Plasmid-mediated acquisition and chromosomal integration of blaCTX-M-14 in a subclade of Escherichia coli ST131-H30 clade C1.

Escherichia coli ST131 is a multidrug-resistant lineage associated with the global spread of extended-spectrum ß-lactamase-producing organisms. Particularly, ST131 clade C1 is the most predominant clade in Japan, harboring blaCTX-M-14 at a high frequency. However, the process of resistance gene acquisition and spread remains unclear. Here, we performed whole-genome sequencing of 19 E. coli strains belonging to 12 STs and 12 fimH types collected between 1997 and 2016. Additionally, we analyzed the full-length genome sequences of 96 ST131-H30 clade C0 and C1 strains, including those obtained from this study and those registered in public databases, to understand how ST131 clade C1 acquired and spread blaCTX-M-14. We detected conjugative IncFII plasmids and IncB/O/K/Z plasmids carrying blaCTX-M-14 in diverse genetic lineages of E. coli strains from the 1990s to the 2010s, suggesting that these plasmids played an important role in the spread of blaCTX-M-14. Molecular phylogenetic and molecular clock analyses of the 96 ST131-H30 clade C0 and C1 strains identified 8 subclades. Strains harboring blaCTX-M-14 were clustered in subclades 4 and 5, and it was inferred that clade C1 acquired blaCTX-M-14 around 1993. All 34 strains belonging to subclade 5 possessed blaCTX-M-14 with ISEcp1 upstream at the same chromosomal position, indicating their common ancestor acquired blaCTX-M-14 in a single ISEcp1-mediated transposition event during the early formation of the subclade around 1999. Therefore, both the horizontal transfer of plasmids carrying blaCTX-M-14 to diverse genetic lineages and chromosomal integration in the predominant genetic lineage have contributed to the spread of blaCTX-M-14.

A molecular epidemiological and transmission analysis of Clostridioides difficile using draft whole-genome sequencing in a single hospital.

BACKGROUND: The nosocomial transmission of toxin-producing Clostridioides difficile is a significant concern in infection control. C. difficile, which resides in human intestines, poses a risk of transmission, especially when patients are in close contact with medical staff. METHODS: To investigate the nosocomial transmission of C. difficile in a single center, we analyzed the genetic relationships of the bacteria. This was done using draft whole-genome sequencing (WGS) and examining single nucleotide polymorphisms (SNPs) in core-genome, alongside data regarding the patient's hospital wards and room changes. Our retrospective analysis covered 38 strains, each isolated from a different patient, between April 2014 and January 2015. RESULTS: We identified 38 strains that were divided into 11 sequence types (STs). ST81 was the most prevalent (n = 11), followed by ST183 (n = 10) and ST17 (n = 7). A cluster of strains that indicated suspected nosocomial transmission (SNT) was identified through SNP analysis. The draft WGS identified five clusters, with 16 of 38 strains belonging to these clusters. There were two clusters for ST81 (ST81-SNT-1 and ST81-SNT-2), two for ST183 (ST183-SNT-1 and ST183-SNT-2), and one for ST17 (ST17-SNT-1). ST183-SNT-1 was the largest SNT cluster, encompassing five patients who were associated with Wards A, B, and K. The most frequent room changer was a patient labeled Pt08, who changed rooms seven times in Ward B. Patients Pt36 and Pt10, who were also in Ward B, had multiple admissions and discharges during the study period. CONCLUSIONS: Additional culture tests and SNP analysis of C. difficile using draft WGS revealed silent transmission within the wards, particularly in cases involving frequent room changes and repeated admissions and discharges. Monitoring C. difficile transmission using WGS-based analysis could serve as a valuable marker in infection control management.

Relapsing bronchopneumonia due to community-associated methicillin-resistant Staphylococcus aureus: a case report.

