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September 22, 2019  |  

Transcriptional landscape of a blaKPC-2 plasmid and response to imipenem exposure in Escherichia coli TOP10.

The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB blaKPC-2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (blaKPC-2, blaTEM and aph(3′)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments.

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September 22, 2019  |  

The plasmid-encoded transcription factor ArdK contributes to the repression of the IMP-6 metallo-ß-lactamase gene blaIMP-6, leading to a carbapenem-susceptible phenotype in the blaIMP-6-positive Escherichia coli strain A56-1S.

Carbapenemase-producing Enterobacteriaceae (CPE) are a global concern because these bacteria are resistant to almost all ß-lactams. Horizontal interspecies gene transfer via plasmid conjugation has increased the global dissemination of CPE. Recently, an Enterobacteriaceae strain positive for carbapenemase gene but showing a carbapenem-susceptible phenotype was identified, suggesting that these susceptible strains may be challenging to detect solely via antimicrobial susceptibility tests without molecular analysis. Here, we isolated a blaIMP-6 carbapenemase-gene positive but imipenem- and meropenem-susceptible Escherichia coli (ISMS-E) strain A56-1S (imipenem and meropenem minimum inhibitory concentration, = 0.125 mg/L), from a human urine specimen in Japan. A56-1S was carbapenemase negative by the Carba NP test, suggesting that IMP-6 production was low or undetectable. Thus, to characterize the mechanism of this phenotype, a meropenem-resistant E. coli A56-1R strain was obtained using meropenem-selection. A56-1R was positive for carbapenemase production by the Carba NP test, and blaIMP-6 transcription in A56-1R was 53-fold higher than in A56-1S, indicating that blaIMP-6 in A56-1S is negatively regulated at the transcriptional level. Comparative genomic analysis between the two strains revealed that the alleviation of restriction of DNA (ardK) gene encoding a putative transcription factor is disrupted by the IS26 insertion in A56-1R. A cotransformation assay of ardK and the regulatory element upstream of blaIMP-6 showed repression of blaIMP-6 transcription, indicating that ArdK negatively modulates blaIMP-6 transcription. ArdK binding and affinity assays demonstrated that ArdK directly binds to the regulatory element upstream of blaIMP-6 with dissociation constant values comparable to those of general transcription factors. The IMP-6 carbapenemase showed low hydrolytic activity against imipenem, resulting in an imipenem-susceptible and meropenem-resistant (ISMR) phenotype (previously reported as a stealth phenotype). However, the low expression of IMP-6 in the A56-1S strain could be a typical characteristic of ISMS-E due to gene repression, indicating that conventional antimicrobial susceptibility tests might be unable to detect such strains even when using both imipenem and meropenem. Bacteria that exhibit the ISMS phenotype could play a potential role as undetectable reservoirs and might facilitate gene transfer to other organisms while avoiding detection.

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September 22, 2019  |  

Emerging multidrug-resistant hybrid pathotype shiga toxin-producing Escherichia coli O80 and related strains of clonal complex 165, Europe.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-?, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum ß-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.

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September 22, 2019  |  

Genotypes and phenotypes of Enterococci isolated from broiler chickens

The objective of this study was to compare the resistance phenotypes to genotypes of enterococci from broiler and to evaluate the persistence and distribution of resistant genotypes in broiler fed bambermycin (BAM), penicillin (PEN), salinomycin (SAL), bacitracin (BAC) or a salinomycin/bacitracin combination (SALBAC) for 35 days. A total of 95 enterococci from cloacal (n=40), cecal (n=38) and litter collected on day 36 (n=17) samples were isolated weekly from day 7 to 36. All isolates were identified by API-20 Strep and their antimicrobial susceptibilities were evaluated using the Sensititre system with the commercially available NARMS’s plates of Gram positive bacteria. Whole genome sequencing (WGS) was used to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. All isolates were further characterized for hemolysin production (HEM), bile salt hydrolysis (BSH) and gelatinase (GEL) activities. Of the 95 isolates, E. faecium (n = 58) and E. faecalis (n = 24) were the most common Enterococcus species identified. Significant differences in the level of resistance for the E. faecium isolates to ciprofloxacin, macrolide, penicillin and tetracycline were observed among treatments. The bcrR, mefA and aac(6) genes were higher in BAM treatment than the other groups whereas bcrR, ermA, ermB, aphA(3) and tetL were more prevalent in PEN and BAC treatments. Overall, E. faecium isolates showed higher prevalence of antimicrobial resistance, but E. faecalis from litter also exhibited a significant level of resistance. A range of 4 to 15 different virulence genes was detected in E. faecalis. All isolates from litter but one (94.1%) showed BSH activities while 52.9% of them produced GEL. HEM activity was observed only in isolates collected on Day 7 (n= 9) and Day 14 (n= 1). This study confirmed that genetically diverse antimicrobial resistant enterococci harboring virulence factors can be promoted by the use of certain antimicrobials in feed and such enterococci could persist in broiler chickens and their litter, potentially contaminating the soil upon land application. This study underscores the need for ongoing monitoring the AMR enterococci.

