Identification of bacterial species is a crucial bottleneck for clinical diagnosis of infectious diseases. Quick and reliable identification is a key factor to provide suitable antibiotherapies and avoid the development of multiple-drug resistance. We propose a novel nuclear magnetic resonance (NMR)-based metabolomics strategy for rapid discrimination and identification of several bacterial species that relies on untargeted metabolic profiling of supernatants from bacterial culture media. We show that six bacterial species (Gram negative: Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis; Gram positive: Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus saprophyticus) can be well discriminated from multivariate statistical analysis, opening new prospects for NMR applications to microbial clinical diagnosis.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an identification procedure for bacteria and fungi. The MALDI-TOF MS-based analysis of resistance to β-lactam antibiotics has been applied to detect hydrolysis of carbapenems by different bacterial strains. However, the detection of enzymatic carbapenem degradation by MALDI-TOF MS lacks well-standardized protocols and several methods and models of interpretation using different calculations of ratio-of-peak intensities have been described in the literature. Here, we used faropenem and ertapenem hydrolysis as model compounds. In an attempt to propose a universal protocol, the hydrolysis was regularly monitored during 24 h using well-characterized bacterial strains producing different types of carbapenemases (KPC, IMP, NDM, VIM, and OXA-48). Variable responses and different timing for detectable hydrolysis, depending on the enzyme produced, were observed. KPC degrades its template antibiotics very quickly (15 min for some KPC producers) compared to other types of enzymes (more than 90 min for other enzymes). Prior bacterial lysis was shown to be of no interest in the modulation or optimization of the hydrolytic kinetics. The adequate detection of carbapenem hydrolysis would, therefore, require several MALDI-TOF MS readouts for the timely detection of rapid hydrolysis without missing slow hydrolysis. This enzymatic constraint limits the implementation of a standard protocol in routine microbiology laboratories.
The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causative Mycobacterium tuberculosis bacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21 M. tuberculosis isolates at 10 5 , 10 3 , and 10 CFU yielded colonies in 5.7 ؎ 1.5 days, 7.6 ؎ 0.8 days, and 10.8 ؎ 1.7 days versus 1.5 ؎ 0.4 days, 3.5 ؎ 0.6 days, and 4.9 ؎ 1 days in MOD9 (P < 0.05, Student's t test). Further, the time to detectable growth of M. tuberculosis was measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P ؍ 0.11, Fisher's exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P ؍ 0.5195, Fisher's exact test). All together, eight M. tuberculosis isolates were cultured solely from chlorhexidine-MOD9, and two M. tuberculosis isolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ؎ 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ؎ 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P < 0.05, Student's t test). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery of M. tuberculosis. P ulmonary tuberculosis caused by Mycobacterium tuberculosis complex (MTC) mycobacteria remains a global public health problem, causing on average 170 deaths every hour worldwide (1). Despite significant advances in the molecular diagnosis of tuberculosis over the past 2 decades (2), culture is still the universal gold standard for the laboratory diagnosis of tuberculosis, enabling complete postculture antimicrobial susceptibility testing and genotyping (3).MTC mycobacteria are fastidious organisms whose growth is routinely detected only after 7 to 12 days using advanced commercially available automated systems (4). These robots use various formulations of the Middlebrook liquid culture medium and indeed reduce the delay in growth detection compared to that with inoculation of standard solid culture media, such as Lowenstein-Jensen (LJ) medium (4). The World Health Organization (WHO) still recommends inoculating specimens in parallel into a liquid medium for accelerated diagnosis of high-titer specimens and onto a solid culture medium to increase the sensitivity of laboratory diagnosis of pulmonary tuberculosis (5).In an effort to conciliate speed and sensitivity of the culturebased diagnosis of tuberculosis, we developed new formulations of solid culture medium and new protocols for incubating and detecting colonies of M. tuberculosis. Recently, we showed that the MOD4 culture medium incorporating blood and egg lecithin, combined with an improved laboratory workflow, s...
Dysentery is a major health threat that dramatically impacts childhood morbidity and mortality in developing countries. Various pathogenic agents cause dysentery such as Shigella spp. and Escherichia coli, which are very closely related if not identical species. Sensitive and precise detection and identification of the infectious agent is important to target the best therapeutic strategy but the differential diagnosis of these two groups remains a challenge using conventional methods. Here we present a nuclear magnetic resonance (NMR) based multivariate classification model employing bacterial metabolic footprints in post-culture growth media with remarkable segregation
The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student’s t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol.
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