Agar Dilution Research Articles

Differentiation and prediction of the in vitro antifungal activity of Brazilian propolis against Fonsecaea pedrosoi using chemometrics and diffuse reflectance infrared Fourier Transform spectroscopy

The in vitro antifungal activity of ethanolic extracts of propolis was evaluated at five different concentrations in twelve Fonsecaea pedrosoi strains using agar dilution. Eight propolis extracts were collected from towns located in the central region of the state of Rio Grande do Sul, Brazil. Diffuse reflectance infrared Fourier Transform spectroscopy (DRIFTS) was employed to analyze the spectra of lyophilized propolis extracts. The use of chemometric tools allowed for the development of models with more relevant factors and with fewer variables. Partial least squares (PLS) regression was applied, allowing for the development of PLS-DRIFTS models to predict antifungal activity of all fungal strains with root mean square error of cross validation (RMSECV) lower to 5.00 mm2 and coefficient correlation of cross-validation (R2) upper to 0.99 with five to eight latent variables at 4000-2400 and 2200-600 cm-1. Concluding, DRIFTS models associated with chemometrics are powerful screening tools of propolis samples with fungistatic properties against Fonsecaea pedrosoi.

Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran

AbstractAntimicrobial disinfectants have been extensively used to control hospital-acquired infections worldwide. Prolonged exposure to bacteria could promote resistance to antimicrobial disinfectants. This study evaluated the antimicrobial activity of four commonly used disinfectants; triclosan, chlorhexidine digluconate, benzalkonium chloride, and formaldehyde against Acinetobacter baumannii clinical isolates. This study also determined the prevalence and association of efflux pumps encoding genes qacE, qacED1, emrA, and aceI with tolerance to disinfectants. A total of 100 A. baumannii isolates were included in the current study. The antimicrobial disinfectants' minimum inhibitory concentration (MIC) was determined using an agar dilution method. Genes involved in resistance to disinfectants were investigated by PCR method. The benzalkonium chloride MICs ranged between 32 and 128 μg mL−1, chlorhexidine digluconate 8–64 μg mL−1, triclosan 1–32 μg mL−1, and formaldehyde 128 μg mL−1. Overall, the highest MIC90 value was identified for formaldehyde (128 μg mL−1), followed by benzalkonium chloride and chlorhexidine digluconate (64 μg mL−1, each one) and triclosan (4 μg mL−1). In the present study, the qacE, qacED1, emrA, and aceI genes were found in 91%, 55%, 100%, and 88% of isolates, respectively. The qacG gene was not identified in our A. baumannii isolates. The qacED1 gene was associated with higher MICs for all disinfectants tested (P < 0.05), while the qacE and aceI genes were associated with higher MICs for benzalkonium chloride and chlorhexidine. This study indicated that triclosan is the most effective disinfectant against A. baumannii isolates.

Antimicrobial resistance and genomic characterization of Campylobacter isolates recovered from retailed poultry meat samples in 20 provinces of China in 2020

Objective: To understand the antimicrobial resistance and genome characteristics of Campylobacter isolates recovered from retailed poultry meat samples in 20 provinces in China in 2020. Methods: In 2020, 265 Campylobacter strains including 244 Campylobacter jejuni and 21 Campylobacter coli collected from retailed poultry meat samples in China were tested for antimicrobial resistance to 9 antimicrobial compounds by using the agar dilution method. Forty-two selected isolates were sent for whole genome sequencing and 38 high-quality genomes were analyzed for their antimicrobial resistance genes, virulence genes, sequence types and genetic diversity. Results: The resistance rates of Campylobacter isolates from poultry meats to tetracycline, nalidixic acid and ciprofloxacin were the highest (84%-100%), with 53.2% of the isolates showing multidrug resistance in this study. The resistance rates of C. coli to erythromycin, azithromycin, telithromycin, gentamicin and clindamycin were significantly higher than those of C. jejuni (P<0.05). The resistance genes conferring resistance to β-lactams (100%, 38/38), quinolones (94.7%, 36/38), tetracycline (81.6%, 31/38) and aminoglycosides (50%, 19/38) were the most frequently detected among 38 Campylobacter genomes. C. jejuni carried more virulence genes than C. coli. In total, 19 and 17 sequence types (ST) were obtained from 20 sequenced C. jejuni and 18 C. coli isolates, respectively, including 5 novel STs. The isolates showed a high genetic diversity based on their sequence types. Conclusion: The phenomenon of antimicrobial resistance in Campylobacter from poultry meat sources in China is relatively serious, and resistance and virulence genes are widely distributed in Campylobacter. There is genetic diversity in Campylobacter.

