Yersinia Pestis CO92 Research Articles

Unraveling virulence determinants in extended-spectrum beta-lactamase-producing Escherichia coli from East Africa using whole-genome sequencing

Escherichia coli significantly causes nosocomial infections and rampant spread of antimicrobial resistance (AMR). There is limited data on genomic characterization of extended-spectrum β-lactamase (ESBL)-producing E. coli from African clinical settings. This hospital-based longitudinal study unraveled the genetic resistance elements in ESBL E. coli isolates from Uganda and Tanzania using whole-genome sequencing (WGS). A total of 142 ESBL multi-drug resistant E. coli bacterial isolates from both Tanzania and Uganda were sequenced and out of these, 36/57 (63.1%) and 67/85 (78.8%) originated from Uganda and Tanzania respectively. Mutations in RarD, yaaA and ybgl conferring resistances to chloramphenicol, peroxidase and quinolones were observed from Ugandan and Tanzanian isolates. We reported very high frequencies for blaCTX−M−15 with 11/18(61.1%), and blaCTX−M−27 with 12/23 (52.1%), blaTEM−1B with 13/23 (56.5%) of isolates originating from Uganda and Tanzania respectively all conferring resistance to Beta-lactam-penicillin inhibitors. We observed chloramphenicol resistance-conferring gene mdfA in 21/23 (91.3%) of Tanzanian isolates. Extraintestinal E. coli sequence type (ST) 131 accounted for 5/59 (8.4%) of Tanzanian isolates while enterotoxigenic E. coli ST656 was reported in 9/34 (26.4%) of Ugandan isolates. Virulence factors originating from Shigella dysenteriae Sd197 (gspC, gspD, gspE, gspF, gspG, gspF, gspH, gspI), Yersinia pestis CO92 (irp1, ybtU, ybtX, iucA), Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (csgF and csgG), and Pseudomonas aeruginosa PAO1 (flhA, fliG, fliM) were identified in these isolates. Overall, this study highlights a concerning prevalence and diversity of AMR-conferring elements shaping the genomic structure of multi-drug resistant E. coli in clinical settings in East Africa. It underscores the urgent need to strengthen infection-prevention controls and advocate for the routine use of WGS in national AMR surveillance and monitoring programs.Availability of WGS analysis pipeline: the rMAP source codes, installation, and implementation manual can free be accessed via https://github.com/GunzIvan28/rMAP.

Cell-Free Protein Systems from Yersinia pestis are Functional and Growth-Temperature Dependent.

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92- are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.

Rapid Diagnosis of Bacterial Pneumonia Using Volatile Organic Compounds (VOCs)

Timely diagnosis and treatment is essential for reducing morbidity, mortality, and inappropriate antibiotic usage often associated with bacterial pneumonia. Volatile organic compounds (VOCs) are a large and chemically diverse group of carbon‐based molecules, generally related by their volatility at ambient temperature. We have demonstrated the diagnostic potential of VOCs derived from biological samples 1– 4, and have established uniquely identifying microbial VOC fingerprints of several bacterial agents of pneumonia, including Francisella tularensis (Rabbit Fever) and Yersinia pestis (Plague). Additionally, we have created VOC fingerprints that distinguish wildtype from genetically engineered kanamycin‐resistant strains of select biothreat agents. We illustrate here the use of our rapid non‐invasive approach to the diagnosis of BALB/c mice infected with either Yersinia pestis CO92 or Francisella tularensis SCHU S4.Support or Funding InformationFunding is provided by DTRA (Grant Number: HDTRA 1‐16‐1‐0040).

Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.

Spectrally-resolved fluorescence cross sections of aerosolized biological live agents and simulants using five excitation wavelengths in a BSL-3 laboratory

A system for measuring spectrally-resolved fluorescence cross sections of single bioaerosol particles has been developed and employed in a biological safety level 3 (BSL-3) facility at Edgewood Chemical and Biological Center (ECBC). It is used to aerosolize the slurry or solution of live agents and surrogates into dried micron-size particles, and to measure the fluorescence spectra and sizes of the particles one at a time. Spectrally-resolved fluorescence cross sections were measured for (1) bacterial spores: Bacillus anthracis Ames (BaA), B. atrophaeus var. globigii (BG) (formerly known as Bacillus globigii), B. thuringiensis israelensis (Bti), B. thuringiensis kurstaki (Btk), B. anthracis Sterne (BaS); (2) vegetative bacteria: Escherichia coli (E. coli), Pantoea agglomerans (Eh) (formerly known as Erwinia herbicola), Yersinia rohdei (Yr), Yersinia pestis CO92 (Yp); and (3) virus preparations: Venezuelan equine encephalitis TC83 (VEE) and the bacteriophage MS2. The excitation wavelengths were 266 nm, 273 nm, 280 nm, 365 nm and 405 nm.

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD50. Intranasal infection of mice with 15 LD50 of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time.

Plague in Guinea Pigs and Its Prevention by Subunit Vaccines

Human pneumonic plague is a devastating and transmissible disease for which a Food and Drug Administration-approved vaccine is not available. Suitable animal models may be adopted as a surrogate for human plague to fulfill regulatory requirements for vaccine efficacy testing. To develop an alternative to pneumonic plague in nonhuman primates, we explored guinea pigs as a model system. On intranasal instillation of a fully virulent strain, Yersinia pestis CO92, guinea pigs developed lethal lung infections with hemorrhagic necrosis, massive bacterial replication in the respiratory system, and blood-borne dissemination to other organ systems. Expression of the Y. pestis F1 capsule was not required for the development of pulmonary infection; however, the capsule seemed to be important for the establishment of bubonic plague. The mean lethal dose (MLD) for pneumonic plague in guinea pigs was estimated to be 1000 colony-forming units. Immunization of guinea pigs with the recombinant forms of LcrV, a protein that resides at the tip of Yersinia type III secretion needles, or F1 capsule generated robust humoral immune responses. Whereas LcrV immunization resulted in partial protection against pneumonic plague challenge with 250 MLD Y. pestis CO92, immunization with recombinant F1 did not. rV10, a vaccine variant lacking LcrV residues 271-300, elicited protection against pneumonic plague, which seemed to be based on conformational antibodies directed against LcrV.

An experimentally-supported genome-scale metabolic network reconstruction for Yersinia pestis CO92

BackgroundYersinia pestis is a gram-negative bacterium that causes plague, a disease linked historically to the Black Death in Europe during the Middle Ages and to several outbreaks during the modern era. Metabolism in Y. pestis displays remarkable flexibility and robustness, allowing the bacterium to proliferate in both warm-blooded mammalian hosts and cold-blooded insect vectors such as fleas.ResultsHere we report a genome-scale reconstruction and mathematical model of metabolism for Y. pestis CO92 and supporting experimental growth and metabolite measurements. The model contains 815 genes, 678 proteins, 963 unique metabolites and 1678 reactions, accurately simulates growth on a range of carbon sources both qualitatively and quantitatively, and identifies gaps in several key biosynthetic pathways and suggests how those gaps might be filled. Furthermore, our model presents hypotheses to explain certain known nutritional requirements characteristic of this strain.ConclusionsY. pestis continues to be a dangerous threat to human health during modern times. The Y. pestis genome-scale metabolic reconstruction presented here, which has been benchmarked against experimental data and correctly reproduces known phenotypes, provides an in silico platform with which to investigate the metabolism of this important human pathogen.

Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague

Deletion of the murein (Braun) lipoprotein gene, lpp, attenuates the Yersinia pestis CO92 strain in mouse models of bubonic and pneumonic plague. In this report, we characterized the virulence of strains from which the plasminogen activating protease (pla)-encoding pPCP1 plasmid was cured from either the wild-type (WT) or the Deltalpp mutant strain of Y. pestis CO92 in the mouse model of pneumonic infection. We noted a significantly increased survival rate in mice infected with the Y. pestis pPCP(-)/Deltalpp mutant strain up to a dose of 5000 LD(50). Additionally, mice challenged with the pPCP(-)/Deltalpp strain had substantially less tissue injury and a strong decrease in the levels of most cytokines and chemokines in tissue homogenates and sera when compared with the WT-infected group. Importantly, the Y. pestis pPCP(-)/Deltalpp mutant strain was detectable in high numbers in the livers and spleens of some of the infected mice. In the lungs of pPCP(-)/Deltalpp mutant-challenged animals, however, bacterial numbers dropped at 48 h after infection when compared with tissue homogenates from 1 h post-infection. Similarly, we noted that this mutant was unable to survive within murine macrophages in an in vitro assay, whereas survivability of the pPCP(-) mutant within the macrophage environment was similar to that of the WT. Taken together, our data indicated that a significant and possibly synergistic attenuation in bacterial virulence occurred in a mouse model of pneumonic plague when both the lpp gene and the virulence plasmid pPCP1 encoding the pla gene were deleted from Y. pestis.

Evaluation of ultrafiltration cartridges for a water sampling apparatus

To determine the efficiency of various ultrafiltration cartridges (UFC) in concentrating test micro-organisms from drinking water. Replicate drinking water samples from three potable water supplies were dosed with Bacillus anthracis Sterne, Francisella tularensis LVS, Yersinia pestis CO92, bacteriophages MS2 and phi-X174, and Cryptosporidium parvum. The test micro-organisms were dosed together in 100 l of water, which was then recirculated through one of five different UFC until the retentate volume was reduced to c. 500 ml. The micro-organisms were assayed before and after ultrafiltration concentration and per cent recoveries were calculated. There were nine statistically significant differences among pairs of filters out of a possible 180 different combinations of UFC, test micro-organisms, and water types. No filter consistently performed better or worse than the others for each test micro-organism in all water samples tested. This study provides performance data on the ability of several different UFC to concentrate a panel of test micro-organisms from three sources of potable water. Water utilities and first responders may use these data when selecting UFC for use in emergency response protocols. This study also provides additional data as to the efficacy of ultrafiltration for recovering bacteria, virus-like particles, and protozoan oocysts from water samples.

Recognizing the pseudogenes in bacterial genomes

Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1–8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as ‘hypothetical’ or ‘unknown’; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes.

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50–96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus ( AJ308438), Photorhabdus luminescens W14 ( AF346499) P. luminescens TTO1 ( BX571873), and Yersinia pestis CO92 ( NC_003143). The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.