BACKGROUND: The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has increased the incidence of community-onset MRSA infection. Respiratory tract infections caused by MRSA has been noted for their severity; however, repeated relapses that require extended antibiotic therapy are rare. CASE PRESENTATION: We report a case of relapsing bronchopneumonia caused by CA-MRSA in a 56-year-old man. The patient responded to antibiotics, but repeatedly relapsed after stopping treatment. MRSA was consistently isolated from airway specimens during each relapse. Extended oral antibiotic treatment with trimethoprim/sulfamethoxazole (TMP/SMX) for 6 months achieved infection control. Whole-genome sequencing of the isolated strain revealed that the causative agent was sequence type (ST)1/staphylococcal cassette chromosome mec (SCCmec) type IVa, a clone that is rapidly increasing in Japan. DISCUSSION AND CONCLUSIONS: This patient had an unusual course of MRSA bronchopneumonia with repeated relapses. Although the choice of antibiotics for long-term use in MRSA respiratory tract infections has not been well established, TMP/SMX was effective and well tolerated for long-term therapy in this case. The clinical course of infections related to the rapid emerging clone, ST1/SCCmec type IVa warrants further attention.

Assessing the discriminability of PCR-based open reading frame typing versus single-nucleotide polymorphism analysis via draft whole-genome sequencing of methicillin-resistant Staphylococcus aureus in nosocomial transmission analysis.

Phylogenetic analysis based on single-nucleotide polymorphism (SNP)-based through whole-genome sequencing is recognized as the standard method for probing nosocomial transmission. However, the application of WGS is constrained by the high cost of equipment and the need for diverse analysis tools, which limits its widespread use in clinical laboratory settings. In Japan, the prevalent use of PCR-based open reading frame typing (POT) for tracing methicillin-resistant Staphylococcus aureus (MRSA) transmission routes is attributed to its simplicity and ease of use. Although POT's discriminatory power is considered insufficient for nosocomial transmission analysis, conclusive data supporting this notion is lacking. This study assessed the discriminatory capabilities of SNP analysis and POT across 64 clinical MRSA strains. All 21 MRSA strains of ST5/SCCmec IIa, having more than 16 SNPs, demonstrated distinct clones. Conversely, two strains shared the same POT number and were identified as group A. Among the 12 MRSA strains of ST8/SCCmec IVl with over nine SNPs, five fell into POT group B, and five into POT group C. All four MRSA strains of ST8/SCCmec IVa were classified into POT group D, although they included strains with more than 30 SNPs. Among the 27 MRSA strains of ST1/SCCmec IVa, 14 were classified into POT group E. However, except for two clusters (each comprising two or three strains), all had SNP counts >10 (Fig. 1-D). SNP analysis of MRSA in CC1/SCCmec IV showed that several strains had the same number of SNPs in POT number (106-183-37), even among bacteria with >100 SNPs, indicating POT's limited use in detailed nosocomial transmission analysis.

Invasive pectoral abscess and costal osteomyelitis with bloodstream infection caused by methicillin-resistant Staphylococcus aureus after nasal septoplasty in an immunocompetent adult patient.

There are few reports of multilocus sequence type (ST) 5/staphylococcal cassette chromosome (SCC) mec type IVc/toxic shock syndrome toxin (TSST)-1- positive methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections. We report a case of community-onset MRSA (CO-MRSA) bloodstream infection in a healthy 41-year-old Japanese man after nasal septoplasty, followed by pectoral abscess and costal osteomyelitis. The patient presented with right anterior chest pain and fever. After admission, MRSA was isolated from two sets of blood cultures, and vancomycin was administered. On the fifth day, contrast-enhanced computed tomography (CT) scan and contrast-enhanced magnetic resonance imaging (MRI) scan showed an abscess in the right anterior chest to the right subpleural region. The dosage of vancomycin (4 g/day) did not reach the effective blood concentration; therefore, there was a switch to daptomycin. On the 23rd day, contrast-enhanced MRI revealed osteomyelitis of the right first rib, and as a result, linezolid was initiated. Two weeks later, contrast-enhanced CT of the chest showed improvement in the abscess. The patient was treated for 6 weeks during hospitalization and then switched to minocycline for 10 weeks. Molecular characterization of this isolate showed that it was ST5/SCCmec type IVc/TSST-1-positive/Panton-Valentine leucocidin (PVL)-negative. PVL-negative CO-MRSA can lead to hematogenous osteomyelitis and abscess even if the patient is immunocompetent, and if isolated from blood cultures, it is important to repeat imaging studies, even if the initial imaging studies were normal. It is possible that this strain contributes to the pathogenesis of invasive CO-MRSA, but further case accumulation is needed.

A case of Bacillus subtilis var. natto bacteremia caused by ingestion of natto during COVID-19 treatment in a maintenance hemodialysis patient with multiple myeloma.

A 70-year-old woman, who started on hemodialysis 7 months before for end-stage renal disease due to diabetic nephropathy and was diagnosed with symptomatic multiple myeloma 1 month before, was admitted to our hospital with critical coronavirus disease 2019 and treated with long-term immunosuppressive therapy such as steroids and tocilizumab. During treatment, Bacillus subtilis was detected in the blood cultures. We could not exclude the association of natto (fermented soybeans) with B. subtilis var. natto, which the patient had been eating every day from 8 days after admission. She was prohibited from eating natto and treated with vancomycin. Later, B. subtilis detected in the blood culture was identified as B. subtilis var. natto, which was identical with those contained in the natto that the patient consumed daily using a next-generation sequencer. Gut dysbiosis due to old age, malignant tumor, diabetes mellitus, end-stage renal disease, and intestinal inflammation caused by severe acute respiratory syndrome coronavirus 2 increased intestinal permeability and the risk of bacterial translocation, causing B. subtilis var. natto bacteremia. Therefore, careful consideration might be given to the intake of fermented foods containing live bacteria in patients with severe immunocompromised conditions.

Cellulitis with bacteremia due to multidrug-resistant Campylobacter jejuni in a case of agammaglobulinemia and bronchiectasis.

This case report demonstrates successful treatment outcomes without recurrence using fosfomycin for cellulitis with bacteremia caused by Campylobacter jejuni resistant to macrolides, fluoroquinolones, and tetracyclines in agammaglobulinemia and bronchiectasis. Whole-genome sequencing indicated the presence of ST137 harboring bla OXA-61 and tet(O), with mutations in the 23S rRNA and gyrA genes.

Molecular epidemiological and antimicrobial-resistant mechanisms analysis of prolonged Neisseria gonorrhoeae collection between 1971 and 2005 in Japan.

Objectives: As antimicrobial-resistant (AMR) Neisseria gonorrhoeae strains have emerged, humans have adjusted the antimicrobials used to treat infections. We identified shifts in the N. gonorrhoeae population and the determinants of AMR strains isolated during the recurring emergence of resistant strains and changes in antimicrobial therapies. Methods: We examined 243 N. gonorrhoeae strains corrected at the Kanagawa Prefectural Institute of Public Health, Kanagawa, Japan, these isolated in 1971-2005. We performed multilocus sequence typing and AMR determinants (penA, mtrR, porB, ponA, 23S rRNA, gyrA and parC) mainly using high-throughput genotyping methods together with draft whole-genome sequencing on the MiSeq (Illumina) platform. Results: All 243 strains were divided into 83 STs. ST1901 (n = 17) was predominant and first identified after 2001. Forty-two STs were isolated in the 1970s, 34 in the 1980s, 22 in the 1990s and 13 in the 2000s, indicating a decline in ST diversity over these decades. Among the 29 strains isolated after 2001, 28 were highly resistant to ciprofloxacin (MIC ≥ 8 mg/L) with two or more amino-acid substitutions in quinolone-resistance-determining regions. Seven strains belonging to ST7363 (n = 3), ST1596 (n = 3) and ST1901 (n = 1) were not susceptible to cefixime, and six strains carried penA alleles with mosaic-like penicillin-binding protein 2 (PBP2; penA 10.001 and 10.016) or PBP2 substitutions A501V and A517G. Conclusions: We observed a significant reduction in the diversity of N. gonorrhoeae over 35 years in Japan. Since 2001, ST1901, which is resistant to ciprofloxacin, has superseded previous strains, becoming the predominant ST population.

