Previous Breakpoints Research Articles

A-285 Adoption of 2023 CLSI Revised Aminoglycoside Clinical Breakpoints on Carbapenem Resistant Enterobacterales Isolates Identified at a Commercial Reference Laboratory in Korea, Republic of

Abstract Background The incidence of carbapenem resistant Enterobacterales (CRE) has been rapidly increasing as a nationally notifiable communicable disease since 2017 in Korea. The therapeutic options available for these pathogens are extremely limited. Several new drugs developed to treat CRE infections was recently introduced in Korea, but was not widely used. Instead, aminoglycoside is used as one of current available antimicrobials in CRE infections. The Clinical and Laboratory Standards Institute (CLSI) publishes revised aminoglycoside breakpoints in Enterobacterales in 2023. The purpose of this study was to evaluate the adoption of changing the aminoglycoside breakpoint in a commercial reference laboratory. Methods We retrospectively evaluated aminoglycosides (amikacin, gentamicin, tobramycin) for 1,393 CRE isolates not including surveillance culture identified by MALDI-TOF MS (Bruker Daltonik, Bremen, Germany) from January to December in 2023 at a commercial reference laboratory in Korea. Antimicrobial susceptibility testing was performed using a N415 card on the VITEK2 XL (bioMerieux, France). Susceptibilities of aminoglycoside were assessed based on previous breakpoints in CLSI M100-S32 in 2022 as well as the current breakpoints from the most recent CLSI MIC breakpoints in 2023. Results We evaluated 1,393 CRE isolates. Most isolates were Klebsiella pneumoniae [n=876 (62.9%)] and Escherichia coli [n=322 (23.1%)] followed by Citrobacter koseri [n=76 (5.5%)] The new aminoglycoside breakpoints reduced the percentage of strains that were previously judged to be susceptible by CLSI M100-S32 from 87.5% (1,219/1,393) to 71.2% (992/1,393) in amikacin, from 45.5 (634/1,393) to 44.8% (624/1,393) in gentamicin and from 23.3% (323/1,385) to 21.4% (297/1,385) in tobramycin, respectively. In meropenem resistant 56 isolates with MIC≤8 mg/L available of meropenem in combination with a second agent, frequently polymyxins or aminoglycosides, the new aminoglycoside breakpoints reduced the percentage of strains that were previously judged to be susceptible from 92.9% (52/56) to 64.3% (36/56) in amikacin and from 64.3% (36/56) to 60.7% (34/56) in gentamicin, respectively. There was no difference in tobramycin. Conclusions Our results show decreased susceptibility of aminoglycoside especially for amikacin in CRE isolates tested. Amikacin is commonly used to treat CRE infections, either alone or as part of combination therapy. Although our study has no correlation with clinical outcomes, we found microbiology laboratories should adopt the updated breakpoints.

Comprehensive Analysis of Equid Herpesvirus Recombination: An Insight Into the Repeat Regions

High-throughput sequencing of genomes has expanded our knowledge of the Alphaherpesvirinae, a widely extended subfamily of DNA viruses that recombine to increase their genetic diversity. It has been acknowledged that equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4), two alphaherpesviruses with an economic impact on the horse industry, can recombine. This work aimed to analyze interspecific recombination between all equid alphaherpesvirus species, using genomes of EHV-1, EHV-3, EHV-4, EHV-6, EHV-8, and EHV-9 available in GenBank. 14 events of recombination by RDP4 and Simplot between EHV-1 x EHV-4, EHV-1 x EHV-9, EHV-8 x EHV-1, and EHV-8 x EHV-9 were identified. Ten out of 14 events involved ORF64, a double-copy gene located at the repeat regions that codifies for the infected cell protein 4 (ICP4). Among the ICP4, recombination can be found between EHV-1 X EHV-9, EHV-8 X EHV-9, and EHV-1 X EHV-4, the former affects zebra-borne genotypes, a type of EHV-1 that infect wild equids, and the latter match with previous breakpoints reported in fields isolates. Consequently, these findings strongly suggest that ICP4 is a hotspot for recombination. This work describes novel recombination events and is the first genome-wide recombination analysis using all available equid alphaherpesvirus species genomes.

