High Minimum Inhibitory Concentration Research Articles

Role of Efflux Pumps in Reduced Susceptibility to Tigecycline among Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a very well-known emerging pathogen and has become a major burden on healthcare system especially in intensive care units (ICUs). Tigecycline is the last resort drug for treatment of multidrug-resistant A. baumannii infections. However, non-susceptibility to this drug is a rising problem. Resistance to tigecycline is mediated by Resistance-nodulation-cell division (RND) efflux pumps. This study was done to detect efflux pump genes (adeABC) and regulator genes (adeS,adeR) responsible for tigecycline resistance among the clinical isolates of A. baumannii. A total of 150 OXA-51 confirmed clinical isolates were subjected for tigecycline susceptibility test by broth microdilution (BMD) method. All isolates irrespective of their MIC were subjected to conventional PCR for detection of efflux genes (adeABC) and regulator genes (adeRS). Prevalence of tigecycline resistance was found to be 14 (9.33%) by the reference broth microdilution method (BMD). Overall prevalence of efflux genes was highest for adeB (69%) and lowest for adeR (29%). Combination of genes especially three, four or five were found more prevalent among resistant isolates with higher minimum inhibitory concentration (MIC). Combination of efflux genes confer higher MIC and can be a major contributor for resistance to tigecycline.

Analysis of In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Gram-Negative Bacilli by Broth Microdilution and Disk Diffusion Method: A Single-Center Study in Odisha, India

IntroductionCefiderocol (CFDC), a novel semi-synthetic siderophore cephalosporin has been developed to combat the menace of infections caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) including Carbapenem-resistant Enterobacterales (CRE) and Carbapenem-resistant Nonfermenting Gram-negative bacilli (CR-NFGNB).MethodsWe determined the in vitro activity of CFDC against a contemporary collection of 503 CR-GNB isolates by the reference broth microdilution method (BMD) using Iron depleted cation adjusted Mueller-Hinton broth (ID-CAMHB). Performance of CFDC disk diffusion (DD) was evaluated against the reference BMD, as an alternative convenient testing method. Molecular characterization of carbapenemase in CR-GNB was performed by PCR targeting blaNDM-1, blaOXA-48like alleles, blaKPC, blaIMP, and, blaVIM. Minimum inhibitory concentration (MIC) distribution of CFDC in CR-GNB harbouring different carbapenemase enzymes was also analyzed.ResultsIn our study, 81.7% (411/503) of CR-GNB isolates [81.3%, (278/342) CRE and 82.6% (133/161) CR-NFGNB] were susceptible to CFDC (p>0.05). Categorical agreement (CA) of DD ranged from 79.8% to 87.5%, Minor error (mE) ranged from 0 to 14%, Major error (ME) ranged from 0 to 3.5%, and Very Major error (VME) ranged from 0 to 12.5% with variations by species tested. Overall CFDC MIC50 and MIC90 values of CR-GNB isolates without any carbapenemase genes were higher as compared to those with the presence of carbapenemase genes (4 µg/mL and 128 µg/mL versus 2 µg/mL and 16 µg/mL respectively).DiscussionCFDC is not yet available for clinical use in India. Hence, multicentric studies are the need of the hour in India for standardization of CFDC susceptibility using disks and CAMHB from different manufacturers as well as understanding mechanisms of high MIC values.

Antifungal susceptibility of the endophytic fungus Rhinocladiella similis (URM 7800) isolated from the Caatinga dry forest in Brazil.

The present study reports a new occurrence of Rhinocladiella similis isolated as an endophytic fungus in the Caatinga dry tropical forest in Brazil and describes its antifungal susceptibility. The isolate R. similis URM 7800 was obtained from leaves of the medicinal plant Myracrodruon urundeuva. Its morphological characterization was performed on potato dextrose agar medium and molecular analysis using the ITS rDNA sequence. The antifungal susceptibility profile was defined using the Clinical and Laboratory Standards Institute (CLSI) protocol M38-A2. The colony of isolate URM 7800 showed slow growth, with an olivaceous-gray color and powdery mycelium; in microculture, it showed the typical features of R. similis. In the antifungal susceptibility test, isolate URM 7800 showed high minimal inhibitory concentration (MIC) values for amphotericin B (>16 μg/mL), voriconazole (16 μg/mL), terbinafine (>0.5 μg/mL), and caspofungin (>8 μg/mL), among other antifungal drugs. Pathogenic melanized fungi are frequently isolated in environments where humans may be exposed, and these data show that it is essential to know if these isolates possess antifungal resistance.

Discerning the role of polymyxin B nonapeptide in restoring the antibacterial activity of azithromycin against antibiotic-resistant Escherichia coli.

Antimicrobial resistance is a global public health threat. Antibiotic development pipeline has few new drugs; therefore, using antibiotic adjuvants has been envisioned as a successful method to preserve existing medications to fight multidrug-resistant (MDR) pathogens. In this study, we investigated the synergistic effect of a polymyxin derivative known as polymyxin B nonapeptide (PMBN) with azithromycin (AZT). A total of 54 Escherichia coli strains were first characterized for macrolide resistance genes, and susceptibility to different antibiotics, including AZT. A subset of 24 strains was then selected for synergy testing by the checkerboard assay. PMBN was able to re-sensitize the bacteria to AZT, even in strains with high minimum inhibitory concentrations (MIC: 32 to ≥128 μg/ml) for AZT, and in strains resistant to the last resort drugs such as colistin and meropenem. The fractional inhibitory concentration index was lower than 0.5, demonstrating that PMBN and AZT combinations had a synergistic effect. The combinations worked efficiently in strains carrying mphA gene encoding macrolide phosphotransferase which can cause macrolide inactivation. However, the combinations were inactive in strains having an additional ermB gene encoding macrolide methylase which causes ribosomal drug target alteration. Killing kinetics study showed a significant reduction of bacterial growth after 6 h of treatment with complete killing achieved after 24 h. Transmission electron microscopy showed morphological alterations in the bacteria treated with PMBN alone or in combination with AZT, with evidence of damage to the outer membrane. These results suggested that PMBN acted by increasing the permeability of bacterial outer membrane to AZT, which was also evident using a fluorometric assay. Using multiple antimicrobial agents could therefore be a promising strategy in the eradication of MDR bacteria. PMBN is a good candidate for use with other antibiotics to potentiate their activity, but further studies are required in vivo. This will significantly contribute to resolving antimicrobial resistance crisis.

