High MIC Values Research Articles

Serotypes, Antibiotic Susceptibility, Genotypic Virulence Profiles and SpaA Variants of Erysipelothrix rhusiopathiae Strains Isolated from Pigs in Poland.

The aim of the study was phenotypic and genotypic characterization of Erysipelothrix rhusiopathiae strains isolated from diseased pigs in Poland and comparison of the SpaA (Surface protective antigen A) sequence of wild-type strains with the sequence of the R32E11 vaccine strain. The antibiotic susceptibility of the isolates was assessed using the broth microdilution method. Resistance genes, virulence genes, and serotype determinants were detected using PCR. The gyrA and spaA amplicons were sequenced to determine nonsynonymous mutations. The E. rhusiopathiae isolates (n = 14) represented serotypes 1b (42.8%), 2 (21.4%), 5 (14.3%), 6 (7.1%), 8 (7.1%), and N (7.1%). All strains were susceptible to β-lactams, macrolides and florfenicol. One isolate showed resistance to lincosamides and tiamulin, and most strains were resistant to tetracycline and enrofloxacin. High MIC values of gentamicin, kanamycin, neomycin, trimethoprim, trimethoprim/sulfadiazine, and rifampicin were recorded for all isolates. Phenotypic resistance was correlated with the presence of the tetM, int-Tn, lasE, and lnuB genes. Resistance to enrofloxacin was due to a mutation in the gyrA gene. All strains contained the spaA gene and several other genes putatively involved in pathogenesis (nanH.1, nanH.2, intl, sub, hlyA, fbpA, ERH_1356, cpsA, algI, rspA and rspB) Seven variants of the SpaA protein were found in the tested strains, and a relationship between the structure of SpaA and the serotype was noted. E. rhusiopathiae strains occurring in pigs in Poland are diverse in terms of serotype and SpaA variant and differ antigenically from the R32E11 vaccine strain. Beta-lactam antibiotics, macrolides, or phenicols should be the first choice for treatment of swine erysipelas in Poland. However, due to the small number of tested strains, this conclusion should be approached with caution.

A critical issue on microbiological cut-off value of ampicillin resistance in Lactiplantibacillus plantarum.

Comprehensive evaluation of antibiotic susceptibility patterns in Lactiplantibacillus plantarum strains isolated from grape marc, based on genomic and phenotypic assessment. We assessed the antibiotic resistance-susceptibility patterns of 20 L. plantarum strains for 16 antibiotics. Genomes of relevant strains were sequenced for in silico assessment and comparative genomic analysis. Results showed high MIC values for spectinomycin, vancomycin, and carbenicillin, indicating natural resistance to these antibiotics. Besides, these strains revealed MIC values for ampicillin higher than previously established by the EFSA, indicating the possible presence of acquired resistance genes in the genomes. However, genomic analysis by complete genome sequencing did not reveal presence of ampicillin resistance genes. Comparative genomic analysis between our strains and other L. plantarum genomes present in the literature showed several substantial genomic differences, and suggested the need to adjust the cut-off value for ampicillin in L. plantarum. However, further sequence analysis will reveal how these strains have acquired antibiotic resistance.

Antibiotic Susceptibility and Clarithromycin Resistance Determinants in Helicobacter pylori in the Northeast of Spain: A One-Year Prospective Study.

Helicobacter pylori is one of the most widespread infections, and it is reaching alarming resistance levels worldwide. The recommended first-line empirical treatment differs according to the local rate of clarithromycin resistance. Macrolide resistance is mainly associated with three point mutations in the 23S rRNA gene. The aim of this study was to describe the antibiotic susceptibility of H. pylori in our healthcare area and the main mechanisms involved in clarithromycin resistance. Gastric biopsies (n = 641) were collected and cultured in a one-year prospective study. Antibiotic susceptibility testing was performed by gradient diffusion. A multiplex real-time PCR test (AllplexTMH.pylori & ClariR Assay, Seegene) was used to detect the most frequent mutations associated with clarithromycin resistance. Overall, 141 isolates were available for antibiotic susceptibility testing. The highest resistance rates were detected in metronidazole and levofloxacin. The rate of clarithromycin resistance was 12.1%, and the associated mutations were A2143G and A2142G. More than half of the clarithromycin-resistant isolates presented high MIC values (>256 mg/L). Tetracycline resistance was not detected, suggesting that therapies that contain tetracycline could be a suitable option. The low clarithromycin resistance rate coupled with the high rates of metronidazole resistance may support the recovery of the classical triple therapy in our healthcare area.

In Vitro Antibacterial and Phytochemical Screening of Hypericum perforatum Extract as Potential Antimicrobial Agents against Multi-Drug-Resistant (MDR) Strains of Clinical Origin.