Occurrence of aflatoxigenic fungi and aflatoxins in maize grains and associated awareness and handling practices among farmers and traders in South Sudan

Maize is a popular staple food among the urban population of South Sudan. However, due to inadequate food safety surveillance and enforcement systems, there is limited information on the aflatoxin safety of maize grains at farm and market levels. Therefore, this study aimed at assessing aflatoxins awareness and handling practices of maize grains among randomly selected farmers (n=30) and traders (n=30) as well as determining the moisture content, Aspergillus species counts and aflatoxins contamination. A cross-sectional descriptive study was carried out in Magwi maize producing areas, Juba retail/wholesale markets and Nimule border points of South Sudan. Moisture content, fungal and aflatoxins contamination in maize grain samples were determined using dry air oven method, dilution plating technique, and High-Performance Liquid Chromatography (HPLC), respectively. The respondent results showed that farmers (97%) and traders (83%) were unaware of aflatoxins. About 83% of the farmers dried their maize grains on tarpaulins, for 4 - 5 days (77%). In addition, most farmers (77%) stored their maize grain bags on raised platforms, whereas most traders (73%) stored grains on bare ground. All the maize grains met the required moisture content limit (below 13.5%). Maize from Nimule main park had the highest levels of contamination with Aspergillus flavus (9 log CFU/g), Aspergillus parasiticus (12 log CFU/g), aflatoxin B1 (505.56 µg/kg) and total aflatoxins (1,032.19 µg/kg). Maize from Gudele market was contaminated with Aspergillus parasiticus (12 log CFU/g), and aflatoxin B1 (76.55 µg/kg), and had a total aflatoxin content of 94.09 µg/kg. Omeo farmers’ maize grains had the least levels of contamination of Aspergillus parasiticus (6 log CFU/g), aflatoxin B1 (4.39 µg/kg), and total aflatoxins (7.83 µg/kg). In addition, Aspergillus flavus was not detected from Omeo farmers’ maize grains and no aflatoxins were detected from Agoro and Paluonganyi farmers’ grains. This study recommends wider aflatoxins awareness and regular aflatoxin screening of maize grains by the relevant stakeholders in South Sudan. Key words: aflatoxins, aflatoxigenic fungi, awareness, farmers, handling practices, maize, South Sudan, traders

An insight into genes responsible for fosfomycin resistance among uropathogens of asymptomatic bacteriuria during pregnancy: A North Indian study.

Asymptomatic bacteriuria (ASB) is a common finding during pregnancy. Effective antibiotic treatment could reduce its adverse effects on both mother and fetus. However, emerging antimicrobial resistance limits the treatment options. Fosfomycin might be a promising drug in this regard, as its resistance is still low. The aim of the study was to determine the antimicrobial susceptibility pattern of fosfomycin in isolates causing ASB by disc diffusion and agar dilution (in selected isolates), determine minimum inhibitory contribution (MIC) by agar dilution in isolates resistant by disc diffusion and detect the genes responsible for fosfomycin resistance. This was a 2-year study carried in the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital (JNMCH), Aligarh Muslim University (AMU), Aligarh. A total of 10 252 urine samples from asymptomatic pregnant females (18-45 years) attending the antenatal care (ANC) outpatient department (OPD) were submitted. Identification of pathogen and antimicrobial susceptibility testing (AST) was carried out as per standard methods of CLSI. There was phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA) and other Staphylococcus species (MRSS), high-level aminoglycoside resistance (HLAR), vancomycin resistant Enterococci (VRE) and S. aureus (VRSA), extended spectrum β-lactamase (ESBL) and carbapenem-resistant Enterobacterales (CRE). All the fosfomycin-resistant isolates (by disk diffusion) were tested by agar dilution. Conventional PCR was performed for murA, fosA, uhpT and glpT genes on all resistant isolates. In this study, the prevalence of ASB among pregnant females was 1173(11.4 %), in which Escherichia coli 495(42 %) was the predominant organism. The overall sensitivity of fosfomycin among Gram-positive cocci (GPC) and Gram-negative bacilli (GNB) was 99 % and 97.6 %, respectively. MRSA and MRSS accounted for 50 (66.6 %) and 71 (76 %), respectively. The highest rates of MIC >2048 µg ml-1 were shown by most isolates (mainly E. coli ) on agar dilution. PCR studies revealed four E. coli strains possessed both murA (also present in one K. pneumoniae strain) and glpT genes. While only one isolate ( E. faecalis ) was positive for fosA gene. But none of the strain possessed the uhpT gene. According to this study, murA and glpT genes were more frequent than fosA. We cannot comment on the prevalence and regional distribution of fosfomycin-resistant genes based on this preliminary study. Therefore, more Indian studies should be carried out to create awareness about the presence of genes in a particular area.

In Vitro Evaluation of Corn Silk Extract as a Potential Alternative Therapy for Vulvovaginal Candidiasis

Abstract Aim To determine the effectivity of corn silk extract in inhibiting the growth of C. albicans. Materials and Methods Corn silk used in this study was Zea mays L. var. indurata and was collected from Desa Suka Makmur, Deli Serdang, Sumatera Utara Province, Indonesia. Tested C. albicans is C. albicans from VVC patients, obtained from the Microbiology Laboratory of Faculty of Medicine, Universitas Sumatera Utara. Preparation of this study included corn silk extraction by maceration method with 96% ethanol, followed by dilution with 10% DMSO (Dimethyl Sulfoxide) into concentrations of 75%, 50%, 25%, and 12.5%. Well diffusion method was used to detect growth inhibition of C. albicans and agar dilution was used to determine MIC (Minimum Inhibitory Concentration) and MFC (Minimum Fungicidal Concentration) of corn silk extract. Data were statistically analyzed with Kruskal-Wallis analysis. Results From this study, corn silk extract of all concentrations showed an inhibitory effect against C. albicans. The mean diameter of inhibition zones for each concentration respectively from highest to lowest concentration were 27.4875 ± 0.3838 mm, 26.7250 ± 0.2533 mm, 25.7250 ± 0.2598 mm, and 24.9375 ± 0.2462 mm. Kruskal-Wallis analysis showed significant results (p-value 0.001). MIC and MFC were 1.5625% and 6.25%, respectively. Conclusions Corn silk extract of concentrations 75%, 50%, 25%, and 12,5% have antifungal activity against C. albicans. The higher the concentration of corn silk extract, the higher the efficacy in inhibiting the growth of C. albicans.

Assessment of the gradient diffusion method for fosfomycin susceptibility testing in Staphylococcus spp. and Enterococcus spp. isolated from the urine of companion dogs in Thailand.

The agar dilution method is the approved method for determining the minimum inhibitory concentration (MIC) in fosfomycin susceptibility testing, whereas the broth dilution method is not recommended. This study aimed to investigate the potential of the gradient diffusion method as a more convenient alternative to agar dilution method for MIC evaluation, particularly for the susceptibility testing of Staphylococcus spp. and Enterococcus spp. to fosfomycin. A total of 194 isolates of Staphylococcus spp. and Enterococcus spp. were collected from urine samples of dogs diagnosed with bacterial cystitis. Bacterial identification and susceptibility to multiple antibiotics were tested using the Vitek 2 automated system. The susceptibility to fosfomycin was compared between agar dilution (reference method) and the gradient diffusion method. We assessed the agreement rates and errors between the two approaches by analyzing the MIC data. Staphylococcus pseudintermedius (98.7%) and Enterococcus faecalis (80.0%) exhibited high fosfomycin susceptibility rates, whereas Enterococcus faecium exhibited a lower susceptibility rate (38.5%). The gradient diffusion method demonstrated unacceptably low essential agreement (EA) rates (>90%) but acceptable categorical agreement (CA) rates (≥ 90%) for S. pseudintermedius (83.54% EA and 97.47% CA) and coagulase-negative staphylococci (CoNS) such as Staphylococcus chromogenes, Staphylococcus hominis, and Staphylococcus simulans (85.00% EA and 95.00% CA). Enterococcus spp. had an acceptable EA of 93.75%, but an unacceptably low CA rate of 82.81%, with a minor error rate of 17.19%. No significant errors were observed for Staphylococcus and Enterococcus spp. The gradient diffusion method reliably determines MICs and interpretative breakpoints (S, I, R) for S. pseudintermedius. However, its applicability to CoNS and enterococci may be limited due to unacceptable errors.

Fosfomycin activity in vitro against Escherichia coli strains isolated from urine specimens

Abstract Urinary tract infections are caused mostly by uropathogenic Escherichia coli strains. The aim of this paper was to assess the activity of fosfomycin in vitro against 74 E. coli strains isolated from urine samples of outpatients with acute cystitis. Minimum inhibitory concentration (MIC) was determined by the recommended method of serial drug dilution in Mueller-Hinton agar supplemented with glucose-6-phosphate (25 mg/L). The estimated MIC values were in the range of 1-32 mg/L. The prevalence of fosfomycin-sensitive strains was 45.9%, while that of fosfomycin-resistant strains – 54.1%. The time-kill assay was performed for a chosen clinical strain (MIC = 1 mg/L) in Mueller-Hinton broth also supplemented with glucose-6-phosphate in the presence of various concentrations of fosfomycin (0.5-64 mg/L). The bacterial population density was expressed as log CFU (colony forming units)/mL. The decrease of the bacterial population viability after 6h incubation was found with ∆log CFU/mL = 1.05-5.02, depending on fosfomycin concentration. However, bactericidal effect of this antibiotic (∆log CFU/mL &gt;3) was observed only at the highest concentrations (32-64 mg/L). Moreover, after prolonged incubation (24 h), the re-growth of bacterial population revealed as the increase of its viability (∆log CFU/mL = 1.03-3.03) was observed at fosfomycin concentration in the range 0.5-32 mg/L, but not at 64 mg/L. This phenomenon may be due the presence of subpopulations of spontaneous mutants sensitive only to higher concentrations of this antibiotic comparing to MIC. The presented data confirm a need to monitor the sensitivity of uropathogenic E. coli strains to fosfomycin.

