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July 7, 2019

ICESag37, a novel integrative and conjugative element carrying antimicrobial resistance genes and potential virulence factors in Streptococcus agalactiae.

ICESag37, a novel integrative and conjugative element carrying multidrug resistance and potential virulence factors, was characterized in a clinical isolate of Streptococcus agalactiae. Two clinical strains of S. agalactiae, Sag37 and Sag158, were isolated from blood samples of new-borns with bacteremia. Sag37 was highly resistant to erythromycin and tetracycline, and susceptible to levofloxacin and penicillin, while Sag158 was resistant to tetracycline and levofloxacin, and susceptible to erythromycin. Transfer experiments were performed and selection was carried out with suitable antibiotic concentrations. Through mating experiments, the erythromycin resistance gene was found to be transferable from Sag37 to Sag158. SmaI-PFGE revealed a new SmaI fragment, confirming the transfer of the fragment containing the erythromycin resistance gene. Whole genome sequencing and sequence analysis revealed a mobile element, ICESag37, which was characterized using several molecular methods and in silico analyses. ICESag37 was excised to generate a covalent circular intermediate, which was transferable to S. agalactiae. Inverse PCR was performed to detect the circular form. A serine family integrase mediated its chromosomal integration into rumA, which is a known hotspot for the integration of streptococcal ICEs. The integration site was confirmed using PCR. ICESag37 carried genes for resistance to multiple antibiotics, including erythromycin [erm(B)], tetracycline [tet(O)], and aminoglycosides [aadE, aphA, and ant(6)]. Potential virulence factors, including a two-component signal transduction system (nisK/nisR), were also observed in ICESag37. S1-PFGE analysis ruled out the existence of plasmids. ICESag37 is the first ICESa2603 family-like element identified in S. agalactiae carrying both resistance and potential virulence determinants. It might act as a vehicle for the dissemination of multidrug resistance and pathogenicity among S. agalactiae.

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July 7, 2019

pirAB(vp) -bearing Vibrio parahaemolyticus and Vibrio campbellii pathogens isolated from the same AHPND-affected pond possess highly similar pathogenic plasmids.

Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease originally shown to be caused by virulent strains of Vibrio parahaemolyticus (VPAHPND). Rare cases of AHPND caused by Vibrio species other than V. parahaemolyticus were reported. We compared an AHPND-causing V. campbellii (VCAHPND) and a VPAHPND isolate from the same AHPND-affected pond. Both strains are positive for the virulence genes pirAB(vp) . Immersion challenge test with Litopenaeus vannamei indicated the two strains possessed similar pathogenicity. Complete genome comparison showed that the pirAB(vp) -bearing plasmids in the two strains were highly homologous, and they both shared high homologies with plasmid pVA1, the reported pirAB(vp) -bearing plasmid. Conjugation and DNA-uptake genes were found on the pVA1-type plasmids and the host chromosomes, respectively, which may facilitate the dissemination of pirAB(vp) . Novel variations likely driven by ISVal1 in the genetic contexts of the pirAB(vp) genes were found in the two strains. Moreover, the VCAHPND isolate additionally contains multiple antibiotic resistance genes, which may bring difficulties to control its future outbreak. The dissemination of the pirAB(vp) in non-parahaemolyticus Vibrio also rises the concern of missing detection in industrial settings since the isolation method currently used mainly targeting V. parahaemolyticus. This study provides timely information for better understanding of the causes of AHPND and molecular epidemiology of pirAB(vp) and also appeals for precautions to encounter the dissemination of the hazardous genes.

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July 7, 2019

Widespread distribution of mcr-1-bearing bacteria in the ecosystem, 2015 to 2016.