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September 22, 2019  |  

A novel probiotic, Lactobacillus johnsonii 456, resists acid and can persist in the human gut beyond the initial ingestion period.

Probiotics are considered to have multiple beneficial effects on the human gastrointestinal tract, including immunomodulation, pathogen inhibition, and improved host nutrient metabolism. However, extensive characterization of these properties is needed to define suitable clinical applications for probiotic candidates. Lactobacillus johnsonii 456 (LBJ 456) was previously demonstrated to have anti-inflammatory and anti-genotoxic effects in a mouse model. Here, we characterize its resistance to gastric and bile acids as well as its ability to inhibit gut pathogens and adhere to host mucosa. While bile resistance and in vitro host attachment properties of LBJ 456 were comparable to other tested probiotics, LBJ 456 maintained higher viability at lower pH conditions compared to other tested strains. LBJ 456 also altered pathogen adhesion to LS 174T monolayers and demonstrated contact-dependent and independent inhibition of pathogen growth. Genome analyses further revealed possible genetic elements involved in host attachment and pathogen inhibition. Importantly, we show that ingestion of Lactobacillus johnsonii 456 over a one week yogurt course leads to persistent viable bacteria detectable even beyond the period of initial ingestion, unlike many other previously described probiotic species of lactic acid bacteria.

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September 22, 2019  |  

Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains.

The recent discovery of the plasmid-mediated mcr-1 gene conferring resistance to colistin is of clinical concern. The worldwide screening of this resistance mechanism among samples of different origins has highlighted the urgent need to improve the detection of colistin-resistant isolates in clinical microbiology laboratories. Currently, phenotypic methods used to detect colistin resistance are not necessarily suitable as the main characteristic of the mcr genes is the low level of resistance that they confer, close to the clinical breakpoint recommended jointly by the CLSI and EUCAST expert systems (S?=?2?mg/L and R?>?2?mg/L). In this context, susceptibility testing recommendations for polymyxins have evolved and are becoming difficult to implement in routine laboratory work. The large number of mechanisms and genes involved in colistin resistance limits the access to rapid detection by molecular biology. It is therefore necessary to implement well-defined protocols using specific tools to detect all colistin-resistant bacteria. This review aims to summarize the current clinical microbiology diagnosis techniques and their ability to detect all colistin resistance mechanisms and describe new tools specifically developed to assess plasmid-mediated colistin resistance. Phenotyping, susceptibility testing, and genotyping methods are presented, including an update on recent studies related to the development of specific techniques.

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September 22, 2019  |  

Description of Schaedlerella arabinophila gen. nov., sp. nov., a D-arabinose utilizing bacterium isolated from feces of C57BL/6J mice and a close relative of Clostridium sp. ASF 502

The use of gnotobiotics has gained large interest in recent years due to technological advances that have revealed the importance of host-associated microbiomes for host physiology and health. One of the oldest and most important gnotobiotics mouse model, the Altered Schaedler Flora (ASF) has been used for several decades. ASF comprises eight different bacterial species, which have been characterized to different extent, but only few are available through public strain collections. Here, the isolation of a close relative to one of the less studied ASF strains, Clostridium sp. ASF 502, is reported. Isolate TLL-A1, which shares 99.6% 16S rRNA gene sequence identity with Clostridium sp. ASF 502, was obtained from feces of C57BL/6J mice where is was detectable at a relative abundance of less than one percent. D-arabinose was used as sole carbon source in the anaerobic cultivation medium. Growth experiments with TLL-A1 on different carbon sources and analysis of its ~6.5 gigabase genome indicate that TLL-A1 harbors a large gene repertoire to utilize different carbohydrates for growth. Comparative genome analyses of TLL-A1 and Clostridium sp. ASF 502 reveal differences in genome content between the two strains, in particular with regards to carbohydrate activating enzymes. Based on physiology and genomic analysis it is proposed to name TLL-A1 to gen. nov. sp. nov Schaedlerella arabinophila TLL-A1 (DSMZ 106076T; KCTC 15657T). The closely related Clostridium sp. ASF 502 is proposed to be renamed to Schaedlerella arabinophila to reflect its taxonomic standing and to keep textquoterightASF 502textquoteright as strain designation.