Feline sporotrichosis caused by Sporothrix schenckii sensu stricto in Southern Thailand: phenotypic characterization, molecular identification, and antifungal susceptibility.

Feline sporotrichosis caused by the Sporothrix schenckii complex is a global subcutaneous mycosis, having higher prevalence in Latin America and Malaysia. However, its etiological agents have not been elucidated in Thailand, a neighboring country of Malaysia, where the cases are increasing. This study identified 38 feline isolates of S. schenckii from Southern Thailand, collected between 2018 and 2021, using phenotypic characterization and molecular identification using polymerase chain reaction (PCR)-sequencing of partial calmodulin (CAL) and ß-tubulin (Bt2) genes. Phenotypic characteristics proved that the isolates were S. schenckii sensu lato, with low thermotolerance. Based on partial CAL and Bt2-PCR sequencing, all isolates were identified as S. schenckii sensu stricto. Phylogenetic analyses revealed that the isolates were clustered with S. schenckii sensu stricto isolated from the cats in Malaysia. A low degree of genetic diversity was observed among the Thai feline isolates. The antifungal susceptibility of these isolates to antifungal agents, including itraconazole (ITC), ketoconazole (KTC), fluconazole (FLC), and amphotericin B (AMB), was investigated according to the M27-A3 protocol of the Clinical and Laboratory Standards Institute. Results showed low ITC, KTC, and AMB activities against S. schenckii sensu stricto isolates, with high minimum inhibitory concentration (MIC) ranges of 1-8, 1-8, and 2-16μg/ml, respectively, whereas FLC exhibited MICs of 64 and>64μg/ml. This study indicated that S. schenckii sensu stricto is the causative agent responsible for feline sporotrichosis in Southern Thailand. Their phenotypic characteristics and in vitro antifungal susceptibility profiles will help to improve our understanding of this mycosis in Thailand.

P288 Rhinocerebral mucormycosis due to Saksenaea vasiformis in a Sri Lankan patient: A rare fungal infection

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMBackground: Rhizopus, Rhizomucor, and Mucor species are renowned agents causing rhinocerebral mucormycosis; a disease with a high mortality rate. Saksenaea vasiformis is extremely rarely observed in this clinical entity. Minimal sporulation in common laboratory media might be responsible for the under-reporting of this organism. Here we present an uncommon case of rhinocerebral mucormycosis due to S. vasiformis. in a Sri Lankan patient with diabetes mellitus.Case History: A 66-year-old female with diabetes mellitus was admitted with frontal headache, right-sided nasal block, anosmia, and right-side facial swelling. Examination revealed facial edema, maxillary sinus tenderness, and a white patch over the hard palate. Thick pus in postnasal space, growth in posterior tongue base, inflamed palate, and oropharynx, were revealed by rigid nasal endoscopy. She developed ophthalmoplegia along with right V and XII cranial nerve palsies irrespective of antibacterial therapy. Right side pansinusitis was observed in non-contrast computed tomography. She was subjected to right-side full house functional endoscopic sinus surgery with right orbital and optic nerve decompression. Irregular wide, ribbon-like, non-septate hyphae suggestive of Zygomycete fungi were observed in the direct microscopy of a deep surgical tissue sample and started with intravenous amphotericin B. After 5 days of incubation, the culture grew a zygomycetes-like mold with a lack of sporulation on Sabouraud dextrose agar, potato dextrose agar, and slide culture. However, the floating agar technique succeeded in producing flasks-shaped sporangium on a short sporangiophore with brown pigmented rhizoids after 14 days of incubation. The phenotypic features were suggestive of S. vasiformis. She was subjected to repeated debridement surgeries and treatment with amphotericin B was continued. She was clinically improving however, refractory hypokalemia along with hypernatremia interrupted her antifungal therapy resulting return of severe symptoms. Contrast-enhanced computed tomography of the brain revealed multiple micro-abscesses in the right temporal lobe. She clinically deteriorated and succumbed to the illness.Discussion: Saksenaea vasiformis, a member of Mucorales, is largely bounded to cutaneous and subcutaneous infections. Rhino-orbital-cerebral infection is relatively uncommon and most of the reported cases were fatal irrespective of optimal therapy. Prompt diagnosis through fungal investigations of deep biopsy is mandatory. The delayed identification of this organism is attributed to its nature of poor sporulation on routine media. Special culture techniques and nutritionally deprived media enhance sporulation. Saksenaea vasiformis is usually sensitive to amphotericin B, high minimum inhibitory concentration has been reported though.ConclusionSpecific culture techniques should be used to induce sporulation if non-sporulating mucormycetes are encountered. Rhinocerebral mucormycosis is associated with a high degree of mortality even with effective antifungal therapy.

