Susceptibility evaluation of novel beta-lactam/beta-lactamase inhibitor combinations against carbapenem-resistant Klebsiella pneumoniae from bloodstream infections in hospitalized patients in Brazil.

Novel beta-lactams/beta-lactamase inhibitors (BIBLI) combinations are commercially available and they have been used for treating carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Continuous surveillance of susceptibility profile and resistance mechanisms identification are necessary to monitor the evolution of resistance as these agents are used. The purpose of this study was to evaluate susceptibility rates to ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in CRKP isolates from patients with bloodstream infection screened for a randomized clinical trial in Brazil. Minimum inhibitory concentration (MIC) was determined by gradient diffusion strip method for meropenem, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam. Carbapenemase genes were detected by multiplex qPCR. KPC-producing isolates showing resistance to any BLBLI and NDM-producing isolates showing susceptibility to any BLBLI were further submitted to whole genome sequencing. From a total of 69 CRKP isolates, 39 were positive for blaKPC, 19 for blaNDM and 11 for blaKPC and blaNDM. KPC-producing isolates demonstrated susceptibility rates above 94% for all BLBLI. Two isolates with resistance to meropenem/vaborbactam showed a Gly and Asp duplication at OmpK36 protein and truncated ompK35 genes. All NDM-producing isolates, including KPC and NDM coproducers, demonstrated susceptibility rates for ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam of 0%, 9.1 to 21.1% and 9.1 to 26.3%, respectively. Five NDM-producing isolates that presented susceptibility to BLBLI also demonstrated alterations in porins. This study demonstrated that, although high susceptibility rates to the BLBLI were found, KPC-2 isolates can also demonstrate resistance due to porin mutations. Additionally, NDM-1 isolates can demonstrate susceptibility in vitro to the BLBLI.

Detection of KPC directly from positive blood cultures by MALDI-TOF: From research to the clinical microbiology laboratory routine.

Bloodstream infections (BSI) caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) are a subject of major clinical concern, mainly those associated with carbapenemase-producing isolates. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed to detect specific ß-lactamases, including KPC. We aimed to detect KPC enzyme directly from positive blood cultures using MALDI-TOF MS. Overall, 146 clinical Gram-negative bacilli (46 CR-GNB) recovered from consecutive blood cultures were evaluated. Proteins were extracted using formic acid, isopropyl alcohol, and water and spotted onto a steel target plate using the double-layer sinapinic acid method. The relative ions intensity ≥120 arbitrary units (a.u.) of a peak close to 28,700 m/z indicated the presence of KPC. The results were compared to HRM-qPCR methodology. This specific peak was observed in 11/14 blood bottles with blaKPC positive isolates (78.6% sensitivity), with 3 false-positive results (97.7% specificity). Analysis from colonies reached identical sensitivity (78.6%), but higher specificity (100%). The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid. It's excellent specificity indicates that positive results are consistently associated with the presence of a KPC producer in positive blood culture.

Detection of Carbapenem Resistance in Enterobacterales Directly From Positive Blood Cultures Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.

CONTEXT.­: Carbapenem-resistant Enterobacterales are disseminated worldwide and associated with infections with high rates of morbidity and mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful tool for identification of pathogens directly from blood cultures in clinical microbiology laboratories. Furthermore, it has been applied for the detection of carbapenemase production, by evaluating carbapenem hydrolysis. OBJECTIVE.­: To determine meropenem hydrolysis to detect carbapenemase production directly from positive blood cultures, using logRQ to establish a quantitative measure of hydrolysis. DESIGN.­: We evaluated 100 Enterobacterales from positive blood cultures, with 81 carrying a carbapenemase gene (blaKPC, blaGES, blaNDM-1, blaIMP, blaVIM, and blaOXA-48-like), as determined by real-time multiplex polymerase chain reaction with high-resolution melting (HRM-qPCR). Bacterial proteins extracted from positive blood culture bottles were incubated in a meropenem solution (2-4 hours) followed by centrifugation for MALDI-TOF MS analysis. The intensity of peaks of the hydrolyzed and nonhydrolyzed forms were used to calculate the logRQ value. RESULTS.­: Overall, sensitivity was 86.8% and specificity, 89.5%. Of note, sensitivity varied depending on enzyme type. For blaKPC-positive isolates, sensitivity was 97.9%, while it reduced significantly for blaNDM-1 and blaOXA-48-like isolates: 62.5% (10 of 16) and 66.7% (6 of 9), respectively. Indeed, logRQ was higher in blaKPC-positive isolates (0.37-1.97) than in blaNDM-1 (-1.37 to 0.83) and blaOXA-48-like isolates (-1.08 to 1.79). CONCLUSIONS.­: This is an inexpensive and rapid test to identify carbapenemase activity directly from blood culture bottles, which contributes to early adequate antimicrobial therapy and implementation of infection control measures.

Evaluation of the ceftazidime/avibactam and meropenem susceptibility by MALDI-TOF MS directly from positive blood cultures.

The purpose of this study was to evaluate the MBT-ASTRA to determine susceptibility to ceftazidime/avibactam (CZA) and meropenem (MEM) of Enterobacterales directly from positive blood cultures (BC). Bacterial suspension was incubated with antibiotic and analyzed by MALDI-TOF MS. The relative growth was calculated and cutoff values were determined to categorize isolates as "S," "I," and "R." Klebsiella spp. with CZA 20/8 mg/L and 1.5-h incubation presented 1 (5.9%) major discrepancy and 96.3% category agreement; other species required 2.5 h for 100% category agreement. For MEM, 4 mg/L and 1.5h were necessary, demonstrating 2 (6.67%) minor discrepancies and 93.3% categorical agreement.

Evaluation of Aztreonam and Ceftazidime/Avibactam Synergism against Klebsiella pneumoniae by MALDI-TOF MS.

INTRODUCTION: Resistance to carbapenems due to the co-production of NDM and ESBL or NDM and KPC is increasing. Therefore, combined therapy with aztreonam (ATM) plus ceftazidime/avibactam (CZA) has been recommended. Then, it is necessary to develop and evaluate fast and simple methods to determine synergism in vitro in microbiology laboratories. OBJECTIVE: To develop a method to determine the synergism of ATM and CZA by MALDI-TOF MS (SynMALDI). METHOD: Klebsiella pneumoniae (n = 22) isolates with blaNDM and/or blaKPC genes were tested. The time-kill curve assay was performed for four isolates (three positives for blaNDM and blaKPC and one positive for blaNDM only). For SynMALDI, each isolate was incubated for 3 h in 4 tubes containing brain-heart infusion broth with the following: (1) no antibiotic; (2) ATM at 64 mg/L; (3) CZA at 10/4 mg/L; and (4) ATM at 64 mg/L plus CZA at 10/4 mg/L. After incubation, the bacterial protein extract was analyzed by MALDI-TOF MS, and the relative growth (RG) was determined for each isolate, considering intensities of the peaks of the bacterium incubated with antibiotic (tubes 2, 3, and 4) to the same bacterium incubated without antibiotic (tube 1), as follows: RG = IntensityWith antibiotic/IntensityWithout antibiotic. The combination was determined as synergistic when there was an RG decrease of 0.3 in the antibiotic combination in relation to the RG of the most active antibiotic alone. RESULTS: The combination of ATM plus CZA proved to be synergic by time-kill curve assay. All isolates tested with the SynMALDI method also presented synergism. CONCLUSIONS: Detection of synergism for ATM plus CZA combination can be determined by MALDI-TOF MS, providing fast results in order to improve patient treatment.

Direct Detection of KPC Peak from Positive Blood Cultures Using MALDI-TOF MS: Are We There Yet?

Detecting carbapenemase-associated carbapenem resistance is a subject of major clinical and epidemiological concern as it influences therapeutic choice. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a means to assess bacterial resistance mechanisms. We aimed to detect the KPC enzyme directly from positive blood cultures using MALDI-TOF MS. To do so, 102 clinical Enterobacteria were evaluated, including 59 blaKPC positives. Proteins were extracted using formic acid, isopropyl alcohol, and water (17:33:50) and spotted onto a steel target plate using the double-layer sinapinic acid technique. Two parameters were considered: (i) the visual detection of a clear peak with the expected KPC m/z and (ii) the evaluation of the relative intensity of the ions in the peak. A peak was observed in 56/59 blaKPC-positive isolates (94.9% sensitivity), with no false-positive results (100% specificity). When considering intensity, with a cut-off ≥120 (a.u.), sensitivity was 94.9% and specificity was 95.3%. We proposed a "buffer" zone, with intermediate values of intensity (115 to 125) reaching 100% sensitivity and specificity. The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid, which may improve appropriate patient therapy and antimicrobial stewardship.

Evaluation of Early Reading of Broth Microdilution Technique for Polymyxin B.

