Latency of Klebsiella species and Escherichia coli strains that harbor a conjugative IncA/C type plasmid carrying both rmtB and bla_<CTX-M-14>

Six high-level aminoglycoside and third generated cephalosporin of cefotaxime resistance Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli isolated from clinical specimens in the same Japanese hospital since 2001. These 6 strains were resistant to all 4,6-disubstituted deoxystreptamine including albekacin. The results of multiplex PCR for 16S rRNA methylase and bla_, bla_, bla_ specific PCR reaction show that these strains harbor 16S rRNA methylase of rmtB and β-lactamase resistance gene of bla_ group in the same large plasmid. Sequence analysis of bla_ group PCR products shows this β-lactamase is bla_. rmtB-harbored plasmids are classified the IncA/C by PCR and restriction pattern by EcoRI show only one or two band differences. Moreover, southern hybridization of EcoRI digested rmtB harbored plasmid by rmtB probe show the same hybridization pattern of 4.8 kbp bands. PFGE fingerprinting analysis of four K. pneumoniae revealed two fingerprinting patterns. These facts suggested that rmtB-positive starains not only spread horizontal transfer of rmtB-harbored plasmid but also clonal spread of the same strain

Population-level transition of capsular polysaccharide types among sequence type 1 group B Streptococcus isolates with reduced penicillin susceptibility during a long-term hospital epidemic

Over a 35-month period, group B Streptococcus isolates with reduced penicillin susceptibility (PRGBS) were detected from elderly patients at a regional hospital in Japan, accompanying population-level transition of PRGBS serotypes. The genetic relatedness of 77 non-duplicate PRGBS from 73 patients was analysed. Serotype III PRGBS predominated (16 serotype III/1 serotype Ib) in the first 9 months (period I), then 3 serotype Ib isolates appeared transiently for the next 3 months (period II), which was replaced predominantly by serotype Ia (20 serotype Ia/1 serotype III/1 non-typeable) for 9 months (period III). In the last 14 months (period IV), besides 25 serotype Ia isolates, 10 serotype III were also identified. Serotypes III and Ia isolates, belonging to ST1, shared G329V, G398A, V405A and G429D substitutions in penicillin-binding protein 2X. Of three strains subjected to whole-genome sequencing, serotype III strain SU12 (period I) had a higher degree of genomic similarity with serotype Ia strain SU97 (period III) than serotype Ib strain SU67 (period II) based on average nucleotide identity and single nucleotide polymorphisms. Analysis of the cps gene clusters and the upstream and downstream flanking sequences revealed that disruption of the hyaluronidase gene located upstream of cpsY by insertion of IS 1548 was found in strain SU12, whereas Delta ISSag8 was inserted between tRNA-Arg and rpsA genes located downstream of cpsL in strain SU97. Interestingly, most serotype III PRGBS re-emerging in period IV had this tRNA-Arg-Delta ISSag8-rpsA region. Capsular switching and nosocomial transmission may possibly contribute to population-level serotype replacement among ST1 PRGBS isolates. (c) 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.ArticleINTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS.53(3):203-210(2019)journal articl

Global Spread of Multiple Aminoglycoside Resistance Genes

Emergence of the newly identified 16S rRNA methylases RmtA, RmtB, and ArmA in pathogenic gram-negative bacilli has been a growing concern. ArmA, which had been identified exclusively in Europe, was also found in several gram-negative pathogenic bacilli isolated in Japan, suggesting global dissemination of hazardous multiple aminoglycoside resistance genes

Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer

Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by de novo sequencing using a next-generation DNA sequencer on total RNAs extracted from an autopsy lung of a patient who died of viral pneumonia with A/H1N1/2009. Among a total of 9.4×106 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than ×200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly172Glu and Gly239Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases

16S rRNA Methylase–producing, Gram-Negative Pathogens, Japan

To investigate the exact isolation frequency of 16S rRNA methylase–producing, gram-negative pathogenic bacteria, we tested 87,626 clinical isolates from 169 hospitals. Twenty-six strains from 16 hospitals harbored 16S rRNA methylase genes, which suggests sparse but diffuse spread of pan-aminoglycoside–resistant microbes in Japan

Complete Sequencing of the blaNDM-1-Positive IncA/C Plasmid from Escherichia coli ST38 Isolate Suggests a Possible Origin from Plant Pathogens

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with blaCMY-2-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The blaNDM-1 gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the blaNDM-1 gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the blaNDM-1-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the blaNDM-1 gene. The complete sequence of pNDM-1_Dok01 suggests that the blaNDM-1 gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate

Differential Expression of Type III Effector BteA Protein Due to IS481 Insertion in Bordetella pertussis

BACKGROUND: Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s) involved in the type shift. METHODOLOGY/PRINCIPAL FINDING: Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 5' untranslated region of bteA, -136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion. CONCLUSION/SIGNIFICANCE: The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis

Prevalence and Genetic Characterization of Pertactin-Deficient Bordetella pertussis in Japan

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions

Global population structure and evolution of Bordetella pertussis and their relationship with vaccination.

Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape