In vitro activities of broad-spectrum cephalosporins against nonmeningeal isolates of Streptococcus pneumoniae: MIC interpretation using NCCLS M100-S12 recommendations

Publication of the NCCLS M100-S12 document in January 2002 introduced ceftriaxone and cefotaxime MIC interpretative breakpoints of < or =1 microg/ml (susceptible), 2 microg/ml (intermediate), and > or =4 microg/ml (resistant) for nonmeningeal isolates of Streptococcus pneumoniae. To estimate the effect of these breakpoint changes on clinical laboratory susceptibility testing results, nonmeningeal pneumococcal isolate (blood and respiratory) data from The Surveillance Network Database-USA, an electronic surveillance database, for the years 1996 to 2000 were collated and studied. Of 9,863 nonmeningeal isolates tested against ceftriaxone, 82.7% were susceptible, 13.2% were intermediate, and 4.1% were resistant by the M100-S11 NCCLS breakpoints (2001); by M100-S12 breakpoints, 95.9% of the isolates were susceptible, 3.1% were intermediate, and 1.0% were resistant. Of 10,777 nonmeningeal isolates tested against cefotaxime, 79.2% were susceptible, 14.3% were intermediate, and 6.5% were resistant by M100-S11 breakpoints; by M100-S12 breakpoints, 93.5% were susceptible, 4.2% were intermediate, and 2.3% were resistant. Overall, the new M100-S12 ceftriaxone and cefotaxime interpretative breakpoints for nonmeningeal isolates of S. pneumoniae decreased the number of isolates interpreted as intermediate by 10% and as resistant by 3 to 4%.     

In vitro activity of imipenem/relebactam,meropenem/vaborbactam and comparators against Enterobacterales from patients with intra-abdominal infections: Results of the study for Monitoring Antimicrobial Resistance Trends (SMART) in Taiwan, 2020

BackgroundMulti-drug resistant Enterobacterales is a growing health threat. Imipenem/relebactam and meropenem/vaborbactam, are not clinically used in Taiwan and the susceptibility is lack from routine laboratory tests.MethodsBroth microdilution method was used to determine the minimum inhibitory concentrations (MICs) and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints criteria. Isolates that were not susceptible to imipenem (MIC ≥ 2 mg/L), imipenem/relebactam (MIC ≥ 2 mg/L), or ceftolozane-tazobactam (MIC ≥ 4 mg/L) were selected for further molecular testing for genes encoding extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases by multiplex PCR assays.ResultsA total of 290 Enterobacterales isolates from 9 participating hospitals were collected in 2020. Escherichia coli (n = 135, 46.6%) and Klebsiella pneumoniae (n = 88, 30.3%) were two leading pathogens of all Enterobacterales isolates. The antimicrobial agents with susceptibility rates more than 90% included amikacin (99.3%, 288/290), ertapenem (90.0%, 261/290), meropenem (97.2%, 282/290), imipenem/relebactam (94.8%, 275/290) and meropenem/vaborbactam (99.3%, 288/290). K. pneumoniae isolates were less susceptible to ertapenem, imipenem, meropenem, piperacillin-tazobactam and ceftozolane/tazobactam than E. coli (all p < 0.05). ESBL, AmpC, and carbapenemase were detected in 40.5% (17/42), 45.2% (19/42) and 11.9% (5/42) among tested isolates, respectively. The 5 carbapenemase genes included 4 bla_KPC and 1 bla_IMP. The imipenem-non-susceptible isolates (n = 30) had higher susceptibility rates to meropenem/vaborbactam (93.3%, 28/30) than imipenem/relebactam (50%, 12/30) (p < 0.05).ConclusionsImipenem/relebactam and meropenem/vaborbactam had excellent efficacy against Enterobacterales isolates. Meropenem/vaborbactam allowed better salvage therapy for carbapenem-resistant Enterobacterales infections.     

