The aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in ...routine microbiology laboratories.
The immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric β-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2).
The overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6–89.2%)/100% (CI 91.8–100%) for RESIST-4, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for CARBA-5, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for Xpert Carba-R, 73.7% (CI 66.2–80.0%)/100% (CI 93.4–99.0%) for β-CARBA, and 97.4% (CI 87.9–99.6%)/97.7% (CI 87.9–99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for β-CARBA (76.6% (CI 68.4–83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3–98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed.
Except for β-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.
The recommended technique for colistin susceptibility testing by both EUCAST and CLSI is broth microdilution (BMD). However, many routine laboratories still use other methods such as gradient strips ...or semi-automated systems. The objective of this study was to compare six of the most widespread commercial products for colistin susceptibility testing in Europe with in-house prepared BMD.
A collection of 325 carbapenemase-producing Enterobacterales was tested for colistin susceptibility with three semi-automated systems (Vitek 2, BD Phoenix, MicroScan WalkAway), one gradient-strip test (Etest®) and two commercial BMD products (MICRONAUT-S, TREK Sensititre). BMD, in-house prepared according to ISO standard 20776-1, served as reference.
The MICRONAUT-S BMD performed best with only one false-resistant (major error, ME) and four false-susceptible (very major error, VME) results while the TREK BMD performed poorer with 16 ME and seven VME. The semi-automated systems Vitek 2 and Phoenix performed poorly with 31 and 26 VME, respectively. The WalkAway semi-automated system showed 16 and 13 false results, depending on the inoculation method. The Etest® showed six ME and 10 VME.
This study shows that colistin susceptibility testing remains a challenging task for laboratories. It emphasizes the need for selecting the most reliable test method to advocate proper treatment and shows that critical evaluation and precautious usage of colistin susceptibility testing results is constantly required.
We aimed to evaluate the effect of ampC derepression on the cefepime MIC in different species of Enterobacterales with chromosomally encoded inducible AmpC β-lactamase.
We analysed a large number of ...wild-type/mutant pairs (n = 1030 in total). Cefepime MICs were determined by broth microdilution according to EUCAST recommendations.
ampC derepression led to increases in MIC by up to 10 dilutions, and significant increases by > 2 MIC dilutions were common across species (744/1030 mutants (72.2%) in total). Interestingly, the frequency of cefepime S→I/S→R transitions varied considerably between species: 66.3% in Enterobacter cloacae complex (167/252 mutants), 1.1% in Klebsiella aerogenes (2/180 mutants), 18.1% in Citrobacter freundii complex (50/277 mutants), 36.4% in Hafnia alvei (59/162 mutants), 19.0% in Providencia rettgeri (4/21 mutants), 22.9% in Providencia stuartii (11/48 mutants), 12.3% in Serratia marcescens (7/57 mutants), 20.0% in Serratia liquefaciens (6/30 mutants) and 0% in Morganella morganii (0/3 mutants).
Our data show that the cefepime MIC is often increased by ampC derepression. However, the risk of S→I/S→R transition is dependent on the species.
•In vitro susceptibility of nitroxoline and nitrofurantoin was compared in 3012 isolates.•Nitroxoline showed MICs well below the EUCAST breakpoint for E. coli for most uropathogens.•Pseudomonas ...aeruginosa isolates showed elevated MICs both to nitroxoline and nitrofurantoin.•Low pH in urine supports nitroxoline activity, in contrast to nitrofurantoin.
The spread of antimicrobial resistance challenges the empirical treatment of urinary tract infections (UTIs). Among others, nitrofurantoin is recommended for first-line treatment, but acceptance among clinicians is limited due to chronic nitrofurantoin-induced lung toxicity and insufficient coverage of Enterobacteriaceae other than Escherichia coli. Nitroxoline appears to be an alternative to nitrofurantoin owing to its favourable safety profile, however data on its current in vitro susceptibility are sparse. In this study, susceptibility to nitroxoline was tested against 3012 urinary clinical isolates (including multidrug-resistant bacteria and Candida spp.) by disk diffusion test and/or broth microdilution. At least 91% of all Gram-negatives (n = 2000), Gram-positives (n = 403) and yeasts (n = 132) had inhibition zone diameters for nitroxoline ≥18 mm. Except for Pseudomonas aeruginosa, nitroxoline MIC90 values were ≤16 mg/L and were 2- to >16-fold lower compared with nitrofurantoin. In extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA), MIC90 values of nitroxoline were two-fold higher compared with non-ESBL-producing enterobacteria and methicillin-susceptible S. aureus (MSSA). The in vitro efficacies of nitroxoline and nitrofurantoin against ATCC strains of E. coli, Enterococcus faecalis and Proteus mirabilis were compared by time–kill curves in Mueller–Hinton broth and artificial urine. Nitroxoline was non-inferior against E. coli, P. mirabilis and E. faecalis in artificial urine. In conclusion, nitroxoline showed a broad antimicrobial spectrum, with inhibition zone diameters and MICs of nitroxoline well below the EUCAST breakpoint for E. coli for most organisms, and thus may also be a target for therapy of uncomplicated UTIs.
To analyse the mechanisms responsible for carbapenem resistance in one Acinetobacter baumannii isolate recovered from a patient transferred to Germany from an Egyptian hospital.
PCR and sequencing ...were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of the isolate.
Sequencing of the PCR product obtained using primers for bla(NDM-1) revealed a variant of NDM-1 that had a C to G substitution at position 82 resulting in an amino acid substitution of proline to alanine at position 28. This variant was designated NDM-2. Genes encoding extended-spectrum β-lactamases or 16S RNA methylase were not detected. The strain lacked detectable plasmids and bla(NDM-2) was not transferred by conjugation. MLST showed that the isolate belonged to a new ST, ST103.
This work further underlines the spread of NDM carbapenemases in A. baumannii, and the spread of the corresponding gene in the Middle East. It also describes the first variant of NDM-1.
In total, 494 isolates of coagulase-negative staphylococci (CoNS) were identified to the species level by biochemical tests and sodA sequencing. Erythromycin resistance phenotypes were determined and ...specific resistance genes were identified by PCR. The prevalence of erythromycin resistance varied widely among staphylococcal species, from 0% in Staphylococcus lugdunensis to almost 90% in Staphylococcus haemolyticus. Most (63%) erythromycin-resistant isolates carried constitutively expressed erm(C) as the sole resistance determinant, with the notable exception of Staphylococcus hominis subsp. hominis, which carried inducible erm(C). The erm(A) and erm(B) determinants were comparatively rare. The msr(A) gene was carried by 20–30% of all erythromycin-resistant isolates, with little variation among species, and was combined in 16.7% of isolates with mph(C), a resistance gene of unknown clinical relevance found previously in isolates of veterinary origin. No erythromycin resistance that could not be attributed to the genes investigated was detected. It was concluded that the presence of methylases cannot be assumed in CoNS isolates that appear erythromycin-resistant and clindamycin-susceptible; thus, methods that detect the export mechanism should be used with clinically significant isolates to indicate whether use of clindamycin may be effective. In Staphylococcus epidermidis and S. haemolyticus, 46% and 66%, respectively, of erythromycin-resistant, clindamycin-susceptible isolates were susceptible to clindamycin therapy.
Penicillinase testing is required for Staphylococcus aureus isolates with a penicillin MIC of ≤0.12 mg/L. This study compared five phenotypic assays with a PCR for blaZ when testing 197 S. aureus ...isolates. The starch-iodine plate method and nitrocefin tests had low sensitivities of 42.9% and 35.7%, respectively. The cloverleaf assay and the penicillin zone-edge determination method had sensitivities of 67.8% and 71.4%, respectively, and these methods might be appropriate in many circumstances, but were not as sensitive as blaZ PCR.
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.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced a few years ago as a new method for bacterial identification. A variety of studies have been ...published concerning MALDI-TOF MS-based identification, most of them using culture collections for the validation of the respective databases in a retrospective manner in favor of a parallel investigation. The score cutoff value is of special importance for reliable species identification in the Biotyper database. The score cutoff values suggested by the manufacturer have been validated using a previously published formic acid extraction protocol. In most of the previously published studies investigating the Biotyper database, only little information was given concerning species-specific score values. In addition, the mass spectrometer instruments, the number of replicates, the number of spectra used to calculate a sum-spectrum by the supplied software, and the score cutoff values which have been applied varied within these studies. In this study, we compared a straightforward direct smear preparation and measurement without replicate testing to defined biochemical identifications in a parallel manner. In addition, we described new species-specific score cutoff values for the identification of certain bacteria.
The aim of this study was to assess the vancomycin MIC distribution for MRSA blood culture isolates over a period of six years in Germany. The study examined 287 MRSA isolates from blood cultures ...collected at several hospitals in two German cities between 2004 and 2009. The vancomycin MIC was determined by Etest. Genotypic features of the MRSA strains with vancomycin MIC ≥ 1 mg/L were determined by semiautomated repetitive-sequence-based polymerase chain reaction. The range of vancomycin MIC as determined by Etest was 0.25 to 2.0 mg/L. The geometric mean MIC increased by 1.34-fold in city A over the study period (
p
< 0.05), but there was no meaningful change in city B (a 1.09-fold increase,
p
> 0.05). Furthermore, in city A a shift in vancomycin MICs occurred as an increase in the percentage of isolates with MIC ≥ 1 mg/L from period one (2004–2006) to period two (2007–2009) (
p
< 0.0001). Typing results showed that in city A a single clone was predominant (55% of the creep isolates). In this study, the creep phenomenon seems to be a regional problem. We suggest that all hospitals should monitor their local status of elevated vancomycin MICs in invasive MRSA isolates.
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