scholarly journals Evaluation of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Samples from Patients with Cystic Fibrosis in the United States

2017 ◽  
Vol 55 (5) ◽  
pp. 1469-1477 ◽  
Author(s):  
Rongpong Plongla ◽  
Clair L. Preece ◽  
John D. Perry ◽  
Peter H. Gilligan
Keyword(s):  
Cystic Fibrosis ◽  
Burkholderia Cepacia ◽  
Nontuberculous Mycobacteria ◽  
Culture Method ◽  
The United States ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Selective Agar ◽  
Tof Ms

ABSTRACTA novel selective agar (RGM medium) has been advocated for the isolation of rapidly growing mycobacteria from the sputa of cystic fibrosis (CF) patients. The aim of this study was to compare RGM medium toBurkholderia cepaciaselective agar (BCSA) and a standard acid-fast bacillus (AFB) culture method for the isolation of nontuberculous mycobacteria (NTM) from patients with CF. The applicability of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for the identification of NTM isolated on RGM medium was also assessed. Respiratory samples (n= 869) were collected from 487 CF patients and inoculated directly onto RGM medium and BCSA. Cultures were incubated at 30°C and examined for up to 28 days. A subset of 212 samples (from 172 patients) was also cultured by using a mycobacterial growth indicator tube (MGIT) and on Lowenstein-Jensen medium following dual decontamination. By using a combination of all methods, 98 mycobacteria were isolated from 869 samples (11.3%). The sensitivity of RGM medium (96.9%) was significantly higher than that of BCSA (35.7%) for the isolation of mycobacteria (P< 0.0001). The sensitivity of RGM medium was also superior to that of standard AFB culture for the isolation of mycobacteria (92.2% versus 47.1%;P< 0.0001). MALDI-TOF MS was effective for the identification of mycobacteria in RGM medium. RGM medium offers a simple and highly effective tool for the isolation of NTM from patients with CF. Extended incubation of RGM medium for 28 days facilitates the isolation of slow-growing species, including members of theMycobacterium aviumcomplex (MAVC).

scholarly journals Evaluation of Various Culture Media for Detection of Rapidly Growing Mycobacteria from Patients with Cystic Fibrosis

2016 ◽  
Vol 54 (7) ◽  
pp. 1797-1803 ◽  
Author(s):  
Clair L. Preece ◽  
Thomas A. Wichelhaus ◽  
Audrey Perry ◽  
Amanda L. Jones ◽  
Stephen P. Cummings ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Burkholderia Cepacia ◽  
Nontuberculous Mycobacteria ◽  
Culture Media ◽  
Culture Method ◽  
Content Type ◽  
Selective Media ◽  
Rapidly Growing Mycobacteria ◽  
Selective Agar ◽  
Bacteria And Fungi

Isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation onBurkholderia cepaciaselective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation ofBurkholderia cepaciacomplex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation ofB. cepaciacomplex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P= 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF.

scholarly journals First Report of Wohlfahrtiimonas chitiniclastica Isolation from a Patient with Cellulitis in the United States

2015 ◽  
Vol 53 (12) ◽  
pp. 3942-3944 ◽  
Author(s):  
Angel de Dios ◽  
Seby Jacob ◽  
Amit Tayal ◽  
Mark A. Fisher ◽  
Tanis C. Dingle ◽  
...  
Keyword(s):  
United States ◽  
16S Rrna Gene Sequencing ◽  
The United States ◽  
Superior Performance ◽  
Rrna Gene ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

We report the first documented isolation ofWohlfahrtiimonaschitiniclasticafrom a human in the United States. Initially misidentified asAcinetobacter lwoffiiby Vitek-2, the isolate was subsequently identified asW.chitiniclasticaby matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. While the clinical significance of the isolate in this case is unclear, it highlights the superior performance of MALDI-TOF MS for bacterial identification.

scholarly journals Epidemiology of Nontuberculous Mycobacteria in French Polynesia

2015 ◽  
Vol 53 (12) ◽  
pp. 3798-3804 ◽  
Author(s):  
Michael Phelippeau ◽  
Djaltou Aboubaker Osman ◽  
Didier Musso ◽  
Michel Drancourt
Keyword(s):  
Nontuberculous Mycobacteria ◽  
Sequence Similarity ◽  
Gene Sequencing ◽  
French Polynesia ◽  
Rrna Gene ◽  
Mycobacterium Fortuitum ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