S4.1d In vitro activity of eight antifungals against 3343 filamentous pathogenic fungi collected during 18 years at the French National Reference Center for invasive mycosis and antifungals

S4.1 Treatment of rare mold infections in 2021: the role of new and old antifungals, September 22, 2022, 10:30 AM - 12:00 PM Background & ObjectivesThe NRCMA oversees the surveillance of invasive fungal diseases in France. As part of our expertise, we perform antifungal susceptibility testing based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology on all isolates. The antifungal profiles help us monitor the emergence of resistant isolates, determine the susceptibility pattern of wild-type strains to new antifungals, and in specific cases, determine the relationship between clinical failure and selection/emergence of a less susceptible isolate.We review the Minimum inhibitory concentrations (MIC) distribution of eight antifungals on clinical isolates of filamentous fungi identified at the NRCMA from 2003 to 2021.MethodsSpecies identification was performed by a combination of morphological features and multilocus sequencing. Only strains that produced enough conidia or spores were tested. In vitro susceptibility testing was performed according to the EUCAST procedure. Eight antifungal agents were used: triazoles [itraconazole, voriconazole, posaconazole, isavuconazole (since 2015)], echinocandins (caspofungin, micafungin), amphothericin B, and terbinafine. The concentrations inhibiting 50% (MIC50) and 90% (MIC90) of the isolates were determined for species with at least 5 and 10 isolates, respectively. For Aspergillus fumigatus isolates exhibiting high azole MICs, we sequenced the cyp51A gene for mutation screening.ResultsMICs were obtained for 3343 pathogenic strains. We identified Aspergillus spp. including cryptic species (32%), Fusarium spp. (21%), Mucorales (18%), phaeohyphomycetes (10%), i.e., Alternaria spp., Curvularia spp., Fonsecaea, Exophiala, Phaeoacremonium, rare hyphomycetes (9%), i.e., Rasamsonia, Paecilomyces, Trichoderma, Scopulariopsis. Scedosporium spp. (7%) and emergent pathogens such as Nannizziopsis obscura (1%), dimorphic fungi (1%), or to a less extent basidiomycetous molds. Examples of MIC results are presented in Table 1.For the nine genera of the order Mucorales, amphotericin B (AmB) exhibited low MICs except for Cunninghamella species and Saksenaea vasiformis. Posaconazole had variable activity depending on the species (high MIC values were observed for Mucor species) while voriconazole and echinocandins had none.In section Fumigati, amphotericin B MIC values were high due to the intrinsic resistance of cryptic species Aspergillus lentulus and A. udagawae. Other cryptic species were identified such as A. hiratsukae isolates which exhibited low MICs to all antifungals.Members of Aspergillus section Nidulantes such as A. nidulans and A. quadrilineatus showed variable MICs to caspofungin and amphotericin B.Among melanized fungi, Alternaria infectoria and A. alternata species group displayed low MICs of all azoles with the exception of voriconazole.Four Exophiala species were analyzed and had similar azole MICs, E. spinifera being the species with the lowest values.Multi-drug resistant profiles were observed especially in species belonging to Microascales such as Lomentospora prolificans, Microascus cirrosus, and Scopulariopsis brevicaulis. Members of Fusarium species complexes had predictably high MIC values to all antifungals including isavuconazole. Only Fusarium dimerum species complex had low MICs to amphotericin B.Further distribution analysis is ongoing. These large datasets provide a baseline for monitoring the emergence of antifungal resistance in our country and supports the fact that MIC determination should be performed for rare/emergent species and also in case of infections due to cryptic species, which would benefit patient management.

P076 The promising antimycotic activities of a novel cyclic antifungal lipopeptide against Human Dermatophyte Isolates

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo determine the minimum inhibitory concentrations (MICs) of a novel antifungal lipopeptide against clinical isolates of dermatophytes of human origin.MethodsTo perform antifungal susceptibility testing (AFST) by CLSI microbroth dilution method, a reversed-phase high-performance liquid chromatography (RP-HPLC) purified lipopeptide of 1071.4 Da from a wild-type soil isolate Bacillus subtilis was used and compared with the standard allylamine terbinafine MICs. Briefly, 1200 ml of cell-free culture supernatant was extracted using a solvent mixture and silica gel (230-400 mesh size)-based adsorption chromatography. The semi-preparative RP-HPLC system consisted of an Agilent quaternary pump and a variable wavelength detector equipped with a Phenomenex Luna C18 column (10 mm × 250 mm, 5 μm). The solvent system for RP- HPLC was (A) water with 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile containing 0.1% TFA. The gradient of solvent B used for purification was as follows: 0%-54% for 0-20 min at the flow rate of 1 ml/min, 54%-60% from 20-48 min at 0.5 ml/min, 60%-100% from 48-58 min at 0.5 ml/min, 100%-0% from 58-65 min at 1 ml/min and monitored at 210 nm.ResultsSuperficial skin infections are caused by dermatophytes including Trichophyton spp. Nowadays, resistance to terbinafine in Trichophyton spp. isolates with higher MICs have been documented in India. We report here the antifungal prowess of a novel small antifungal lipopeptide against 20 clinical isolates of Trichophyton spp. of human origins (from human skin scrapings and nails) with the clinical diagnosis of tinea corporis/cruris and tinea unguium. The representative photographs of T. tonsurans, T. rubrum, and T. mentagrophytes complex are provided below. A total of 6 isolates of T. mentagrophytes and T. rubrum carry point mutations at F397L of squalene epoxidase (SQLE) protein. The in vitro antifungal efficacies determined by AFST revealed that the lipopeptide showed less or equivalent MICs (100% inhibition) in the case of five dermatophytes. The lipopeptide drug has exhibited improved MICs against two T. mentagrophytes complex and two T. rubrum isolates with amino acid substitution F397L in SQLE protein. Trichophyton mentagrophytes complex and T. rubrum with F397L mutations were inhibited at MICs 4-32 μg/mL of terbinafine. In comparison, the lipopeptide showed 4-16 μg/ml (100% inhibition). In the case of all four Trichophyton tonsurans, the MICs ranged between 2-4 μg/ml for the lipopeptide.ConclusionsThe broad-spectrum lipopeptide showed promising antifungal activity against dermatophytes and may be considered for nano-emulsion formulation and tested for topical application in a mice model.

