fluconazole and nystatin. Two missense mutations in the CDR2 gene were shared among C. albicans isolates resistant to nystatin. Most of the children's C. albicans isolates had MIC values similar to those from their mothers, and 70% remained stable on antifungal medications from 0 to 2 years. For caspofungin, 29% of the children's isolates showed an increase in MIC values from 0 to 2 years. Results of the longitudinal cohort indicated that clinically used oral nystatin was ineffective in reducing the carriage of C. albicans in children; novel antifungal regimens in infants are needed for better oral yeast control.

Subspecies Distribution and Antimicrobial Susceptibility Testing of Mycobacterium abscessus Clinical Isolates in Madrid, Spain: a Retrospective Multicenter Study.

Mycobacterium abscessus (MABS) is the most pathogenic and drug-resistant rapidly growing mycobacteria. However, studies on MABS epidemiology, especially those focusing on subspecies level, are scarce. We aimed to determine MABS subspecies distribution and its correlation with phenotypic and genotypic antibiotic profiles. A retrospective multicenter study of 96 clinical MABS isolates in Madrid between 2016 to 2021 was conducted. Identification at the subspecies level and resistance to macrolides and aminoglycosides were performed by the GenoType NTM-DR assay. The MICs of 11 antimicrobials tested against MABS isolates were determined using the broth microdilution method (RAPMYCOI Sensititer titration plates). Clinical isolates included 50 (52.1%) MABS subsp. abscessus; 33 (34.4%) MABS subsp. massiliense; and 13 (13.5%) MABS subsp. bolletii. The lowest resistance rates corresponded to amikacin (2.1%), linezolid (6.3%), cefoxitin (7.3%), and imipenem (14.6%), and the highest to doxycycline (100.0%), ciprofloxacin (89.6%), moxifloxacin (82.3%), cotrimoxazole (82.3%), tobramycin (81.3%), and clarithromycin (50.0% at day 14 of incubation). Regarding tigecycline, although there are no susceptibility breakpoints, all strains but one showed MICs ≤ 1 µg/mL. Four isolates harbored mutations at positions 2058/9 of the rrl gene, one strain harbored a mutation at position 1408 of the rrl gene, and 18/50 harbored the T28C substitution at erm(41) gene. Agreement of the GenoType results with clarithromycin and amikacin susceptibility testing was 99.0% (95/96). The rate of MABS isolates showed an upward trend during the study period, being M. abscessus subsp. abscessus the most frequently isolated subspecies. Amikacin, cefoxitin, linezolid, and imipenem showed great in vitro activity. The GenoType NTM-DR assay provides a reliable and complementary tool to broth microdilution for drug resistance detection. IMPORTANCE Infections caused by Mycobacterium abscessus (MABS) are increasingly being reported worldwide. Identifying MABS subspecies and assessing their phenotypic resistance profiles are crucial for optimal management and better patient outcomes. M. abscessus subspecies differ in erm(41) gene functionality, which is a critical determinant of macrolide resistance. Additionally, resistance profiles of MABS and the subspecies distribution can vary geographically, highlighting the importance of understanding local epidemiology and resistance patterns. This study provides valuable insights into the epidemiology and resistance patterns of MABS and its subspecies in Madrid. Elevated resistance rates were observed for several recommended antimicrobials, emphasizing the need for cautious drug use. Furthermore, we assessed the GenoType NTM-DR assay, which examines principal mutations in macrolides and aminoglycosides resistance-related genes. We observed a high level of agreement between the GenoType NTM-DR assay and the microdilution method, indicating its usefulness as an initial tool for early initiation of appropriate therapy.

Residual phenotypic susceptibility to doravirine in multidrug-resistant HIV-1 from subjects enrolled in the PRESTIGIO Registry.