The perennial plant Hypericum perforatum is widely distributed around the world. It has been used for many years in conventional medicine to treat a variety of illnesses, including stress, mild to moderate depression, and minor injuries. This study examined the antimicrobial activity of the H. perforatum total extract and its fractions (n-hexane, ethyl acetate, chloroform, and aqueous) against multi-drug-resistant (MDR) isolates that were gathered from clinical samples, including methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Escherichia coli, and Klebsiella pneumonia. Aerial parts of H. perforatum were collected and extracted using various solvents and were tested versus different isolated bacterial species. The inhibition zone of tested extracts was detected using an agar diffusion assay, and MICs were measured. Phytochemical analysis of promising H. perforatum extract was done using LC-ESI-MS/MS. Ultrastructure examination for the most altered bacteria used transmission electron microscopy. Antioxidant assays were done using DPPH and ABTS scavenging capacity methods. Cytotoxicity was reported versus Vero cells. Different extracts of H. perforatum showed promising antibacterial activity against the pathogens. While the subfractions of the total extract were observed to show lesser inhibition zones and higher MIC values than the total extract of H. perforatum against MDR strains, the total extract of H. perforatum demonstrated the most potent antimicrobial action with an inhibition zone range of 17.9-27.9 mm. MDR-K. pneumoniae was discovered to be the most susceptible strain, which is consistent with the antibacterial inhibitory action of H. perforatum whole extract. Additionally, after treatment at the minimum inhibitory concentration (MIC 3.9 μg/ml), the transmission electron microscope showed alterations in the ultrastructure of the K. pneumoniae cells. Methanol extract from H. perforatum has a CC50 value of 976.75 μg/ml. Future inhibitors that target MDR strains may be revealed by these findings. Additionally, the extracts that were put to the test demonstrated strong antioxidant effects as shown by DPPH or ABTS radical-scavenging assays.

In Silico and In Vitro Analyses Reveal Promising Antimicrobial Peptides from Myxobacteria

Antimicrobial resistance (AMR) is a global concern, and as soon as new antibiotics are introduced, resistance to those agents emerges. Therefore, there is an increased appetite for alternative antimicrobial agents to traditional antibiotics. Here, we used in silico methods to investigate potential antimicrobial peptides (AMPs) from predatory myxobacteria. Six hundred seventy-two potential AMP sequences were extracted from eight complete myxobacterial genomes. Most putative AMPs were predicted to be active against Klebsiella pneumoniae with least activity being predicted against Staphylococcus aureus. One hundred seventeen AMPs (defined here as ‘potent putative AMPs’) were predicted to have very good activity against more than two bacterial pathogens, and these were characterized further in silico. All potent putative AMPs were predicted to have anti-inflammatory and antifungal properties, but none was predicted to be active against viruses. Twenty six (22%) of them were predicted to be hemolytic to human erythrocytes, five were predicted to have anticancer properties, and 56 (47%) were predicted to be biofilm active. In vitro assays using four synthesized AMPs showed high MIC values (e.g. So_ce_56_913 250 µg/ml and Coral_AMP411 125 µg/ml against E. coli). However, antibiofilm assays showed a substantial reduction in numbers (e.g. Coral_AMP411 and Myxo_mac104 showed a 69% and 73% reduction, respectively, at the lowest concentration against E. coli) compared to traditional antibiotics. Fourteen putative AMPs had high sequence similarity to proteins which were functionally associated with proteins of known function. The myxobacterial genomes also possessed a variety of biosynthetic gene clusters (BGCs) that can encode antimicrobial secondary metabolites, but their numbers did not correlate with those of the AMPs. We suggest that AMPs from myxobacteria are a promising source of novel antimicrobial agents with a plethora of biological properties.

125. Induction of Resistance Against Antipseudomonal Agents: Comparison of Novel b-Lactam/b-Lactamase Inhibitor (BL/BLI) Combinations and Other b-lactam Agents

Abstract Background The acquisition of mutations is the main driver of β-lactam resistance in Pseudomonas aeruginosa isolates. New BL/BLIs, such as ceftazidime-avibactam (CAZ-AVI), ceftolozane-tazobactam (C/T), and imipenem-relebactam (IMI-REL), are active against most P. aeruginosa isolates from US hospitals, but the ability of these agents to induce resistance have not been explored. We subjected 8 P. aeruginosa isolates, including ATCC 27853 and 7 clinical isolates, to a 10-day serial passage with 6 antipseudomonal agents to evaluate resistance levels and mechanisms in terminal mutant strains. Fold change in MIC results from parent isolate to terminal mutant Methods Serial passaging was performed in broth microdilution (BMD) for CAZ-AVI, IMI-REL, C/T, meropenem (MEM), cefepime (FEP), and piperacillin-tazobactam (P/T). The MIC of the terminal mutants was determined after 2X passaging on drug-free agar. Parent strains and terminal mutants were subjected to short-read whole genome sequencing (WGS) at 100X coverage. Parent isolates were sequenced using long-read WGS and the data was combined with short-reading sequencing for single nucleotide polymorphism (SNP) analysis. Results Overall, IMI-REL (1- to 4-fold) and CAZ-AVI (2- to 8-fold) displayed lower fold increases in MIC values when compared to other agents tested (Figure). Of the CAZ-AVI terminal mutants, 3 displayed a nalD regulator alteration, and 1 of these had a clpA chaperone missense substitution. FEP terminal mutants exhibited alterations in ampD, mexB, and the TetR family transcriptional regulator AmrR. C/T mutants had ampG and ftsI missense alterations. MEM mutants had nalC, ftsI, and phoP missense alterations. Mutations in merR, nalC, and ampD were observed in the P/T terminal mutants. Among 2 IMI-REL terminal mutants displaying a SNP alteration, 1 displayed a nonsense mutation in pilF, a pilus forming protein. Many terminal mutants displayed alterations in genes not commonly associated to β-lactam resistance. Conclusion MEM, FEP, and P/T terminal mutants displayed high MIC values compared to those obtained after exposure to C/T, CAZ-AVI, and IMI-REL. This data might indicate a benefit of using these newer agents to prevent the emergence of high-level resistance. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support Timothy Doyle, MS, AbbVie: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Jessica Ewald, PhD, AbbVie: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support.

