E-test Method Research Articles

In Vitro Activity of Single and Combined Antibiotics against Carbapenem Resistant Enterobacteriaceae Clinical Isolates in Relation to their Resistance Genes

Background: Mortality due to infection with carbapenem-resistant Enterobacteriaceae (CRE) is reported globally and carbapenemase production is the main mechanism of resistance in these isolates. The detection and treatment of carbapenemase-producing Enterobacteriaceae (CPE) is a major challenge in health care facilities.
 Objectives: The aim of the current study was to evaluate the in-vitro effect of different single and combined antibiotic agents against CRE clinical isolates.
 Methodology: Fifty CRE isolates were detected using disk diffusion test as a screening test. Species identification and antibiotic susceptibility testing was done using Vitek 2 system. Carbapenemase enzyme production was confirmed by Carba NP test. Multiplex PCR was done to detect carbapenem resistance genes. Antibiotics were tested in the form of single agents (colistin and tigecycline) and combined (tigecycline/ colistin, doripenem/ colistin and dual carbapenem therapy (ertapenem and doripenem) against CRE isolates using E-test method.
 Results: Most of the CRE isolates were K. pneumoniae, 68%, followed by E. coli, 22%, S. marcescens, 4%, E. cloacae, 4% and C. freundii, 2%. CPE was confirmed in 46 isolates by multiplex PCR; blaNDM-like was the main carbapenem resistance gene in (84%) of the isolates, followed by blaOXA-48-like (6%) and blaKPC-like (2%). Carba NP test detected 90% of CPE isolates. Single use of colistin and tigecycline showed 100% sensitivity against all tested CRE isolates except in blaNDM-like (83%). Combination of colistin/tigecycline showed synergetic activity in 18% of CRE that was correlated to their carbapenemase R genes showing a significant increase in blaOXA-48-like and blaKPC-like positive isolates (100%) compared to blaNDM-like (7%). Other combinations showed indifferent effect whereas antagonism was not detected in any of the tested combinations.
 Conclusions: blaNDM-like is the main carbapenemase-producing gene detected among our CPE isolates followed by blaOXA-48-like. Colistin and tigecycline are still effective when used as single agents, and may offer effective treatment options when used in combination for CRE infections. Characterization of carbapenemases is crucial in determining treatment options. There is urgent demand for the development of novel therapeutic agents against NDM-producing CPE isolates.

Investigation of the Defective Growth Pattern and Multidrug Resistance in a Clinical Isolate of Candida glabrata Using Whole-Genome Sequencing and Computational Biology Applications.

ABSTRACTCandida glabrata is increasingly isolated from blood cultures, and multidrug-resistant isolates have important implications for therapy. This study describes a cholesterol-dependent clinical C. glabrata isolate (ML72254) that did not grow without blood (containing cholesterol) on routine mycological media and that showed azole and amphotericin B (AmB) resistance. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and whole-genome sequencing (WGS) were used for species identification. A modified Etest method (Mueller-Hinton agar supplemented with 5% sheep blood) was used for antifungal susceptibility testing. WGS data were processed via the Galaxy platform, and the genomic variations of ML72254 were retrieved. A computational biology workflow utilizing web-based applications (PROVEAN, AlphaFold Colab, and Missense3D) was constructed to predict possible deleterious effects of these missense variations on protein functions. The predictive ability of this workflow was tested with previously reported missense variations in ergosterol synthesis genes of C. glabrata. ML72254 was identified as C. glabrata sensu stricto with MALDI-TOF, and WGS confirmed this identification. The MICs of fluconazole, voriconazole, and amphotericin B were >256, >32, and >32 μg/mL, respectively. A novel frameshift mutation in the ERG1 gene (Pro314fs) and many missense variations were detected in the ergosterol synthesis genes. None of the missense variations in the ML72254 ergosterol synthesis genes were deleterious, and the Pro314fs mutation was identified as the causative molecular change for a cholesterol-dependent and multidrug-resistant phenotype. This study verified that web-based computational biology solutions can be powerful tools for examining the possible impacts of missense mutations in C. glabrata.IMPORTANCE In this study, a cholesterol-dependent C. glabrata clinical isolate that confers azole and AmB resistance was investigated using artificial intelligence (AI) technologies and cloud computing applications. This is the first of the known cholesterol-dependent C. glabrata isolate to be found in Turkey. Cholesterol-dependent C. glabrata isolates are rarely isolated in clinical samples; they can easily be overlooked during routine laboratory procedures. Microbiologists therefore need to be alert when discrepancies occur between microscopic examination and growth on routine media. In addition, because these isolates confer antifungal resistance, patient management requires extra care.

Helicobacter pylori Eradication Efficacy of Therapy Based on the Antimicrobial Susceptibility in Children with Gastritis and Peptic Ulcer in Mekong Delta, Vietnam

Background: The efficacy of Helicobacter pylori (H. pylori) eradication therapy for children is currently low, and antibiotic resistance is a significant cause of treatment failure. The purpose of this study was to evaluate the H. pylori eradication efficacy of therapy based on antimicrobial susceptibility in pediatric patients with gastritis and peptic ulcer. Methods: This study was conducted at Can Tho Children’s Hospital and Can Tho University of Medicine and Pharmacy Hospital between March 2019 and April 2022. We performed an upper gastrointestinal endoscopy, cultured H. pylori from biopsies of gastric mucosa, determined antibiotic sensitivities to H. pylori by the E-test method, and treated eradication based on the antibiotic susceptibilities of bacteria. After at least 4 weeks of eradication therapy, we assessed the effectiveness of treatment with a breath test. Results: Among 237 children recruited in this study, 48.9% were boys and 51.1% were girls, and the mean age was 10.03 ± 2.53 years. We determined that 80.6% of H. pylori were resistant to clarithromycin, as well as amoxicillin, metronidazole, levofloxacin, and tetracycline, at 71.7%, 49.4%, 45.1%, and 11.4%, respectively. The overall eradication rate of H. pylori was 83.1% (172/207). Among therapies tailored to antimicrobial susceptibility, the bismuth quadruple regimen achieved the greatest success, but the efficacy of triple therapy with esomeprazole + AMX + CLR/MTZ was low. Conclusions: Tailored eradication therapy was highly successful in our study but did not achieve over 90%. We recommend that in countries with a high prevalence of antibiotic resistance in H. pylori strains, particularly where the amoxicillin-resistance rate of H. pylori is high, therapy tailored to antimicrobial susceptibility should be used as first-line therapy, and bismuth and tetracycline should be added to enhance the eradication efficacy in children.

Molecular Identification and Pathogenic Potential of Clinical Bacillus Sp.20

Antibiotic resistance bacteria has become a worldwide problem. The shortage of new antibiotics has prompted research into chemicals that could act as adjuvants and enhance the efficacy of available antibiotics. Bacterial susceptibility to antibiotics was determined using the e-test method to evaluate the effect of commonly used antibiotics on the selected isolate (S20). The isolate displayed resistance to cefoxitin, benzylpenicillin, oxacillin, erythromycin, ciprofloxacin, and azithromycin. Whereas it was susceptible to the rest antibiotics. In this study, we reported the draft genome sequence of S20, isolated from a patient. The genome consists of 4,557,070 bp, with a GC of 46.4%. It has 228 RNA reads that protein-coding genes encoding multidrug resistance transporters, virulence factors. The draft genome sequences project was deposited in GenBank under accession no.. PRJNA480592. The version described in this paper is the first version. To estimate the phylotypes in the selected genomes, the 16S rRNA gene sequences were retrieved from the RAST annotation and used as a query against the SILVA reference database with the threshold set to above 97%. Bacillus mycoides was the closest genus to our isolate (100%).

Antimicrobial Susceptibility Testing Patterns of Neisseria gonorrhoeae from Patients Attending Sexually Transmitted Infections Clinics in Six Regions in Tanzania.