Revision of fluoroquinolone breakpoints used for interpretation of antimicrobial susceptibility testing of canine bacterial isolates.

The fluoroquinolone antimicrobial agents, enrofloxacin and marbofloxacin, were US Food and Drug Administration (FDA) approved in the United States for use in dogs in 1988 and 1999, respectively. There have been many advances since then concerning the pharmacokinetic-pharmacodynamic (PK-PD) evaluation of fluoroquinolones, and there are data available on the susceptibility of targeted pathogens since the original approval. Using this information, the Clinical and Laboratory Standards Institute (CLSI) Veterinary Antimicrobial Susceptibility Testing Subcommittee (VAST) revised its antimicrobial susceptibility testing breakpoints. The previous breakpoints (used in older editions of CLSI standards) for enrofloxacin in dogs were susceptible (S), ≤ 0.5 µg/mL, intermediate (I) 1-2 µg/mL, and resistant (R) ≥ 4 µg/mL. The new breakpoints are S ≤ 0.06 µg/mL for a dose of 5 mg/kg, 0.12 µg/mL for a dose of 10 mg/kg, 0.25 µg/mL for a high dose of 20 mg/kg, and R ≥ 0.5 µg/mL. The breakpoints of 0.12 and 0.25 µg/mL represent a new susceptible-dose dependent (SDD) category. For marbofloxacin, previous breakpoints were S, ≤ 1 µg/mL, I 2 µg/mL, and R ≥ 4 µg/mL. The new breakpoints are S ≤ 0.12 µg/mL for a dose of 2.8 mg/kg, 0.25 µg/mL for a dose of 5.5 mg/kg (SDD), and R ≥ 0.5 µg/mL. The new breakpoints will be published in the next edition of CLSI-Vet01(S) and deviate considerably from the prior breakpoints. Laboratories are encouraged to revise their testing standards. These changes will likely reduce the unnecessary use of these fluoroquinolones in dogs.

A Cautionary Note Regarding the Use of Cumulative Burnt Areas for the Determination of Fire Danger Index Breakpoints

Identifying the links between fire danger metrics and fire activity is critical in various operational and research fields. A common methodology consists in analysing the relationship between cumulative burnt areas and fire danger metrics. Building on this approach, it has been proposed that fuel moisture content (FMC) drives fire activity in some ecosystems through between one and three breakpoints corresponding to the onset or saturation of fire activity. We demonstrate, through two different approaches, that this methodology is incorrect, because it is biased by the frequency distribution of FMC values. From comparison with a neutral fire distribution and correction for the frequency bias, we show that cumulative burnt area breakpoints are spurious: an upper breakpoint might exist (but would be higher than expected), while no evidence of reduced fire danger was detected for the lowest values of FMC (on the contrary, a secondary increase was detected). Our findings clearly suggest that previous breakpoints resulting from this methodology should be considered with caution, as erroneous conclusions regarding fire danger breakpoints could have major consequences for both fire safety and science outcomes. Finally, we discuss widespread confusion between fire danger breakpoints and fire danger levels, which explains most previous erroneous conclusions.

Antimicrobial susceptibility of 6 antimicrobial agents in Helicobacter pylori clinical isolates by using EUCAST breakpoints compared with previously used breakpoints

IntroductionThe aim of this study was to determine the differences in percentage resistance in H. pylori clinical isolates using EUCAST breakpoints compared with previously used breakpoints. MIC value distribution in H. pylori clinical isolates was also studied. MethodsSusceptibility to amoxicillin, tetracycline, metronidazole, clarithromycin, rifampicin and levofloxacin was performed by E-test in 824 H. pylori clinical isolates. EUCAST and previous breakpoints defined resistance as follows: MIC >0.12mg/L and ≥2mg/L for amoxicillin, >8mg/L and ≥8mg/L for metronidazole, >0.5mg/L and ≥1mg/L for clarithromycin, >1mg/L and ≥32mg/L for rifampicin, and >1mg/L and ≥4mg/L for tetracycline and >1mg/L levofloxacin. ResultsOverall resistance rate by EUCAST and by previous breakpoints was 8.5% and 3.2% for amoxicillin, 0.6% and 0.1% for tetracycline, 39.2% and 39.7% for metronidazole, 51.2% and 51.2% for clarithromycin, 32% and 3.1% for rifampicin, and 6.7% and 6.7% for levofloxacin. ConclusionsWhen using the different breakpoints for antimicrobial susceptibility testing, similar results were found with most antibiotics tested (tetracycline, metronidazole, clarithromycin, and levofloxacin), except for amoxicillin and rifampicin.

Antimicrobial susceptibility of 6 antimicrobial agents in Helicobacter pylori clinical isolates by using EUCAST breakpoints compared with previously used breakpoints

IntroductionThe aim of this study was to determine the differences in percentage resistance in H. pylori clinical isolates using EUCAST breakpoints compared with previously used breakpoints. MIC value distribution in H. pylori clinical isolates was also studied. MethodsSusceptibility to amoxicillin, tetracycline, metronidazole, clarithromycin, rifampicin and levofloxacin was performed by E-test in 824 H. pylori clinical isolates. EUCAST and previous breakpoints defined resistance as follows: MIC >0.12mg/L and ≥2mg/L for amoxicillin, >8mg/L and ≥8mg/L for metronidazole, >0.5mg/L and ≥1mg/L for clarithromycin, >1mg/L and ≥32mg/L for rifampicin, and >1mg/L and ≥4mg/L for tetracycline and >1mg/L levofloxacin. ResultsOverall resistance rate by EUCAST and by previous breakpoints was 8.5% and 3.2% for amoxicillin, 0.6% and 0.1% for tetracycline, 39.2% and 39.7% for metronidazole, 51.2% and 51.2% for clarithromycin, 32% and 3.1% for rifampicin, and 6.7% and 6.7% for levofloxacin. ConclusionsWhen using the different breakpoints for antimicrobial susceptibility testing, similar results were found with most antibiotics tested (tetracycline, metronidazole, clarithromycin, and levofloxacin), except for amoxicillin and rifampicin.

Helicobacter pylori resistance to six antibiotics by two breakpoint systems and resistance evolution in Bulgaria

Background: Helicobacter pylori resistance to antibiotics is the main cause for eradication failures. Methods: Antibiotic resistance in 299 H. pylori strains from 233 untreated adults, 26 treated adults, and 40 untreated children was assessed by E tests and, for metronidazole, by breakpoint susceptibility testing and two breakpoint systems. Results: Using EUCAST breakpoints (EBPs) and previous breakpoints (PBPs), overall resistance rates were: amoxicillin 4.0 and 0.6%, metronidazole 33.8 and 33.8%, clarithromycin 28.1 and 27.4%, levofloxacin 19.4 and 19.4%, tetracycline 3.7 and 1.5%, respectively, and rifampin 8.3% (EBP). Multidrug resistance was detected in treated and untreated adults and an untreated child and included 17 (EBPs) and 15 strains (PBPs). Differences between susceptibility categories were found for amoxicillin (3.5% of strains), clarithromycin (0.7%), and tetracycline (2.2%). Using PBPs, from 2005–2007 to 2010–2015, overall primary clarithromycin resistance continued to increase (17.9–25.6%) as noted in our previous study. However, in 2010–2015, overall primary metronidazole (24.0–31.5%) and fluoroquinolone (7.6–18.3%) resistance rates also increased. Primary resistance rates in children and adults were comparable. Conclusions: Briefly, differences in resistance rates by the two breakpoint systems affected the results for three antibiotics. National antibiotic consumption was linked to macrolide resistance in adults. Current primary H. pylori resistance to three antibiotics increased in all untreated patients and in the untreated adults, with the sharpest rise for the fluoroquinolones. The presence of fivefold H. pylori resistance to metronidazole, clarithromycin, tetracycline, levofloxacin, and amoxicillin according to EBPs is alarming.

Trends in Klebsiella pneumoniae carbapenemase-positive K. pneumoniae in US hospitals: report from the 2007–2009 SENTRY Antimicrobial Surveillance Program

We report the prevalence of carbapenemase-positive Klebsiella pneumoniae among clinical isolates collected from US medical centers (n = 42) from 2007–2009 through the SENTRY Antimicrobial Surveillance Program. Isolates with imipenem or meropenem MIC ≥2 μg/mL were screened by PCR for various carbapenemase genes. Of 2049 K. pneumoniae isolates, 126 (6.1%) were non-susceptible to imipenem or meropenem. blaKPC was identified in 113 isolates (5.5%). No other carbapenemase genes were identified. For US regions combined, prevalence of K. pneumoniae carbapenemase (KPC)-positive isolates were 5.9% in 2007, 4.9% in 2008, and 5.7% in 2009. Rates were highest in the Mid-Atlantic region (28.6% overall), with fluctuation over time (29%, 23%, and 33% from 2007–2009), followed by the East North Central region (2.4% overall), with a slightly increasing trend (nil, 3.1%, 3.8% from 2007–2009). All KPC-positive organisms were carbapenem non-susceptible according to updated CLSI breakpoints, although all but one was similarly classified according to previous breakpoints.

Three cases of IMP-type metallo-β-lactamase-producing Enterobacter cloacae bloodstream infection in Japan

We report three cases of IMP-type metallo-β-lactamase-producing Enterobacter cloacae bloodstream infection, which showed minimum inhibitory concentration values for imipenem with 2μg/ml in all isolates. Although carbapenems were initiated empirically in all cases, two of three cases died. The Clinical and Laboratory Standards Institute lowered the breakpoints of carbapenems for Enterobacteriaceae in 2010. However, the previous breakpoints are still used in many clinical laboratories, which can result in failure to detect carbapenem-resistant Enterobacteriaceae. Therefore, lower breakpoints of carbapenems should be used in clinical settings, and alternative tests for detecting metallo-β-lactamase such as polymerase chain reaction and immunochromatographic assays may contribute to better detection of carbapenem-resistant isolates.

Prevalence of Serotype and Multidrug-Resistance of Streptococcus pneumoniae Respiratory Tract Isolates in 265 Adults and 36 Children in Korea, 2002–2005

In total, 301 isolates of Streptococcus pneumoniae collected from patients with respiratory tract infections admitted at primary clinics during 2002-2005 were tested for multidrug-resistance (MDR) phenotypes and their serotypes in Korea. The predominant serotypes were 19F, 19A, 23F, 11A, 3, 6A, and 6B, accounting for 67.8% of all isolates. Their serotype coverage by 23-valent polysaccharide vaccine and 7-valent conjugation vaccine was 73.1% and 39.2%, respectively. For the application of Clinical and Laboratory Standards Institute's new breakpoint for penicillin, the resistance rate of penicillin was 27.9% (but the penicillin resistance was 80.4% based on the previous breakpoint for penicillin of Clinical and Laboratory Standards Institute). Actually, the full resistance rate was only 4.0% (minimum inhibitory concentration >or=8 mg/L). Resistances to erythromycin, clindamycin, and tetracycline were very high (82.9%, 79.4%, and 71.7%, respectively). Especially, 56.1% of all the isolates were MDR, defined as resistant to three or more of the following agents: penicillin, erythromycin, clindamycin, cefotaxime, tetracycline, and levofloxacin. MDR strains were relatively associated with serotypes 19F, 19A, 23F, and 11A, accounting for 58.0% of the isolates. Their serotype coverage by 23-valent polysaccharide vaccine and 7-valent conjugation vaccine was 79.5% and 45.9%, respectively. Levofloxacin, as a representative fluoroquinolone, was active against 88.2% of all MDR isolates. Of particular concern was the high prevalence of MDR pneumococci in non-PCV7 serotypes with an MDR serotype 19A, 11A, 3, and 6A being mostly responsible. It would be prudent to consider more efficient protective strategies for people at high risk for pneumococcal diseases in regions with a high prevalence of MDR pneumococci.

NCCLS perspectives in changing susceptibility breakpoints for antimicrobial drugs

The spread of resistance to many antimicrobial agents in various microbial species has been highlighted by the World Health Organisation and many government agencies around the world. The reasons for this increase and spread are complex and are discussed. A number of surveillance studies has monitored the increase in resistance among isolates of Streptococcus pneumoniae to various important classes of antimicrobials. These results are discussed with particular reference to penicillins, macrolides and fluoroquinolones. Although there is evidence that in vitro resistance to macrolides and more recently to fluoroquinolones may be associated with a reduced clinical efficacy, there is no such clear association with resistance to β-lactams and lack of clinical efficacy in non-meningeal infections. Resistance or susceptibility to an antimicrobial agent is based on breakpoints and these are set in the US by the National Committee of Clinical and Laboratory Standards. In response to these recent clinical studies showing that non-meningeal pneumococcal infections with strains classified as resistant in vitro still responded well to treatment with various β-lactams, new breakpoints have been set. Results are presented showing that using these breakpoints, the levels of resistance to the third generation cephalosporins, ceftriaxone and cefotaxime against a range of non-meningeal pneumococcal isolates were lower than those obtained using the previous breakpoints. The excellent pharmacokinetic and pharmacodynamic properties of these agents are believed to contribute to their good activity in the clinic.

A gene for nonsyndromic mental retardation maps to chromosome 3p25-pter.

To establish genetic linkage between polymorphic microsatellite loci and a disease locus responsible for an autosomal recessive type of nonsyndromic mental retardation (MR). Although MR is the most common developmental disability in the United States, the etiologies of most nonsyndromic cases are not known. A genealogic database provided information to reconstruct the relationships between 32 individuals from five nuclear families in a single pedigree with 10 affected individuals with nonsyndromic MR. To find a MR disease locus in this population, we performed a genome-wide search using genetic loci spaced at 10- to 20-cM intervals. Pairwise linkage analysis, multipoint linkage analysis, and haplotype reconstruction were used to localize the disease gene. Genetic linkage between a MR disease locus and locus D3S3050 on chromosome 3p25-pter was established with a Zmax = 9.18 at theta = 0.00. Fine mapping this region delimited a 13. 47-cM candidate interval defined by key recombinants at loci D3S3525 and D3S1304. Multipoint linkage analysis refined the critical region to a 6.71-cM interval flanked by loci D3S3525 and D3S1560. Evidence that a gene for MR resides in this location is supported by previous breakpoint deletion mapping studies performed in the chromosome 3p- syndrome. These results suggest that a gene on the subtelomeric region of chromosome 3p contributes to general intelligence. The genes for the cell adhesion L1-like molecule (CALL), the inositol triphosphate receptor (ITPR1), and the AD neuronal thread protein (AD7c-NTP) are leading positional candidates because of their role in brain development, neuronal signaling, and structure.

Correlation of Oxacillin MIC with mecA Gene Carriage in Coagulase-Negative Staphylococci

The National Committee for Clinical Laboratory Standards has recently changed the oxacillin breakpoint from >/=4 mg/liter to >/=0. 5 mg/liter to detect methicillin-resistant coagulase-negative staphylococci (CoNS) because the previous breakpoint lacked sensitivity. To determine the correlation between the new oxacillin breakpoint and the presence of the mecA gene, 493 CoNS of 11 species were tested. The presence of the mecA gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0. 125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS strains with mecA as methicillin resistant and strains of Staphylococcus epidermidis, S. haemolyticus, and S. hominis without mecA as methicillin susceptible. The breakpoint of >/=0.5 mg/liter was not specific for S. cohnii, S. lugdunensis, S. saprophyticus, S. warneri, and S. xylosus, in that it categorized 70 of 74 strains of these species without mecA (94.6%) as methicillin resistant. The results of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with mecA but is not specific for strains of certain species of CoNS without mecA.