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July 7, 2019

Characterization of oqxAB in Escherichia coli isolates from animals, retail meat, and human patients in Guangzhou, China.

The purpose of this study was to investigate the prevalence and genetic elements of oqxAB among Escherichia coli isolates from animals, retail meat, and humans (patients with infection or colonization) in Guangzhou, China. A total of 1,354 E. coli isolates were screened for oqxAB by PCR. Fifty oqxAB-positive isolates were further characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), S1-PFGE, genetic environment analysis, plasmid replicon typing, and plasmid sequencing. oqxAB was detected in 172 (33.79%), 60 (17.34%), and 90 (18.07%) E. coli isolates from animal, food, and human, respectively. High clonal diversity was observed among oqxAB-positive isolates. In 21 oqxAB-containing transformants, oqxAB was flanked by two IS26 elements in the same orientation, formed a composite transposon Tn6010 in 19 transformants, and was located on plasmids (33.3~500 kb) belonging to IncN1-F33:A-:B- (n = 3), IncHI2/ST3 (n = 3), F-:A18:B- (n = 2), F-:A-:B54 (n = 2), or others. Additionally, oqxAB was co-located with multiple resistance genes on the same plasmid, such as aac(6′)-Ib-cr and/or qnrS, which were identified in two F-:A18:B- plasmids from pigs, and blaCTX-M-55, rmtB, fosA3, and floR, which were detected in two N1-F33:A-:B- plasmids from patients. The two IncHI2/ST3 oqxAB-bearing plasmids, pHNLDF400 and pHNYJC8, which were isolated from human patient and chicken meat, respectively, contained a typical IncHI2-type backbone, and were similar to each other with 2-bp difference, and also showed 99% identity to the Salmonella Typhimurium oqxAB-carrying plasmids pHXY0908 (chicken) and pHK0653 (human patient). Horizontal transfer mediated by mobile elements may be the primary mechanism underlying oqxAB spread in E. coli isolates obtained from various sources in Guangzhou, China. The transmission of identical oqxAB-carrying IncHI2 plasmids between food products and humans might pose a serious threat to public health.

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July 7, 2019

Characterization of four multidrug resistance plasmids captured from the sediments of an urban coastal wetland.

Self-transmissible and mobilizable plasmids contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance. The objective of this study was to capture and characterize self-transmissible and mobilizable resistance plasmids from a coastal wetland impacted by urban stormwater runoff and human wastewater during the rainy season. Four plasmids were captured, two self-transmissible and two mobilizable, using both mating and enrichment approaches. Plasmid genomes, sequenced with either Illumina or PacBio platforms, revealed representatives of incompatibility groups IncP-6, IncR, IncN3, and IncF. The plasmids ranged in size from 36 to 144 kb and encoded known resistance genes for most of the major classes of antibiotics used to treat Gram-negative infections (tetracyclines, sulfonamides, ß-lactams, fluoroquinolones, aminoglycosides, and amphenicols). The mobilizable IncP-6 plasmid pLNU-11 was discovered in a strain of Citrobacter freundii enriched from the wetland sediments with tetracycline and nalidixic acid, and encodes a novel AmpC-like ß-lactamase (blaWDC-1), which shares less than 62% amino acid sequence identity with the PDC class of ß-lactamases found in Pseudomonas aeruginosa. Although the IncR plasmid pTRE-1611 was captured by mating wetland bacteria with P. putida KT2440 as recipient, it was found to be mobilizable rather than self-transmissible. Two self-transmissible multidrug-resistance plasmids were also captured: the small (48 kb) IncN3 plasmid pTRE-131 was captured by mating wetland bacteria with Escherichia coli HY842 where it is seemed to be maintained at nearly 240 copies per cell, while the large (144 kb) IncF plasmid pTRE-2011, which was isolated from a cefotaxime-resistant environmental strain of E. coli ST744, exists at just a single copy per cell. Furthermore, pTRE-2011 bears the globally epidemic blaCTX-M-55 extended-spectrum ß-lactamase downstream of ISEcp1. Our results indicate that urban coastal wetlands are reservoirs of diverse self-transmissible and mobilizable plasmids of relevance to human health.

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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

Rapid gene turnover as a significant source of genetic variation in a recently seeded population of a pathogen.

Genome sequencing has been useful to gain an understanding of bacterial evolution. It has been used for studying the phylogeography and/or the impact of mutation and recombination on bacterial populations. However, it has rarely been used to study gene turnover at microevolutionary scales. Here, we sequenced Mexican strains of the human pathogen Acinetobacter baumannii sampled from the same locale over a 3 year period to obtain insights into the microevolutionary dynamics of gene content variability. We found that the Mexican A. baumannii population was recently founded and has been emerging due to a rapid clonal expansion. Furthermore, we noticed that on average the Mexican strains differed from each other by over 300 genes and, notably, this gene content variation has accrued more frequently and faster than the accumulation of mutations. Moreover, due to its rapid pace, gene content variation reflects the phylogeny only at very short periods of time. Additionally, we found that the external branches of the phylogeny had almost 100 more genes than the internal branches. All in all, these results show that rapid gene turnover has been of paramount importance in producing genetic variation within this population and demonstrate the utility of genome sequencing to study alternative forms of genetic variation.

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July 7, 2019

Repetitive sequences in malaria parasite proteins.