In Vitro Combination of Terbinafine with Ketoconazole Against Aspergillus Species with Terbinafine High MIC Values Isolated From Otomycosis.

Otomycosis is a common mycotic infection of the external auditory canal, and Aspergillus species are one of the most frequent causative agents worldwide. The limited antifungal arsenal, the high toxicity and side effects of antifungal agents, and the growing resistance to the currently available antifungals underscore the need for new therapeutic strategies. The present study aimed to evaluate the combined in vitro efficacy of terbinafine and ketoconazole against Aspergillus species with terbinafine high MIC values isolated from patients with otomycosis.84 Aspergillus species with high MIC values to terbinafine (≥ 4µg/ml), consisting of A. flavus, A. tubingensis, A. niger, and A. terreus, were included in this study. The checkerboard microdilution method evaluated the in vitro interactions using the CLSI reference technique. Synergistic effects were observed for 66.67% (56/84) of all isolates (FICI ranging from 0.19 to 0.5). However, the interactions of terbinafine and ketoconazole exhibited indifference in 33.33% (28/84) of the isolates, and no antagonism was observed for any combination. The interaction of terbinafine and ketoconazole showed synergistic activity against Aspergillus species with high MIC values, suggesting that this is an alternative and promising approach for treating otomycosis.

Computational and data mining studies to understand the distribution and dynamics of Temoneria (TEM) β-lactamase and their interaction with β-lactam and β-lactamase inhibitors

β-lactams are large group of antibiotics widely used to suppress the bacterial growth by inhibiting cell wall synthesis. Bacterial resistance against β-lactam antibiotics is primarily mediated through the production of Temoneria (TEM) β-lactamase (BLs), with almost 474 variants identified in Lactamase Engineering Database (LacED). The present study aims to develop a model to track the evolution of TEM BLs and their interactions with β-lactam and BLs inhibitors through data mining and computational approaches. Further, the model will be used to predict the effective combinations of β-lactam and BLs inhibitors to treat the bacterial infection harbouring emerging variants of β-lactamase. The molecular docking study results demonstrated that most TEM mutants recorded the least binding energy to penicillin and cephalosporin (I/II/III/IV/V generations) class of antibiotics. On the contrary, the same mutants recorded higher binding energy to carbapenem and Monobactam class of antibiotics. Among the BLs inhibitors, tazobactam recorded the least binding energy against most of the TEM mutants, indicating that it can lower the catalytic activity of TEM BLs, thereby potentiating antibiotic action. Similarly, data mining work has assisted us in creating a database of TEM mutants that has comprehensive data on mutations, bacterial diversity, Km, MIC, and IRT types. It has been noted that earlier released antibiotics like amoxicillin and ampicillin had lower Km and higher MIC values, which indicates the prevalence of bacterial resistance. By analysing the differential binding energy (ΔBE) of the selected TEM mutants against β-lactam and BLs inhibitors, the most effective combination of β-lactam (carbapenem and monobactam class of antibiotics) and BLs inhibitors (tazobactam) was identified, to cure bacterial diseases/infections and to prevent similar antibiotic resistance outbreaks. Therefore, our study opens a new avenue in developing strategies to manage antibiotic resistance in bacteria.

Emergence of a floR-carrying plasmid in extended spectrum β-lactamase (ESBL) producing Pasteurella aerogenes, isolated from an avian species in China

Identification and analysis of the antimicrobial resistance of Pasteurella aerogenes (P. aerogenes) isolated from poultry. For susceptibility testing in accordance with the CLSI, plasmids were extracted via alkaline lysis and transferred by CaCl2 treatment. Genomic DNA of a representative P. aerogenes isolate was subjected to whole genome sequencing. CCCP was utilized to determine whether SF190908 contains an efflux pump. The blaVEB gene was ligated with the pET-28 plasmid and transferred to Escherichia coli to verify it as an ESBL gene. SF190908 isolated from poultry was identified as P. aerogenes based upon biochemical and 16s rRNA results. The isolate showed high MIC values for eight antimicrobials. Sequencing results showed that the mobile element-mediated antimicrobial resistance gene cluster conferred antimicrobial resistance on the strain, and a single 5,105-bp plasmid, designated pRCAD0752PA-1, was isolated. Four antimicrobial resistance gene clusters were identified in the SF190908 chromosome; one antimicrobial resistance gene cluster carried the blaVEB gene, which was verified as ESBL according to the CLSI and was detected in Pasteurellaceae for the first time, to the best of our knowledge. The efflux pump may confer antimicrobial resistance to SF190908. P. aerogenes isolated from poultry showed resistance genes encoded in mobile elements that confer multi-antimicrobial resistance to SF190908. The antimicrobial-resistant plasmid pRCAD0752PA-1 was isolated in SF190908 and conferred resistance to florfenicol. This study indicates an urgent need to increase efforts to monitor the spread of P. aerogenes multi-antimicrobial-resistant strains and plasmids, especially in newly discovered at-risk species such as poultry.

S4.3d Mechanism of fluconazole resistance in Candida vulturna, a member of the Candida haemulonii complex of multidrug - resistant yeasts

Emergent species of the Candida genus, September 22, 2022, 10:30 AM - 12:00 PM Objetives Candida vulturna is an emerging yeast firstly isolated in 2016 from flowers and from a blood sample in Malaysia. It is a member of the Candida haemulonii species complex. This complex has 9 species within which C. haemulonii (including the vulnera variety), C. pseudohemulonii, C. duobushaemulonii, C. auris, and now C. vulturna are considered human pathogens. Like the other members of the haemulonii complex, the identification of C. vulturna is difficult. Due to the scarcity of strains to study, the susceptibility pattern of this species is unknown. The objectives of this work were to study C. vulturna strains isolated from clinical samples from different cities of Colombia, to study their susceptibility patterns, and to establish their mechanisms of azole resistance.MethodsFive C. vulturna strains isolated from urine (n = 1) and blood (n = 4) in three different cities in Colombia were studied. Strains were identified as C. duobushaemulonii (n = 4) and C. haemulonii (n = 1) by means of VITEK2 (software version 9.02) and as C. pseudohaemulonii (n = 5) by using Bruker's MALDI-TOF in Colombia. Upon arrival in Argentina, isolates were molecularly identified by sequencing the ITS regions (rDNA). Antifungal susceptibility testing was performed following the CLSI´s M27 4th ed document. Amphotericin B, anidulafungin, caspofungin, 5-flucytosine, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole were tested. In addition, ERG11 genes of the five strains were amplified and sequenced.ResultsThe sequences of the ITS regions were analyzed and the 5 strains were identified as C. vulturna (100% homology). All the strains presented low echinocandins MICs (0.06-0.12 μg/ml), 5-flucytosine (0.06-0.12 μg/ml) and triazoles with the exception of fluconazole (isavuconazole: <0.015 -0.12 μg/ml, itraconazole: 0.06-0.50 μg/ml, posaconazole: <0.015-0.015 μg/ml and voriconazole: 0.03-0.50 μg/ml). In the case of amphotericin B and fluconazole, 4 of the 5 strains presented high MICs for both antifungals (>8 μg/ml and >64 μg/ml), while the remaining one presented MIC values = 1 μg/ml for the polyene and for the azole (cross amphotericin B and fluconazole high MICs). The strain with low fluconazole and amphotericina B MICs was the one isolated from urine. ERG11 sequences analysis showed that the 4 strains with high fluconazole MICs (blood isolates) harbor five amino acid substitutions: S119A, P135S, D180E, K266S, N326K (using C. albicans numbering). Only the P135 residue is highly conserved among ERG11 from different species (Fig. 1, box shows the substitution). In addition, this substitution is present in the Erg11p of fluconazole-resistant Debaryomyces hansenii and the P135 residue is located near the Erg11p´s Heme group where azole drugs bind (Fig. 2: Saccharomyces cereviciae Erg11p complexed with voriconazole 3D structure. In yellow the region between Y131 and P135 residues. In grey voriconazole binding the Heme group).ConclusionThis emerging multi-resistant pathogen may be underdiagnosed by the methods commonly used in clinical laboratories. The only method capable of identifying this species is DNA sequencing. The strains studied presented very high MIC values for amphotericin B and fluconazole. The P135S substitution could be responsible for the high fluconazole MIC values observed in 4 of the 5 strains of C. vulturna.

P024 Genetic determinants of antifungal drug resistance in Fusarium solani species complex

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMDue to its challenging diagnosis and treatment, fungal keratitis is one of the most serious kinds of corneal infection. The Fusarium solani species complex is responsible for nearly half of all fungal keratitis cases. Fusarium infection is difficult to treat because of the increased antifungal resistance of Fusarium species.ObjectiveTo check the antifungal susceptibility in keratitis-causing isolates of F. solani species complex.To find the genetic determinants of resistance using whole genome sequencing.MethodologyPrior to AFST and according to CLSI, clinical isolates of Fusarium species (n = 5) were cultured on potato dextrose agar for 7 days at room temperature. Fungal spores were harvested using 0.8% NaCl and antifungal drug susceptibility testing of 6 different antifungal drugs were tested using CLSI standards broth microdilution method and minimum inhibitory concentrations were obtained. After 24 and 48 h, broth microdilution plates were manually examined. The wells for growth control were also examined. The minimum inhibitory concentration of anti-fungal drugs was the lowest dose that inhibits growth completely (compared to the growth control well). Minimal effective concentration (MEC) was also determined against antifungal drugs. Whole genome sequencing was done using Illumina Hiseqx10. Bioinformatics analysis was done using different bioinformatics tools and software (FASTQC, SPAdes, OmicsBox, AUGUSTUS, etc.).ResultsIn this study the antifungal drug susceptibility from average MIC value were found as fluconazole (512 μg/ml) > itraconazole (32 μg/ml) > amphotericin B (8 μg/ml) > natamycin (8 μg/ml) > posaconazole (2 μg/ml) > voriconazole (1 μg/ml). Fluconazole had a higher MIC value (512 g/ml) in all isolates, however, voriconazole was shown to be more sensitive, with a lower MIC range (0.25-4 g/ml). Glyoxalase/Bleomycin resistance protein, mfs—multidrug resistance transporter, fusaric acid resistance protein, efflux pump antibiotic resistance protein, and copper resistance protein were found by genome-based analysis.ConclusionAs a conclusion of this study, we observed antifungal drug resistance in Fusarium spp., which is often used to treat keratitis in patients, employing fluconazole, itraconazole, and amphotericin B. Resistance to first-line azoles was as a gene variant which contributed to antifungal drug resistance. This study using the phenotypic and genotypic characterization of drug resistance patterns will help to combat antifungal drug resistance.

P279 Invasive Trichosporonosis: an emerging blood stream fungal infection in immunocompromised patients

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Trichosporon is a genus of yeast-like fungi. It is perhaps most widely known as the cause of white piedra, a benign superficial mycosis seen in immunocompetent individuals in tropical and subtropical regions. However, the incidence of invasive trichosporonosis has increased in immunocompromised patients, most notably those with hematologic malignancies. Invasive infections due to Trichosporon species are considered rare so far, but during the past two decades, they have emerged as important opportunistic pathogens in immunocompromised individuals. All patients with blood culture positive for Trichosporon species from January 2020 to August 2020 at Woodlands Multispecialty Hospital, Kolkata, India were evaluated. In vitro susceptibility testing was performed using the reference broth micro-dilution method. Clinical co-relation was done with the positive culture report and patient's clinical condition to rule out colonization/contamination. A total of 7 patients were found to have blood culture positive for Trichosporon species among which five were true infections. Various predisposing factors previously reported to be associated with invasive trichosporonosis were also considered in the present study. All the cases were associated with either post radiotherapy/chemotherapy with renal failure requiring dialysis via hemodialysis catheter or use of central venous catheter or chemoport. Underlying malignancy was found in all patients. Susceptibility testing has been performed for 5-FC, amphotericin B, and Azoles. Azoles had good in vitro activity, whereas amphotericin B had higher MIC values. The all-cause mortality rate was 43%. Invasive Trichosporon infection is a rare, life-threatening infection in immunocompromised patients. Our study highlights the association of central venous catheter and hemodialysis catheter with Trichosporon fungemia and its high mortality. Therefore, strict infection control measures while handling these devices are recommended to prevent these infections. Species identification and susceptibility testing are to be attempted for all relevant clinical isolates in view of demonstrable resistance to certain antifungal drugs. Echinocandins are not effective in the treatment of trichosporon infection. Amphotericin B should also be avoided. Azoles, in particular, voriconazole is the drug of choice.

P051 A case of recalcitrant sporotrichosis by infection of Sporothrix globosa

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesSporotrichosis is the leading subcutaneous mycosis caused by the Sporothrix (S.) schenckii complex. S. globosa is the causative organism of fixed sporotrichosis in Korea. The preferred regimen of cutaneous sporotrichosis is itraconazole for 3-6 months, however, there were few studies for recalcitrant sporotrichosis.MethodsIn 2018, we performed a histological examination of a patient who suffered sporotrichosis for 3 years and cultured part of the specimen. Despite various regimens for years, improvement and exacerbation were repeated, so we took another skin biopsy and cultured it in 2021. Isolates from the 2018 and 2021 lesions were identified as S. globosa by ribosomal DNA ITS sequencing (GenBank accession number: MH499862 and MH499863). The in vitro antifungal sensitivity tests were performed by broth microdilution method according to CLSI M38-A2 guidelines or Sensititre YeastOne® manufacturer's instructions. They were incubated at 30°C in a non-CO2 incubator for 7 days.ResultsIn 2018, histologically, we observed chronic inflammatory granuloma comprising lymphocytes, histiocytes, and giant cells, and several spores with periodic acid-Schiff (PAS) staining. Microscopic findings and ITS sequences of rRNA gene were identical with S. globosa. The antifungal susceptibility profile in 2018 revealed sensitive to terbinafine (0.125 μg/ml), and moderate to high MIC values for amphotericin B (2 μg/ml), itraconazole (>16 μg/ml), voriconazole (>16 μg/ml), and echinocandins (>16 μg/ml). Treatment with terbinafine, itraconazole, or amphotericin B, the skin lesions were partially improved, but were not cured. In 2021, we took another skin biopsy and culture specimen. Histopathological and mycological examination results were the same as before. The antifungal susceptibility profile revealed sensitive to itraconazole (0.5/ml), and high MIC for others. Clinically, skin lesions were not improved with the use of itraconazole 200 mg/d. Itraconazole 400 mg/d with local heating induced moderate improvement. There was no evidence of immune deficiency.ConclusionWe experienced recalcitrant sporotrichosis that did not respond to itraconazole and terbinafine, and the sensitivity of antifungal was changed. In this case, the combination treatment including local heating, saturated KI may be considered, and frequent antifungal susceptibility tests are needed.

