Multicenter Evaluation of the Novel ETEST Fosfomycin for Antimicrobial Susceptibility Testing of Enterobacterales, Enterococcus faecalis, and Staphylococcus Species.

Fosfomycin is a phosphonic acid derivative active against a wide spectrum of Gram-positive and Gram-negative pathogens. It is used for the treatment of uncomplicated urinary tract infections (uUTI) or severe infections by oral or intravenous (i.v.) administration. In order to improve its performance and robustness, the fosfomycin strip, an antibiotic gradient diffusion strip, was redeveloped and evaluated in the multicenter study summarized in this paper. ETEST fosfomycin (ETEST FO) clinical performance was evaluated by three study sites on 152 Enterococcus faecalis, 100 Staphylococcus spp. and 330 Enterobacterales in comparison with the CLSI and EUCAST agar dilution reference method. Referring to FDA performance criteria, the ETEST FO achieved 91.0% of essential (EA) and 99.0% of categorical agreement (CA) for Escherichia coli. In addition, 98.0% EA and 93.4% CA were achieved for E. faecalis, with no very major errors (VME) or major errors (ME). According to EUCAST breakpoints for intravenous fosfomycin use, Enterobacterales and Staphylococcus spp. also met ISO acceptance criteria for EA and CA (EA 91.5%, 94.0%, respectively, and CA 98.0% for both). A VME rate of 8.8% was observed for Enterobacterales but the MICs were within EA. A trend to predict lower MICs for Citrobacter spp., E. coli and Salmonella enterica and to predict higher MICs for Klebsiella pneumoniae MICs was observed, while ETEST FO should not be used for Enterobacter cloacae, because of low EA and a high VME rate. The study results support the efficiency of the novel ETEST FO, making it an easy-to-handle tool as a substitute to the classical agar dilution method.

Delafloxacin In Vitro Broth Microdilution and Disk Diffusion Antimicrobial Susceptibility Testing Guidelines: Susceptibility Breakpoint Criteria and Quality Control Ranges for an Expanded-Spectrum Anionic Fluoroquinolone.

Delafloxacin, a recently approved anionic fluoroquinolone, was tested within an international resistance surveillance program. The in vitro susceptibilities of 7,914 indicated pathogens causing acute bacterial skin and skin structure infections (ABSSSI) were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution MIC testing methods. The U.S. Food and Drug Administration (FDA) susceptibility testing breakpoints and quality control ranges for routine broth microdilution and disk diffusion methods were confirmed. The delafloxacin MIC50/90 (% susceptibility) results were as follows: Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), 0.008/0.25 µg/ml (92.8%); Staphylococcus lugdunensis, 0.016/0.03 µg/ml (99.3%); Streptococcus pyogenes, 0.016/0.03 µg/ml (100.0%); Streptococcus anginosus group, 0.008/0.016 µg/ml (100.0%); Enterococcus faecalis, 0.12/1 µg/ml (66.2%); and Enterobacteriaceae, 0.12/4 µg/ml (69.5%). The FDA clinical breakpoints were used to assess intermethod test agreement between delafloxacin MIC and disk diffusion methods for the indicated pathogens. The intermethod susceptibility test categorical agreement for delafloxacin was acceptable, with only 0.4% very major, false-susceptible errors among S. aureus strains. Across all FDA-indicated species, the selected breakpoints produced only 0.0 to 1.7% rates of serious (very major and major errors) intermethod error. Quality control ranges for these standardized delafloxacin susceptibility test methods were calculated from three multilaboratory (12 total sites) studies for six control organisms. In conclusion, the application of FDA MIC breakpoints for delafloxacin against contemporary (2014 to 2016) isolates of ABSSSI pathogens provides additional support for the use of delafloxacin in the treatment of adults with ABSSSI. Delafloxacin MIC and disk diffusion susceptibility testing methods have been standardized for clinical application, achieving high intermethod categorical agreement.

Ceftolozane/tazobactam activity against drug-resistant Enterobacteriaceae and Pseudomonas aeruginosa causing healthcare-associated infections in the Asia-Pacific region (minus China, Australia and New Zealand): report from an Antimicrobial Surveillance Programme (2013-2015).

The aim of this study was to evaluate the in vitro activity of ceftolozane/tazobactam and comparator agents tested against Enterobacteriaceae and Pseudomonas aeruginosa isolates from patients in the Asia-Pacific (APAC) region with healthcare-associated infections. Ceftolozane/tazobactam is an antipseudomonal cephalosporin combined with a well-established ß-lactamase inhibitor. A total of 1963 Gram-negative organisms (489 P. aeruginosa and 1474 Enterobacteriaceae) were consecutively collected using a prevalence-based approach from 14 medical centres in the APAC region. Antimicrobial susceptibility testing was performed by broth microdilution method as described by the CLSI and the results were interpreted according to EUCAST and CLSI breakpoint criteria. Ceftolozane/tazobactam [MIC50/90, 0.25/4 µg/mL; 89.2/85.8% susceptible (CLSI/EUCAST)] and meropenem [MIC50/90, ≤0.06/≤0.06 µg/mL; 96.3/96.5% susceptible (CLSI/EUCAST)] were the most active compounds tested against Enterobacteriaceae. Isolates displayed susceptibility rates to other ß-lactam agents ranging from 85.8/81.0% for piperacillin/tazobactam to 74.4/72.7% for cefepime and 72.8/68.1% for ceftazidime using CLSI/EUCAST breakpoints. Among the Enterobacteriaceae isolates, 3.6% were carbapenem-resistant Enterobacteriaceae (CRE) and 25.6% exhibited an extended-spectrum ß-lactamase (ESBL) non-CRE phenotype. Ceftolozane/tazobactam showed good activity against ESBL non-CRE phenotype strains of Enterobacteriaceae (MIC50/90, 0.5/16 µg/mL), but not against isolates with a CRE phenotype (MIC50/90, >32/>32 µg/mL). Ceftolozane/tazobactam was the most potent (MIC50/90, 0.5/4 µg/mL) ß-lactam agent tested against P. aeruginosa isolates, inhibiting 90.8% at an MIC of ≤4 µg/mL. Pseudomonas aeruginosa exhibited high rates of susceptibility to amikacin [91.2/89.4% (CLSI/EUCAST)] and colistin [98.4/100.0% (CLSI/EUCAST)]. Ceftolozane/tazobactam was the most active ß-lactam agent tested against P. aeruginosa and demonstrated higher in vitro activity than available cephalosporins when tested against Enterobacteriaceae.

Ceftolozane-tazobactam activity against drug-resistant Enterobacteriaceae and Pseudomonas aeruginosa causing healthcare-associated infections in Australia and New Zealand: Report from an Antimicrobial Surveillance Program (2013-2015).

OBJECTIVES: To evaluate the in vitro activity of ceftolozane-tazobactam and comparator agents tested against isolates of Enterobacteriaceae and Pseudomonas aeruginosa from patients in Australia and New Zealand with healthcare-associated infection. METHODS: A total of 1459 gram-negative organisms (440 P. aeruginosa and 1019 Enterobacteriaceae) were consecutively collected from 11 medical centers located in Australia and New Zealand. The organisms were tested for susceptibility by broth microdilution methods as described by the CLSI M07-A10 document and the results interpreted according to EUCAST and CLSI breakpoint criteria. RESULTS: Ceftolozane-tazobactam (MIC50/90, 0.25/0.5µg/mL; 97.7/95.9% susceptible [CLSI/EUCAST]), meropenem (MIC50/90, ≤0.06/≤0.06µg/mL; 99.8/99.9% susceptible [CLSI/EUCAST]) and amikacin (MIC50/90, 2/4µg/mL; 99.8/99.6% susceptible [CLSI/EUCAST]) were the most active compounds tested against Enterobacteriaceae. Enterobacteriaceae isolates displayed susceptibility rates to other ß-lactam agents ranging from 95.3/94.4% for cefepime, 94.1/91.4% for piperacillin-tazobactam, and 93.3/91.5% for ceftazidime using CLSI/EUCAST breakpoints. Among the Enterobacteriaceae isolates tested, 0.1% were CRE and 6.6% exhibited an ESBL non-CRE phenotype. Whereas ceftolozane-tazobactam showed good activity against ESBL non-CRE phenotype strains of Enterobacteriaceae (MIC50/90, 0.5/2µg/mL), it lacked useful activity (MIC, >32µg/mL) against the single isolate with a CRE resistant phenotype. Ceftolozane-tazobactam was the most potent (MIC50/90, 0.5/2µg/mL) ß-lactam agent tested against P. aeruginosa isolates, inhibiting 95.7% at an MIC of ≤4µg/mL. CONCLUSIONS: Ceftolozane-tazobactam was the most active ß-lactam agent tested against P. aeruginosa and demonstrated higher in vitro activity than currently available cephalosporins and piperacillin-tazobactam when tested against Enterobacteriaceae.

International dissemination of Escherichia coli strains with discrepant behaviour in phenotypic antimicrobial susceptibility tests.

Preventing the dissemination of antimicrobial resistance depends on appropriate antibiotic stewardship and accurate antimicrobial susceptibility testing (AST). We report the international dissemination of Escherichia coli strains, showing discrepancies between reference methods when phenotypically tested for susceptibility to piperacillin/tazobactam (TZP). We demonstrate that these related strains are predisposed to problematic TZP AST interpretations.

Comparison of three reference methods for testing susceptibility of staphylococci to trimethoprim-sulfamethoxazole.

Three reference MIC methods approved by the Clinical and Laboratory Standards Institute were compared by testing 567 staphylococci against trimethoprim-sulfamethoxazole. Category agreement ranged from 94.9% (broth macrodilution versus broth microdilution) to 98.6% (agar dilution versus broth microdilution). Twenty-seven strains resistant by broth macrodilution were susceptible by broth microdilution.

Development of a standardized susceptibility test for campylobacter with quality-control ranges for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem.

A standardized agar dilution susceptibility testing method was developed for Campylobacter that consisted of testing on Mueller-Hinton medium supplemented with 5% defibrinated sheep blood in an atmosphere of 10% CO2, 5% O2, and 85% N2. Campylobacter jejuni ATCC 33560 was identified as a quality-control (QC) strain. Minimal inhibitory concentration (MIC) QC ranges were determined for two incubation time/temperature combinations: 36 degrees C for 48 hr and 42 degrees C for 24 hr. Quality-control ranges were determined for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem. For all antimicrobial agents tested at both temperatures, 95-100% of the QC MIC results fell within recommended QC ranges. Twenty-one Campylobacter clinical isolates, encompassing five species of Campylobacter (C. jejuni, C. coli, C. jejuni, subsp. doylei, C. fetus, and C. lari) were tested in conjunction with the C. jejuni QC strain. While C. jejuni and C. coli could be reliably tested under both test conditions, growth of C. jejuni subsp. doylei, C. fetus, and C. lari isolates was inconsistent when incubated at 42 degrees C. Therefore, it is recommended that these species only be tested at 36 degrees C.

PCR-RFLP typing of ureC from Helicobacter pylori isolated from gastric biopsies during a European multi-country clinical trial.

A multi-country clinical trial was conducted in ten European countries to determine the efficacy of clarithromycin-omeprazole dual therapy for treating Helicobacter pylori infection in peptic ulcers. Gastric biopsies were cultured for H. pylori before and after treatment. PCR-RFLP was used to determine the genetic heterogeneity of 100 H. pylori isolates from pretreatment and posttreatment biopsies. An 820 bp amplified fragment of the ureC gene was digested with the restriction enzymes Sau3A and Hhal. Fourteen different Sau3A patterns and 15 different Hhal patterns were identified among the pretreatment isolates. In combination, 42 different RFLP types were identified. Comparison of isolates before treatment with those after treatment showed that five of ten patients on clarithromycin-omeprazole dual therapy had the same RFLP type and that all 12 patients on omeprazole therapy alone had the same RFLP type. All isolates were susceptible to clarithromycin prior to treatment, while seven of ten patients on clarithromycin-omeprazole therapy had H. pylori that was resistant to clarithromycin after therapy and 11 of 12 patients on omeprazole therapy had isolates susceptible to clarithromycin after treatment. In addition to PCR-RFLP typing, the presence of the cytotoxin-associated gene (cagA) and the vacuolating gene (vacA) was determined; 79% of the isolates were cagA-positive and all were vacA-positive. The results of this study indicate that infection of H. pylori in Europe is not restricted to a few RFLP types.

Point mutations in the 23S rRNA gene of Helicobacter pylori associated with different levels of clarithromycin resistance.

Fifty-four of 59 (91.5%) clarithromycin-resistant isolates of Helicobacter pylori from different patients possessed either the A2143G (formerly A2058G) or the A2144G (formerly A2059G) mutation in the gene encoding 23S rRNA. The A2143G mutation was significantly more likely to occur in isolates with MICs exceeding 64 mg/L (65% versus 30% with the A2144G mutation; P = 0.01). The majority (26 of 31; 83.9%) of isolates with the A2143G mutation had MICs exceeding 64 mg/L. Peptic ulcer disease recurred in a substantial proportion of patients infected with H. pylori strains containing either the A2143G or the A2144G mutation.

PCR-RFLP typing of ureC from Helicobacter pylori isolated in Argentina from gastric biopsies before and after treatment with clarithromycin.

A clinical trial was conducted in Argentina to determine the efficacy of clarithromycin plus lansoprazole for the treatment of Helicobacter pylori in duodenal ulcers and non-ulcer dyspepsia. PCR-RFLP was conducted on an 820-bp amplified product of the ureC gene of H. pylori to determine the genetic heterogeneity of 83 pretreatment and 21 post-treatment isolates. Twelve different restriction patterns were observed when digested with Sau 3A or Hha I, resulting in 40 different RFLP types. Comparison of isolates before treatment to after treatment showed that 20 of 20 patients had the same RFLP type. In addition, the presence of the cytotoxin-associated gene (cagA) and the vacuolating gene (vacA) were determined. All pretreatment isolates were positive for vacA whereas 75% of the pretreatment isolates were positive for cagA. The results of this study indicate that a high degree of heterogeneity exists among H. pylori and that infection is not limited to a small number of RFLP types.

A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clarithromycin-resistant Helicobacter pylori.

We have developed a rapid PCR-oligonucleotide ligation assay that can discriminate single base substitutions that are associated with clarithromycin resistance in Helicobacter pylori. Susceptible isolates were wild type at positions 2143 and 2144 (cognate to 2058 and 2059 in Escherichia coli), while 93% of the resistant isolates contained A-to-G mutations at either position and 7% of the isolates contained A-to-C mutations at position 2143. In addition, the MIC for 86% of the resistant isolates with an A2143 mutation was > or = 64 micrograms per ml, and that for 89% of the resistant isolates with an A2144 mutation was < or = 32 micrograms per ml.

Identification of a 23S rRNA gene mutation in clarithromycin-resistant Helicobacter pylori.

BACKGROUND: Transition mutations (A-G) at residue 2143, cognate to position 2058 in the Escherichia coli 23S rRNA gene, have been shown to confer resistance to macrolides in Helicobacter pylori. This study reports the finding that transversion mutations (A-C) can occur at 2143 as well. MATERIALS AND METHODS: Three clarithromycin-resistant H. pylori isolated from three different patients after treatment with clarithromycin were analyzed for point mutations by cycle sequencing of a 163-bp amplified region surrounding residue 2143 within the conserved loop of the 23S rRNA gene. RESULTS: Nucleotide sequence comparisons of a 163-bp amplified product revealed that A-C transversion mutations occurred at position 2143. H. pylori isolated from the patients prior to treatment were susceptible to clarithromycin and displayed no polymorphism at 2143. CONCLUSION: This is the first report to show that A-C transversion mutations at position 2143 can confer resistance to clarithromycin in H. pylori and further support the role that mutations at position 2143 play in conferring macrolide resistance in H. pylori.

Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.

Twelve clarithromycin-resistant Helicobacter pylori isolates (100% of resistant isolates examined) from seven different patients each contained an A-->G transition mutation within a conserved loop of 23S rRNA. A-->G transition mutations at positions cognate with Escherichia coli 23S rRNA positions 2058 and 2059 were identified. Clarithromycin-susceptible H. pylori isolates from 14 different patients displayed no polymorphisms in a conserved loop within domain V of 23S rRNA. The study is the first to report mutations in H. pylori associated with resistance to an antimicrobial agent used in established peptic ulcer treatment regimens.