Gradient Strip Test Research Articles

Molecular Characterization of Colistin- and Carbapenem-Resistant Klebsiella pneumoniae: mgrB Mutations and Clonal Diversity in Pediatric Intensive Care Isolates.

Colistin- and carbapenem-resistant Klebsiella pneumoniae (ColR CrKp) cause important health problems in pediatric intensive care units (PICUs) due to its ability to harbor multiple resistance genes and spread of high-risk clones. In this study, molecular epidemiological characteristics, transferable resistance genes, and mgrB alterations of ColR CrKp isolated from PICU were investigated. Isolates were identified by MALDI-TOF MS, and antimicrobial susceptibility tests were performed using disk diffusion method, gradient strip test, and broth microdilution method. Extended spectrum beta-lactamase, AmpC beta-lactamase, carbapenemase, 16S rRNA methyltransferase, plasmid-mediated quinolone resistance, and mcr-1 to -5 genes were investigated by polymerase chain reaction. Sanger sequencing was performed to obtain blaOXA-48-like and mgrB sequences. Pulsed-field gel electrophoresis and multilocus sequence typing were used to determine the clonal spread of the isolates. Ten ColR CrKp harboring blaOXA-48 (70%), blaOXA-232 (20%), blaCTX-M (90%), armA (20%), qnrB (20%), and qnrS (50%) were identified. No mcr genes were found, whereas mgrB mutations through modifications (A7T, C88T, and A121G) and insertion of an IS-1-like insertion sequence were determined. Isolates belonged to ST 14, ST 37, ST 101, ST 147, ST 661, ST 985, and ST 2096. It is crucial to determine the antimicrobial resistance properties and the clonal spread of the isolates to guide the treatment decisions, implement effective infection control measures, and develop novel antimicrobial strategies.

Investigation of colistin heteroresistance and the colistin resistance genes mcr-1 to mcr-5 in Escherichia coli and Klebsiella pneumoniae isolates in a tertiary hospital in Turkey

Introduction: Heteroresistance is not detected by traditional antimicrobial susceptibility testing methods and may lead to treatment failures. Investigating the presence of plasmid-mediated colistin resistance genes is important because of the horizontal transmission of the relevant genes between bacterial species. This study aimed to investigate the presence of colistin heteroresistance and the colistin resistance genes mcr-1 to mcr-5 in Escherichia coli and Klebsiella pneumoniae isolates. Methodology: A total of 254 isolates, including 100 E. coli and 154 K. pneumoniae strains isolated from clinical samples, were included in the study. Colistin susceptibility was evaluated using the broth microdilution method for all strains. Heteroresistance screening was performed using the gradient strip test. Eight strains were evaluated for heteroresistance by population analysis profiling (PAP). The colistin resistance genes mcr-1 to mcr-5 were investigated by multiplex polymerase chain reaction (PCR) in colistin-resistant K. pneumoniae isolates. Multilocus sequence typing (MLST) analysis was performed on two K. pneumoniae strains. Results: Colistin resistance was not detected in the E. coli isolates and was detected in 16.23% (25/154) of the K. pneumoniae isolates. No heteroresistant bacteria were detected by the gradient strip test or by PAP. All colistin-resistant isolates were negative for the mcr genes. The two isolates analyzed by MLST were ST14 and ST2096. Conclusions: Periodic follow-up of colistin heteroresistance is useful for administering appropriate antibiotic therapy. In addition, the investigation of colistin resistance genes is important for infection control measures.

Evaluation of Antimicrobial Susceptibility and Resistance Patterns of Treponema denticola Isolated From Periodontal Disease: An In Vitro Study.

Background Periodontal disease poses a significant oral health challenge, involving inflammatory conditions impacting tooth-supporting structures. Treponema denticola, a "red complex" organism, plays a crucial role in periodontal pathogenesis, forming biofilms in subgingival environments and contributing to dysbiosis. Antimicrobial therapy is pivotal in managing periodontal disease, requiring a nuanced understanding of susceptibility patterns exhibited by key pathogens like T. denticola. Aims and objectives This study aims to investigate the antimicrobial susceptibility and resistance profiles of Treponema denticola, a prominent bacterium in periodontal disease, by examining its responses to various antimicrobial agents commonly used in periodontal therapy. Methodology Plaque samples were meticulously collected from individuals diagnosed with periodontal disease to ensure a diverse representation of the oral microbiome. All the samples were cultured, and red complex bacteria were isolated under anaerobic culture. Treponema denticola isolates were cultured from these samples under anaerobic conditions, and molecular techniques were employed for species identification. A comprehensive panel of antimicrobial agents was selected to assess the response of Treponema denticola. In vitro antimicrobial susceptibility testing (AST) was conducted using the antimicrobial gradient method, employing a hybrid approach combining elements of disk-diffusion and dilution methods. Results Treponema denticola had exhibitedresistance to metronidazole, a commonly used antibiotic effective against anaerobic bacteria, emphasizing limitations in its applicability. However, the bacterium displayed sensitivity to tetracycline, imipenem, cefoperazone, chloramphenicol, clindamycin, and moxifloxacin, offering diverse therapeutic options. The antimicrobial gradient strip test provided detailed minimum inhibitory concentration (MIC) values, contributing to a nuanced understanding of susceptibility and resistance patterns. Conclusion This study significantly advances our understanding of Treponema denticola's antimicrobial susceptibility and resistance profiles in the context of periodontal disease. The findings underscore the importance of tailored treatment strategies and contribute to broader efforts in antimicrobial stewardship, aligning with global initiatives to combat antibiotic resistance. This research lays the foundation for more effective and personalized approaches to periodontal care, emphasizing the intricate microbial dynamics associated with periodontal health and disease.

Rapid Detection of Ceftazidime/Avibactam Susceptibility/Resistance in Enterobacterales by Rapid CAZ/AVI NP Test.

We developed a novel culture-based test, the Rapid CAZ/AVI NP test, for rapid identification of ceftazidime/avibactam susceptibility/resistance in Enterobacterales. This test is based on glucose metabolization upon bacterial growth in the presence of a defined concentration of ceftazidime/avibactam (128/53 μg/mL). Bacterial growth is visually detectable by a red to yellow color change of red phenol, a pH indicator. A total of 101 well characterized enterobacterial isolates were used to evaluate the test performance. This test showed positive percent agreement of 100% and negative percent agreement of 98.5% with overall percent agreement of 99%, by comparison with the MIC gradient strip test (Etest) taken as the reference standard method. The Rapid CAZ/AVI NP test had only 1.5% major errors and 0% extremely major errors. This test is rapid (result within 2 hours 45 minutes), reliable, affordable, easily interpretable, and easy to implement in clinical microbiology laboratories without requiring any specific equipment.

Azithromycin susceptibility testing for salmonella enterica isolates: Comparing disk diffusion results with MIC gradient strips

Background: Enteric fever remains an imperative public health problem in developing countries. After the emergence of cephalosporin resistance in Salmonella enterica subsp. enterica serovar Typhi, azithromycin is increasingly being used for oral treatment of enteric fever. Reports of sporadic azithromycin resistance have been reported across the country, additionally, misuse of azithromycin during the COVID-19 pandemic has concerns regarding emerging azithromycin resistance. This study evaluated the reliability of the disc diffusion method as a screening test for detecting azithromycin resistance by comparing it with the minimum inhibitory concentrations (MIC) gradient strip results, in 231 typhoidal salmonellae. Material and Methods: This prospective study was conducted in the Section of microbiology of the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore from March 2022 to March 2023. Isolates recovered from blood cultures of patients, suffering from enteric fever were selected. Azithromycin susceptibility testing was performed both by disk diffusion and as well as gradient strips and their results were compared. Results: Among typhoidal salmonellae, a significant portion consisted of extensively drug resistant Salmonella Typhi (61.9%). Only one XDR S. Typhi was found to be resistant to azithromycin both by disk diffusion method and MIC gradient strip method, with a MIC value of 64µg/ml. The study found no discrepancy between the disk diffusion and gradient strip methods. Conclusion: The current study found no discordance between disk diffusion and gradient strip test methods for evaluating azithromycin susceptibility among typhoidal salmonellae. Keywords: Azithromycin, disk diffusion testing, enteric fever, Minimum inhibitory concentration, Salmonella Typhi

Evaluation of antibiotic susceptibility in enterococci isolated from blood culture samples

Aims: Increased vancomycin resistance in enterococci is an important cause of life-threatening bloodstream infections in hospitalized patients. The aim of this study is to determine the antibiotic susceptibility rates of Enterococcus strains isolated from blood cultures in hospitalized patients in Antalya Training and Research Hospital. 
 Methods: The antibiotic resistance rates of Enterococcus strains isolated from blood cultures of patients hospitalized in the service and intensive care units (ICU) between 1 January 2018 and 30 December 2022 were examined retrospectively. Blood samples were studied with the BacT/ALERT 3D culture system (Biomerieux, France). Bacterial identification was performed using conventional methods, Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometer (MALDI-TOF MS) and VITEK 2 (Biomerieux, France) systems. Antimicrobial susceptibility tests were performed with VITEK 2 (Biomerieux, France) systems. Ampicillin, vancomycin, teicoplanin, high-level gentamicin resistance (HLGR) and linezolid susceptibility of isolated strains were evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Vancomycin minimal inhibitory concentration (MIC) values of vancomycin resistant strains were studied by microdilution gradient strip test (Bioanalyse).
 Results: A total of 623 strains of enterococci were isolated from blood culture samples. Of the enterococci, 305 (48.9%) were identified as Enterococcus faecalis, 281 (45.6%) Enterococcus faecium, 12 (1.9%) Enterococcus avium, 11 (1.8%) Enterococcus gallinarum, 7 (1.2%) Enterococcus casseliflavus, 2 (0.4%) Enterococcus durans and 1 Enterococcus hirae (0.2%). Ampicillin and HLGR resistance rates of isolated E. faecalis strains were 11 (3.6%) and 72 (23.6%), respectively, and all strains were found to be susceptible to vancomycin, teicoplanin and linezolid. The ampicillin, vancomycin, teicoplanin and HLGR resistance rates of E. faecium strains were determined as 229 (81.5%), 36 (12.8%), 30 (10.7%) and 142 (50.5%), respectively, and all strains were found to be susceptible to linezolid. 
 Conclusion: In infections caused by enterococci, identification and determination of antibiotic susceptibility rates according to culture antibiogram results would be the right approach. Knowing the current susceptibility rates of enterococci isolated from blood culture samples in our hospital will contribute for clinicians' planning of empirical treatment.

Antimicrobial susceptibility surveillance and antimicrobial resistance in Neisseria gonorrhoeae in Africa from 2001 to 2020: A mini-review.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG), compromising gonorrhea treatment, is a global public health concern. Improved, quality-assured NG AMR monitoring at the global level is essential. This mini-review examined NG AMR susceptibility surveillance and AMR data from the African continent from 2001 to 2020. Eligible peer-reviewed publications (n = 30) containing NG AMR data for antimicrobials currently recommended for gonorrhea treatment were included. Overall, very limited NG surveillance and AMR data was available. Furthermore, the NG AMR surveillance studies varied greatly regarding surveillance protocols (e.g., populations and samples tested, sample size, antimicrobials examined), methodologies (e.g., antimicrobial susceptibility testing method [agar dilution, minimum inhibitory concentration (MIC) gradient strip test, disc diffusion test] and interpretative criteria), and quality assurance (internal quality controls, external quality assessments [EQA], and verification of AMR detected). Moreover, most studies examined a suboptimal number of NG isolates, i.e., less than the WHO Global Gonococcal Antimicrobial Surveillance Program (GASP) and WHO Enhanced GASP (EGASP) recommendations of ≥100 isolates per setting and year. The notable inter-study variability and frequently small sample sizes make appropriate inter-study and inter-country comparisons of AMR data difficult. In conclusion, it is imperative to establish an enhanced, standardized and quality-assured NG AMR surveillance, ideally including patient metadata and genome sequencing as in WHO EGASP, in Africa, the region with the highest gonorrhea incidence globally. This will enable the monitoring of AMR trends, detection of emerging AMR, and timely refinements of national and international gonorrhea treatment guidelines. To achieve this aim, national and international leadership, political and financial commitments are imperative.

Characterization of coagulase-negative staphylococci and macrococci isolated from cheese in Germany

Cheese, especially ripened varieties, harbor a very complex and heterogeneous microbiota. In addition to the desired microorganisms (starter cultures) added during cheese production, potentially harmful bacteria may also enter the production chain. Regarding the latter, the focus of this study was on coagulase-negative staphylococci (CNS) and Macrococcuscaseolyticus. Both are known to harbor a variety of genes coding for antibiotic resistance, including mecA, mecB, mecC, and mecD. Coagulase-negative staphylococci or macrococci carrying such genes or other virulence factors should not be present in cheese. Cheese samples (101 in total) were collected from retail sources. Coagulase-negative staphylococci and M. caseolyticus were isolated utilizing selective agars, and species were identified by phenotypical tests and partial sequencing of the sodA gene. The results allowed identification of 53 CNS strains and 19 M. caseolyticus strains. Among the CNS, 11 isolates of Staphylococcus saprophyticus and one Staphylococcus epidermidis isolate were obtained. Both species are potential human pathogens and may thus adversely affect the safety of these food products. Screening for antimicrobial resistance was performed by application of disc diffusion tests, a gradient strip-test, and 14 different PCR tests. Evidence for methicillin resistance (by either positive disc diffusion assay for cefoxitin or by mec PCR) was found in CNS isolates and M. caseolyticus (9 isolates each). Regarding other virulence factors, no genetic determinants for coagulase or the most common staphylococcal enterotoxins sea, seb, sec, sed, and see were detected in any of the CNS or M. caseolyticus isolates by PCR testing. In conclusion, the presence of facultatively pathogenic CNS and carriers of genes for antibiotic resistance in both groups of microorganisms, especially mec genes, and the respective food safety issues need further evaluation and surveillance.

Nadir Bir Enfeksiyon Etkeni: Elizabethkingia anophelis; Türkiye’den Bildirilen İkinci Olgu

Elizabethkingia anophelis is a Gram-negative, aerobic, nonmotile bacillus belonging to the Flavobacteriaceae family. In recent years, it has emerged as a cause of life-threatening infections, especially in immunocompromised patients. In this study, a 6-month-old baby patient with E. anophelis growth in simultaneous tracheal aspirate and urine culture samples sent to investigate the etiology of fever while being followed in the intensive care unit due to the diagnosis of optic glioma is presented. Bacteria identification was performed using the VITEK MS® system, antibiotic susceptibility tests were performed using the VITEK 2 automated system and gradient strip test. Our case is the second E.anophelis case reported from Turkey. This case showed that this bacterium would start to appear as a factor in our country. More studies are needed to obtain more detailed information about this bacterium, to determine its transmission routes and resistance mechanisms and to establish appropriate treatment protocols.

Investigation of Antimicrobial Susceptibilities and Resistance Genes of Campylobacter Isolates from Patients in Edirne, Turkey.

Background:We aimed to determine the susceptibility of Campylobacter isolates obtained from patients to various antimicrobial agents and to investigate some related antimicrobial resistance genes.Methods:Fifty-six Campylobacter isolates obtained from fecal specimens by conventional methods at the Trakya University Health Center for Medical Research and Practice, Department of Medical Microbiology in Edirne, Turkey, from 2017–2017 were included. Antimicrobial susceptibilities were investigated by the gradient strip test method, and species determination was made by multiplex polymerase chain reaction (mPCR). The presence of the erm(B) gene and tet(O) gene was investigated in all isolates by PCR. DNA sequence analysis was performed to detect the presence of mutations in the 23S rRNA positions 2074 and 2075 in five isolates, including two erythromycin resistant isolates. The gyrA gene mutation was investigated by the mismatch amplification mutation assay (MAMA)-PCR.Results:In 54 C. jejuni isolates, resistance to erythromycin was 3.7%; to tetracycline, 59.3%; and to ciprofloxacin, 74.1%. Phenotypically, the tet(O) gene was detected in 33 tetracycline-resistant isolates, but no erm(B) gene was found in any of the Campylobacter isolates. As a result of the DNA sequencing, it was found no mutations in the 23S rRNA gene at the 2074 and 2075 positions. The gyrA mutation was observed in all 41 ciprofloxacin resistant Campylobacter isolates.Conclusion:Among the antimicrobial agents tested, ciprofloxacin had the highest resistance rate, and erythromycin had the lowest. Antimicrobial resistance in Campylobacter increased significantly compared with previously studies in our region as well as in the entire world. Monitoring the resistance to antimicrobial agents used to treat Campylobacter infections is important in determining empiric antimicrobial treatment.

Detection and Characterization of Clinical Bordetella trematum Isolates from Chronic Wounds

Bordetella trematum is a relatively newly discovered and potentially frequently overlooked Bordetella species, mostly isolated from chronic wounds and predominantly in those of the lower extremities. Its susceptibility profile and clinical significance is still debated, given the limited amount of available data. We contribute providing a molecular and phenotypical analysis of three unique clinical B. trematum isolates detected between August 2019 and January 2020 to aid the matter. Cryo-conserved isolates were subcultured and re-identified using various routine means of identification. Bacterial genomes were fully Illumina-sequenced and phenotypical susceptibility was determined by broth microdilution and gradient-strip tests. All isolates displayed increased susceptibility to piperacillin–tazobactam (<2/4 mg/L), imipenem (<1 mg/L), and meropenem (<0.047 mg/L), whereas they displayed decreased susceptibility to all tested cephalosporins and fluoroquinolones (according to PK-PD, EUCAST 10.0 2020). One isolate carried a beta-lactamase (EC 3.5.2.6) and a sulfonamide resistance gene (sul2) and cells displayed resistance to ampicillin, ampicillin/sulbactam, and trimethoprim/sulfamethoxazole. All isolates carried genes conferring decreased susceptibility to aminoglycosides (aadA), fosfomycin (fosA) and fluoroquinolones (gyrB EC 5.99.1.3). Awareness that B. trematum can be resistant to trimethoprim/sulfamethoxazole is warranted.

Disbalancing Envelope Stress Responses as a Strategy for Sensitization of Escherichia coli to Antimicrobial Agents

Disbalancing envelope stress responses was investigated as a strategy for sensitization of Escherichia coli to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σE, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (ΔpspA and ΔpspB mutants), cefepime (ΔpspB and ΔpspC mutants) and ertapenem (ΔpspB mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log10 (ΔpspA mutant) and 0.9 log10 (ΔpspB mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log10 (ΔpspA mutant) and 4 log1010 (ΔpspB mutant) was shown after 4–6 h. For cefepime, 4.2 log10 (ΔpspB mutant) and 2.6 log10 (ΔpspC mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log10 (ΔpspB mutant) was observed at 24 h. A deficient Psp envelope stress response increased E. coli susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.

False amoxicillin/clavulanic acid susceptibility in Bacteroides fragilis using gradient strip tests

BackgroundRepeatedly, too low MIC results were obtained in Bacteroides fragilis quality assessment strains, using gradient strip tests with a ratio of amoxicillin:clavulanic acid of 2:1. We aimed to find the most accurate available gradient strip tests for susceptibility testing of amoxicillin/clavulanic acid in B. fragilis in comparison with agar dilution with EUCAST methodology and breakpoints. MethodsTwenty-seven clinical B. fragilis isolates were investigated using gold standard EUCAST amoxicillin/clavulanic acid agar dilution (fixed clavulanic acid concentration at 2 mg/L, with increasing amoxicillin concentrations) as well as three commercial gradient strip tests: XL (ratio), AUG (ratio) or AMC (fixed concentration). ResultsUsing agar dilution (fixed concentration), 19 isolates were susceptible, 1 isolate was susceptible increased exposure (I) and 7 isolates were resistant. Categorical agreement of the gradient strip tests with agar dilution (fixed concentration) was 70% for XL (ratio), 71% for AUG (ratio) and 89% for AMC (fixed concentration). Very major error rates in comparison with agar dilution (fixed concentration) were 100%, 0%, and 0%, respectively. ConclusionsEUCAST breakpoint usage in amoxicillin/clavulanic acid susceptibility tests for B. fragilis should be accompanied by EUCAST methodology. When using alternative methods such as gradient strip tests, a higher degree of alignment with EUCAST methodology, such as using fixed clavulanic acid concentrations, improves precision.

High susceptibility to zoliflodacin and conserved target (GyrB) for zoliflodacin among 1209 consecutive clinical Neisseria gonorrhoeae isolates from 25 European countries, 2018.

Novel antimicrobials for treatment of gonorrhoea are imperative. The first-in-class spiropyrimidinetrione zoliflodacin is promising and currently in an international Phase 3 randomized controlled clinical trial (RCT) for treatment of uncomplicated gonorrhoea. We evaluated the in vitro activity of and the genetic conservation of the target (GyrB) and other potential zoliflodacin resistance determinants among 1209 consecutive clinical Neisseria gonorrhoeae isolates obtained from 25 EU/European Economic Area (EEA) countries in 2018 and compared the activity of zoliflodacin with that of therapeutic antimicrobials currently used. MICs of zoliflodacin, ceftriaxone, cefixime, azithromycin and ciprofloxacin were determined using an agar dilution technique for zoliflodacin or using MIC gradient strip tests or an agar dilution technique for the other antimicrobials. Genome sequences were available for 96.1% of isolates. Zoliflodacin modal MIC, MIC50, MIC90 and MIC range were 0.125, 0.125, 0.125 and ≤0.004-0.5 mg/L, respectively. The resistance was 49.9%, 6.7%, 1.6% and 0.2% to ciprofloxacin, azithromycin, cefixime and ceftriaxone, respectively. Zoliflodacin did not show any cross-resistance to other tested antimicrobials. GyrB was highly conserved and no zoliflodacin gyrB resistance mutations were found. No fluoroquinolone target GyrA or ParC resistance mutations or mutations causing overexpression of the MtrCDE efflux pump substantially affected the MICs of zoliflodacin. The in vitro susceptibility to zoliflodacin was high and the zoliflodacin target GyrB was conserved among EU/EEA gonococcal isolates in 2018. This study supports further clinical development of zoliflodacin. However, additional zoliflodacin data regarding particularly the treatment of pharyngeal gonorrhoea, pharmacokinetics/pharmacodynamics and resistance selection, including suppression, would be valuable.

Evaluation of Fluoroquinolone Resistance in Clinical Avian Pathogenic Escherichia coli Isolates from Flanders (Belgium).

Fluoroquinolones are frequently used antimicrobials for the treatment of avian pathogenic Escherichia coli (APEC) infections. However, rapid development and selection of resistance to this class of antimicrobial drugs is a significant problem. The aim of this study was to investigate the occurrence and mechanisms of antimicrobial resistance against enrofloxacin (ENRO) in APEC strains in Flanders, Belgium. One hundred and twenty-five APEC strains from broilers with clinical colibacillosis were collected in Flanders from November 2017 to June 2018. The minimum inhibitory concentration (MIC) of all strains and the mutant prevention concentration (MPC) of a sample of sensitive isolates were determined using a commercial gradient strip test and via the agar dilution method, respectively. Non-wild type (NWT) isolates were further characterized using polymerase chain reaction (PCR), gel electrophoresis and gene sequencing. Forty percent of the APEC strains were NWT according to the epidemiological cut-off (ECOFF) measure (MIC > 0.125 μg/mL). With respect to clinical breakpoints, 21% were clinically intermediate (0.5 ≤ MIC ≤ 1 μg/mL) and 10% were clinically resistant (MIC ≥ 2). The MPC values of the tested strains ranged from 0.064 to 1 μg/mL, resulting in MPC/MIC ratios varying from 4 to 32. The majority (92%) of the NWT strains carried one or two mutations in gyrA. Less than a quarter (22%) manifested amino acid substitutions in the topoisomerase IV parC subunit. Only three of the NWT strains carried a mutation in parE. Plasmid mediated quinolone resistance (PMQR) associated genes were detected in 18% of the NWT strains. In contrast to the relatively large number of NWT strains, only a small percentage of APEC isolates was considered clinically resistant. The most common MPC value for sensitive strains was 0.125 μg/mL. Some isolates showed higher values, producing wide mutant selection windows (MSW). Chromosomal mutations in DNA gyrase and topoisomerase IV were confirmed as the main source of decreased antimicrobial fluoroquinolone susceptibility, de-emphasizing the role of PMQR mechanisms.

Agreement of Quantitative and Qualitative Antimicrobial Susceptibility Testing Methodologies: The Case of Enrofloxacin and Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the causal agent of colibacillosis, one of the most common bacterial infections in the poultry sector. Antimicrobial susceptibility testing (AST) is essential for rational and prudent antimicrobial therapy. Subsequently, uniformity in test results from the various testing methodologies used in diagnostic laboratories is pivotal. The aim of this study was therefore to evaluate the agreement between different AST methods in determining fluoroquinolone resistance in APEC. Twenty APEC isolates were selected and subjected to four different susceptibility tests: the quantitative microbroth dilution, agar dilution and gradient strip tests, and the qualitative disk diffusion method. The experiments were performed in triplicate. Categorical agreement, essential agreement and different errors were assessed. Moreover, agreement was also evaluated by calculating intraclass correlation coefficients (ICCs) for the quantitative tests and determining the Pearson correlation coefficients for the agreement between the disk diffusion method and the quantitative tests. Categorical agreement and essential agreement when compared with the microbroth technique ranged from 85–95% and 85–100%, respectively. No very major errors (false susceptible) and only one major error (false resistant) and minor errors (results involving an intermediary category) were detected. The calculated ICC values of the three quantitative tests fluctuated around 0.970 (range 0.940–0.988). There was a high negative correlation between the disk diffusion method and the other tests (correlation coefficients ranging from −0.979 to −0.940), indicating a clear inverse relationship between the minimum inhibitory concentration value and the zone diameter of growth inhibition. In conclusion, the overall agreement between the four different testing methodologies was very high. These results confirm the reliability of the disk diffusion and gradient strip test methods as substantiated alternatives, next to the gold standard agar and microbroth dilution, for fluoroquinolone susceptibility testing of APEC isolates.

Investigation of Carbapenem resistance mechanisms in Klebsiella pneumoniae by using phenotypic tests and a molecular assay.

The aim of this study was to investigate the presence of carbapenemase production and carbapenem resistance mechanisms in 47 carbapenem resistant Klebsiella pneumoniae isolates by phenotypic confirmatory tests and molecular assay. Carbapenem resistance genes KPC, OXA-48 and NDM were investigated with the BD MAX CRE assay kit in the BD MAX real time PCR instrument. Modified Hodge test, MBL gradient strip test, D70C Carbapenemase Detection Set, Temocillin gradient strip test methods were used as phenotypic confirmatory tests. Clonal relationship between study isolates was investigated with pulsed-field gel electrophoresis. Analysis with BD MAX CRE assay revealed OXA-48 positivity in 17 (36%) strains, NDM positivity in 6 (13%) strains and coexistence of OXA-48 + NDM positivity in 8 (17%) strains. In 16 (34%) strains, none of the KPC, OXA-48 and NDM genes were detected. While MHT was the most sensitive phenotypic confirmatory test, D70C disc set had not been considered as a useful tool to assist the search for carbapenemase production. Temocillin gradient test alone could not be considered as sufficient to detect the presence of OXA-48. PFGE analyses revealed that 23 of 31 carbapenemase producing strains were in three major PFGE genotypes (A, B and C). This study revealed that carbapenem resistance observed in K. pneumoniae isolates was mainly due to OXA-48 and NDM genes and the increase of carbapenem resistance among K. pneumoniae strains in our hospital was due to the interhospital spread of especially 3 epidemic clones.

Evaluation of six commercial products for colistin susceptibility testing in Enterobacterales

ObjectivesThe recommended technique for colistin susceptibility testing by both EUCAST and CLSI is broth microdilution (BMD). However, many routine laboratories still use other methods such as gradient strips or semi-automated systems. The objective of this study was to compare six of the most widespread commercial products for colistin susceptibility testing in Europe with in-house prepared BMD. MethodsA collection of 325 carbapenemase-producing Enterobacterales was tested for colistin susceptibility with three semi-automated systems (Vitek 2, BD Phoenix, MicroScan WalkAway), one gradient-strip test (Etest®) and two commercial BMD products (MICRONAUT-S, TREK Sensititre). BMD, in-house prepared according to ISO standard 20776-1, served as reference. ResultsThe MICRONAUT-S BMD performed best with only one false-resistant (major error, ME) and four false-susceptible (very major error, VME) results while the TREK BMD performed poorer with 16 ME and seven VME. The semi-automated systems Vitek 2 and Phoenix performed poorly with 31 and 26 VME, respectively. The WalkAway semi-automated system showed 16 and 13 false results, depending on the inoculation method. The Etest® showed six ME and 10 VME. ConclusionsThis study shows that colistin susceptibility testing remains a challenging task for laboratories. It emphasizes the need for selecting the most reliable test method to advocate proper treatment and shows that critical evaluation and precautious usage of colistin susceptibility testing results is constantly required.

Antimicrobial susceptibility pattern of oral isolates of Aggregatibacter actinomycetemcomitans.

Background:Aggregatibacter actinomycetemcomitans is involved in the etiology of localized aggressive periodontitis (LAP), a condition that frequently requires supplemental antibiotic therapy. Information on antimicrobial susceptibility pattern and guidelines for oral antibiotic therapy are not available on Indian patients.Aim:The main aim of the present study was to screen clinical isolates on a panel of antibiotics commonly used for oral/systemic therapy.Materials and Methods:The study included 40 strains of A. actinomycetemcomitans isolated from patients with LAP. The subgingival plaque was plated onto Trypticase Soy Serum Bacitracin Vancomycin Agar medium and incubated for 72 h, and suspected colonies were confirmed by phenotypic tests. Each isolate was tested against a panel of 12 antibiotics using MIC gradient strip test. ATCC strains of A. actinomycetemcomitans serotype A and C were used as standards. Performance and interpretation of the test were done according to the manufacturers’ instructions. Distribution of MICs among isolates (n = 40) were used to calculate concentrations inhibiting 50% (MIC50) and 90% (MIC90) of strains.Results:Moxifloxacin, cefotaxime and ceftriaxone showed excellent activity with 100% growth inhibition followed by amoxicillin, amoxiclav and doxycycline (>90% activity). The bacterial strains were moderately susceptible to cefuroxime, cefazolin and tetracycline but displayed poor susceptibility to clindamycin and azithromycin. All isolates were resistant to metronidazole.Conclusion:The isolates of A. actinomycetemcomitans displayed a high level of resistance to azithromycin and clindamycin. Development of resistance against tetracycline also appears to be significant. Variable resistance among the different members of the cephalosporin group is a factor to be investigated further since susceptibility profile against these antibiotics and interpretative criteria for oral bacteria are not available.

Tigecycline activity: low resistance rates but problematic disc breakpoints revealed by a multicentre sentinel survey in the UK

A multicentre surveillance of tigecycline activity against relevant pathogens in the UK. Forty-three representative UK hospitals were each asked to test 150 consecutive, clinically significant isolates from hospitalized patients, excluding those from urine or faeces. The sentinel laboratories tested all these isolates against a panel of antibiotics by the BSAC disc method and a selection of isolates were retested centrally by the BSAC agar dilution method. The isolates collected comprised Staphylococcus aureus (39.9%), Escherichia coli (13.3%), streptococci (11.9%), enterococci (6.3%), Klebsiella (6.1%), coagulase-negative staphylococci (6.1%), Enterobacter spp. (3.9%), Proteus spp. (2.2%) and other Gram-negative species (10.3%). The laboratories' disc susceptibility testing found that 4% of isolates were resistant to tigecycline, using the zone breakpoints in place at that time. However, centralized retesting by agar dilution showed that many of these 'resistant' isolates were susceptible, with resistance only confirmed in a range of Gram-negative isolates and one S. aureus. These findings reduced the estimated resistance rate to 2.4%, or to 0.8% if Proteus spp. were ignored as intrinsically resistant to tigecycline. Sixty-two percent of the isolates found resistant by the disc method but susceptible by agar dilution had zones of inhibition within experimental error (4 mm) of the published breakpoints. Tigecycline resistance was rare in isolates causing clinically significant infections in the UK and was overestimated ∼2-fold by the BSAC disc method. Adjustment of the breakpoints might overcome this problem but at the risk of increasing the false susceptibility rate. The best advice is to perform dilution tests, e.g. by gradient strip test on isolates with borderline results, especially in severe infection and when tigecycline use is intended.