Genomic characterization of Staphylococcus aureus isolated from patients admitted to intensive care units of a tertiary care hospital: epidemiological risk of nasal carriage of virulent clone during admission.

We conducted a molecular epidemiological study of Staphylococcus aureus using whole-genome sequence data and clinical data of isolates from nasal swabs of patients admitted to the intensive care unit (ICU) of Hiroshima University hospital. The relationship between isolate genotypes and virulence factors, particularly for isolates that caused infectious diseases during ICU admission was compared with those that did not. The nasal carriage rates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in patients admitted to the ICU were 7.0% and 20.1%, respectively. The carriage rate of community-acquired (CA)-MRSA was 2.3%, accounting for 32.8% of all MRSA isolates. Whole-genome sequencing analysis of the MRSA isolates indicated that most, including CA-MRSA and healthcare-associated (HA)-MRSA, belonged to clonal complex (CC) 8 [sequence type (ST) 8] and SCCmec type IV. Furthermore, results for three disease foci (pneumonia, skin and soft tissue infection, and deep abscess) and the assessment of virulence factor genes associated with disease conditions [bacteremia, acute respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC), and septic shock] suggested that nasal colonization of S. aureus clones could represent a risk for patients within the ICU. Particularly, MRSA/J and MSSA/J may be more likely to cause deep abscess infection; ST764 may cause ventilation-associated pneumonia, hospital-acquired pneumonia and subsequent bacteremia, and ARDS, and tst-1-positive isolates may cause DIC onset.IMPORTANCENasal colonization of MRSA in patients admitted to the intensive care unit (ICU) may predict the development of MRSA infections. However, no bacteriological data are available to perform risk assessments for Staphylococcus aureus infection onset. In this single-center 2-year genomic surveillance study, we analyzed all S. aureus isolates from nasal swabs of patients admitted to the ICU and those from the blood or lesions of in-patients who developed infectious diseases in the ICU. Furthermore, we identified the virulent clones responsible for causing infectious diseases in the ICU. Herein, we report several virulent clones present in the nares that are predictive of invasive infections. This information may facilitate the design of preemptive strategies to identify and eradicate virulent MRSA strains, reducing nosocomial infections within the ICU.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Coordination of prophage and global regulator leads to high enterotoxin production in staphylococcal food poisoning-associated lineage.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP. IMPORTANCE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

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.

National genomic surveillance integrating standardized quantitative susceptibility testing clarifies antimicrobial resistance in Enterobacterales.

Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the blaCTX-M extended-spectrum ß-lactamase genes, blaCTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC ß-lactamase genes were blaCMY-2 and blaDHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of blaCMY-2 and blaDHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

Characterization of 29 newly isolated bacteriophages as a potential therapeutic agent against IMP-6-producing Klebsiella pneumoniae from clinical specimens.

Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria globally. Phage therapy has emerged as an effective strategy for the treatment of CPE infections. In western Japan, the rise of Klebsiella pneumoniae strains harboring the pKPI-6 plasmid encoding bla IMP-6 is of increasing concern. To address this challenge, we isolated 29 phages from Japanese sewage, specifically targeting 31 K. pneumoniae strains and one Escherichia coli strain harboring the pKPI-6 plasmid. Electron microscopy analysis revealed that among the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) phages belonged to myovirus, siphovirus, and podovirus morphotypes, respectively. Host range analysis showed that 18 Slopekvirus strains within the isolated phages infected 25-26 K. pneumoniae strains, indicating that most of the isolated phages have a broad host range. Notably, K. pneumoniae strain Kp21 was exclusively susceptible to phage øKp_21, whereas Kp22 exhibited susceptibility to over 20 phages. Upon administering a phage cocktail composed of 10 phages, we observed delayed emergence of phage-resistant bacteria in Kp21 but not in Kp22. Intriguingly, phage-resistant Kp21 exhibited heightened sensitivity to other bacteriophages, indicating a "trade-off" for resistance to phage øKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain prevalent in Japan, underscoring the potential of a well-designed phage cocktail in mitigating the occurrence of phage-resistant bacteria. IMPORTANCE The emergence of Klebsiella pneumoniae harboring the bla IMP-6 plasmid poses an escalating threat in Japan. In this study, we found 29 newly isolated bacteriophages that infect K. pneumoniae strains carrying the pKPI-6 plasmid from clinical settings in western Japan. Our phages exhibited a broad host range. We applied a phage cocktail treatment composed of 10 phages against two host strains, Kp21 and Kp22, which displayed varying phage susceptibility patterns. Although the phage cocktail delayed the emergence of phage-resistant Kp21, it was unable to hinder the emergence of phage-resistant Kp22. Moreover, the phage-resistant Kp21 became sensitive to other phages that were originally non-infective to the wild-type Kp21 strains. Our study highlights the potential of a well-tailored phage cocktail in reducing the occurrence of phage-resistant bacteria.