Delay in the results of standard phenotypic susceptibility tests is the main obstacle to adequate antibiotic treatment. For this reason, the European Committee for Antimicrobial Susceptibility Testing has proposed the Rapid Antimicrobial Susceptibility Testing for the disk diffusion method directly from blood culture. However, to date, there are no studies evaluating early readings of polymyxin B broth microdilution (BMD), the only standardized methodology for assessing susceptibility to polymyxins. This study aimed to evaluate modifications in the BMD technique for polymyxin B using fewer antibiotic dilutions and reading after an incubation time of 8-9 hr (early reading) in comparison to 16-20 hr of incubation (standard reading) for isolates of Enterobacterales, Acinetobacter baumannii complex, and Pseudomonas aeruginosa. A total of 192 isolates of gram-negative bacteria were evaluated and the minimum inhibitory concentrations were read after early and standard incubations. The early reading presented 93.2% of essential agreement and 97.9% of categorical agreement with the standard reading of BMD. Only three isolates (2.2%) presented major errors and only one (1.7%) presented a very major error. These results indicate a high agreement between the early and the standard reading times of BMD of polymyxin B.

A Rapid and Easy Method of MALDI Biotyper Antibiotic Susceptibility Test Rapid Assay To Provide Early Meropenem Susceptibility Profile in Enterobacterales.

Antimicrobial susceptibility testing (AST) that can provide faster results is necessary. The MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) can provide early AST results but still needs to be simplified in order to facilitate its execution by microbiology laboratories. The aim of this study was to evaluate an adaptation of MBT-ASTRA. Isolates of Enterobacterales were tested for meropenem susceptibility by MBT-ASTRA using a solution prepared from meropenem disks and performing a manual spectrum analysis. The relative growth (RG) was calculated for each isolate, and a cutoff value was established to determine the susceptibility profile of the isolates. Results of the adapted method were compared with the standard susceptibility method (broth microdilution). An adapted method of MBT-ASTRA was developed. The RG cutoff values for meropenem were ≤0.1510 for susceptibility and >0.6272 for resistance, presenting 95.65% categorical agreement, with 2.9% (2/69) minor discrepancy and 3.23% (1/31) very major discrepancy. MBT-ASTRA can be used to provide rapid AST results with a simpler and more accessible protocol, especially regarding spectrum analysis. IMPORTANCE The simplification of the MBT-ASTRA technique, especially in spectrum analysis, can considerably allow more laboratories to rapidly determine antimicrobial susceptibility profiles.

Treated tannery effluent and its impact on the receiving stream water: physicochemical characterization and cytogenotoxic evaluation using the Allium cepa test.

Tanneries are considered some of the most polluting industries due to the heavy use of toxic compounds, most of which are released into water bodies, thus exerting adverse effects on aquatic biota. However, the effects on organisms of treated effluents when released into the natural environment are rarely evaluated. This study aims to assess the physicochemical parameters of a tannery effluent after treatment (TE) at a Common Effluent Treatment Plant as well as the water of the receiving stream and to evaluate cytogenotoxic effects in Allium cepa. Three sampling sites (A: TE discharge point; B: 100 m downstream from site A along the receiving stream; C: 100 m upstream from site A along the stream) were selected. Onion bulbs were exposed to TE (100%, 80%, 60% v/v), water samples from sites B and C, and tap water for 72 h. Chromosomal aberration and mitotic index were analyzed on the root cells of A. cepa. The TE was above the standard limits for ammoniacal nitrogen, COD, and total nitrogen. No cytogenotoxicity was observed in A. cepa exposed to samples from sites A and C. However, the stream water sampled downstream from the TE discharge site significantly reduced the mitotic index, indicating a cytotoxic effect. Therefore, this demonstrates the effects of interactions between the receiving water and the complex chemical mixtures in the TE. The findings thus showed that the toxicity assessment of treated effluents along with the receiving water body would provide valuable and more realistic information about the joint toxicity of chemical pollutants in aquatic environments.

MALDI-TOF mass spectrometry for direct KPC detection among Enterobacterales.

In this study, we evaluate a method for the KPC enzyme detection, using MALDI-TOF MS, for Enterobacterales. A total of 300 clinical Enterobacterales isolates were selected. The collection included 259 carbapenemase-producing (157 KPC and 102 non-KPC) and 41 carbapenemase non-producing isolates. Bacterial proteins were extracted from Mueller-Hinton agar plates using formic acid, isopropyl alcohol, and water (17:33:50). Samples were prepared with a double layer of synapinic acid. Analyses were performed using a Microflex LT mass spectrometer (Bruker Daltonics) and flexAnalysis 4.0 software (Bruker Daltonics). Statistical analyses were performed using SPSS Software. A distinctive peak at m/z 28,643-28,731 was found in all 157 KPC-producing isolates, and it was consistently absent in the 143 KPC non-producing group. KPC-producing peak intensities ranged from 77 to 3893. Considering an intensity cutoff value ≥ 120 for the presence of KPC, this methodology presented 98.09% and 97.90% of sensitivity and specificity, respectively.

