Dose-dependent Susceptibility Research Articles

Antimicrobial Resistance in Sepsis Cases Due to Escherichia coli and Klebsiella pneumoniae: Pre-Pandemic Insights from a Single Center in Southwestern Romania.

To determine the resistance profile of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) strains isolated in sepsis cases diagnosed at the Infectious Diseases Clinic in Craiova, Romania. The bacteria responsible for sepsis cases were identified using the Vitek 2 Systems version 06.01, which was then employed to assess their antimicrobial susceptibility (Global CLSI and Phenotypic 2017). We have identified 989 patients diagnosed with bacterial sepsis. Among these, 953 cases were caused by Gram-negative rods, with 415 attributed to E. coli and 278 to K. pneumoniae. High levels of resistance to ampicillin were recorded for E. coli strains isolated in sepsis cases (64.6%); adding sulbactam lowers the level of resistance to 41.8%. Resistance to 3rd generation cephalosporins varied between 7.47 and 14.6% and another 3.41 to 11.1% are dose-dependent susceptibility strains. Resistance to carbapenems (i.e., ertapenem, meropenem) is low-2.18-2.42%. More than 95% of the tested K. pneumoniae strains were resistant to ampicillin and adding sulbactam as a β-lactamase inhibitor only halves that level. Resistance to 3rd generation cephalosporins varied between 20.7% and 22.5%; resistance levels for K. pneumoniae were notably higher than those for E. coli. Over 95% of K. pneumoniae strains showed resistance to ampicillin, and resistance to 3rd generation cephalosporins varied between 20.7% and 22.5%. Additionally, K. pneumoniae exhibited higher resistance to carbapenems (13.7-19.5%) compared to E. coli (2.18-2.42%). Antimicrobial resistance levels are generally lower than continental and national data, except for ampicillin and carbapenems (meropenem and ertapenem). K. pneumoniae strains are significantly more resistant than E. coli strains.

Comparison of BD Phoenix and disk diffusion to broth microdilution for determining cefepime susceptibility among carbapenem-resistant Enterobacterales.

There are increasing reports of carbapenem-resistant Enterobacterales (CRE) that test as cefepime-susceptible (S) or susceptible-dose dependent (SDD). However, there are no data to compare the cefepime testing performance of BD Phoenix automated susceptibility system (BD Phoenix) and disk diffusion (DD) relative to reference broth microdilution (BMD) against carbapenemase-producing (CPblaKPC-CRE) and non-producing (non-CP CRE) isolates. Cefepime susceptibility results were interpreted according to CLSI M100Ed32. Essential agreement (EA), categorical agreement (CA), minor errors (miEs), major errors (MEs), and very major errors (VMEs) were calculated for BD Phoenix (NMIC-306 Gram-negative panel) and DD relative to BMD. Correlates were also analyzed by the error rate-bounded method. EA and CA for CPblaKPC-CRE isolates (n = 64) were <90% with BD Phoenix while among non-CP CRE isolates (n = 58), EA and CA were 96.6%, and 79.3%, respectively. CA was <90% with DD for both cohorts. No ME or VME was observed for either isolate cohort; however, miEs were >10% for CPblaKPC-CRE and non-CP CRE with BD Phoenix and DD tests. For error rate-bounded method, miEs were <40% for IHigh + 1 to ILow - 1 ranges for CPblaKPC-CRE and non-CP CRE with BD Phoenix. Regarding disk diffusion, miEs were unacceptable for all MIC ranges among CPblaKPC-CRE. For non-CP CRE isolates, only IHigh + 1 to ILow - 1 range was acceptable at 37.2%. Using this challenge set of genotypic-phenotypic discordant CRE, the BD Phoenix MICs and DD susceptibility results trended higher (toward SDD and resistant phenotypes) relative to reference BMD results yielding lower CA. These results were more prominent among CPblaKPC-CRE than non-CP CRE.

Three cases of vulvovaginal candidiasis due to Candida nivariensis

Candida nivariensis is emerging as a highly resistant species of the Candida glabrata complex causing invasive and mucocutaneous infections. In this study, three cases of vulvovaginal candidiasis caused by C. nivariensis are described and identified by Internal Transcribed Spacer 1–2 sequencing. All isolates were susceptible in vitro to anidulafungin, micafungin, caspofungin, 5-flucytosine, posaconazole, voriconazole, itraconazole, amphotericin B, and showed dose-dependent susceptibility to fluconazole. In two patients, three doses of oral fluconazole were effective, while one patient developed clinical fluconazole resistance with a new relapse after 6 months. Increasing the weekly dose of fluconazole showed to be effective in this patient.

