Susceptibility Testing Criteria Research Articles

A comparative study of VITEK-2, Double Disc Synergy and Combined Disc Methods for detection of ESBL (Extended Spectrum Beta-Lactamase) production in Escherichia coli and Klebsiella pneumoniae strains

Aim: In this prospective study we aimed to compare the effectiveness of VITEK-2 (bioMérieux, France) automated system, double-disc synergy (DDST) versus combined disc test (CDT) in detecting the Extended-Spectrum Beta-Lactamase (ESBL) positivity in Escherichia coli and Klebsiella pneumoniae strains isolated from various clinical samples. Material and Method: E.coli and K. pneumoniae strains inoculated on Mueller Hinton Agar plate. Susceptibility tests were performed with the VITEK 2 (BioMérieux, France) system before. Afterward, EBSL positivity was investigated manually DDST and CDT. Minimal inhibitor concentration (MIC) results of three tests were compared with each other according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Results: 184 E. coli and K.pneumoniae strains, elevated. 92.9% of 98 patients with VITEK 2 positive results were positive with combined disc and DDS method, 100% of the 86 patients with negative results of VITEK 2 were negative with combined disc and MDC. Conclusion: VITEK 2 was found to have a sensitivity of 100%, a specificity of 92.4%, a positive predictive value of 92.8% and a negative predictive value of 100%. VITEK 2 was found to be compatible with validation tests for ESBL positivity.

INVESTIGATION OF ANTIBIOTIC AND ANTISEPTIC RESISTANCE GENES IN METHICILLIN-RESISTANT AND METHICILLIN-SUSCEPTIBLE STAPHYLOCOCCUS AUREUS ISOLATES

Aim: This study aimed to determine the antibiotic susceptibility of Staphylococcus aureus isolated from different clinical materials sent to the Microbiology Laboratory of Hatay Mustafa Kemal University Hospital, to investigate the mechanisms mediating antibiotic and antiseptic resistance, to determine the SCCmec type of methicillin-resistant isolates. Materials and Method: Overall, 187 S. aureus were included in the study. Antibiotic susceptibilities of the isolates were performed by the disc diffusion method and evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Antibiotic resistance, antiseptic resistance, and Staphylococcal Cassette Chromosome mec (SCCmec) types in MRSA strains were investigated using polymerase chain reaction (PCR). Results: While all of the isolates were found to be susceptible to linezolid and vancomycin; various rates of resistance for penicillin (87.1%), cefoxitin (49.93%), erythromycin (19.79%), ciprofloxacin (13.37%), tetracycline (11.23%), clindamycin (10.16%), trimethoprim-sulfamethoxazole (8.02%), gentamicin (17.82%), fusidic acid (64.2%) and rifampin (1.07%) were determined. A statistically significant difference was found between MRSA and MSSA strains in terms of MDR phenotype rates (p=0.001). Among S. aureus isolates, single resistance genes or various combinations of resistance genes were detected. SCCmec type III (52.4%) was the most common SCCmec type. Conclusions: The results of this study indicated that current control strategies should be revised to minimize antibiotic resistance and periodic surveillance studies must be carried out.

Addressing the Antimicrobial Resistance of Ruminant Mycoplasmas Using a Clinical Surveillance Network.

Antimicrobial resistance (AMR) surveillance of mycoplasmas of veterinary importance has been held back for years due to lack of harmonized methods for antimicrobial susceptibility testing (AST) and interpretative criteria, resulting in a crucial shortage of data. To address AMR in ruminant mycoplasmas, we mobilized a long-established clinical surveillance network called “Vigimyc.” Here we describe our surveillance strategy and detail the results obtained during a 2-year monitoring period. We also assess how far our system complies with current guidelines on AMR surveillance and how it could serve to build epidemiological cut-off values (ECOFFs), as a first attainable criterion to help harmonize monitoring efforts and move forward to clinical breakpoints. Clinical surveillance through Vigimyc enables continuous collection, identification and preservation of Mycoplasma spp. isolates along with metadata. The most frequent pathogens, i.e., M. bovis and species belonging to M. mycoides group, show stable clinicoepidemiological trends and were included for annual AST. In the absence of interpretative criteria for ruminant mycoplasmas, we compared yearly minimum inhibitory concentration (MIC) results against reference datasets. We also ran a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis on the overall service provided by our AMR surveillance strategy. Results of the 2018–2019 surveillance campaign were consistent with the reference datasets, with M. bovis isolates showing high MIC values for all antimicrobial classes except fluoroquinolones, and species of the Mycoides group showing predominantly low MIC values. A few new AMR patterns were detected, such as M. bovis with lower spectinomycin MICs. Our reference dataset partially complied with European Committee on Antimicrobial Susceptibility Testing (EUCAST) requirements, and we were able to propose tentative epidemiological cut-off values (TECOFFs) for M. bovis with tilmicosin and spectinomycin and for M. mycoides group with tilmicosin and lincomycin. These TECOFFs were consistent with other published data and the clinical breakpoints of Pasteurellaceae, which are often used as surrogates for mycoplasmas. SWOT analysis highlighted the benefit of pairing clinical and antimicrobial resistance surveillance despite the AST method-related gaps that remain. The international community should now direct efforts toward AST method harmonization and clinical interpretation.

Prevalence of multidrug-resistant and extended-spectrum beta-lactamase-producing Escherichia coli in urban community wastewater

Antibiotic resistance (ABR) and the spread of multidrug-resistant and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli via wastewater to environmental compartments are of rapidly growing global health concern. Health care facilities, industries and slaughterhouses discharge high loads of ABR bacteria with their wastewater. However, the general community is often the biggest indirect discharger. Yet, research focusing explicitly on this important diffuse source is rather scarce raising questions about variations in the occurrence of ESBL-producing E. coli in wastewater from different communities and over time.Between April 2019 and March 2020, wastewater from three socio-spatially different districts in the Ruhr Metropolis, Germany, and the receiving wastewater treatment plant was sampled monthly and analysed for the occurrence of ESBL-producing E. coli via culture-based methods. Isolates were validated with matrix assisted laser desorption ionization time of flight mass spectrometry and antibiotic resistance profiles were analysed via microdilution. Results were interpreted using the European Committee on Antimicrobial Susceptibility Testing criteria. The German Commission for Hospital Hygiene and Infection Prevention criteria were used for multidrug-resistance categorization.Phenotypic ESBL-producing E. coli could be isolated from every wastewater sample demonstrating that the general community is an important indirect discharger. The socio-spatially disadvantaged area displayed higher absolute loads of ESBL-producing E. coli compared to the other two areas, as well as higher adjusted loads for domestic discharge and inhabitants, particularly during winter, indicating a higher ABR burden. Thirty-two isolates (28.6%) were characterized as multidrug-resistant Gram-negative bacteria (3MRGN). Resistance profiles varied only for those antibiotics, which can be administered in outpatient care. Resistance levels tended to be around 10% lower in the socio-spatially advantaged area. This study shows that spatial and seasonal influences regarding the occurrence of ESBL-producing E. coli in wastewater from socio-spatially different communities are identifiable.

Establishment of sentinel surveillance of human clinical campylobacteriosis in Ireland.

The aim of this work was the establishment of a national laboratory sentinel surveillance service for human clinical Campylobacter in Ireland. This included detailed genomic molecular epidemiology of Campylobacter for 2019. For February-December 2019, 24 clinical microbiology laboratories in Ireland submitted all PCR/culture-positive clinical Campylobacter spp. specimens to Public Health Laboratory (PHL) Dublin oneweek out of every four. Antimicrobial susceptibility testing (AST) according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria was carried out for Campylobacter spp. isolates for ciprofloxacin, tetracycline and erythromycin. Batch whole genome sequencing (WGS) was carried out on cultures and analysis was performed to determine species, genotype, identify antimicrobial resistance (AMR) and virulence determinants and identify clusters. A total of 75 isolates and 366 PCR-positive stools were received, and 277 isolates recovered (55.7% recovery from stools). Of 257 isolates characterized by WGS, 86.4% (n=222) were Campylobacter jejuni, 11.7% (n=30) Campylobacter coli and 1.9% (n=5) Campylobacter lari. There were 20 clonal complexes with ST-21 clonal complex most prevalent at 26.8% (n=69). 50.5% (n=140) of isolates were susceptible to all three antimicrobials tested. 39.3% (n=109) isolates were ciprofloxacin resistant, 26.3% (n=73) tetracycline resistant and two isolates erythromycin resistant. Congruence between phenotypic and genotypic AST was observed. There was 95.9% and 95.6% sensitivity and specificity for WGS to predict ciprofloxacin sensitivity and 98.6% and 99.5% sensitivity and specificity for WGS to predict tetracycline sensitivity. Virulence factors flaA, racR, ciaB and cdtB were detected in all isolates. WGS identified 31 potential clusters for public health alert. This sentinel surveillance of human campylobacteriosis in Ireland establishes the basis for a national reference service. Linking with other partners in a 'One Health' framework will help us better understand sources of infection to reduce disease burden and the threat of AMR.

A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination.

Criteria defining bedaquiline resistance for tuberculosis have been proposed addressing an emerging concern. We evaluated bedaquiline phenotypic drug susceptibility testing (pDST) criteria using drug-resistant tuberculosis clinical isolates tested at five reference laboratories. Isolates were tested at the proposed bedaquiline MGIT960 and 7H11 agar proportion (AP) critical concentrations and also at higher dilutions. The epidemiological cutoff value for the broth microdilution (BMD) plates (frozen and dry) was investigated. Sanger sequencing was performed (atpE and Rv0678 genes) for any isolate testing resistant. The composite reference standard (CRS) defined susceptibility or resistance as is if all pDST methods agreed. If the pDST result was discordant, sequencing results were used for final classification. Geographically diverse and bedaquiline-unexposed isolates were tested (n = 495). The epidemiological cutoff value for BMD was confirmed to be 0.12 μg/ml. The majority of isolates were determined to be susceptible by all methods (467/495; 94.3%), and 28 were determined to be resistant by at least one method; 4 of these were determined to be resistant by all methods. Of the 28 resistant isolates, 12 harbored Rv0678 mutations exclusively. Isolates with insertions/deletions were more likely to be determined to be resistant by more than one method (5/7) compared to isolates with a single nucleotide polymorphism (1/5). Applying the CRS to 24 discordant pDST, BMD dry correctly detected most (15/24; 63%), followed by MGIT960 and BMD frozen (13/24; 61%) and lastly AP (12/24; 50%). Applying the CRS, the prevalence of bedaquiline resistance was 2.2% and ranged from 1.4 to 3.4%, depending on the method used. All methods performed well for bedaquiline susceptibility determination; however, resistance detected should be investigated by a second, alternative method.

Investigation of Plasmid Mediated mcr Colistin Resistance Gene in Clinical Enterobacterales Isolates

Carbapenems are used in the treatment of infections caused by multidrug-resistant bacteria and colistin (polymyxin E) is used as the last choice of antimicrobial agent in those resistant to carbapenems. The worldwide and increased use of colistin, which causes cell death by disrupting the permeability of the cytoplasmic membrane of gram-negative bacteria, raised the problem of resistance. The transferable colistin resistance enzyme mcr, is a phosphoethanolamine transferase that adds phosphoethanolamine to lipid A and modifies lipopolysaccharides, leading to polymyxin resistance. The aim of this study was to investigate some of the most prevalent plasmid mediated colistin and carbapenemase resistance genes in colistin resistant Enterobacterales isolates. Enterobacterales isolates which were isolated in the samples of patients treated in the clinical units between October 2016 and September 2018 in the Karadeniz Technical University Faculty of Medicine Farabi Hospital Medical Microbiology Laboratory were included in the study. In addition to conventional methods, isolates were identified to the species level by MALDI-TOF MS (Bruker Daltonics, Germany). The antibiotic susceptibilities of Enterobacterales isolates were studied by an automated microbiology system (Phoenix, Becton Dickinson, USA) and evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. In isolates that are resistant to colistin, and the isolates that are found to be sensitive but should be included in the patient report of the colistin susceptibility test, colistin susceptibility tests were repeated with liquid microdilution method in accordance with EUCAST standards. The presence of extended spectrum beta-lactamase (ESBL), AmpC beta-lactamase and carbapenemase were determined by phenotypic methods according to EUCAST recommendations in colistin resistant Enterobacterales isolates. Furthermore, resistance genes of mcr-1-5, blaOXA-48, blaKPC, blaNDM, blaVIM, blaIMP were detected by polymerase chain reaction (PCR) method, followed by nucleotide sequence analysis of the amplified products. In our study, 14657 Enterobacterales isolates belonging to 7535 patients treated in different clinical units were examined retrospectively. Escherichia coli 61.2% (n= 8968), Klebsiella pneumoniae 22.7% (n= 3334) and Enterobacter cloacae 6.9% (n= 1005) were the most prevalent isolates. Carbapenem resistance was detected in 894 isolates, and 5.8% (n= 412) of 7135 isolates isolated between October 2016 and September 2017; 6.4% (n= 482) of 7522 isolates between October 2017 and September 2018 were found to be resistant. Considering all isolates, colistin resistant isolates were 65 (0.9%) between October 2016 and September 2017 and 97 (1.3%) between October 2017 and September 2018. By including only the first isolates in the study for the same agent growths in different samples of the same patient, 46 colistin resistant isolates were selected. Six isolates which could not be cultivated from stock cultures were excluded from the study material. Thirteen (32.5%) of the 40 colistin resistant Enterobacterales isolates were isolated in 2017 and 27 (67.5%) were isolated in 2018. ESBL was detected in 22, AmpC beta-lactamase was detected in 6, carbapenem resistance was detected in 15 of them by phenotypic methods. As a result of PCR analysis, mcr-1 gene detected in 2 isolates, blaOXA-48 in 2 isolates, blaVIM in 1 isolate, blaKPC and blaOXA-48 in 1 isolate, blaNDM and blaOXA-48 in 5 isolates. These results were confirmed by sequencing of the PCR products. The mcr-1 genes were found in E.coli isolates grown in urine culture samples of 2 women over 65 years of age treated in our hospital. Among the antibiotics tested, only ampicillin resistance was observed in 1 of the patients, whereas ampicillin, amoxicillin-clavulanate and ciprofloxacin resistance were detected in the other. In conclusion, as far as we can reach in the literature our publication is the first study showing the presence of mcr-1 gene in clinical samples in our country and confirmed by DNA sequence analysis. The detection of mcr gene in isolates without multidrug resistance showed once again the importance of colistin susceptibility testing in the laboratories. In addition, the presence of isolates containing more than one resistance genes in our study, suggests that the spread of carbapenem and colistin resistance may be faster than expected.