P156 Oral Candidiasis among inpatients with COVID-19 in the North of Iran

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesWe aimed to characterize oral candidiasis epidemiology, species distribution, and antifungal susceptibility profiles among COVID-19 patients.MethodsThis observational cross-sectional study enrolled patients ˃18 years old with confirmed oral candidiasis admitted at Razi teaching hospital (a reference infectious disease center in Mazandaran Province). Oral samples from patients with suspected oral candidiasis infection were collected and were confirmed for oral candidiasis by microscopic examination and fungal culture. Fungal isolates were identified using Multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. In vitro susceptibility testing for amphotericin B, fluconazole, voriconazole, and micafungin of all identified isolates was performed using broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) broth microdilution guideline (M27-A3 and M60) standard.ResultsAmong 4133 COVID-19 admitted patients during a year in Razi hospital, 120 patients had confirmed oral candidiasis. Totally 172 Candida isolates causing oral candidiasis isolated from these patients were identified. Most patients were infected with a single Candida species; however, it is notable that mixed Candida species caused oral candidiasis in 46 patients. Candida albicans (60.46%) was the most common species. Among non-albicans Candida species, C. glabrata (17.44%) was the most isolates, followed by C. tropicalis (11.62%), C. kefyr (7.55%), and C. krusei (2.9%). Although non-albicans Candida species, including C. glabrata and C. krusei demonstrated high minimum inhibitory concentration (MIC) against azole drugs, in terms of MIC90 values, all tested drugs exhibited superior activity against C. albicans. In terms of MIC Geometric mean, amphotericin B and micafungin were more potent than all comparator drugs.ConclusionsOur study described the high incidence of oral candidiasis caused by non-albicans Candida species in COVID-19 patients; most of them, including C. glabrata and C. krusei exhibits intrinsic decreased susceptibility to the azole class of antifungals. Further studies should design an appropriate prophylaxis program to prevent oral candidiasis in COVID-19 patients.

P001 Characteristics and dynamics of azole-resistant Aspergillus fumigatus variants emerging over a 28-year period in the Netherlands

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMBackground Aspergillus fumigatus, a globally distributed opportunistic pathogen, is the main cause of invasive aspergillosis, especially in immunocompromised patients with high mortality. The emergence of azole-resistant A. fumigatus isolates has been a significant concern worldwide and an important clinical problem.ObjectivesWe aim to determine the presence of variants in a large collection of clinical A. fumigatus isolates from the Netherlands, if the number of variants increased over time and if the presence of additional short nucleotide polymorphisms (SNPs) or tandem repeats (TR) variations impacted on the triazole phenotype.MethodsThe Radboud University Medical Center has collected 11 813 clinical A. fumigatus isolates since 1994. The collection includes isolates cultured from patients admitted to our own center, isolates sent from other hospitals for identification and in vitro susceptibility testing, and isolates sent from five university medical centers and five teaching hospitals that contribute to the national Aspergillus resistance surveillance. The genotypes were detected by Cyp51A Sanger sequencing. All isolates were subjected to in vitro susceptibility testing using the EUCAST microdilution reference method. Minimal inhibitory concentrations (MICs) were determined for itraconazole, voriconazole, posaconazole, in all isolates and for isavuconazole in isolates cultured in 2015 and thereafter.ResultsIn total, 1826 A. fumigatus isolates harbored azole-resistant mutations in the Cyp51A-gene with 92 genotypes. Tandem Repeat-associated resistance genotypes accounted for 55.43% of the variants and were involved in 1728 isolates (94.63%). TR34/L98H and TR46/Y121F/T289A resistance mutations remained dominant, and increasingly additional SNPs in the Cyp51A-gene or changes to the gene promoter were observed. The G448S mutation was relatively common and present in various genetic backgrounds. This SNP was most often found in isolates harboring the TR46 resistance mechanism (8 variants) and was also observed in two variants in the TR34 genetic background. TR34 and TR46 resistance mutations are associated with 1170 (64.07%) isolates that exhibited a pan-azole resistance phenotype, 547 (29.96%) a multi-azole resistance phenotype, and 75 (4.11%) resistance to a single azole. TR34/L98H confers high itraconazole resistance, while T289A confers high voriconazole resistance in the TR46 background. Isolates with a G448S point mutation show high MICs for both voriconazole and itraconazole. The TR34/L98H/T289A/G448S isolate showed low itraconazole MICs but high voriconazole resistance, and mutations in the promoter region, TR34/C-86 G/L98H, and (T-66 G)/TR34/L98H variants, showed increased voriconazole and isavuconazole MIC compared with the parent phenotype. TR46/Y121F/M172I/T289A/G448S variant was observed with an increased itraconazole (GM MIC 16 mg/L, 1→16 mg/l) and decreased voriconazole (GM MIC 18.664 mg/l, 4→16 mg/l) compared with the parent MIC of TR46/Y121F/T289A, while TR92/Y121F/M172I/T289A/G448S and TR46/Y121F/ T289A/G448S variants showed the consistent MIC distribution with parent genotype. The variants with more combination mutations showed pan-azole resistance with increased MIC distribution.ConclusionOur survey showed a significant increase in resistance genotypes in clinical A. fumigatus over a period of 28 years. Azoles resistance phenotypes vary from resistant variants in clinical isolates; it is an implication for clinical A. fumigatus infection treatment options and antifungal stewardship.

P240 Candida nivariensis infection in South East Asia: series of case reports from Selayang Hospital, Malaysia and South East Asia Region

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Introduction Candida nivariensis, first described from Spain in 2005 is an emerging fungal pathogen. Malaysia has captured a total of 11 cases since its first reported here in 2014. Indonesia, Thailand, and Vietnam are among South East Asia countries that reported its occurrence too.ObjectivesThe occurrence and characteristics of Candida nivariensis in South East Asia are studied. We would like to highlight two cases from Selayang Hospital, Malaysia that occurred during pandemic COVID-19 in 2020 and 2022.Case DescriptionIn July 2020, a 60-year-old lady underlying poorly controlled diabetes mellitus, CKD stage V approaching ESRD, congestive cardiac failure, hypertension, and dyslipidemia was diagnosed as complicated MSSA bacteremia secondary to right gluteal carbuncle. Appropriate antibiotics were commenced. Aggressive source control including incision and drainage, cauterization of right gluteal carbuncle, and multiple wound debridement for the infected gluteal wound was performed. She underwent a trephine sigmoid loop colostomy. Tracheostomy was done following prolong ventilation. Anidulafungin as empirical fungal coverage was commenced in ICU as she further deteriorated. Candida nivariensis was isolated later from her blood culture.In February 2022, a 74-year-old man underlying Diabetes mellitus, chronic kidney disease, hypertension, ischemic heart disease with two vessels disease stented, post-Whipple's procedure due to necrotizing pancreatitis in 2014 complicated with chronic abdominal pain with hyperalgesia admitted to the ward for acute right lacunar infarct with failed swallowing test and neurogenic bladder. His general condition deteriorated with worsening of biochemical and septic parameters. His blood culture grew C. nivariensis following which anidulafungin was started. No distant seeding and no signs of endophthalmitis. He then required mechanical ventilatory support complicated with multiorgan failure, leading to ICU admission.MethodsFor both cases, yeast isolates were subcultured on Sabouraud dextrose agar and CHROMagar. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) with molecular testing was performed for confirmation of isolates. Phylogenetic tree among isolates from Malaysia was analyzed. Antifungal susceptibility test (AFST) to study the minimum inhibitory concentration (MIC) among treatment options was performed using the e-test method.ResultsWide use of MALDI-TOF MS with molecular testing increased the detection rate of the species. Isolation sites in Malaysia and South East Asia region include blood culture, peritoneal fluid, high vaginal swab, and oropharyngeal swab. Risk factors namely immunocompromised status, indwelling vascular catheter, abdominal surgeries, renal failure or on dialysis support, polymicrobial usage, and prolonged stay in ICU. AFST established susceptibility towards fluconazole, high MIC to itraconazole and low MIC against amphotericin B, anidulafungin and voriconazole for both patients who unfortunately succumbed to their illness.ConclusionDiagnosing C. nivariensis is challenging due to its closely related phylogenetic with C. glabrata, and C. bracarensis, thus molecular testing is vital. Inadequate antifungal coverage or delay of treatment could be detrimental to patient's outcome. Continuous epidemiological surveillance is crucial to address this potential invasive fungal pathogen and to observe emerging of drug resistance.

Molecular characteristics of fluoroquinolone-resistant Escherichia coli isolated from suckling piglets with colibacillosis

ObjectivesColibacillosis is a frequent enteric disease in the pig industry that causes significant economic losses. The objective of this study was to investigate the molecular characteristics of fluoroquinolone (FQ)-resistant E. coli isolates from suckling piglets with colibacillosis.ResultsA total of 43 FQ-resistant E. coli isolates were tested in this study and all isolates showed multi-drug resistance (MDR) and mutations in quinolone resistance determining regions (gyrA or parC). Especially, FQ-resistant E. coli isolates with double mutations in both gyrA and parC were shown a high FQs minimum inhibitory concentration (≥ 64 mg/L for ciprofloxacin, ≥ 128 mg/L for enrofloxacin, and ≥ 256 mg/L for norfloxacin). Among 43 FQ-resistant E. coli isolates, 12 (27.9%) were showed plasmid-mediated quinolone resistance (PMQR) positive E. coli. Prevalence of PMQR gene, aac(6’)-Ib-cr, qnrS, and qepA, were identified in 7, 3, and 2 E. coli isolates, respectively. We identified the following in PMQR-positive E. coli isolates: the tetracycline resistance genes tetD (12 isolates, 100.0%), tetE (12 isolates, 100.0%), tetA (11 isolates, 91.7%), and tetB (1 isolate, 8.3%); β-lactamases–encoding blaCMY-2 (10 isolates, 83.3%), blaTEM-1 (7 isolates, 58.3%), blaOXA-1 (7 isolates, 58.3%), blaSHV-1 (3 isolates, 16.7%), and blaAAC-2 (1 isolate, 8.3%); and the chloramphenicol resistance genes (10 isolates, 83.3%); the sulfonamide resistance genes sul1 (9 isolates, 75.0%) and sul2 (10 isolates, 83.3%); the aminoglycoside modifying enzyme gene aac(3)-II (2 isolates, 16.7%). The F4 (7 isolates, 58.3%), LT:STb:EAST1 (5 isolates, 41.7%), and paa (3 isolates, 25.0%) were most common fimbrial antigen, combinations of toxin genes, and non-fimbrial adhesins genes, respectively. All PMQR-positive E. coli carried class I integrons but only 4 isolates carried the gene cassette. The most prevalent plasmid replicon was FIB (9 isolates, 75.0%), followed by FIC, HI1, and N (7 isolates, 58.3%), respectively.ConclusionsBecause FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs, this represents a public health hazard.

