High MIC Values Research Articles

Combining Fluconazole with Benzo[a]phenoxazine Derivatives as a Promising Strategy Against Fluconazole-Resistant Candida Species.

The rise in non-albicans Candida species, exhibiting unpredictable antifungal resistance, complicates treatment and contributes to the growing threat of invasive, life-threatening infections. This study evaluates the antifungal activity of four benzo[a]phenoxazine derivatives (C34, C35, A42, and A44) against 14 Candida strains following EUCAST standards. Fluconazole interactions are analysed through fractional inhibitory concentration index (FICI) calculation and response surface analysis based on the Bliss model. Macrophage-like J774A.1 cells are used to assess Candida killing in the presence of synergistic compounds. The MIC values against the different strains vary, with C34 showing the strongest activity, followed by C35, while A42 has the highest MIC values, indicating lower efficacy. However, A42 demonstrates the best synergy with fluconazole against fluconazole-resistant Candida strains. Cytotoxicity assays reveal that the chloropropyl group present in C35 and A42 enhances cytocompatibility. Co-culture with macrophages shows significant yeast killing for C. albicans and C. auris when fluconazole and A42 are combined, requiring concentrations 4 and 16 times lower than their MIC values, enhancing antifungal activity. Given fluconazole's fungistatic nature and the emergence of drug-resistant strains, benzo[a]phenoxazine derivatives' ability to enhance fluconazole's efficacy present a promising strategy to address antifungal resistance in critical pathogens. These findings align with global research priorities, offering new potential avenues for developing more effective antifungal therapies.

Genomic epidemiology and resistant genes of Acinetobacter baumannii clinical strains in Vietnamese hospitals.

Introduction. Acinetobacter baumannii is a common cause of multidrug-resistant (MDR) nosocomial infections worldwide, including Vietnam.Hypothesis. Analysis of crucial genetic factors may link to epidemiological characteristics and antibiotic resistance of A. baumannii clinical strains in Vietnamese hospitals.Methodology. Fifty-one A. baumannii clinical strains from six different tertiary hospitals in Vietnam were analysed using whole genome sequencing (WGS), between 2017 and 2019.Results. Eleven sequence types (STs) were identified, including four STs reported for the first time in Vietnam based on the PubMLST database and three new STs not previously documented. ST1336, ST1260 and ST575 were found exclusively in Vietnam. These STs were widely distributed in all hospitals in Vietnam, with ST2 and ST571 being the most dominant. Resistant rates to eight antibiotics, belonging to four antibiotic groups, were very high (72.5-94.1 %) with high MIC values, while resistance to colistin was 29.4%. Fifty-one isolates were identified as MDR, with 100% (51/51) isolates carrying antimicrobial-resistant (AMR) genes, and 52 antibiotic-resistant genes were detected among these strains, including β-lactam (22 genes), chloramphenicol (5 genes), lincosamide (2 genes), aminoglycoside (11 genes), rifampicin (1 gene), quinolone (2 genes), sulfonamide and trimethoprim (4 genes) and tetracycline (5 genes) resistance. The most commonly found mobile structures carried partial or complete transposons: ISaba24/ISEc29/ISEc35 contains a series of antibiotic-resistant genes.Conclusion. The WGS results of the 51 strains of A. baumannii provided important information regarding the distribution of STs and associated antibiotic-resistant genes among A. baumannii strains.

In-silico and in-vitro study of novel antimicrobial peptide AM1 from Aegle marmelos against drug-resistant Staphylococcus aureus

Antimicrobial peptides have garnered increasing attention as potential alternatives due to their broad-spectrum antimicrobial activity and low propensity for developing resistance. This is for the first time; proteome sequences of Aegle marmelos were subjected to in-silico digestion and AMP prediction were performed using DBAASP server. After screening the peptides on the basis of different physiochemical property, peptide sequence GKEAATKAIKEWGQPKSKITH (AM1) shows the maximum binding affinity with − 10.2 Kcal/mol in comparison with the standard drug (Trimethoprim) with − 7.4 kcal/mol and − 6.8 Kcal/mol for DHFR and SaTrmK enzyme respectively. Molecular dynamics simulation performed for 300ns, it has been found that peptide was able to stabilize the protein more effectively, analysed by RMSD, RMSF, and other statistical analysis. Free binding energy for DHFR and SaTrmK interaction from MMPBSA analysis with peptide was found to be -47.69 and − 44.32 Kcal/mol and for Trimethoprim to be -13.85 Kcal/mol and − 11.67 Kcal/mol respectively. Further in-vitro study was performed against Methicillin Susceptible Staphylococcus aureus (MSSA), Methicillin Resistant Staphylococcus aureus (MRSA), Multi-Drug Resistant Staphylococcus aureus (MDR-SA) strain, where MIC values found to be 2, 4, and 8.5 µg/ml lesser in comparison to trimethoprim which has higher MIC values 2.5, 5, and 9.5 µg/ml respectively. Thus, our study provides the insight for the further in-vivo study of the peptides against multi-drug resistant S. aureus.