Development of Novel Spray-dried Microparticles to Treat Cystic Fibrosis: A Tri-drug Approach.

Cystic fibrosis is the predominant autosomal recessive disorder known to reduce life expectancy. Research findings indicate that around 60 to 70% of adult individuals with this condition carry infections of Pseudomonas aeruginosa. The ongoing research investigates the potential synergy of merging ivacaftor and ciprofloxacin to address bacterial infections. The two drugs were spray-dried into microparticles, which were then coated with Lsalbutamol and were to be delivered by a dry powder inhaler. Microparticles were generated by applying the spray drying method, utilizing bovine serum albumin and L-leucine in their preparation. Additionally, L-salbutamol was mixed and adsorbed onto the surface of the spray-dried microparticles, and it acted as a bronchodilator. The microparticles produced via spray drying exhibited a particle size measuring 1.6 ± 0.04 μm, along with a polydispersity ratio of 0.33. Their zeta potential measured -27.3 ± 1.1 mV, while the mass median aerodynamic diameter of these microparticles was 3.74 ± 0.08 μm. SEM, XRD, and FTIR studies confirmed the entrapment of ivacaftor and ciprofloxacin. The morphology was observed by SEM and TEM scans. Antibacterial synergy was confirmed through the agar broth and dilution method, and the formulation's safety was established based on the outcomes of the MTT assay. Using spray-dried microparticles containing ciprofloxacin, ivacaftor, and L-salbutamol presents a novel approach to the treatment of cystic fibrosis.

Preliminarily investigation on the In-vitro Antimicrobial, Antioxidant and Antidiabetic Activities of Sorindeia warneckei Extracts

Abstract Phytochemicals that are naturally occurring in plants are known to be potentially bioactive, having anti-oxidative, antimicrobial and enzyme inhibiting potentials. This study investigated the antimicrobial, antioxidant and antidiabetic activities of the methanol and dichloromethane extracts of the leaf and stem Sorindeia warneckei. The plant extract showed the presence of saponins, flavonoids, terpenoids, tannins, glycosides, coumarins, steroids, alkaloids, anthraquinones and reducing sugars. The antioxidant potential was evaluated using DPPH radical scavenging assay while the antidiabetic activity was evaluated using an in-vitro α-amylase inhibitory assay. The DPPH free radical scavenging activity was expressed in % inhibition with L Ascorbic acid as standard showing IC50 0.69μg/ml with methanol extract while dichloromethane leaf extract having the highest activity at 0.45μg/ml. The α-amylase assay showed that the extracts exhibited potent alpha amylase inhibitory activity in a dose dependent manner with the leaf methanol extract having an IC50 of 2.33µg/L. The antimicrobial screening using the agar diffusion and agar dilution methods against the bacteria isolates Staphylococcus aureus, Salmonella typhirimurium, Escherichia coli and Streptococcus mutans and the fungal isolates Candida albicans, Aspergillus niger, Aspergillus flavus and Rhizopus oligosporus showed a good positive result. Preliminary investigation reveals that Sorindeia warneckei, contain bioactive compounds that possess promising pharmaceutical potentials that could be exploited.

Molecular Characteristics, Antimicrobial Susceptibility, Biofilm-Forming Ability of Clinically Invasive Staphylococcus aureus Isolates.

This study aimed to investigate the molecular characteristics, antimicrobial resistance, and biofilm-forming ability of Staphylococcus aureus isolates from invasive infections. A total of 92 non-repetitive S. aureus isolates from invasive infections were analyzed by Multi-locus Sequence Typing (MLST), spa typing, and chromosomal cassette mec (SCCmec) typing. Antibiotic susceptibility testing was performed using the disk diffusion and agar dilution methods. Biofilm-forming ability was assessed using crystal violet assay. The presence and expression of biofilm-associated genes were examined using PCR and RT-qPCR. Among the 55 Methicillin-resistant S. aureus (MRSA) and 41 Methicillin-sensitive S. aureus (MSSA) isolates, ST59 (43.6%) predominated in MRSA, while ST7 (39.0%) was most common in MSSA. As expected, MRSA exhibited higher antibiotic resistance rates compared to MSSA isolates. Biofilm formation assays revealed that the majority of isolates (88.5%) produced biofilms, with 26.0% classified as strong producers (OD570 ≥ 1.0) and 62.5% as weak producers (0.2 ≤ OD570<1.0). MSSA exhibited a higher biofilm-forming ability than MRSA (P < 0.01), with variations across clones. Notably, ST7 isolates displayed greater biofilm-forming ability than other sequence types (ST59, ST5, and ST239). RT-qPCR results revealed that ST7 isolates exhibited higher expression levels of icaA compared to other sequence types. This study revealed significant molecular heterogeneity among invasive S. aureus isolates, with ST59 and ST7 as dominant clones. The strong biofilm-forming capacity of ST7 merits concern given its rising prevalence regionally. Continuous surveillance of emerging successful lineages is critical to help guide infection control strategies against invasive S. aureus infections.