Gram-Negative Bacteria and Antibiotic Resistances Isolated from Endotracheal Aspirate Samples of Patients Hospitalized in the Intensive Care Unit: 3-Years Retrospective Analysis

Intensive care units (ICUs) are the most frequent department where hospital-acquired infections caused by multi-resistant microorganisms. Ventilator-associated pneumonia (VAP) is one of the most common serious infections in intensive care units with high mortality rate. In this study, it was aimed to determine the microorganisms and their antibiotic resistance rates of endotracheal aspirate (ETA) samples taken from patients who applied mechanical ventilation in our hospital's ICU. Gram negative microorganisms isolated from ETA samples sent from patients hospitalized at ICU between January 2016 and December 2018 were included in this study. For identification of the growing strains, matrix-mediated laser desorption ionization-flight time mass spectrometry (MALDI-TOF) (VITEK-MS, bioMérieux, France), VITEK 2 automated system (bioMérieux, France) were used to determine antibiotic susceptibility. The antibiotic susceptibility of the strains was evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. A total of 385 Gram negative microorganisms were isolated from ETA specimens with significant growth over the three-year period. When analyzing the distribution of microorganisms, A. baumannii (34.2%) was in the first place, followed by P. aeruginosa and K. pneumoniae. There was no significant difference in bacterial distribution in the three-year period. A. baumannii strains showed high levels of carbapenem resistance (94%), while the most effective antibiotics were colistin and tigecycline. Carbapenem and colistin resistance in P. aeruginosa strains were determined as 27% and 6.5%, respectively. Tigecycline was found to be the most effective antibiotic with 8% resistance rate in K. pneumoniae isolates. The high carbapenem resistance seen in A. baumannii strains isolated in our study is remarkable. Increased colistin resistance in K. pneumoniae shows that the last line drugs are also in danger. It is thought that following the antimicrobial resistance patterns of the ICU regularly, updating the treatment protocols in this direction and applying the infection control measures carefully will contribute to both the success of the treatment and the reduction of the resistance rates. Keywords: Endotracheal aspirate, intensive care units, antibiotic resistance. Special Issue of Health Sciences DOI: 10.7176/JSTR/6-03-31

1556. Assessment of Translational In Vitro and Animal Pharmacokinetic–Pharmacodynamic Data Used to Support Drug Development of Recent Tetracycline Derivatives

BackgroundNonclinical (animal and in vitro) models are commonly used during the development of antibacterial drugs. Pharmacokinetic (PK) and pharmacodynamic (PD) data obtained from these nonclinical models are used to generate a PK-PD target, which can then be bridged to humans in a probability of PK-PD target attainment (PTA) analysis to support selection of the dose regimen for phase 3 trials and in vitro susceptibility testing criteria (breakpoints) to guide clinical usage.MethodsTwo recently approved tetracycline antibacterial drugs, eravacycline (ERV) and omadacycline (OMD), were evaluated. PK-PD data from nonclinical models and clinical microbiological response were collected from each of the respective clinical pharmacology reviews and assessments published by FDA and EMA, respectively. The highest MICs (minimum inhibitory concentrations) reflecting 80% success in the ability of the drug to inhibit growth in the target bacteria were identified from clinical and nonclinical data and termed the MIC cutoff. The nonclinical MIC cutoff was obtained from the PTA analysis using the PK-PD targets from animal studies. The clinical MIC cutoff was obtained from microbiological response (microbiological intent-to-treat population) data from clinical trial experience. The ratios of the clinical and nonclinical MIC cutoffs were calculated and used to evaluate potential discrepancies between the animal model prediction and clinical trial experience.ResultsThe drug development programs for ERV and OMD included murine infection models and in vitro models to characterize PK-PD. The clinical to nonclinical MIC cutoff ratios ranged from 4 to 32. Higher values of the MIC cutoff signify that the drug can treat larger proportions of the bacterial population; therefore, high clinical to nonclinical MIC cutoff ratios signify that the drugs had more activity in reducing the bacterial population in clinical than in nonclinical studies.ConclusionThus, the nonclinical models for ERV and OMD under-predicted microbiological response and breakpoints. While nonclinical models are generally useful, more characterization of translational factors may be needed to allow nonclinical models to be more predictive of clinical trial outcomes.Disclosures All authors: No reported disclosures.

Multicentre study to establish interpretive criteria for clofazimine drug susceptibility testing.

<sec id="st1"> <title>OBJECTIVE</title> To conduct a multicentre study to establish the critical concentration (CC) for clofazimine (CFZ) for drug susceptibility testing (DST) of Mycobacterium tuberculosis on the MGIT™960™ system using the distribution of minimum inhibitory concentrations (MIC) and genotypic analyses of Rv0678 mutations. </sec> <sec id="st2"> <title>DESIGN</title> In phase I of the study, the MIC distribution of laboratory strains (H37Rv and in vitro-selected Rv0678 mutants) and clinical pan-susceptible isolates were determined (n = 70). In phase II, a tentative CC for CFZ (n = 55) was proposed. In phase III, the proposed CC was validated using clinical drug-resistant tuberculosis (DR-TB) isolates stratified by Rv0678 mutation (n = 85). </sec> <sec id="st3"> <title>RESULTS AND CONCLUSION</title> The MIC distribution of CFZ for laboratory and clinical pan-susceptible strains ranged between 0.125 μg/ml and 0.5 μg/ml. As the MIC values of DR-TB isolates used for phase II ranged between 0.25 μg/ml and 1 μg/ml, a CC of 1 μg/ml was proposed. Validation of the CC in phase III showed that probably susceptible and probably resistant Rv0678 mutants overlapped at 1 μg/ml. We therefore recommend a CC of 1 μg/ml, with additional testing at 0.5 μg/ml to define an intermediate category. This was the first comprehensive study to establish a CC for routine phenotypic DST of CFZ using the MGIT960 system to guide therapeutic decisions. </sec>.

Temporal and Geographic Variation in Antimicrobial Susceptibility and Resistance Patterns of Enterococci: Results From the SENTRY Antimicrobial Surveillance Program, 1997-2016.

BackgroundThe SENTRY Antimicrobial Surveillance Program was established in 1997 and presently encompasses more than 750 000 bacterial isolates from over 400 medical centers worldwide. Among these pathogens, enterococci represents a prominent cause of bloodstream (BSIs), intra-abdominal (IAIs), skin and skin structure, and urinary tract infections (UTIs). In the present study, we reviewed geographic and temporal trends in Enterococcus species and resistant phenotypes identified throughout the SENTRY Program.MethodsFrom 1997 to 2016, a total of 49 491 clinically significant enterococci isolates (15 species) were submitted from 298 medical centers representing the Asia-Pacific (APAC), European, Latin American (LATAM), and North American (NA) regions. Bacteria were identified by standard algorithms and matrix-assisted laser desorption ionization–time of flight mass spectrometry. Susceptibility (S) testing was performed by reference broth microdilution methods and interpreted using Clinical and Laboratory Standards Institute/US Food and Drug Administration and European Committee on Antimicrobial Susceptibility Testing criteria.ResultsThe most common Enterococcus species in all 4 regions were Enterococcus faecalis (64.7%) and E. faecium (EFM; 29.0%). Enterococci accounted for 10.7% of BSIs in NA and was most prominent as a cause of IAIs (24.0%) in APAC and of UTIs (19.8%) in LATAM. A steady decrease in the susceptibility to ampicillin and vancomycin was observed in all regions over the 20-year interval. Vancomycin-resistant enterococci (VRE) accounted for more than 8% of enterococcal isolates in all regions and was most common in NA (21.6%). Among the 7615 VRE isolates detected, 89.1% were the VanA phenotype (91.0% EFM) and 10.9% were VanB. Several newer antimicrobial agents demonstrated promising activity against VRE, including daptomycin (99.6–100.0% S), linezolid (98.0%–99.6% S), oritavancin (92.2%–98.3% S), tedizolid (99.5%–100.0% S), and tigecycline (99.4%–100.0% S).ConclusionsEnterococci remained a prominent gram-positive pathogen in the SENTRY Program from 1997 through 2016. The overall frequency of VRE was 15.4% and increased over time in all monitored regions. Newly released agents with novel mechanisms of action show promising activity against VRE.

Antibiotic Susceptibility of Cronobacter spp. Isolated from Clinical Samples.

Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for blaTEM, blaSHV, and blaCTX-M β-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.

2396. Fosfomycin Resistance Among Carbapenem-Resistant Enterobacteriaceae Clinical Isolates in Connecticut, 2017

BackgroundFosfomycin is among the limited treatment options for carbapenem-resistant Enterobacteriaceae (CRE) infections. Despite its use, prevalence of fosfomycin resistance among CRE in the United States is largely unknown. In 2017, submission of Enterobacteriaceae isolates resistant to ≥1 carbapenem became mandated in Connecticut (CT), allowing further characterization at the state public health laboratory (SPHL). We analyzed fosfomycin resistance among CRE isolates in CT during 2017, and explored demographic and molecular factors potentially associated with resistance.MethodsAfter confirming carbapenem resistance, SPHL tests fosfomycin susceptibility using disk diffusion. For each CRE patient, the isolate most resistant to fosfomycin was included in this analysis. We used the Clinical and Laboratory Standards Institute (CLSI) fosfomycin breakpoint for Escherichia coli (nonsusceptible <16 mm) to evaluate associations among fosfomycin resistance and demographic factors, carbapenemase activity (modified carbapenem inactivation method, mCIM) and carbapenemase genes tested at SPHL. We report fosfomycin resistance rate by European Committee on Antimicrobial Susceptibility Testing (EUCAST, resistance <24 mm for E. coli) criteria for comparison.ResultsAmong 138 CRE isolates, 39 (28.3%) were fosfomycin nonsusceptible by CLSI criteria. Most nonsusceptible isolates were Enterobacter cloacae (18; 46.2%) or Klebsiella pneumoniae (17; 43.6%). Isolates from patients aged ≥65 years were more likely to be fosfomycin nonsusceptible than isolates from patients aged <65 years (χ2 = 3.8; P = 0.050). No other demographic characteristics were statistically significant. Of fosfomycin nonsusceptible isolates, 12 (30.8%) produced a carbapenemase (mCIM-positive), and 9 (23.1%) had the blaKPC gene. By EUCAST criteria, 96 (69.6%) CRE isolates were fosfomycin resistant.ConclusionA substantial proportion of CRE in CT during 2017 were fosfomycin nonsusceptible, and nonsusceptibility was associated with older patient age. Fosfomycin resistance risk factors and molecular mechanisms need further exploration. The substantial proportion of isolates with results falling between CLSI and EUCAST breakpoints warrants evaluation.Disclosures All authors: No reported disclosures.

Antimicrobial resistance of Escherichia coli isolates from outpatient urinary tract infections in women in six European countries including Russia

In the Northern Dimension Antibiotic Resistance Study (NoDARS), Finland, Germany, Latvia, Poland, Russia and Sweden collected urine samples from outpatient women (aged 18-65years) with symptoms of uncomplicated urinary tract infection (UTI) to investigate the levels of antimicrobial resistance (AMR) among Escherichia coli isolates. A total of 775 E. coli isolates from 1280 clinical urine samples were collected from October 2015 to January 2017. Antimicrobial susceptibility testing was performed and the results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Overall AMR rates to the commonly used antibiotics nitrofurantoin, fosfomycin and mecillinam (except for Germany that was missing a result for mecillinam) were 1.2%, 1.3% and 4.1%, respectively. The highest overall resistance rates were determined for ampicillin (39.6%), trimethoprim (23.8%), trimethoprim/sulfamethoxazole (22.4%), amoxicillin/clavulanic acid (16.7%) and ciprofloxacin (15.1%), varying significantly between countries. The rate of extended-spectrum β-lactamase (ESBL) production was 8.7%. None of the isolates showed resistance to meropenem. In most cases, low AMR rates were detected against the first-line antibiotics recommended in national UTI treatment guidelines, giving support to their future use. These results also support the European Association of Urology guidelines stating that nitrofurantoin, fosfomycin and mecillinam are viable treatment options for uncomplicated UTI.

Time for Precision: A World Without Susceptibility Breakpoints.

Interpretive criteria for in vitro susceptibility testing criteria, “susceptibility breakpoints,” underpin the evaluation and selection of antimicrobial regimens. However, despite their strengths, susceptibility breakpoints are a relatively blunt instrument employed to address an extremely complex question—what is the likelihood of treatment success for individual patients? With regard to evaluating patients on a case-by-case basis, breakpoints merely allow us to account for pathogen susceptibility. This approach precludes consideration of drug exposures achieved in patients, thus overlooking half of the equation for predicting treatment success. Herein, we propose the framework for considering both pathogen- and patient-specific information to provide clinicians a means of evaluating antimicrobial regimens for individual patients through tools automating pharmacokinetic-pharmacodynamic target attainment analyses. Implementing these tools along with their acceptance by professional organizations will allow for a shift in the paradigm for how antimicrobials are selected and dosed—toward patient-centered care through precision medicine.

Surveillance of iclaprim activity: in vitro susceptibility of Gram-positive skin infection pathogens collected from 2015 to 2016 from North America and Europe

Iclaprim is a diaminopyrimidine, which inhibits bacterial dihydrofolate reductase, and surveillance data prior to 2006 suggested that iclaprim was active against Gram-positive pathogens including emerging drug-resistant pathogens. In an era of increasing antimicrobial resistance, we undertook testing iclaprim and comparators against 931 Gram-positive clinical isolates from the United States and Europe collected between 2015 and 2016. Susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) interpretations were based on CLSI and European Committee on Antimicrobial Susceptibility Testing criteria. MIC50/MIC90 was 0.03/0.12 for all Staphylococcus aureus, 0.06/0.06 for methicillin-susceptible S. aureus, 0.03/0.12 for methicillin-resistant S. aureus, 0.12/0.5 for Streptococcus agalactiae, ≤0.015/≤0.015 for Streptococcus anginosus, 0.03/0.06 for Streptococcus dysgalactiae, and ≤0.015 /0.03 μg/mL for Streptococcus pyogenes. Iclaprim was active against a contemporary collection (2015–2016) of Gram-positive bacteria isolated from the skin or soft tissue from patients with SSSI from the United States and Europe.

Comparative activity of ceftobiprole against coagulase-negative staphylococci from the BSAC Bacteraemia Surveillance Programme, 2013\u20132015

Coagulase-negative staphylococci (CoNS) are a significant cause of bacteraemia, the treatment of which is becoming increasingly complex due to the emergence of multidrug-resistant strains. This study aimed to evaluate the in vitro activity of ceftobiprole, an advanced-generation cephalosporin, as compared with other antimicrobial agents against CoNS from patients with bacteraemia. As part of the British Society for Antimicrobial Chemotherapy (BSAC) Bacteraemia Surveillance Programme, 650 blood isolates of CoNS were obtained from patients with bacteraemia at 74 centres throughout the UK and Ireland for the years 2013–2015. Minimum inhibitory concentrations (MICs) of ceftobiprole and other antimicrobial agents were determined using the BSAC agar dilution method. Susceptibility was assessed by European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. The majority of the isolates (63.2%) were Staphylococcus epidermidis. Overall, methicillin resistance, as determined by oxacillin susceptibility testing, was observed in 64.2% of isolates. The MIC50/90 of ceftobiprole was 1/2 mg/L, and 100% of CoNS isolates were inhibited at the EUCAST ceftobiprole non-species-specific pharmacokinetic/pharmacodynamic breakpoint of 4 mg/L. Only one isolate was resistant to vancomycin. Overall rates of resistance to ciprofloxacin, clindamycin, erythromycin and teicoplanin were 50.5, 25.1, 68.2 and 20.9%, respectively. In S. epidermidis, resistance to oxacillin was associated with increased resistance to other antimicrobials. Ceftobiprole demonstrated in vitro activity against all CoNS species isolated from patients with bacteraemia and was active against species resistant to other antistaphylococcal antimicrobials. The collection of clinical data regarding the efficacy of ceftobiprole in treating CoNS bacteraemia is warranted.

Aerococcus urinae, Alloscardovia omnicolens, and Actinotignum schaalii: the AAA Minor League Team of Urinary Tract Infection Pathogens

In clinical microbiology laboratories, advancements in the methods used for routine organism identification have facilitated more accurate species level resolution. This, along with increasing knowledge regarding “new” pathogens, has provided new insights into biology and has revealed clinical associations not previously known. Aerococcus urinae, Alloscardovia omnicolens, and Actinotignum schaalii are Gram-positive bacteria associated with urinary tract infections but can also be members of the urinary tract microbiota. In addition, invasive infections have been reported for the three bacteria, usually with a urinary tract source. Importantly, the use of routine biochemical testing methods for identification of these bacteria can result in misidentification or the incorrect determination that they are not clinically significant. Of these bacteria, only A. urinae has antimicrobial susceptibility testing criteria and interpretative breakpoints. Microbiologists and clinicians need to be aware of these urinary tract pathogens and how to correctly identify them.

Susceptibility testing and detection of β-lactam resistance mechanisms in Enterobacteriaceae: a multicentre national proficiency study

A nationwide study was developed to evaluate the ability of 60 Spanish clinical microbiology laboratories to predict the underlying β-lactam resistance mechanisms of 12 Enterobacteriaceae strains (CCS01–CCS12). Results obtained by two reference laboratories were compared with those reported by the participant laboratories that used their own routine susceptibility testing methodology. Clinical and Laboratory Standards Institute (CLSI) interpretive criteria were used in 53.3% of centres, whilst 46.7% used European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Overall categorical agreement (CA) was 85.5%. Rates of very major errors (VME), minor errors (MinE) and major errors (ME) were 5%, 9.7% and 5.5%, respectively. The lowest CA values were obtained for carbapenems (56.7–78.1%). β-Lactam/β-lactamase inhibitors were also problematic compounds: VME for amoxicillin/clavulanic acid were 8% (EUCAST criteria) and MinE for piperacillin/tazobactam were 45.7% (CLSI criteria). OXA-48 (CCS02, CCS10, CCS11), VIM-1 (CCS09) and KPC-3 (CCS05) were correctly identified by 75–87%, 65% and 71% of centres, respectively. Strains with an extended-spectrum β-lactamase (ESBL) plus a carbapenemase (CCS03, CCS04, CCS06) were correctly detected in 32–68% of centres. Seven percent correct identifications were recorded for CCS08 (chromosomal K1 β-lactamase hyperexpression plus IMP-8). Strains with permeability deficiencies [CCS07 (ACT-1 plus porin deficit) and CCS12 (TEM-24 plus porin deficit)] were correctly detected in 17% and 10% of centres, respectively. The TEM-1-producer (CCS01) was detected in 40% of centres. Microbiologists should be aware of new antibiotic resistance mechanisms, and these surveillance studies are particularly useful for this purpose and for the communication of such traits.

Surveillance of iclaprim activity: In vitro susceptibility of gram-positive pathogens collected from 2012 to 2014 from the United States, Asia Pacific, Latin American and Europe

Iclaprim is a diaminopyrimidine, which inhibits bacterial dihydrofolate reductase, and it is highly active against Gram-positive pathogens including emerging drug-resistant pathogens. In vitro activity of iclaprim and comparators against 2814 Gram-positive clinical isolates from the United States, Asia Pacific, Latin American and Europe collected between 2012 and 2014 were tested. Susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) interpretations were based on CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. MIC50/MIC90 for all S. aureus, methicillin susceptible S. aureus, methicillin resistant S. aureus, beta-hemolytic streptococci, and Streptococcus pneumoniae were 0.06/0.12, 0.06/0.12, 0.06/0.5, 0.06/0.25, and 0.06/2μg/mL, respectively. Iclaprim was 8 to 32-fold more potent than trimethoprim, the only FDA approved dihydrofolate reductase inhibitor, against all Gram-positive isolates including resistant phenotypes. The MIC90 of iclaprim was also lower than most of the comparators including linezolid and vancomycin against Gram-positive pathogens. Iclaprim demonstrated potent activity against a contemporary collection (2012–2014) of Gram-positive clinical isolates from the United States, Asia Pacific, Latin America and Europe.