OBJECTIVES: Doravirine shows a rather distinct resistance profile within the nonnucleoside reverse transcriptase inhibitor (NNRTI) class. This study aimed to evaluate the phenotypic susceptibility to doravirine, rilpivirine and etravirine in a panel of multidrug-resistant (MDR) HIV-1 isolates collected from people living with HIV (PLWH) enrolled in the PRESTIGIO Registry. METHODS: Recombinant viruses expressing PLWH-derived protease, reverse transcriptase coding regions were generated from plasma samples at virological failure with documented resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors, NNRTIs and integrase strand transfer inhibitors. In vitro susceptibility was assessed through a phenotypic assay measuring fold-change values with respect to the reference NL4-3 virus. Genotypic susceptibility was computed by the Stanford HIVdb algorithm 8.9-1. RESULTS: Plasma samples were collected from 22 PLWH: 20 (91%) were male, median age 55 years (IQR 50-58), time since HIV-1 diagnosis 27 years (23-31) and time on antiretroviral treatment 23 years (22-26). Median doravirine, etravirine and rilpivirine fold-change values were 9.8 (2.9-40.4), 42.9 (3.1-100.0) and 100.0 (17.9-100.0), respectively. According to the fold-change cut-offs, full susceptibility was observed in five (23%), four (18%) and one (5%) cases with doravirine, etravirine and rilpivirine, respectively. Irrespective of the presence of specific doravirine mutations, higher numbers of NNRTI mutations correlated with higher fold-change values for doravirine. By comparing the distribution of fold-change values with the Stanford HIVdb predicted susceptibility, a significant correlation was detected for doravirine and rilpivirine but not etravirine. CONCLUSION: Despite extensive cross-resistance among NNRTIs, doravirine can be a valid option in a proportion of PLWH with MDR HIV-1. Doravirine activity appeared to be inferred with fair accuracy by the HIVdb algorithm.

Oomicidal activity of polypyrrole nanoparticles against Pythium insidiosum.

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 µg mL-1, and the MIC90 and MIC50 were 16 µg mL-1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.

Dihydroorotate dehydrogenase inhibitor olorofim has potent in vitro activity against Microascus/Scopulariopsis, Rasamsonia, Penicillium and Talaromyces species.

BACKGROUND: Treatment options against infections caused by rare but emerging moulds may be limited by their reduced susceptibility or resistance to clinically available antifungals. The investigational antifungal olorofim, which targets the biosynthesis of pyrimidines within fungi, has activity against different species of filamentous fungi, including Aspergillus and Scedosporium/Lomentospora prolificans isolates that are resistant to available antifungals. OBJECTIVE: We evaluated the in vitro activity of olorofim against 160 isolates within the genera Microascus/Scopulariopsis, Penicillium, Talaromyces and the Rasamsonia argillacea species complex. METHODS: One hundred sixty clinical isolates that had previously been identified to the species level by DNA sequence analysis were included. Antifungal susceptibility testing was performed by CLSI M38 broth microdilution for olorofim, amphotericin B, caspofungin, posaconazole and voriconazole. RESULTS: Olorofim demonstrated in vitro activity against each of the genera tested. Overall, olorofim MICs ranged from ≤0.008 to 0.5 mg/L against all isolates tested, with MIC90 and modal MIC values ranging from ≤0.008 to 0.25 mg/L and ≤0.008 to 0.03 mg/L, respectively. This activity was also maintained against individual isolates that had reduced susceptibility to or in vitro resistance against amphotericin B, posaconazole and/or voriconazole. CONCLUSIONS: The investigational agent olorofim demonstrated good in vitro activity against clinical isolates of emerging mould pathogens, including those with reduced susceptibility or resistance to clinically available antifungals. Further studies are warranted to determine how well this in vitro activity translates into in vivo efficacy against infections caused by these fungi.

In vitro susceptibility of common Enterobacterales to eravacycline in Taiwan.

BACKGROUND: New tetracycline derivatives exhibit broad-spectrum antimicrobial activities. This study aimed to assess the in vitro activity of eravacycline against common Enterobacterales. METHODS: Clinical Enterobacterales isolates were collected between 2017 and 2021. The minimum inhibitory concentration (MIC) was determined using a broth microdilution test. RESULTS: We identified Klebsiella pneumoniae (n = 300), Escherichia coli (n = 300), Klebsiella oxytoca (n = 100), Enterobacter cloacae complex (n = 100), Citrobacter freundii (n = 100), and Proteus mirabilis (n = 100). All P. mirabilis strains were resistant to eravacycline. Excluding P. mirabilis, the susceptibility rates to eravacycline, omadacycline, and tigecycline were 75.2%, 66.9%, and 73%, respectively. The MIC50 and MIC90 (mg/L) of eravacycline were 0.5 and 4 for K. pneumoniae, 0.5 and 1 for E. coli, 0.5 and 1 for K. oxytoca, 0.5 and 2 for E. cloacae complex, and 0.25 and 1 for C. freundii. In cefotaxime non-susceptible and meropenem susceptible Enterobacterales, excluding P. mirabilis, the susceptibility rates of eravacycline, omadacycline, and tigecycline were 69.7%, 57.1%, and 66.2%. We found decreased susceptibility rates of three new tetracycline derivatives against meropenem non-susceptible Enterobacterales (eravacycline: 47.1%, omadacycline: 39.4%, and tigecycline: 39.4%). Eravacycline showed a high susceptibility rate against cefotaxime non-susceptible and meropenem susceptible K. oxytoca (100%), C. freundii (93.2%), E. coli (85.9%), and meropenem non-susceptible E. coli (100%). CONCLUSION: This study provides the MIC and susceptibility rate of eravacycline for common Enterobacterales. Eravacycline could be a therapeutic choice for cefotaxime non-susceptible or meropenem non-susceptible Enterobacterales, especially K. oxytoca, C. freundii, and E. coli.

Reviving the role of mecillinam against extended spectrum beta-lactamase producing enterobacterales.

Background and Objectives: This study was designed to determine the in vitro efficacy of mecillinam against extended spectrum beta lactamse producing Enterobacterales. Materials and Methods: After proper permission from Ethical Review Committee of the Institute, all samples yielding growth of ESBL producing Enterobacterales were part of the study and were processed according to routine microbiological procedures. Routine antibiotic sensitivity testing was done on Muller Hinton Agar by Modified Kirby Bauer Method. All Gram negative isolates were subjected to concomitant detection of ESBL production by double disc synergy method. All ESBL producers were then subjected to the mecillinam Minimum Inhibitory Concentration (MIC) determination by E test. The results were interpreted as per CLSI Guidelines. Results: A total of 120 ESBL producing Enterobacterales isolates were included in the study. The mean age of patients with ESBL infection was 45 ± 18.7 years. There were 44% male and 55% female patients. Majority of the ESBL producing Enterobacterales were isolated from urine samples (56%), followed by pus. Among the isolated organisms, Escherichia coli (45%) was the most frequently isolated organism followed by Klebsiella spp. (22%). Overall 83% of the isolates turned out to be sensitive to mecillinam. MIC50 of mecillinam against ESBL producing Gram negative rods (GNR) turned out to be 1 ug/ml and MIC90 turned out to be 2 ug/ml. Conclusion: Mecillinam shows good in vitro efficacy against ESBL producing Enterobacterales in our study. Further studies with more sample size and from diverse areas across the country should be done to evaluate its efficacy.

Assessment of the Susceptibility of Clinical Gram-Negative and Gram-Positive Bacterial Strains to Fosfomycin and Significance of This Antibiotic in Infection Treatment.