Comparison of disk diffusion,Etest and VITEK2 for detection of carbapenemase‐producing Klebsiella pneumoniae with the EUCAST and CLSI breakpoint systems

The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion carbapenem breakpoints for the detection of carbapenemase‐producing Klebsiella pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20) carbapenemases were characterized by disk diffusion (Oxoid) and Etest (bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux, five different cards). Extended‐spectrum β‐lactamase (ESBL) testing was performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson) and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints, respectively, 11 and seven of the strains were susceptible to imipenem, 12 and eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological cut‐off (ECOFF) values for meropenem and ertapenem identified all carbapenemase producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase producers were detected with EUCAST disk diffusion breakpoints for ertapenem and meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem) isolates as susceptible. When cards with a single carbapenem were used, detection failures with VITEK2 were four for imipenem, none for meropenem and one for ertapenem. Cards containing all three carbapenems had one to two failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM producers were positive. With VITEK2, no VIM producers and between none and seven KPC producers were ESBL‐positive. All carbapenemase producers were detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem detected all carbapenemase producers. VITEK2 had between none and four failures in detecting carbapenemase producers, depending on the antibiotic card.     

Cefixime disk susceptibility test criteria.

A total of 583 bacterial isolates was tested for susceptibility to cefixime by broth microdilution and by disk agar diffusion with 5-, 10-, and 30-microgram disks. At MIC breakpoints of less than or equal to 1.0 and greater than or equal to 4 micrograms/ml for susceptible and resistant, respectively, the 5-microgram disk showed slightly better discrimination. The 5-microgram cefixime disk is recommended with proposed interpretive breakpoint criteria of: less than or equal to 17 mm, resistant; 18 to 20 mm, intermediate; and greater than or equal to 21 mm, susceptible.     

Effects of Efflux Pump Inhibitors on Erythromycin, Ciprofloxacin, and Tetracycline Resistance in Campylobacter spp. Isolates

The aim was to assess the potency of the efflux pump inhibitors (EPIs) phenylalanine-arginine ?-naphthylamide (PA?N) and 1-(1-naphthylmethyl)-piperazine (NMP) and the putative natural EPI phenolic (-)-epigallocatechin gallate (EGCG) for the reversal of erythromycin, ciprofloxacin, and tetracycline resistance in Campylobacter jejuni and Campylobacter coli isolates. We investigated target mutations and resistant genes involved in erythromycin and tetracycline resistance and determined the roles of the bacterial drug efflux systems (cmeB, cmeF, and cmeR) in antimicrobial resistance. Our data show that most of the high-level erythromycin resistance and all of the tetracycline resistance can be explained through mutations in 23S rRNA and the presence of the tetO gene, respectively. The EPIs show the ability to partly reverse drug resistance in these Campylobacter isolates. Based on a fourfold or greater reduction in the erythromycin minimal inhibitory concentration (MIC), PA?N and NMP had clear effects in almost of all of the isolates tested. PA?N had a highly selective action on the ciprofloxacin and tetracycline MICs. Inactivation of cmeB increased susceptibility to all of the antimicrobials tested, whereas inactivation of cmeF and cmeR had no effects. A notable decrease in resistance to erythromycin and ciprofloxacin in the presence of subinhibitory concentrations of EGCG demonstrates the resistance-modifying activities of this natural EPI, and indicates its potential use in the control of Campylobacter spp. in the food chain.     

The roles of mutations in gyrA,parC, and ompK35 in fluoroquinolone resistance in Klebsiella pneumoniae

In a survey of 541 Klebsiella pneumoniae isolates from 44 hospitals in Taiwan, three distinct populations were identified by the disk diffusion method according to the disribution of zone diameters of ciprofloxacin. Isolates with resistant, reduced-susceptible, and susceptible to fluoroquinolone were defined as CIP zone diameters of < or = 15 mm, 16-26 mm, and > or = 27 mm, respectively. Thus, in addition to 38 (7%) resistant isolates, there were 30 (5.5%) reduced-susceptible isolates and 473 (87.5%) susceptible isolates. A total of 34 isolates consisting of nine resistant, 13 reduced-susceptible, and 12 susceptible isolates were assessed for point mutations in gyrA and parC and the outer membrane profiles. The susceptibility to fluoroquinolone of 13 reduced-susceptible isolates was not altered in the presence of carbonyl cyanide m-chlorophenylhydrazone, an efflux inhibitor, showing that efflux is not a major contributor to reduced susceptibility. In addition to single mutation in gyrA, OmpK35 porin loss can also be the first step for developing fluoroquinolone resistance. No strain possesses a parC mutation without the simultaneous presence of a gyrA mutation, suggesting that mutations in parC play a complementary role for higher-level of fluoroquinolone resistance and fluoroquinolone resistance is a multistep process.     

Predicting Pseudomonas aeruginosa susceptibility phenotypes from whole genome sequence resistome analysis

ObjectivesThe aim was to develop and validate a Pseudomonas aeruginosa genotypic resistance score, based on analysis of the whole genome sequence resistome, to predict antimicrobial susceptibility phenotypes.MethodsA scoring system based on the analysis of mutation-driven resistance in 40 chromosomal genes and horizontally acquired resistance (Resfinder) was developed for ceftazidime, ceftolozane/tazobactam, meropenem, ciprofloxacin and tobramycin. Resistance genes/mutations were scored from 0 (no effect) to 1 (EUCAST clinical resistance). One hundred wild-type strains obtained from 51 different hospitals during a 2017 multicentre study were fully sequenced and analysed in order to define a catalogue of natural polymorphisms in the 40 chromosomal resistance genes. The capacity of genotypic score to predict the susceptibility phenotype was tested in 204 isolates randomly selected from the 51 hospitals (four from each hospital).ResultsThe analysis of the 100 wild-type isolates yielded a catalogue of 455 natural polymorphisms in the 40 genes involved in mutational resistance. However, resistance mutations and high-risk clones (such as ST235) were also documented among a few wild-type isolates. Overall, the capacity of the genotypic score (<0.5) for predicting phenotypic susceptibility (S + I in the case of meropenem) was very high (95–100%). In contrast, the capacity of the genotypic score to predict resistance (≥1) was far more variable depending on the agent. Prediction of meropenem clinical resistance was particularly low (18/39, 46.1%), whereas it classified clinical ceftolozane/tazobactam resistance in 100% (7/7) of cases.DiscussionAlthough a margin for improvement was evidenced in this proof of concept study, an overall good correlation between the genotypic resistance score and the susceptibility profile was documented. Further refining of the scoring system, automatization and testing of large international cohorts should follow.     

The relative contribution of efflux and target gene mutations to fluoroquinolone resistance in recent clinical isolates of Pseudomonas aeruginosa

The clinical utility of fluoroquinolones (FQs) for the treatment of Pseudomonas aeruginosa (PA) and other serious Gram-negative infections is currently decreasing due to the rapid emergence of resistance. Because previous studies have shown that efflux is a common mechanism contributing to FQ resistance in PA, one suggested approach to extend the longevity of this class of drugs is combination therapy with an efflux pump inhibitor (EPI). In order to determine the viability of this approach, it is necessary to understand the relative contribution of efflux- vs. target-mediated mechanisms of FQ resistance in the clinic. A set of 26 recent PA clinical isolates were characterized for antibiotic resistance profiles, efflux pump expression, topoisomerase mutations, and FQ susceptibility with and without an EPI. The contribution of OprM to the overall antibiotic resistance was assessed in a subset of these strains. Our results suggest that the co-administration of an EPI with FQs or other antibiotics currently in use would not be sufficient to combat the complexity of resistance mechanisms now present in many clinical isolates.     