Molecular and Clinical Features of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus in Tertiary Care Hospitals in South India

Objectives: This study aimed to detect heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) among methicillin-resistant S. aureus (MRSA) isolated from healthcare-associated infections and identify staphylococcal cassette chromosome mec (SCCmec) types. Methods: This study was conducted from February 2019 to March 2020 and included patients admitted in 4 tertiary care hospitals in Karnataka, India. Isolation and identification of MRSA were done using standard bacteriological methods. Antimicrobial susceptibility testing was done using Kirby–Bauer disc diffusion; macrolide-lincosamide-streptogramin B phenotypes were identified using the D test. The minimum inhibitory concentration (MIC) of vancomycin was determined using agar dilution. hVISA were confirmed by the modified population analysis profile-area under the curve test. SCCmec types and the Panton- Valentine leukocidin (pvl) gene were detected using multiplex polymerase chain reaction. Results: Of 220 MRSA stains, 14 (6.4%) were hVISA. None of the MRSA isolates was vancomycin-intermediate or -resistant and all hVISA were susceptible to linezolid and teicoplanin. The macrolide-streptogramin B phenotype was present in 42.9% of hVISA; 92.9% of the hVISA strains had vancomycin MIC in the range of 1–2 μg/mL. Majority of the hVISA and vancomycin-susceptible MRSA were isolated from patients with skin and soft tissue infections. SCCmec III and IV were present in 50% and 35.7% of hVISA, respectively; 14.3% of the hVISA harboured SCCmec V. Conclusion: The prevalence rate of hVISA among MRSA was 6.4%. Therefore, MRSA strains should be tested for hVISA before starting vancomycin treatment. None of the isolates was vancomycin-intermediate or -resistant and all the hVISA strains were susceptible to linezolid and teicoplanin. The majority of the hVISA were isolated from patients with skin and soft tissue infections and harboured SCCmec III and IV. Keywords: MRSA; Hospital Infection; Molecular Typing; Vancomycin; India.

Formulation and evaluation of herbal toothpaste containing eupatorium triplinervisleaf extract

Eupatorium triplinervis (Asteraceae),popularly known as Ayapana , is widely used in folk medicine, due to its antibacterial, antifungal, analgesic, antianorexic, antiparasitic, anthelmintic and sedative properties. The present study was focused on evaluating the anti-bacterial action of the formulated herbal toothpaste containing Eupatorium triplinervis leaf extract and determining the best formula for preparing the toothpaste. The extraction was conducted by the soxhlet apparatus using methanol as solvent and was subjected to preliminary phytochemical screening. The antimicrobial activity of the extracts was performed and the minimum inhibitory concentration (MIC) values of the extract were reported against three organisms- Staphylococcus aureus ( gram positive), E. coli ( gram negative) and oral flora by agar dilution assay. Also, the antibacterial activity by agar well diffusion method was determined for the prepared toothpaste against all the above three bacteria. The phytochemical screening shows the presence of coumarins in the methanolic extract which were responsible for the antibacterial activity of the extract. The formulations shows considerable zoneof inhibition when compared with a marketed toothpaste formulation (Dant kanti). Thus, this toothpaste preparation has antibacterial properties and so can be used for gingivitis and other bacterial disorders of the oral cavity.

In vitro susceptibility test of Cladosporium cladosporioides isolates from Argentinian tomato crops against commercial fungicides

Tomato cultivation is an important agricultural activity in northeast of Argentina, and disease control is indispensable for its production. The purpose of the current study was to identify two fungi isolated from tomato plants cultivated in greenhouses, with symptoms of leaf mould disease and a strain of the genus Cladosporium from a culture collection, and evaluate their in vitro susceptibility to four commercial fungicides. Macro and microscopic examination, molecular characterisation and sequence analysis were applied for identification. Broth dilution and spread plate methods were used to determine the minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC). The active ingredients of the products were azoxystrobin+difenoconazole, trifloxystrobin+tebuconazole, chlorothalonil and metalaxyl-M+mancozeb. The results were processed using the Kruskal-Wallis method. The isolates were identified as Cladosporium cladosporioidesa-c; consequently, lesions found on tomato plants did not corrspond to Cladosporium fulvum. There was a significant statistical difference between the obtained values. Qualitatively, the three strains had a similar behaviour for chlorothalonil (MIC values: 0.25 - 0.5 μg/ml, MFC values: 4 μg/ml). In all cases, tests with metalaxyl-M+mancozeb yielded higher values than those achieved for chlorothalonil (MIC values: 8 μg/ml, MFC values: 8- 32 μg/ml). trobilurin-formulated fungicides were less effective against C. cladosporioidesa-b (MIC values: 16-256 μg/ml, MFC values: &gt;64 μg/ml). C. cladosporioidesc was the most sensitive isolate. The information about the presence of a non-frequent fungus and its fungicide susceptibility, would be useful for establishing control strategies and enhance production.

565. Antimicrobial susceptibility profiles of Acinetobacter species in blood: 10-year experience from a major United States reference laboratory

Abstract Background Antimicrobial resistance (AMR) is a threat to healthcare, and infections with antimicrobial resistant organisms carry significant morbidity and mortality. Owing to high rates of AMR, the Centers for Disease Control and Prevention listed Acinetobacter baumannii as a top priority pathogen for research and development of new antimicrobial agents. Antimicrobial susceptibility testing is challenging for this organism, and susceptibility profiles are variable, leading to need of a potential guidance document with assistance in selecting effective empiric therapeutic regimens. Methods We retrospectively reviewed the microbiology and antimicrobial susceptibility testing (AST) results for Acinetobacter spp. from blood culture isolates submitted to Mayo Clinic Laboratories from December 2012 to 2022. AST was performed using agar dilution and interpretations following Clinical &amp; Laboratory Standards Institute (CLSI) guidelines. Isolates were identified by MALDI mass spectrometry and/or 16S sequencing, as available (Figure 1).Figure 1:Isolate identification Isolate identification description. Isolates were analyzed based on this classification used in out laboratory. Results 804 blood culture isolates were included. A. baumannii/calcoaceticus complex (27%) was most frequently isolated, followed by A. lwoffii (22%), and showed the highest resistance across all antimicrobials (73% susceptible or below) (Figure 2). A. baumannii/calcoaceticus complex showed rising resistance rates over time from 2012 to 2022 for meropenem, minocycline and ampicillin-sulbactam (Figure 3). Cefiderocol non-susceptible rate was 35% for A. baumannii/calcoaceticus complex.Figure 2:Antimicrobial susceptibility profiles for Acinetobacter spp. Description of susceptibility profiles for Acinetobacter spp. isolates, based on the classification used by our laboratory. Antimicrobials are presented at the top and isolates on the left. Number of isolates analyzed are presented in the table with the percentage of the isolates susceptible to each antibiotic class in parenthesis.Figure 3:Acinetobacter baumannii/calcoaceticus complex isolates and resistance, trend Trend of isolates per year and resistance percentage to meropenem, colistin, minocycline and amp-sulb. Number of isolates per year at the base of each bar; percentage of resistance for antimicrobials expressed on lines. Amp-sulb = ampicillin-sulbactam. Conclusion A. baumannii/calcoaceticus complex showed high levels of resistance ( &amp;gt;75%) across multiple antimicrobials and increasing rates of resistance over time from 2012 to 2022. A high number of cefiderocol non-susceptible organisms is also concerning (Figure 2). Disclosures Audrey N. Schuetz, MD, Merck: Advisor/Consultant

682. Novel rpoB mutations and trends in clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility

Abstract Background Fidaxomicin use is increasing to treat Clostridioides difficile infection (CDI) following clinical treatment guideline updates placing it as first-line therapy. This puts a selection pressure on the development of fidaxomicin resistance, which has been associated with in vitro fitness costs compared to wildtype isolates. Here we aimed to assess the epidemiology of reduced susceptibility (RS) to fidaxomicin and prevalence of rpoB mutations in a clinical cohort. Methods Stool samples from patients with CDI were collected from two different healthcare systems in Houston, TX between 2016-21. Fidaxomicin minimum inhibitory concentrations (MIC) were determined via agar dilution susceptibility testing following CLSI standards. RS was defined as MIC &amp;gt; 0.5 mg/L based on the EUCAST [T]ECOFF. A subgroup of 40 isolates with RS underwent Sanger sequencing to detect rpoB single nucleotide polymorphisms (SNPs). Results Of 535 patients included, 114 (21.3%) had infecting strains with fidaxomicin RS (MIC50 = 0.5 mg/L, MIC90 = 1.0 mg/L). Fidaxomicin RS was more common in RT 027 (49.4%, 41/83) compared to non-027 strains (16%, 68/425; P &amp;lt; 0.001). There were no differences in the proportions demonstrating RS across collection years (P=0.18) or based on location in or out of the Texas Medical Center (P=0.29). Mean MICs were similar between isolates collected before and after the IDSA/SHEA guideline update in 2018 (P =0.1). rpoB SNP mutations were detected in 12.5% (5/40) of isolates at 7 novel base positions; no SNPs were detected at previously reported position 3428. Conclusion RS to fidaxomicin occurred in one fifth of clinical isolates with no changes in incidence between 2016-21. Several new rpoB SNPs were observed in isolates with RS. Studies are needed to determine potential association(s) between fidaxomicin exposure and MIC changes. Disclosures Anne J. Gonzales-Luna, PharmD, BCIDP, Cidara Therapeutics: Grant/Research Support|Ferring Pharmaceuticals: Personal Fees|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support Kevin Garey, PharmD, MS, BCIDP, Acurx Pharmaceuticals: Grant/Research Support|Cidara Therapeutics: Grant/Research Support|Ferring Pharmaceuticals: Advisor/Consultant|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support|Vedanta Therapeutics: Grant/Research Support

Exploring Alternative Treatment Choices for Multidrug-Resistant Clinical Strains of Helicobacter pylori in Mongolia.

Helicobacter pylori is a pathogen related to severe diseases such as gastric cancer; because of rising antimicrobial-resistant strains, failure to eradicate H. pylori with antibiotics has increased worldwide. Multidrug-resistant H. pylori and gastric cancer is common in Mongolia; therefore, we aimed to explore alternative antimicrobial treatments and the genomes of resistant strains in this country. A total of 361 H. pylori strains isolated from patients in Mongolia were considered. Minimal inhibitory concentrations for two fluoroquinolones (ciprofloxacin and moxifloxacin), rifabutin, and furazolidone were determined via two-fold agar dilution. Genomic mutations in antibiotic-resistant strains were identified by next-generation sequencing using the Illumina Miseq platform and compared with genes from a reference H. pylori strain (26695). The resistance rate of H. pylori strains to quinolones was high (44% to ciprofloxacin and 42% to moxifloxacin), and resistance to rifabutin was low (0.5%); none were resistant to furazolidone. Most quinolone-resistant strains possessed gyrA gene mutations causing amino acid changes (e.g., N87K, A88P, and D91G/Y/N). While one rifabutin-resistant strain had amino acid-substituting mutations in rpoB (D530N and R701C), the other had three novel rpoB mutations; both rifabutin-resistant strains were sensitive to furazolidone. Overall, our findings suggest that rifabutin and/or furazolidone may be an alternative, effective H. pylori treatment in patients who have failed to respond to other treatment regimens.

Molecular epidemiology, phenotypic and genomic characterization of antibiotic-resistant enterococcal isolates from diverse farm animals in Xinjiang, China

Multidrug-resistant (MDR) bacteria in farm environments can be transferred to humans through the food chain and occupational exposure. Enterococcus infections caused by linezolid resistant enterococci (LRE) are becoming more challenging to treat as their resistance to antibiotics intensifies. Therefore, this study investigated the molecular epidemiology, phenotypic and genomic characterization of enterococci in seven species of farm animals (sheep, chicken, swine, camel, cattle, equine, pigeon) anal swab from Xinjiang, China by agar dilution method, polymerase chain reaction (PCR), whole-genome sequencing (WGS) and bioinformatics analysis. A total of 771 samples were collected, 599 (78 %) were contaminated with Enterococcus spp., among which Enterococcus faecalis (350/599) was dominant. Antimicrobial susceptibility testing showed that high resistance was observed in rifampicin (80 %), tetracycline (71 %), doxycycline (71 %), and erythromycin (69 %). The results of PCR showed the highest prevalent antibiotic resistance genes (ARGs) were aac(6′)-aph(2″) (85 %), followed by tet(M) (73 %), erm(B) (62 %), and aph(3′)-IIIa (61 %). Besides, 29 optrA-carrying E. faecalis isolates belonging to 13 STs (including 3 new alleles) were detected, with ST714 (31 %, 9/29) being the dominant ST type. The phylogenetic tree showed that optrA-carrying E. faecalis prevalent in the intensive swine farm is mainly caused by clonal transmission. Notably, optrA gene in Enterococcus spp. isolate from camel was first characterized here. WGS of E. faecalis F109 isolate from camel confirmed the colocalization of optrA with other five ARGs in the same plasmid (pAFL-109F). The optrA-harboring genetic context is IS1216E-fexA-optrA-erm(A)-IS1216E. This study highlights the prevalence of MDR Enterococcus (≥88 %) and four ARGs (≥75 %) in swine (intensive farming), cattle (commercial farming), and chickens (backyard farming) are high and also highlights that optrA-carrying E. faecalis of farm animals incur a transmission risk to humans through environment, food consumption and others. Therefore, antibiotic-resistant bacteria (ARB) monitoring and effective control measures should be strengthened and implemented in diverse animals.