Five species of parasite cause malaria in humans with the most severe disease caused by Plasmodium falciparum. Many of the proteins encoded in the P. falciparum genome are unusually enriched in repetitive low-complexity sequences containing a limited repertoire of amino acids. These repetitive sequences expand and contract dynamically and are among the most rapidly changing sequences in the genome. The simplest repetitive sequences consist of single amino acid repeats such as poly-asparagine tracts that are found in approximately 25% of P. falciparum proteins. More complex repeats of two or more amino acids are also common in diverse parasite protein families. There is no universal explanation for the occurrence of repetitive sequences and it is possible that many confer no function to the encoded protein and no selective advantage or disadvantage to the parasite. However, there are increasing numbers of examples where repetitive sequences are important for parasite protein function. We discuss the diverse roles of low-complexity repetitive sequences throughout the parasite life cycle, from mediating protein-protein interactions to enabling the parasite to evade the host immune system.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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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

Legionnaires’ disease outbreakcaused by endemic strain of Legionella pneumophila, New York, New York, USA, 2015.

During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires’ disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires’ disease.

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July 7, 2019

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori.

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ?modH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

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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

Comparative transcriptome profiling of virulent and non-virulent Trypanosoma cruzi underlines the role of surface proteins during infection.

Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving several morphologically and biochemically distinct stages that establish intricate interactions with various insect and mammalian hosts. It has also a heterogeneous population structure comprising strains with distinct properties such as virulence, sensitivity to drugs, antigenic profile and tissue tropism. We present a comparative transcriptome analysis of two cloned T. cruzi strains that display contrasting virulence phenotypes in animal models of infection: CL Brener is a virulent clone and CL-14 is a clone that is neither infective nor pathogenic in in vivo models of infection. Gene expression analysis of trypomastigotes and intracellular amastigotes harvested at 60 and 96 hours post-infection (hpi) of human fibroblasts revealed large differences that reflect the parasite’s adaptation to distinct environments during the infection of mammalian cells, including changes in energy sources, oxidative stress responses, cell cycle control and cell surface components. While extensive transcriptome remodeling was observed when trypomastigotes of both strains were compared to 60 hpi amastigotes, differences in gene expression were much less pronounced when 96 hpi amastigotes and trypomastigotes of CL Brener were compared. In contrast, the differentiation of the avirulent CL-14 from 96 hpi amastigotes to extracellular trypomastigotes was associated with considerable changes in gene expression, particularly in gene families encoding surface proteins such as trans-sialidases, mucins and the mucin associated surface proteins (MASPs). Thus, our comparative transcriptome analysis indicates that the avirulent phenotype of CL-14 may be due, at least in part, to a reduced or delayed expression of genes encoding surface proteins that are associated with the transition of amastigotes to trypomastigotes, an essential step in the establishment of the infection in the mammalian host. Confirming the role of members of the trans-sialidase family of surface proteins for parasite differentiation, transfected CL-14 constitutively expressing a trans-sialidase gene displayed faster kinetics of trypomastigote release in the supernatant of infected cells compared to wild type CL-14.

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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

Streptococcal toxic shock syndrome caused by the dissemination of an invasive emm3/ST15 strain of Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen that causes a wide spectrum of clinical manifestations. Although invasive GAS (iGAS) infections are relatively uncommon, emm3/ST15 GAS is a highly virulent, invasive, and pathogenic strain. Global molecular epidemiology analysis has suggested that the frequency of emm3 GAS has been recently increasing.A 14-year-old patient was diagnosed with streptococcal toxic shock syndrome and severe pneumonia, impaired renal function, and rhabdomyolysis. GAS was isolated from a culture of endotracheal aspirates and designated as KS030. Comparative genome analysis suggested that KS030 is classified as emm3 (emm-type) and ST15 (multilocus sequencing typing [MLST]), which is similar to iGAS isolates identified in the UK (2013) and Switzerland (2015).We conclude that the global dissemination of emm3/ST15 GAS strain has the potential to cause invasive disease.

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July 7, 2019

A novel Tn1696-like composite transposon (Tn6404) harboring bla IMP-4 in a Klebsiella pneumoniae isolate carrying a rare ESBL gene bla SFO-1.

Genetic determinants of a clinical Klebsiella pneumoniae isolate (KP1814) coproducing IMP-4 and a rare ESBL gene SFO-1 was investigated. KP1814 belongs to a novel sequence type (ST) assigned to ST2270. WGS identified four circular DNA sequences in KP1814, including two multidrug-resistance (MDR) plasmids, one virulence plasmid, and one circular form. The MDR plasmid pKP1814-1 (299.9 Kb) is untypeable, and carries two large mosaic multiresistance regions (MRRs). bla SFO-1 and bla IMP-4 co-exists on MRR1, and bla SFO-1 is associated with an IS/Tn-independent genetic context. bla IMP-4 is carried by a novel In804-like integron (intlI-bla IMP-4-Kl.pn.I3-qacG2-aacA4-catB3?) associated with a novel Tn1696-like transposon (designed Tn6404) flanked by IS5075. The other MDR plasmid pKP1814-3 is a 95,701-bp IncFII plasmid, and is a hybrid of a Shigella flexneri plasmid pSF07201 and an E. coli plasmid pCA08. All resistance genes of pKP1814-3 were detected in a ~16-kb IS26-flanked composite transposon carried by a Tn5396 transposon. The circular form (18.3 Kb) was composed of two parts belonging to pKP1814-1 and pKP1814-3, respectively. The plasmid pKP1814-2, carrying multiple virulence factors, encodes IncFIBK and IncFIIK replicons with a size of 187,349?bp. The coexistence of MDR and virulence plasmids largely enhances the bacterial fitness in the host and environment.

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