The recently discovered colistin resistance-encoding element, mcr-1, adds to the list of mobile resistance genes whose products rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics, but also the last line agents of carbapenems and colistin. The relative prevalence of mcr-1-bearing strains in various ecological niches including 1,371 food samples, 480 animal faecal samples, 150 human faecal samples and 34 water samples was surveyed using a novel in-house method. Bacteria bearing mcr-1 were commonly detected in water (71% of samples), animal faeces (51%), food products (36%), and exhibited stable carriage in 28% of human subjects surveyed. Such strains, which exhibited variable antibiotic susceptibility profiles, belonged to various Enterobacteriaceae species, with Escherichia coli being the most dominant in each specimen type. The mcr-1 gene was detectable in the chromosome as well as plasmids of various sizes. Among these, two conjugative plasmids of sizes ca?33 and ca?60 kb were found to be the key vectors that mediated mcr-1 transmission in organisms residing in various ecological niches. The high mcr-1 carriage rate in humans found in this study highlights the importance of continued vigilance, careful antibiotic stewardship, and the development of new antimicrobials.

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July 7, 2019

Integrated genomic and proteomic analyses of high-level chloramphenicol resistance in Campylobacter jejuni.

Campylobacter jejuni is a major zoonotic pathogen, and its resistance to antibiotics is of great concern for public health. However, few studies have investigated the global changes of the entire organism with respect to antibiotic resistance. Here, we provide mechanistic insights into high-level resistance to chloramphenicol in C. jejuni, using integrated genomic and proteomic analyses. We identified 27 single nucleotide polymorphisms (SNPs) as well as an efflux pump cmeB mutation that conferred modest resistance. We determined two radical S-adenosylmethionine (SAM) enzymes, one each from an SNP gene and a differentially expressed protein. Validation of major metabolic pathways demonstrated alterations in oxidative phosphorylation and ABC transporters, suggesting energy accumulation and increase in methionine import. Collectively, our data revealed a novel rRNA methylation mechanism by a radical SAM superfamily enzyme, indicating that two resistance mechanisms existed in Campylobacter. This work provided a systems biology perspective on understanding the antibiotic resistance mechanisms in bacteria.

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July 7, 2019

Complete genome sequencing and genomic characterization of two Escherichia coli strains co-producing MCR-1 and NDM-1 from bloodstream infection.

We previously described the discovery of two Escherichia coli isolates (EC1002 and EC2474) co-harbouring mcr-1 and bla NDM-1 genes, which were recovered from bloodstream infection in China. More importantly, these antibiotic resistance genes were located on different plasmids and signaling the potential spread of pandrug-resistant bacteria. Here, the complete genome sequences of both isolates were determined using Pacbio RS II and Illumina HiSeq2000 systems. The genome of EC1002 consists of a 5,177,501 base pair chromosome and four circular plasmids, while the genome of EC2474 consists of a 5,013,813 base pair chromosome and three plasmids. The plasmid replicon type of pEC1002_NDM and pEC2474_NDM were identified as IncA/C2 and IncF, respectively. The genetic environment of bla NDM-1 in this study was similar to bla NDM-carrying plasmids detected in China, although the overall nucleotide identity and query coverage were variable. The plasmid replicon type of pEC1002_MCR and pEC2474_MCR were identified as IncI2 and IncHI2, respectively. Two different genetic strategies for mcr-1 gene spread were observed in this study and bla NDM-1 genes were also found transferred by two different mobile genetic elements in two plasmids. The findings of this study further support that the diversified transfer mechanisms of bla NDM-1 and mcr-1 present in Enterobacteriaceae.

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July 7, 2019

Complete genome sequences of Clostridium perfringens Del1 strain isolated from chickens affected by necrotic enteritis.

Clostridium perfringens is ubiquitous in nature. It is a normal inhabitant in the intestinal tract of animals and humans. As the primary etiological agent of gas gangrene, necrosis and bacteremia, C. perfringens causes food poisoning, necrotic enteritis (NE), and even death. Epidemiology research has indicated that the increasing incidence of NE in poultry is associated with the withdrawal of in-feed antibiotic growth promoters in poultry production in response to government regulations. The recent omics studies have indicated that bacterial virulence is typically linked to highly efficient conjugative transfer of toxins, or plasmids carrying antibiotic-resistance traits. Currently, there is limited information on understanding of host-pathogen interaction in NE caused by virulent strains of C. perfringens. Elucidating such pathogenesis has practical impacts on fighting infectious diseases through adopting strategies of prophylactic or therapeutic interventions. In this report, we sequenced and analyzed the genome of C. perfringens Del1 strain using the hybrid of PacBio and Illumina sequencing technologies.Sequence analysis indicated that Del1 strain comprised a single circular chromosome with a complete 3,559,163 bp and 4 plasmids: pDel1_1 (82,596 bp), pDel1_2 (69,827 bp), pDel1_3 (49,582 bp), and pDel1_4 (49,728 bp). The genome had 3361 predicted coding DNA sequences, harbored numerous genes for pathogenesis and virulence factors, including 6 for antibiotic and antimicrobial resistance, and 3 phage-encoded genes. Phylogenetic analysis revealed that Del1 strain had similar genome and plasmid sequences to the CP4 strain.Complete chromosomal and plasmid sequences of Del1 strain are presented in this report. Since Del1 was isolated from a field disease outbreak, this strain is a good source to identify virulent genes that cause many damaging effects of Clostridial infections in chicken gut. Genome sequencing of the chicken pathogenic isolates from commercial farms provides valuable insights into the molecular pathogenesis of C. perfringens as a gastrointestinal pathogen in food animals. The detailed information on gene sequencing of this important field strain will benefit the development of novel vaccines specific for C. perfringens-induced NE in chickens.

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July 7, 2019

RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum.

The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq).We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745.Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.

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July 7, 2019

Phenotypic and genotypic features of a Salmonella Heidelberg strain isolated in broilers in Brazil and their possible association to antibiotics and short-chain organic acids resistance and susceptibility.

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to aBacillus subtilis-based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibioticsin vitro, and (iii) SCOAin vivo; and (iv) relate these phenotypic observations with its genome characteristics. Twoin vivotrials used 1-day-old chicks housed for 21?days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 107?CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2?×?2 factorial design. Zootechnical parameters were unaffected (P?>?0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge (P?>?0.05). SH UFPR1 was susceptible to amikacin, amoxicillin?+?clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin?+?sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in anin vitrominimum inhibitory concentration model using Mueller-Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321?bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related tobgt, bgr, andrpoSgenes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry.

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July 7, 2019

Comparative genomics reveals specific genetic architectures in nicotine metabolism of Pseudomonassp. JY-Q.

Microbial degradation of nicotine is an important process to control nicotine residues in the aqueous environment. In this study, a high active nicotine degradation strain namedPseudomonassp. JY-Q was isolated from tobacco waste extract (TWE). This strain could completely degrade 5.0 g l-1nicotine in 24 h under optimal culture conditions, and it showed some tolerance even at higher concentrations (10.0 g l-1) of nicotine. The complete genome of JY-Q was sequenced to understand the mechanism by which JY-Q could degrade nicotine and tolerate such high nicotine concentrations. Comparative genomic analysis indicated that JY-Q degrades nicotine through putative novel mechanisms. Two candidate gene cluster duplications located separately at distant loci were predicted to be responsible for nicotine degradation. These two nicotine (Nic) degradation-related loci (AA098_21325-AA098_21340, AA098_03885-AA098_03900) exhibit nearly completely consistent gene organization and component synteny. The nicotinic acid(NA)degradation gene cluster (AA098_17770-AA098_17790) andNic-like clusters were both predicted to be flanked by mobile genetic elements (MGE). Furthermore, we analyzed the regions of genomic plasticity (RGP) in the JY-Q strain and found a dynamic genome carrying a type VI secretion system (T6SS) that promotes nicotine metabolism and tolerance based on transcriptomics and usedin silicomethods to identify the T6SS effector protein. Thus, a novel nicotine degradation mechanism was elucidated forPseudomonassp. JY-Q, suggesting its potential application in the bioremediation of nicotine-contaminated environments, such as TWEs.

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July 7, 2019

Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors.

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including a-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.

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July 7, 2019

Study of mesophilic Aeromonas salmonicida A527 strain sheds light on the species’ lifestyles and taxonomic dilemma.

The Gram-negative bacterium Aeromonas salmonicida contains five subspecies: salmonicida, smithia, achromogenes, masoucida and pectinolytica. Pectinolytica is a mesophilic subspecies with the ability to thrive at a wide range of temperatures, including 37°C, while the four other subspecies are psychrophilic, restricted to lower temperatures. The psychrophilic subspecies are known to infect a wide range of fishes. However, there is no evidence of pathogenicity for the mesophilic subspecies pectinolytica. Study of the differences between the mesophilic and psychrophilic subspecies is hampered by the lack of completely sequenced and closed genomes from the mesophilic subspecies. A previous study reported that insertion sequences, which can induce genomic rearrangements at temperatures around 25°C, could be one of the determinants explaining the differences in lifestyle (mesophilic or psychrophilic) between the subspecies. In this study, the genome of mesophilic strain A527 of A. salmonicida was sequenced, closed and analyzed to investigate the mesophilic-psychrophilic discrepancy. This reference genome supports the hypothesis that insertion sequences are major determinants of the lifestyle differences between the A. salmonicida subspecies. Moreover, the phylogenetic analysis performed to position strain A527 within the taxonomy raises an issue regarding the intraspecies structure of A. salmonicida.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

A blaOXA-181-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance.

Carbapenem resistant Klebsiella pneumoniae (CR-KP) infections are an ever-increasing global issue, especially in the Indian subcontinent. Here we report genetic insight into a blaOXA-181 harbouring Klebsiella pneumoniae, belonging to the pandemic lineage ST147, that represents an intermediate stage towards pan-drug resistance. The CR-KP isolate DA48896 was isolated from a patient from Pakistan and was susceptible only to tigecycline and colistin. It harboured blaOXA-181 and was assigned to sequence type ST147. Analysis from whole genome sequencing revealed a very high sequence similarity to the previously sequenced pan-resistant K. pneumoniae isolate MS6671 from the United Arab Emirates. The two isolates are very closely related with only 46 chromosomal nucleotide differences, 14 indels and differences in plasmid content. Both carry a substantial number of plasmid-borne and chromosomally encoded resistance determinants. Interestingly, the two differences in susceptibility between the isolates could be attributed to DA48896 lacking an insertion of blaOXA-181 into the mgrB gene that results in colistin resistance in MS6671 and SNPs affecting AcrAB efflux pump expression likely to result in tigecycline resistance. These differences between the otherwise very similar isolates indicate that strong selection has occurred for resistance towards these last-resort drugs and illustrates the trajectory of resistance evolution of OXA-181-producing versions of the ST147 international risk clone.

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July 7, 2019

Characterization of Fusobacterium varium Fv113-g1 isolated from a patient with ulcerative colitis based on complete genome sequence and transcriptome analysis.

Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn’s disease and ulcerative colitis (UC). Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb) isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb) and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS) and 13 Fusobacterium adhesion (FadA) paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium.

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July 7, 2019

Genomic characterization of a local epidemic Pseudomonas aeruginosa reveals specific features of the widespread clone ST395.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with several clones being frequently associated with outbreaks in hospital settings. ST395 is among these so-called ‘international’ clones. We aimed here to define the biological features that could have helped the implantation and spread of the clone ST395 in hospital settings. The complete genome of a multidrug resistant index isolate (DHS01) of a large hospital outbreak was analysed. We identified DHS01-specific genetic elements, among which were identified those shared with a panel of six independent ST395 isolates responsible for outbreaks in other hospitals. DHS01 has the fifth largest chromosome of the species (7.1 Mbp), with most of its 1555 accessory genes borne by either genomic islands (GIs,n=48) or integrative and conjugative elements (ICEs,n=5). DHS01 is multidrug resistant mostly due to chromosomal mutations. It displayed signatures of adaptation to chronic infection in part due to the loss of a 131 kbp chromosomal fragment. Four GIs were specific to the clone ST395 and contained genes involved in metabolism (GI-4), in virulence (GI-6) and in resistance to copper (GI-7). GI-7 harboured an array of six copper transporters and was shared with non-pathogenicPseudomonassp. retrieved from copper-contaminated environments. Copper resistance was confirmed phenotypically in all other ST395 isolates and possibly accounted for the spreading capability of the clone in hospital outbreaks, where water networks have been incriminated. This suggests that genes transferred from copper-polluted environments may have favoured the implantation and spread of the international cloneP. aeruginosaST395 in hospital settings.

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