Comprehensive Genomic Characterization of Staphylococcus aureus Isolated from Atopic Dermatitis Patients in Japan: Correlations with Disease Severity, Eruption Type, and Anatomical Site.

Atopic dermatitis (AD) shows frequent recurrence. Staphylococcus aureus is the primary microbial component in AD and is associated with disease activity. However, traditional typing methods have failed to characterize virulent AD isolates at the clone level. We conducted a comprehensive genomic characterization of S. aureus strains isolated from the skin of AD patients and healthy donors, comparing the whole-genome sequences of the 261 isolates with anatomical and lesional (AD-A)/nonlesional (AD-NL)/healthy sites, eruption types, clinical scores, virulence, and antimicrobial resistance gene repertoires in Japan. Sequence type (ST) diversity was lost with worsening disease activity; ST188 was the most frequently detected ST in AD-A and had the strongest correlation with AD according to the culture rate and proportion with worsening disease activity. ST188 and ST20 isolates inhabited all skin conditions, with significantly higher proportions in AD skin than in healthy skin. ST8, ST15, and ST5 proportions were equivalent for all skin conditions; ST30 was detected only in healthy skin; and ST12 was detected only in AD skin. ST97 detected in AD-A and healthy skin was clearly branched into two subclades, designated ST97A and ST97H. A comparison of two genomes led to the discovery that only ST97A possessed the complete trp operon, enabling bacterial survival without exogenous tryptophan (Trp) on AD skin, where the Trp level was significantly reduced. Primary STs showing an AD skin inhabitation trend (ST188, ST97A, ST20, and ST12) were all trp operon positive. The predominant clones (ST188 and ST97) possessed almost no enterotoxin genes, no mecA gene, and few other antimicrobial resistance genes, different from the trend observed in Europe/North America. IMPORTANCE While Staphylococcus aureus is a member of the normal human skin flora, its strong association with the onset of atopic dermatitis (AD) has been suggested. However, previous studies failed to assign specific clones relevant to disease activities. Enterotoxins produced by S. aureus have been suggested to aggravate and exacerbate the inflammation of AD skin, but their role remains ambiguous. We conducted a nuanced comprehensive characterization of isolates from AD patients and healthy donors, comparing the whole-genome sequences of the isolates with anatomical and lesional/nonlesional/healthy sites, eruption types, clinical scores, virulence, and antimicrobial resistance gene repertoires in Japan. We demonstrate that specific clones are associated with disease severity and clinical manifestations, and the dominant clones are devoid of enterotoxin genes and antimicrobial resistance genes. These findings undermine the established notion of the pathophysiological function of S. aureus associated with AD and introduce a new concept of S. aureus colonization in AD.

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.

Diversity and Standard Nomenclature of Staphylococcus aureus Hyaluronate Lyases HysA and HysB.

Bacterial hyaluronate lyases (Hys) are enzymes that degrade hyaluronic acid in their host and are known to contribute to the pathogenesis of several illnesses. The first two identified Hys genes in Staphylococcus aureus were registered as hysA1 and hysA2. However, their annotations have been mistakenly reversed in some registered assembly data, and different abbreviations (hysA and hysB) in some reports complicates comparative analysis of Hys proteins. We investigated the hys loci of S. aureus genome sequences registered in public databases, analyzed the homology, and defined hysA as hys located in the core genome surrounded by a lactose metabolic operon and a ribosomal protein cluster present in almost all strains and hysB as that located on the genomic island νSaß of the accessory genome. Homology analysis of the amino acid sequences of HysA and HysB revealed that they are conserved among clonal complex (CC) groups with a few exceptions. Thus, we propose a new nomenclature for S. aureus Hys subtypes: HysACC*** for HysA and HysBCC*** for HysB, with the asterisks representing the clonal complex number of the S. aureus strain producing the Hys subtype. The application of this proposed nomenclature will facilitate the intuitive, straightforward, and unambiguous designation of Hys subtypes and contribute to enhancing comparative studies in this regard. IMPORTANCE Numerous whole-genome sequence data for Staphylococcus aureus harboring two hyaluronate lyase (Hys) genes have been registered. However, the assigned gene names for hysA1 and hysA2 are incorrect in some assembled data, and in some cases, the genes are annotated differently as hysA and hysB. This creates confusion with respect to the nomenclature of Hys subtypes and complicates analysis involving Hys. In this study, we compared the homology of Hys subtypes and observed that to some extent, their amino acid sequences are conserved in each clonal complex group. Hys has been implicated as an important virulence factor, but relative sequence heterogeneity among S. aureus clones raises the question of whether Hys activities are different among these clones. Our proposed Hys nomenclature will facilitate comparison of the virulence of Hys, as well as discussions of the subject.