P039 Cross-resistance to clinical and agricultural azoles among Aspergillus fumigatus strains isolated from humans and environment in Italy

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesIn Italy, a prevalence of 16.9% of resistance to clinical azoles was observed among Aspergillus fumigatus isolates from an agricultural environment. This spread of azole-resistance is attributed to the widespread use of 14a-demethylase inhibitors (DMIs).The aims of the present study were to investigate: the DMIs resistance in Italian A. fumigatus strains of clinical and environmental origin, both susceptible and resistant to clinical azoles; the molecular mechanism of resistance in strains susceptible to clinical azoles but resistant to at least one of the tested DMIs; the in vitro DMI resistance induced by prolonged exposure to DMIs in susceptible clinical and environmental strains, and the molecular mechanism of resistance.MethodsA total of 54 A. fumigatus strains were selected: 23 susceptible to clinical azoles (CAS) and 31 resistant (CAR) with and without mutations in the CYP51A gene (TR34/L98H, F219I, G54R, G54E, D269Y, M220I, or F46Y/M172V/N248T/D255E/E427K). Antifungal susceptibility testing was performed for 8 DMIs (tebuconazole, epoxiconazole, difenoconazole, propiconazole, tetraconazole, flusilazole, fenbuconazole, and prochloraz) using broth microdilution method according to EUCAST and CLSI methods. Mutations in CYP51A, CYP51B, and HMG1 genes were investigated in CAS with DMI high MIC values. In vitro induction of resistance was performed using the 8 DMIs on 11 (6 clinical and 5 environmental) A. fumigatus strains susceptible both to clinical azoles and DMIs. A suspension of 106 conidia was inoculated on glucose-yeast extract-peptone agar plates containing different DMIs at different concentrations and incubated at 37°C for 72 h for six repeated passages.ResultsComparable results were obtained using EUCAST and CLSI methods.Resistance (MIC ≥16) to tetraconazole and fenbuconazole was observed in 100% of isolates, both CAR and CAS. On the contrary, a statistically significant difference in tebuconazole, epoxiconazole, difenoconazole, propiconazole, and flusilazole MICs between CAR strains and CAS strains was observed with higher geometric means (GM) in CAR (range 4.9-9.3 mg/L) than in CAS (1.5-2.7 mg/L) strains. Prochloraz showed the lowest GMs: 0.6 and 0.25 mg/L in CAR and CAS strains, respectively.A significant difference of the GMs for all the DMIs tested, except prochloraz, was observed between the isolates harboring a TR34/L98H or a M220I mutation (GM range 10.4-16 mg/L) and those with other CYP51A mutations (GM range 1-4.6 mg/L).In the CAS showing high DMI MICs, the absence of CYP51A mutations was confirmed, while a synonymous mutation P394P, was identified in CYP51B. No mutations in HMG1 gene were found.In the induction tests, the prolonged exposure to DMIs showed an induced phenotypic resistance of 100% (11/11 isolates) for epoxiconazole, of 72.7% (8/11) for propiconazole, of 54% (6/11) for tebuconazole and difenoconazole, and of 9.1% (1/11) for prochloraz.Molecular analysis to understand if the phenotypic resistance corresponds to induced mutations in CYP51A, CYP51B, and HMG1 genes is in progress.ConclusionsPreliminary results confirm cross-resistance between clinical azoles and DMIs, with MIC differences between CAR and CAS and between strains with different mutations in the CYP51A gene. Furthermore, the ability of DMIs to induce resistance in vitro was highlighted.

Microbial contamination, antimicrobial resistance and biofilm formation of bacteria isolated from a high-throughput pig abattoir.

The aim of this work was to assess the level of microbial contamination and resistance of bacteria isolated from a highthroughput heavy pig slaughterhouse (approx. 4600 pigs/day) towards antimicrobials considered as critical for human, veterinary or both chemotherapies. Samples, pre-operative and operative, were obtained in 4 different surveys. These comprised environmental sampling, i.e. air (ntotal = 192) and surfaces (ntotal = 32), in four different locations. Moreover, a total of 40 carcasses were sampled in two different moments of slaughtering following Reg. (CE) 2073/2005. Overall, 60 different colonies were randomly selected from VRBGA plates belonging to 20 species, 15 genera and 10 families being Enterobacteriaceae, Moraxellaceae and Pseudomonadaceae the most represented ones. Thirty-seven isolates presented resistance to at least one molecule and seventeen were classified as multi-drug resistant. Enterobacteriaceae, particularly E. coli, displayed high MIC values towards trimethoprim, ampicillin, tetracycline and sulphametoxazole with MICmax of 16, 32, 32 and 512 mg/L, respectively. Moreover, isolated Pseudomonas spp. showed high MIC values in critical antibiotics such as ampicillin and azithromycin with MICmax of 32 and 64 mg/L, respectively. Additionally, in vitro biofilm formation assays demonstrated that fifteen of these isolates can be classified as strong biofilm formers. Results demonstrated that a high diversity of bacteria containing antibiotic resistant and multiresistant species is present in the sampled abattoir. Considering these findings, it could be hypothesised that the processing environment could be a potential diffusion determinant of antibiotic resistant bacteria through the food chain and operators.

#13 Dalbavancin Activity against Bacterial Isolates from Pediatric Patients: Results from the International Dalbavancin Evaluation of Activity (IDEA) Program for the United States and Europe (2019-2021)

Abstract Background Dalbavancin is a lipoglycopeptide approved in the United States (US; 2014) and Europe (2015) to treat skin and skin structure infections in adults with a convenient parenteral administration of either a single dose of 1500 mg or a dose of 1000 mg followed by 500 mg a week later. We evaluated dalbavancin in vitro spectrum and potency against Gram-positive isolates from pediatric patients. Method 4,487 consecutive, unique pediatric patient isolates of clinical significance were collected from 32 US and 34 European medical centers and tested for susceptibility to dalbavancin and comparators by broth microdilution. The most common organisms were Staphylococcus aureus (n=2,633), Streptococcus pneumoniae (n=689), and beta-hemolytic streptococci (BHS; n=584). Susceptibility results were analyzed by patient age group: ≤1 years old (yo; n=1,459), 2-5 (1,106), 6-12 (1,164), and 13-17 (758). Results Dalbavancin showed complete activity against S. aureus (100% susceptibility), with a highest MIC value of 0.12 mg/L and consistent activity against all age groups (MIC50/90 of 0.03/0.03 mg/L for the US and Europe as well as the MSSA and MRSA subsets). MRSA rates were higher in the US (32.4% overall) than Europe (14.0%). In the US, MRSA rates were 38.7%, 34.2%, 28.4%, and 25.0% in the ≤1, 2-5, 6-12, and 13-17 yo groups, respectively; whereas, in Europe, it was slightly lower in the 2-5 yo group (11.5%) compared to the other age groups (14.1-14.8%). Ceftaroline was consistently active against S. aureus isolates from all age groups, with susceptibility rates ranging from 99.4% (13-17 yo) to 99.8% (2-5 yo) in the US and from 95.3% (≤1 yo) to 100.0% (2-5 yo) in Europe. Dalbavancin showed consistent activity against S. pneumoniae from all age groups (MIC50/90 of 0.008/0.015 mg/L for all age groups in the US and Europe) with a highest MIC value of 0.06 mg/L (1 isolate). All S. pneumoniae isolates were susceptible to ceftaroline, and susceptibility to ceftriaxone (at ≤1 mg/L; non-meningitis breakpoint) ranged from 92.7% (13-17 yo) to 100.0% (≤1 yo), or 97.8% overall. Dalbavancin was also highly active against BHS (n=584; MIC50/90, 0.008/0.015 mg/L; highest MIC, 0.06 mg/L), coagulase-negative staphylococci (n=240; MIC50/90, 0.03/0.06 mg/L; highest MIC, 0.25 mg/L), Enterococcus faecalis (n=200; MIC50/90, 0.03/0.06 mg/L; highest MIC, 0.06 mg/L), viridans group streptococci (n=102; MIC50/90, 0.008/0.03 mg/L; highest MIC, 0.12 mg/L), and vancomycin-susceptible E. faecium (n=26; MIC50/90, 0.03/0.12 mg/L; highest MIC, 0.25 mg/L), but showed limited activity against vancomycin-resistant E. faecium (VREFM; n=13 [0.3% of the organism collection]; MIC50/90, &amp;gt;2/&amp;gt;2 mg/L). Conclusion Dalbavancin showed potent in vitro activity against a large collection of Gram-positive isolates from pediatric patients, independent of patient age and geographic region (US and Europe). Only 13 of 4,487 (0.3%) isolates exhibited a dalbavancin MIC &amp;gt;0.25 mg/L, all of which were VREFM. These in vitro results support further clinical development of dalbavancin for treatment of Gram-positive infections in children.

Genetic Structure and Triazole Antifungal Susceptibilities of Alternaria alternata from Greenhouses in Kunming, China.

ABSTRACTAlternaria alternata is an opportunistic human fungal pathogen and a ubiquitous phytopathogen capable of causing diseases to >100 agricultural crops and ornamental plants. To control plant diseases caused by A. alternata, triazole fungicides have been widely used both in open crop and vegetable fields and in indoor growth facilities such as greenhouses. At present, the effect of fungicide use on triazole resistance development in A. alternata populations is not known. Here, we isolated 237 A. alternata strains from nine greenhouses around metropolitan Kunming in Yunnan, southwest China, determined their genotypes using 10 short tandem repeat markers, and quantified their susceptibility to four triazoles (difenoconazole, tebuconazole, itraconazole, and voriconazole). Abundant allelic and genotypic diversities were detected among these A. alternata strains. Significantly, over 17% of the strains were resistant to difenoconazole, and both known and new drug-resistance mutations were found in the triazole target gene cyp51. Our findings of high-level genetic variation of A. alternata in greenhouses coupled with high-frequency fungicide resistance call for greater attention to continued monitoring and to developing alternative plant fungal disease management strategies in greenhouses.IMPORTANCE Alternaria alternata is among the most common fungi in our environments, such as indoor facilities, the soil, and outdoor air. It can cause diseases in >100 crop and ornamental plants. Furthermore, it can cause human infections. However, our understanding of its genetic diversity and antifungal susceptibility is very limited. Indeed, the critical threshold values for resistance have not been defined for most antifungal drugs in this species. Greenhouses are known to have heavy applications of agricultural fungicides. In this study, we analyzed strains of A. alternata from nine greenhouses near metropolitan Kunming in southwestern China. Our study revealed very high genetic diversity and identified strains with high MIC values against two agricultural and two medical triazole antifungals within each of the nine greenhouses. Our study calls for greater attention to this emerging threat to food security and human health.