Antimicrobial resistance (AMR) is global health threat that is on the increase, and it has been adversely affecting the proper management of sexually transmitted infections (STI). Data on antimicrobial susceptibility testing patterns of N. gonorrhoeae are limited in local settings. We determined in vitro antimicrobial susceptibility and phenotypic profiles of N. gonorrhoeae isolated from STI/Outpatient Department (OPD) clinics. Minimum Inhibitory Concentrations (MIC) (µg/mL) were determined using E-Test and agar dilution methods for previously and currently recommended antimicrobial agents. A total of 164 N. gonorrhoeae isolates from urethral discharge and endocervical swabs were tested. The prevalence of resistant N. gonorrhoeae to tetracycline, norfloxacin, penicillin and ciprofloxacin were 98.6%, 82.2%, 84.3% and 75.6%, respectively. None of the isolates was resistant to kanamycin. Penicillinase producing N. gonorrhoeae (PPNG) was found to be 73.7%, with 56.7% and 43.3% observed among isolates from women and men, respectively. Tetracycline resistant-N. gonorrhoeae (TRNG) was found to be 34.0%, and QRNG with HLR to ciprofloxacin was 79.9%. The overall MDR-NG was 79.9%, and XDR-NG was 3.6%. MIC50 and MIC90 were 4.0 and 8.0 and 2.0 and 4.0 µg/mL for ciprofloxacin and norfloxacin, respectively. Dendrograms showed that 44 phenotypic groups are associated with a high rate of AMR among high MDR-NG and moderate XDR-NG isolates. The predominant groups of quinolone-resistant N. gonorrhoeae (QRNG)+PPNG (34.7%) and QRNG+PPNG+TRNG (32.9%) were observed among the isolates having HLR to ciprofloxacin. We reported a high prevalence of AMR (>90%) to previously recommended antimicrobials used for the treatment of gonorrhoea. Multidrug resistant N. gonorrhoeae (MDR-NG) was highly reported, and extensively drug resistant (XDR-NG) has gradually increased to the currently recommended cephalosporins including ceftriaxone and cefixime. Heterogeneous groups of QRNG+PPNG+ and QRNG+PPNG+TRNG were highly resistant to penicillin, tetracycline, ciprofloxacin and norfloxacin. A surveillance program is imperative in the country to curb the spread of AMR.

Studies on the Efficacy of Various Antimycotic Drugs on Emerging and Reemerging, Superficial, Cutaneous and Subcutaneous Mycotic Infections

Introduction: The efficacy of five systemic and topical antifungal medications, Voriconazole, clotrimazol, beclometasone, Itraconazole, and Fluconazole, on dermatomycosis, which affects the superficial layers of the skin, nails, foot, and hair, was tested with 180 patients.
 Methods: Included were specimen collection, processing, microscopy, and culture, as well as antifungal susceptibility testing using the E-test method. The Candida species were confirmed and their susceptibility to Voriconazole and Fluconazole was tested using the automated Vitek 2.
 Results: The final strain identification indicated 41 dermatophytes (69.49%), 11 non-dermatophytic molds (NDM) (18.64%), and 7 yeasts (11.87%). (candida). Candida was the most prevalent nondermatophyte species found. Trichophyton rubrum was the most prevalent species isolated in Tinea corporis, T. cruris, T. capitis, and T. faciei. When tested with the E strips, all dermatophyte strains showed the greatest vulnerability to beclometasone and clotrimazole (MIC range of 0.04–0.64), but homogeneous resistance to Fluconazole (i.e. MIC 32 g/ml).
 Conclusion: Variation in species distribution was shown to be statistically significant (p = 0.001) in terms of clinical presentation.

Antimicrobial susceptibility pattern of ceftazidime-avibactam against Eschericia Coli.