摩根摩根菌中质粒介导KPC-2型碳青霉烯酶的检测

目的 研究碳青霉烯耐药摩根摩根菌的分子流行病学及其耐药机制.方法 2010年10月-2011年2月从杭州市中医院分离到7株碳青霉烯不敏感的摩根摩根菌.脉冲场凝胶电泳(PFGE)分析菌株之间的同源性;琼脂稀释法测定抗生素对细菌的最低抑菌浓度(MIC);接合试验、质粒图谱分析、特异性PCR扩增和序列分析等研究细菌对碳青霉烯耐药的分子机制.结果 分离自急诊监护病房的6株摩根摩根菌PFGE条带完全相同或相差1～3个条带;分离自重症监护病房的1株摩根摩根菌与其他6株PFGE条带差异明显.7株摩根摩根菌的耐药模式基本相同.亚胺培南、美罗培南和厄他培南的MIC值差异较大,分别为8μg/ml(耐药)、1μg/ml(敏感)和0.25～0.50μg/ml(敏感或中介耐药).7株摩根摩根菌对青霉素类、氨曲南和环丙沙星耐药,对头孢菌素类耐药或敏感,对阿米卡星敏感.接合试验使大肠埃希菌EC600对碳青霉烯类抗生素由敏感变为耐药,对其他β-内酰胺类抗生素也均耐药.摩根摩根菌及转移接合子均含有一个约为60kb的质粒.PCR扩增及测序表明摩根摩根菌及转移接合子均产KPC-2型碳青霉烯酶,且携带qnrS1基因.结论 首次在摩根摩根菌中检测到KPC-2型碳青霉烯酶,KPC-2是引起摩根摩根菌对碳青霉烯类不敏感的主要原因.     

The antimicrobial susceptibility in adult invasive pneumococcal disease in the era of pneumococcus vaccination: A hospital-based observational study in Taiwan

BackgroundA regional antibiotic susceptibility data of common pathogens is crucial to first-line physician for clinical judgment and appropriate selection of antimicrobial agents. The aim of this study is to update the epidemiology data of drug resistance of pneumococcus causing invasive pneumococcal disease (IPD) in adults.MethodsFrom the logbooks of microbiology laboratories, we retrospectively retrieved Streptococcus pneumoniae isolates, collected from normally sterile sites in adult patients in three hospitals in Taiwan from July 2011 to June 2015. Antibiotic resistance and serotypes of the isolates and clinical manifestations were further analyzed.ResultsA total of 150 non-duplicated isolates were collected. According to CLSI meningitis breakpoint, the proportion of ceftriaxone non-susceptible pneumococcus (CNSP) showed an increasing trend from 4.5% in 2011 to > 40% in 2013–2015 (p = 0.007). Serotypes 19A and 23F were significantly associated with CNSP. Imipenem and meropenem had a relative low susceptible rate of 36.7% and 50.7%, respectively. Serotypes 6A, 14, 19A and 19F were significantly associated with the non-susceptibility to these carbepanems.ConclusionThe increase in the prevalence of CNSP using meningitis breakpoint was observed. For treating pneumococcal meningitis, empirical monotherapy with ceftriaxone might not be adequate. Imipenem and meropenem might not be a good choice for empirical treatment of adult IPDs. Antibiotic resistance of pneumococcus to ceftriaxone, cefepime, imipenem and meropenem were associated with 13-velent pneumococcal conjugate vaccine serotypes.     

Activity of meropenem against imipenem-resistant bacteria and selection in vitro of carbapenem-resistantEnterobacteriaceae

The activity of meropenem against 106 imipenem-resistant (MIC 8 mg/l) clinical isolates, and the frequency of resistance to meropenem and imipenem among 24Enterobacteriaceae was determined. Both agents selected colonies on agar but 20–80 % were susceptible after one subculture and 72 % of the mutants reverted to susceptibility 1 to 6 months after selection. All isolates and stable mutants were inhibited by > 1 mg/l meropenem, although the MIC of imipenem was 4–16 mg/l. Three of sixXanthomonas maltophilia isolates were susceptible to meropenem (MICs 2–4 mg/l).Pseudomonas aeruginosa lacking outer membrane protein D2 were resistant to meropenem, although isolates with substantially reduced expression of this protein were susceptible. None of the imipenem-resistant gram-positive bacteria were susceptible to meropenem. There was no clear correlation between altered outer membrane protein expression and decreased susceptibility to carbapenems, and there was no apparent involvement of plasmid or chromosomal -lactamase.     

Molecular characterization of clinical Streptococcus pneumoniae isolates with reduced susceptibility to fluoroquinolones emerging in Italy

Fifteen Streptococcus pneumoniae clinical isolates with reduced fluoroquinolone susceptibility (defined as a ciprofloxacin MIC of > or = 4 microg/ml), all collected in Italy in 2000-2003, were typed and subjected to extensive molecular characterization to define the contribution of drug target alterations and efflux mechanisms to their resistance. Serotyping and pulsed-field gel electrophoresis analysis indicated substantial genetic unrelatedness among the 15 isolates, suggesting that the new resistance traits arise in multiple indigenous strains rather than through clonal dissemination. Sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE demonstrated that point mutations producing single amino acid changes were more frequent in topoisomerase IV (parC mutations in 14 isolates and parE mutations in 13) than in DNA gyrase subunits (gyrA mutations in 7 isolates and no gyrB mutations observed). No isolate displayed a quinolone efflux system susceptible to carbonyl cyanide m-chlorophenylhydrazone; conversely, four-fold or greater MIC reductions in the presence of reserpine were observed in all 15 isolates with ethidium bromide, in 13 with ulifloxacin, in 9 with ciprofloxacin, in 5 with norfloxacin, and in none with five other fluoroquinolones. The effect of efflux pump activity on the level and profile of fluoroquinolone resistance in our strains was minor compared with that of target site modifications. DNA mutations and/or efflux systems other than those established so far might contribute to the fluoroquinolone resistance expressed by our strains. Susceptibility profiles to nonquinolone class antibiotics and resistance-associated phenotypic and genotypic characteristics were also determined and correlated with fluoroquinolone resistance. A unique penicillin-binding protein profile was observed in all five penicillin-resistant isolates, whereas the same PBP profile as S. pneumoniae R6 was exhibited by all six penicillin-susceptible isolates. This is the first attempt to molecularly characterize clinical isolates of S. pneumoniae with reduced susceptibility to fluoroquinolones emerging in Italy.     

Prevalence and role of efflux pump activity in ciprofloxacin resistance in clinical isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

We investigated the prevalence and role of efflux pump activity and possible drug influx resistance in ciprofloxacin susceptibility amongst 26 distinct clinical isolates of Klebsiella pneumoniae of varying ciprofloxacin susceptibilities and known quinolone resistance-determining region (QRDR) genotypes. Cellular [¹⁴C]ciprofloxacin accumulation patterns and the amount of cell-associated [¹⁴C]ciprofloxacin of mid-logarithmic phase cells were determined before and after challenging with the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Most isolates (24/26), and all with ciprofloxacin minimum inhibitory concentrations (MICs) >1 μg/ml, had efflux activity that could extrude up to 90% of cell-associated [¹⁴C]ciprofloxacin; none had significant influx resistance. In isolates with no QRDR mutations, efflux alone reduced ciprofloxacin susceptibility. In isolates with QRDR mutations, the efflux activity varied: in one isolate with no efflux activity, the most common fluoroquinolone resistance-causing QRDR mutation did not bring about clinically significant ciprofloxacin resistance; isolates with multiple mutations had high MICs and, usually, high levels of efflux activity. Fluoroquinolone efflux activity is much more common in clinical isolates of K. pneumoniae than previously reported and it can contribute to decreased ciprofloxacin susceptibility.     

Mechanisms of resistance to carbapenems in meropenem- resistant Acinetobacter isolates from clinical samples

Purpose: To analyze the resistance mechanisms in Acinetobacter species by phenotypic methods. Methods: Antibiotic susceptibility profile for 150 clinical isolates of Acinetobacter was determined by the standard disk diffusion method. Isolates detected to be meropenem resistant were tested further by broth microdilution minimum inhibitory concentration (MIC) for meropenem. The resistant isolates were also tested for metallo β-lactamase (MBL) production by the double-disk approximation test, for AmpC beta-lactamase production and efflux pump detection by agar microdilution MIC with and without reserpine. Results: Twenty-one isolates were found resistant to meropenem by the standard disk diffusion method. Nine samples were from patients admitted in intensive care units (ICUs). Broth microdilution MICs of the isolates revealed low-level resistance to meropenem. MBL was not produced by any of these isolates. AmpC β-lactamases were produced by nine (43%) isolates. ‘Efflux pump’-mediated resistance to meropenem was detected in two out of nine random isolates tested for the same. Conclusions: Carbapenem resistance is not uncommon in Acinetobacter isolates. AmpC production may cause carbapenem resistance. MBL and efflux pump may not be important causes of carbapenem resistance.     

Penicillin susceptibility breakpoints for Streptococcus pneumoniae and their effect on susceptibility categorisation in Germany (1997–2013)

Continuous nationwide surveillance of invasive pneumococcal disease (IPD) was conducted in Germany. From July 1, 1997, to June 30, 2013, data on penicillin susceptibility were available for 20,437 isolates. 2,790 of these isolates (13.7 %) originate from patients with meningitis and 17,647 isolates (86.3 %) are from non-meningitis cases. A slight decline in isolates susceptible at 0.06 and 0.12 μg/ml can be noticed over the years. Overall, 89.1 % of the isolates had minimum inhibitory concentrations (MICs) of ≤0.015 μg/ml. In 2012/2013, the first three isolates of Streptococcus pneumoniae with MICs of 8 μg/ml were found. The application of different guidelines with other MIC breakpoints for the interpretation of penicillin resistance leads to differences in susceptibility categorisation. According to the pre-2008 Clinical and Laboratory Standards Institute (CLSI) interpretive criteria, 5.3 % of isolates overall were intermediate and 1.4 % were resistant to penicillin. Application of the 2008–2014 CLSI interpretive criteria resulted in 7.6 % resistance among meningitis cases and 0.5 % intermediate resistance in non-meningitis cases. Referring to the 2009–2014 European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, 7.6 % of the isolates in the meningitis group were resistant to penicillin. In the non-meningitis group, 6.1 % of the isolates were intermediate and 0.5 % were resistant. These differences should be kept in mind when surveillance studies on pneumococcal penicillin resistance are compared.     

Multilaboratory evaluation of disk diffusion antimicrobial susceptibility testing of Neisseria meningitidis isolates

In 2005, the Clinical and Laboratory Standards Institute published MIC interpretive criteria for 13 antimicrobial agents used for either therapy or prophylaxis of Neisseria meningitidis infections. The MIC method includes the use of lysed horse blood-supplemented Mueller-Hinton broth with incubation in 5% CO2 for 20 to 24 h. Since some clinical laboratories might prefer the option of disk diffusion testing for infrequently encountered isolates a multicenter collaborative study was conducted to evaluate the reproducibility of a disk diffusion method for testing isolates of N. meningitidis. Interpretive criteria were developed for 12 antimicrobial agents. Four laboratories tested a common collection of 50 meningococcal strains and then tested 25 unique isolates per laboratory. Isolates were tested using Mueller-Hinton sheep blood agar plates incubated for 20 to 24 h in 5% CO2; they were also tested by the reference broth microdilution method in parallel. Pooling of the MIC and disk diffusion data from the common and unique isolates provided a sufficient sample size to develop susceptible, intermediate, and resistant zone diameter interpretive criteria using the error rate-bounded method for the following agents: chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and rifampin. Due to the lack of resistant strains at the present time, "susceptible only" interpretive criteria were proposed for cefotaxime, ceftriaxone, meropenem, azithromycin, and minocycline. The numbers of minor interpretive errors with penicillin and ampicillin disk tests were unacceptably high and precluded recommended testing of those agents by the disk method. However, amdinocillin, an agent that preferentially binds to the altered penicillin binding protein responsible for diminished penicillin susceptibility, has potential utility as a surrogate screening reagent for ampicillin resistance. A disk diffusion breakpoint was derived for nalidixic acid to serve as a surrogate marker for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Disk diffusion testing with meningococci can be performed in a reproducible manner with several antimicrobial agents and represents a practical and cost-effective option for testing sporadic clinical isolates or for surveillance purposes by resource-limited laboratories.     

Comparison of Meropenem MICs and Susceptibilities for Carbapenemase-Producing Klebsiella pneumoniae Isolates by Various Testing Methods

We describe the levels of agreement between broth microdilution, Etest, Vitek 2, Sensititre, and MicroScan methods to accurately define the meropenem MIC and categorical interpretation of susceptibility against carbapenemase-producing Klebsiella pneumoniae (KPC). A total of 46 clinical K. pneumoniae isolates with KPC genotypes, all modified Hodge test and bla_KPC positive, collected from two hospitals in NY were included. Results obtained by each method were compared with those from broth microdilution (the reference method), and agreement was assessed based on MICs and Clinical Laboratory Standards Institute (CLSI) interpretative criteria using 2010 susceptibility breakpoints. Based on broth microdilution, 0%, 2.2%, and 97.8% of the KPC isolates were classified as susceptible, intermediate, and resistant to meropenem, respectively. Results from MicroScan demonstrated the most agreement with those from broth microdilution, with 95.6% agreement based on the MIC and 2.2% classified as minor errors, and no major or very major errors. Etest demonstrated 82.6% agreement with broth microdilution MICs, a very major error rate of 2.2%, and a minor error rate of 2.2%. Vitek 2 MIC agreement was 30.4%, with a 23.9% very major error rate and a 39.1% minor error rate. Sensititre demonstrated MIC agreement for 26.1% of isolates, with a 3% very major error rate and a 26.1% minor error rate. Application of FDA breakpoints had little effect on minor error rates but increased very major error rates to 58.7% for Vitek 2 and Sensititre. Meropenem MIC results and categorical interpretations for carbapenemase-producing K. pneumoniae differ by methodology. Confirmation of testing results is encouraged when an accurate MIC is required for antibiotic dosing optimization.Carbapenems are considered first-line therapy for infection with multidrug-resistant Enterobacteriaceae (14). However, the increasing emergence of serine-based carbapenemase-producing Klebsiella pneumoniae (KPC) worldwide is of growing concern. This problem is particularly worrisome due to the fact that this K. pneumoniae is one of the leading causes of hospital-acquired infections in severely ill patients, and few antibiotics retain microbiological activity against isolates that produce bla_KPC (15). Additionally, studies have demonstrated increased mortality rates in patients infected with carbapenem-resistant Enterobacteriaceae compared with those infected with susceptible strains (1, 12, 13).Detection of KPC based strictly on susceptibility testing is challenging due mostly to the heterogeneous expression of β-lactam resistance (15). Many automated systems report KPC as susceptible to meropenem, and while some isolates truly are, the MICs for most KPC are above the Food and Drug Administration (FDA) susceptibility breakpoint (4 μg/ml) (11). To address testing and reporting issues, the Clinical Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing changed the susceptibility breakpoint for meropenem, imipenem, and doripenem to ≤1 μg/ml against Enterobacteriaceae in January 2010 (8). At the time of writing, the FDA breakpoint remained at ≤4 μg/ml for meropenem.Given the lack of options for antibiotics that retain susceptibility against pathogens that produce KPC, selection of a dosing regimen that could potentially treat infections caused by these organisms depends on the ability to accurately determine the antibiotic MIC. With respect to KPC, the accurate determination of the meropenem MIC may permit the application of pharmacodynamic principles to dosing regimen optimization by administering higher doses and using prolonged or continuous infusions, as has been accomplished against other resilient bacteria (3, 10, 14).Herein, we describe the levels of agreement between commonly used testing methods (broth microdilution [BMD], Etest, Vitek 2, Sensititre, and MicroScan) in their abilities to accurately determine the meropenem MIC and further classify categorical susceptibilities of carbapenemase-producing K. pneumoniae isolates based on the 2010 CLSI breakpoints compared with FDA breakpoints.