The two-component regulatory systems GraRS and SrrAB mediate Staphylococcus aureus susceptibility to Pep5 produced by clinical isolate of Staphylococcus epidermidis.

Staphylococcus aureus is a common bacterium on the skin and in the nose that sometimes causes severe illness. Bacteriocins, antimicrobial peptides, or proteins produced by bacteria are candidates for the treatment of S. aureus infection. In this study, we found that a clinical Staphylococcus epidermidis strain, KSE112, produced the lantibiotic Pep5, which showed anti-S. aureus activity. The complete nucleotide sequence of the Pep5-encoding plasmid was determined. Several S. aureus two-component regulatory systems (TCSs) are known to be involved in bacteriocin susceptibility. Therefore, susceptibility tests were performed using TCS-inactivated S. aureus mutants to determine which TCS is responsible for Pep5 susceptibility; the ΔgraRS mutant exhibited increased susceptibility to Pep5, while the ΔsrrAB mutant exhibited decreased susceptibility. GraRS is known to regulate dltABCD and mprF in concert with vraFG, and Pep5 susceptibility was significantly increased in the ΔdltABCD, ΔmprF, and ΔvraFG mutants. Regarding the ΔsrrAB mutant, cross-resistance to aminoglycosides was observed. As aminoglycoside activity is known to be affected by aerobic respiration, we focused on qoxABCD and cydAB, which are quinol oxidase genes that are necessary for aerobic respiration and have downregulated the expression in the ΔsrrAB mutant. We constructed ΔqoxABCD and ΔcydAB mutants and found that qoxABCD inactivation decreased susceptibility to Pep5 and aminoglycosides. These results indicate that reduced aerobic respiration due to the reduced qoxABCD expression in the ΔsrrAB mutant decreased Pep5 activity.IMPORTANCEThe emergence of drug-resistant bacteria, including MRSA, is a severe health problem worldwide. Thus, the development of novel antimicrobial agents, including bacteriocins, is needed. In this report, we found a Pep5-producing strain with anti-S. aureus activity. We determined the complete sequence of the Pep5-encoding plasmid for the first time. However, in S. aureus, GraRS and its effectors conferred decreased susceptibility to Pep5. We also revealed that another TCS, SrrAB, affects susceptibility Pep5 and other lantibiotics by controlling aerobic respiration. In our study, we investigated the efficacy of Pep5 against S. aureus and other Gram-positive bacteria and revealed that respiratory constancy regulated by TCS is required for the antimicrobial activity of nisin, nukacin, and Pep5. These findings provide important information for the clinical application of bacteriocins and suggest that they have different properties among similar pore-forming lantibiotics.

Antibiotic susceptibility and genome analysis of Enterococcus species isolated from inpatients in one hospital with no apparent outbreak of vancomycin-resistant Enterococcus in Japan.

To prevent nosocomial infection, it is important to screen for potential vancomycin-resistant Enterococcus (VRE) among patients. In this study, we analyzed enterococcal isolates from inpatients in one hospital without any apparent outbreak of VRE. Enterococcal isolates were collected from inpatients at Hiroshima University Hospital from April 1 to June 30, 2021 using selective medium for Enterococci. Multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. A total of 164 isolates, including Enterococcus faecium (41 isolates), Enterococcus faecalis (80 isolates), Enterococcus raffinosus (11 isolates), Enterococcus casseliflavus (nine isolates), Enterococcus avium (12 isolates), Enterococcus lactis (eight isolates), Enterococcus gallinarum (two isolates), and Enterococcus malodoratus (one isolate), were analyzed. We found one vanA-positive E. faecium, which was already informed when the patient was transferred to the hospital, nine vanC-positive E. casseliflavus, and two vanC-positive E. gallinarum. E. faecium isolates showed resistance to ampicillin (95.1%), imipenem (95.1%), and levofloxacin (87.8%), and E. faecalis isolates showed resistance to minocycline (49.4%). Ampicillin- and levofloxacin-resistant E. faecium had multiple mutations in penicillin-binding protein 5 (PBP5) (39/39 isolates) and ParC/GyrA (21/36 isolates), respectively. E. raffinosus showed resistance to ampicillin (81.8%), imipenem (45.5%), and levofloxacin (45.5%), and E. lactis showed resistance to ampicillin (37.5%) and imipenem (50.0%). The linezolid resistance genes optrA and cfr(B) were found only in one isolate of E. faecalis and E. raffinosus, respectively. This study, showing the status of enterococci infection in hospitalized patients, is one of the important information when considering nosocomial infection control of VRE.

