European Committee For Antimicrobial Susceptibility Testing Research Articles

Sythesis, characterisation, anticancer and antimicrobial activity of Ag-N-heterocyclic carbene complexes containing benzimidazole derivatives

To prepare a novel series of silver(I) complexes, the interaction of benzimidazolium salts having their two nitrogen atoms substituted by bulky groups with Ag2O in DCM has been carried out. Their structures were characterized by elemental analyses, 1H NMR, 13C NMR, elemental analysis, and IR spectroscopy techniques. Further, the antimicrobial and antifungal activities properties of both the salts and their silver(I)–NHC complexes were tested against positive and negative bacteria using the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) respectively. The results show that silver complexes are effective against E. coli, P. aeruginosa and S. aureus bacterial species with moderate to high activity, and their minimum inhibitory concentrations ranging from 25 to 400 µg/Ml. It was also observed that 3b compound, which has the best antifungal properties among the synthesized compounds, showed high antimicrobial activity when all bacterial species were taken into account. In addition, the salts and complexes were then evaluated for their ability to inhibit the proliferation of four different cancer cell lines (A549, MCF-7, HCT116, SH-SY5Y and BEAS-2B cells lines). AlamarBlue assay demonstrated that the salts and complexes exhibited potent cytotoxicity towards all cancer lines. 2c compound showed the highest cytotoxic activity against BEAS-2B, A549 and MCF-7 cell lines. Except for 2c, compounds 3d, 3e and 3f showed cytotoxic activity at the lowest concentrations against MCF7 cell line.The study highlights the importance of N-heterocyclic carbene ligands in the development of silver-based drugs with anticancer properties.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil

We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

Evaluation of the feasibility of EUCAST RAST using antimicrobial disks available in Japan

BackgroundIn November 2018, the European Committee for Antimicrobial Susceptibility Testing (EUCAST) established rapid antimicrobial susceptibility testing (RAST), which could be performed directly on positive blood culture samples. Although concentrations of antimicrobial agents in several antimicrobial disks available in Japan are different from those recommended by the EUCAST, the feasibility of EUCAST RAST using antimicrobial disks available in Japan remains to be evaluated. MethodsBlood culture bottles spiked with 127 clinical isolates (65 Escherichia coli and 62 Klebsiella pneumoniae) were tested by RAST for cefotaxime (CTX), ceftazidime (CAZ), meropenem, and ciprofloxacin using antimicrobial disks available in Japan, and compared with a reference AST method using automated AST instrument (VITEK®2). ResultsThe overall category agreement (CA) for RAST using antimicrobial disks available in Japan was 96.3%, 96.8%, and 95.6% after 4, 6, and 8 h of incubations, respectively.However, the CAZ RAST for E. coli showed major error of 8.2% (8 h incubation) for the Sensi disk, 14.3% (6 h incubation), and 24.5% (8 h incubation) for the KB disk.The CTX RAST for K. pneumoniae showed 25% (4 h incubation) and 31.3% (4 h incubation) of very major error for the Sensi and KB disks, respectively. ConclusionsThe EUCAST RAST results for E. coli and K. pneumoniae using antimicrobial disks available in Japan suggest their usefulness, although modified RAST breakpoints are required for several antimicrobial agents.

Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

Epidemiological cut-off values for non-O1/ non-O139 Vibrio cholerae disc diffusion data generated by standardised methods

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

P041 Preliminary evaluation of gradient concentration strips for detection of terbinafine resistance in Trichophyton spp.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesDermatophytosis is the most common superficial fungal infection. Trichophyton rubrum and T. mentagrophytes are the most frequently isolated species, but their incidence varies according to geographical regions. Terbinafine is the main molecule used to treat this type of infection. In recent years, a high incidence of chronic infections, reinfections, and treatment failures due to a newly described specie, T. indotineae, have been reported in India and recently described in Europe. It is currently a public health problem for the management of these infections in this country.Until now, the monitoring of dermatophyte susceptibility to antifungals was rarely performed due to the lack of standardized in vitro tests. Since then, an in vitro technique has been standardized by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to test terbinafine and other antifungals. Recently, a gradient concentration strip method has been marketed.The aim of this study was to compare terbinafine susceptibility testing by the gradient concentration strip (GCS) method and the EUCAST standardized method.MethodsA panel of 47 molecularly identified isolates of T. interdigitale, T. mentagrophytes, and T. indotineae was used. The panel included 39 terbinafine- susceptible isolates and 8 terbinafine resistant isolates for which the squalene epoxidase gene was sequenced.Minimum inhibitory concentration (MIC) of terbinafine was determined using EUCAST microdilution broth method for dermatophytes. Inoculum was supplemented with cycloheximide and chloramphenicol. Final drug concentrations ranged from 0.008 to 8 μg/ml and microtiter plates were incubated at 25°C for 5 days. The MIC was determined spectrophotometrically with a 90% growth inhibition endpoint.MIC of terbinafine was also determined using GCS (Terbinafine Ezy MIC™ Strip, HiMedia, India) on RPMI agar. The plates were incubated for 5 days at 25°C. After incubation, MIC was read by using a complete inhibition endpoint. Isolates were considered wild-type when MIC was ≤ 0.125 μg/ml.ResultsEUCAST MIC values ranged from 0.008 to 0.0625 μg/mL and from 0.25 to 16 μg/ml for susceptible and resistant isolates, respectively.GCS MIC values ranged from 0.002 to 0.03 μg/ml and 0.125 to >32 for susceptible and resistant isolates, respectively.The categorical agreement (percentage of strains found in the same category) by the two techniques was 98%.ConclusionThese preliminary results show that GCS can detect resistance to terbinafine and could be used as a screening method. These results must be confirmed on a larger panel of isolates.

Bacteriological quality of community well water and public health concerns in Enugu urban, Nigeria

Background: Water is a basic necessity used by humans for both domestic and industrial uses. Next to air, water is essential to life. It takes up about 71% of the earth’s surface. The objective of this study is to determine the bacteriological quality of well water in Enugu urban, Nigeria
 Methodology: A total of 60 domestic wells were selected from Abakpa, Obiagu and Achara layouts in Engu urban, Nigeria by stratified random sampling method, with 20 wells selected from each area based on location of well sites and construction parameters. Water samples were collected from each well using a sterile 200ml plastic bottle for bacteriological analysis to estimate total bacteria count in colony forming unit (cfu)/ml, total coliform count in most probable number (mpn)/100ml, and faecal coliform count in most probable number (mpn)/100ml. Bacterial isolates were identified using Gram reaction and conventional biochemical tests including catalase and coagulase for Gram positive bacteria, and oxidase, citrate utilization, hydrogen sulfide, indole, urease, methyl red, Voges Proskauer, and sugar fermentation tests for Gram negative bacteria. Antibiotic susceptibility testing (AST) of each isolate was performed by the disk diffusion method against selected antibiotics including penicillin G (10µg), ciprofloxacin (5µg), streptomycin (10µg), amoxicillin-clavulanic acid (20/10µg), and trimethoprim-sulfamethoxazole (25µg), and result interpreted using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) break points. Comparative statistics of the data was performed using analysis of variance (ANOVA) with p<0.05 considered statistically significant.
 Results: The well water in the three layouts were heavily contaminated as shown by comparatively high mean total bacteria counts of 0.8825±0.66x104 cfu/ml, 0.8435±0.6413x104 cfu/ml, and 0.8384±0.5948x104 cfu/ml for Abakpa, Obiagu and Achara layouts respectively (p=0.9714). The mean total coliform counts were 5.15±5.284, 5.45±4.31 and 5.05±4.763 mpn/100ml (p=0.8038), and the mean faecal coliform counts were 2.4±3.393, 2.65±2.796 and 2.05±2.35 mpn/100ml (p=0.9631) for Abakpa, Obiagu and Achara layouts respectively. A total of 50 pathogenic bacterial isolates were identified; Klebsiella pneumoniae 21 (43.8%), Escherichia coli 13 (30.0%), Proteus spp 6 (12.5%), Pseudomonas aeruginosa 6 (12.5%), and Staphylococcus aureus 2 (4.2%). The AST result shows that 75% of K. pneumoniae, E. coli, Proteus spp and S. aureus were resistant to all five antibiotics tested.
 Conclusion: These findings showed high faecal contamination of domestic well water sources, which poses a significant infection risk to the community. Proper water treatment measures and personal hygiene practices are recommended, and well sites should be located at a safe distance from septic tanks, pit latrines, flowing gutters and refuse dump sites.

In-Vitro Efficacy of Cefiderocol in Carbapenem-Non-Susceptible Gram-Negative Bacilli of Different Genotypes in Sub-Region of North Rhine Westphalia, Germany.

In the last two decades, the worldwide dissemination of multidrug-resistant Gram-negative bacteria (MDR-GNB) has continued. Therapy options for such infections caused by MDR-GNB remain scarce, and only few new antimicrobial agents have been granted market approval. Cefiderocol has been approved for the treatment of infections associated with aerobic GNB with limited therapy options. This study evaluated the in vitro efficacy of cefiderocol against carbapenem-non-susceptible clinical GNB isolates from Germany. A total of 115 non-duplicate carbapenem-nonsusceptible GNB isolates, 61 (53.05%) of which were Enterobacterales species and 54 (46.95%) were non-fermenters (Acinetobacter baumanii and Pseudomonas aeruginosa), were investigated for their cefiderocol susceptibility. Minimum inhibitory concentrations (MICs) for cefiderocol were determined by disk diffusion, according to EUCAST (European committee for antimicrobial susceptibility testing). Susceptibility rates were based on EUCAST breakpoints. In the absence of a species-specific breakpoint, pharmacokinetic/-dynamic breakpoints were used. The most common pathogen was A. baumannii (33.91%), followed by Klebsiella pneumoniae (31.3%), P. aeruginosa (13.04%) and Escherichia coli (9.57%). Overall, 83.6% (51/61) of the Enterobacterales and 81.48% (44/54) of the non-fermenters were susceptible towards cefiderocol. In total, 20 species of Enterobacterales and non-fermenting GNB were resistant towards cefiderocol, irrespective of the isolation year (2014 to 2021). Moreover, the majority of the resistant isolates were among the OXA-23 producing A. baumannii (n = 7/26; 26.92%) from patients hospitalized during 2018 and 2019. Cefiderocol demonstrated high in vitro susceptibility rates against a wide range of carbapenem-non-susceptible GNB, including carbapenemase-producing isolates. Cefiderocol exhibited stability against hydrolysis by all carbapenemases, including metallo-β-lactamases (MBLs), except that few OXA-producing isolates exhibited resistance towards cefiderocol.

Stable prevalence of triazole-resistance in Aspergillus fumigatus complex clinical isolates in a Belgian tertiary care center from 2016 to 2020

BackgroundPrevalence reports of triazole-resistance in Aspergillus fumigatus differ between countries and centers and may likewise vary over time. Continuous local surveillance programs to establish the evolving epidemiology of triazole-resistance in A. fumigatus are crucial to guide therapeutic recommendations. Here, we determined the prevalence of triazole-resistance in A. fumigatus complex culture-positive patients at the tertiary care center University Hospitals Leuven in Belgium in clinical isolates from 2016 to 2020. MethodsAll A. fumigatus complex isolates cultured from UZ Leuven patients between 2016 and 2020 were screened for triazole-resistance. Confirmation of resistance to voriconazole, posaconazole and itraconazole was performed with the European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. Mutations in the cyp51A gene in triazole-resistant isolates were determined by sequencing. Patients were classified as susceptible or resistant cases based on their isolate's susceptibility phenotype. ResultsWe screened 2494 A. fumigatus complex isolates from 1600 patients (320 ± 38 [SD] patients per year). The prevalence of triazole-resistance in patients was 8.3% (28/337), 6.7% (26/386), 7.0% (21/301), 7.1% (21/294) and 7.4% (21/282) in 2016, 2017, 2018, 2019 and 2020 respectively, with an overall triazole-resistance prevalence of 7.1% (85/1192; 95% CI 6.6–7.7%). The TR34/L98H mutation was the most prevalent (83.0%, 78/94) with most isolates displaying resistance to all triazole antifungals tested (94.8%, 74/78). ConclusionThe prevalence of triazole-resistance in A. fumigatus has remained stable from 2016 to 2020 in our center ranging between 6.7 and 8.3%, with an overall five-year prevalence of 7.1%. The environmentally associated cyp51A gene mutations were most prevalent amongst triazole-resistant isolates and conferred resistance to all antifungals tested in 73% of the isolates.

Environmental Clonal Spread of Azole-Resistant Candida parapsilosis with Erg11-Y132F Mutation Causing a Large Candidemia Outbreak in a Brazilian Cancer Referral Center.

Clonal outbreaks due to azole-resistant Candida parapsilosis (ARCP) isolates have been reported in numerous studies, but the environmental niche of such isolates has yet to be defined. Herein, we aimed to identify the environmental niche of ARCP isolates causing unremitting clonal outbreaks in an adult ICU from a Brazilian cancer referral center. C. parapsilosis sensu stricto isolates recovered from blood cultures, pericatheter skins, healthcare workers (HCW), and nosocomial surfaces were genotyped by multilocus microsatellite typing (MLMT). Antifungal susceptibility testing was performed by the EUCAST (European Committee for Antimicrobial Susceptibility Testing) broth microdilution reference method and ERG11 was sequenced to determine the azole resistance mechanism. Approximately 68% of isolates were fluconazole-resistant (76/112), including pericatheter skins (3/3, 100%), blood cultures (63/70, 90%), nosocomial surfaces (6/11, 54.5%), and HCW’s hands (4/28, 14.2%). MLMT revealed five clusters: the major cluster contained 88.2% of ARCP isolates (67/76) collected from blood (57/70), bed (2/2), pericatheter skin (2/3), from carts (3/7), and HCW’s hands (3/27). ARCP isolates were associated with a higher 30 day crude mortality rate (63.8%) than non-ARCP ones (20%, p = 0.008), and resisted two environmental decontamination attempts using quaternary ammonium. This study for the first time identified ARCP isolates harboring the Erg11-Y132F mutation from nosocomial surfaces and HCW’s hands, which were genetically identical to ARCP blood isolates. Therefore, it is likely that persisting clonal outbreak due to ARCP isolates was fueled by environmental sources. The resistance of Y132F ARCP isolates to disinfectants, and their potential association with a high mortality rate, warrant vigilant source control using effective environmental decontamination.

Punica granatum L. Inhibits the Growth of Microorganisms Associated with Bovine Mastitis

Background: The current research aimed to explore secondary metabolites and antibacterial activity against bacterial agents associated with bovine mastitis from extracts prepared from the peel of fruits of Punica granatum L.. Mastitis is an inflammation in the mammary gland mainly due to infection of bacteria and can be presented clinically or subclinically. The infectious bovine mastitis disease is associated with the reduction and changes in the composition of the milk, as well as in the early disposal of the animal. Objective: We describe the bactericidal activity of ethanolic extracts of the fruit peel of P. granatum, against etiological agents of bovine mastitis (Escherichia coli, Staphylococcus aureus and Streptococcus agalactiae). Method: The bactericidal activity was evaluated according to the standards of the European Committee for Antimicrobial Susceptibility Testing and by the broth microdilution methodology according to the standards of the Clinical and Laboratory Standards Institute. The fruit peel extracts of P. granatum were prepared with ethanol solvent (90%). Results: The best results of total inhibition of bacterial growth of the three strains tested were obtained with the concentrations of 75mg/mL of the extract after 1 and 3 hours of treatment. Cell membrane integrity analysis by flow cytometry showed that 1 h of contact there was loss of integrity of the wall and cell membranes. Conclusion: We can suggest that the ethanolic extract of the fruit peel of P. granatum can be an important natural coadjutant compound in the treatment of mastitis as well as being used in the formulation of sanitizers.

Occurrence of Campylobacter jejuni and Campylobacter coli in Cattle and Sheep in Northern Spain and Changes in Antimicrobial Resistance in Two Studies 10-years Apart.

A cross-sectional survey was conducted in 2014–2016 in 301 ruminant herds to estimate C. jejuni and C. coli prevalence, and investigate their susceptibility to antimicrobials. Risk of shedding C. jejuni was higher in cattle than sheep (81.2% vs. 45.2%; ORadj = 5.22, p < 0.001), whereas risk of shedding C. coli was higher in sheep than in cattle (19.1% vs. 11.3%; ORadj = 1.71, p = 0.128). Susceptibility to six antimicrobials was determined by broth microdilution using European Committee for Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values. C. coli exhibited higher resistance (94.1%, 32/34) than C. jejuni (65.1%, 71/109), and resistance was more widespread in isolates from dairy cattle than beef cattle or sheep. Compared to results obtained 10-years earlier (2003–2005) in a similar survey, an increase in fluoroquinolone-resistance was observed in C. jejuni from beef cattle (32.0% to 61.9%; OR = 3.45, p = 0.020), and a decrease in tetracycline-resistance in C. jejuni from dairy cattle (75.0% to 43.2%; OR = 0.25, p = 0.026). Resistance to macrolides remained stable at low rates and restricted to C. coli from dairy cattle, with all macrolide-resistant C. coli showing a pattern of pan-resistance. Presence of the single nucleotide polymorphisms (SNPs) associated to quinolone and macrolide resistance was confirmed in all phenotypically resistant isolates. The increase in fluoroquinolone resistance is worrisome but susceptibility to macrolides is reassuring.

In vitro activity of tigecycline and comparators against a European collection of anaerobes collected as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) 2010–2016

The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) is a global program that aims to monitor the in vitro antimicrobial activities of current therapeutic agents against clinical isolates. This study presents surveillance data for Gram-positive and Gram-negative anaerobic isolates (N = 7008) collected from nine European countries between 2010 and 2016. Presented in this study are antimicrobial susceptibility data, according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) breakpoints, and minimum inhibitory concentration (MIC) distributions. The antimicrobial agents tested were cefoxitin (Gram-negative isolates only), clindamycin, meropenem, metronidazole, penicillin (Gram-positive isolates only), piperacillin-tazobactam and tigecycline. Among all Gram-positive and Gram-negative anaerobes, the lowest rates of resistance were to meropenem and metronidazole (0.0%–1.7% and 0.0%–1.9%, respectively). High rates of resistance were reported to clindamycin, in particular among isolates of the Bacteroides fragilis group (22.1%–48.1%) and Prevotella spp. (10.9%–32.2%). The majority of MIC distributions were unimodal, with the exception of clindamycin, which were mostly bimodal. Fifty percent of Gram-negative isolates gave tigecycline MICs between 0.06 and 1 mg/L, and 50% of Gram-positive isolates exhibited tigecycline MICs between 0.06 and 0.25 mg/L. The findings of this study suggest that the majority of anaerobic isolates were susceptible to meropenem and metronidazole, and that tigecycline remained active, but clindamycin resistance is a cause for concern in Europe. Surveillance studies, such as T.E.S.T., provide information on changes in the susceptibility of clinically important pathogens to commonly prescribed antimicrobial agents, and can highlight problems of antimicrobial resistance that need to be addressed.

Antibacterial potential of Croatian honey against antibiotic resistant pathogenic bacteria.

Aim To determine antimicrobial activity of honey against clinical bacterial strains and their respective reference strains. Methods Twelve samples of Croatian honey from various botanical origin were evaluated for their antimicrobial activity against four clinical antibiotic resistant pathogens and their respective reference strains: Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli. Antibacterial susceptibility was checked out by using broth microdilution method and interpreted according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) recommendations. Results Significant differences in the antibacterial activity of tested honey samples were noticed. Fir honeydew honey and Mint honey showed the best antibacterial potential, while the Locust tree honey, Rapeseed honey and Spring pasture honey expressed the weakest antimicrobial activity. Conclusion Croatian honey, prominently honeydew honey, has the potential to become an important additive to therapeutic techniques available to a medical practitioner against resistant pathogens, but the exact mechanisms of its activity should be investigated further.

Assessment of the comparability of CLSI, EUCAST and Stokes antimicrobial susceptibility profiles for Escherichia coli uropathogenic isolates

Background: As many clinical laboratories convert between Stokes, Clinical and Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) methods, the problem of comparing differently derived sets of antimicrobial susceptibility testing (AST) data with each other arises, owing to a scarcity of knowledge of inter-method comparability. The purpose of the current study was to determine the comparability of CLSI, EUCAST and Stokes AST methods for determining susceptibility of uropathogenic Escherichia coli to ampicillin, amoxicillin-clavulanate, trimethoprim, cephradine/cephalexin, ciprofloxacin and nitrofurantoin.Methods: A total of 100 E. coli isolates were obtained from boric acid urine samples from patients attending GP surgeries. For EUCAST and CLSI, the Kirby-Bauer disc diffusion method was used and results interpreted using the respective breakpoint guidelines. For the Stokes method, direct susceptibility testing was performed on the urine samples.Results: The lowest levels of agreement were for amoxicillin-clavulanate (60%) and ciprofloxacin (89%) between the three AST methods, when using 2017 interpretive guidelines for CLSI and EUCAST. A comparison of EUCAST and CLSI without Stokes showed 82% agreement for amoxicillin-clavulanate and 94% agreement for ciprofloxacin. Discrepancies were compounded by varying breakpoint susceptibility guidelines issued during the period 2011–2017, and through the inclusion of a definition of intermediate susceptibility in some cases.Conclusions: Our data indicate that the discrepancies generated through using different AST methods and different interpretive guidelines may result in confusion and inaccuracy when prescribing treatment for urinary tract infection.

In Vitro Activity of Oral Antimicrobial Agents against Pathogens Associated with Community-Acquired Upper Respiratory Tract and Urinary Tract Infections: A Five Country Surveillance Study.

IntroductionBacterial infections that cause community-acquired urinary tract infections (CA-UTI) and upper respiratory tract infections (CA-URTI) are most frequently treated empirically. However, an increase in antimicrobial resistance has become a problem when treating outpatients.MethodsThis study determined the in vitro activities of oral antibiotics among 1501 pathogens from outpatients with CA-UTI and CA-URTI in medical centers during 2012 and 2013 from Argentina, Mexico, Venezuela, Russia, and the Philippines. Minimal inhibitory concentrations (MICs) were determined using broth microdilution and susceptibility defined by Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) criteria.ResultsCeftibuten (MIC50, ≤0.25 mg/L) was more potent in vitro compared to other β-lactams against Enterobacteriaceae from CA-UTI. Susceptibility to fluoroquinolones using CLSI criteria varied: Argentina and Mexico (50%), the Philippines (60%), Venezuela (70%), and Russia (80%). Fosfomycin susceptibility was >90% against Enterobacteriaceae in each country. Susceptibility among Enterobacteriaceae to trimethoprim-sulfamethoxazole was 30.6–75.6% and nitrofurantoin susceptibility also varied among the countries and was higher when EUCAST breakpoints were applied (65–>90%) compared to CLSI (52–84%). All Haemophilus influenzae isolates from CA-URTI were susceptible to ceftibuten, cefixime, cefpodoxime, and cefuroxime using CLSI breakpoint criteria. EUCAST criteria produced intermediate and resistant MIC values for these oral cephalosporins. Country-specific susceptibility variation for fluoroquinolones, macrolides, and trimethoprim-sulfamethoxazole was observed among Streptococcus pneumoniae and Streptococcus pyogenes from CA-URTI.ConclusionThis study demonstrated that antimicrobial susceptibility patterns varied in the five countries investigated among pathogens from CA-UTI and CA-URTI.FundingMerck & Co. Inc., Kenilworth, New Jersey, USA.

EUCAST recommendations for antimicrobial susceptibility testing applied to the three main Campylobacter species isolated in humans

Antimicrobial susceptibility testing of Campylobacter isolates is of great importance for treatment options especially in systemic diseases. The European Committee for Antimicrobial Susceptibility Testing (EUCAST) recently proposed epidemiological cut-offs (ECOFFs) for a limited number of antimicrobial compounds and for Campylobacter jejuni and Campylobacter coli only. In the present study, the EUCAST method was used after minor modifications to define antimicrobial susceptibility patterns for, 1997 C. jejuni, 419 C. coli and 100 Campylobacter fetus strains received at the French National Reference Center for Campylobacters and Helicobacters. Our results show that the ECOFFs defined by EUCAST for tetracycline and ciprofloxacin can be used for C. jejuni and C. coli. The same ECOFF can be used for erythromycin for the three species. The C. jejuni and C. coli ECOFFs for ciprofloxacin however cannot be applied to C. fetus. We also provide data to categorise two 2 β-lactams of interest for systemic diseases, ampicillin and amoxicillin+clavulanate, for the three species.