Objectives: To identify the susceptibility of Ceftazidime-avibactam against Escherichia coli and their frequency in different clinical specimens. To correlate the susceptibility pattern of ceftazidime-avibactam against Escherichia coli in different age groups and gender. To evaluate susceptibility of drugs among Escherichia coli as compared to traditional antibiotics. Study Design: Cross-sectional Study. Setting: PNS Shifa Hospital Karachi. Period: September 2019 to May 2020. Material & Methods: The sample size was 150. The age group 10-50 years. Ethical permission was taken. Informed consent was taken. The specimens were inoculated on Blood and MacConkey’s agar culture plates. The culture plate was inoculated at 37℃ in incubator for 24 to 48 hours. Identification of Enterobacteriaceae was done by colony morphology, gram staining, biochemical tests, and API 20E. After identification, the susceptibility profile of conventional antibiotics was identified. Mueller Hinton agar was used to check the antibiotic susceptibility of Ceftazidime-avibactam by disk diffusion method. Results: Ceftazidime-avibactam shows sensitivity against 82.7% of the isolates while 17.3 % isolates were resistant. The minimum inhibitory concentration of microorganisms is measured by E-test method that revealed most of the isolate show sensitivity less than 1 µg/mL concentration whereas few of them showed susceptibility on 2-8 µg/mL concentration. We compared our results with other classes of antibiotics used commonly This gives an insight for improved treatment methodologies for future prospects particularly diseases caused by members of Enterobacteriaceae. Conclusion: It was concluded that Ceftazidime-avibactam is a novel drug combination that shows high sensitivity against Escherichia coli.

Antibiotic Resistance Patterns of Listeria Species Isolated from Broiler Abattoirs in Lusaka, Zambia.

L. monocytogenes is a public health threat linked to fast foods such as broiler chickens. This study aimed to verify the occurrence of Listeria species in chickens from abattoirs and evaluate their antimicrobial resistance. In total, 150 broiler carcass swabs distributed as cloacal (n = 60), exterior surface (n = 60), and environmental (n = 30) were collected. Listeria species were characterized using biochemical tests and PCR. We conducted antibiotic resistance tests using the disc diffusion and Etest (Biomerieux, Durham, NC, USA) methods. Overall isolation of Listeria species was 15% (23/150) 95% CI (10.16–22.33), 2% (3/150) 95% CI (0.52–6.19) and 13% (20/150) 95% CI (8.53–20.08) came from environmental swabs and carcass swabs, respectively. Proportions of positive Listeria isolates were L. monocytogenes 74% (17/23), L. welshimeri 22% (5/23), and L. innocua 4% (1/23). Listeria species from the exterior carcass swabs was 61% (14/23), cloacal swabs 26% (6/23), and environmental swabs 3% (3/23). L. monocytogenes had the greatest resistance percentage to the following antibiotics: clindamycin (61%, 10/23), tetracycline 30% (7/23), and erythromycin 13%, (3/23). Isolation of L. monocytogenes in relatively high numbers, including the antimicrobial profiles, suggests a potential risk of the pathogen remaining viable in the food continuum and a public health risk to would-be consumers.

Risk factors and lethality associated with Candidemia in severe COVID-19 patients.

Candidemia remained important in the intensive care units (ICU) during the COVID-19 pandemic. This study aimed to investigate the clinical and laboratory data on candidemia in COVID-19 patients. The baseline characteristics, as well as laboratory and clinical findings of candidemia and non-candidemia patients, were compared. Candidemia was defined as the isolation of Candida spp. from blood cultures. The isolates were identified by VITEK® 2 (bioMérieux, France) commercial method. Antifungal susceptibility was assessed using the E-test method. Univariate and multiple binary logistic regression analyses were performed to compare the variables. In total, 126 patients with the COVID-19 disease were included. Candidemia was diagnosed in 44 (35%) of the patients. The number of patients with diabetes mellitus and chronic renal failure was higher in the candidemia group. In the candidemia group, the duration of ICU stay of patients, the 30-day mortality rate, mechanical ventilation therapy, and systemic corticosteroids (Prednisone) usage were significantly higher in candidemia patients. Moreover, the median white blood cell, neutrophils, and lactate dehydrogenase were higher in the candidemia group.Univariate and multiple binary logistic regression analyses were performed to compare the variables. Isolated species were identified as Candida albicans (n=12, 41%), Candida parapsilosis (n=7, 24%), Candida glabrata (n=6, 21%), Candida tropicalis (n=3, 10%), and Candida dublinensis (n=1, 3%). In total, three isolates of six C. glabrata species had dose-dependent sensitivity to fluconazole, and one C. parapsilosis was determined to be resistant. The COVID-19 patients who are admitted to ICU have many risk factors associated with candidemia. The most common risk factors for the development of candidemia were mechanical ventilation, diabetes mellitus, neutrophilia, and low hemoglobin level. The most frequently isolated species was C. albicans. Moreover, caspofungin was found to be the most effective drug in vitro. No significant resistance pattern was detected against the isolated species. It should be noted that risk-stratified antifungal prophylaxis in the ICU is possible.