Evolutionary dynamics of the novel ST22-PT methicillin-resistant Staphylococcus aureus clone co-harbouring Panton-Valentine leucocidin and duplicated toxic shock syndrome toxin 1 genes.

OBJECTIVES: Globally, the isolation of community-associated methicillin-resistant Staphylococcus aureus (MRSA) harbouring both the Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) genes is rare. However, we encountered an outbreak of the ST22-PT clone exhibiting this phenotype in Japan. Notably, the TSST-1 gene was duplicated in most of the strains. This study aimed to elucidate the mechanisms underlying this gene duplication. METHODS: A total of 90 MRSA isolates were collected from the skin of outpatients in Fukuoka City, Japan, between 2017 and 2019. Whole-genome sequencing was performed on MRSA strains that were PVL and TSST-1 positive. RESULTS: A total of 43 (47.8%) strains produced TSST-1, 20 (22.2%) produced PVL, and 16 (17.8%) produced both. Fifteen isolates were classified as ST22/SCCmec type IVa (ST22-PT clone) and one as ST1/SCCmec type V (ST1-PT clone). Three distinct ST22-PT clones were identified: Fukuoka clone I (one PVL gene and one TSST-1 gene), Fukuoka clone II (addition of a TSST-1 gene to Fukuoka clone I), and Fukuoka clone III (marked by a chromosomal inversion in a large region from Fukuoka clone II). DISCUSSION: Fukuoka clone I may have integrated a novel pathogenicity island bearing the TSST-1 gene, leading to the emergence of Fukuoka clone II with a duplicated TSST-1 gene. This duplication subsequently instigated a chromosomal inversion in a large region owing to the homologous sequence surrounding TSST-1, giving rise to Fukuoka clone III. These findings provide crucial insights into the genetic evolution of MRSA.

Analysis of the stepwise acquisition of blaCTX-M-2 and subsequent acquisition of either blaIMP-1 or blaIMP-6 in highly conserved IncN-pST5 plasmids.

Objectives: ESBL and carbapenemase genes in Enterobacterales spread via plasmids. Nosocomial outbreaks caused by Enterobacterales producing both CTX-M-2 and either IMP-1 or IMP-6-type carbapenemases have been reported. These organisms carry the incompatibility type N plasmid belonging to plasmid ST 5 (IncN-pST5). We investigated the construction process of the ESBL and carbapenemase genes co-carrying IncN-pST5. Methods: We retrospectively performed draft WGS analysis for blaIMP- or blaCTX-M-positive Enterobacterales in our strain collection (n = 281). Results: We selected four types of Escherichia coli plasmids for our study: type A, which carries both blaCTX-M-2 and blaIMP-1 (n = 6); type B, which carries both blaCTX-M-2 and blaIMP-6 (n = 2); type C, which carries blaCTX-M-2 (n = 10); and type D, which carries no ß-lactamase genes (n = 1). It should be noted that type D plasmid was only detected in E. coli TUM2805, which carries the blaCTX-M-14 on the IncB/O/B/Z plasmid. Long-read sequencing using MinION revealed that all types of IncN-pST5 were highly conserved and carried a class 1 integron. Integron numbers were type A for In798, type B for In1690, type C for In127 and type D for In207. Because the gene cassettes downstream of blaIMP were different between In798 and In1690, the change from blaIMP-1 to blaIMP-6 by point mutation was unlikely. Representative plasmids from types A, B and C were conjugatively transferred with quite a high frequency between 1.3 × 10-1 and 2.5 × 10-2. Conclusions: This study suggested that IncN-pST5 acquired blaCTX-M-2 by ISEcp1 in a stepwise manner, followed by either blaIMP-1 or blaIMP-6 into a class 1 integron.

Whole-Genome Sequences of Three Psychrotolerant Mycolicibacterium sp. Strains Isolated from Antarctic Soil.

We report the whole-genome sequences of three psychrotolerant Mycolicibacterium strains, TUM20983, TUM20984, and TUM20985, isolated from Antarctic soils. Taxonomic analyses indicate that these strains are putative new species. These genome sequences may provide insight into the cold adaptation mechanisms of Mycolicibacterium spp. through future comparative genomic studies.

