Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates.

c Carbapenemase-producing Enterobacteriaceae (CPE) are an increasing problem worldwide, and rectal swab surveillance is recommended as a component of infection control programs. The performance of a prototype chromogenic medium (chromID CARBA) was evaluated and compared with media tested by four other screening methods: (i) overnight selective enrichment in 5 ml tryptic soy broth with a 10-g ertapenem disk followed by plating onto MacConkey agar (CDC-TS), (ii) short selective enrichment in 9 ml brain heart infusion broth with a 10-g ertapenem disk followed by plating onto chromID ESBL medium (ESBL-BH), (iii) direct plating onto chromID ESBL, and (iv) direct plating onto MacConkey agar supplemented with meropenem (1 g/ml) (MCM). The screening methods were applied to detect CPE in 200 rectal swab specimens taken from different hospitalized patients. Identification and antimicrobial susceptibility were performed by the Vitek 2 system. Carbapenem MICs were checked by Etest. Carbapenemase production was confirmed using the modified Hodge test, combined-disk tests, and PCR assays. In total, 133 presumptive CPE strains were detected. Phenotypic and genotypic assays confirmed 92 strains to be CPE (56 KPC-positive Klebsiella pneumoniae, 29 VIM-positive K. pneumoniae, and 7 KPC-positive Enterobacter aerogenes strains) recovered from 73 patients, while the remaining 41 strains were confirmed to be CPE negative (19 ESBL producers and 22 nonfermenters). chromID CARBA, ESBL-BH, and chromID ESBL exhibited the highest sensitivity (92.4%), followed by CDC-TS and MCM (89.1%) (P ‫؍‬ 0.631). The specificity was greater for chromID CARBA (96.9%) and ESBL-BH (93.2%) than for CDC-TS (86.4%), MCM (85.2%), and chromID ESBL (84.7%) (P ‫؍‬ 0.014). In conclusion, chromID CARBA was found to be a rapid and accurate culture screening method for active CPE surveillance.

Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted "revolution" was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches.

Recently, plasmid-mediated and, therefore, transferable bacterial polymyxin resistance was discovered in strains from both humans and animals. Such a trait may widely spread geographically, while simultaneously crossing microbial species barriers. This may ultimately render the Blast resort^polymyxin antibiotics therapeutically useless. Colistin is currently used to treat infections caused by Gram-negative carbapenemase producers and colistin resistance may lead to practical panantibiotic resistance. We here analyzed the medical and diagnostic consequences of (emerging) colistin resistance and propose pathways toward adequate diagnostics for timely detection of both asymptomatic carriage and infection. Culture-based testing using chromogenic and selective media for screening clinical (and veterinary) specimens may constitute key tools for that purpose. Relevant molecular tests are also discussed.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.