Unpredictable susceptibility of emerging clinical moulds to tri-azoles: review of the literature and upcoming challenges for mould identification.

Tri-azoles represent the front-line drugs for the treatment of mould diseases; nevertheless, some emerging moulds, such as Fusarium spp., Scedosporium spp., Mucorales and others, may be less susceptible or resistant to these antifungals. A review of the literature was conducted on the susceptibility of rare moulds to the tri-azoles itraconazole, posaconazole and voriconazole. Particular attention was paid to isolates identified by molecular analyses. The range of susceptibility values described for the three tri-azoles was frequently large (from 0.06 to >16), and a high variability was found within each species; isolates were rarely reported as entirely susceptible to all tri-azoles. In addition, the susceptibility of 76 emerging moulds from our collection (including Hypocreales, Dothideomycetes, Scedosporium spp., Mucorales and rare Aspergillus spp.) to itraconazole and voriconazole was determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 and European Committee for Antimicrobial Susceptibility Testing (EUCAST) methods. Susceptibility discrepancies (of two dilutions) were found comparing CLSI and EUCAST for Dothideomycetes; the values for the remaining moulds were similar. More practical, faster and inexpensive susceptibility tools are welcome for testing emerging moulds, as these tests still represent a critical tool to support clinicians on the selection of proper antifungal treatment. The susceptibility of emerging moulds to tri-azoles cannot be predicted exclusively following mould identification, as the isolates' susceptibilities showed highly variable values. Some emerging moulds still remain very difficult to identity, even following standard molecular analyses which result in complex fungal collections. This fact limits the definition of epidemiological cut-offs and clinical breakpoints that are still imperative for emerging moulds.

Dynamics of the sensitivity of N. gonorrhoeae strains found in the territory of the Russian Federation in 2010-2013 to antimicrobial drugs used for the treatment of gonococcal infection

Goal of the study. To study the sensitivity of N. gonorrhoeae strains found in the territory of the Russian Federation in 2010- 2013 to antimicrobial drugs. Materials and methods. During the monitoring of antibiotic resistance of N. gonorrhoeae in 2010-2013, phenotypic properties of N. gonorrhoeae strains to antimicrobial drugs (penicillin, tetracycline, ciprofloxacin, spectinomycin, azithromycin and ceftriaxone) were examined by the agar serial dilution technique. As many as 1,305 viable N. gonorrhoeae strains from different regions of the russian Federation were examined. The results were evaluated according to the criteria such as CLSI (Clinical and Laboratory Standards Institution) and EUCAST (European Committee for Antimicrobial Susceptibility Testing). The results were processed by using the WHONET software, version 5.4. Results. According to the results of a study of N. gonorrhoeae antibiotic resistance, N. gonorrhoeae is highly resistant to penicillin (in 2010 - 72.4%, in 2011 - 50.6%, in 2012 - 51.2%, in 2013 - 49.3%), to tetracycline (in 2010 - 68.5%, in 2011 - 55%, in 2012 - 61.9%, in 2013 - 49.3%) and ciprofloxacin (in 2010 - 56.2%, in 2011 - 37.1%, in 2012 - 35%, in 2013 - 24.6%) for the entire observation period without any substantial trend towards its reduction. During studies of the resistance of the gonococcal infection pathogen to spectinomycin, the share of non-sensitive strains to the antibiotic reduced from 16.7% in 2010 to 0.7% in 2013. A slight reduction in the share of non-sensitive N. gonorrhoeae strains to azithromycin was also observed (in 2010 - 15.3%, in 2011 - 25.3%, in 2012 - 17.5%, in 2013 - 9.9%) yet these data substantially exceed the level recommended by the WHO for prescribing an antimicrobial therapy. A high level of N. gonorrhoeae sensitivity to ceftriaxone (100%) was revealed in 2010-2013. Conclusions. According to the analysis of resistance of N. gonorrhoeae strains to antimicrobial drugs found in the territory of the russian Federation in 2010-2013, there is a trend towards the reduction in the number of non-sensitive N. gonorrhoeae strains to penicillin, tetracycline, ciprofloxacin, spectinomycin and azithromycin, which can confirm the efficacy of the Neisseria gonorrhoeae antibiotic resistance monitoring measures taken by State research Center of Dermatovenereology and Cosmetology since 2002.