2115. Rezafungin Activity against Invasive Candidiasis Isolates Globally: Results from the 2022 Rezafungin Surveillance Program

Abstract Background Rezafungin (RZF) is a new echinocandin (ECH) approved by the US FDA to treat candidemia and invasive candidiasis (IC). Fluconazole (FLC) resistance (R) is a raising concern to treat IC, and ECHs are often used as first-line therapy. We evaluated the in vitro activity of RZF, caspofungin (CSF), micafungin (MCF), anidulafungin (ANF), and azoles against a global collection of 500 Candida isolates causing IC. Methods C. albicans (CA; 233 isolates; 46.6%), C. glabrata (CG; 102; 20.4%), C. parapsilosis (CP; 82; 16.4%), C. tropicalis (CT; 41; 8.2%), C. krusei (CK; 18; 3.6%), C. dubliniensis (CD; 15; 3.0%), and C. auris (CARS; 9; 1.8%) isolates were collected (1/patient) in 2022 from 52 medical centers located in Europe (EU; n=203; 19 centers), North America (NA; n=167; 14 centers), Asia-Pacific (AP; n=63; 8 centers), and Latin America (LA, n=67; 7 centers), identified by MALDI-TOF MS and/or sequencing and tested by CLSI broth microdilution. CLSI breakpoints (BP) were applied where available. CARS CDC tentative BPs were applied. Results RFZ exhibited activity against CA (Table) inhibiting 99.1% of the isolates overall, and 98.6%, 99.0%, 100%, and 100% of isolates from NA, EU, AP, and LA. Only 1 CA isolate (from US) displayed a FLC MIC value within the susceptible-dose dependent (SDD) category. CSP, ANF, and MCF showed S rates of 99.1% against CA. All but 1 CG isolate was susceptible (S) to RZF (99.0%S). CG S rates to CSP, ANF, MCF, and FLC were 98.0%, 98.0%, 97.1%, and 96.1% (SDD), respectively. All CP isolates were S to RZF, CSF, and MCF. However, only 84.1% of CP isolates were S to FLC. RZF and other ECHs inhibited all FLC-R isolates (10 total, 9 EU and 1 US). All CT and CK isolates were S to RZF and other ECHs. All CT were also S to azoles, and 94.4% of CK were S to VRC. Only RZF BPs are available by CLSI against CD (86.7%S). All CARS isolates (9 total; 4 EU, 3 NA, and 2 LA) were FLC-R; but 77.8% and 88.9% were S to RZF and other ECHs, respectively. Conclusion RZF demonstrated potent in vitro activity against invasive candidiasis isolates regardless of the region, and remained active against FLC-R CP and most of the FLC-R CARS isolates. Based on MIC50 and MIC90 results, RZF had similar activity to the other ECHs overall. Disclosures Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Paul Rhomberg, BS, MT(ASCP), bioMerieux: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Abby Klauer, BS, Melinta: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

Revision of fluoroquinolone breakpoints used for interpretation of antimicrobial susceptibility testing of canine bacterial isolates.

The fluoroquinolone antimicrobial agents, enrofloxacin and marbofloxacin, were US Food and Drug Administration (FDA) approved in the United States for use in dogs in 1988 and 1999, respectively. There have been many advances since then concerning the pharmacokinetic-pharmacodynamic (PK-PD) evaluation of fluoroquinolones, and there are data available on the susceptibility of targeted pathogens since the original approval. Using this information, the Clinical and Laboratory Standards Institute (CLSI) Veterinary Antimicrobial Susceptibility Testing Subcommittee (VAST) revised its antimicrobial susceptibility testing breakpoints. The previous breakpoints (used in older editions of CLSI standards) for enrofloxacin in dogs were susceptible (S), ≤ 0.5 µg/mL, intermediate (I) 1-2 µg/mL, and resistant (R) ≥ 4 µg/mL. The new breakpoints are S ≤ 0.06 µg/mL for a dose of 5 mg/kg, 0.12 µg/mL for a dose of 10 mg/kg, 0.25 µg/mL for a high dose of 20 mg/kg, and R ≥ 0.5 µg/mL. The breakpoints of 0.12 and 0.25 µg/mL represent a new susceptible-dose dependent (SDD) category. For marbofloxacin, previous breakpoints were S, ≤ 1 µg/mL, I 2 µg/mL, and R ≥ 4 µg/mL. The new breakpoints are S ≤ 0.12 µg/mL for a dose of 2.8 mg/kg, 0.25 µg/mL for a dose of 5.5 mg/kg (SDD), and R ≥ 0.5 µg/mL. The new breakpoints will be published in the next edition of CLSI-Vet01(S) and deviate considerably from the prior breakpoints. Laboratories are encouraged to revise their testing standards. These changes will likely reduce the unnecessary use of these fluoroquinolones in dogs.

