Masahiro Suzuki

The genus Aeromonas is increasingly implicated in human infections. However, accurate species-level identification remains challenging, particularly in clinical microbiology laboratories. This study aimed to develop a multiplex polymerase chain reaction (PCR) assay to identify four Aeromonas species-Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii, and Aeromonas dhakensis-most frequently associated with human infectious diseases. A total of 788 whole genome sequencing (WGS) data sets from 31 Aeromonas species were analyzed to identify open reading frames (ORFs) specifically present in A. hydrophila, A. caviae, A. veronii, and A. dhakensis. Primer sets were designed based on sequences of ORFs specific to each species to develop a multiplex PCR assay. To validate the efficacy of the assay, 256 clinical Aeromonas isolates were tested, and the results were compared with taxonomic affiliation inferred by WGS data, along with 19 type strains. The multiplex PCR successfully identified all strains of the four target species and produced no amplification in non-target species strains except the band for internal control. The multiplex PCR enables rapid and reliable identification of four Aeromonas spp. commonly involved in human infectious diseases.IMPORTANCEThe multiplex PCR assay facilitates accurate identification of clinically important Aeromonas spp. in clinical microbiology laboratories, providing crucial information to guide appropriate antimicrobial therapy and advance understanding of the epidemiology of Aeromonas spp.

INTRODUCTION: Although skin and soft tissue infections (SSTIs) of the extremities are primarily caused by Gram-positive cocci (GPC), some cases are caused by Gram-negative rod (GNR). In addition, malignancy is a recognized risk factor for GNR infection. Nevertheless, information on the clinical and microbiologic characteristics of SSTIs of the extremities caused by GNR in patients with malignancy is limited. METHODS: Clinical and microbiological characteristics of patients with malignancy who developed bacteremic SSTIs of the extremities at a single cancer center over eight years were reviewed. In addition, whole-genome sequencing of the GNR isolates causing necrotizing fasciitis was conducted. RESULTS: Of 42 cases identified, 32 cases (76.2%) and 10 cases (23.8%) were caused by GPC and GNR, respectively. Four cases in the GNR group were due to Escherichia coli, and the remaining cases were caused by diverse species. The majority of cases in the GNR group were hospital-onset and the lesions were limited to a single extremity. Chronic liver disease, cellular immunodeficiency, or anatomic abnormalities of the gastrointestinal, biliary, or urinary tract underlay seven GNR cases (70%). Inappropriate empiric therapy was numerically more common in the GNR group compared to the GPC group (33.3% vs. 9.4%, p = 0.107). Whole-genome sequencing analysis revealed that two cases of GNR necrotizing fasciitis were caused by E. coli ST1193-fimH64 and Klebsiella pneumoniae K2-ST86. CONCLUSIONS: GNR organisms are a significant cause of SSTIs of the extremities in patients with malignancy and may be associated with inappropriate empiric therapy.

Bartonella henselae, a Gram-negative facultative intracellular bacterium, is the etiological agent of cat-scratch disease and also causes bacillary angiomatosis in immunocompromised individuals. Although the ability to promote vascular endothelial cell proliferation differs among Bartonella species, variations among strains within B. henselae remain unclear. Bartonella angiogenic factor A (BafA) and Bartonella adhesin A (BadA) have been identified as autotransporters of B. henselae that are involved in endothelial cell proliferation. Although strain-specific differences in the expression of BadA and the VirB/D4 type IV secretion system have been reported, BafA expression among B. henselae strains has yet to be examined. Therefore, the present study investigated the proliferation-promoting ability of 13 B. henselae strains from several sources in human umbilical vein endothelial cells (HUVECs). We identified BafA variants 1 and 2 based on the deduced amino acid sequences of its passenger domain. The recombinant proteins of both variants exhibited similar proliferation activity against HUVECs. However, BafA variant 2 strains showed cytotoxicity at a high bacterial inoculum in a direct coculture with HUVECs, which was attenuated in an indirect coculture. These strains, in contrast to BafA variant 1 strains, highly expressed BadA and exhibited bacterial aggregation. Based on a core genome SNP analysis of 50 B. henselae strains, the BafA variant types corresponded to clades 1-4. These results indicate that vasoproliferative traits differ among B. henselae clades based on the variant types. Therefore, this study provides a new conceptual framework in which the clades of B. henselae may predict their pathogenicity in humans.IMPORTANCEBartonella species including Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis cause vasoproliferative lesions. Their proliferation-promoting ability in vascular endothelial cells differs among Bartonella species; however, it is unclear whether these differences exist among B. henselae strains. We herein showed that B. henselae strains exhibited variable proliferation-promoting ability and cytotoxicity in vascular endothelial cells, which corresponded to the bafA gene variants possessed by the strains. The expression levels of Bartonella angiogenic factor A (BafA) and Bartonella adhesin A, as well as the degree of proliferation-promoting ability and cytotoxicity in endothelial cells, varied among the strains. A core genome SNP analysis of strains using whole genome sequencing data divided B. henselae strains into four clades, with each clade corresponding to BafA variants 1-4. These results suggest the differential vasoproliferative potency of B. henselae, with potential implications in clinical management, including risk stratification and predictions of the clinical course.

A 70-year-old Japanese man with well-controlled diabetes mellitus and chronic kidney disease was hospitalized for an examination of acute renal failure and elevated inflammatory reactions. He had a history of Klebsiella pneumoniae bacteremia without extended-spectrum β-lactamase (ESBL) production five months earlier. The patient was found to have bacteremia due to hypermucoviscous ESBL-producing Klebsiella pneumoniae, and developed septic shock, multiple cerebral infarctions, and an abscess in the left masticatory muscle space. The causative organism was resistant to ampicillin-sulbactam and piperacillin-tazobactam, which were used for empiric therapy, and the patient died despite subsequent definitive treatment with meropenem. Whole genome sequencing analysis showed that the strain of K. pneumoniae was ST412 with capsular genotype K57 and carried virulence genes iroBCDN, iucABCD, iutA, mrkABCDFHIJ, rmpA2, ybtAEPQSTUX. The strain also carried the blaCTX-M-15 ESBL gene. Although the antimicrobial susceptibility of the causative organisms of hvKp infections in Japan has been favorable in most cases, severe infections caused by ESBL-producing hvKp may increase in the near future considering the recent increase in ESBL-producing K. pneumoniae.

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains ('major group' and 'minor group'). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.