Establishing a quantitative index of meropenem hydrolysis for the detection of KPC- and NDM-producing bacteria by MALDI-TOF MS.

BACKGROUND: Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), commonly used for microorganism identification, can also be applied for the detection of carbapenemase-producing bacteria by the evaluation of carbapenem hydrolysis. Since KPC- and NDM-producing bacteria are related to high mortality rates, diagnostic assays for its detection are essential. The aim of this study was to develop and evaluate a method to establish a quantitative measure (hydrolysis index - HI) to detect meropenem hydrolysis by MLADI-TOF MS. METHODS: blaKPC and blaNDM positive and negative Klebsiella pneumoniae isolates and Escherichia coli ATCC 25922 (control) were incubated in a meropenem solution for 2 h. Protein extraction from these suspensions were submitted to MALDI-TOF MS analysis. The intensity of peaks at 384 m/z and 379 m/z of each isolate were used to establish the HI as follows: HI = (Peak intensity384 Test / Peak intensity379 Test) / (Peak intensity384 Control / Peak intensity379 Control). Receiver Operating Characteristic curve was used to determine a cutoff value to differentiate carbapenemase-producing from carbapenemase non-producing bacteria. RESULTS: As all carbapenemase-producing K. pneumoniae presented HI ≤0.55 and all carbapenemase non-producing isolates presented a HI ≥0.57, the index of 0.56 was established as a cutoff value to differentiate carbapenemase (KPC and NDM) producing and non-producing bacteria.

Antifungal potential against Sclerotinia sclerotiorum (Lib.) de Bary and plant growth promoting abilities of Bacillus isolates from canola (Brassica napus L.) roots.

Endophytic bacteria show important abilities in promoting plant growth and suppressing phytopathogens, being largely explored in agriculture as biofertilizers or biocontrol agents. Bacteria from canola roots were isolated and screened for different plant growth promotion (PGP) traits and biocontrol of Sclerotinia sclerotiorum. Thirty isolates belonging to Bacillus, Paenibacillus, Lysinibacillus, and Microbacterium genera were obtained. Several isolates produced auxin, siderophores, hydrolytic enzymes, fixed nitrogen and solubilized phosphate. Five isolates presented antifungal activity against S. sclerotiorum by the dual culture assay and four of them also inhibited fungal growth by volatile organic compounds production. All antagonistic isolates belonged to the Bacillus genus, and had their genomes sequenced for the search of biosynthetic gene clusters (BGC) related to antimicrobial metabolites. These isolates were identified as Bacillus safensis (3), Bacillus pumilus (1), and Bacillus megaterium (1), using the genomic metrics ANI and dDDH. Most strains showed several common BGCs, including bacteriocin, polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS), related to pumilacidin, bacillibactin, bacilysin, and other antimicrobial compounds. Pumilacidin-related mass peaks were detected in acid precipitation extracts through MALDI-TOF analysis. The genomic features demonstrated the potential of these isolates in the suppression of plant pathogens; however, some aspects of plant-bacterial interactions remain to be elucidated.

Assessment of DNA damage in floriculturists in southern Brazil.

The aim of this study was to assess possible genotoxic effects on floriculturists in a region of the state of Rio Grande do Sul, in the south of Brazil, using the micronucleus test (MN) and comet assay. Thirty-seven floriculturists and 37 individuals not exposed to pesticides participated in the study. The micronucleus test was performed with epithelial cells of the oral mucosa. In the microscopic analysis, 2000 cells were evaluated per subject, verifying the frequency of MN and the frequency of other nuclear abnormalities (nuclear buds, binucleated cells, and karyorrhexis). For the comet assay in the peripheral blood lymphocytes, 100 cells were classified in five classes, according to the migration of DNA fragments, thereby generating the frequency of damaged cells and the damage index. There was no difference between the exposed and control groups in the frequencies of MN and other nuclear abnormalities in the epithelial cells of the oral mucosa. However, the comet assay showed that both the frequency of DNA damaged cells and the damage index were significantly greater in the exposed group. The results therefore indicate that floriculturists are exposed to mixtures of pesticides with genotoxic potential.