E-test Method Research Articles

English

Susceptibility of Streptococcus pneumoniae (pneumococci) to oxacillin and ceftriaxone was compared using disk diffusion and E-test methods. A total of 206 children attending Gertrude’s Children’s Hospital (GCH) were recruited. Sterile Copan Flocked Swabs were used to obtain nasopharyngeal swabs. Samples were inoculated on gentamicin blood agar and initial identification was done on the basis of colony morphology. Optochin test was performed to definitively identify the isolates as pneumococci. Antibiotic testing was done using disk diffusion and E-test methods on Muller Hinton agar enriched with 5% defibrinated sheep blood. A total of 42 (20%) isolates were recovered from the samples. Based on disk diffusion method, 74 and 40% of the isolates were resistant to oxacillin and ceftriaxone while; on the basis of E-test, 45 and 14% of the isolates were resistant to oxacillin and ceftriaxone, respectively. About 40% (n=17) of isolates that had zone diameters ≥ 20 mm which is considered susceptible to oxacillin by disk diffusion, had MICs ≤0.06 µg/ml correspondingly susceptible by E-test. Most isolates deemed susceptible to (≤0.5 µg/ml) ceftriaxone by E-test (72%, n=30) also exhibited susceptibility (≥27mm) to the antimicrobial agent by disk diffusion. E-test presents more sensitive results compared to disk diffusion. Isolates that exhibit resistance to penicillin and ceftriaxone by disk diffusion should be confirmed by E-test before being reported as resistant.   Key words: E-test, disk diffusion, antibiotic resistance, ceftriaxone and oxacillin.

Vancomycin-resistant Enterococcus faecium in Algeria: phenotypic and genotypic characterization of clinical isolates.

vancomycin-resistant Enterococcus faecium (VREfm) is a major public health problem worldwide. The aim of our study was to determine the microbiological, epidemiological and molecular characteristics of VREfm isolated in north-central, eastern and western Algeria. a collection of 48 VREfm isolated from September 2010 to April 2017 in several Algerian hospitals were studied. Minimum inhibitory concentrations (MICs) were determined by E-test method according to CLSI guidelines. the detection of van genotype of all strains was performed by PCR. Clonal relationship of five VREfm targeted by region were characterized using multilocus sequence typing (MLST). All isolates have multidrug-resistance (MDR) and were resistant to at least five classes of antibiotics; however, all were susceptible to tigecycline and daptomycin with MIC50 at 0.094 µg/mL and 2 µg/mL respectively. All strains belonged to vanA genotype and have high level of resistance to vancomycin and teicoplanin. MLST revealed two sequence types (STs): ST80 (from the four regions of Algeria) and ST789, both belonging to the former hospital-adapted clonal complex CC17. the alarming dissemination of MDR E. faecium vanA and the ST80 in several regions of Algeria suggest a clonal spread of VREfm strains, which urgently require implementation of adequate infection control measures.

Categorizing Susceptibility of Clinical Isolates of Candida auris to Amphotericin B, Caspofungin, and Fluconazole by Use of the CLSI M44-A2 Disk Diffusion Method.

We evaluated the CLSI M44ed3E disk diffusion method compared with the CLSI M27ed4 broth microdilution method for caspofungin and fluconazole and the Etest method for amphotericin B to categorize susceptibility of 347 clinical isolates of Candida auris Utilizing the zone diameter cutoffs established here, we observed overall categorical agreement between the two methods. For caspofungin, concordant results were observed for 98% of isolates, with <1% very major and 1% major errors. For fluconazole, concordant results were observed for 91% of isolates, with 1% very major and 8% major errors. For amphotericin B, concordant results were observed for 74% of isolates, with <1% very major errors and 25% major errors. The disk diffusion approach provides an accurate method for determining the susceptibility of C. auris for caspofungin and fluconazole and for identification of at least 75% of amphotericin B-susceptible isolates.

Antifungal Susceptibility Testing of Dermatophytes by ABDD and E-Test, a Comparative Study