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September 22, 2019  |  

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

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September 22, 2019  |  

Comparative Analysis of the Genetic Basis of Branched Nonylphenol Degradation by Sphingobium amiense DSM 16289T and Sphingobium cloacae JCM 10874T.

Branched nonylphenol (BNP), a degradation product of nonylphenol polyethoxylates, exerts estrogenic effects on various organisms. The genes underlying BNP degradation by Sphingobium amiense DSM 16289T were analyzed by complete genome sequencing and compared with those of the versatile BNP-degrading Sphingobium cloacae JCM 10874T. An opdA homolog (opdADSM16289) encoding BNP degradation activity was identified in DSM 16289T, in contrast with JCM 10874T, possessing both the opdA homolog and nmoA. The degradation profile of different BNP isomers was examined by Escherichia coli transformants harboring opdADSM16289, opdAJCM10874, and nmoAJCM10874 to characterize and compare the expression activities of these genes.

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September 22, 2019  |  

Mutators as drivers of adaptation in Streptococcus and a risk factor for host jumps and vaccine escape

Heritable hypermutable strains deficient in DNA repair genes (mutators) facilitate microbial adaptation as they may rapidly generate beneficial mutations. Mutators deficient in mismatch (MMR) and oxidised guanine (OG) repair are abundant in clinical samples and show increased adaptive potential in experimental infection models but their role in pathoadaptation is poorly understood. Here we investigate the role of mutators in epidemiology and evolution of the broad host pathogen, Streptococcus iniae, employing 80 strains isolated globally over 40 years. We determine phylogenetic relationship among S. iniae using 10,267 non-recombinant core genome single nucleotide polymorphisms (SNPs), estimate their mutation rate by fluctuation analysis, and detect variation in major MMR (mutS, mutL, dnaN, recD2, rnhC) and OG (mutY, mutM, mutX) genes. S. iniae mutation rate phenotype and genotype are strongly associated with phylogenetic diversification and variation in major streptococcal virulence determinants (capsular polysaccharide, hemolysin, cell chain length, resistance to oxidation, and biofilm formation). Furthermore, profound changes in virulence determinants observed in mammalian isolates (atypical host) and vaccine-escape isolates found in bone (atypical tissue) of vaccinated barramundi are linked to multiple MMR and OG variants and unique mutation rates. This implies that adaptation to new host taxa, new host tissue, and to immunity of a vaccinated host is promoted by mutator strains. Our findings support the importance of mutation rate dynamics in evolution of pathogenic bacteria, in particular adaptation to a drastically different immunological setting that occurs during host jump and vaccine escape events.Importance Host immune response is a powerful selective pressure that drives diversification of pathogenic microorganisms and, ultimately, evolution of new strains. Major adaptive events in pathogen evolution, such as transmission to a new host species or infection of vaccinated hosts, require adaptation to a drastically different immune landscape. Such adaptation may be favoured by hypermutable strains (or mutators) that are defective in normal DNA repair and consequently capable of generating multiple potentially beneficial and compensatory mutations. This permits rapid adjustment of virulence and antigenicity in a new immunological setting. Here we show that mutators, through mutations in DNA repair genes and corresponding shifts in mutation rate, are associated with major diversification events and virulence evolution in the broad host-range pathogen Streptococcus iniae. We show that mutators underpin infection of vaccinated hosts, transmission to new host species and the evolution of new strains.

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September 21, 2019  |  

Functional analysis of the first complete genome sequence of a multidrug resistant sequence type 2 Staphylococcus epidermidis.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. The ST2 lineage is frequently multidrug resistant and accounts for most of the clinical disease worldwide. However, there are no publically available, closed ST2 genomes and pathogenesis studies have not focused on these strains. We report the complete genome and methylome of BPH0662, a multidrug resistant, hospital adapted, ST2 S. epidermidis, and describe the correlation between resistome and phenotype, as well as demonstrate its relationship to publically available, international ST2 isolates. Furthermore, we delineate the methylome determined by the two type I restriction modification systems present in BPH0662 through heterologous expression in Escherichia coli, allowing the assignment of each system to its corresponding target recognition motif. As the first complete ST2 S. epidermidis genome, BPH0662 provides a valuable reference for future genomic studies of this clinically relevant lineage. Defining the methylome and the construction of these E. coli hosts provides the foundation for the development of molecular tools to bypass restriction modification systems in this lineage that has hitherto proven intractable.

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September 21, 2019  |  

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7.

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

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September 21, 2019  |  

Mistranslation drives the evolution of robustness in TEM-1 ß-lactamase.

How biological systems such as proteins achieve robustness to ubiquitous perturbations is a fundamental biological question. Such perturbations include errors that introduce phenotypic mutations into nascent proteins during the translation of mRNA. These errors are remarkably frequent. They are also costly, because they reduce protein stability and help create toxic misfolded proteins. Adaptive evolution might reduce these costs of protein mistranslation by two principal mechanisms. The first increases the accuracy of translation via synonymous “high fidelity” codons at especially sensitive sites. The second increases the robustness of proteins to phenotypic errors via amino acids that increase protein stability. To study how these mechanisms are exploited by populations evolving in the laboratory, we evolved the antibiotic resistance gene TEM-1 in Escherichia coli hosts with either normal or high rates of mistranslation. We analyzed TEM-1 populations that evolved under relaxed and stringent selection for antibiotic resistance by single molecule real-time sequencing. Under relaxed selection, mistranslating populations reduce mistranslation costs by reducing TEM-1 expression. Under stringent selection, they efficiently purge destabilizing amino acid changes. More importantly, they accumulate stabilizing amino acid changes rather than synonymous changes that increase translational accuracy. In the large populations we study, and on short evolutionary timescales, the path of least resistance in TEM-1 evolution consists of reducing the consequences of translation errors rather than the errors themselves.

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September 21, 2019  |  

Decreased fitness and virulence in ST10 Escherichia coli harboring blaNDM-5 and mcr-1 against a ST4981 strain with blaNDM-5.

Although coexistence of blaNDM-5 and mcr-1 in Escherichia coli has been reported, little is known about the fitness and virulence of such strains. Three carbapenem-resistant Escherichia coli (GZ1, GZ2, and GZ3) successively isolated from one patient in 2015 were investigated for microbiological fitness and virulence. GZ1 and GZ2 were also resistant to colistin. To verify the association between plasmids and fitness, growth kinetics of the transconjugants were performed. We also analyzed genomic sequences of GZ2 and GZ3 using PacBio sequencing. GZ1 and GZ2 (ST10) co-harbored blaNDM-5 and mcr-1, while GZ3 (ST4981) carried only blaNDM-5. GZ3 demonstrated significantly more rapid growth (P < 0.001) and overgrew GZ2 with a competitive index of 1.0157 (4 h) and 2.5207 (24 h). Increased resistance to serum killing and mice mortality was also identified in GZ3. While GZ2 had four plasmids (IncI2, IncX3, IncHI2, IncFII), GZ3 possessed one plasmid (IncFII). The genetic contexts of blaNDM-5 in GZ2 and GZ3 were identical but inserted into different backbones, IncX3 (102,512 bp) and IncFII (91,451 bp), respectively. The growth was not statistically different between the transconjugants with mcr-1 or blaNDM-5 plasmid and recipient (P = 0.6238). Whole genome sequence analysis revealed that 28 virulence genes were specific to GZ3, potentially contributing to increased virulence of GZ3. Decreased fitness and virulence in a mcr-1 and blaNDM-5 co-harboring ST10 E. coli was found alongside a ST4981 strain with only blaNDM-5. Acquisition of mcr-1 or blaNDM-5 plasmid did not lead to considerable fitness costs, indicating the potential for dissemination of mcr-1 and blaNDM-5 in Enterobacteriaceae.

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