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July 7, 2019

Genome comparisons of two Taiwanese community-associated methicillin-resistant Staphylococcus aureus ST59 clones support the multi-origin theory of CA-MRSA.

Sequence type (ST) 59 is an epidemic lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Asia. Two ST59 clones are prevalent in Taiwan: the Taiwan clone (TW) causes severe infections, whereas the Asian-Pacific clone (AP) is usually commensal. In this study, we sequenced the genome and transcriptome of the representative strains of these two clones and found their differences to focus on three mobile genetic elements: TW carries SCCmec Type VT, Panton-Valentine leucocidin (PVL)-encoding prophage FSa2, whereas AP carries SCCmec Type IV and staphylokinase (SAK)-encoding prophage FSa3. The anti-virulent role of SAK was confirmed using murine skin and bloodstream infection models. FSa3 usually integrates into the hlb gene, but in AP was found to be integrated at the genomic island ?Saß. The mutation of the attB site “TGTATCCAAACTGG” to “TGTATCCGAATTGG” led to a failure in the integration of FSa3 in hlb, prompting atypical integration at other sites. The sak gene possessed remarkably different patterns of distribution among the different STs of S. aureus. We conclude that the atypical integration of FSa3 may help S. aureus adapt to the human host habitat and that the subsequent loss of FSa3 contributes toward the development of a virulent CA-MRSA lineage for wider horizontal transmission. Copyright © 2017. Published by Elsevier B.V.

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July 7, 2019

Characterization of NDM-5-positive extensively resistant Escherichia coli isolates from dairy cows.

The aim of this study was to investigate the prevalence of blaNDM-5 gene in Escherichia coli isolates from dairy cows and to characterize the molecular traits of the blaNDM-5-positive isolates. A total of 169 cows were sampled (169 feces and 169 raw milk samples) in three dairy farms in Jiangsu Province and 203 E. coli isolates were recovered. Among these strains, three isolates carried blaNDM-5 gene, including one co-harboring mcr-1, which belonged to sequence type 446 and the other two belonged to ST2. Susceptibility testing revealed that the three blaNDM-5-positive isolates showed extensive resistance to antimicrobials. The blaNDM-5 gene was located on a ~46-kb IncX3 transferrable pNDM-MGR194-like plasmid in all three isolates, while mcr-1 was located on a ~260-kb IncHI2 plasmid pXGE1mcr. Competition experiments revealed that acquisition of blaNDM-5 or mcr-1-bearing plasmid can incur fitness cost of bacterial host, however, plasmid stability testing showed that both blaNDM-5 and mcr-1-carrying plasmid maintained stable in the hosts after ten passages without antimicrobial selection. Whole genome sequencing revealed that the mcr-1 gene coexisted with multiple resistance genes in pXGE1mcr and the backbone of this plasmid was similar to that of previously reported mcr-1-positive plasmid pHNSHP45-2. Moreover, pXGE1mcr could be conjugated into clinical NDM-5-positive E. coli isolates in vitro, thereby generating strains that approached pan-resistance. Active surveillance efforts are imperative to monitor the prevalence of blaNDM-5 and mcr-1 in carbapenem-resistant Enterobacteriaceae from dairy farms throughout China. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Clostridium chauvoei, an evolutionary dead-end pathogen.

Full genome sequences of 20 strains of Clostridium chauvoei, the etiological agent of blackleg of cattle and sheep, isolated from four different continents over a period of 64 years (1951-2015) were determined and analyzed. The study reveals that the genome of the species C. chauvoei is highly homogeneous compared to the closely related species C. perfringens, a widespread pathogen that affects human and many animal species. Analysis of the CRISPR locus is sufficient to differentiate most C. chauvoei strains and is the most heterogenous region in the genome, containing in total 187 different spacer elements that are distributed as 30 – 77 copies in the various strains. Some genetic differences are found in the 3 allelic variants of fliC1, fliC2 and fliC3 genes that encode structural flagellin proteins, and certain strains do only contain one or two alleles. However, the major virulence genes including the highly toxic C.chauvoei toxin A, the sialidase and the two hyaluronidases are fully conserved as are the metabolic and structural genes of C. chauvoei. These data indicate that C. chauvoei is a strict ruminant-associated pathogen that has reached a dead end in its evolution.

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July 7, 2019

Candida auris for the clinical microbiology laboratory: Not your grandfather’s Candida species

Candida auris is a newly emerging species that was first identified in Asia in 2009 but has rapidly spread across the world. C. auris differs from most other Candida species in that antifungal resistance is the norm rather than the exception, it is a commensal of human skin rather than the human gut, and it can be easily transmitted from person to person in a health care setting. This review discusses the emergence of C. auris, global epidemiology, identification, antifungal susceptibility testing, and precautions to be taken when it is identified from a patient specimen.