Aim: The objective of this study was to isolate, identify, and explore the in-vitro antifungal susceptibility pattern of dermatophytes isolated from clinically suspected cases of dermatophytosis (tinea infections) attending the Dermatology Department at J.S.S Hospital. Methods: This study was conducted at JSS Medical College and Hospital from December 2016 to December 2017. Clinical samples (e.g., skin scrapings and hair stumps) were collected under aseptic precautions. The identification of dermatophytes was performed through microscopic examination using 10%, 20% & 40% potassium hydroxide (KOH) and culture on Sabouraud dextrose agar (SDA), SDAac, PDA and Dermatophyte test medium (DTM). All dermatophytes isolates were subjected to antifungal susceptibility testing using the agar-based disk diffusion (ABDD) and E-test method against Terbinafine, Itraconazole, Fluconazole, and Griseofulvin. Data were analyzed by using Chi square test. Results: A 100 samples were studied, 46% tinea corporis, 2%tinea cruris, 9% tinea pedis, 5% tinea faciei. The dermatophytes isolated were Trichophyton rubrum 11 (35%), Trichophyton mentagrophyte 8 (25%), Trichophyton tonsurans 5 (16%), Microsporum gypseum 3 (10%), Trichophyton verrucosum 2 (6%), Trichophyton violaceum 1 (3%) and Microsporum audouinii 1 (3%). Out of 31 dermatophytes 17 were sensitive to all four antifungal agents within the range of FLC (2 - 6 mcg/ml), ITR (0.125 - 2), TER (0.125 - 2), and GRI (0.125 - 2), 5 isolates were resistant in which 2 were resistant to FLC (64.256 mcg/ml), 2 isolates were resistant to TER 2 (32.38 mcg/ml), 1 isolate was resistant to both GRI and TER (16.32 mcg/ml) and 9 isolates were within the intermediate range. Conclusion: Every patient with a tinea infection should be properly studied for a mycological examination and should be treated accordingly. Dermatophyte test medium is more useful as an identification medium in the isolation of dermatophytes. The ABDD method appears to be a simple, cost-effective, and promising method for the evaluation of antifungal susceptibility of dermatophytes. E-test method is the most sensitive method due to the fact that quantitative MICs can be obtained directly from the E-strip. However, the E-test method is expensive and difficult in defining the precise borders of the inhibition zones in dermatophytes.

Comparative Assessment of Disk Diffusion, E-test and Broth Dilution Methods for Determining Colistin Susceptibility in Acinetobacter Species

Introduction: Colistin is the ultimate reserve drug for gram- negative bacilli especially non fermenting bacilli like Acinetobacter species and only therapeutic option available against carbapenem resistant bacteria. Clinical and Laboratory Standards Institute (CLSI) recommended broth dilution method for colistin susceptibility testing but this method require expertise and very time consuming and is also not feasible to routinely perform in clinical laboratories. In the background of ever expanding problems of drug resistance coupled with increasing use of colistin, it becomes essential to find a testing method which is faster, accurate and can be used as an alternative to broth dilution method. Aim: To assess disk diffusion, E-test and broth dilution method to determine colistin susceptibility in Acinetobacter species. Materials and Methods: This cross-sectional observational study was done in the Department of Microbiology, ABVIMS and Dr RML Hospital, New Delhi, India, from November 2016 to March 2018. Various clinical specimens were received from wards and Intensive Care Unit (ICU) and Acinetobacter species were isolated and identified following standard protocol. All strains were screened by disc diffusion test for various antibacterial drugs and among them 50 MDR strains were selected. Colistin susceptibility by disc diffusion and Epsilometer test (E-test) were done and compared against the reference broth dilution method following. Minimum Inhibitory Concentration (MIC) of colistin for the isolates were determined using macro broth dilution method using colistin sulphate. Chi-square test was used to investigate distributions of categorical variables. Results: All 50 strains were tested for colistin susceptibility by disc diffusion method. Among the 50 isolates, 10 isolates (20%) showed resistance to colistin by disc diffusion method while none of the isolate was found resistant by E-test or broth dilution method. High error rate and poor concordance of disc diffusion with reference to broth dilution method rules out the utility of disk diffusion method in clinical laboratories. Conclusion: A complete agreement between the results of E-test and broth dilution method makes E-test a suitable alternative to broth dilution test in clinical laboratories.

Point Mutations at gyrA and gyrB Genes of Levofloxacin Resistant Helicobacter pylori Strains and Dual Resistance with Clarithromycin.