Effect of Voriconazole on Biofilm of Filamentous Species Isolated from Keratitis

Background: Biofilm formation is a vital feature of the pathogenesis in filamentous fungi. Objectives: Herein, we investigated in vitro antifungal pattern of biofilm of filamentous species keratitis isolates, and the effect of different concentrations of voriconazole on biofilm morphology changes using scanning electron microscopy. Methods: Here 25 ocular fungal isolates were included (Fusarium solani; (n = 15) and Aspergillus flavus; (n = 10). We determined the biofilm formation of isolates in terms of their susceptibilities to different antifungals on sessile cells. This procedure was performed according to CLSI-M38-3rd. Biofilm morphology changes revealed with scanning electron microscopy (SEM). Results: Twelve F. solani strains displayed the capacity to form biofilms from patients wearing contact lenses, while 8 A. flavus strains were recovered from cornea scrapings of trauma. Itraconazole, posaconazole and natamycin had the maximum activity against biofilms of all tested filamentous strains (MIC ranging from 0.031 - 0.25 µg/mL, 0.031 - 0.25 µg/mL and 2 - 4 µg/mL), respectively. Three F. solani and one A. flavus strains showed the high MIC values against voriconazole (MIC ≥ 1 µg/mL). Posaconazole revealed the lowest MIC values against biofilms of two strains (MIC ranging from 0.031 - 0.25 µg/mL); however, no significant difference was observed for itraconazole (P &gt; 0.05). The efficacy of 16 µg/mL and 4 µg/mL of voriconazole was confirmed for biofilm of F. solani and A. flavus, respectively. The considerable changes in the morphologies of improved biofilms were seen by SEM. Conclusions: For the successful treatment of fungal biofilm infections, it was necessary to have knowledge of the mechanism of action, penetration rates, and therapeutic concentrations of drugs.

Identification of a 3-(5-methyl-2-thiazolylamino)phthalide as a new minor groove agent

A new 3-(5-methyl-2-thiazolylamino)phthalide molecule, 3-((5-methylthiazol-2-yl)amino)isobenzofuran-1(3H)-one, was synthesized and characterized experimentally by FT-IR, NMR, UV-Vis, and single-crystal X-ray analysis and theoretically by quantum chemical calculations. The single-crystal X-ray studies revealed that the compound crystallizes in the monoclinic space group P-21/c with unit-cell parameters a = 8.0550(6) Å, b = 6.1386(3) Å, c = 23.3228(18) Å, β = 97.724(6)° and Z = 4. Optimized geometries and the vibrational frequencies were studied at the density functional theory (DFT) level by using the hybrid functional B3LYP with a 6-311 G (d,p) basis set. The title compound was evaluated for its anti-quorum sensing (anti-QS) activity on Chromobacterium violaceum 12472 and additionally for its antibacterial activity against Staphylococcus aureus 29213, Staphylococcus epidermidis 12228, Pseudomonas aeruginosa 27853, Escherichia coli 25922, and Proteus mirabilis 14153. The lowest MIC value was 0.24 μg/mL for S. aureus 29213 and the highest MIC value was 30.75 μg/mL for E. coli 25922. While anti-bacterial activity was observed in those other than the S. epidermidis and P. Mirabilis, anti-QS activity wasn’t detected. Investigations on dsDNA binding affinity indicate that the title compound binds to dsDNA via the groove binding mode. Molecular docking calculations and molecular dynamics simulations results showed also that the title compound prefers binding to the minor groove of dsDNA and remains stable in the minor groove throughout the molecular dynamics simulation. Communicated by Ramaswamy H. Sarma

A Facile Synthesis of New Substituted Thiazol-2-amine Derivatives as Potent Antimicrobial Agent

A facile synthesis of a new thiazol-2-amine derivative as antimicrobial agent and condensation with substituted thiourea compounds to afford the corresponding substituted thiazole derivatives in excellent yields. All the reactions were completed within less reaction time at reflux temperature. According to the result obtained, the compounds 3b, 3d, 3e, 3f, 3g, 3i, 3l, and 3m were found the leader antimicrobial activity with the highest MIC values. The synthesized compound can be considered to develop new antimicrobial drug candidates. Amongst these, many compounds show better antibacterial and antifungal activity. The compounds 3b, 3d, 3e, 3l, and 3m exhibited significant antibacterial activity against B. subtilis, S. aureus, and E. coli. The compounds 3f, 3g, 3i show significant antifungal against fungal strains i.e., C. albicans, A. flavus and A. niger.