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July 7, 2019

Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure.

The natural environment serves as a reservoir of opportunistic pathogens. A well-established method for studying the epidemiology of such opportunists is multilocus sequence typing, which in many cases has defined strains predisposed to causing infection. Burkholderia multivorans is an important pathogen in people with cystic fibrosis (CF) and its epidemiology suggests that strains are acquired from non-human sources such as the natural environment. This raises the central question of whether the isolation source (CF or environment) or the multilocus sequence type (ST) of B. multivorans better predicts their genomic content and functionality. We identified four pairs of B. multivorans isolates, representing distinct STs and consisting of one CF and one environmental isolate each. All genomes were sequenced using the PacBio SMRT sequencing technology, which resulted in eight high-quality B. multivorans genome assemblies. The present study demonstrated that the genomic structure of the examined B. multivorans STs is highly conserved and that the B. multivorans genomic lineages are defined by their ST. Orthologous protein families were not uniformly distributed among chromosomes, with core orthologs being enriched on the primary chromosome and ST-specific orthologs being enriched on the second and third chromosome. The ST-specific orthologs were enriched in genes involved in defense mechanisms and secondary metabolism, corroborating the strain-specificity of these virulence characteristics. Finally, the same B. multivorans genomic lineages occur in both CF and environmental samples and on different continents, demonstrating their ubiquity and evolutionary persistence.

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July 7, 2019

Genomic analysis of factors associated with low prevalence of antibiotic resistance in extraintestinal pathogenic Escherichia coli sequence type 95 strains.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains belonging to multilocus sequence type 95 (ST95) are globally distributed and a common cause of infections in humans and domestic fowl. ST95 isolates generally show a lower prevalence of acquired antimicrobial resistance than other pandemic ExPEC lineages. We took a genomic approach to identify factors that may underlie reduced resistance. We fully assembled genomes for four ST95 isolates representing the four major fimH-based lineages within ST95 and also analyzed draft-level genomes from another 82 ST95 isolates, largely from the western United States. The fully assembled genomes of antibiotic-resistant isolates carried resistance genes exclusively on large (>90-kb) IncFIB/IncFII plasmids. These replicons were common in the draft genomes as well, particularly in antibiotic-resistant isolates, but we also observed multiple instances of a smaller (8.3-kb) ampicillin resistance plasmid that had been previously identified in Salmonella enterica. Among ST95 isolates, pansusceptibility to antibiotics was significantly associated with the fimH6 lineage and the presence of homologs of the previously identified 114-kb IncFIB/IncFII plasmid pUTI89, both of which were also associated with reduced carriage of other plasmids. Potential mechanistic explanations for lineage- and plasmid-specific effects on the prevalence of antibiotic resistance within the ST95 group are discussed. IMPORTANCE Antibiotic resistance in bacterial pathogens is a major public health concern. This work was motivated by the observation that only a small proportion of ST95 isolates, a major pandemic lineage of extraintestinal pathogenic E. coli, have acquired antibiotic resistance, in contrast to many other pandemic lineages. Understanding bacterial genetic factors that may prevent acquisition of resistance could contribute to the development of new biological, medical, or public health strategies to reduce antibiotic-resistant infections.

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July 7, 2019

Complete genome sequences of three Salmonella enterica subsp. enterica serovar Saintpaul isolates associated with a 2013 multistate outbreak in the United States.

In 2013, a multistate outbreak of Salmonella enterica subsp. enterica serovar Saintpaul from cucumber caused 84 cases of salmonellosis in the United States. In this announcement, we report the complete genome sequences of three clinical Salmonella Saintpaul isolates associated with the 2013 outbreak. Copyright © 2017 Yao et al.

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July 7, 2019

Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line.

Babesia bovis is an intra-erythrocytic tick-transmitted apicomplexan protozoan parasite. It has a complex lifestyle including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family, are differentially expressed during tick and mammalian stages of the parasite’s life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of the phenotypically distinct B. bovis Mo7clonal line.In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5′ and 3′ regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1a promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites was performed and in vitro inhibition assays using anti 6-Cys E antibodies.Full genome sequencing of 6-Cys EKO-cln B. bovis demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild-type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays confirmed the ability of anti-6 Cys E antibodies to inhibit the growth of the wild-type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites.Overall, the data suggest that the anti-6 Cys E antibody neutralising effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages.