Spontaneous point mutations in genes encoding gyrA/B subunits of DNA gyrase are responsible for fluoroquinolone resistance. We aimed to determine the clarithromycin and levofloxacin resistance phenotypically in H. pylori strains and to investigate the mutations responsible for levofloxacin resistance and the effects of these mutations on dual antibiotic resistance. A total of 65 H. pylori isolates were included. The E-test method was used for the clarithromycin and le-vofloxacin antimicrobial susceptibility test. Real-time PCR was used to detect the point mutations. Twenty-four (36.9%) of 65 H. pylori strains were phenotypically resistant to clarithromycin and 14 (21.5%) to levofloxacin. The phenotypic levofloxacin resistance rate of strains with Asn87Lys and Asp91Asn mutations were significantly higher (gyrA gene) (p < 0.05). The phenotypic levofloxacin resistance rate of strains with Arg484Lys and Asp481Glu mutations were significantly higher (gyrB gene) (p < 0.05). The Asn87Lys mutation increased the risk of phenotypes being resistant to levofloxacin 70.156 times and Asp91Asn mutation increased 125,427 times higher. Seven (10.8%) of 65 H. pylori strains showed dual resistance to both levofloxacin and clarithromycin. The rate of being dual resistant with A2143G mutation (clarithromycin resistance) was found to be significantly higher (p < 0.05). The Asn87Lys and Asp91Asn mutations in the gyrA gene had a phenotypically enhancing effect on levofloxacin resistance, while the presence of Asp481Glu and Arg484Lys mutations in the gyrB gene did not. The existence of dual resistance was developed with the increase in clarithromycin and levofloxacin resistance rates.

Antimicrobial resistance patterns of Acinetobacter baumannii using the E-test method.

The opportunistic pathogen, Acinetobacter baumannii is a gram negative, obligate aerobic, non-fermentative coccobacillus responsible for a variety of infections. It has the ability to develop resistance to many antibiotics. Antimicrobial resistance of this organism has become a worldwide problem that limited therapeutic options. Surveillance of antimicrobial drug resistance is substantially a great issue to guide empirical treatment. This study aimed to determine the antimicrobial resistance pattern of Acinetobacter baumannii. In this retrospective study, the antibiotic resistance of 88 Acinetobacter isolates to 12 antibiotics was measured during one year using the E-Test (MIC, France) method in Mueller Hinton agar (Conda, Spain) plates. Species identification was determined by VITEK2 automated system (bioMerieux, Inc. Durham, NC27712 USA). The data was analyzed using Spss version 22 software (SPSS Inc. Chicago, IL). The most isolated Acinetobacter baumannii was isolated from the wound (98.9%). The frequency of antibiotic resistance in Acinetobacter isolates was as follows: Ticarcillin (96.6%), Ceftazidime (96.6%), Cefepime (96.6%), Piperacillin/Tazobactam (95.5%), Meropenem (94.3%), Ciprofloxacin (94.3%), Levofloxacin (93.1%), Trimethoprim/Sulfamethoxazole (89.8%), Gentamicin (86.4%), Tobramycin (79.5%), Rifampicin (38.7%), Colistin (7%). Of all the isolates 97.7% were identified as having a MDR phenotype based on the definition that 86 isolates of 88 Acinetobacter baumannii isolates exhibited resistance to carbapenem or resistance to at least one agent in three or more antibiotic classes. Acinetobacter baumannii isolates showed the highest sensitivity to Colistin and the lowest sensitivity to Ticarcillin, Ceftazidime and Cefepime. It has high resistance [96.6%] to: Ticarcillin, Ceftazidime and Cefepime in Acinetobacter baumannii isolates. According to this study we suggest that we could use Colistin and rifampicin to empirical treatment infections caused by Acinetobacter baumannii.

1586. A Comparative Analysis of In Vitro Susceptibilities of Vancomycin-Resistant Enterococci Against Doxycycline, Minocycline, Tigecycline, Eravacycline and Omadacycline

BackgroundVancomycin-resistant Enterococci (VRE) are nosocomial pathogens that cause significant morbidity and mortality especially among patients with chronic medical conditions, critical illness and prolonged hospitalizations. Majority of human infections are caused by two species— E. faecium and E. faecalis, and which can acquire resistance to ampicillin, aminoglycosides, and vancomycin. Our aim was to study the susceptibility profile of the VRE strains to the tetracycline group of antibiotics on isolates collected from our local hospital. Older tetracyclines and their novel derivatives are included in this study.MethodsEighty preserved isolates of VRE were tested against five tetracyclines, i.e. doxycycline, minocycline, tigecycline, eravacycline and omadacycline. Antimicrobial susceptibility testing was performed using the E-test method in accordance to CLSI guidelines. Isolates were then classified as either susceptible, intermediately susceptible or resistant based on established breakpoints.ResultsEighty isolates (54 VRE. faecium and 26 VRE. faecalis) were included in the study. Out of 54 E. faecium isolates, 52 (96.3%) were susceptible to tigecycline, 15 (27.8%) were susceptible to minocycline, 14 (25.9%) were susceptible to doxycycline, 42 (77.8 %) were susceptible to omadacycline, and 52 (96.3%) were susceptible to eravacycline. Out of 26 E. faecalis isolates, 26 (100%) were susceptible to tigecycline, 2 (7.6%) were susceptible to minocycline, 2 (7.6%) were susceptible to doxycycline, 2 (7.6%) were susceptible to omadacycline, and 25 (96.15%) were susceptible to eravacycline.ConclusionTigecycline and eravacycline exhibited better in vitro antimicrobial activity against vancomycin-resistant E. faecium and E. faecalis when compared to doxycycline and minocycline. Omadacycline showed a relatively favorable susceptibility profile for E. faecium, but less favorable for E. faecalis. Results of this study will be useful to incorporate in the local antibiogram and will guide antimicrobial stewardship efforts. These findings will not only add to existing knowledge on susceptibility profiles of the novel tetracyclines, i.e. eravacycline and omadacycline, but also on their applicability in the clinical setting.Disclosures All Authors: No reported disclosures

Molecular Epidemiology and Risk Factors of Clostridium difficile ST81 Infection in a Teaching Hospital in Eastern China.

BackgroundThe prevalence of Clostridium difficile causes an increased morbidity and mortality of inpatients, especially in Europe and North America, while data on C. difficile infection (CDI) are limited in China.MethodsFrom September 2014 to August 2019, 562 C. difficile isolates were collected from patients and screened for toxin genes. Multilocus sequence typing (MLST) and antimicrobial susceptibility tests by E-test and agar dilution method were performed. A case group composed of patients infected with sequence type (ST) 81 C. difficile was compared to the non-ST81 infection group and non CDI diarrhea patients for risk factor and outcome analyses.ResultsThe incidence of inpatients with CDI was 7.06 cases per 10,000 patient-days. Of the 562 C. difficile isolates, ST81(22.78%) was the predominant clone over this period, followed by ST54 (11.21%), ST3 (9.61%), and ST2 (8.72%). Toxin genotype tcdA+tcdB+cdt- accounted for 50.18% of all strains, while 29.54% were tcdA-tcdB+cdt- genotypes. Overall, no isolate was resistant to vancomycin, teicoplanin or daptomycin, and resistance rates to meropenem gradually decreased during these years. Although several metronidazole-resistant strains were isolated in this study, the MIC values decreased during this period. Resistance rates to moxifloxacin and clindamycin remained higher than those to the other antibiotics. Among CDI inpatients, longer hospitalization, usage of prednisolone, suffering from chronic kidney disease or connective tissue diseases and admission to emergency ward 2 or emergency ICU were significant risk factors for ST81 clone infection. All-cause mortality of these CDI patients was 4.92%(n=18), while the recurrent cases accounted for 5.74%(n=21). The 60-day mortality of ST81-CDI was significantly higher than non-ST81 infected group, while ST81 also accounted for most of the recurrent CDI cases.ConclusionThis study revealed the molecular epidemiology and risk factors for the dominant C. difficile ST81 genotype infection in eastern China. Continuous and stringent surveillance on the emerging ST81 genotype needs to be initiated.

Detection of vancomycin susceptibility among methicillin resistant staphylococcus aureus in a tertiary care hospital

Introduction: Staphylococcus aureus infections in current times have become challenging to treat because of advent of Methicillin Resistant Staphylococcus aureus (MRSA) strains which are concurrently resistant to a wide panel of drugs and posing a threat to clinicians and microbiologists globally. The optimal drug for treatment of such MRSA infections is vancomycin but strains with augmented Minimum Inhibitory concentration (MIC) for this drug also have surfaced. Objectives: To know the frequency of MRSA isolates in various clinical samples with their antimicrobial sensitivity patterns and to equate agar dilution and E-test methods for MIC determination of vancomycin to MRSA strains. Materials and Methods: A total of 50 non repeat clinical isolates of staphylococcus aureus isolates were collected from various clinical specimens and were tested for methicillin resistance using the cefoxitin disc diffusion test (30mg). All MRSA isolates were tested for specific MIC by agar dilution and E-test methods. Results: 29 (58%) isolates were resistant to cefoxitin (MRSA). 13.8% isolates had MIC of 4mg/ml for vancomycin (VISA) by both agar dilution and E-test methods. However by agar dilution method 25 (86.2%) isolates exhibited vancomycin MIC of ? 2 mg/ml and by E-test 68.9% of the isolates showed MIC ? 2 mg/ml. Conclusion: Multidrug resistant MRSA strains are on the rise and alternate drug of choice for these infections; vancomycin also is showing increased MIC so prudent use of this drug is advocated. E-test can detect MRSA strains with intermediate MIC values useful for detection of MIC creep so that vancomycin can be used rationally.

Current trends of antifungal susceptibility pattern of dermatomycosis in a tertiary care hospital by Etest and VITEK-2 methodologies

Dermatomycosis affect the superficial layers of the skin, nails and hair and are often caused by dermatophytic molds, candida & non dermatophytic molds. However, over the last decade, an increasing number of non– dermatophyte filamentous fungi & non albicans Candida have been recognized as agents of skin and nail infections in humans, producing lesions clinically similar to those caused by dermatophytes. Although tissue invasion by them is a rare possibility, they are important as a public health problem particularly in the immunocompromised. There are limited studies on the efficacy of antifungal agents against dermatophytes. This study was conducted to test the efficacy of 3 systemic antifungal agents viz. Voriconazole, Itraconazole & Fluconazole using the E-test method and 2 systemic antifungals viz. Itraconazole and Voriconazole by VITEK-2. Three different species of dermatophytes which were isolated from the clinically suspected cases were Trichophyton mentagrophytes, T.rubrum and M.gypseum. According to the obtained results, Itraconazole and Voriconazole showed the lowest MIC range while Fluconazole had the highest MIC range for most fungi tested. Determining the resistance pattern is especially necessary to assist clinicians in treating superficial fungal infections more effectively. This will minimize side effects and prevent development of antifungal resistance & treatment failures. Keywords: Dermatomycosis, Antifungal resistance, Etest, VITEK-2, Voriconazole, Itraconazole, Fluconazole.

Prevalence of virulence factors and antifungal susceptibility testing of Candida species isolated from clinical specimens

Introduction: The main aim of our study is to isolate and identify Candida species from clinical samples and to detect the virulence markers and also to determine their susceptibilities to various antifungal agents. Materials and Methods: Eighty Candida isolates from respiratory samples were included in the study. Detection of various virulence markers of candida spp such as Phospholipase, Proteinase, Hemolysin and Biofilm production was carried out by Phenotypic methods. Antifungal susceptibility testing of the isolated yeasts for Amphotericin B, Itraconazole, Fluconazole, Caspofungin and Voriconazole was determined by E-test methods. Results: In our study, most common spp isolated was Candida albicans(65%) followed by Candida tropicalis(15%) and Candida glabrata(10%).Prevalence percentage of virulence factors such as Hemolysin, Proteinase, Biofilm and Phospholipase production in Candida spp were 85%, 77.5%, 68.8% and 45% respectively. The susceptibility pattern of Candida isolates to antifungal agents showed highest resistant rate of 33.8% for Itraconazole and lowest resistant rate of 2.5% for Caspofungin. Conclusion: Presence of virulence factors in Candida species might indicate invasiveness and its relation with infection. Hence the significance of virulence determination and antifungal susceptibility testing should be adopted as a routine procedure in the laboratory. Keywords: Candida species, E-test, Hemolysin Biofilm formation, Proteinase, phospholipase activity.

Antibiotic susceptibility pattern among clinical isolates of staphylococcus aureus with special reference to vancomycin

Introduction: Staphylococcus is a major pathogen of community and hospital acquired infections. Vancomycin is used in MRSA caused infections. Emergence of VISA and VRSA has been of great concern in clinical aspects. Materials and Methods: All clinical samples were processed in the laboratory according to standard procedure. Inoculated plates were incubated at 37? C for 24-48 hours. Only S. aureus isolates were included in the present study. Antibiotic susceptibility testing was done by Kirby-Bauer disc diffusion method using a panel of drugs. Cefoxitin disc was used to identify methicillin resistance. The MIC of vancomycin for MRSA isolates was carried out by Agar dilution method and E-test according to standard methods. Heteroresistance to vancomycin was detected by using BHI screen agar. Results: 190 S. aureus were isolated from various clinical samples. Most of the isolates were resistant to amoxyclav (96.2%) followed by ciprofloxacin (84.2%), erythromycin (33.2%), Clindamycin (31.2%), Cotrimoxazole (14.6%), Teicoplanin (4.2%), Mupirocin (2.1%) and none of the isolates were resistant to linezolid. Out of 190 S. aureus isolates, 97 (51.1%) were identified as MRSA. None of the isolates were resistant to vancomycin by agar dilution method and E-test method. Four out of 97 (4.1%) MRSA isolates showed intermediate susceptibility to vancomycin. Among the isolates with MIC of 2µg/ml, 5 (19.2%) showed heteroresistance to vancomycin by BHI screen agar method. Conclusion: Rapid identification of patients harboring VRSA, VISA or hVISA and adherence to infection control protocols are very important in controlling the dissemination of these pathogens. Keywords: MRSA, VISA, VRSA, Heterointermediate resistant Staphylococcus aureus (hVISA), Agar dilution method, E-test.

Antimicrobial Resistance of Lactobacillus johnsonii and Lactobacillus zeae in Raw Milk

Lactobacillus johnsonii and Lactobacillus zeae are among the lactobacilli with probiotic properties, which occur in sour milk products, cheeses, and to a lesser extent in raw milk. Recently, resistant strains have been detected in various species of lactobacilli. The aim of the study was to determine the incidence of resistant Lactobacillus johnsonii and Lactobacillus zeae strains in various types of raw milk. A total of 245 isolates were identified by matrix-assisted laser desorption/ionization mass spectrometry and polymerase chain reaction methods as Lactobacillus sp., of which 23 isolates of Lactobacillus johnsonii and 18 isolates of Lactobacillus zeae were confirmed. Determination of susceptibility to selected antibiotics was performed using the E-test and broth dilution method, where 7.3% of lactobacilli strains were evaluated as ampicillin-resistant, 14.7% of isolates as erythromycin-resistant, and 4.9% of isolates as clindamycin-resistant. The genus Lactobacillus johnsonii had the highest resistance to erythromycin (34.8%), similar to Lactobacillus zeae (33.3%). Of the 41 isolates, the presence of the gene was confirmed in five Lactobacillus johnsonii strains and in two strains of Lactobacillus zeae. The presence of resistant strains of Lactobacillus johnsonii and Lactobacillus zeae is a potential risk in terms of spreading antimicrobial resistance through the food chain.

Antibiotic analysis and whole genome sequencing of two nocardia farcinica strains causing joint infection

Objective: To analyze the molecular characteristics and antibiotic susceptibility of two strains of Nocardia farcinica isolated from patients with joint infection using whole genome sequencing. Methods: Two strains of Nocardia farcinica causing knee-joint infections in two elderly patients were collected in January 2020. Whole genome sequencing was used to determine the nocardia species. Drug sensitivity test was performed using the micro-broth dilution and E-test method according to CLSI M24 guideline. ABRicate was used to analyze drug resistance and virulence genes. Snippy and other bioinformatic tools were used for genomic comparison, and to construct SNP homologous tree. Results: The clinical isolates in this study were both Nocardia farcinica. Antimicrobial susceptibility test showed the isolates were resistant to ceftriaxone, cefepime, cefotaxime and trimethoprim/sulfamethoxazole (TMP/SMX). Imipenem, linezolid and amoxicillin-clavulanic acid showed good activity. Four antibiotic resistance genes including class A β-lactamase gene far-1, RNA polymerase binding protein gene RbpA, multi-drug resistance efflux pump transcription activator gene MtrA and regulatory transcription factor gene vanR-O were identified in the Nocardia farcinica genomes, which conferred resistance to beta-lactams, rifampicin, macrolides and vancomycin respectively. No acquired TMP/SMX resistance genes were identified. There are multiple missense mutations in the dihydrofolate reductase family genes. Four virulence genes of icl, mbtH, phoP, and relA that are homologous to Mycobacterium tuberculosis were found. SNP homologous tree analysis showed the two Nocardia strains were closely related, and there were only ten SNP sites, six compound substitutions and one deletion mutation between them. Conclusions: Whole genome sequencing technology is helpful to explore the molecular characteristics and resistance mechanisms of Nocardia species. Nocardia farcinica has a trend of spreading in China. Resistance to TMP/SMX is worthy of attention. The mutation of genes involved in the metabolic pathway of dihydrofolate might be one of multiple TMP/SMX resistance mechanisms.

Changing trends of antimicrobial susceptibility and resistance mechanisms to quinolones in typhoidal salmonellae isolated from India in last 5 years

