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Acinetobacter baumannii is a leading pathogen responsible for nosocomial infections. The emergence of carbapenem-resistant A. baumannii (CRAB) has left few effective antibiotics for clinicians to use. To investigate the temporal evolutionary relationships among CRAB strains, we collected 248 CRAB isolates from a Chinese burns institute over 3 years. The prevalence of the OXA-23 gene was detected by polymerase chain reaction. Multilocus sequence typing was used to type the CRAB strains and eBURST was used to analyze their evolutionary relationships. Wound surfaces (41%), sputa (24%), catheters (15%), and bloods (14%) were the four dominant isolation sources. Except for minocycline (33.5%) and sulbactam/cefoperazone (74.6%), these CRAB strains showed high resistance rates (>90%) to 16 tested antibiotics. The 248 isolates fall into 26 sequence types (STs), including nine known STs and 17 unknown STs. The majority (230/248) of these isolates belong to clonal complex 92 (CC92), including eight isolates belonging to seven unreported STs. A new CC containing 11 isolates grouped into four new STs was identified. The OXA-23 gene was detected at high prevalence among the CRAB isolates and the prevalence rate among the various STs differed. The majority of the isolates displayed a close evolutionary relationship, suggesting that serious nosocomial spreading and nosocomial infections of CRAB have occurred in the burn unit. In conclusion, the main CC for CRAB in this Chinese burn unit remained unchanged during the 3-year study period, and a new CC was identified. CC92 was the dominant complex, and more attention should be directed toward monitoring the new CC we have identified herein. [ABSTRACT FROM AUTHOR]

We investigated global patterns of variation in 157 whole-genome sequences of Vibrio parahaemolyticus, a free-living and seafood associated marine bacterium. Pandemic clones, responsible for recent outbreaks of gastroenteritis in humans, have spread globally. However, there are oceanic gene pools, one located in the oceans surrounding Asia and another in the Mexican Gulf. Frequent recombination means that most isolates have acquired the genetic profile of their current location. We investigated the genetic structure in the Asian gene pool by calculating the effective population size in two different ways. Under standard neutral models, the two estimates should give similar answers but we found a 27-fold difference. We propose that this discrepancy is caused by the subdivision of the species into a hundred or more ecotypes which are maintained stably in the population. To investigate the genetic factors involved, we used 51 unrelated isolates to conduct a genome-wide scan for epistatically interacting loci. We found a single example of strong epistasis between distant genome regions. A majority of strains had a type VI secretion system associated with bacterial killing. The remaining strains had genes associated with biofilm formation and regulated by cyclic dimeric GMP signaling. All strains had one or other of the two systems and none of isolate had complete complements of both systems, although several strains had remnants. Further "top down" analysis of patterns of linkage disequilibrium within frequently recombining species will allow a detailed understanding of how selection acts to structure the pattern of variation within natural bacterial populations. [ABSTRACT FROM AUTHOR]

Yersinia pestis, the causative agent of plague, poses a serious health threat to rodents and human beings. TyrR is a transcriptional regulator (TyrR) that controls the metabolism of aromatic amino acids in Escherichia coli. In this paper, TyrR played an important role in Y. pestis virulence. Inactivation of tyrR did not seem to affect the in vitro growth of this organism, but resulted in at least 10,000-fold attenuation compared with the wild-type (WT) strain upon subcutaneous infection to mice. In addition, loads of tyrR mutant within mice livers and spleens significantly decreased compared with the WT strain. Transcriptome analysis revealed that TyrR, directly or indirectly, regulated 29 genes encoded on Y. pestis chromosome or plasmids under in vitro growth condition. Similar to the regulatory function of this protein in E. coli, five aromatic-pathway genes (aroF-tyrA, aroP, aroL, and tyrP) were significantly reduced upon deletion of the tyrR gene. Two genes (glnL and glnG) that encode sensory histidine kinase and regulator in a two-component regulatory system involved in nitrogen assimilation were downregulated in the tyrR mutant. Several genes encoding type III secretion proteins were transcribed by 2.0-4.2-fold in a tyrR mutant relative to the WT strain. Interestingly, the acid-stressed genes, hdeB and hdeD, were downregulated, and such downregulation partly accounted for the decrease in tolerance of the tyrR mutant under acidic conditions. In conclusion, regulation of TyrR in Y. pestis is similar to, but distinct from, that in E. coli. TyrR is a metabolic virulence determinant in Y. pestis that is important for extracellular survival and/or proliferation. [ABSTRACT FROM AUTHOR]