Multidrug resistance of bacteria has prompted intensive development work on new medicines, but also the search for effective options among the oldest antibiotics. Although intravenous fosfomycin (IVFOS) seems to be an interesting proposal, the recommended agar dilution method for susceptibility determination poses a major problem in routine diagnostic testing. As a consequence, there is a lack of comprehensive data on the frequency of isolation of susceptible or resistant strains. This fact triggered the disposition of EUCAST concerning the revision of IVFOS breakpoints (BPs), including withdrawal of BPs for Enterobacterales (excluding E. coli) and coagulase-negative staphylococci. Therefore, the aim of this study was to assess the activity of fosfomycin against numerous clinical strains using recommended methods. Materials and methods: A total of 997 bacterial strains were tested from the following genera: Enterobacterales, Pseudomonas spp., Staphylococcus spp., Acinetobacter spp., and Enterococcus spp., for which there are currently no BPs. The strains were isolated from various clinical materials from patients hospitalized in five hospitals. During the investigation, the recommended agar dilution method was used. Susceptibility to other antibiotics and resistance mechanisms were determined using an automatic method (Phoenix) the disk diffusion method, and E-tests. MIC values of fosfomycin were estimated for all strains and for susceptible and multidrug-resistant (MDR) strains individually. Results: Except for Acinetobacter and Enterococcus, 83% of the strains were susceptible to IVFOS, including the largest percentage of S. aureus and E. coli. Klebsiella spp. turned out to be the least susceptible strains (66%). The highest proportion of susceptibility to fosfomycin was found among strains that were sensitive to other antibiotics (80.9%), and the lowest was found among Gram-negative carbapenemase-producing bacteria (55.6%) and ESBL+ bacteria (61.6%). The MIC evaluation revealed the lowest MIC50 and MIC90 values for S. aureus (0.5 mg/L and 1 mg/L, respectively) and E. coli (4 mg/L and 32 mg/L, respectively). The highest values of MIC50 were found for Acinetobacter spp. (256 mg/L), while the highest values of MIC90 were found for Acinetobacter spp. and Klebsiella spp. (256 mg/L and 512 mg/L, respectively). Conclusions: IVFOS appears to be suitable for the treatment of many infections, including the empirical treatment of polymicrobial infections and those caused by MDR strains, since the sensitivity of the studied strains to this antibiotic in different groups ranged from 66% to as much as 99%. Sensitivity to fosfomycin was also demonstrated by 60% of carbapenem-resistant strains; therefore, IVFOS is one of the few therapeutic options that can be effective against the most resistant Gram-negative rods. In light of the general consultation posted by EUCAST, obtaining data such as IVFOS MIC value distributions may be vital for the decision of implementing fosfomycin into breakpoint tables.

The Arylamidine T-2307 as a Novel Treatment for the Prevention and Eradication of Candida tropicalis Biofilms.

Candida tropicalis is an emerging pathogen with a high mortality rate due to its virulence factors, including biofilm formation, that has important repercussions on the public health system. The ability of C. tropicalis to form biofilms, which are potentially more resistant to antifungal drugs and the consequent increasing antimicrobial resistance, highlights an urgent need for the development of novel antifungal. The present study analyzed the antibiofilm capacity of the arylamidine T-2307 on two strains of Candida tropicalis. Antimicrobial activity and time-killing assays were performed to evaluate the anticandidal effects of T-2307, the antibiofilm ability on biomass inhibition and eradication was evaluated by the crystal violet (CV) method. Furthermore, in Galleria mellonella infected larvae an increased survival after pre-and post- treatment with T-2307 was observed. The MTT test was used to determine the viability of immortalized human prostate epithelial cells (PNT1A) after exposure to different concentrations of T-2307. Levels of interleukin IL-4, IL-8, IL-10 were quantified after Candida infection of PNT1A cells and treatment. Active doses of T-2307 did not affect the viability of PNT1A cells, and drug concentrations of 0.005 or 0.01 µg mL-1 inhibited the secretion of inflammatory cytokines. Taken together, these results provide new information on T-2307, indicating this drug as a new and promising alternative therapeutic option for the treatment of Candida infections.

Initial Results of the International Efforts in Screening New Agents against Candida auris.

BACKGROUND: Candida auris is an emergent fungal pathogen and a global concern, mostly due to its resistance to many currently available antifungal drugs. OBJECTIVE: Thus, in response to this challenge, we evaluated the in vitro activity of potential new drugs, diphenyl diselenide (PhSe)2 and nikkomycin Z (nikZ), alone and in association with currently available antifungals (azoles, echinocandins, and polyenes) against Candida auris. METHODS: Clinical isolates of C. auris were tested in vitro. (PhSe)2 and nikZ activities were tested alone and in combination with amphotericin B, fluconazole, or the echinocandins, micafungin and caspofungin. RESULTS: (PhSe)2 alone was unable to inhibit C. auris, and antagonism or indifferent effects were observed in the combination of this compound with the antifungals tested. NikZ appeared not active alone either, but frequently acted cooperatively with conventional antifungals. CONCLUSION: Our data show that (PhSe)2 appears to not have a good potential to be a candidate in the development of new drugs to treat C. auris, but that nikZ is worthy of further study.