Primary bacterial intercostal pyomyositis diagnosis: A case report.

RATIONALE: Pyomyositis is a microbial infection of the muscles and contributes to local abscess formation. Staphylococcus aureus frequently causes pyomyositis; however, transient bacteremia hinders positive blood cultures and needle aspiration does not yield pus, especially at the early disease stage. Therefore, identifying the pathogen is challenging, even if bacterial pyomyositis is suspected. Herein, we report a case of primary pyomyositis in an immunocompetent individual, with the identification of S aureus by repeated blood cultures. PATIENT CONCERNS: A 21-year-old healthy man presented with fever and pain from the left chest to the shoulder during motion. Physical examination revealed tenderness in the left chest wall that was focused on the subclavicular area. Ultrasonography showed soft tissue thickening around the intercostal muscles, and magnetic resonance imaging with short-tau inversion recovery showed hyperintensity at the same site. Oral nonsteroidal anti-inflammatory drugs for suspected virus-induced epidemic myalgia did not improve the patient's symptoms. Repeated blood cultures on days 0 and 8 were sterile. In contrast, inflammation of the soft tissue around the intercostal muscle was extended on ultrasonography. DIAGNOSES: The blood culture on day 15 was positive, revealing methicillin-susceptible S aureus JARB-OU2579 isolates, and the patient was treated with intravenous cefazolin. INTERVENTIONS: Computed tomography-guided needle aspiration from the soft tissue around the intercostal muscle without abscess formation was performed on day 17, and the culture revealed the same clone of S aureus. OUTCOMES: The patient was diagnosed with S aureus-induced primary intercostal pyomyositis and was successfully treated with intravenous cefazolin for 2 weeks followed by oral cephalexin for 6 weeks. LESSONS: The pyomyositis-causing pathogen can be identified by repeated blood cultures even when pyomyositis is non-purulent but suspected based on physical examination, ultrasonography, and magnetic resonance imaging findings.

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.

A Fatal Case of Disseminated Infection Caused by Community-Associated Methicillin-Resistant Staphylococcus aureus USA300 Clone.

Methicillin-resistant Staphylococcus aureus (MRSA) USA300 is a representative community-associated MRSA (CA-MRSA) clone worldwide. Herein, we report the case of a patient with USA300 clone infection who could not be salvaged. A 25-year-old man who had sex with men presented with symptoms including fever persisting for one week and skin lesions located on the buttocks. Computed tomography imaging showed multiple nodules and consolidations, especially in the peripheral lung fields, right iliac vein thrombosis, and pyogenic myositis of medial thighs bilaterally. Blood cultures revealed MRSA bacteremia. The patient's condition deteriorated rapidly, complicated by acute respiratory distress syndrome and infective endocarditis. Despite the intubation on the 6th hospital day, he died on the 9th day. Multilocus sequence typing of this patient's MRSA strain revealed sequence type 8 with a staphylococcal cassette chromosome of mec type IVa, Panton-Valentine leukocidin gene, and the arginine catabolic mobile element, indicating presence of the USA300 clone. Patients with CA-MRSA skin lesions presenting with furuncles or carbuncles on the lower body are at a higher risk of severe disease. The patient's background, appearance, and location of skin lesions are critical for the early diagnosis of severe CA-MRSA infection.

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.

New Molecular Mechanism of Superbiofilm Elaboration in a Staphylococcus aureus Clinical Strain.

Previously, we reported a novel regulator of biofilm (rob) with a nonsense mutation in the superbiofilm-elaborating strain JP080. Intriguingly, the complementation of JP080 with wild-type rob did not completely abolish its superbiofilm-elaborating phenotype. Therefore, we searched for other possible mutation(s) using complete genome sequence data and found a missense mutation in the gene icaR, which altered its 35th amino acid (Ala35Thr). To further study the mechanism of superbiofilm elaboration in JP080, we reconstructed the same mutations of rob and icaR in the strain FK300 and analyzed the phenotypes. The mutation of rob (A331T) increased biofilm elaboration, as previously demonstrated; similarly, an icaR mutation increased poly-N-acetylglucosamine and biofilm production in strain FK300. Furthermore, our analyses indicated that the double mutant of rob and icaR produced significantly more biofilms than the single mutants. Additionally, gel shift analysis revealed that the icaR from JP080 lost its ability to bind to the ica promoter region. These findings suggest that the icaR mutation in JP080 may result in a nonfunctional protein. We compared ica operon expression in an icaR single mutant, rob single mutant, and rob and icaR double mutant to the wild type. The rob and icaR mutants showed increased ica operon transcription by approximately 19- and 79-fold, respectively. However, the rob and icaR double mutant showed an approximately 350-fold increase, indicating the synergistic effects of icaR and rob on JP080 biofilm elaboration. Consequently, we concluded that the double mutations rob and icaR synergistically increased ica operon transcription, resulting in a superbiofilm phenotype in Staphylococcus aureus. IMPORTANCE Poly-N-acetylglucosamine (PNAG) is a major component of S. aureus biofilm. PNAG production is mediated by the products of four genes, icaADBC encoded in the ica operon, and the major negative regulator of this operon is IcaR encoded just upstream of icaADBC. Previously, we reported another negative regulator, Rob, through gene expression analysis of clinically isolated superbiofilm-elaborating strain JP080. The rob gene is encoded at different loci distant from the ica operon. Here, we report that JP080 also carried a mutation in icaR and demonstrated that IcaR and Rob synergistically regulate PNAG production. We successfully reconstructed these mutations in a wild type, and the double mutant resulted in superbiofilm-elaborating phenotype. We clearly show that loss of function of both IcaR and Rob is the very reason that JP080 is showing the superbiofilm-elaborating phenotype. This study clearly demonstrated there are at least two independent regulators synergistically fine-tuning PNAG production and suggested the complex regulatory mechanism of biofilm production.

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.

First Isolation of Vancomycin-Resistant Enterococcus faecium Carrying Plasmid-Borne vanD1.

Vancomycin-resistant Enterococcus faecium carrying the vanD1 gene on plasmid pEF-D was isolated from a fecal sample of a hospitalized patient in Japan. The strain JH5687 showed moderate resistance to vancomycin (MIC, 16 µg/mL) but remained susceptible to teicoplanin (MIC, 1 µg/mL). The backbone gene organization of pEF-D was highly homologous to that of conjugative plasmid pMG1 or pHTß. The calculated conjugation frequency of JH5687 was 10-4 to 10-5 per donor cell.

Complete genome sequences of two Escherichia coli clinical isolates from Egypt carrying mcr-1 on IncP and IncX4 plasmids.

Colistin is a last-resort antibiotic used in the treatment of multidrug resistant Gram-negative bacteria. However, the activity and efficacy of colistin has been compromised by the worldwide spread of the mobile colistin resistance genes (mcr-1 to mcr-10). In this study, two clinical Escherichia coli strains, named EcCAI51, and EcCAI73, harbored mcr-1, showed multidrug-resistant phenotypes (with colistin MIC = 4 µg/ml), and belonged to phylogroup D: multilocus sequence type 1011 (ST1011) and phylogroup A: ST744, respectively. Findings revealed the existence of mcr-1 gene on two conjugable plasmids, pAMS-51-MCR1 (∼122 kb IncP) and pAMS-73-MCR1 (∼33 kb IncX4), in EcCAI51, and EcCAI73, respectively. The mcr-1-pap2 element was detected in the two plasmids. Additionally, the composite transposon (ISApl1-IS5D-pap2-mcr-1-ISApl1) was identified only in pAMS-51-MCR1 suggesting the potential for horizontal gene transfer. The two strains carried from 16 to 18 different multiple acquired antimicrobial resistance genes (ARGs). Additionally, two different multireplicon virulence plasmids (∼117 kb pAMS-51-Vr and ∼226 kb pAMS-73-Vr) carrying the sit operon, the Salmochelin siderophore iroBCDE operon and other several virulence genes were identified from the two strains. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of EcCAI73, and EcCAI51 with global E. coli lineages at HC levels of 50 (HC50) to 100 (HC100) core genome allelic differences. To the best of our knowledge, this study presented the first complete genomic sequences of mcr-1-carrying IncP and IncX4 plasmids from human clinical E. coli isolates in Egypt. In addition, the study illustrated the mcr-1 broad dissemination in diverse plasmids and dissimilar E. coli clones.