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July 7, 2019

Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain.

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5?144?392?bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2?803?932?bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577?351?bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157?:?H7; therefore, it is not an ancestral state for the species.

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July 7, 2019

Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen.

Neisseria meningitidis (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasive meningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, US_NmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of US_NmUC isolates was an IS1301 element in the intergenic region separating the capsule ctr-css operons and adjacent deletion of cssA/B/C and a part of csc, encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an ~20-kb sequence near the cps locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification norB-aniA gene cassette, and strains grow well anaerobically. The cc11 US_NmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the Neisseria gonorrheae norB-aniA cassette promoting anaerobic growth.

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July 7, 2019

Characterization of miR-122-independent propagation of HCV.

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.

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July 7, 2019

Phylogeography of Burkholderia pseudomallei isolates, Western Hemisphere.

The bacterium Burkholderia pseudomallei causes melioidosis, which is mainly associated with tropical areas. We analyzed single-nucleotide polymorphisms (SNPs) among genome sequences from isolates of B. pseudomallei that originated in the Western Hemisphere by comparing them with genome sequences of isolates that originated in the Eastern Hemisphere. Analysis indicated that isolates from the Western Hemisphere form a distinct clade, which supports the hypothesis that these isolates were derived from a constricted seeding event from Africa. Subclades have been resolved that are associated with specific regions within the Western Hemisphere and suggest that isolates might be correlated geographically with cases of melioidosis. One isolate associated with a former World War II prisoner of war was believed to represent illness 62 years after exposure in Southeast Asia. However, analysis suggested the isolate originated in Central or South America.

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July 7, 2019

Metabolic diversity of the emerging pathogenic lineages of Klebsiella pneumoniae.

Multidrug resistant and hypervirulent clones of Klebsiella pneumoniae are emerging pathogens. To understand the association between genotypic and phenotypic diversity in this process, we combined genomic, phylogenomic and phenotypic analysis of a diverse set of K. pneumoniae and closely related species. These species were able to use an unusually large panel of metabolic substrates for growth, many of which were shared between all strains. We analysed the substrates used by only a fraction of the strains, identified some of their genetic basis, and found that many could not be explained by the phylogeny of the strains. Puzzlingly, few traits were associated with the ecological origin of the strains. One noticeable exception was the ability to use D-arabinose, which was much more frequent in hypervirulent strains. The broad carbon and nitrogen core metabolism of K. pneumoniae might contribute to its ability to thrive in diverse environments. Accordingly, even the hypervirulent and multidrug resistant clones have the metabolic signature of ubiquitous bacteria. The apparent few metabolic differences between hypervirulent, multi-resistant and environmental strains may favour the emergence of dual-risk strains that combine resistance and hypervirulence.© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019

A novel disrupted mcr-1 gene and a lysogenized phage P1-like sequence detected from a large conjugative plasmid, cultured from a human atypical enteropathogenic Escherichia coli (aEPEC) recovered in China.

Sir,The recent description of the plasmid-mediated colistin resistance gene, mcr-1, in bacterial isolates cultured in China has triggered several retrospective studies investigating this gene.1The mcr-1 gene has so far been reported to be associated with various plas- mid replicon types, and was found only rarely to be chromoso- mally encoded.2,3However, no report of a directly inactivated mcr-1 gene has been described to date. In this study, we present the complete nucleotide sequence of an ESBL-producing atypical enteropathogenic Escherichia coli (aEPEC) isolate, SLK172, one of whose plasmids carried a uniquely disrupted mcr-1 gene, being inactivated following the insertion of an ISApl1 element (Figure1a).

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July 7, 2019

Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection.

Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish long-lasting chronic infections(1-5), creating an infectious reservoir to sustain transmission(1,6). It is widely accepted that the maintenance of chronic infection involves evasion of adaptive immunity by antigenic variation(7). However, genes involved in this process have been identified in only two of five human-infecting species: Plasmodium falciparum and Plasmodium knowlesi. Furthermore, little is understood about the early events in the establishment of chronic infection in these species. Using a rodent model we demonstrate that from the infecting population, only a minority of parasites, expressing one of several clusters of virulence-associated pir genes, establishes a chronic infection. This process occurs in different species of parasites and in different hosts. Establishment of chronicity is independent of adaptive immunity and therefore different from the mechanism proposed for maintenance of chronic P. falciparum infections(7-9). Furthermore, we show that the proportions of parasites expressing different types of pir genes regulate the time taken to establish a chronic infection. Because pir genes are common to most, if not all, species of Plasmodium(10), this process may be a common way of regulating the establishment of chronic infections.

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