Whole-Genome Sequencing and Drug-Susceptibility Analysis of Serial Mycobacterium abscessus Isolates from Thai Patients.

Simple SummaryWe undertook genetic diversity analysis between subspecies, mutation-rate estimation and identification of drug-resistant mutations analysis of Mycobacterium abscessus. We differentiated between re-infection and persistent infection of M. abscessus based on genetic distance using WGS analysis. We found a high mutation rate of M. abscessus during the initial infection process, followed by a dramatic reduction in this rate in serial isolates separated by more than 180 days. Mutation rate also differed between the two subspecies that we included: subsp. abscessus (MAB) and subsp. massilense (MMAS). MMAS strains show more susceptibility to clarithromycin, amikacin and linezolid than MAB. This can inform choice of an appropriate regimen for patient management and reduce the speed of development of drug resistance for M. abscessus patients.Mycobacterium abscessus is an important pathogen that can cause serious human diseases and is difficult to treat due to antibiotic resistance. In this study, we analyzed, using whole-genome sequence (WGS) data, M. abscessus strains serially isolated from patients at various time intervals. We undertook genetic diversity analysis between subspecies, mutation-rate estimation and identification of drug-resistant mutations with minimum inhibitory concentration (MIC) analysis. Clonal isolates of M. abscessus:—subsp. abscessus (MAB) and subsp. massiliense (MMAS)—causing persistent infection through time, differed by 0–7 and 0–14 SNPs, respectively, despite being isolated 1 to 659 days apart. Two cases caused by MMAS differed by ≥102 SNPs at 350 days apart and were regarded as examples of reinfection. Isolates collected ≤7 days apart exhibited a high mutation rate (133.83 ± 0.00 SNPs/genome (5 Mb)/year for MMAS and 127.75 SNPs/genome (5 Mb)/year for MAB). Mutation rates declined in a time-dependent manner in both subspecies. Based on isolates collected > 180 days apart, MMAS had a significantly higher average mutation rate than MAB (2.89 ± 1.02 versus 0.82 ± 0.83 SNPs/genome (5 Mb)/year, (p = 0.01), respectively). All well-known drug-resistance mutations were found to be strongly associated with high MIC levels for clarithromycin and ciprofloxacin. No known mutations were identified for strains resistant to linezolid and amikacin. MAB strains in the study were susceptible to amikacin, while most MMAS strains were susceptible to clarithromycin, amikacin and linezolid. No hetero-resistance was found in the strains analyzed. Our study reports the genetic diversity and mutation rate of M. abscessus between the two major subspecies and confirms the drug resistance-associated mutations. Information about drug-resistance and associated mutations can be applied in diagnosis and patient management.

Detection and characterisation of Bordetella hinzii in line-related bacteraemia and respiratory tract infection in Australia

Bordetella hinzii has emerged as an unusual cause of infection in immunocompromised patients, previously linked to zoonotic transmission. Antimicrobial susceptibility and genetic diversity of B.hinzii are poorly understood. This study reports phenotypic and genomic characteristics of the first four Australian isolates of B.hinzii obtained from elderly immunocompromised patients. Bordetella hinzii isolates were identified by MALDI-TOF and whole genome sequencing (WGS). Antibiotic susceptibility testing was performed using disk diffusion or E-test. Genomes of B.hinzii were analysed in global context. A phylogenetic tree was constructed of all isolates using Roary and a maximum-likelihood tree was generated from the core-snp alignment. Bordetella hinzii minimum inhibitory concentrations (MICs) were largely uniform with high MICs to ampicillin, ceftriaxone and ciprofloxacin and low MICs to meropenem and piperacillin-tazobactam. Genomic analysis of isolate sequences divided strains analysed into two phylogenetically distinct groups, with one Australian B.hinzii isolate (AUS-4) assigned to Group 1, and the remaining isolates (AUS1-AUS3 and AUS-5) to Group 2. Single nucleotide polymorphism (SNP) analysis revealed two isolates, AUS-1 and AUS-2, were closely related with 14 SNP differences between them. All other Australian isolates were unrelated to each and all other isolates from the international dataset. Bordetella hinzii appears to pose a risk to immunocompromised individuals but remains susceptible to extended spectrum β-lactam and carbapenem antibiotics. Genomic analysis suggested a dissemination of genetically distinct strains.

Azole Resistance and cyp51A Mutation of Aspergillus fumigatus in a Tertiary Referral Hospital in Taiwan.

