Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.

Ireland bT he use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for microbial identification in clinical microbiology in recent years. This development can be attributed to its low operating cost and to its speed and reliability of identification for many bacteria and fungi. However, the use of MALDI-TOF MS for mycobacteriology has been less satisfactory, mainly owing to nonstandardized preparatory methods, poor reproducibility, and comparatively inferior results (1). The accurate and rapid identification of mycobacteria to the species level has implications for effective patient treatment and implementation of timely infection control measures.Our laboratory set about validating the Bruker Biotyper for the identification of mycobacteria using wild-type Mycobacterium spp. previously identified using genotype CM/AS and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing by the Irish Mycobacterial Reference Laboratory, St. James's Hospital, Dublin, Ireland, and Bruker's Mycobacteria Extract Method (MycoEX) version 3 (Fig. 1). Heat inactivation coupled with ethanol treatment (as detailed in Fig. 1) proved efficacious in the inactivation of mycobacteria; following extended incubation, there was no growth. Preliminary procedures using Bruker's recommended protocol yielded poor results. Therefore, we set about improving the results attained.Cell disruption has previously been shown to be a key step in liberating mycobacterial protein for analysis (2). We designed a two-step cell disruption protocol (Fig. 1) and found that the combined use of 0.5-mm-diameter silica/zirconia beads and sonication for 15 min produced greatly improved results (Table 1). A direct comparison between our method and MycoEX showed a marked improvement for Mycobacterium tuberculosis complex isolates using our method; 100% achieved successful identification to the genus level, with 85% achieving a reliable identification to the species level (log score of greater than 2.0). MycoEX resulted in successful identification to the genus level of 79% of isolates, with 62% achieving reliable identification to the species level. The number of results showing no identifications was reduced from 7 (22%) to 0 using the two-step cell disruption protocol. Similar improvements were shown for mycobacteria other than tuberculosis isolates, using the two-step cell disruption protocol; 87.5% achieved a log score of 1.7 or greater and 62.5% achieved a log score of greater than 2 compared to MycoEX, and 67% of isolates achieved a log score of 1.7 or greater, with 29% achieving a log score of greater than 2.0.In conclusion, using the described two-step cell disruption protocol, there was a 9-fold decrease in failures to achieve correct

We examined whether survival of different rat strains administered anthrax lethal toxin is genetically determined. A reproducible test population of first filial generation hybrid rats was bred based on the susceptibility of progenitors to anthrax lethal toxin and to maximize genetic diversity across the strains. These rats were then tested with varying doses of anthrax lethal toxin. We found that all 'sensitive' strains died within 2 h following systemic administration of 240 mg/kg lethal toxin, while one strain survived following a five times higher dose (1.4 mg/kg). The ability of lethal toxin to lyse macrophage cultures derived from the bone marrow of these strains corresponded with in vivo results. We conclude that a rat test population can detect strain differences in response to anthrax lethal toxin. Survival is influenced by the host genome background and is likely due to a single gene with a recessive mode of inheritance.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2–98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

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The field of clinical microbiology has been revolutionised by genomic and proteomic methods, which have facilitated more rapid diagnosis and characterisation of infection in many cases. In contrast, mycobacteriological evolution has tended to retain the traditional methods of smear microscopy for detection of acid-fast bacilli to indicate mycobacteria, along with culture, and in synergy with more modern molecular methods. Thus, efforts have been focused on reducing the time to diagnosis of infection, while increasing the amount of diagnostic information available, including more definitive speciation, and more rapid susceptibility test results. Although smear microscopy remains a mainstay for the laboratory-based diagnosis of mycobacterial infection, molecular testing has vastly reduced the time needed for identification of Mycobacterium tuberculosis in particular, when compared with traditional culture-based techniques. Molecular methods may also yield antimicrobial susceptibility results through testing for the most common resistance-inducing mutations to some of the antimicrobial agents of choice. However, the diversity of resistance mutations already characterised suggests that these currently-available molecular detection systems should be accompanied by culture-based susceptibility testing. This review compares the efficacy of microscopic, phenotypic, proteomic and genotypic methods available for mycobacterial diagnosis. The diversity of methods currently in use reflects the complexity of this area of diagnostic microbiology.

AimsCampylobacter fetus subsp fetus (CFF) can cause intestinal illness, particularly in immunocompromised humans, with the potential to cause severe systemic infections. CFF is a zoonotic pathogen with a broad host range among farm animals and humans, inducing abortion in sheep and cows. The current paper describes a strain of CFF isolated from a patient with prosthetic valve endocarditis in Mercy University Hospital, Cork, Ireland, during 2017. Only five cases of C. fetus as a cause of prosthetic valve endocarditis have been reported in the literature, with no reports of biofilm formation within the species.MethodsThe aetiological strain was speciated and subspeciated by the VITEK 2 NH card and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. CFF biofilm formation was analysed using a crystal violet staining method. C. jejuni National Collection of Type Cultures (NCTC) 11168 was used as a positive control organism. Strains were incubated statically in Mueller-Hinton broth and Mueller-Hinton broth supplemented with 0.025% sodium deoxycholate for 3 and 7 days at 37°C, microaerobically.ResultsThe CFF strain formed stronger attached biofilms on polystyrene plates on day 3 (72 hours) than the C. jejuni NCTC 11168 control strain, but were weaker than the control strain on day 7 in Mueller-Hinton broth. Monoculture of this C. fetus isolate was found to exist in three defined forms of biofilms (attached, air–liquid interface and floccules).ConclusionsThis clinically significant C. fetus isolate showed considerable biofilm-forming capability, which we suggest conferred a survivalist advantage, contributing to the genesis of infective prosthetic valve endocarditis.