Ursoricin, a bacteriocin of Streptococcus ursoris, has potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.

The emergence of drug-resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has increased the need to discover novel antimicrobial agents that are effective against these species. Here, we describe the identification and purification of the mutacin BHT-B-like gene locus and bacteriocin peptide from Streptococcus ursoris, which is closely related to Streptococcus ratti; hence, we named this bacteriocin ursoricin. Ursoricin is a cationic, chromosome-encoded peptide that has potent antimicrobial effects against Gram-positive pathogens, including MRSA and VRE, with minimum inhibitory concentrations in the micromolar range. Ursoricin also inhibits the biofilm formation of high biofilm-forming S. aureus. Antibacterial activity was retained after treatment at 100°C for 60 min at a pH range of 3-9 and was partially reduced by treatment with proteinase K for 2 h (63% residual activity). The potent anti-MRSA, anti-VRE, and antibiofilm effects of ursoricin suggest that it is a possible candidate for the treatment of MRSA, VRE, and biofilm-associated infections. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has posed a significant public health threat and economic burdens that make the identification and development of novel antimicrobial agents urgent. Bacteriocins are promising new agents that exhibit antibacterial activity against a wide range of human pathogens. In this study, we report that the bacteriocin produced by Streptococcus ursoris showed good antibacterial activity against a wide range of Staphylococcus aureus and enterococcus strains, particularly methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and high biofilm-forming S. aureus. Interestingly, this bacteriocin had a stronger effect on S. aureus than on Staphylococcus epidermidis, which is a major commensal bacterium in human skin; this result is important when considering the disturbance of bacterial flora, especially on the skin, mediated by the application of antibacterial agents.

Triterpenoid saponin from Panax ginseng increases the sensitivity of methicillin-resistant Staphylococcus aureus to ß-lactam and aminoglycoside antibiotics.

The triterpenoid saponins, ginsenosides, are the major bioactive compound of red ginseng and can exert various physiological activities. In the present study, we examined whether red ginseng extract (RGE) exerts antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). RGE had no bactericidal activity, at least in the range of dissolvable concentration. However, RGE reduced 0.03-0.25-fold the minimum inhibitory concentration (MIC) values of ß-lactam antibiotics (oxacillin, ampicillin, carbenicillin, and cefazolin) and aminoglycoside antibiotics (kanamycin and gentamicin) against the two laboratory strains of MRSA. Moreover, the fractional inhibitory concentration index indicated the synergistic activity of RGE with each of the antibiotics. RGE also increased the kanamycin sensitivity of 15 MRSA strains isolated from human volunteers and increased the ampicillin sensitivity of five MRSA strains isolated from dairy cows diagnosed with bovine mastitis. In contrast, RGE did not alter the MIC values of fosfomycin, tetracycline, and erythromycin, suggesting that RGE acts selectively. In contrast, Triton X-100, which was reported to reduce the MIC value of ß-lactam antibiotics to MRSA by increasing membrane permeability, reduced the MIC values of fosfomycin and tetracycline. These results indicate that RGE increases the bactericidal effect of antibiotics via a mechanism different from that used by Triton X-100. We found that ginsenoside Rg3 (Rg3), a component of RGE, was an essential compound that exhibits synergy activity with antibiotics. Furthermore, the non-natural compound K, which contains a common protopanaxadiol aglycon moiety with Rg3, also showed synergistic activity with antibiotics. Thus, Rg3 and compound K are potentially new antibiotic adjuvants against MRSA.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant organism that is prevalent worldwide. Therefore, the research and development of new agents against MRSA are required. We first found that ginsenoside Rg3 (Rg3) in red ginseng, made from the roots of Panax ginseng C. A. Meyer, increased the sensitivity of ß-lactam antibiotics and aminoglycoside antibiotics to MRSA. Furthermore, we identified that compound K, an unnatural ginsenoside analog, also increased the sensitivity of antibiotics to MRSA, similar to Rg3. By contrast, neither Rg3 nor compound K increased the sensitivity of fosfomycin, tetracycline, and erythromycin to MRSA, suggesting that these act selectively. In the present study, the natural compound Rg3 and its structural isomer, compound K, are potentially new antibiotic adjuvants against MRSA. Currently, multiple antibiotics are used to treat MRSA, but the use of these adjuvants is expected to enable the treatment of MRSA with a single antibiotic and low concentrations of antibiotics.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

