- Volume 3, Issue 2, 2020

Introduction. Rapid differentiation between Staphylococcus aureus (SA) and coagulase-negative staphylococci (CoNS) is critical in clinical infection. Direct matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification from blood culture is highly accurate, but is associated with a significant failure rate, delaying identification. However, MALDI-TOF failure may itself be indicative of CoNS infection.

Aim. We sought to examine whether failure of MALDI-TOF direct ID was indicative of CoNS infection and could be used as a diagnostic tool to promote antimicrobial stewardship.

Methodology. Results of Gram stains, MALDI-TOF identification and formal identification were extracted from the large, multi-centre RAPIDO trial. All blood cultures with presumed staphylococci were included. MALDI-TOF performance (correct identification, incorrect identification, failed identification) was calculated for each sample and across sites. Risk of SA disease was calculated for each group (correct, incorrect, failed) and across sites. Logistic regression was used to identify if clinical features are associated with MALDI-TOF performance.

Results. In the RAPIDO trial, 4312 patients were allocated to the MALDI-TOF arm. After exclusions, 880 patients were eligible and had a blood culture with a Gram stain consistent with presumed staphylococci. In total, 204 of these (23.2 %) were ultimately identified as SA. MALDI-ID was successful 83.9 % of the time, and was 100 % accurate when successful. Failure was more common in CoNS isolates (124/641, 19.3 %) than in SA (13/191, 6.4 %). When MALDI-TOF failed, the risk of SA disease was 9.2 % across the whole cohort, although failure rates and risk of SA disease varied significantly between centres. MALDI-TOF failure was independent of clinical characteristics.

Conclusion. Presumed staphylococci that fail direct MALDI-TOF identification from blood culture are significantly more likely to be CoNS isolates than SA. In low-risk or low-prevalence settings, SA therapy can be withheld if MALDI-TOF is unsuccessful.

Azithromycin is increasingly being used for the treatment of shigellosis despite a lack of interpretative guidelines and with limited clinical evidence. The present study determined azithromycin susceptibility and correlated this with macrolide-resistance genes in Shigella spp. isolated from stool specimens in Vellore, India. The susceptibility of 332 Shigella isolates to azithromycin was determined using the disc diffusion method. Of these, 31 isolates were found to be azithromycin resistant. The azithromycin minimum inhibitory concentration (MIC) was determined using the broth microdilution method. In addition, isolates were screened for mphA and ermB genes using conventional PCR. Furthermore, an isolate that was positive for resistance genes was subjected to complete genome analysis, and was analysed for mobile genetic elements. The azithromycin MIC for the 31 resistant Shigella isolates ranged between 2 and 16 mg l−1. PCR results showed that a single isolate of Shigella sonnei carried a mphA gene. Complete genome analysis revealed integration of an IncFII plasmid into the chromosome of S. sonnei , which was also found to carry the following resistance genes: sul1, bla DHA1, qnrB4, mphA, tetR. Mutations in the quinolone-resistance-determining region (QRDR) were also observed. Additionally, prophages, insertion sequences and integrons were identified. The novel finding of IncFII plasmid integration into the chromosome of S. sonnei highlights the potential risk of Shigella spp. becoming resistance to azithromycin in the future. These suggests that it is imperative to monitor Shigella susceptibility and to study the resistance mechanism of Shigella to azithromycin considering the limited treatment choices for shigellosis.

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in S. aureus and should dramatically improve the scalability of gene-function studies.

Tsukamurella pulmonis ( Actinobacteria ), a Gram-positive, obligate aerobic and weakly or variably acid-fast bacterium, is an opportunistic pathogen. Here we report two cases of conjunctivitis caused by T. pulmonis . Both patients had a previous history of nasolacrimal duct obstruction (NLDO). Isolation of T. pulmonis was performed on chocolate, tryptic soy blood and Columbia nalidixic agars. After 24 h of incubation, odourless, white-greyish, membrane-like colonies were observed. The VITEK-2 bacterial identifier system failed to identify the species, while Vitek-MS matrix-assisted laser desorption ionization time-of-flight technology, successfully identified the isolate from case 2 but not from case 1. Final identification was verified using 16S rRNA gene sequencing. An antibiogram was performed and according to the results cefazoline in addition to vancomycin eye drops for 5 days, were suggested as a treatment in case 1. In case 2 the infection was ended without treatment. This is the first report of Tsukamurella as a pathogen that causes conjunctivitis in patients with NLDO.

Introducing creative workshops in higher education curricula, in addition to formal lectures, is an excellent way of reinforcing knowledge and encouraging creative thinking. In particular, the use of card games as a tool for inducing student engagement and enthusiasm has been reported to be a very effective approach. Here, we report an innovative card game-based workshop for use at the intermediate undergraduate level. The name of the game is Microbes Against Humanity and has been adapted from the widely known party game Cards Against Humanity, which is freely available under a creative commons licence. Overall, 64 students and two academics participated in this 2 h workshop. Our students found the workshop to be very enjoyable, considered it to be helpful for their learning and suggested interesting ideas for further improvement. In conclusion, it was shown that such exciting workshops can trigger students’ enthusiasm for microbiology and enhance their learning potential.