Understanding chromoblastomycosis in Sri Lanka; insights into fungal aetiology and antifungal susceptibility patterns

Introduction: Chromoblastomycosis is a chronic disfiguring fungal infection of the subcutaneous tissue found mainly in the socio-economically marginalized population in the tropical and sub-tropical regions, characterized by the presence of sclerotic cells. This study was done with the aim of identifying the antifungal sensitivity patterns of the causative agents of chromoblastomycosis in Sri Lanka from 1992 to 2019.Methodology: Study was done on the stored isolates received from patients with chromoblastomycosis since 1992 to 2019, to the Mycology Reference Laboratory of Medical Research Institute, Colombo. Isolates were revived by subculture on Potato dextrose agar. Identification was done phenotypically. Antifungal sensitivity testing was performed by E test method. Available demographic and clinical data were analysed.Results: Of the 74 isolates, 71 (96%) were caused by Fonsecaea pedrosoi. Mean MIC for the antifungals tested were lowest for posaconazole 0.015 μg/ml followed by itraconazole 0.225 μg/ml, and voriconazole 0.768 μg/ml while amphotericin B demonstrated complete resistance with mean MIC of 30.591 μg/ml. No statistically significant differences were noted on the antifungal sensitivity of the isolates from 1992 to 2004 and 2005 to 2019.Conclusions: Fonsecaea pedrosoi was the commonest causative agent causing chromoblastomycosis in the studied population. Posaconazole demonstrated the lowest MIC values for the tested isolates followed by itraconazole and voriconazole. Amphotericin B demonstrated very high MIC values which were likely to result in clinical failure upon treatment. MIC values demonstrated minimal fluctuations throughout the tested time.

505 Fusarium Isolates in Burn-injured Patients: Clinical Characteristics and Susceptibility Patterns

Abstract Introduction Fusarium species are ubiquitous in the environment and can cause opportunistic infections in burn-injured patients. Patients with larger burns, prolonged lengths of stay, concomitant antimicrobial use and invasive lines are at higher risk for Fusarium infections. Little data exists on clinical characteristics and in vivo antifungal susceptibilities in burn-injured patients. Methods All positive Fusarium species cultures were retrospectively identified between November 2017 to June 2023 at a regional burn center. Demographic, clinical and microbiologic susceptibility data was collected from the electronic medication record. Descriptive statistics were used to describe the cohort. Results A total of 18 patients had positive Fusarium wound cultures during the study period. The average age was 38.4 ± 11.9 years. The average %TBSA was 54.5 ± 23.4 % and R-Baux score of 92.9 ± 29.3. The most common mechanism of injury was thermal burn (17 patients, 94%). This cohort experienced prolonged ICU (68.9 ± 31.9 days) and hospital (73.6 ± 38.2 days) lengths of stay. The average time from burn injury to positive Fusarium cultures was 19.6 ± 9.0 days. Mechanical ventilation was prolonged (46.1 ± 23.3 days). The average central line duration prior to Fusarium infection was 9.8 ± 10.2 days. Susceptibility data was obtained for voriconazole in 15 patients and for amphotericin in 14 patients. For voriconazole, 5/15 tested isolates had an MIC of 4, 1/15 had an MIC of 8, and 9/15 (60%) isolates returned with MIC ≥ 16. A majority (13/14, 93%) of the MICs tested for amphotericin were ≤ 1. Patients were taken to the OR for source control in all cases. Ten of 18 patients with Fusarium positive infections survived to hospital discharge (55%). The most common cause of death was multi-system organ failure and sepsis (88%). Recorded adverse drug events possibly due to voriconazole and posaconazole included transaminitis (4 cases), QTc prolongation (1 case), and hallucinations (1 case). Amphotericin was suspected of contributing to an acute kidney injury in 1 case. Conclusions This case series reports the clinical characteristics and susceptibility data on Fusarium isolates in a burn-injured population. This cohort was critically ill based on the %TBSA, R-Baux score, and high rate of mechanical ventilation. The MIC results obtained for voriconazole demonstrated a majority of isolates with relatively high MIC values ≥ 16. A majority of the MICs for amphotericin were < 1. It is important to note that no established breakpoints exist for Fusarium species and clinical outcomes are dependent on a variety of factors beyond the susceptibility data. Applicability of Research to Practice Empiric double coverage with voriconazole and amphotericin or amphotericin monotherapy should be considered when Fusarium is a suspected primary pathogen.