We report Salmonella enterica serotype Typhi strains with a nonclassical quinolone resistance phenotype (i.e., decreased susceptibility to ciprofloxacin but with susceptibility to nalidixic acid) associated with a nonsynonymous mutation at codon 464 of the gyrB gene. These strains, not detected by the nalidixic acid disk screening test, can result in fluoroquinolone treatment failure. [ABSTRACT FROM AUTHOR]

Background. Primary pneumonic plague (PPP) caused by Yersinia pestis is the most threatening clinical form of plague. An outbreak was reported in July 2009 in Qinghai Province, China. Methods. This outbreak was investigated by clinical, epidemiological, bacteriological, and immunological methods. Multilocus variable number tandem repeat analysis (MLVA) was used to track the source of the outbreak. Results. The index case, a patient with PPP, contaminated 11 close contacts. All the 12 cases, including the index patient, experienced sudden onset of fever, headache, and productive coughing with bloody sputum. Three of them died. Nevertheless, another 61 direct and 256 indirect contacts were not infected during the 2-week quarantine. Antibodies to F1 antigen were detected in 9 survival cases, with a 4-fold increase in titers in serum samples collected at different periods. Seven strains of Y. pestis were isolated from dogs and patients. Field investigation and MLVA of the isolated strains revealed that this outbreak was started by a deceased dog. Conclusion. Dogs are believed to be an indicator animal for plague surveillance, but their association with PPP is rare. Our results provide evidence for this possibility, which suggests the public health significance of dogs as a source of plague. [ABSTRACT FROM AUTHOR]

Objectives: Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. [ABSTRACT FROM PUBLISHER]

Background: Salmonella Typhi (S. Typhi) causes typhoid fever, which remains an important public health issue in many developing countries. Kathmandu, the capital of Nepal, is an area of high incidence and the pediatric population appears to be at high risk of exposure and infection. Methods: We recently defined the population structure of S. Typhi, using new sequencing technologies to identify nearly 2,000 single nucleotide polymorphisms (SNPs) that can be used as unequivocal phylogenetic markers. Here we have used the GoldenGate (Illumina) platform to simultaneously type 1,500 of these SNPs in 62 S. Typhi isolates causing severe typhoid in children admitted to Patan Hospital in Kathmandu. Results: Eight distinct S. Typhi haplotypes were identified during the 20-month study period, with 68% of isolates belonging to a subclone of the previously defined H58 S. Typhi. This subclone was closely associated with resistance to nalidixic acid, with all isolates from this group demonstrating a resistant phenotype and harbouring the same resistance-associated SNP in GyrA (Phe83). A secondary clone, comprising 19% of isolates, was observed only during the second half of the study. Conclusions: Our data demonstrate the utility of SNP typing for monitoring bacterial populations over a defined period in a single endemic setting. We provide evidence for genotype introduction and define a nalidixic acid resistant subclone of S. Typhi, which appears to be the dominant cause of severe pediatric typhoid in Kathmandu during the study period. [ABSTRACT FROM AUTHOR]

The article examines the interaction between Yersinia pestis and its host at the molecular level during pneumonic plague development. It states that pneumonic plague that can be transmitted via respiratory route is more acute than bubonic one with higher mortality. During the study, 32 genes of Y. pestis were selected to determine the gene expression. The expression of each gene was quantified by matching their standard curves. All the data were then normalized to the expression data of 16S rRNA and then transformed as the ratio against the corresponding in vitro sample,

The catalase or catalase-peroxidase activity commonly exists in many pathogens and plays an important role in resisting the oxidative burst of phagocytes helping the pathogen persistently colonize in the host. Yersinia pestis is a facultative pathogen and the causative agent of plague. KatY has been identified as a thermosensing antigen with modest catalase activity in this pathogen. Here Y. pestis KatA and KatY were experimentally confirmed as a monofunctional catalase and bifunctional catalase-peroxidase, respectively. Their expression induced by H2O2 was proven to be mediated by the oxidative regulator, OxyR. Expression of KatA changed with growth phases and was crucial to its traditional physiological role in protecting Y. pestis cells against toxicity of exogenous H2O2. KatY was regulated by temperature and H2O2, two major elements of phagolysosomal microenvironments. Consistent with the above results, gene expression of katY increased significantly during intracellular growth of Y. pestis compared with that in vitro growth. However, a ΔkatY mutant was fully virulent to mice, suggesting that KatY is not required for Y. pestis virulence. [ABSTRACT FROM AUTHOR]

Background: The transcription regulator PhoP has been shown to be important for Y. pestis survival in macrophages and under various in vitro stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in Y. pestis. A series of biochemical experiments were done herein to study members of the PhoP regulon of Y. pestis biovar Microtus. Results: By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in Y. pestis. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation. Conclusion: The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which Y. pestis survives the antibacterial strategies employed by host macrophages. [ABSTRACT FROM AUTHOR]

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Background: Environmental modulation of gene expression in Yersinia pestis is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions in vitro. Results: To provide us with a comprehensive view of environmental modulation of global gene expression in Y. pestis, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of Y. pestis were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA). Conclusion: The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in Y. pestis, it also serves as a basis for integrating increasing volumes of microarray data using existing methods. [ABSTRACT FROM AUTHOR]

Plague is a deadly disease caused by Yersinia pestis. Human plague can be effectively controlled by timely antibiotic administration, chloramphenicol being a drug of choice. In this study, a DNA microarray was used to investigate the gene expression profile of Y. pestis in response to chloramphenicol. Seven hundred and fifty-five genes were differentially expressed on chloramphenicol treatment: 364 genes were up-regulated and 391 were down-regulated. In addition to a large number of genes encoding unknown or unassigned functions, genes encoding the components of the translation apparatus, cell envelope and transport/binding functions were strongly represented amongst the induced genes. Genes encoding proteins involved in energy metabolism and synthesis and modification of macromolecules were strongly represented amongst the down-regulated genes. A number of heat-shock genes were also repressed. These global transcriptional changes provide an insight into the mechanisms of action of chloramphenicol against Y. pestis. [ABSTRACT FROM AUTHOR]

Genomics research provides an unprecedented opportunity for us to probe into the pathogenicity and evolution of the world's most deadly pathogenic bacterium, Yersinia pestis, in minute detail. In our present work, extensive microarray analysis in conjunction with PCR validation revealed that there are considerable genome dynamics, due to gene acquisition and loss, in natural populations of Y. pestis. We established a genomotyping system to group homologous isolates of Y. pestis, based on profiling or gene acquisition and loss in their genomes, and then drew an outline of parallel microevolution of the Y. pestis genome. The acquisition of a number of genomic islands and plasmids most likely induced Y. pestis to evolve rapidly from Yersinia pseudotuberculosis to a new, deadly pathogen. Horizontal gene acquisition also plays a key role in the dramatic evolutionary segregation of Y. pestis lineages (biovars and genomovars). In contrast to selective genome expansion by gene acquisition, genome reduction occurs in Y. pestis through the loss of DNA regions. We also theorized about the links between niche adaptation and genome microevolution. The transmission, colonization, and expansion of Y. pestis in the natural foci of endemic plague are parallel and directional and involve gradual adaptation to the complex of interactions between the environment, the hosts, and the pathogen itself. These adaptations are based on the natural selections against the accumulation of genetic changes within genome. Our data strongly support that the modern plague originated from Yunnan Province in China, due to the arising of biovar orientalis from biovar antiqua rather than mediaevalis. [ABSTRACT FROM AUTHOR]

Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically-dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis. Multiple genes encoded on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold-tolerance in in vitro phenotypic assays. Further, silencing of virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions. [ABSTRACT FROM AUTHOR]

Innate immune cells, including neutrophils and macrophages, are critically involved in host antimicrobial defense responses. Intrinsic regulatory mechanisms controlling neutrophil and macrophage activities are poorly defined. In this study,we found that IL-17A, a natural signal factor, could provide protection against early pneumonic plague inflammation by coordinating the functions of neutrophils and programming of macrophages. The IL-17A level is promptly increased during the initial infection. Importantly, abrogation of IL-17A or IL-17AR significantly aggravated the infection, but mIL-17A treatment could significantly alleviate inflammatory injury, revealing that IL-17A is a critical requirement for early protection of infection. We also demonstrated that IL-17A was predominantly produced by CD11b+Ly6G+ neutrophils. Although IL-17A could not significantly affect the antimicrobial responses of neutrophils, it could target the proinflammatory macrophage (M1) programming and potentiate the M1's defense against pneumonic plague. Mechanistically, IFN-γ treatment or IFN-γ-activated M1 macrophage transfer could significantly mitigate the aggravated infection of IL-17A-/- mice. Finally, we showed that IL-17A and IFN-γ could synergistically promote macrophage anti-infection immunity. Thus, our findings identify a previously unrecognized function of IL-17A as an intrinsic regulator in coordinating neutrophil and macrophage antimicrobial activity to provide protection against acute pneumonic plague. [ABSTRACT FROM AUTHOR]

Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions. [ABSTRACT FROM AUTHOR]

Yersinia pestis is one of the most dangerous bacterial pathogens. PhoP and cyclic AMP receptor protein (CRP) are global regulators of Y. pestis, and they control two distinct regulons that contain multiple virulence-related genes. The PhoP regulator and its cognate sensor PhoQ constitute a two-component regulatory system. The regulatory activity of CRP is triggered only by binding to its cofactor cAMP, which is synthesized from ATP by adenylyl cyclase (encoded by cyaA). However, the association between the two regulatory systems PhoP/PhoQ and CRP-cAMP is still not understood for Y. pestis. In the present work, the four consecutive genes YPO1635, phoP, phoQ, and YPO1632 were found to constitute an operon, YPO1635-phoPQ-YPO1632, transcribed as a single primary RNA, whereas the last three genes comprised another operon, phoPQ-YPO1632, transcribed with two adjacent transcriptional starts. Through direct PhoP-target promoter association, the transcription of these two operons was stimulated and repressed by PhoP, respectively; thus, both positive autoregulation and negative autoregulation of PhoP/PhoQ were detected. In addition, PhoP acted as a direct transcriptional activator of crp and cyaA. The translational/transcriptional start sites, promoter - 10 and -35 elements, PhoP sites, and PhoP box-like sequences were determined for these PhoP-dependent genes, providing a map of the PhoP-target promoter interaction. The CRP and PhoP regulons have evolved to merge into a single regulatory cascade in Y. pestis because of the direct regulatory association between PhoP/PhoQ and CRP-cAMP. [ABSTRACT FROM AUTHOR]

Evaluates the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in identifying Staphylococcus aureus and in determining its methicillin resistance. Heterogeneous nature of methicillin resistance in aureus; Replicate spectral profiles of isolate; Polymerase chain reaction amplification.

We deciphered the genome of Yersinia pestis strain 2501, isolated from the Junggar Basin, a newly discovered great gerbil plague focus in Xinjiang, China. The total length of assembly was 4,597,322 bp, and 4,265 coding sequences were predicted within the genome. It is the first Y. pestis genome from this plague focus. [ABSTRACT FROM AUTHOR]

Legionella (Fluoribacter) dumoffii is one of the agents causing Legionnaires' disease. Here, we used Illumina second-generation sequencing technology to decipher for the first time the whole-genome sequences of two strains of this species, TEX-KL and NY-23. The assembly results for both strains consist of one chromosome and two plasmids. [ABSTRACT FROM AUTHOR]

Here we report the draft genome sequence of one Acinetobacter genomic species 3 strain, D499, which harbors the blaNDM-1 gene. The total length of the assembled genome is 4,103,824 bp, and 3,896 coding sequences (CDSs) were predicted within the genome A previously unreported blaNDM-1-bearing plasmid was identified in this strain. [ABSTRACT FROM AUTHOR]

To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague. [ABSTRACT FROM AUTHOR]

The ferric uptake regulator (Fur) is a predominant bacterial regulator controlling the iron assimilation functions in response to iron availability. Our previous microarray analysis on Yersinia pestis defined the iron-Fur modulon. In the present work, we reannotated the iron assimilation genes in Y. pestis, and the resulting genes in complementation with those disclosed by microarray constituted a total of 34 genome loci (putative operons) that represent the potential iron-responsive targets of Fur. The subsequent real-time reverse transcription-PCR (RT-PCR) in conjunction with the primer extension analysis showed that 32 of them were regulated by Fur in response to iron starvation. A previously predicted Fur box sequence was then used to search against the promoter regions of the 34 operons; the homologue of the above box could be predicted in each promoter tested. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified His6 tag-fused Fur protein was able to bind in vitro to each of these promoter regions. Therefore, Fur is a global regulator, both an activator and a repressor, and directly controls not only almost all of the iron assimilation functions but also a variety of genes involved in various non-iron functions for governing a complex regulatory cascade in Y. pestis. In addition, real-time RT-PCR, primer extension, EMSA, and DNase I footprinting assay were used to elucidate the Fur regulation of the ybt locus encoding a virulence-required iron uptake system. By combining the published data on the YbtA regulation of ybt, we constructed a concise Fur/YbtA regulatory network with a map of the Fur-promoter DNA interactions within the ybt locus. The data presented here give us an overview of the iron-responsive Fur regulon in Y. pestis. [ABSTRACT FROM AUTHOR]