Background: Antimicrobial resistance in enteric fever is a major therapeutic challenge and there is a need to monitor the pattern of resistance to the antityphoidal agents and detection of molecular mechanisms of resistance.The study was designed to monitor the antimicrobial susceptibility of Salmonella enterica serovars Typhi and Paratyphi A isolated from patients of enteric fever during 2014 to 2019 and detection of antimicrobial resistance mechanisms to quinolones. Methods and materials: Total 281 strains isolated from January 2014 to September 2019 were selected for the study. Antimicrobial susceptibility was done as per CLSI guidelines (2019) for amoxicillin, chloramphenicol, co-trimoxazole, ciprofloxacin, ceftriaxone and azithromycin. Minimum inhibitory concentration (MIC) determination for ciprofloxacin, ceftriaxone and azithromycin were done by E-Test method according to manufacturer's (ABs Biodisk, Sweden) instructions. DNA sequencing was done for gyrA, gyrB, parC and parE genes to find mutations in quinolone resistance determining region (QRDR). Results: Out of total 281 strains included in the study 214 (76.2%) were S. Typhi and 67 (23.8%) were S. Paratyphi A. As per CLSI 2019, antimicrobial susceptibility for chloramphenicol, ampicillin and cotrimoxazole was 0.7% (2/281), 1.42% (4/281) and 1.06% (3/281) respectively. Only 6/214 (2.8%) were susceptible for S. Typhi and no strain was found to be susceptible in S. Paratyphi A. Azithromycin susceptibility was 100% in S. Typhi, for S. Paratyphi there is no breakpoint available. Ciprofloxacin MIC50 and MIC90 values found to be 0.25 μg/mL and 1 μg/mL in S. Typhi and 0.5 μg/mL and 6 μg/mL in S. Paratyphi A. Ceftriaxone MIC50 and MIC90 values found to be 0.023 μg/mL and 0.5 μg/mL in both S. Typhi and S. Paratyphi A. For azithromycin MIC50 and MIC90 were 2 μg/mL and 6 μg/mL for S. Typhi and 4 μg/mL and 12 μg/mL for S. Paratyphi A respectively. All quinolone resistance strains had mutations in QRDR of gyrA and parC genes. Conclusion: Ciprofloxacin is no longer a drug of choice for treatment of enteric fever in India. Although susceptibility to ceftriaxone is 100% but MIC is creeping towards resistance. Azithromycin can be a promising treatment option for uncomplicated enteric fever.

Molecular Characterization of Ciprofloxacin Resistant Escherichia coli from Ghana

Aim: This study aimed to characterize ciprofloxacin-resistance genes in clinical Escherichia coli isolates obtained from a six-month antimicrobial resistance (AMR) surveillance from Ghana.&#x0D; Methods: Eighty-three of 440 archived E. coli isolates were confirmed by biochemical reactions and resistance profiles by the disc diffusion method. These isolates were cultured from urine (42), stool (23), vaginal swabs (12), wounds (5) and heart valve (1) during AMR surveillance. Minimum Inhibition Concentration (MIC) by E-test method was performed on all E. coli isolates that were resistant to ciprofloxacin by the disc diffusion method. Additionally, all isolates with reduced MIC to ciprofloxacin (&gt;32 µg/ml) were selected for molecular assays. Three chromosomal and nine plasmid-mediated resistance genes were screened in all Ciprofloxacin resistant E. coli (CRE) by polymerase chain reaction (PCR). Randomly selected amplified genes were commercially sequenced and analyzed.&#x0D; Results: In total, 47/83 (56.6%) E. coli isolates were resistant to ciprofloxacin and 29 (61.7%) had MIC values greater than 32 µg/ml. Chromosomal mediated genes (gyrA, gyrB and parC) were present in all 29 CRE isolates (100%). Distribution of the plasmid-mediated genes were as follows; qnrA 16/29 (55.1%), qnrB 16/29(55.1%), qnrC 22/29(75.8%), qnrS 26/29(89.6%), qepA 5/29(17.2%) and oqxB 19/29(65.5%). Genes encoding for altered aminoglycoside acetyltransferase [aac(6’)1bcr] were also present in all 29 CRE isolates. The majority (72.4%) of the CRE isolates had gyrA mutations at codons 83 and 87. In parC, the mutations were at codons 71 and 80. Five isolates had mutations at codon 56 and four each had mutations at positions 79 and 80.&#x0D; Conclusion: In this study, fluoroquinolone resistance genes were identified in all CRE isolates, mostly with putative mutations in the Quinolone Resistance Determining Region (QRDR). These chromosomal and plasmid-mediated genes may be widespread in Ghana and associated with CRE from the AMR surveillance. Although new mutations points were identified in parC, they may not be linked to the CRE.

Prevalence of carbapenem-resistant Enterobacteriaceae and emergence of high rectal colonization rates of blaOXA-181-positive isolates in patients admitted to two major hospital intensive care units in Kuwait.