The yeast genus Tardiomyces gen. nov. with one new species and two new combinations

PurposeRare yeasts species are increasingly reported as causative agents of invasive human infection. Proper identification and antifungal therapy are essential to manage these infections. Candida blankii is one of these emerging pathogens and is known for its reduced susceptibility to multiple antifungals.MethodsTo obtain more insight into the characteristics of this species, 26 isolates reported as C. blankii were investigated using genetic and phenotypical approaches.ResultsAmong the 26 isolates, seven recovered either from blood, sputum, urine, or the oral cavity, displayed substantial genetic and some phenotypical differences compared to the other isolates, which were confirmed as C. blankii. We consider these seven strains to represent a novel species, Tardiomyces depauwii. Phylogenomics assigned C. blankii, C. digboiensis, and the novel species in a distinct branch within the order Dipodascales, for which the novel genus Tardiomyces is erected. The new combinations Tardiomyces blankii and Tardiomyces digboiensis are introduced. Differences with related, strictly environmental genera Sugiyamaella, Crinitomyces, and Diddensiella are enumerated. All three Tardiomyces species share the rare ability to grow up to 42 °C, display slower growth in nutrient-poor media, and show a reduced susceptibility to azoles and echinocandins. Characteristics of T. depauwii include high MIC values with voriconazole and a unique protein pattern.ConclusionWe propose the novel yeast species Tardiomyces depauwii and the transfer of C. blankii and C. digboiensis to the novel Tardiomyces genus.

Quantitative Phytochemical Analysis and Antimicrobial Activity of Ricinus communis Linn. Seed oil

Ricinus communis (R. communis) is a plant in the spurge family that has been traditionally used to treat numerous ailments. The study aims to evaluate the phytochemicals present in R. communis seed oil and its antimicrobial efficacy. The phytochemicals were quantified using standard procedures, and antimicrobial activity was carried out using broth dilution method at -100, 50, 25, and 12.5 mg/ml of the seed oil against Staphylococcus aureus, Escherichia coli, Salmonella sp., Aspergillus niger, and Aspergillus flavus. Alkaloids, flavonoids, phenols, saponins, and tannins were detected with varying concentrations. The highest MIC value, 100 mg/ml, was recorded against fungal isolates, while the lowest, 12.5 mg/ml, was recorded against S. aureus. Similarly, the highest MLC was recorded against the fungal isolates, and the lowest value of 50 mg/ml was recorded against all the bacterial species. The seed oil contained an appreciable amount of phytochemicals and exhibited antimicrobial activity against the tested isolates.

Impact of the phenotypic expression of temocillin resistance in Escherichia coli on temocillin efficacy in a murine peritonitis model.

Temocillin is a narrow spectrum β-lactam active against MDR Enterobacterales. Mechanisms of acquired resistance to temocillin are poorly understood. We analysed resistance mechanisms in clinical isolates of Escherichia coli and evaluated their impact on temocillin efficacy in vitro and in a murine peritonitis model. Two sets of isogenic clinical E. coli strains were studied: a susceptible isolate (MLTEM16S) and its resistant derivative, MLTEM16R (mutation in nmpC porin gene); and temocillin-resistant derivatives of E. coli CFT073: CFT-ΔnmpC (nmpC deletion), CFTbaeS-TP and CFTbaeS-AP (two different mutations in the baeS efflux-pump gene).Fitness cost, time-kill curves and phenotypic expression of resistance were determined. Temocillin efficacy was assessed in a murine peritonitis model. MICs of temocillin were 16 and 64 mg/L for MLTEM16S and MLTEM16R, respectively, and 8, 128, 256 and 256 mg/L for E. coli-CFT073, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP, respectively. No fitness cost of resistance was evidenced. All resistant strains showed heteroresistant profiles, except for CFTbaeS-AP, which displayed a homogeneous pattern. In vitro, temocillin was bactericidal against MLTEM16R, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP at 128, 256, 512 and 512 mg/L, respectively. In vivo, temocillin was as effective as cefotaxime against MLTEM16R, CFT-ΔnmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality). Heteroresistant NmpC porin alteration and active efflux modification do not influence temocillin efficacy despite high MIC values, unfavourable pharmacokinetic/pharmacodynamic conditions and the absence of fitness cost, whereas homogeneously expressed BaeS efflux pump alteration yielding similar MICs leads to temocillin inefficacy. MIC as sole predictor of temocillin efficacy should be used with caution.

Antifungal Susceptibility of Saccharomyces cerevisiae Isolated from Clinical Specimens.

(1) Background: Despite being considered a non-pathogenic yeast, recently, a growing occurrence of Saccharomyces cerevisiae infections has been noted. There is little knowledge about the drug susceptibility of this species. Therefore, the objective of this research was to expand it and determine the drug susceptibility profile of a local collection of clinical isolates of this species. (2) Methods: This study contained 55 clinical isolates identified as Saccharomyces cerevisiae using the MALDI-TOF method. The susceptibility of Saccharomyces cerevisiae was tested to 10 antifungals (amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, micafungin, anidulafungin, caspofungin, and itraconazole) using MICRONAUT-AT tests and manogepix, a new drug, using the microdilution method according to EUCAST. (3) Results: Overall, most strains were classified as sensitive to amphotericin B and flucytosine (MIC ranges of ≤0.03-1 and ≤0.06-0.125, respectively) and also to echinocandins. However, five isolates expressed high MIC values for all of the tested azoles, indicating cross-resistance. The MIC range for manogepix was 0.001-0.125 mg/L, with an MIC50 of 0.03 mg/L and an MIC90 of 0.06 mg/L. (4) Conclusions: The occurrence of resistance to azoles may be a concerning problem and therefore should be investigated further. However, the new antifungal manogepix appears to be an interesting new therapeutic option for treating such infections.

Evaluating the Antimicrobial Efficacy of Apple Cider Vinegar on Bacteria and Fungi Isolated from Vaginitis

Vaginitis is a prevalent medical illness that affects a substantial proportion of women, mostly attributed to the excessive proliferation of bacteria and fungi. The objective of this study was to assess the antimicrobial effectiveness of Apple Cider Vinegar against bacteria and fungi that were obtained from cases of vaginitis. 50 vaginal swabs were collected from vaginitis patients at private clinics in Kirkuk City, Iraq (10 pregnant women and 40 non-pregnant women). The research included the isolation and identification of bacterial and fungal isolates. All isolates performed the Minimum Inhibitory Concentration and disc diffusion techniques tests. Different concentrations of apple cider vinegar (20%, 30%, 40%, 50% and 100%) were tested against the isolates and Standard antibiotic drugs were used as positive controls. The results of 50 patients were examined, showing that 22 (44%) had bacterial vaginitis and 39 (78%) had fungal vaginitis. Vaginitis was more prevalent in pregnant women compared with non-pregnant. Staphylococcus aureus recorded 14 (24.9%) out of the 54 bacterial isolates, followed by Echerichia coli, Group B- Streptococcus, Klebsiella pneumoniae, Staphylococcus epidermidis and Pseudomonas aeruginosa respectively. whereas Candida albicans recorded 30 (61.2%) out of the 49 fungal isolates, followed by Candida tropicalis and Candida parapsilosis, respectively. The findings showed a range of sensitivity to apple cider vinegar among the isolated samples. The efficacy of apple cider vinegar was found to be greater against fungal isolates compared to bacterial isolates, with a higher effectiveness observed against gram-negative bacteria in comparison to gram-positive bacteria. The highest MIC values were found for Gram-positive bacteria, which recorded 125 (µg/mL), followed by Gram-negative bacteria, 62.5 μg/ml. While the lowest MIC values for the isolated fungi were (31.25 μg/ml). A comparison to traditional antimicrobial drugs has highlighted ACV's potential as an alternative treatment for vaginitis. According to these findings, ACV may be an effective treatment choice for vaginitis.

Activity of Bangle Rhizome Extract (zingiber cassumunar roxb.) Inhibits the Growth of Trichophyton rubrum and Trichophyton mentagrophytes

Bangle rhizome extract (zingiber purpureum roxb.) is a herbal plant that is widely consumed in Indonesia. Bangle rhizome extract (zingiber purpureum roxb.) has broad medical properties such as diuretic, anti-hypertension, brochodilator, immunomodulator, analgesic, anti-inflammatory, anti-oxidant, anti-tumor, anti-diabetic, anti-microbial and anti-fungal. The aim of this research was to determine the yield results of extracts using the solvents Aquadest, Aceton, Ethanol 70%, Ethanol 96% and Methanol. And to find out the results of phytochemical screening tests and active ingredient content using GCMS. The stages of this research include the extraction process, phytochemical screening test and GCMS. The results of research on the yield of Bangle rhizome extract with the five solvents obtained the largest yield, namely with distilled water, 7.2 percent. Based on the phytochemical screening test, Bangle rhizome extract was positive for containing secondary metabolites such as tannins, alkaloids, triterpenoids, steroids and saponins. The lowest MIC results for Bangle extract were in Aquadest solvent at 6.25 mg/mL with a value of 0.049 mg/mL and continued in 70% Ethanol solvent with a value of 3.125 mg/mL in the fungus Trichophyton rubrum and in the fungus Trichophyton mentagrophytes with an MIC value of 3.125 mg/mL. while the highest MIC value was obtained in the solvent acetone, Ethanol 96%, Methanol with a value of 0.049 ml/ML for both fungi.

Fungal diversity and drug susceptibility of the oral mycobiome of domestic dogs.

The purpose of this study was to characterize the variety and diversity of the oral mycobiome of domestic dogs and to identify the commensal and potentially pathogenic fungi present. Two hundred fifty-one buccal swabs from domestic dogs were obtained and struck onto a chromogenic fungal growth medium that distinguishes between fungal species based on colony color and morphology. After isolating and harvesting single colonies, genomic DNA was extracted from pure cultures. PCR was used to amplify a fungal-specific variable rDNA region of the genome, which was then sent for sequencing. Sequencing results were input into the NCBI BLAST database to identify individual components of the oral mycobiome of tested dogs. Of the 251 dogs swabbed, 73 had cultivable fungi present and 10 dogs had multiple fungal species isolated. Although the dogs did not show signs of oral infections at the time, we did find fungal species that cause pathogenicity in animals and humans. Among fungal isolates, Malassezia pachydermatis and species from the genus Candida were predominant. Following fungal isolate identification, antifungal drug susceptibility tests were performed on each isolate toward the medically important antifungal drugs including fluconazole, ketoconazole, and terbinafine. Drug susceptibility test results indicated that a large number of isolates had high MIC values for all three drugs. Exploring the oral mycobiome of dogs, as well as the corresponding drug susceptibility profiles, can have important implications for canine dental hygiene, health, and medical treatment. Identifying the microorganisms within the canine mouth can illustrate a common pathway for fungal pathogens of One Health concern to spread from our canine companions to humans.

Molecular distribution of biocide resistance genes and susceptibility to biocides among vancomycin resistant Staphylococcus aureus (VRSA) isolates from intensive care unit (ICU) of cardiac hospital- A first report from Pakistan

BackgroundThe study was conducted with the aim to investigate the VRSA isolates in terms of their susceptibility to routinely used biocides influenced by the co-occurrence of biocide resistant gene (BRGs) and efflux pumps genes. MethodologyFrequently touched surfaces within intensive care unit (ICU) of cardiac hospital were classified into three primary sites i.e., structure, machines and miscellaneous. Over a period of six months (January 2021 to July 2021) twenty three swabs samples were collected from these sites. Subsequently, these samples underwent both phenotypic and molecular methods for VRSA isolation and identification. Susceptibility and efficacy testing of biocides (benzalkonium chloride (BAC), cetrimide (CET) and chlorhexidine gluconate (CHG)) were evaluated using microdilution broth and suspension method. Furthermore, specific primers were used for singleplex PCR targeting BRGs (cepA, qacA, and qacE) and efflux pump (norA, norB, norC, sepA, mepA and mdeA) associated genes. ResultsWe found that 72.2 % S. aureus demonstrate the presence of vanA or vanB genes with no significant difference among three sites (p > 0.05). cepA is the most dominant BRGs followed by qacA and qacE from structure site as compared to other sites (p < 0.05). BAC showed reduced biocide susceptibility and MIC50. There was no significant difference between presence or absence of BRGs and high MIC values of VRSA isolates from all three sites. However, efflux pump genes (EFPGs) particularly norA and norA + sepA had a significant association with BRGs and reduced biocide. ConclusionBAC is the most effective disinfectant against VRSA. Proper and controlled use of BAC is required to overcome the VRSA contamination. We recommend continuous monitoring of the BRGs prevalence for better prevention of microorganism dissemination and infection control in hospitals.

Surveillance of Amphotericin B and Azole Resistance in Aspergillus Isolated from Patients in a Tertiary Teaching Hospital.

The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives.

Evaluation of in vitro pharmacodynamic drug interactions of ceftazidime/avibactam and fosfomycin in Escherichia coli.

Combination therapy can increase efficacy of antibiotics and prevent emergence of resistance. Ceftazidime/avibactam and fosfomycin may be empirically combined for this purpose, but a systematic and quantitative evaluation of this combination is needed. In this study, a systematic analysis of the pharmacodynamic interactions of ceftazidime/avibactam and fosfomycin in clinical and isogenic Escherichia coli strains carrying genes coding for several carbapenemases or ESBLs was performed and pharmacodynamic interactions were quantified by modelling and simulations. Pharmacodynamic interactions were evaluated in 'dynamic' chequerboard experiments with quantification of viable bacteria in eight isogenic and six clinical E. coli strains. Additionally, supplemental time-kill experiments were performed and genomic analyses were conducted on representative fosfomycin-resistant subpopulations. Models were fitted to all data using R and NONMEM®. Synergistic drug interactions were identified for 67% of the clinical and 75% of the isogenic isolates with a mean EC50 reduction of >50%. Time-kill experiments confirmed the interactions and modelling quantified EC50 reductions up to 97% in combination and synergy prevented regrowth of bacteria by enhanced killing effects. In 9 out of 12 fosfomycin-resistant mutants, genomic analyses identified previously reported mutations. The broad synergistic in vitro activity of ceftazidime/avibactam and fosfomycin confirms the potential of the application of this drug combination in clinics. The substantial reduction of the EC50 in combination may allow use of lower doses or treatment of organisms with higher MIC values and encourage further research translating these findings into the clinical setting.

Antifungal susceptibility patterns for Aspergillus, Scedosporium and Exophiala isolates recovered from cystic fibrosis patients against amphotericin B, and three triazoles and their impact after long term therapies.

In cystic fibrosis (CF) patients, fungal colonization of the respiratory tract is frequently found. Aspergillus fumigatus, Scedosporium genus and Exophiala dermatitidis are the most commonly isolated molds from the respiratory tract secretions of patients with this genetic disorder. The aim of this 5-year surveillance study was to identify trends in species distribution and susceptibility patterns of 212 mold strains identified as Aspergillus spp., Scedosporium spp. and Exophiala spp., isolated from sputum of 63 CF patients who received long term therapy with itraconazole and/or voriconazole. The Aspergillus spp. isolates were identified as members of the sections Fumigati (n=130), Flavi (n=22), Terrei (n=20), Nigri (n=8), Nidulantes (n=1) and Usti (n=1). Among the 16 species of the genus Scedosporium, 9 were S. apiospermum, 3 S. aurantiacum and 4 S. boydii. Among the 14 Exophiala species, all were molecularly identified as E. dermatitidis. Overall, 94% (15/16) of Scedosporium spp., 50% (7/14) of E. dermatitidis, and 7.7% (14/182) of Aspergillus spp. strains, showed high MIC values (≥ 8 µg/ml) for at least one antifungal. Particularly, 8.9% (19/212) of isolates showed high MIC values to amphotericin B, 11.7% (25/212) itraconazole, 4.2% (9/212) to voriconazole and 3.3% (7/212) to posaconazole. In some cases, such as some A. fumigatus and E. dermatitidis isolates recovered from the same patient, susceptibility to antifungal azoles decreased over time. We show that the use of azoles for a long time in CF patients causes the selection/isolation of mold strains with higher MIC values.

