The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed Cap-sidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

The Gram-positive human pathogen Staphylococcus aureus is a leading cause of severe bacterial infections. Recent studies have shown that various cell types could readily internalize S. aureus and infected cells have been proposed to serve as vehicle for the systemic dissemination of the pathogen. Here we focused on the intracellular behavior of the Community-Associated Methicillin-Resistant S. aureus strain USA300. Supporting earlier observations, we found that wild-type S. aureus strain USA300 persisted for longer period within endothelial cells than within macrophages and that a mutant displaying the small colony variant phenotype (ΔhemDBL) had increased intracellular persistence. Time-lapse microscopy revealed that initial persistence of wild-type bacteria in endothelial cells corresponded to distinct single cell events, ranging from active intracellular bacterial proliferation, leading to cell lysis, to non-replicating bacterial persistence even 1 week after infection. In sharp contrast, ΔhemDBL mutant bacteria were essentially non-replicating up to 10 days after infection. These findings suggest that internalization of S. aureus in endothelial cells triggers its persistence and support the notion that endothelial cells might constitute an intracellular persistence niche responsible for reported relapse of infection after antibiotic therapy.

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Overuse of antibiotics in clinical medicine has contributed to the global spread of multidrug-resistant bacterial pathogens, including Acinetobacter baumannii. We present a case of an 88-year-old Chinese man who developed hospital-acquired pneumonia caused by carbapenem-resistant A. baumannii (CRAB). A personalized lytic pathogen-specific single-phage preparation was nebulized to the patient continuously for 16 days in combination with tigecycline and polymyxin E. The treatment was well tolerated and resulted in clearance of the pathogen and clinical improvement of the patient’s lung function.

Background Chronic lung infection in cystic fibrosis (CF) patients by Staphylococcus aureus is a well-established epidemiological fact. Indeed, S. aureus is the most commonly identified pathogen in the lungs of CF patients. Improving our understanding of the mechanisms associated with the persistence of S. aureus is therefore an important issue. Methods We selected pairs of sequential S. aureus isolates from 3 patients with CF and from 1 patient with non-CF chronic lung disease. We used a combination of genomic, proteomic, and metabolomic approaches with functional assays for in-depth characterization of S. aureus long-term persistence. Results In this study, we show that late S. aureus isolates from CF patients have an increased ability for intracellular survival in CF bronchial epithelial-F508del cells compared to ancestral early isolates. Importantly, the increased ability to persist intracellularly was confirmed for S. aureus isolates within the own-patient F508del epithelial cells. An increased ability to form biofilm was also demonstrated. Furthermore, we identified the underlying genetic modifications that induce altered protein expression profiles and notable metabolic changes. These modifications affect several metabolic pathways and virulence regulators that could constitute therapeutic targets. Conclusions Our results strongly suggest that the intracellular environment might constitute an important niche of persistence and relapse necessitating adapted antibiotic treatments.

Colanic acid has promising applications in food, cosmetic, and healthcare fields. In this study, a recombinant WQM003/pRAU was constructed by deleting genes lon and hns and overexpressing genes rcsA and galU in E. coli MG1655Δ(L-Q). After systematic optimization of fermentation conditions, colanic acid yield in WQM003/pRAU reached 19.79 g/L, the highest yield reported so far. The colanic acid produced by WQM003/pRAU was purified and its structure and physical properties were determined. This colanic acid shows a triple-helical structure and is stable up to 102 °C, and its melting temperature is 253.9 °C. This colanic acid shows a sphere-like chain conformation in aqueous solution. The viscosity of this colanic acid solution is related to concentration, shear rate, salt, temperature, and pH. At high concentrations, this colanic acid shows both viscous and elastic behaviors. These results suggest that the colanic acid produced by WQM003/pRAU has broad application prospects.

The annual prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysia has been estimated to be 30 % to 40 % of all S. aureus infections. Nevertheless, data on the antimicrobial resistance and genetic diversity of Malaysian MRSAs remain few. In 2009, we collected 318 MRSA strains from various wards of our teaching hospital located in Kuala Lumpur, the capital city of Malaysia, and performed antimicrobial susceptibility testing on these strains. The strains were then molecularly characterized via staphylococcal cassette chromosome (SCC) mec and virulence gene (cna, sea, seb, sec, sed, see, seg, seh, sei, eta, etb, Panton-Valentine leukocidin and toxic shock syndrome toxin-1) typing; a subset of 49 strains isolated from the intensive care unit was also typed using PFGE. Most strains were found to be resistant to ciprofloxacin (92.5 %), erythromycin (93.4 %) and gentamicin (86.8 %). The majority (72.0 %) of strains were found to harbour SCCmec type III-SCCmercury with the presence of ccrC, and carried the sea+cna gene combination (49.3 %), with cna as the most prevalent virulence gene (94.0 %) detected. We identified four PFGE clusters, with pulsotype C (n=19) as the dominant example in the intensive care unit, where this pulsotype was found to be associated with carriage of SCCmec type III and the sea gene (P=0.05 and P=0.02, respectively). In summary, the dominant MRSA circulating in our hospital in 2009 was a clone that was ciprofloxacin, erythromycin and gentamicin resistant, carried SCCmec type III-SCCmercury with ccrC and also harboured the sea+cna virulence genes. This clone also appears to be the dominant MRSA circulating in major hospitals in Kuala Lumpur.

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. In the current study, we focused on one key PPP enzyme, transketolase (TKT). We showed that inactivation of the unique gene encoding TKT activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared with wild-type strain in early intracellular proliferation and in the ability to colonize kidneys. Transcriptional activity of the 2 master regulators sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription suggests that TKT—or a functional PPP—strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.