Oral and rectal colonization of methicillin-resistant Staphylococcus aureus in long-term care facility residents and their association with clinical status.

Staphylococcus aureus is a commensal bacterium in humans, but it sometimes causes opportunistic infectious diseases such as suppurative skin disease, pneumonia, and enteritis. Therefore, it is important to determine the prevalence of S. aureus and methicillin-resistant S. aureus (MRSA) in individuals, especially older adults. In this study, we investigated the prevalence of S. aureus and MRSA in the oral cavity and feces of residents in long-term care facilities (LTCFs). S. aureus was isolated from the oral cavity of 61/178 (34.3%) participants, including 28 MRSA-positive participants (15.7%), and from the feces of 35/127 (27.6%) participants, including 16 MRSA-positive participants (12.6%). S. aureus and MRSA were isolated from both sites in 19/127 individuals (15.0%) and 10/127 individuals (7.9%), respectively. Among 19 participants with S. aureus isolation from both sites, 17 participants showed the same sequence type (ST) type. Then, we analyzed the correlation of S. aureus and MRSA in the oral cavity and rectum with the participant's condition. S. aureus and MRSA positivity in the oral cavity was significantly related to tube feeding, while there was no correlation of rectal S. aureus/MRSA with any factors. Our findings regarding the oral inhabitation of MRSA and its risk factors indicate the importance of considering countermeasures against MRSA infection in LTCFs.

Isolation of Streptococcus mutans temperate bacteriophage with broad killing activity to S. mutans clinical isolates.

Bacteriophages are expected to be therapeutic agents against infectious diseases. Streptococcus mutans are involved in dental plaque formation related to dental caries and periodontitis. In S. mutans, lytic bacteriophages have been isolated previously, but the isolation of temperate bacteriophage has not been reported although their presence in the genome has been confirmed. Here, we report the isolation of temperate bacteriophage, φKSM96, from S. mutans. φKSM96 has a circular DNA 39,820 bp long and reveals Siphoviridae morphology. φKSM96 shows a broad range of susceptibility against S. mutans strains with different serotypes. By the addition of φKSM96, S. mutans growth and biofilm formation were significantly inhibited. In cocultures of S. mutans with other bacterial species, the proportion of S. mutans significantly decreased in the presence of φKSM96. In summary, φKSM96 shows selective anti-S. mutans activity. The isolation of temperate bacteriophage is important for future genetic manipulation to create more efficient bacteriophages.

Complete genome sequence of cfr(B)-carrying Enterococcus raffinosus isolated from bile in a patient in Japan.