Azole resistance in Aspergillus fumigatus has increasingly been reported worldwide. Its major mechanism of resistance is mediated by mutations in cyp51A. The objective of this study was to test the antifungal susceptibilities of A. fumigatus isolates from Chang Gung Memorial Hospital (CGMH), the largest tertiary referral hospital in Taiwan, and to investigate cyp51A mutations in azole-resistant strains. A. fumigatus isolates preserved in the Research Laboratory of Medical Mycology of CGMH from 2015 to 2021 were used. Antifungal susceptibility testing was performed using the YeastOneTM method. Isolates with high minimal inhibitory concentrations (MICs) against antifungals were further tested using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. Mutations in the cyp51A in azole-resistant strains were detected by Sanger sequencing. The overall prevalence of azole-resistant isolates was 1.77% (two out of 113 isolates). The two azole-resistant strains had tandem repeats (TR) in the promoter region and mutations in the cyp51A gene (TR34/L98H and TR34/L98H/S297T/F495I). One strain showed intermediate susceptibility to voriconazole, and its Cyp51A protein had five amino acid substitutions (F46Y/M172V/N248T/D255E/E427K). TR34/L98H and TR34/L98H/S297T/F495I are the most prevalent cyp51A mutations in Taiwan, mediating azole resistance based on current publications and our results. YeastOneTM was validated as a rapid tool for the antifungal susceptibility test; however, further confirmation by CLSI should be considered when MIC values of voriconazole, posaconazole, and amphotericin B are close to the clinical breakpoints or ecological cutoff values.

Invasive Group A Streptococcal Penicillin Binding Protein 2× Variants Associated with Reduced Susceptibility to β-Lactam Antibiotics in the United States, 2015-2021.

All known group A streptococci [GAS] are susceptible to β-lactam antibiotics. We recently identified an invasive GAS (iGAS) variant (emm43.4/PBP2x-T553K) with unusually high minimum inhibitory concentrations (MICs) for ampicillin and amoxicillin, although clinically susceptible to β-lactams. We aimed to quantitate PBP2x variants, small changes in β-lactam MICs, and lineages within contemporary population-based iGAS. PBP2x substitutions were comprehensively identified among 13,727 iGAS recovered during 2015-2021, in the USA. Isolates were subjected to antimicrobial susceptibility testing employing low range agar diffusion and PBP2x variants were subjected to phylogenetic analyses. Fifty-five variants were defined based upon substitutions within an assigned PBP2x transpeptidase domain. Twenty-nine of these variants, representing 338/13,727 (2.5%) isolates and 16 emm types, exhibited slightly elevated β-lactam MICs, none of which were above clinical breakpoints. The emm43.4/PBP2x-T553K variant, comprised of two isolates, displayed the most significant phenotype (ampicillin MIC 0.25 μg/ml) and harbored missense mutations within 3 non-PBP genes with known involvement in antibiotic efflux, membrane insertion of PBP2x, and peptidoglycan remodeling. The proportion of all PBP2x variants with elevated MICs remained stable throughout 2015-2021 (<3.0%). The predominant lineage (emm4/PBP2x-M593T/ermT) was resistant to macrolides/lincosamides and comprised 129/340 (37.9%) of isolates with elevated β-lactam MICs. Continuing β-lactam selective pressure is likely to have selected PBP2x variants that had escaped scrutiny due to MICs that remain below clinical cutoffs. Higher MICs exhibited by emm43.4/PBP2x-T553K are probably rare due to the requirement of additional mutations. Although elevated β-lactam MICs remain uncommon, emm43.4/PBP2x-T553K and emm4/PBP2x-M593T/ermT lineages indicate that antibiotic stewardship and strain monitoring is necessary.

In vitro Susceptibility of Nontuberculous Mycobacteria to Tedizolid.

ObjectiveNontuberculous mycobacteria (NTM) can cause pulmonary and extrapulmonary diseases. Tedizolid (TZD) is a new oxazolidinone with in vitro activity against NTM such as Mycobacterium avium complex (MAC), Mycobacterium fortuitum, and Mycobacterium abscessus complex. The aim of this study was to evaluate the TZD susceptibility profiles of clinical isolates of NTM.MethodsThe microdilution method was used to identify the minimum inhibitory concentration (MIC) of TZD and linezolid (LZD) for 133 clinical NTM isolates. Broth microdilution chequerboard assays were used to investigate the synergistic effects of TZD and three antibiotics on two reference isolates and eleven clinical isolates of NTM.ResultsThe TZD MIC50 and MIC90 for M. abscessus complex were 2 and 4 μg/mL, 16 and >32 μg/mL for MAC, respectively. TZD exhibited lower MICs than that of LZD for most NTM, which were positively correlated. Due to the high MIC values of TZD against MAC, it is necessary to conduct drug sensitivity tests before TZD administration. TZD-clarithromycin combination had synergistic response on M. abscessus complex in 3 of the 8 isolates, which lasted only 3–5 days. TZD-cefoxitin had synergistic effect against all five M. fortuitum isolates.ConclusionOur study demonstrates that TZD had greater in vitro potency than LZD, and synergy studies suggested that TZD may be an important component of multi-drug treatment regimen.

Molecular characterization of florfenicol and oxazolidinone resistance in Enterococcus isolates from animals in China.

Florfenicol is widely used for the treatment of bacterial infections in domestic animals. The aim of this study was to analyze the molecular mechanisms of florfenicol and oxazolidinone resistance in Enterococcus isolates from anal feces of domestic animals. The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method. Polymerase chain reaction (PCR) was performed to analyze the distribution of the resistance genes. Whole-genome sequencing and comparative plasmid analysis was conducted to analyze the resistance gene environment. A total of 351 non-duplicated enteric strains were obtained. Among these isolates, 22 Enterococcus isolates, including 19 Enterococcus. faecium and 3 Enterococcus. faecalis, were further studied. 31 florfenicol resistance genes (13 fexA, 3 fexB, 12 optrA, and 3 poxtA genes) were identified in 15 of the 19 E. faecium isolates, and no florfenicol or oxazolidinone resistance genes were identified in 3 E. faecalis isolates. Whole-genome sequencing of E. faecium P47, which had all four florfenicol and oxazolidinone resistance genes and high MIC levels for both florfenicol (256 mg/L) and linezolid (8 mg/L), revealed that it contained a chromosome and 3 plasmids (pP47-27, pP47-61, and pP47-180). The four florfenicol and oxazolidinone resistance genes were all related to the insertion sequences IS1216 and located on two smaller plasmids. The genes fexB and poxtA encoded in pP47-27, while fexA and optrA encoded in the conjugative plasmid pP47-61. Comparative analysis of homologous plasmids revealed that the sequences with high identities were plasmid sequences from various Enterococcus species except for the Tn6349 sequence from a Staphylococcus aureus chromosome (MH746818.1). The current study revealed that florfenicol and oxazolidinone resistance genes (fexA, fexB, poxtA, and optrA) were widely distributed in Enterococcus isolates from animal in China. The mobile genetic elements, including the insertion sequences and conjugative plasmid, played an important role in the horizontal transfer of florfenicol and oxazolidinone resistance.

Microwave-assisted synthesis, characterizations, antimicrobial activities, and DFT studies on some pyridine derived Schiff bases

This study reports a joint experimental, theoretical and microbiological investigation on the (E)-N,N-dimethyl-4-((pyridine-2-ylmethylene)amino)aniline (5), (E)-N,N-dimethyl-4-((pyridine-4-ylmethylene)amino)aniline (6) and (E)-N,N-dimethyl-4-((pyridine-3-ylmethylene)amino)aniline (7). These compounds were synthesized with microwave method and their structures characterized by FT-IR, 1H-NMR, 13C-NMR, and elemental analysis tecniques. In the theoretical studies, torsional barriers analysis, ground state structure, Fourier Transform Infrared spectra (FT-IR), and Nuclear Magnetic Resonance spectra (NMR) of 5, 6, and, 7 were calculated by Density Functional Theory (DFT) computations. The conformers obtained from the torsional barrier scanning were optimized by B3LYP/6-31G(d,p) level. The harmonic vibrational frequencies, potential energy distribution (PED), infrared intensities, and NMR chemical shifts of the most stable conformers were determined using the B3LYP/6-311++G(d,p). Theoretically, predicted spectral data were compared with experimental results. Antimicrobial studies of the synthesized compounds were performed against various microbial strains. Antimicrobial activities of 5, 6, and, 7 were tested against selected bacteria and yeast through minimum inhibitory concentration (MIC) and diffusion method. Compound 7 was found to be the most active against bacteria and yeast, while compound 5 was found to be moderately active. Compounds 6 (against S. aureus and C. albicans) and, 7 were found to have a very high minimum inhibitory concentration, ranging between 1.95 and 7.81 g/mL (against P. aeruginosa and E. coli). Compounds (6 and 7) showed zone of inhibition values in the range of 10–20 mm against other bacteria except L. monocytogenes and S. thyphimurium.

Prevalence and Antimicrobial Susceptibility of Bovine Mycoplasma Species in Egypt.

Simple SummaryBovine Mycoplasma species, particularly antimicrobial resistant Mycoplasma bovis are important causes of bovine respiratory disease (BRD) in cattle, which causes major economic losses worldwide. Thus, the current study aimed to determine the prevalence and antimicrobial resistance profiles of bovine Mycoplasma spp. isolated from cattle’s respiratory tracts, in addition to evaluating the fluoroquinolone resistance in the recovered isolates using broth microdilution and conventional PCR techniques in Egypt. Our result showed that M. bovis was the most common spp. (61%), followed by M. bovirhinis (15%). In total, mycoplasma isolates were more prevalent among all examined lung tissues (38%), followed by nasal swabs (35%), tracheal tissues (28%), and tracheal swabs (27%). All the examined mycoplasma isolates (n = 76) were 100% susceptible to spectinomycin, tulathromycin, spiramycin, and tylosin, but high doxycycline and enrofloxacin minimum inhibitory concentrations (MICs) values were observed among 43.4% and 60.5% of the tested isolates, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%), meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, understanding antimicrobial resistance mechanisms is a significant tool for the future development of genetic-based diagnostic techniques for the rapid detection of resistant mycoplasma strains.Among many bovine Mycoplasma species (spp.), Mycoplasma bovis is recognized as a significant causative agent of respiratory diseases in cattle. In recent years, resistant M. bovis isolates, especially to fluoroquinolones, have been reported globally as a result of the extensive usage of antimicrobials in the treatment of bovine pneumonia. Therefore, the aim of this study is to investigate the prevalence and antimicrobial susceptibility patterns of bovine Mycoplasma spp. isolated from the respiratory tracts of cattle in Egypt and to assess the fluoroquinolones resistance in the recovered mycoplasma isolates via broth microdilution and conventional PCR techniques. Conventional phenotypic methods identified 128 mycoplasma isolates (32%) from 400 different samples, with M. bovis being the predominant spp. (61%), followed by M. bovirhinis (15%). Of note, mycoplasma isolates were rarely isolated from total healthy lung tissues (7/55, 12.7%), but they were frequently isolated from pneumonic lungs (31/45, 68.9%). All the examined mycoplasma isolates (n = 76) were sensitive to tilmicosin, tylosin, tulathromycin, spiramycin, and spectinomycin (100% each), while 60.5% and 43.4% of the examined isolates had high minimum inhibitory concentration (MIC) values to enrofloxacin and doxycycline, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%); meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, our findings revealed alarming resistance to enrofloxacin and doxycycline antibiotics; thus, antimicrobial usage must be restricted and molecular techniques can help in the rapid detection of the resistant strains.