The Impact of Colistin Resistance on the Activation of Innate Immunity by Lipopolysaccharide Modification.

Colistin resistance is acquired by different lipopolysaccharide (LPS) modifications. We proposed to evaluate the of effect in vivo colistin resistance acquisition on the innate immune response. We used a pair of ST11 clone Klebsiella pneumoniae strains: an OXA-48, CTX-M-15 K. pneumoniae strain susceptible to colistin (CS-Kp) isolated from a urinary infection and its colistin-resistant variant (CR-Kp) from the same patient after prolonged treatment with colistin. No mutation of previously described genes for colistin resistance (pmrA, pmrB, mgrB, phoP/Q, arnA, arnC, arnT, ugdH, and crrAB) was found in the CR-Kp genome; however, LPS modifications were characterized by negative-ion matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The strains were cocultured with human monocytes to determine their survival after phagocytosis and induction to apoptosis. Also, monocytes were stimulated with bacterial LPS to study cytokine and immune checkpoint production. The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A of CR-Kp accounted for the colistin resistance. CR-Kp survived significantly longer inside human monocytes after being phagocytosed than did the CS-Kp strain. In addition, LPS from CR-Kp induced both higher apoptosis in monocytes and higher levels of cytokine and immune checkpoint production than LPS from CS-Kp. Our data reveal a variable impact of colistin resistance on the innate immune system, depending on the responsible mechanism. Adding Ara4N to LPS in K. pneumoniae increases bacterial survival after phagocytosis and elicits a higher inflammatory response than its colistin-susceptible counterpart.

Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms.

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Outbreak by KPC-62-producing ST307 Klebsiella pneumoniae isolates resistant to ceftazidime/avibactam and cefiderocol in a university hospital in Madrid, Spain.

OBJECTIVES: Ceftazidime/avibactam and cefiderocol are two of the latest antibiotics with activity against a wide variety of Gram-negatives, including carbapenem-resistant Enterobacterales. We sought to describe the phenotypic and genotypic characteristics of ceftazidime/avibactam- and cefiderocol-resistant KPC-Klebsiella pneumoniae (KPC-Kp) detected during an outbreak in 2020 in the medical ICU of our hospital. METHODS: We collected 11 KPC-Kp isolates (6 clinical; 5 surveillance samples) resistant to ceftazidime/avibactam and cefiderocol from four ICU patients (November 2020 to January 2021), without prior exposure to these agents. All patients had a decontamination regimen as part of the standard ICU infection prevention protocol. Additionally, one ceftazidime/avibactam- and cefiderocol-resistant KPC-Kp (June 2019) was retrospectively recovered. Antibiotic susceptibility was determined by broth microdilution. ß-Lactamases were characterized and confirmed. WGS was also performed. RESULTS: All KPC-Kp isolates (ceftazidime/avibactam MIC  ≥16/4 mg/L; cefiderocol MIC ≥4 mg/L) were KPC + CTX-M-15 producers and belonged to the ST307 high-risk-clone (ST307-HRC). KPC-62 (L168Q) was detected in all isolates involved in the 2020 outbreak, contained in January 2021. KPC-31 (D179Y) was identified in the KPC-Kp from 2019. Cloning experiments demonstrated that both blaKPC-62 and blaKPC-31 were responsible for ceftazidime/avibactam resistance (MIC >16 mg/L) and an increased cefiderocol MIC. Additionally, mutations in OmpA and EnvZ/OmpR porin proteins (in KPC-62-Kp) and in PBP2 (in KPC-31-Kp) were found and may be involved in cefiderocol resistance. CONCLUSIONS: The emergence of resistance to both ceftazidime/avibactam and cefiderocol in KPC-Kp-HRCs, together with the diversification of novel KPC enzymes displaying different antibiotic resistance phenotypes, is an epidemiological and clinical risk.

Impact of Ceftazidime-Avibactam Treatment in the Emergence of Novel KPC Variants in the ST307-Klebsiella pneumoniae High-Risk Clone and Consequences for Their Routine Detection.

The emergence of Klebsiella pneumoniae isolates carrying novel blaKPC variants conferring ceftazidime-avibactam (CAZ/AVI) resistance is being increasingly reported. We evaluated the accuracy of phenotypic methods commonly used in routine clinical laboratories in the detection of novel K. pneumoniae carbapenemase (KPC) enzymes. Additionally, we characterized by whole-genome sequencing (WGS) the KPC-ST307-K. pneumoniae isolates recovered in our hospital before and after CAZ/AVI therapy. Rectal colonization or infection by carbapenem-resistant KPC-3 K. pneumoniae isolates (imipenem MIC, 16 mg/L; meropenem MIC, 8 to >16 mg/L) and CAZ/AVI-susceptible isolates (CAZ/AVI MIC, 1 to 2 mg/L) were first detected in three intensive care unit (ICU) patients admitted between March 2020 and July 2020. KPC K. pneumoniae isolates with increased CAZ/AVI MICs (8 to 32 mg/L) and carbapenem susceptibility (imipenem and meropenem MIC, <1 mg/L) were recovered within 6 to 24 days after CAZ/AVI treatment. WGS confirmed that all KPC K. pneumoniae isolates belonged to the sequence type 307 (ST307) high-risk clone and carried identical antimicrobial resistance genes and virulence factors. The presence of the novel blaKPC-46, blaKPC-66, and blaKPC-92 genes was confirmed in the K. pneumoniae isolates with increased CAZ/AVI MICs and restored carbapenem activity. KPC production was confirmed by immunochromatography, the eazyplex Superbug CRE system, and the Xpert Carba-R assay in all KPC K. pneumoniae isolates, but not in any isolate using chromogenic agar plates for carbapenemase producers (ChromID-CARBA), the KPC/MBL/OXA-48 Confirm kit, and the ß-CARBA test. Nevertheless, all grew in chromogenic agar plates for extended-spectrum ß-lactamase (ESBL) producers (ChromID-ESBL). We report the failure of the most common phenotypic methods used for the detection of novel KPC carbapenemases but not of rapid molecular or immunochromatography assays, thus highlighting their relevance in microbiology laboratories.

Time kill-assays of antibiotic combinations for multidrug resistant clinical isolates of OXA-48 carbapenemase producing Klebsiella pneumoniae.

Treatment of infections caused by OXA-48 carbapenemase producing multidrug-resistant isolates often necessitates combination therapy. In vitro effect of different antibiotic combinations against multidrug-resistant (MDR) Klebsiella pneumoniae isolates were evaluated in this study.Meropenem-tobramycin (MER+TOB), meropenem-ciprofloxacin (MER+CIP), colistin-meropenem (COL+MER), colistin-ciprofloxacin (COL+CIP) and colistin-tobramycin (COL+TOB) combinations were tested by time kill-assays. Each antibiotic alone and in combination at their Cmax values were tested against 4 clinical K. pneumoniae isolates at 1, 2, 4, 6, 8, 12 and 24 h. Effect of colistin and its associations were also assessed at 30 min. Bactericidal activity was defined as ≥3log10 CFU mL-1 decrease compared with initial inoculum. Synergy was defined as ≥2log10CFU mL-1 decrease by the combination compared with the most active single agent. Presence of blaOXA-48, blaNDM, blaVIM, blaIMP, blaKPC and blaCTX-M-1 genes was screened by PCR using specific primers.The blaOXA-48 gene was identified together with blaCTXM-1 group gene in all isolates. COL+MER demonstrated to be synergistic and bactericidal. MER+TOB showed synergistic and bactericidal effect on two strains although, regrowth was seen on other two strains at 24 h. MER+CIP exhibited indifferent effect on the strains.Combination therapy could be a potential alternative to treat MDR K. pneumoniae infections. This combination might prevent resistance development and secondary effects of colistin monotherapy. MER+TOB and MER+CIP might have an isolate-dependent effect, that may not always result in synergism.

Characterization of carbapenemase-producing Serratia marcescens and whole-genome sequencing for plasmid typing in a hospital in Madrid, Spain (2016-18).

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) are increasingly recognized in nosocomial infections, also affecting ICU patients. We aimed to characterize the carbapenemase-producing Serratia marcescens (CPSm) isolates recovered in our hospital in Madrid (Spain) between March 2016 and December 2018. METHODS: Overall, 50 isolates from clinical and epidemiological surveillance samples were recovered from 24 patients admitted to the medical ICU and 10 non-ICU-related patients based on their phenotypic resistance. Carbapenemase characterization, antibiotic susceptibility, PFGE clonal relatedness, plasmid characterization, WGS (Illumina-NovaSeq 6000) and phylogenetic analysis were performed. RESULTS: A single isolate was finally considered for each patient, except for Patient 8 that was colonized by two different isolates (n = 35). Isolates were characterized as VIM-1 (n = 29) or OXA-48 producers (n = 6). Up to seven genetic lineages were found by PFGE, with dominance of two clones. Plasmid characterization confirmed that almost all CPSm carried the same ∼60 kb IncL OXA-48- or VIM-1-encoding plasmid, which was related to the globally disseminated IncL-pOXA-48a. WGS allowed plasmid reconstruction with two variants: IncL-pVIM-1 (∼65 kb) and IncL-pOXA-48 (∼62 kb). blaOXA-48-Tn1999 (∼5 kb) was the unique antibiotic resistance gene in pOXA-48, whereas pVIM-1 plasmids (∼8 kb) harboured a class 1 integron containing 5'-blaVIM-1+aacA4+dfrB1+aadA1+catB2+qacEDelta1+sul1-3'. CONCLUSIONS: Our results confirm the dissemination of CPSm within our institution in both ICU and non-ICU environments, representing two prevalent CPSm clones, and the same IncL-pOXA-48 plasmid previously described in other Enterobacterales, but containing the blaVIM-1 gene. This also reinforces the relevance of species different from Klebsiella pneumoniae or Escherichia coli in the CPE landscape and circulating lineages and plasmids in local CPE epidemiology.

Murepavadin antimicrobial activity against and resistance development in cystic fibrosis Pseudomonas aeruginosa isolates.

BACKGROUND: Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. OBJECTIVES: To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. METHODS: MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time-kill assays. Resistant mutants were studied by WGS. RESULTS: The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1-5 h. Murepavadin MICs were 2-9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8×MIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). CONCLUSIONS: Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

Anti-biofilm activity of murepavadin against cystic fibrosis Pseudomonas aeruginosa isolates.

OBJECTIVES: To determine the activity of murepavadin in comparison with tobramycin, colistin and aztreonam, against cystic fibrosis (CF) Pseudomonas aeruginosa isolates growing in biofilms. The biofilm-epidemiological cut-off (ECOFF) values that include intrinsic resistance mechanisms present in biofilms were estimated. METHODS: Fifty-three CF P. aeruginosa isolates from respiratory samples were tested using the Calgary (closed system) device, while 4 [2 clinical (one smooth, one mucoid) and 2 reference strains] were tested using the BioFlux, a microfluidic open model of biofilm testing. Biofilm was stained with SYTO9® and propidium iodide. The minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) were determined. The MBIC-ECOFF and the MBEC-ECOFF were calculated. RESULTS: Colistin, tobramycin and murepavadin presented similar MBIC50/MBIC90 values (4/32, 8/64 and 2/32, respectively). Murepavadin exhibited the lowest MBEC90 (64 mg/L). Aztreonam MBIC and MBEC values were higher than those of the other antibiotics tested. Tobramycin and murepavadin had the lowest MBEC-ECOFF (64 and 128 mg/L, respectively), while those of aztreonam and colistin exceeded 512 mg/L. Using the BioFlux, for the PAO1, PAO mutS and the smooth clinical strain, a significant difference (P < 0.0125) was observed when comparing the fluorescence of treated and untreated biofilms. For the mucoid strain, only the biofilm treated with aztreonam (MBIC and MBEC) and tobramycin (MBEC) showed differences with respect to the untreated biofilm. CONCLUSIONS: Murepavadin demonstrated good activity against P. aeruginosa biofilms both in open and closed systems. The MBIC-ECOFF and the MBEC-ECOFF are proposed as new parameters to estimate the activity of antibiotics on biofilms.

Impact of De-escalation on Prognosis of Patients With Bacteremia due to Enterobacteriaceae: A Post Hoc Analysis From a Multicenter Prospective Cohort.

BACKGROUND: More data are needed about the safety of antibiotic de-escalation in specific clinical situations as a strategy to reduce exposure to broad-spectrum antibiotics. The aims of this study were to investigate predictors of de-escalation and its impact on the outcome of patients with bloodstream infection due to Enterobacteriaceae (BSI-E). METHODS: A post hoc analysis was performed on a prospective, multicenter cohort of patients with BSI-E initially treated with ertapenem or antipseudomonal ß-lactams. Logistic regression was used to analyze factors associated with early de-escalation (EDE) and Cox regression for the impact of EDE and late de-escalation (LDE) on 30-day all-cause mortality. A propensity score (PS) for EDE vs no de-escalation (NDE) was calculated. Failure at end of treatment and length of hospital stay were also analyzed. RESULTS: Overall, 516 patients were included. EDE was performed in 241 patients (46%), LDE in 95 (18%), and NDE in 180 (35%). Variables independently associated with a lower probability of EDE were multidrug-resistant isolates (odds ratio [OR], 0.50 [95% confidence interval {CI}, .30-.83]) and nosocomial infection empirically treated with imipenem or meropenem (OR, 0.35 [95% CI, .14-.87]). After controlling for confounders, EDE was not associated with increased risk of mortality; hazard ratios (HR) (95% CIs) were as follows: general model, 0.58 (.25-1.31); model with PS, 0.69 (.29-1.65); and PS-based matched pairs, 0.98 (.76-1.26). LDE was not associated with mortality. De-escalation was not associated with clinical failure or length of hospital stay. CONCLUSIONS: De-escalation in patients with monomicrobial bacteremia due to Enterobacteriaceae was not associated with a detrimental impact on clinical outcome.

Use of Calgary and Microfluidic BioFlux Systems To Test the Activity of Fosfomycin and Tobramycin Alone and in Combination against Cystic Fibrosis Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa is a major cause of morbidity and mortality in chronically infected cystic fibrosis patients. Novel in vitro biofilm models which reliably predict the therapeutic success of antimicrobial therapies against biofilm bacteria should be implemented. The activity of fosfomycin, tobramycin, and the fosfomycin-tobramycin combination against 6 susceptible P. aeruginosa strains isolated from respiratory samples from cystic fibrosis patients was tested by using two in vitro biofilm models: a closed system (Calgary device) and an open model based on microfluidics (BioFlux). All but one of the isolates formed biofilms. The fosfomycin and tobramycin minimal biofilm inhibitory concentrations (MBIC) were 1,024 to >1,024 µg/ml and 8 to 32 µg/ml, respectively. According to fractional inhibitory concentration analysis, the combination behaved synergistically against all the isolates except the P. aeruginosa ATCC 27853 strain. The dynamic formation of the biofilm was also studied with the BioFlux system, and the MIC and MBIC of each antibiotic were tested. For the combination, the lowest tobramycin concentration that was synergistic with fosfomycin was used. The captured images were analyzed by measuring the intensity of the colored pixels, which was proportional to the biofilm biomass. A statistically significant difference was found when the intensity of the inoculum was compared with the intensity of the microchannel in which the MBIC of tobramycin, fosfomycin, or their combination was used (P < 0.01) but not when the MIC was applied (P > 0.01). Fosfomycin-tobramycin was demonstrated to be synergistic against cystic fibrosis P. aeruginosa strains in the biofilm models when both the Calgary and the microfluidic BioFlux systems were tested. These results support the clinical use of this combination.

First Report of an OXA-48- and CTX-M-213-Producing Kluyvera Species Clone Recovered from Patients Admitted in a University Hospital in Madrid, Spain.

Enterobacterales species other than Klebsiella pneumoniae also contribute to OXA-48 carbapenemase endemicity. We studied the emergence of an OXA-48-producing Kluyvera species clone, which expresses the novel CTX-M-213 enzyme, colonizing patients in our hospital. Rectal swabs from patients admitted in four wards (March 2014 to March 2016; R-GNOSIS project) were seeded onto Chromo ID-ESBL) and Chrom-CARB/OXA-48 chromogenic agar plates. Carbapenemases and extended-spectrum ß-lactamases (ESBLs) were characterized (PCR, sequencing, cloning, and site-directed mutagenesis), and antibiotic susceptibility was determined. Clonal relatedness was established (XbaI pulsed-field gel electrophoresis [XbaI-PFGE]), and plasmid content was studied (transformation, S1 nuclease digestion-PFGE, SB-hybridization, restriction fragment length polymorphism [RFLP] analysis [DraI and HpaI], and PCR [incompatibility group and repA, traU, and parA genes]). Whole-genome sequencing (WGS) (Illumina HiSeq-2500) and further bioinformatics analysis of plasmids (PLACNET and plasmidSPAdes) were performed. Patients' charts were reviewed. Six unrelated patients (median age, 75 years [range, 59 to 81 years]; 4/6 male patients) colonized with OXA-48-producing Kluyvera species isolates (>95% similarity of the PFGE pattern) were identified. Nosocomial acquisition was demonstrated. In two patients, OXA-48-producing Kluyvera species isolates coexisted with OXA-48-producing Raoultella ornithinolytica, K. pneumoniae, and Escherichia coli The blaOXA-48 gene was located on an ∼60-kb IncL plasmid related to IncL/M-pOXA-48a and the novel blaCTX-M-213 gene in a conserved chromosomal region of Kluyvera species isolates. CTX-M-213, different from CTX-M-13 (K56E) but conferring a similar ß-lactam resistance profile, was identified. Genomic analysis also revealed a 177-kb IncF plasmid (class I integron harboring sul1 and aadA2) and an 8-kb IncQ plasmid (IS4-blaFOX-8). We describe the first blaOXA-48 plasmid in Kluyvera spp. and the novel chromosomal CTX-M-213 enzyme and highlight further nosocomial dissemination of blaOXA-48 through clonal lineages or plasmids related to IncL/M-pOXA-48a.

Etest® versus broth microdilution for ceftaroline MIC determination with Staphylococcus aureus: results from PREMIUM, a European multicentre study.

OBJECTIVES: To compare the concordance of ceftaroline MIC values by reference broth microdilution (BMD) and Etest (bioMérieux, France) for MSSA and MRSA isolates obtained from PREMIUM (D372SL00001), a European multicentre study. METHODS: Ceftaroline MICs were determined by reference BMD and by Etest for 1242 MSSA and MRSA isolates collected between February and May 2012 from adult patients with community-acquired pneumonia or complicated skin and soft tissue infections; tests were performed across six European laboratories. Selected isolates with ceftaroline resistance in broth (MIC >1 mg/L) were retested in three central laboratories to confirm their behaviour. RESULTS: Overall concordance between BMD and Etest was good, with >97% essential agreement and >95% categorical agreement. Nevertheless, 12 of the 26 MRSA isolates found resistant by BMD scored as susceptible by Etest, with MICs ≤1 mg/L, thus counting as very major errors, whereas only 5 of 380 MRSA isolates found ceftaroline susceptible in BMD were miscategorized as resistant by Etest. Twenty-one of the 26 isolates with MICs of 2 mg/L by BMD were then retested twice by each of three central laboratories: BMD MICs of 2 mg/L were consistently found for 19 of the 21 isolates. Among 147 Etest results for these 21 isolates (original plus six repeats per isolate) 112 were >1 mg/L. CONCLUSIONS: BMD and Etest have good overall agreement for ceftaroline against Staphylococcus aureus; nevertheless, reliable Etest-based discrimination of the minority of ceftaroline-resistant (MIC 2 mg/L) MRSA is extremely challenging, requiring careful reading of strips, ideally with duplicate testing.

[Dalbavancin breakpoints and recommendations for in vitro study of its activity]. / Puntos de corte de dalbavancina y recomendaciones para el estudio de su actividad in vitro.

Dalbavancin is a semisynthetic lipoglycopeptide approved for the treatment of acute skin and soft tissue infections due to Gram-positive microorganisms susceptible to this antimicrobial agent. The FDA (Food and Drug Administration) and the EUCAST (European Committee on Antimicrobial Susceptibility Testing) have established clinical breakpoints to interpret the results of the antibiogram (expressed as MIC [minimum inhibitory concentration]) with approved doses (1g intravenously [IV] followed by 0.5g IV at day 8 or 1.5g IV in a single dose). The EUCAST has also determined PK/PD (pharmacokinetic/pharmacodynamic) breakpoints -susceptible, ≤ 0.25mg/L; resistant, > 0.25mg/L-, established recommendations for in vitro susceptibility testing (addition of polysorbate-80 to the growth media) and subrogate values based on the vancomycin-susceptible category to interpret dalbavancin susceptibility in the absence of in vitro study of this antimicrobial.

Is a New Standard Needed for Diffusion Methods for In Vitro Susceptibility Testing of Fosfomycin against Pseudomonas aeruginosa?

We analyzed fosfomycin susceptibility results in Pseudomonas aeruginosa clinical isolates obtained by MIC gradient strips and disk diffusion methods using two different inocula, 10(8) and 10(6) CFU/ml, and compared them to the agar dilution reference method. Essential and categorical agreements were 93.6% and 95%, respectively, for the 10(6) CFU/ml alternative inoculum, and they were 67.6% and 78.2%, respectively, for the standard inoculum (10(8) CFU/ml). The use of the 10(6) CFU/ml inoculum improves the agreement values and inhibition zone readings.

Detection of Carbapenemase Production in a Collection of Enterobacteriaceae with Characterized Resistance Mechanisms from Clinical and Environmental Origins by Use of Both Carba NP and Blue-Carba Tests.

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producing Enterobacteriaceae from hospital (n = 102) and environmental (n = 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

Antimicrobial Activity of Fosfomycin-Tobramycin Combination against Pseudomonas aeruginosa Isolates Assessed by Time-Kill Assays and Mutant Prevention Concentrations.

The antibacterial activity of fosfomycin-tobramycin combination was studied by time-kill assay in eight Pseudomonas aeruginosa clinical isolates belonging to the fosfomycin wild-type population (MIC = 64 µg/ml) but with different tobramycin susceptibilities (MIC range, 1 to 64 µg/ml). The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined in five of these strains (tobramycin MIC range, 1 to 64 µg/ml) in aerobic and anaerobic conditions simulating environments that are present in biofilm-mediated infections. Fosfomycin-tobramycin was synergistic and bactericidal for the isolates with mutations in the mexZ repressor gene, with a tobramycin MIC of 4 µg/ml. This effect was not observed in strains displaying tobramycin MICs of 1 to 2 µg/ml due to the strong bactericidal effect of tobramycin alone. Fosfomycin presented higher MPC values (range, 2,048 to >2,048 µg/ml) in aerobic and anaerobic conditions than did tobramycin (range, 16 to 256 µg/ml). Interestingly, the association rendered narrow or even null MSWs in the two conditions. However, for isolates with high-level tobramycin resistance that harbored aminoglycoside nucleotidyltransferases, time-kill assays showed no synergy, with wide MSWs in the two environments. glpT gene mutations responsible for fosfomycin resistance in P. aeruginosa were determined in fosfomycin-susceptible wild-type strains and mutant derivatives recovered from MPC studies. All mutant derivatives had changes in the GlpT amino acid sequence, which resulted in a truncated permease responsible for fosfomycin resistance. These results suggest that fosfomycin-tobramycin can be an alternative for infections due to P. aeruginosa since it has demonstrated synergistic and bactericidal activity in susceptible isolates and those with low-level tobramycin resistance. It also prevents the emergence of resistant mutants in either aerobic or anaerobic environments.

Emergence of cfr-Mediated Linezolid Resistance in a Methicillin-Resistant Staphylococcus aureus Epidemic Clone Isolated from Patients with Cystic Fibrosis.

Resistance to linezolid (LZD) in methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with cystic fibrosis (CF) is due mainly to ribosomal mutations. We report on four CF patients with LZD-resistant MRSA bronchopulmonary infections by strains carrying the cfr gene. Strains from one patient also harbored the G2576U mutation (23S rRNA) and the G139R substitution (L3 protein). All strains belonged to the epidemic clone ST125 MRSA IVc. Our results support the monitoring of LZD resistance emergence in CF and non-CF MRSA isolates.

Evaluation of the eazyplex® SuperBug CRE system for rapid detection of carbapenemases and ESBLs in clinical Enterobacteriaceae isolates recovered at two Spanish hospitals.

OBJECTIVES: To evaluate the performance of the eazyplex(®) SuperBug CRE system, a loop-mediated isothermal amplification (LAMP)-based system, for confirming the presence of carbapenemases in addition to CTX-M-type ESBLs in previously genotypically and/or phenotypically characterized clinical Enterobacteriaceae isolates recovered in two centres in Spain. METHODS: A collection of 94 carbapenemase-producing strains previously characterized by conventional PCR and sequencing and a total of 45 prospectively collected isolates with phenotypes compatible with the presence of a carbapenemase were tested with the eazyplex(®) SuperBug CRE system. In both cases, the presence of an ESBL was also assessed. Results were evaluated to establish the accuracy of this rapid LAMP-based system as well as to determine the concordance between all approaches. RESULTS: The eazyplex(®) SuperBug CRE system correctly detected bla carbapenemase genes with or without blaCTX-M genes in 100% of the molecularly characterized strains. Absolute concordance (100%) was also observed in the case of isolates with phenotypes compatible with the presence of a carbapenemase with or without an ESBL inferred by susceptibility patterns and phenotypic inhibitory profiles. Determinations performed with the eazyplex(®) SuperBug CRE system took 15 min. CONCLUSIONS: The eazyplex(®) SuperBug CRE system proved to be a powerful tool for the detection of different carbapenemases as well as CTX-M-type ESBLs in Enterobacteriaceae with a rapid resolution time. The test has the high-performance parameters attributable to the sensitivity and specificity already demonstrated by LAMP-based assays. These results assure the usefulness of this test for routine rapid confirmation of carbapenemase-producing Enterobacteriaceae.

Performance of EUCAST and CLSI approaches for co-amoxiclav susceptibility testing conditions for clinical categorization of a collection of Escherichia coli isolates with characterized resistance phenotypes.

OBJECTIVES: There are different methodological recommendations for in vitro testing of the co-amoxiclav combination. Performance of co-amoxiclav MIC testing for Escherichia coli by the standard ISO microdilution method (ISO 20776-1) was compared using EUCAST (fixed 2 mg/L clavulanate concentration) and CLSI (2 : 1 ratio) interpretive criteria. METHODS: MICs were determined by broth microdilution using a 2 : 1 ratio and fixed clavulanate concentrations (2 and 4 mg/L) for 160 clinical E. coli isolates with characterized resistance mechanisms. Essential agreements, categorical agreements and relative errors were determined. RESULTS: For all isolates, essential agreement between microdilution using 2 mg/L clavulanate and a 2 : 1 ratio was 25.6%. For ESBL-producing isolates, considering EUCAST breakpoints, 55% of isolates tested with 2 mg/L clavulanate were classified as resistant; conversely, 95% of isolates tested with 4 mg/L clavulanate were susceptible. When using CLSI breakpoints and a 2 : 1 ratio, 90% of isolates were susceptible and 10% were intermediate. CONCLUSIONS: Variation in the clavulanate concentration gave different susceptibility testing results, particularly among ESBL-producing E. coli isolates. The in vitro concentration of clavulanate that better correlates with clinical outcome is still under debate and should be established.

Evaluation of a loop-mediated isothermal amplification-based methodology to detect carbapenemase carriage in Acinetobacter clinical isolates.

Carbapenem-resistant Acinetobacter baumannii is a major source of nosocomial infections worldwide and is mainly associated with the acquisition of OXA-type carbapenemases and, to a lesser extent, metallo-ß-lactamases (MBLs). In this study, 82 nonepidemiologically related Acinetobacter strains carrying different types of OXA or MBL enzymes were tested using the Eazyplex system, a loop-mediated isothermal amplification (LAMP)-based method to rapidly detect carbapenemase carriage. The presence/absence of carbapenem-hydrolyzing enzymes was correctly determined for all isolates in <30 min.