As few data are available in the Pacific countries and territories of the Oceania region regarding nontuberculous mycobacteria, we retrospectively identified 87 such isolates from French Polynesia from 2008 to 2013 by hybridization using DNA-strip, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and partialrpoBgene sequencing. PartialrpoBgene sequencing classified 42/87 (48.3%) isolates in theMycobacterium fortuitumcomplex, 28 (32.2%) in theMycobacterium abscessuscomplex, 8 (9.2%) in theMycobacterium mucogenicumcomplex, and 5 (5.7%) in theMycobacterium aviumcomplex. Two isolates were identified asMycobacterium acapulcensisandMycobacteriumcosmeticumby partial 16S rRNA gene sequencing. One isolate, unidentified by MALDI-TOF MS and yielding less than 92% and 96% sequence similarity withrpoBandhsp65reference sequences, respectively, was regarded as a potentially new species. Samples from three patients exhibiting ≥2Mycobacterium porcinumisolates and from one patient with emphysema and a lung abscess exhibiting 2Mycobacterium senegalenseisolates fulfilled the American Thoracic Society microbiological criteria for nontuberculous mycobacterial lung infection. Remote geographic areas, such as French Polynesia, are potential sources for the discovery of new mycobacterial species.

scholarly journals Evaluation of MALDI Biotyper Interpretation Criteria for Accurate Identification of Nontuberculous Mycobacteria

2020 ◽  
Vol 58 (10) ◽  
Author(s):  
David Rodriguez-Temporal ◽  
Belén Rodríguez-Sánchez ◽  
Fernando Alcaide
Keyword(s):  
Liquid Medium ◽  
Solid Medium ◽  
Protein Extraction ◽  
Nontuberculous Mycobacteria ◽  
Cutoff Score ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Interpretation Criteria ◽  
Tof Ms

ABSTRACT Identification of mycobacteria by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) requires not only a good protein extraction protocol but also an adequate cutoff score in order to provide reliable results. The aim of this study was to assess the cutoff scores proposed by the MALDI-TOF MS system for mycobacterial identification. A total of 693 clinical isolates from a liquid medium and 760 from a solid medium were analyzed, encompassing 67 different species of nontuberculous mycobacteria (NTM). MALDI-TOF MS identified 558 (80.5%) isolates from the liquid medium and 712 (93.7%) isolates from the solid medium with scores of ≥1.60. Among these, four (0.7%) misidentifications were obtained from the liquid medium and four (0.5%) from the solid medium. With regard to species diversity, MALDI-TOF MS successfully identified 64 (95.5%) different species, while PCR-reverse hybridization (GenoType Mycobacterium CM and AS assays) identified 24 (35.8%) different species. With MALDI-TOF MS scores of ≥2, all isolates were correctly identified, and with scores in the range from 1.60 to 1.99, most isolates were correctly identified, except for Mycobacterium angelicum, M. parascrofulaceum, M. peregrinum, M. porcinum, and M. gastri. In conclusion, MALDI-TOF MS is a useful method for identifying a large diversity of NTM species. A score threshold of 1.60 proved useful for identifying almost all the isolates tested; only a few species required a higher score (≥2.00) to obtain a valid definitive identification.

scholarly journals Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Jie Lin ◽  
Chunquan Xu ◽  
Renchi Fang ◽  
Jianming Cao ◽  
Xiucai Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Population Analysis ◽  
Colistin Resistance ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Expression Levels ◽  
Maldi Tof ◽  
Broth Microdilution Method ◽  
Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

scholarly journals Performance of Five Commercial Identification Platforms for Identification of Staphylococcus delphini

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Matthew C. Canver ◽  
Tsigereda Tekle ◽  
Samantha T. Compton ◽  
Katrina Callan ◽  
Eileen M. Burd ◽  
...  
Keyword(s):  
Distinct Species ◽  
Human Infection ◽  
Species Level ◽  
Accurate Identification ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Staphylococcus Intermedius ◽  
Animal Pathogens ◽  
Tof Ms

ABSTRACT The Staphylococcus intermedius group (SIG) is a collection of coagulase-positive staphylococci consisting of four distinct species, namely, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus intermedius, and Staphylococcus pseudintermedius. SIG members are animal pathogens and rare causes of human infection. Accurate identification of S. pseudintermedius has important implications for interpretation of antimicrobial susceptibility testing data and may be important for other members of the group. Therefore, we sought to evaluate the performance of five commercially available identification platforms with 21 S. delphini isolates obtained from a variety of animal and geographic sources. Here, we show that automated biochemical platforms were unable to identify S. delphini to the species level, a function of its omission from their databases, but could identify isolates to the SIG level with various degrees of success. However, all automated systems misidentified at least one isolate as Staphylococcus aureus. One matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system was able to identify S. delphini to the species level, suggesting that MALDI-TOF MS is the best option for distinguishing members of the SIG. With the exception of S. pseudintermedius, it is unclear if other SIG members should be routinely identified to the species level; however, as our understanding of their role in animal and human diseases increases, it may be necessary and important to do so.

scholarly journals Candida auris Clinical Isolates from South Korea: Identification, Antifungal Susceptibility, and Genotyping

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Yong Jun Kwon ◽  
Jong Hee Shin ◽  
Seung A Byun ◽  
Min Ji Choi ◽  
Eun Jeong Won ◽  
...  
Keyword(s):  
Antifungal Susceptibility ◽  
Diagnostic Tools ◽  
Candida Auris ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Fluconazole Resistant ◽  
Vitek 2 ◽  
Vitek Ms ◽  
Tof Ms

ABSTRACT Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.

scholarly journals Rapid Identification of Clinically Relevant Members of the Genus Exophiala by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and Description of Two Novel Species, Exophiala campbellii and Exophiala lavatrina

2017 ◽  
Vol 55 (4) ◽  
pp. 1162-1176 ◽  
Author(s):  
Andrew M. Borman ◽  
Mark Fraser ◽  
Adrien Szekely ◽  
Daniel E. Larcombe ◽  
Elizabeth M. Johnson
Keyword(s):  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

ABSTRACT Exophiala is a ubiquitous pleomorphic genus comprising at least 40 species, many of which have been associated with superficial, visceral, or systemic infections in humans, other mammals, or cold-blooded animals. In this study, we investigated the potential of matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) for the identification of Exophiala species. A total of 89 isolates (including 50 human and 4 animal clinical isolates) stored in the National Collection of Pathogenic Fungi were identified by PCR amplification and sequencing of internal transcribed spacer region 1. Eighty-three of the isolates corresponded to 16 known species within Exophiala/Rhinocladiella . The remaining six isolates are shown by phylogenetic analyses based on four loci to represent two novel Exophiala species. Four isolates from domestic bathrooms which form a sister species with Exophiala lecanii-corni are described here as Exophiala lavatrina sp. nov. The remaining two isolates, both from subcutaneous infections, are distantly related to Exophiala oligosperma and are described here as Exophiala campbellii sp. nov. The triazoles and terbinafine exhibited low MICs against all Exophiala isolates in vitro . MALDI-TOF MS successfully distinguished all 18 species and identified all isolates after appropriate reference spectra were created and added to commercial databases. Intraspecific mean log scores ranged from 1.786 to 2.584 and were consistently significantly higher than interspecific scores (1.193 to 1.624), with the exception of E. lecanii-corni and E. lavatrina , for which there was considerable log score overlap. In summary, MALDI-TOF MS allows the rapid and accurate identification of a wide range of clinically relevant Exophiala species.

scholarly journals Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from “Pseudo-Cronobacter” to New Enterobacteriaceae Genera

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Barbora Svobodová ◽  
Jiří Vlach ◽  
Petra Junková ◽  
Ludmila Karamonová ◽  
Martina Blažková ◽  
...  
Keyword(s):  
Intact Cell ◽  
Foodborne Pathogen ◽  
Principal Component ◽  
Powdered Infant Formula ◽  
Content Type ◽  
Reliable Identification ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Maldi Biotyper ◽  
Tof Ms

ABSTRACT In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter. Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method. IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter. While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

scholarly journals Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates

2015 ◽  
Vol 53 (11) ◽  
pp. 3438-3447 ◽  
Author(s):  
In Kwon Park ◽  
Amy P. Hsu ◽  
Hervé Tettelin ◽  
Shamira J. Shallom ◽  
Steven K. Drake ◽  
...  
Keyword(s):  
Acid Fast Bacillus ◽  
Mycobacterium Abscessus ◽  
Colony Morphology ◽  
Culture Methods ◽  
Genetic Changes ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

The smooth-to-rough colony morphology shift inMycobacterium abscessushas been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistentM. abscessusinfection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level offadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed asM. abscessussubspeciesabscessusand were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations inmps1andmps2, 13% displayed previously unreported mutations inmmpL4aandmmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression offadD23, sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection withM. abscessus. GPL loss alone may not account for all traits associated with rough morphology.

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