Evaluation of Minimum Inhibitory Concentration of Heavy Metals Contained in Packaging Material Digest on Prominent Gut Microbiota.

Several scientific investigations have revealed that the leaching of metals from packaging material into the packed food is an unavoidable process. Hence, this study is aimed at investigating the effect of leached heavy metals from food packing materials on normal human gut flora. We analysed the effect of vanadium, arsenic, cadmium, and mercury present in digested packaging materials (DPM) on standard strains of Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70063, and Enterococcus faecalis ATCC 29212. The minimum inhibitory concentration (MIC) of laboratory-grade heavy metal salts and heavy metals present in DPM was determined by the agar dilution method. For all four bacteria, the MIC of cadmium and arsenic in the DPM was 7 μg/ml and 1.6 μg/ml, respectively. The MIC of mercury in DPM was 1.6 μg/ml for E. coli, K. pneumoniae, and E. faecalis and 1.4 μg/ml for P. aeruginosa. MIC of vanadium for E. coli, P. aeruginosa, and E. faecalis was 2.2 μg/ml, and for K. pneumoniae was 2.0 μg/ml. The difference in MICs of heavy metals in DPMs and heavy metal salts was not statistically significant. MICs were within CODEX-specified permissible levels. Though heavy metals in packaging material have not shown a deleterious effect on representative human gut flora, there is scope to study their effect on the gut microbiome. Thus, understanding the risk of heavy metal ingestion through unknown sources and avoiding any possible ingestion is crucial to preventing chronic diseases.

Bacitracin enhances ceftriaxone susceptibility of the high-level ceftriaxone-resistant gonococcal FC428 clone

ABSTRACT Neisseria gonorrhoeae is a multidrug-resistant bacterial pathogen for which ceftriaxone mono-antimicrobial therapy and ceftriaxone plus azithromycin dual-antimicrobial therapy are the only remaining effective therapies. However, ceftriaxone efficacy is threatened by the global dissemination of the high-level ceftriaxone-resistant FC428 clone, while azithromycin has in recent years been removed from the recommended dual therapy in some countries as a result of increasing (high-level) azithromycin resistance. Inclusion of an alternative second antimicrobial could be an effective strategy to protect ceftriaxone from (further) resistance development. In this study, we investigated gonococcal susceptibility to bacitracin and determined its synergistic anti-gonococcal activity with ceftriaxone. Gonococcal susceptibility to bacitracin was investigated for 449 contemporary clinical isolates using the agar dilution method. Bacitracin displayed consistent activity against the N. gonorrhoeae strain collection, with a minimum inhibitory concentration (MIC) range of 2–32 mg/L and a MIC 90 of 32 mg/L. Furthermore, bacitracin was bactericidal in time-kill assays, particularly against the tested FC428-associated isolate, which showed 10 5 -fold inactivation after 8 hours of exposure to a 4× MIC dose. Importantly, bacitracin and ceftriaxone displayed synergistic activity, with a fractional inhibitory concentration index ≤0.5 for the majority of tested strains. Finally, spot assays showed strong synergistic activity against the FC428-associated isolates, with up to 200-fold reduced plating efficacy for bacitracin combined with ceftriaxone compared with ceftriaxone only. In conclusion, bacitracin shows synergistic anti-gonococcal activity with ceftriaxone and might therefore be an interesting candidate for inclusion in a dual-antimicrobial therapy with ceftriaxone. IMPORTANCE Ceftriaxone-based antimicrobial therapies for gonorrhea are threatened by waning ceftriaxone susceptibility levels and the global dissemination of the high-level ceftriaxone-resistant gonococcal FC428 clone. Combination therapy can be an effective strategy to restrain the development of ceftriaxone resistance, and for that purpose, it is important to find an alternative antimicrobial to replace azithromycin, which has recently been removed in some countries from the recommended ceftriaxone plus azithromycin dual-antimicrobial therapy. Ideally, the second antimicrobial should display synergistic activity with ceftriaxone. We hypothesized that bacitracin might display synergistic activity with ceftriaxone because of their distinct mechanisms targeting bacterial cell wall synthesis. In this study, we showed that bacitracin indeed displays synergistic activity with ceftriaxone against Neisseria gonorrhoeae . Importantly, strains associated with the FC428 clone appeared to be particularly susceptible to the bacitracin plus ceftriaxone combination, which might therefore be an interesting dual therapy for further in vivo testing.