OBJECTIVES: Linezolid is an antibiotic used to treat infectious diseases caused by vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Recently, Enterococcus Spp.-carrying mobile linezolid resistance genes were reported. Herein, we report the complete genome sequence of Enterococcus raffinosus JARB-HU0741, which was isolated from a bile sample of a patient in Japan on May 5, 2021, and carries a linezolid resistance gene, cfr(B). Nevertheless, this isolate was susceptible to linezolid. METHODS: Whole-genome sequencing was performed using HiSeq X FIVE (Illumina) and GridION (Oxford Nanopore Technologies). The sequence reads were assembled using Unicycler v0.4.8, and the complete genome was annotated using DFAST v1.2.18. Antimicrobial resistance genes were detected with Abricate v1.0.1, using the ResFinder database. The minimum inhibitory concentrations (MICs) were determined using broth microdilution and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: E. raffinosus JARB-HU0741 contained a 3 248 808-bp chromosome and a 1 156 277-bp megaplasmid. cfr(B) was present in the Tn6218-like transposon, which was inserted into a gene encoding a PRD domain-containing protein present in the megaplasmid, but the isolate was susceptible to linezolid (MIC, 0.5 µg/mL). The Tn6218-like transposon was similar to the Tn6218 of Clostridioides difficile Ox3196 and the Tn6218-like transposon of Enterococcus faecium UW11733; however, three genes encoding a topoisomerase, an S-adenosylmethionine-dependent methyltransferase, and a TetR family transcriptional regulator were present in the previous Tn6218- or Tn6218-like transposon. CONCLUSION: This is the first report of the complete genome sequence of E. raffinosus carrying cfr(B). E. raffinosus carrying cfr(B) without linezolid resistance poses a threat, as it could serve as a reservoir for mobile linezolid resistance genes.

Complete genome sequence of optrA-carrying Enterococcus faecalis isolated from open pus in a Japanese patient.

OBJECTIVES: The occurrence of linezolid resistance in enterococci has recently increased. Here, we report the genomic characterization of Enterococcus faecalis strain JARB-HU0796-isolated from the open pus of a patient in Hiroshima, Japan-which shows nonsusceptibility to linezolid (MIC of 4 µg/mL). METHODS: JARB-HU0796 whole-genome sequencing was performed using short-read sequencing with Illumina Hiseq X Five and long-read sequencing using GridION. These reads were collected using the assembly pipeline Unicycler and annotated with DFAST. Antimicrobial resistance genes were detected using the Abricate and ResFinder databases, and the sequence type identified using PubMLST. The antimicrobial susceptibility of JARB-HU0796 was determined with the Eiken dry-plate QH02 system. RESULTS: The JARB-HU0796 complete genome contained a circular chromosome (2 722 585 bp) and two circular plasmids (85 996 bp and 58 872 bp). The chromosome harbours the optrA gene, which confers resistance to oxazolidinones and phenicols. JARB-HU0796 showed nonsusceptibility to linezolid and multidrug resistance to other antibiotics. MLST analysis identified JARB-HU0796 as ST476, similar to the optrA-positive E. faecalis ST476 isolates from swine (South Korea, 2020) and pet food (Switzerland, 2022). The optrA region of JARB-HU0796 is nearly identical to that of ST476 E. faecalis strain TZ2, isolated from humans (China, 2013). CONCLUSIONS: To the best of our knowledge, this is the first report of the complete genome sequence of E. faecalis ST476 carrying optrA on a chromosome isolated from a patient in Japan. The strain may have originated in animals, suggesting that the organisms acquired resistance to linezolid because the optrA gene may be closely spread between animals and humans.

Comprehensive Analysis of Bacteriocins Produced by the Hypermucoviscous Klebsiella pneumoniae Species Complex.

Klebsiella pneumoniae produces several kinds of bacteriocins that have antimicrobial effects against closely related species, but few studies have comprehensively reported bacteriocin distribution among the Klebsiella population. In this study, we identified bacteriocin genes in 180 K. pneumoniae species complex genomes, including 170 hypermucoviscous isolates, and investigated the antibacterial activity against 50 strains, including antimicrobial-resistant organisms, belonging to multiple species, namely, Klebsiella spp., Escherichia coli, Pseudomonas spp., Acinetobacter spp., Enterobacter cloacae, Stenotrophomonas maltophilia, Chryseobacterium indologenes, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus mutans. Our study determined that 32.8% (59/180) of isolates carried at least one bacteriocin type. Different types of bacteriocin were usually present in different specific sequence types (STs); meanwhile, bacteriocins were not detected in certain STs. Microcin E492 was the most prevalent bacteriocin (14.4%), mostly in ST23 isolates, and displayed a wide spectrum of activity, including against Klebsiella spp., E. coli, Pseudomonas spp., and Acinetobacter spp. Cloacin-like bacteriocin was detected in 7.2% of strains, all of which were non-ST23 isolates, and exhibited inhibitory activity against closely related species, mainly Klebsiella spp. Klebicin B-like bacteriocin was detected at a rate of 9.4%, although 82.4% of these strains carried a disrupted bacteriocin gene, and an inhibitory effect could not be observed from the intact-gene-carrying isolates. Other bacteriocins, such as microcin S-like, microcin B17, and klebicin C-like, were detected at lower rates and had limited inhibitory activity. Our findings suggested that Klebsiella strains that carry different bacteriocin types may affect the composition of the surrounding bacterial community. IMPORTANCE 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. This work was the first comprehensive report of bacteriocin distribution among the hypermucoviscous K. pneumoniae species complex population and the inhibitory activity of each bacteriocin type against various species, including multidrug-resistant strains. Our findings provide a foundation for future studies on the K. pneumoniae species complex, including studies on the competition within the microflora and the potential applications of bacteriocins in treating multidrug-resistant bacteria.

Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates.

Clinical isolates of Clostridioides difficile sometimes exhibit multidrug resistance and cause diarrhea after antibiotic administration. Metronidazole and vancomycin are often used as therapeutic agents, but resistance to these antibiotics has been found clinically. Therefore, the development of alternative antimicrobial agents is needed. Nisin A, produced by Lactococcus lactis, has been demonstrated to be effective against C. difficile infection. In this study, we evaluated the susceptibility of 11 C. difficile clinical isolates to nisin A and found that they could be divided into 2 groups: high and low susceptibility. Since CprABC and DltDABC, which are responsible for nisin A efflux and cell surface charge, respectively, have been reported to be related to nisin A susceptibility, we investigated the expression of cprA and dltA among the 11 strains. cprA expression in all strains was induced by nisin A, but dltA expression was not. The expression levels of both genes did not correlate with nisin A susceptibility in these clinical isolates. To evaluate cell surface charge, we performed a cytochrome C binding assay and found no relationship between charge and nisin A susceptibility. Then, we determined the whole genome sequence of each clinical isolate and carried out phylogenetic analysis. The 11 isolates separated into two major clusters, which were consistent with the differences in nisin A susceptibility. Furthermore, we found common differences in several amino acids in the sequences of CprA, CprB, and CprC between the two clusters. Therefore, we speculated that the different amino acid sequences of CprABC might be related to nisin A susceptibility. In addition, C. difficile strains could be divided in the same two groups based on susceptibility to epidermin and mutacin III, which are structurally similar to nisin A. These results suggest that genotypic variations in C. difficile strains confer different susceptibilities to bacteriocins.

Disinfectant Susceptibility of Third-Generation-Cephalosporin/Carbapenem-Resistant Gram-Negative Bacteria Isolated from the Oral Cavity of Residents of Long-Term-Care Facilities.