Comparative qualitative and phytochemical composition and inhibitory potential of ethanolic seed extract of buchholzia coriacea (wonderful kola) on selected pathogens

The emergence of drug-resistant bacteria and the attendant effects on medical health has aggravated attention on traditional herbs as alternative remedy to conventional antibiotics in use. In this study, the comparative qualitative phytochemical screening and antimicrobial activity of aqueous, ethanolic and methanolic extract of Buchholzia coriacea seed on Staphylococcus aureus, Escherichia coli and Salmonella typhi was evaluated using standard methods for phytochemical screening and agar well diffusion method. The findings revealed that the ethanolic extract of B. coriacea seed had higher yield (6.90%) of the extract compared to the aqueous and methanolic extracts. The extracts revealed the presence of saponins, flavonoids, alkaloids, tannins, anthraquinone, terpenoids, steroids and glycosides. The ethanolic extracts showed significant dose dependent susceptibility among the solvents at varying concentrations (400 mg/ml, 200 mg/ml 100 mg/ml) against Staph aureus, S. typhi and E. coli ranging from 10.00mm to 18.00mm but less potent compared to the antibiotic control. The organisms studied were susceptible to the seed extracts, however, least antibacterial activity was observed with the aqueous extract of B. coriacea against S. typhi (12 mm) at 400mg/ml. This study suggests that these plant extracts which proved to be potentially effective against these organisms can be used as natural alternative preventives to control infections caused by these agents. However, further analysis of its biosafety and toxicity is needed to ascertain usage.

Cefepime in vivo activity against carbapenem-resistant Enterobacterales that test as cefepime susceptible or susceptible-dose dependent in vitro: implications for clinical microbiology laboratory and clinicians.

Carbapenem-resistant Enterobacterales (CRE) are a public health concern. Among these isolates, there are reports of isolates that test as cefepime susceptible or susceptible-dose dependent (SDD) in vitro despite presence of a carbapenemase. This study aimed to evaluate the pharmacokinetic/pharmacodynamic profile of cefepime against carbapenemase-producing (CP-CRE) and non-producing (non-CP-CRE) isolates with a range of cefepime MICs. Reference broth microdilution and modified carbapenem inactivation method (mCIM) were performed on genotypically characterized clinical CRE isolates. Ultimately, CP-CRE (n = 21; blaKPC) and non-CP-CRE (n = 19) isolates with a distribution of cefepime MICs (≤0.5 to >256 mg/L) were utilized in the murine thigh infection model. Mice were treated with cefepime human-simulated regimens (HSRs) representative of a standard dose (1 g q12h 0.5 h infusion) or the SDD dose (2 g q8h 0.5 h infusion). Efficacy was assessed as the change in bacterial growth at 24 h compared with 0 h control, where ≥1 log bacterial reduction is considered translational value for clinical efficacy. Among both cohorts of CRE isolates, i.e. CP-CRE and non-CP-CRE, that tested as SDD to cefepime in vitro, 1 log bacterial reduction was not attainable with cefepime. Further blunting of cefepime efficacy was observed among CP-CRE isolates compared with non-CP-CRE across both susceptible and SDD categories. Data indicate to avoid cefepime for the treatment of serious infections caused by CRE isolates that test as cefepime susceptible or SDD. Data also provide evidence that isolates with the same antibiotic MIC may have different pharmacokinetic/pharmacodynamic profiles due to their antimicrobial resistance mechanism.

Effiectiveness of Etiotropic Therapy in Patients with Chronic Recurrent Oral Candidiasis: a Randomized Controlled Clinical Trial

Background. Effective treatment of oral candidiasis is especially relevant due to the increase in patients with recurrent fungal lesions of the oral cavity caused by the growing number of Candidaspp. strains refractory to antimycotic drugs. The paper presents the results of the study on the clinical efficacy of etiotropic therapy in patients with chronic recurrent course of oral candidiasis.Objective. To assess the effectiveness of etiotropic therapy in patients with chronic recurrent oral candidiasis.Methods. A randomized controlled clinical trial was conducted in 56 patients with chronic recurrent oral candidiasis aged 45 to 74 years. Examination and treatment of patients was performed at the Department of Dentistry of Volgograd State Medical University and at Volgograd Regional Clinical Dental Polyclinic (Volgograd, Russia). Study timeline: September 2020 — November 2022. Patients were divided into 2 groups depending on the antimycotic drug used: in the main group, patients took voriconazole, in the control group — fluconazole. In addition to etiotropic therapy, patients of both groups were prescribed complex treatment aimed at stimulating the immune protection of the body, restoring vitamin and mineral deficiency, and correcting the imbalance of the oral ecosystem. The patients underwent local symptomatic therapy: antiseptic treatment with 0.06% chlorhexidine bigluconate solution, anesthesia with 15% lidocaine gel, and 3.44% retinol oil solution to stimulate epithelization. Oral sanitation and professional hygiene were recommended for patients to prevent oral candidiasis. In order to restore chewing function, patients were referred to prosthodontic treatment. The main reference point of the study was to assess the effectiveness of the therapy, both by clinical criteria and by evaluating the results of bacterial culture test in order to record the quantitative, species and susceptibility profile of Candidaspp. strains in 3 weeks, 6 and 12 months after the end of treatment. Analysis and statistical processing of the obtained data were performed using Excel program to MS Windows 10 (Microsoft Corp., USA).Results. The most common pathogenic agent of chronic recurrent forms of oral candidiasis is C. albicans (69.6%), followed by C. non-albicans species — C. krusei (10.7%), C. tropicalis (10.7%) and C. glabrata (8.9%). Analysis of the quantitative characteristics of Candidaspp. using bacterial culture test revealed a high and moderate number of Candidaspp. CFU in most clinical isolates before treatment, and no such cases in patients of the main group after treatment. The susceptibility test of yeast fungi to fluconazole and itraconazole showed the presence of isolates with dose-dependent susceptibility, and the resistance of individual samples C. krusei to fluconazole and C. glabrata (16.7% and 60%, respectively) and to itraconazole (33.3% and 100%, respectively). All Candidaspp. isolates were found to be highly susceptible to voriconazole. The efficacy of the combined treatment of an episode with voriconazole is 95.4%. Continued suppressive therapy with voriconazole resulted in relapse prevention in 98.3% of patients. Etiotropic therapy with voriconazole provided a stable clinical effect (92.7%) after a year of follow-up.Conclusion. The results of the study confirm the effectiveness of etiotropic therapy with voriconazole for chronic oral candidiasis complicated by a recurrent course, especially in case of detected C. non-albicans or fluconazole- and itraconazole-resistant C. albicans, or if it is impossible to identify the pathogenic agent.

Fungal Spectrum and Susceptibility Against Nine Antifungal Agents in 525 Deep Fungal Infected Cases.

Deep fungal infection has become an important cause of infection and death in hospitalized patients, and this has worsened with increasing antifungal drug resistance. A 3-year retrospective study was conducted to investigate the clinical characteristics, pathogen spectrum, and drug resistance of deep fungal infection in a regional hospital of Guangzhou, China. Non-duplicate fungi isolates recovered from blood and other sterile body fluids of in-patients of the clinical department were identified using biochemical tests of pure culture with the API20C AUX and CHROMagar medium. Antifungal susceptibilities were determined by Sensititre YeastOne® panel trays. In this study, 525 patients (283 female, 242 male) with deep fungal infection were included, half of them were elderly patients (≥60 years) (54.67%, n=286). A total of 605 non-repetitive fungi were finally isolated from sterile samples, of which urine specimens accounted for 66.12% (n=400). Surgery, ICU, and internal medicine were the top three departments that fungi were frequently detected. The mainly isolated fungal species were Candida albicans (43.97%, n=266), Candida glabrata (20.00%, n=121), and Candida tropicalis (17.02%, n=103), which contributed to over 80% of fungal infection. The susceptibility of the Candida spp. to echinocandins, 5-fluorocytosine, and amphotericin B remained above 95%, while C. glabrata and C. tropicalis to itraconazole were about 95%, and the dose-dependent susceptibility of C. glabrata to fluconazole was more than 90%. The echinocandins had no antifungal activity against Trichosporon asahi in vitro (MIC90>8 μg/mL), but azole drugs were good, especially voriconazole and itraconazole (MIC90 = 0.25 μg/mL). The main causative agents of fungal infection were still the genus of Candida. Echinocandins were the first choice for clinical therapy of Candida infection, followed with 5-fluorocytosine and amphotericin B. Azole antifungal agents should be used with caution in Candida glabrata and Candida tropicalis infections.

In-vitro evaluation of virulence markers and antifungal resistance of clinical Candida albicans strains isolated from Karachi, Pakistan

Candidiasis is a significant fungal infection with high mortality and morbidity rates worldwide. Candida albicans is the most dominant species responsible for causing different manifestations of candidiasis. Certain virulence traits as well as its resistance to antifungal drugs contribute to the pathogenesis of this yeast. This study was designed to determine the production of some virulence factors, such as biofilm formation and extracellular hydrolytic enzymes (esterase, coagulase, gelatinase, and catalase) by this fungus, as well as its antifungal resistance profile. A total of 304 clinical C. albicans isolates obtained from different clinical specimens were identified by a conventional diagnostic protocol. The antifungal susceptibility of C. albicans strains was determined by disk diffusion technique against commercially available antifungal disks, such as nystatin 50 μg, amphotericin B 100 unit, fluconazole 25 μg, itraconazole 10 μg, ketoconazole 10 μg, and voriconazole 1 μg. The assessment of biofilm formation was determined by the tube staining assay and spectrophotometry. Gelatinase, coagulase, catalase, and esterase enzyme production was also detected using standard techniques. A total of 66.1% (201/304) and 28.9% (88/304) of C. albicans strains were susceptible-dose dependent (SDD) to nystatin and itraconazole, respectively. Among the antifungal drugs, C. albicans strains showed high resistance to ketoconazole 24.7% (75/304); however, no statistically significant relationship between the clinical origin of C. albicans isolates and antifungal drug resistance pattern was detected. For virulence factors, the majority of the C. albicans strains actively produced biofilm and all hydrolytic enzymes. Biofilm formation was demonstrated by 88% (267/304) of the strains with a quantitative mean value 0.1762 (SD ± 0.08293). However, 100% (304/304) of isolates produced catalase enzyme, 69% (211/304) produced coagulase, 66% (197/304) produced gelatinase, and 52% (157/304) produced esterase enzyme. A significant relationship between the source of specimens and biofilm formation by C. albicans was observed; nevertheless, there was no significant relationship between different sources of C. albicans strains and the production of different enzymatic virulence factors. The study found that C. albicans strains have excellent potential to produce virulence markers and resistance to antifungals, which necessitates surveillance of these opportunistic pathogens to minimize the chances of severe invasive infections.

Phytochemical analysis, antiproliferative and antifungal activities of different Syzygium aromaticum solvent extracts

The outbreaks of fungal resistance to the different antifungal agents represent apublic health problem resulting in a significant morbidity and mortality. Furthermore, chemotherapy represents a serious issue in the cancer treatment worldwide. Accordingly, the efficiency of clove extracts as anticandidal and antitumoral agents was evaluated. Disk diffusion and 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) techniques were achieved to evaluate the antimycotic and antiproliferative activities of different clove extracts. The active phytochemicals of clove extracts were analyzed using gas chromatography mass spectrometry. Resistance ofCandida glabratastrain to clotrimazole was obvious whileC. albicansandC. tropicalisexpressed dose dependent susceptibility. In contrast,the clove acetonic extract expressed the highest antimycotic proficiency againstC. glabrata, C. albicans and C. tropicalisstrains with suppressive zones of 18.54 ± 0.24, 19.86 ± 0.34 and 22.6 ± 0.32 mm respectively. While, the ethanolic clove extract displayed the uppermost antiproliferative efficiency with relative IC50of6.80 µg/mL.Phytochemical analysis showed that eugenol compound was the most prominent active ingredient of clove acetonic and ethanolic extracts with corresponding proportions of46.53 and 54.71 % respectively.Anticandidal activity of the main active ingredient, eugenol, was significantly higher than that of clotrimazole, used as control, against C. glabrata strain (P ≤ 0.05). The proficiency of antimicrobial activity of clove and its major constituent, eugenol, againstcandidal strains supports utilizing these extracts in fabrication of anticandidal drugs especially against the resistantC. glabratastrain. Also, these extracts could be a source of adjuvant anticancer therapies due to their potent antiproliferative activity.

P088 Molecular identification and antifungal susceptibility of pathogenic yeasts from the China Antifungal Resistance Surveillance Trial (CARST-fungi) Study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesInvasive fungal diseases (IFDs) caused by yeast species have considerable morbidity and mortality, especially in immunocompromised hosts, and those with antifungal resistance represent a major clinical challenge. In order to have a comprehensive understanding of the characteristics of epidemiology and antifungal susceptibilities in clinical yeasts, the China Antifungal Resistance Surveillance Trial (CARST-fungi) study, a prospective national surveillance program for IFDs in mainland China, was conducted.MethodsThe CARST-fungi study encompassed nine ‘rank-A tertiary’ hospitals distributed throughout different cities in China in the year 2019-2020. All yeast isolates recovered from various clinical samples were subcultured and identified by sequencing of the internal transcribed spacer (ITS), 28S ribosomal subunit (D1/D2), and the intergenic spacer (IGS, for Trichosporon spp. and Cryptococcus spp.). Antifungal susceptibilities of fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (POS), caspofungin (CAS), anidulafungin (ANF), micafungin (MCF), and amphotericin B (AMB) against the yeast isolates were performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A4 broth microdilution method.ResultsA total of 269 nonduplicate yeast isolates from 261 patients were collected. About half of the yeast isolates (127, 47.9%) were recovered from blood, followed by ascetic fluid (46, 17.4%). C. albicans remained the most prevalent (120, 44.6%), followed by C. parapsilosis complex (50, 18.5%), C. tropicalis (40, 14.9%), and C. glabrata (36, 13.4%). Among C. albicans, 5 (4.2%), 11 (9.2%), 6 (5%), 10 (8.4%) isolates were resistant/non-wide-type (NWT) to FLC, ITC, VRC, and POS, respectively, and 9 (7.5%) isolates were cross-resistant to triazoles. As for C. parapsilosis complex, only 1 (2.4%) isolate of C. parapsilosis sensu stricto was cross-resistant to FLC and POS, while all the 9 C. metapsilosis isolates were wide-type (WT) to triazoles. However, only 45% (18/40) C. tropicalis were susceptible/WT to triazoles, and 12 (30%), 3 (7.5%), 8 (20%), 19 (47.5%) isolates were resistant/NWT to FLC, ITC, VRC, and POS, respectively, and 8 (20%) isolates were cross-resistant to triazoles. Among C. glabrata, 2 (5.6%) isolates were resistant to FLC and the remaining 34 isolates were susceptible-dose dependent (SDD), 20 (55.6%), and 8 (22.2%) isolates were resistant/NWT to VRC and POS, respectively, and 4 (10.3%) isolates were cross-resistant to triazoles. One isolate of Meyerozyma guilliermondii was NWT to POS. Except for 3 isolates of C. tropicalis exhibiting intermediate to CAS and ANF, and 2 isolates of C. glabrata were cross-resistant to CAS, MCF, ANF, which were also NWT to POS and defined as multidrug-resistant, other isolates of common Candida species were all susceptible to echinocandins. All yeast isolates tested in this study were WT to AMB (MICs ≤ 2 μg/ml). For less common species, 1 isolate of Rhodotorula mucilaginosa exhibited high MICs to echinocandins and FLC, and 1 isolate of Trichosporon asahii showed high MICs to all the antifungals tested except AMB.ConclusionAmong 269 yeast isolates from the CARST-fungi study, C. albicans remain the most predominant, followed by C. parapsilosis complex, C. tropicalis, and C. glabrata. Triazole-resistance is notable among C. tropicalis and C. glabrata. Multidrug-resistant isolates of C. glabrata and less common yeast have been emerging.

Focused Commentary; About Revision of CLSI Antimicrobial Breakpoints, 2018-2021

In recent years, antimicrobial resistance has been on the rise and infectious disease specialists and other clinicians face several challenges when treating patients with antimicrobial resistant infections. Clinical and Laboratory Standards Institute (CLSI) annually publishes guidelines for antimicrobial resistance tests, including revised antimicrobial breakpoints, because of the rise in antimicrobial resistance and changes to nomenclature owing to advances in whole genome sequencing technology. However, many laboratories use historical guidelines and do not follow the revised breakpoints. Moreover, the current breakpoints have limitations. This study evaluated the changes in antimicrobial breakpoints over the last 4 years, from 2018 to 2021. Here, I describe the microbiological and clinical background of the CLSI breakpoint revision and evaluate the advantages and limitations of new breakpoints with a review of large study data. In addition, I reviewed problems associated with each antimicrobial breakpoint and made suggestions for how they might be improved, for example, increasing or decreasing the minimum inhibitory concentration (MIC) or zone diameter, deleting or adding an S, I, or R category, introducing new concepts (such as susceptible-dose dependent (SDD)), and requesting more evaluation methods. Conclusions; CLSI annually publishes guidelines for antimicrobial resistance tests. I reviewed problems associated with each antimicrobial breakpoint for last 4 years, and made suggestions for how they might be improved.

Investigation of fluconazole susceptibility to Candida albicans by MALDI-TOF MS and real-time PCR for CDR1, CDR2, MDR1 and ERG11

BackgroundC. albicans is a pathogenic yeast that is the most common cause of fungal infections in humans. Unfortunately, the yeast’s resistance to the antifungal medication fluconazole (FLC) is increasing; furthermore, testing its susceptibility to FLC by conventional methods takes time, resulting in treatment failure. The susceptibility of C. albicans to FLC was investigated using MALDI-TOF Mass Spectrometry and Real-time PCR tests for CDR1, CDR2, MDR1 and ERG11. Overall, 32 C. albicans strains made up of four reference strains (three FLC susceptible [S] and one FLC resistant [R], one spontaneous mutant strain [FLC susceptible-dose-dependent (SDD)] and 27 clinical strains obtained from two Thai University Hospitals) were tested for susceptibility to FLC. The following tests were performed: SensititreYeastOne and broth microdilution method, FLC resistant expression mechanism by Real-time PCR, and the major peak determination by MALDI-TOF MS.ResultsThe change of CDR1 and CDR2 mRNA expression was only significantly observed in SDD and R strains. MALDI-TOF MS was performed after incubation for six hours; the change of mass spectral intensity at range 3376–3382 m/z (major peak) was significantly related to FLC susceptibility as SDD (decreased at 4 µg/mL and increased at 8 µg/mL), S (all increased), and R (all slightly decreased or no change). All 27 clinical strains showed FLC minimum inhibitory concentrations (MIC range 0.25-2 µg/mL), no change in CDR1 and CDR2 expression and S major peak type. The FLC resistant C. albicans with CDR1and CDR2 expression may possibly affect the change of mass spectral intensity at range 3376–3382 m/z.ConclusionsThe MALDI-TOF MS may be used to simultaneously classify and predict FLC resistant C. albicans strains associated with CDR1 and CDR2 expression. Further studies are essential to clarify the methodology and improve the reliability of this assay for routine diagnosis.

Daptomycin susceptibility testing and therapeutic use in enterococcal bloodstream infection (EBSI) in a setting with high rates of vancomycin-resistant Enterococcus faecium (VREfm).

There is in vitro and clinical evidence to suggest daptomycin has good activity against Enterococcus. In 2019, CLSI produced clinical breakpoints for Enterococcus spp. To describe the distribution of MICs of daptomycin for enterococcal bloodstream infection (EBSI) isolates in a large Scottish health board, the indications for local daptomycin susceptibility testing and daptomycin doses used in vancomycin-resistant Enterococcus faecium (VREfm) infection. We investigated all EBSIs over a 21 month period and identified isolates tested against daptomycin. We recorded the distribution of MICs, as well as indications for daptomycin susceptibility testing and information on daptomycin dosing, where it was used. There were 293 blood culture isolates of Enterococcus spp., of which 37 had daptomycin susceptibility testing performed, from 31 individual patients. Of the 293 isolates, 103 were E. faecium, of which 63 were VREfm. Daptomycin testing was indicated by vancomycin resistance in 24/37 isolates. All E. faecium isolates tested were in the CLSI 'susceptible dose-dependent (SDD)' range of MICs. All other Enterococcus spp. tested were in the 'susceptible' range. Twelve patients received daptomycin, and dosing information was recovered for 10. Nine of these patients received 8-12 mg/kg/day dosing. There were no recorded adverse drug reactions. Ten of 12 patients were alive at the time of data collection. Daptomycin MIC distribution for EBSI isolates suggests a high local rate of susceptibility, according to CLSI breakpoints, in a population with high rates of VREfm. CLSI-recommended doses of daptomycin were used, with encouraging survival outcomes.

Understanding and Application of Daptomycin-Susceptible Dose-Dependent Category for Enterococcus: A Mixed-Methods Study.

BackgroundIn 2018, the Clinical Microbiology Laboratory at our institution adopted updated daptomycin Enterococcus–susceptible dose-dependent breakpoints. While the introduction of susceptible dose-dependent (SDD) was intended to guide practice toward optimal dosing, the understanding and application of daptomycin SDD breakpoints for enterococci were unknown.MethodsThis mixed-methods study combined a clinician survey with a retrospective pre–post prescribing analysis. An 8-question survey was distributed to infectious diseases (ID) and internal medicine (IM) clinicians. A retrospective chart review of hospitalized adults with infections due to Enterococcus spp. was conducted before (pre-SDD) and after (post-SDD) adoption of SDD reporting for enterococci.ResultsSurvey response rates were 40 of 98 (41%) for IM and 22 of 34 (65%) for ID clinicians. ID clinicians scored significantly higher than IM clinicians in knowledge of SDD. Chart review of 474 patients (225 pre- vs 249 post-SDD) showed that daptomycin dosage following susceptibility testing was significantly higher post-SDD compared with pre-SDD (8.5 mg/kg vs 6.4 mg/kg; P < .001) with no difference in empiric dosing (6.3 mg/kg vs 6.2 mg/kg; P = .67). Definitive daptomycin use varied between the pre- and post-SDD periods (35.1% vs 16.9%; P < .001).ConclusionsThe survey revealed that ID clinicians placed more importance on and had more confidence in the SDD category over IM clinicians. SDD reporting was associated with a change in definitive daptomycin dosing. ID specialist involvement is recommended in the care of infections due to enterococci for which daptomycin is reported as SDD given their expertise.

Candida species cause vulvovaginitis and resistance to antifungals used for treatment

In this study were identified Candida species from vaginal secretion isolates, evaluated their in vitro antifungal susceptibilities, and correlated these features with antifungal agents prescribed for patients assisted in a primary care service. Species identification by Polymerase Chain Reaction showed that 36.5% of isolates were characterized as non-C. albicans species. In antifungal susceptibility tests most isolates were susceptible to ketoconazole, fluconazole, and itraconazole, although between 40% and 50% of isolates show resistance or dose-dependent susceptibility to miconazole and nystatin, respectively. Analysis of drugs prescribed to patients revealed that 34.2% of the isolates were considered resistant to agents used in treatment. Several Candida species can cause vulvovaginitis and exhibit different susceptibility profiles to antifungal drugs used in treatment. The identification of Candida species is relevant and useful to the epidemiological management of infections. The antifungal susceptibility test may also be useful for choosing most effective drug treatment for each patient.

CORRELATE BETWEEN BIOFILM FORMATION AND AZOLE ANTIFUNGAL SUSCEPTIBILITY AGAINST PLANKTONIC AND SESSILE CANDIDA ALBICANS CLINICAL ISOLATES

Objective: To assess the correlation between biofilm formation and azole antifungal susceptibility against plank tonic and sessile clinical isolates of C.albicans.&#x0D; Study Design: Prospective observational study.&#x0D; Place and Duration of Study: Combined Military Hospital Peshawar, from Jun 2016 to Sep 2017.&#x0D; Methodology: All standard microbiological procedures were carried out according to latest Clinical &amp; laboratory standard institute (CLSI) guidelines. After gram staining and presumptive identification on CHRO Magar Candida, the isolates were biochemically identified by API AUX Candida as C.albicans. Planktonic antifungal susceptibility was carried out by Kirby Bauer disk diffusion method on 300 C.albicans isolates. Broth microdilution method was used to determine Minimum inhibitory concentration (MICs) of plank tonic cells and micro titer assay was used for assessment of biofilm formation by C.albicans.&#x0D; Results: In planktonic antifungal susceptibility, fluconazole was susceptible against 195 (65%) and voriconazole against 241 (80%) C. albicans isolates. C. albicans was found susceptible dose dependent (SDD) to fluconazole in 28 (9%) and to voriconazole in 21 (7%) isolates. Seventy-seven (26%) and 38 (13%) C.albicans isolates were found fluconazole and voriconazole resistant, respectively. Sessile antifungal susceptibility was carried out through broth micro dilution method in which 160 (53%) were susceptible, 42 (14%) were susceptible dose dependent SDD and 98 (33%) were resistance to voriconazole, and 161 (54%) were susceptible, 36 (12%) were SDD and 103 (34%) were found resistant to fluconazole. Biofilm forming isolates of C.albicans were observed to be 285 (95%).The p-value is highly significance i.e. &lt;0.01 between......

Ceftaroline In-vitro Activity against Methicillin Resistant Staphylococcal Isolates in a Rural Tertiary Healthcare Centre

Introduction: The emergence of Methicillin Resistant (MR) staphylococcal infections had led to fewer therapeutic options. Ceftaroline fosamil is the only cephalosporin approved by United States of America (USA) Food and Drug Administration (FDA) till now which has activity against MR staphylococcal isolates. Until now, studies on in-vitro activity of ceftaroline are limited. Aim: To know the susceptibility pattern of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Resistant Coagulase Negative Staphylococci (MRCoNS) against ceftaroline in a rural tertiary health care centre. Materials and Methods: The present cross-sectional study was conducted in a tertiary care hospital in rural part of Karnataka between two months (July and August 2019). A total of 50 consecutive, non repetitive clinical isolates of MRSA and MRCoNS were obtained. Antibiotic susceptibility testing was done according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The Minimum Inhibitory Concentration (MIC) of ceftaroline was detected using E-strips (Biomerieux). The CLSI breakpoints applied for the interpretation of ceftaroline MIC- Sensitive: ≤1 μg/mL; Susceptible Dose Dependent (SDD): 2-4 μg/ mL and resistant ≥8 μg/mL). Staphylococcus aureus American Type Culture Collection (ATCC) 29213 was used as a quality control. Statistical analysis was done using microsoft excel. Percentages were used in this study to analyse variables. Results: Out of 50 MR staphylococcal isolates, 10 (20%) were MRSA and 40 (80%) were MRCoNS. Of the MR staphylococcal isolates tested, 49 (98%) were sensitive to ceftaroline. The MIC 50 and MIC 90 for the 50 MR staphylococci was 0.25 μg/mL and 1 μg/mL, respectively. Conclusion: Ceftaroline demonstrated a potent in-vitro activity against MR staphylococci. So it can be used as an effective drug in the treatment of such infections.