Early Molecular Insights into Thanatin Analogues Binding to A. baumannii LptA.

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro.

Aspergillus Species Causing Invasive Fungal Disease in Queensland, Australia

BackgroundAspergillus species are important causes of invasive fungal disease, particularly among those with an impaired immune system. Increasing reports have revealed a rising incidence of antifungal drug resistance among Aspergillus spp., particularly among cryptic species. Understanding local antifungal susceptibility patterns is paramount to delivering optimal clinical care.MethodsAspergillus spp. recovered from clinical specimens between 2000 and 2021 from Pathology Queensland were collected. Aspergillus spp. were identified routinely morphologically, and where there was ambiguity or a lack of sporulation, by sequencing of the internal transcribed spacer (ITS) region. All Aspergillus spp. that underwent antifungal susceptibility testing according to the CLSI M38-A3 method and were recorded and included in the study. Amphotericin B, voriconazole, posaconazole, isavuconazole, micafungin, caspofungin, and anidulafungin were tested. Pathology Queensland services all public healthcare facilities in Queensland, Australia.Results236 Aspergillus spp. were identified from clinical specimens during the study period. The most frequent species identified were Aspergillus section Fumigati (n = 119), Aspergillus section Flavi (n = 35), Aspergillus terreus (n = 32) and Aspergillus niger (n = 29). Overall, MIC50/90 values for voriconazole, posaconazole, itraconazole, and isavuconazole were 0.25/1, 0.25/0.5, 0.25/0.5, and 0.5/2 mg/L respectively. Echinocandins demonstrated low MIC values overall with micafungin and anidulafungin both having an MIC50/90 of 0.015/0.03 mg/L. A total of 15 cryptic species were identified; high triazole MIC values were observed with a voriconazole MIC50/90 of 2/8 mg/L. From 2017 to 2021 we observed an increase in incidence of isolates with high voriconazole MIC values. There was no difference in voriconazole MIC values between Aspergillus spp. acquired in North Queensland when compared to Southeast Queensland, Australia.ConclusionIncreasing reports of antifungal resistance among Aspergillus spp. is concerning and warrants further investigation both locally and worldwide. Active surveillance of both the emergence of different Aspergillus spp. and changes in antifungal susceptibility patterns over time is crucial to informing clinicians and treatment guidelines.

Comparison of Broth Microdilution, Disk Diffusion and Strip Test Methods for Cefiderocol Antimicrobial Susceptibility Testing on KPC-Producing Klebsiella pneumoniae

The aim of this study was to compare the reference broth microdilution (BMD) method with the Disk Diffusion (DD) test and strip test against a collection of 75 well-characterized Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) clinical strains to assess cefiderocol (CFD) antimicrobial activity. Whole-genome sequencing was performed on KPC-Kp strains by Illumina iSeq100 platform. The Categorical Agreement (CA) between the BMD method and DD test was 92% (69/75) with a Major Error (ME) of 16.7% (6/36). Additionally, the CA between the BMD method and test strip was 90.7% (68/75) with a Very Major Error (VME) of 17.9% (7/39) and 82.7% (62/75) between the strip test and DD with a ME of 30.2%. KPC-Kp strains showing resistance to CFD were 27 out of 75 (36%) by three methods. Specifically, 51.9% (14/27) of KPC-Kp resistant to CFD harbored blaKPC-3, while 48.1% (13/27) harbored mutated blaKPC-3. Moreover, KPC-Kp strains carrying a mutated blaKPC-3 gene exhibited high MIC values (p value < 0.001) compared to wild-type blaKPC-3. In conclusion, the DD test resulted as a valid alternative to the BMD method to determine the in vitro susceptibility to CFD, while the strip test exhibited major limitations.

Fluoroquinolone resistance among fecal extended spectrum βeta lactamases positive Enterobacterales isolates from children in Dar es Salaam, Tanzania

BackgroundFluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria.MethodsA cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing.ResultsA total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacterspp. (100%, 5/5), followed by Klebsiella.pneumoniae (76.1%; 35/46), Escherichiacoli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6’)-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx,qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E.coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E.coli isolates, while ST607 was more common out of 12 sequence types detected among the K.pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids.ConclusionThe ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents.