Background: For residents of long-term care facilities (LTCFs), antimicrobial-resistant bacteria (ARB) are a risk factor, yet their oral colonisation, potentially leading to aspiration pneumonia, remains unclear. This study was undertaken to survey the prevalence, phenotypic characteristics, and molecular epidemiology of antimicrobial-resistant Gram-negative bacteria in the oral cavity of LTCF residents, and to analyse the risk factors for such carriers. Methods: This study involved 98 residents of a LTCF in Hiroshima City, Japan, aged between 55 and 101 years. Oropharyngeal swabs were collected and plated on screening media for ESBL-producing and carbapenem-resistant bacteria; isolates were identified and tested for antibiotic susceptibility; biofilm formation was tested in vitro; identification of epidemic clones were pre-determined by PCR; resistance genes, sequence types, and whole-genome comparison of strains were conducted using draft genome sequences. Demographic data and clinical characterisations were collected and risk factors analysed. Results: Fifty-four strains from 38% of the residents grew on screening media and comprised predominantly of Acinetobacter spp. (35%), Enterobacteriaceae spp. (22%), and Pseudomonas spp. (19%). All Escherichia coli isolates carried CTX-M-9 group and belonged to the phylogroup B2, O25:H4 ST131 fimH30 lineage. Six Acinetobacter baumannii isolates presented identical molecular characteristics and revealed more biofilm production than the others, strongly suggesting their clonal lineage. One Acinetobacter ursingii isolate displayed extensive resistance to various ß-lactams due to multiple acquired resistance genes. One Pseudomonas aeruginosa isolate showed exceptional resistance to all ß-lactams including carbapenems, aminoglycosides, and a new quinolone, showing a multidrug-resistant Pseudomonas aeruginosa (MDRP) phenotype and remarkable biofilm formation. Genome sequence analysis revealed this isolate was the bla_IMP-1-positive clone ST235 in Japan. Strokes (cerebral infarction or cerebral haemorrhage) and percutaneous endoscopic gastrostomy tubes were recognised as risk factors for oral colonisation by ARB in the LTCF residents. Conclusions: ARB, as defined by growth on screening agar plates, which carried mobile resistance genes or elements or conferred high biofilm formation, were already prevalent in the oral cavity of LTCF residents. Health-care workers involved in oral care should be aware of antimicrobial resistance and pay special attention to transmission prevention and infection control measures to diminish ARB or mobile resistance elements dissemination in LTCFs.

Staphylococcus epidermidis is a commensal bacterium in humans. To persist in the bacterial flora of the host, some bacteria produce antibacterial factors such as the antimicrobial peptides known as bacteriocins. In this study, we tried to isolate bacteriocin-producing S. epidermidis strains. Among 150 S. epidermidis isolates from the oral cavities of 287 volunteers, we detected two bacteriocin-producing strains, KSE56 and KSE650. Complete genome sequences of the two strains confirmed that they carried the epidermin-harboring plasmid pEpi56 and the nukacin IVK45-like-harboring plasmid pNuk650. The amino acid sequence of epidermin from KSE56 was identical to the previously reported sequence, but the epidermin synthesis-related genes were partially different. The prepeptide amino acid sequences of nukacin KSE650 and nukacin IVK45 showed one mismatch, but both mature peptides were entirely similar. pNuk650 was larger and had an additional seven ORFs compared to pIVK45. We then investigated the antibacterial activity of the two strains against several skin and oral bacteria and found their different activity patterns. In conclusion, we report the complete sequences of 2 plasmids coding for bacteriocins from S. epidermidis, which were partially different from those previously reported. Furthermore, this is the first report to show the complete sequence of an epidermin-carrying plasmid, pEpi56.

Staphylococcus aureus is a bacterium that mainly colonizes the nasal cavity and skin. To colonize the host, it is necessary for S. aureus to resist many antibacterial factors derived from human and commensal bacteria. Among them are the bacteria-derived antimicrobial peptides (AMPs) called bacteriocins. It was reported that some two-component systems (TCSs), which are signal transduction systems specific to bacteria, are involved in the resistance to several bacteriocins in S. aureus. However, the TCS-mediated resistance is limited to relatively low concentrations of bacteriocins, while high concentrations of bacteriocins still exhibit antibacterial activity against S. aureus. To determine whether we could obtain highly bacteriocin-resistant mutants, we tried to isolate highly nisin A-resistant mutants by exposing the cells to sub-minimum inhibitory concentrations (MICs) of nisin A. Nisin A is one of the bacteriocins produced by Lactococcus lactis and is utilized as a food preservative worldwide. Finally, we obtained highly nisin A-resistant mutants with mutations in one TCS, BraRS, and in PmtR, which is involved in the expression of pmtABCD. Notably, some highly resistant strains also showed increased pathogenicity. Based on our findings, this review provides up-to-date information on the role of TCSs in the susceptibility to antibacterial peptides. Additionally, the mechanism for high antimicrobial peptides resistance and its association with pathogenicity in S. aureus is elucidated.

Klebsiella pneumoniae (Kp) has emerged as a global life-threatening pathogen owing to its multidrug resistance and hypervirulence phenotype. Several fatal outbreaks of carbapenem-resistant hypervirulent Kp have been reported recently. Hypermucoviscosity (HMV) is a phenotype commonly associated with hypervirulence of Kp, which is usually regulated by rmpA or rmpA2 (regulators of the mucoid phenotype). Here, we found that temperature was important in the HMV phenotype of Kp, and the impact of temperature on HMV was not uniform among strains. We investigated the HMV phenotype at 37 °C and room temperature (20–25 °C) in 170 clinically isolated hypermucoviscous Kp strains in Japan and analysed the association between the HMV phenotype, virulence genes and antimicrobial resistance (AMR) genes. String length distribution at different temperatures was correlated with the genomic population of Kp. The strains carrying rmpA/rmpA2 frequently showed the HMV phenotype at 37 °C, while the strains negative for these genes tended to show the HMV phenotype at room temperature. Hypervirulent Kp clusters carrying rmpA/rmpA2 without extended-spectrum beta-lactamases (ESBL)/carbapenemases produced higher string lengths at 37 °C than at room temperature, and were mostly isolated from the respiratory tract. Other HMV strains showed distinct characteristics of not carrying rmpA/rmpA2 but were positive for ESBL/carbapenemases, with a higher string length at room temperature than at 37 °C, and were frequently isolated from bloodstream infections. In total, 21 (13.5 %) HMV isolates carried ESBL and carbapenemases, among which five isolates were carbapenem-resistant hypervirulent Kp with a pLVPK-like plasmid (an epidemic virulence plasmid) and a pKPI-6-like plasmid (an epidemic bla _IMP-6-bearing plasmid in Japan), suggesting the convergence of worldwide hypervirulence and epidemic AMR in Japan.

Klebsiella pneumoniae is a Gram-negative commensal bacterium that asymptomatically colonizes human mucosal membranes, such as the intestinal tract, but it is also a leading cause of health care- and community-associated infections. Additionally, multidrug-resistant K. pneumoniae has been continuously evolving, which significantly challenges the available chemotherapeutic treatment for its infections. K. pneumoniae produces several kinds of antimicrobial peptides known as bacteriocins, which have antibacterial activity against closely related species.