Optimizing Doses of Ceftolozane/Tazobactam as Monotherapy or in Combination with Amikacin to Treat Carbapenem-Resistant Pseudomonas aeruginosa.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a hospital-acquired pathogen with a high mortality rate and limited treatment options. We investigated the activity of ceftolozane/tazobactam (C/T) and its synergistic effects with amikacin to extend the range of optimal therapeutic choices with appropriate doses. The E-test method is used to determine in vitro activity. The optimal dosing regimens to achieve a probability of target attainment (PTA) and a cumulative fraction of response (CFR) of ≥90% were simulated using the Monte Carlo method. Of the 66 CRPA isolates, the rate of susceptibility to C/T was 86.36%, with an MIC50 and an MIC90 of 0.75 and 24 µg/mL, respectively. Synergistic and additive effects between C/T and amikacin were observed in 24 (40%) and 18 (30%) of 60 CRPA isolates, respectively. The extended infusion of C/T regimens achieved a ≥90% PTA of 75% and a 100% fT > MIC at C/T MICs of 4 and 2 µg/mL, respectively. Only the combination of either a short or prolonged C/T infusion with a loading dose of amikacin of 20–25 mg/kg, followed by 15–20 mg/kg/day amikacin dosage, achieved ≥90% CFR. The C/T infusion, combined with currently recommended amikacin dose regimens, should be considered to manage CRPA infections.

Direct Identification, Antimicrobial Susceptibility Testing, and Extended-Spectrum β-Lactamase and Carbapenemase Detection in Gram-Negative Bacteria Isolated from Blood Cultures.

PurposeTo shorten the turnaround time for blood culture (BC) analyses, a rapid method was developed for the direct identification, antimicrobial susceptibility testing (AST), and multidrug resistance testing of bacteria-positive BCs.Materials and MethodsThe mixtures in BC bottles were treated with the multistep centrifugation method developed here and the conventional culture-based method. The bacterial sediment obtained after centrifugation was analyzed directly with MALDI-TOF MS and Vitek 2 Compact, and AST was performed directly with the Kirby–Bauer (K–B) disk diffusion, VITEK 2 Compact, and E-test methods. Extended spectrum lactamases (ESBLs) were detected with discs containing cefotaxime, cefotaxime/clavulanate, ceftazidime, and ceftazidime/clavulanate, and carbapenemase was detected with the modified carbapenem inactivation method (mCIM) and EDTA-mCIM (eCIM).ResultsAll the results of direct testing were compared to those of the conventional methods, to evaluate the accuracy of the direct methods. The accuracies of the direct Vitek 2 Compact and MALDI-TOF MS methods were 95.5% (214/224) and 90.2% (202/224), respectively. Direct AST with K–B, Vitek 2, and E-test showed category agreement of 96.0% (2611/2721), 96.1% (2614/2721), and 97.4% (2650/2721), respectively, and the major errors and very major errors were < 2% for all three methods. In the direct determination of ESBLs, the results for cefotaxime combined with cefotaxime/clavulanate were completely consistent with those after the standard isolation method. The carbapenemase detection rate with direct mCIM and eCIM was exactly the same as that with the standard method.ConclusionThese direct procedures based on multistep centrifugation are not only highly accurate but are appropriate for clinical laboratory use because the turnaround time is shorter.

The using of the polymerase chain reaction for the detection of resistance genes in gram-negative bacteria in routine practice in a pediatric hospital.

Objective - assessment of RT-PCR for the detection of carbapenem-resistance genes in gram-negative bacteria. A total, 499 strains of gram-negative microorganisms isolated in two pediatric hospitals in 2019-2020 were studied. Species identification was performed using MALDI-ToF mass-spectrometry (Bruker Daltonics, Germany). Meropenem and imipenem minimal inhibitory concentration (MIC) was determined by E-test method (BioMerieux, France). The presence of acquired carbapenemase genes of IMP, NDM, VIM, KPC, OXA-48, OXA-23, OXA-40, OXA-58-groups was determined by RT-PCR. Klebsiella pneumoniae (34%), Escherichia coli (4%), Serratia marcescens (6%) and other members of Enterobacterales (6%), also gram-negative non-glucose-fermenting bacteria Acinetobacter baumannii (14%), Pseudomonas aeruginosa (36%) were found among selected strains. Carbapenemase production was found in 385 isolates (77%). The main mechanism determining carbapenem resistance in P. aeruginosa was the production of blaVIM (100%). A. baumanii strains harbored OXA-23 (55%) and OXA-40 (45%) carbapenemases. The major determinant of carbapenem resistance in K. pneumoniae isolates was OXA-48 carbapenemase, detected in 63% strains, 13% of the strains possessed blaNDM-group, 16% isolates had a combination of blaNDM-group and blaOXA-48-like. Carbapenemase of KPC-group was found in 8% K. pneumoniae strains. OXA-48 carbapenemase prevailed (95%) among S. marcescens strains. Most of E. coli isolates harbored metallo-beta-lactamase NDM (89%). Other members of Enterobacterales most often had OXA-48 carbapenemase (57%), 39% of the isolates carried blaNDM-group. In one strain, a combination of blaNDM-group and blaOXA-48-like was discovered. RT-PCR is a fast and reliable method for the detection of acquired carbapenemases and can be recommended for routine use in bacteriological laboratories.

E-Test or Agar Dilution for Metronidazole Susceptibility Testing of Helicobacter pylori: Importance of the Prevalence of Metronidazole Resistance

BackgroundA number of studies have shown that E-test overestimated the presence of Helicobacter pylori resistance compared to agar dilution.ObjectiveThe purpose of this study was to explore whether E-test could be an alternative for agar dilution to detect the metronidazole susceptibility of H. pylori.MethodE-test and agar dilution were used to assess the susceptibility of H. pylori to metronidazole, clarithromycin, and levofloxacin in 281 clinical isolates obtained from China where the resistance was high. Cohen’s kappa analysis, McNemar’s test, and essential and categorical agreement analysis were performed for these two methods.ResultsOverall, the result of the E-test showed a similar prevalence of resistance rate to all antibiotics compared with agar dilution. The essential agreement of the E-test method and agar dilution in the evaluation susceptibility of H. pylori to clarithromycin and levofloxacin was moderate at 89.0 and 79.7%, respectively, but only 45.9% for metronidazole. The results shown by a categorical agreement (CA) between the E-test and agar dilution were 100% for both clarithromycin and levofloxacin. As for metronidazole, the CA was 98.7%, no major error was identified, and the rate of a very major error was 1.8%.ConclusionE-test can be an alternative method to detect the metronidazole susceptibility of H. pylori.

Multicenter Evaluation of a Gradient Diffusion Method for Antimicrobial Susceptibility Testing of Helicobacter pylori.

ABSTRACTHelicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization.IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.

Molecular Epidemiology of Multidrug-Resistant Pneumococci among Ghanaian Children under Five Years Post PCV13 Using MLST.

Antibiotic resistance in pneumococci contributes to the high pneumococcal deaths in children. We assessed the molecular characteristics of multidrug-resistant (MDR) pneumococci isolated from healthy vaccinated children under five years of age in Cape Coast, Ghana. A total of 43 MDR isolates were selected from 151 pneumococcal strains obtained from nasopharyngeal carriage. All isolates were previously serotyped by multiplex PCR and Quellung reaction. Susceptibility testing was performed using either the E-test or disk diffusion method. Virulence and antibiotic resistance genes were identified by PCR. Molecular epidemiology was analyzed using multilocus sequence typing (MLST). Vaccine-serotypes 23F and 19F were predominant. The lytA and pavB virulence genes were present in all isolates, whiles 14–86% of the isolates carried pilus-islets 1 and 2, pcpA, and psrP genes. Penicillin, tetracycline, and cotrimoxazole resistance were evident in >90% of the isolates. The ermB, mefA, and tetM genes were detected in (n = 7, 16.3%), (n = 4, 9.3%) and (n = 43, 100%) of the isolates, respectively. However, >60% showed alteration in the pbp2b gene. MLST revealed five novel and six known sequence types (STs). ST156 (Spain9V-3) and ST802 were identified as international antibiotic-resistant clones. The emergence of international-MDR clones in Ghana requires continuous monitoring of the pneumococcus through a robust surveillance system.

Comparison of Carbapenem Resistance Detected by the BD Phoenix Automated System in Enterobacteriaceae Isolates with E-Test Method

Objective: Automatic identification and antimicrobial susceptibility systems are frequently used to identify clinical isolates in hospitalized patients, but mistakes in these systems can lead to potentially devastating treatment failures for patients. Therefore, the ‘‘Centers for Disease Control and Prevention (CDC)’’ recommends confirming all Carbapenem-resistant and low-susceptibility isolates with a different method. The aim of this study is to compare the Carbapenem susceptibility results of isolates reported as Carbapenem-resistant Enterobacteriaceae according to the BD Phoenix 100 automated system with the E-test method. Materials and Methods: The study included 70 strains of Carbapenem-resistant Enterobacteriaceae members which were isolated and grown from several types of clinical samples in the Medical Microbiology Laboratory. Conventional methods (Gram stain, negative oxidase test) and the BD Phoenix 100 automated system were used to identify the isolates. The susceptibility of all strains to imipenem, ertapenem and meropenem was investigated by E-test method. Automated system results and E-test results were compared. Results: The frequency distribution of all isolated bacterial strains comprised K. pneumoniae in 56 (80%) of the samples included in the study. The automated system test results were correlated with the results of the E-test at a rate of 96.1 % for the imipenem-resistant strains, 84.3% for the meropenem-resistant strains, 84.1% for the ertapenem-resistant strains Conclusions: Automated systems are frequently used in microbiology laboratories to identify isolates. However, automated systems can show a high error rate against some antimicrobials. For this reason, comparing the results of automated system test results with tests such as E-test is very important to prevent both treatment failures and inappropriate antibiotic use that may occur on a patient basis.

Activity of Fosfomycin against the Spectrum of Uropathogens Causing Cystitis

Background: Urinary tract infections (UTIs) are the most frequent bacterial infections commonly seen in females. A high degree of antimicrobial resistance in uropathogens has challenged the use of therapeutic agents. Fosfomycin, which is an old antibiotic with distinctive characteristics, seems to be a promising novel therapeutic agent with a good bactericidal activity towards multi-drug resistant (MDR) uropathogens. Objective: The main objective of the study is to evaluate the antibacterial activity of fosfomycin among uropathogens causing cystitis. Methods: The study was carried out between 2017-2018. A total of 2060 UTI suspects from outpatient departments (OPDs) and inpatient departments (IPDs) were screened. Out of 2060 screened patients, 1658 were IPD patients, and 402 were OPD patients. The patient’s midstream urine samples were collected aseptically and processed according to standard protocols. The frequency of extendedspectrum- beta lactamases (ESBLs) producer and carbapenem resistance were estimated, respectively. Cultures with significant growth of uropathogens were identified, and minimum inhibitory concentration (MIC) to fosfomycin was determined by agar dilution (AD) and by E-test methods. Results: 184 out of 2060 (8.9%) urine samples showed significant growth of uropathogens. Uropathogenic Escherichia coli (UPEC) (64%, 118/184) was found to be the most isolated uropathogen. Among these Gram-negative uropathogens, 80% were ESBLs producers, 43.2% were carbapenemresistant, and 78% isolates were found to be MDR. The fosfomycin susceptibility for UPEC was 95% by the AD method. Conclusions: This study suggests that fosfomycin is reasonably effective and can be used in the treatment of MDR uropathogens along with uncomplicated UTIs.

Antimicrobial Susceptibility of Clinical Oral Isolates of Actinomyces spp.

Actinomyces species play an important role in the pathogenesis of oral diseases and infections. Susceptibility testing is not always routinely performed, and one may oversee a shift in resistance patterns. The aim of the study was to analyze the antimicrobial susceptibility of 100 well-identified clinical oral isolates of Actinomyces spp. against eight selected antimicrobial agents using the agar dilution (AD) and E-Test (ET) methods. We observed no to low resistance against penicillin, ampicillin-sulbactam, meropenem, clindamycin, linezolid and tigecycline (0–2% ET, 0% AD) but high levels of resistance to moxifloxacin (93% ET, 87% AD) and daptomycin (83% ET, 95% AD). The essential agreement of the two methods was very good for benzylpenicillin (EA 95%) and meropenem (EA 92%). The ET method was reliable for correctly categorizing susceptibility, in comparison with the reference method agar dilution, except for daptomycin (categorical agreement 87%). Penicillin is still the first-choice antibiotic for therapy of diseases caused by Actinomyces spp.

Antibiotic Resistance and Molecular Biological Characteristics of Non-13-Valent-Pneumococcal Conjugate Vaccine Serogroup 15 Streptococcus pneumoniae Isolated From Children in China.

Background: The isolation rate of serogroup 15 Streptococcus pneumoniae has been increasing since developing countries began administering the 13-valent pneumococcal conjugate vaccine.Methods: We detected the antibiotic resistance and molecular characteristics of 126 serogroup 15 S. pneumoniae strains isolated from children in China. Serotypes were determined via the Quellung reaction. Antibiotic resistance was tested using the E-test or disc diffusion method. Sequence types were assigned via multilocus sequence typing. Data were analyzed using WHONET 5.6 software.Results: The frequencies of S. pneumoniae serotypes 15A, 15B, 15C, and 15F were 29.37, 40.48, 28.57, and 1.59%, respectively. Continuous-monitoring data from Beijing showed that the annual isolation rates of serogroup 15 S. pneumoniae were 7.64, 7.17, 2.58, 4.35, 3.85, 7.41, and 10.53%, respectively, from 2013 to 2019. All 126 serogroup 15 strains were susceptible to vancomycin and ceftriaxone. The non-susceptibility rate to penicillin was 78.57%. All strains were resistant to erythromycin with high minimum inhibitory concentrations (MICs). The multidrug resistance rate was 78.57%. The most common clonal complexes were CC3397, CC6011, CC10088, CC9785, and ST8589.Conclusion: Serogroup 15 S. pneumoniae is common among children in China, and these strains should be continuously monitored.

Investigation of antimicrobial resistance of ESßL, Amp-C, and carbapenemaseproducing E. coli strains in retail poultry meats

Antimicrobial agents are used for suppressing some bacterial infections during the rearing of poultry. In this study; the antibiotic resistance properties of Extended Spectrum ßeta-lactamase (ESßL), Amp-C, carbapenemase-producing Escherichia coli (E. coli) strains isolated from chicken breast meat were determined by disc diffusion and E-test methods. Additionally, it is aimed to determine antibiotic resistance genes by classical polymerase chain reaction (PCR). In this context, E. coli strains were isolated from 7 of 100 chicken breast meat (fillets) and identified by a rapid identification system (BBL Crystal). Disc diffusion test results of the isolates were evaluated and 100% to tetracycline; ampicillin, ciprofloxacin, cefoxitin, nalidixic acid, oxytetracycline 85.7%; ceftazidime, cefotaxime and meropenem 28.5%; chloramphenicol 71.4%, trimethoprim/sulfamethoxazole 42.8%; 14.2% resistance to gentamicin was observed. Test results of the isolates were evaluated and ESßL activity was found in 2 isolates in the disc diffusion test. Amp-C and carbapenemase activity was detected in 6 isolates in disc diffusion, in all isolates in E-test; 2 isolates in disc diffusion were detected carbapenemase activity, while carbapenemase activity was not detected in E-test, respectively. Classical PCR was performed after the DNAs were isolated for the detection of antibiotic resistance genes. According to classical PCR results; ESßL activity was detected in all isolates; carbapenemase activity was positive in only 1 isolate, while Amp-C activity was positive in 4 isolates. CTXM-1 (71%) and SHV1 (71%), OXA-1 (42.8%), CTXM-9 (28.5%), and TEM (14.2%) were detected by classical PCR, respectively. It has been concluded that E. coli isolated from poultry meat collected from the market may pose a risk in terms of ESßL, Amp-C, and carbapenemase genes, and continuous monitoring is required in this regard.