BackgroundFecal colonization by carbapenem-resistant Enterobacteriaceae (CRE) can be the main reservoir for transmission of these resistant organisms especially in the Intensive Care Units (ICUs).AimThis study was conducted to evaluate the rate of rectal carriage and molecular characterization of CRE in patients hospitalized in the ICUs of 2 major hospitals (Adan and Mubarak Al Kabeer Hospitals) in Kuwait.Materials and methodsRectal swabs were collected from all patients at admission, 48 h after admission and once weekly from April 2017- March 2018. Initial CRE screening was carried out on MacConkey agar on which meropenem disc 10μg was placed. Identification of isolates was by API 20E. Susceptibility testing was performed using the E-test method. Polymerase chain reaction (PCR) was used to detect the carbapenemase-encoding genes. Clonal relationship was investigated by pulsed-field electrophoresis (PFGE). Genes of blaOXA-181 and blaNDM-5–carrying plasmids were detected in some strains.ResultsA total of 590 patients were recruited into the study. Of these, 58 were positive for CRE, giving a prevalence of 9.8%; 25/320 (7.8%) in Adan and 33/270 (12.2%) in Mubarak Al Kabeer Hospitals. All isolates were resistant to multiple antibiotics. Resistance rates to colistin and tigecycline were 17% and 83%, respectively. Single genes of blaOXA-181 were detected in isolates from 38 (65.5%) out of 58 patients and in 5 patients colonized by blaOXA-48-positive CRE. A combination of 2 genes was detected in 12 isolates; 5 blaKPC-2 and blaOXA-181, 4 blaVIM-1 and blaOXA-181, and 3 blaNDM-5 and blaOXA-181. PFGE showed an overall level of similarity of 38%. Southern hybridization studies localized the blaOXA-181 and blaNDM-5 genes to a large plasmid of 200kb in 3 K. pneumoniae isolates and a small plasmid of 80kb in 2 E. coli isolates, respectively.ConclusionThe prevalence of CRE colonization in the 2 hospital ICUs was relatively high and the emergence of blaOXA-181-mediated CRE is a cause for concern as there is the possibility of rapid horizontal spread among hospital patients in Kuwait. Active surveillance of CRE in the ICUs is highly recommended to stem its spread.

E-test Determination of Antifungal Susceptibility of Candida Species Isolated from Turkeys.

IntroductionCandida species are a natural component of the intestinal tract microflora, but in favourable conditions they can cause superficial, mucosal, or even systemic candidiasis. Poultry production might be a source of human drug-resistant yeast infections, including Candida spp. The limited data concerning the antifungal susceptibility of poultry Candida isolates prompted us to carry out research to determine the susceptibility of isolates from turkey intestinal tracts.Method and MaterialsThe beak cavity, crop and cloaca were swabbed of 580 turkeys from 58 flocks in western Poland. The susceptibility tests were conducted using the E-test method with amphotericin B, fluconazole, itraconazole, and voriconazole on 52 isolates of C. albicans, C. catenulata, C. glabrata, C. palmioleophila, C. rugosa, C. krusei and C. lusitaniae.ResultsAll isolates were susceptible to voriconazole. According to the MIC values obtained for amphotericin B and fluconazole, all Candida spp. isolates were classified as susceptible according to the described breakpoints except for C. krusei, which was the only isolate that was amphotericin B-, fluconazole- and itraconazole-resistant. The susceptibility to itraconazole varied: 11 of the Candida isolates were susceptible (21.1%), 29 were dose-dependently susceptible (55.8%), and 12 isolates were resistant (23.1%).ConclusionThere are few resistant strains of Candida in turkeys, and the drug resistance varies. When Candida passes from turkeys to humans, there is a wide range of antifungal treatment options.

First Report of New Delhi Metallo-β-Lactamase-6 (NDM-6) in a Clinical Acinetobacter baumannii Isolate From Northern Spain.

The objective of this study was the phenotypic and genotypic characterization of a carbapenem resistant Acinetobacter baumannii (CRAB) isolate. The isolate, recovered in Northern Spain in 2019, was identified by MALDI-TOF to the species level. Antimicrobial susceptibility testing was performed using the Phoenix BD NMIC-502 Panel, E-test, and broth microdilution methods. The presence of a metallo-β-lactamase (MBL) was verified by PCR and immunochromatographic assays. The genetic location of the MBL was confirmed using S1-pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blot hybridization. Whole genome sequencing (WGS) was completed using the Miseq and MinION platforms, followed by core-genome MLST (cgMLST) and seven-locus MLST analysis. The CRAB was assigned ST85 (Pasteur scheme) and ST957 (Oxford scheme) representing international clone (IC) 9 and harbored the intrinsic β-lactamase OXA-94 with ISAba1 upstream of it, and the MBL blaNDM-6. Hybridization experiments revealed that the blaNDM-6 was encoded on the chromosome. Using WGS the blaNDM-6 environment could be identified arranged in the following order: ISAba14, aphA6, ISAba125, blaNDM-6, bleMBL, trpF, dsbC, cutA, and ISAba14. Downstream, a 10,462 bp duplication was identified, including a second copy of blaNDM-6 in the following genetic composition: ISAba125, blaNDM-6, bleMBL, trpF, dsbC, cutA, and ISAba14. To our knowledge, this is the first description of blaNDM-6 in A. baumannii. The MBL was present in two copies in the chromosome in a new genetic environment associated with IS elements highlighting the contribution of mobile genetic elements in the dissemination of this gene.