We have recently reported the isolation of third-generation-cephalosporin-resistant Gram-negative bacteria from the oral cavity of residents of a long-term-care facility (LTCF). Since disinfectants are often used in the oral cavity, it is important to investigate the disinfectant susceptibility of oral bacteria. Here, we evaluated the susceptibilities of Gram-negative antimicrobial-resistant bacteria (GN-ARB), including Pseudomonas, Acinetobacter, and Enterobacteriaceae, obtained from the oral cavity of residents of LTCFs to povidone-iodine (PVPI), cetylpyridinium chloride (CPC), benzalkonium chloride (BZK), and chlorhexidine chloride (CHX). We also evaluated the susceptibilities of isolates from the rectum to the same agents to compare the susceptibility profiles of oral and rectal isolates. Next, we investigated the relationship between their susceptibility and disinfectant resistance genes delineated by whole-genome sequencing of the isolates. Additionally, we evaluated the correlation between disinfectant-resistant GN-ARB and clinical information. In oral GN-ARB, the MIC of PVPI showed almost identical values across isolates, while the MICs of CPC, BZK, and CHX showed a wide range of variation among species/strains. In particular, Pseudomonas aeruginosa exhibited high-level resistance to CPC and BZK. The disinfectant susceptibility of rectal GN-ARB showed a tendency similar to that of oral GN-ARB. The presence of qacEΔ1 was correlated with CPC/BZK resistance in P. aeruginosa, while other species exhibited no correlation between qacEΔ1 and resistance. Multiple analyses showed the correlation between the presence of CPC-resistant bacteria in the oral cavity and tube feeding. In conclusion, we found that some oral GN-ARB isolates showed resistance to not only antibiotics but also disinfectants. IMPORTANCE Antibiotic-resistant bacteria (ARB) are becoming a serious concern worldwide. We previously reported the isolation of third-generation-cephalosporin-resistant Gram-negative bacteria from the oral cavity of residents of a long-term-care facility (LTCF). To prevent infection with ARB in hospitals and eldercare facilities, we must pay more attention to the use of not only antibiotics but also disinfectants. However, the effect of disinfectants on ARB is unclear. In this study, we evaluated the susceptibility of Gram-negative ARB (GN-ARB) from the oral cavity of residents of LTCFs to some disinfectants that are often used for the oral cavity; we found that some isolates showed resistance to several disinfectants. This is the first comprehensive analysis of the disinfectant susceptibility of oral GN-ARB. These results provide some important information for infection control and suggest that disinfectants should be applied carefully.

Oral and Rectal Colonization by Antimicrobial-Resistant Gram-Negative Bacteria and Their Association with Death among Residents of Long-Term Care Facilities: A Prospective, Multicenter, Observational, Cohort Study.

INTRODUCTION: The prevalence of antimicrobial-resistant bacteria (ARB) in long-term care facilities (LTCFs) remains unclear. Furthermore, the effect of ARB colonization on the clinical outcomes of LTCF residents has not been explored. METHODS: We conducted a prospective multicenter cohort study and investigated the residents (N = 178) of six Japanese LTCFs (three Welfare Facilities for the Elderly Requiring Long-term Care and three Geriatric Health Service Facilities) for oral and rectal carriage of ARB. The clinical outcomes of the residents were evaluated based on isolating bacterial strains and subjecting them to whole-genome sequencing. RESULTS: Of the 178 participants, 32 belonging to Geriatric Health Service Facilities with no information on their clinical outcome were excluded, and the remaining 146 were followed up for at most 21 months. Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales and Pseudomonas aeruginosa were detected in 42.7% (n = 76) and 2.8% (n = 5) of the rectal swabs and 5.6% (n = 10) and 3.4% (n = 6) of the oral swabs, respectively. Detection of ARB in the oral and rectal cavities showed remarkable association with enteral nutrition. Further, P. aeruginosa was significantly associated with an increase in mortality of the residents, but there were not significant association between ESBL-producing Enterobacterales and mortality. Core-genome phylogeny of P. aeruginosa revealed a wide-spread distribution of the isolated strains across the phylogeny, which included a cluster of ST235 strains with substantially higher biofilm formation ability than the other isolated P. aeruginosa strains. DISCUSSION/CONCLUSION: This study is the first to investigate the carriage of both oral and rectal ARB, genomic relatedness and determinants of antimicrobial resistance in isolated strains, and clinical outcomes of LTCF residents. Our study provides the first direct evidence for the burden of antimicrobial resistance in LTCFs.

Periapical lesion following Cnm-positive Streptococcus mutans pulp infection worsens cerebral hemorrhage onset in an SHRSP rat model.

Cerebral hemorrhage severely affects the daily life of affected individuals. Streptococcus mutans and its adhesion factor Cnm increase the adverse effects of cerebral hemorrhages. However, the mechanism by which Cnm-positive bacteria migrate from apical lesions to cerebral hemorrhage sites is unclear. Therefore, we established an S. mutans-infected apical lesion in a rat model of hypertension and investigated the neurological symptoms associated with cerebral hemorrhage. Eighteen 12-week-old stroke-prone spontaneously hypertensive rats were randomly divided into three groups, i.e. the no infection (control), dental infection with S. mutans KSM153 wild type (Cnm positive), and KSM153 Δcnm groups. Immunofluorescent staining was performed to visualize S. mutans protein. Serum interleukin-1ß levels were measured. The adhesion of S. mutans to the extracellular matrix and human fibroblast cells was also analyzed. Serum antibody titers against S. mutans were comparable between Cnm positive and knockout mutants. However, 3-10 days post-infection, neurological symptom scores and cerebral hemorrhage scores were higher in Cnm-positive rats than in knockout mutants. The localization of S. mutans-derived protein was observed in the vicinity of disrupted blood vessels. Serum interleukin-1ß levels significantly increased post-KSM153 WT infection. Cnm-positive S. mutans clinical isolates showed increased adhesion to the extracellular matrix, human dental pulp cells, and human umbilical vein endothelial cells compared with the Cnm-negative S. mutans isolates. In conclusion, Cnm-positive bacteria colonize the apical lesion site using the extracellular matrix as a foothold and affect cerebral hemorrhage via the bloodstream.

Efficiency of Antimicrobial Peptides Against Multidrug-Resistant Staphylococcal Pathogens.

Antibiotics play a vital role in saving millions of lives from fatal infections; however, the inappropriate use of antibiotics has led to the emergence and propagation of drug resistance worldwide. Multidrug-resistant bacteria represent a significant challenge to treating infections due to the limitation of available antibiotics, necessitating the investigation of alternative treatments for combating these superbugs. Under such circumstances, antimicrobial peptides (AMPs), including human-derived AMPs and bacteria-derived AMPs (so-called bacteriocins), are considered potential therapeutic drugs owing to their high efficacy against infectious bacteria and the poor ability of these microorganisms to develop resistance to them. Several staphylococcal species including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus are commensal bacteria and known to cause many opportunistic infectious diseases. Methicillin-resistant Staphylococci, especially methicillin-resistant S. aureus (MRSA), are of particular concern among the critical multidrug-resistant infectious Gram-positive pathogens. Within the past decade, studies have reported promising AMPs that are effective against MRSA and other methicillin-resistant Staphylococci. This review discusses the sources and mechanisms of AMPs against staphylococcal species, as well as their potential to become chemotherapies for clinical infections caused by multidrug-resistant staphylococci.

Gene Rearrangement and Modification of Immunity Factors Are Correlated with the Insertion of Bacteriocin Cassettes in Streptococcus mutans.

Bacteriocins have been applied in the food industries and have become promising next-generation antibiotics. Some bacteria produce bacteriocins and possess immunity factors for self-protection. Nisin A, a bacteriocin produced by Lactococcus lactis, shows broad-spectrum activity. However, the evolution and cross-resistance ability of the immunity factors in some species results in reduced susceptibility to bacteriocins. Here, we investigated the elements responsible for nisin A resistance in Streptococcus mutans and their contribution to mutacins (bacteriocins produced by S. mutans) resistance. We classified the nisin A-resistance regions into six types based on the different combinations of 3 immunity factors, mutFEG, nsrX, and mutHIJ, and the presence of mutacin synthesis operon upstream of mutF. Data shows that NsrX effectively acts against nisin A but not mutacins, while the newly identified ABC transporter MutHIJ acts against three mutacins but not nisin A. Three types of MutFEG are identified based on their amino acid sequences: α (in Nsr-types C and D-I), ß (in Nsr-types B and d-III), and γ (in Nsr-type E). MutFEG-α strongly contributes to mutacin I resistance, while MutFEG-ß and MutFEG-γ strongly contribute to mutacin III, IIIb, and nisin A resistance. Additionally, mutFEG-like structures could be found in various streptococcal species isolated from the oral cavity of humans, chimpanzees, monkeys, bears, and hamsters. Our findings suggest that immunity factors rearrange and adapt in the presence of bacteriocins and could be transferred among closely related species, thus altering the bacterial competition within the microflora. IMPORTANCE Streptococcus mutans is an important organism of oral microbiota and associated with dental caries and systemic diseases such as stroke and endocarditis. They produce bacteriocins known as mutacins to compete with other oral bacteria and possess immune factors for self-protection. We found that the nisin A and mutacins resistance patterns correlated with the immunity components and MutFEG variants, and the genetic difference was driven by the insertion of mutacin-synthesis cassettes. Our study provides an understanding of the development of bacteriocin resistance among streptococcal species, which may alter the bacterial interaction and ecology within the oral biofilm.

Complete sequences of epidermin and nukacin encoding plasmids from oral-derived Staphylococcus epidermidis and their antibacterial activity.

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.

Comprehensive characterization of sortase A-dependent surface proteins in Streptococcus mutans.

Streptococcus mutans, a cariogenic pathogen, adheres to the tooth surface and forms a biofilm. Bacterial cell surface proteins are associated with adherence to substrates. Sortase A (SrtA) mediates the localization of proteins with an LPXTG motif-containing proteins to the cell surface by covalent binding to peptidoglycan. In S. mutans UA159, six SrtA-dependent proteins, SpaP, WapA, WapE, DexA, FruA, and GbpC, were identified. Although some of these proteins were characterized, a comprehensive analysis of the six proteins has not been reported. In this study, we constructed mutants deficient in each of these proteins and the SrtA-deficient mutant. The SrtA-deficient mutant showed drastically decreased binding to salivary components, biofilm formation, bacterial coaggregation activity, hydrophobicity, and cellular matrix binding (collagen type I, fibronectin, and laminin). The SpaP-deficient mutant showed significantly reduced binding to salivary components and partially increased coaggregation with Porphyromonas gingivalis, and decreased hydrophobicity, and collagen binding. The WapA-deficient mutant showed slightly decreased coaggregation with Fusobacterium nucleatum. Although the SrtA-deficient mutant showed drastically altered phenotypes, all SrtA-dependent protein-deficient mutants, except the SpaP-deficient mutant, did not show considerable alterations in binding to salivary components. These results indicate that the six proteins may coordinately contribute to these activities. In addition, using genomic data of 125 S. mutans strains, the amino acid sequences of each surface protein were compared and many variations were found among strains, which may affect the phenotype of cell surface proteins in S. mutans.

Antibacterial Peptides Resistance in Staphylococcus aureus: Various Mechanisms and the Association with Pathogenicity.

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.

Comprehensive analysis of bacteriocins in Streptococcus mutans.

Streptococcus mutans produces bacteriocins that show antibacterial activity against several bacteria. However, comprehensive analysis of these bacteriocins has not been well done. In this study, we isolated 125 S. mutans strains from volunteers and determined their whole genome sequence. Based on the genome analysis, the distribution of each bacteriocin gene (mutacins I-IV, K8 and Smb) was investigated. We found 17, 5, and 2 strains showing 100% matches with mutacin I, mutacin II and mutacin III, respectively. Five mutacin III-positive strains had 2 mismatches compared to mature mutacin III. In 67 mutacin IV-positive strains, 38 strains showed 100% match with mutacin IV, while 29 strains showed some variations. In 23 mutacin K8- and 32 mutacin Smb-positive strains, all except one mutacin K8-positive strain showed 100% match with the mature peptides. Among 125 strains, 84 (65.1%), 26 (20.2%), and 5 (3.9%) strains were positive for one, two and three bacteriocin genes, respectively. Then, the antibacterial activity against oral streptococci and other oral bacterial species was investigated by using bacteriocin gene single-positive strains. Each bacteriocin gene-positive strain showed a different pattern of antibacterial activity. These results speculate that individual S. mutans strains may affect the bacterial composition of dental plaques.