Whole-Genome Sequencing of the Novel Pseudomonas Strain TUM22785 Harboring blaPAM-1, Isolated from a Patient with Urinary Tract Infection in Japan.

Pseudomonas species are Gram-negative aerobic bacteria that cause opportunistic infections. Here, we report the whole-genome sequence of the Pseudomonas sp. strain TUM22785, isolated from an outpatient with a urinary tract infection at a medical institution in Japan. This strain harbors a metallo-ß-lactamase (MBL) blaPAM-1 gene.

Complete Genome Sequence of Polynucleobacter sp. Strain TUM22923, Isolated from Antarctic Lake Sediment.

Here, we report the complete genome sequence of Polynucleobacter sp. strain TUM22923, isolated from Antarctic lake sediment. This strain has a genome of 1,860,127 bp, comprising 1,848 protein-coding sequences. These sequence data could contribute to the elucidation of genome streamlining and low-temperature adaptation in members of Polynucleobacter, a cosmopolitan group of ultramicrobacteria.

Molecular Analysis of blaKPC-2-Harboring Plasmids: Tn4401a Interplasmid Transposition and Tn4401a-Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient.

The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales is a public health concern. KPC-encoding blaKPC is predominantly spread by strains of a particular phylogenetic lineage, clonal group 258, but can also be spread by horizontal transfer of blaKPC-carrying plasmids. Here, we report the transfer of a blaKPC-2-harboring plasmid via mobilization from K. pneumoniae to Citrobacter freundii complex and Morganella morganii strains in a single patient. We performed draft whole-genome sequencing to analyze 20 carbapenemase-producing Enterobacterales strains (15 of K. pneumoniae, two of C. freundii complex, and three of M. morganii) and all K. pneumoniae strains using MiSeq and/or MinION isolated from a patient who was hospitalized in New York and Montreal before returning to Japan. All strains harbored blaKPC-2-containing Tn4401a. The 15 K. pneumoniae strains each belonged to sequence type 258 and harbored a Tn4401a-carrying multireplicon-type plasmid, IncN and IncR (IncN+R). Three of these K. pneumoniae strains also possessed a Tn4401a-carrying ColRNAI plasmid, suggesting that Tn4401a underwent interplasmid transposition. Of these three ColRNAI plasmids, two and one were identical to plasmids harbored by two Citrobacter europaeus and three M. morganii strains, respectively. The Tn4401a-carrying ColRNAI plasmids were each 23,753 bp long and incapable of conjugal transfer via their own genes alone, but they mobilized during the conjugal transfer of Tn4401a-carrying IncN+R plasmids in K. pneumoniae. Interplasmid transposition of Tn4401a from an IncN+R plasmid to a ColRNAI plasmid in K. pneumoniae and mobilization of Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii. IMPORTANCE Plasmid transfer plays an important role in the interspecies spread of carbapenemase genes, including the Klebsiella pneumoniae carbapenemase (KPC)-coding gene, blaKPC. We conducted whole-genome sequencing (WGS) analysis and transmission experiments to analyze blaKPC-2-carrying mobile genetic elements (MGEs) between the blaKPC-2-harboring K. pneumoniae, Citrobacter europaeus, and Morganella morganii strains isolated from a single patient. blaKPC-2 was contained within an MGE, Tn4401a. WGS of blaKPC-2-carrying K. pneumoniae, C. europaeus, and M. morganii strains isolated from one patient revealed that Tn4401a-carrying ColRNAI plasmids were generated by plasmid-to-plasmid transfer of Tn4401a from a multireplicon-type IncN and IncR (IncN+R) plasmid in K. pneumoniae strains. Tn4401a-carrying ColRNAI plasmids were incapable of conjugal transfer in C. europaeus and M. morganii but mobilized from K. pneumoniae to a recipient Escherichia coli strain during the conjugal transfer of Tn4401a-carrying IncN+R plasmid. Therefore, Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii.