In-vitro gene disruption using Tn7-based transposition: evaluation of its utility and efficiency in generating a Brucella virB1 knockout

A Tn7-based transposition system carrying a kanamycin resistance gene has been used to disrupt a known virulence gene of Brucella melitensis 16M and Brucella abortus 2308 in-vitro. The suicide plasmid pBAvirB1::kan-29 was created, in part, by cloning the B. abortus virB1 gene into a vector that is not stably maintained in Brucella spp. The cloned virB1 gene was then mutagenized by in-vitro transposition of a Tn7-based transposon. This construct was used in-vivo to insert a disrupted virB1 gene in place of the wild type virB1 found in B. abortus 2308 and B. melitensis 16M by homologous recombination. The resulting virB1 mutant brucellae exhibited resistance to kanamycin, decreased survival in murine macrophages and attenuated virulence in a BALB/c mouse model. This method is proposed to be a simple, efficient means of generating gene knockouts in Brucella spp., and may be useful in targeting virulence factors for the creation of live, attenuated vaccines. The genus Brucella consists of bacteria that are facultative intracellular pathogens. Individuals in this genus of Gram-negative coccobacilli are responsible for causing late term spontaneous abortion in cattle and goats as well as the human disease brucellosis (undulant fever). The success of Brucella is dependent on their ability to invade, survive and multiply within phagocytes. The virB operon, consisting of eleven genes in most Brucella species, is thought to contribute to a type IV secretion system responsible for maintaining infection in a mammalian host. Further results of this study suggest that the Brucella virB1 gene product, like its Agrobacterium counterpart, may not be essential for virulence; disruption of the Brucella virB1 gene results in a one to two log decrease in intracellular survival at forty-eight hours in an in-vitro macrophage model and by four weeks in an in-vivo mouse model

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay

AbstractLegionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5′-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Genomic heterogeneity differentiates clinical and environmental subgroups of Legionella pneumophila sequence type 1.

Legionella spp. are the cause of a severe bacterial pneumonia known as Legionnaires' disease (LD). In some cases, current genetic subtyping methods cannot resolve LD outbreaks caused by common, potentially endemic L. pneumophila (Lp) sequence types (ST), which complicates laboratory investigations and environmental source attribution. In the United States (US), ST1 is the most prevalent clinical and environmental Lp sequence type. In order to characterize the ST1 population, we sequenced 289 outbreak and non-outbreak associated clinical and environmental ST1 and ST1-variant Lp strains from the US and, together with international isolate sequences, explored their genetic and geographic diversity. The ST1 population was highly conserved at the nucleotide level; 98% of core nucleotide positions were invariant and environmental isolates unassociated with human disease (n = 99) contained ~65% more nucleotide diversity compared to clinical-sporadic (n = 139) or outbreak-associated (n = 28) ST1 subgroups. The accessory pangenome of environmental isolates was also ~30-60% larger than other subgroups and was enriched for transposition and conjugative transfer-associated elements. Up to ~10% of US ST1 genetic variation could be explained by geographic origin, but considerable genetic conservation existed among strains isolated from geographically distant states and from different decades. These findings provide new insight into the ST1 population structure and establish a foundation for interpreting genetic relationships among ST1 strains; these data may also inform future analyses for improved outbreak investigations

Genomic Analysis Reveals Novel Diversity among the 1976 Philadelphia Legionnaires’ Disease Outbreak Isolates and Additional ST36 Strains

<div><p><i>Legionella pneumophila</i> was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires’ disease (LD) at a Pennsylvania veterans’ convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of <i>Legionella</i> epidemiology and scientific research. Only one of the original isolates, “Philadelphia-1”, has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with <i>L</i>. <i>pneumophila</i> isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with <i>L</i>. <i>pneumophila</i> str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of “Broad Street pneumonia”, and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for <i>Legionella</i> outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.</p></div

Nucleotide polymorphisms shared by strains CDC Philadelphia-2, -3, -4, and ATCC Philadelphia-1 relative to the NCBI Philadelphia-1 reference sequence.

<p>Nucleotide polymorphisms shared by strains CDC Philadelphia-2, -3, -4, and ATCC Philadelphia-1 relative to the NCBI Philadelphia-1 reference sequence.</p

Nucleotide polymorphisms shared by all CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.

<p>Nucleotide polymorphisms shared by all CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.</p

Mauve whole-genome alignment of <i>L</i>. <i>pneumophila</i> strains within the Philadelphia clade.

<p>ProgressiveMauve was used to compare the fully assembled sequences of the Philadelphia historical <i>Legionella</i> strains as well as isolate E1-P. The minimum weight for pairwise LCBs (locally collinear blocks), which share common colors across genomes, was set to 100, otherwise, the program was run using default parameters as described in the Methods. The general clade organization, as well as the identity and location of the ~40-kb pP36-Ph and the ~45-kb pLP45 elements are shown. The general, expanded Philadelphia clade organization from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164074#pone.0164074.g001" target="_blank">Fig 1</a> is shown, therefore the phylogenetic distances are not to scale.</p

Nucleotide polymorphisms unique to the CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.

<p>Nucleotide polymorphisms unique to the CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.</p