Post-Operative Poor Sleep Quality and Its Associated Factors Among Non-Small Cell Lung Cancer Patients: A Cross-Sectional Study.

Objective: The study aimed to determine the post-operative prevalence and factors associated to poor sleep quality in non-small cell lung cancer (NSCLC) patients in China. Methods: NSCLC patients (n=307) who underwent thoracoscopic surgery at the Department of Thoracic Surgery of Shanghai Pulmonary Hospital were enrolled in this study. The Pittsburgh Sleep Quality Index (PSQI), Generalized Anxiety Disorder-7 (GAD-7), Patient Health Questionnaire-9 (PHQ-9), Prince Henry Hospital Pain Score and the Six-Minute Walk Test (6MWT), and Forced Expiratory Volume in one second (FEV-1) were used to assess the factors that could lead to poor sleep quality. All assessments were carried out between April 1 and May 30, 2023. Descriptive analyses and stepwise factor regression were employed to determine the impact of various factors on sleep quality. The factors predictive of poor sleep quality were used to develop a predictive nomogram. The Hosmer-Lemeshow test was used to assess the predictive value of the nomogram. Results: The median PQSI score was 8 (interquartile range (IQR) 5-12), and 74.6% of patients had poor sleep quality. The median anxiety and depression scores were 6 (IQR 3-9) and 4 (IQR 2-7), respectively. The PSQI latency dimension had the highest score, while the use of sleep medications dimension had the lowest score. The multivariate analysis revealed that patients who were female (OR, 2.38; 95% CI, 1.40-4.05; P <0.01), had post-secondary education (OR, 0.42; 95% CI, 0.19-0.92; P =0.03), tertiary education (OR, 0.38; 95% CI, 0.17-0.84; P =0.02), comorbidities (OR, 2.57; 95% CI, 1.51-4.39; P <0.01), a pain score 1 (OR, 4.22; 95% CI, 2.37-7.50; P <0.01), and cough (OR, 2.97; 95% CI, 1.63-5.40; P <.001) were more like to experience poor sleep quality. The positive predictive value of the nomogram was 79.80% (p=0.390). Conclusion: Sociodemographic variables, comorbidities, and pain could be used to predict the post-operative sleep quality in NSCLC patients.

Microevolution of Salmonella 4,[5],12:i:- derived from Salmonella enterica serovar Typhimurium through complicated transpositions.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-), derived from S. Typhimurium, has become the dominant serotype causing human salmonellosis. In this study, we define the genetic mechanism of the generation of Salmonella 4,[5],12:i:- from S. Typhimurium through complicated transpositions and demonstrate that Salmonella 4,[5],12:i:- displays more efficient colonization and survival abilities in mice than its parent S. Typhimurium strain. We identified intermediate strains carrying both resistance regions (RRs) and the fljAB operon for the generation of Salmonella 4,[5],12:i:-. The insertion of RR3 into the chromosomal hin-iroB site of S. Typhimurium produced RR3-S. Typhimurium as a primary intermediate. Salmonella 4,[5],12:i:- was then produced by replacing the fljAB operon and/or its flanking sequences through intramolecular transpositions mediated by IS26 and/or IS1R elements in RR3-S. Typhimurium, which was further confirmed both in vitro and in vivo. Overall, we demonstrate the molecular mechanism underlying the origin, generation, and advantage of RRs-Salmonella 4,[5],12:i:- from S. Typhimurium.

The Subjective Will and Psychological Experience of Home-Based Exercise in Lung Cancer Patients During Interval of Chemotherapy: A Qualitative Study.

Purpose: This qualitative study explores the subjective will and psychological experience of home exercise in patients with lung cancer during the interval of chemotherapy. Methods: Semi-structured interviews were conducted with 15 lung cancer patients undergoing chemotherapy. Following the Colaizzi 7-step analysis method, the interview data were read carefully, meaningful statements related to the research questions were extracted, coded, collected and described in detail, and the authenticity of the theme was verified. Results: The analysis revealed the home-based exercise experience of patients' in the interval period of chemotherapy, and identified three themes: 1) the perception experience of home-based exercise (beneficial home-based exercise experience, negative home-based exercise experience); 2) the influencing factors of home-based exercise (exercise rehabilitation knowledge, disease symptoms and adverse effects of chemotherapy, exercise history, exercise self-efficacy, social support, restrictions on objective conditions); 3) Patients with lung cancer expected to get professional guidance about home-based exercise rehabilitation knowledge from medical care providers. Conclusion: Patients' perception and attitude towards home-based exercise behavior is affected by many factors during the interval of chemotherapy, and they expect professional guidance from medical care providers. Medical care providers should know the problems and the influencing factors in the process of home-based exercise of patients, and formulate personalized exercise measures for patients based on their own characteristics and needs, so as to relieve the symptoms of discomfort and improve the quality of life of patients with lung cancer.

Temperature-Responsive Nanocarrier-Regulated Alternative Release of "Cargos" for a Multiplex Photoelectrochemical Bioassay of Antibiotic-Resistant Genes.

A smart temperature stimuli-driven multiplex photoelectrochemical (PEC) assay was constructed for antibiotic resistance genes (ARGs) detection, where the stimuli-responsive gatekeeping by regulating the alternative release of "cargo" allowed for the simultaneous detection of multiple tetracycline resistance gene, using tetA (TDNA1) and tetC (TDNA2) as the model. Dual temperature-responsive nanoassemblies were embedded in the PEC bioassay as signal DNA tages: one thermoresponsive polymer (poly(N-isopropylacrylamide), PNIPAM)-capped mesoporous silica nanoparticles (MSN) with loading the "cargo" of HgO nanoparticles as signal DNA1 tags (SDNA1-PNIPAM@MSN@HgONPs) and the other antimony tartrate (SbT)-anchored silica nanospheres as signal DNA2 tags (SDNA2-SbT@SiO2NSs). At 20 °C, below the lower critical solution temperature (LCST) of PNIPAM, the "gatekeeper" PNIPAM in SDNA1-PNIPAM@MSN@HgONPs was in an ON state, igniting Hg2+ release through the pore of SiO2. While at above LCST (40 °C), it was in an OFF state. Likewise, the thermo-dependent dissociation of SbT endowed the grafted SDNA2 tags switching from the OFF (at 20 °C) to ON state (at 40 °C), igniting SbO+ release. The released Hg2+ and SbO+ triggered the amplified photocurrents due to the structure evolution of the photoactive layer into HgS/ZnS or Sb2S3/ZnS heterostructure, thus achieving sensitive detection of multiple ARGs: tetA, tetC, tetG, tetM, tetO, tetZ, tetX, and tetW. Combined with heat map analysis, rapid screening of the ARGs profiles in 12 samples could be realized. This bioassay is simple and accessible for multiple genes analysis with the detection limit down to 0.50 nM. And it was successfully applied for measuring tetracycline ARGs in real sludge samples.

Enhanced electrochemiluminescence bioassay triggered by intracellular leakage for detection of Escherichia coli.

An intracellular leakage-trigged signal-on solid-state electrochemiluminescent (ECL) assay is developed for the detection of Escherichia coli (E. coli). A self-assembled multilayer ensemble of N, S co-doped carbon dots -poly dimethyl diallylammonium chloride grafted carbon nanospheres is used as ECL luminophores with peroxydisulfate (PS) ions as coreactants. The incorporation of molecularly imprinted electrospun nanofibers with the multilayer ensemble enables a robust, highly selective solid-state ECL probe without using any expensive and fragile biological receptor. Upon the imprinted E. coli exposed to the assay, under bactericidal effects of PS ions by destroying the integrity of E. coli cell membrane, intracellular leakage K+-triggered ECL enhancement is first disclosed via prompting the involved 1O2-mediated ECL process. Benefiting from the ECL enhancement upon increasing the concentration of E. coli, a unique intracellular leakage-trigged signal-on ECL system is created for sensing E. coli. Such a assay is proved to be highly specific and sensitive for sensing E. coli in the concentration range from 5 to 107 cfu mL-1, achieving a detection limit of 1 cfu mL-1 (S/N = 3). This label-free, simple and facile assay provides a promising point-of-care diagnostic tool for pathogen detection.

Genetic Diversity of Listeria monocytogenes Isolates from Invasive Listeriosis in China.

Listeria monocytogenes is a deadly foodborne pathogen, and infections can result in meningoencephalitis and sepsis with mortality rates of up to 30%. In this study, we performed comparative whole-genome analysis of 30 clinical isolates sequenced together with 32 previously sequenced clinical and food isolates from China. The data indicate that L. monocytogenes isolates belonging to the clonal complexes (CC) -1, -8, -9, -87, -121, and -155 are present in human clinical cases. The majority of isolates are from CC-87, 9, and 8 and overlap with those CCs previously reported on the basis of multilocus sequence typing for isolates from Chinese food products. Detailed genome analysis of isolates, representative of CCs in clinical and food products, revealed strong similarities both in their core- and accessory genomes indicating that they are highly related. When compared to genome sequences of isolates of a given CC worldwide, clinical isolates of China were distinct and clustered in unified clades. Our data indicate that epidemic clones of L. monocytogenes (CC-87, 9, and 8) with unusually high occurrence of plasmids are unique to China and suggest that common populations of L. monocytogenes clones are present in both clinical and food products in China.

The Invasion Plasmid Antigen J (IpaJ) from Salmonella Inhibits NF-κB Activation by Suppressing IκBα Ubiquitination.

Salmonella enterica serovar Pullorum is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. In contrast to the strong inflammatory response induced by Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, S Pullorum causes systemic infection with little inflammation. The effector proteins secreted by Salmonella often play a crucial role in modulating host signal transduction and cellular processes to the pathogen's advantage. In the present study, the invasion plasmid antigen J (IpaJ) protein specifically identified in S Pullorum was found to significantly inhibit activation of the key proinflammatory transcription factor, NF-κB, which was induced by tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and lipopolysaccharide (LPS). IpaJ inhibited the NF-κB pathway in cells infected with S Pullorum through the stabilization of IκBα. Deletion of ipaJ in S Pullorum caused a significantly increased level of ubiquitinated IκBα that was subsequently degraded by the proteasome in HeLa cells. Moreover, IpaJ was efficient in the prevention of NF-κB translocation to the nucleus and ultimately interfered with the secretion of the proinflammatory cytokines IL-1ß, IL-6, and IL-8 in infected HeLa cells. Additionally, the transformation of ipaJ into S Enteritidis decreased the secretion of proinflammatory cytokines in HeLa cells through suppression of the NF-κB pathway. The infection of chicken peripheral blood monocyte-derived macrophages (chMDM) confirmed that ipaJ-deleted S Pullorum induced a stronger expression of proinflammatory cytokines than the wild-type and complementary strains. In summary, the present study revealed that IpaJ functions as an important anti-inflammatory protein involved in S Pullorum infection through inhibition of the NF-κB pathway and the subsequent inflammatory response.

Pig as a reservoir of CRISPR type TST4 Salmonella enterica serovar Typhimurium monophasic variant during 2009-2017 in China.

CRISPR-based typing was performed to subtype isolates of S. Typhimurium and its monophasic variant Salmonella 4,[5],12:i:- from humans and animals between 2009 and 2017 in China. CRISPR typing classified all isolates into two lineages and four sub-lineages. All isolates from Lineage II and Lineage IB-1 were Salmonella Typhimurium. All of Salmonella 4,[5],12:i: - isolates were distributed in Lineage IA and Lineage IB-2, which all belonged to ST34 by MLST typing. Only Lineage IB-2 contained ST34 isolates from both Salmonella Typhimurium and Salmonella 4,[5],12:i:-. Among the isolates of ST34, TST4 was identified as the most common CRISPR type representing 86.5% of Salmonella 4,[5],12:i:- and 14.5 % of Salmonella Typhimurium mainly from pigs and humans. This study demonstrated that TST4-ST34 isolates were predominant in Salmonella 4,[5],12:i:-, and pig was the main reservoir for Salmonella 4,[5],12:i:- in China, which might have the potential to transmit to humans by pig production.

A hybrid sub-lineage of Listeria monocytogenes comprising hypervirulent isolates.

The foodborne pathogen Listeria monocytogenes (Lm) is a highly heterogeneous species and currently comprises of 4 evolutionarily distinct lineages. Here, we characterize isolates from severe ovine listeriosis outbreaks that represent a hybrid sub-lineage of the major lineage II (HSL-II) and serotype 4h. HSL-II isolates are highly virulent and exhibit higher organ colonization capacities than well-characterized hypervirulent strains of Lm in an orogastric mouse infection model. The isolates harbour both the Lm Pathogenicity Island (LIPI)-1 and a truncated LIPI-2 locus, encoding sphingomyelinase (SmcL), a virulence factor required for invasion and bacterial translocation from the gut, and other non-contiguous chromosomal segments from another pathogenic species, L. ivanovii. HSL-II isolates exhibit a unique wall teichoic acid (WTA) structure essential for resistance to antimicrobial peptides, bacterial invasion and virulence. The discovery of isolates harbouring pan-species virulence genes of the genus Listeria warrants global efforts to identify further hypervirulent lineages of Lm.

Loss and Gain in the Evolution of the Salmonella enterica Serovar Gallinarum Biovar Pullorum Genome.

Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S Pullorum) is the etiological agent of pullorum disease, causing white diarrhea with high mortality in chickens. There are many unsolved issues surrounding the epidemiology of S Pullorum, including its origin and transmission history as well as the discordance between its phenotypic heterogeneity and genetic monomorphism. In this paper, we report the results of whole-genome sequencing of a panel of 97 S Pullorum strains isolated between 1962 and 2014 from four countries across three continents. We utilized 6,795 core genome single nucleotide polymorphisms (SNPs) to reconstruct a phylogenetic tree within a spatiotemporal Bayesian framework, estimating that the most recent common ancestor of S Pullorum emerged in ∼914 CE (95% confidence interval [95%CI], 565 to 1273 CE). The extant S Pullorum strains can be divided into four distinct lineages, each of which is significantly associated with geographical distribution. The intercontinental transmissions of lineages III and IV can be traced to the mid-19th century and are probably related to the "Hen Fever" prevalent at that time. Further genomic analysis indicated that the loss or pseudogenization of functional genes involved in metabolism and virulence in S Pullorum has been ongoing since before and after divergence from the ancestor. In contrast, multiple prophages and plasmids have been acquired by S Pullorum, and these have endowed it with new characteristics, especially the multidrug resistance conferred by two large plasmids in lineage I. The results of this study provide insight into the evolution of S Pullorum and prove the efficiency of whole-genome sequencing in epidemiological surveillance of pullorum disease.IMPORTANCE Pullorum disease, an acute poultry septicemia caused by Salmonella Gallinarum biovar Pullorum, is fatal for young chickens and is a heavy burden on poultry industry. The pathogen is rare in most developed countries but still extremely difficult to eliminate in China. Efficient epidemiological surveillance necessitates clarifying the origin of the isolates from different regions and their phylogenic relationships. Genomic epidemiological analysis of 97 S Pullorum strains was carried out to reconstruct the phylogeny and transmission history of S Pullorum. Further analysis demonstrated that functional gene loss and acquisition occurred simultaneously throughout the evolution of S Pullorum, both of which reflected adaptation to the changing environment. The result of our study will be helpful in surveillance and prevention of pullorum disease.

Virulence of Salmonella enterica serovar Pullorum isolates compared using cell-based and chicken embryo infection models.

To reveal differences in virulence among strains of Salmonella enterica serovar Pullorum (S. Pullorum), we used 2 cell-based infection models and a chicken embryo infection model in this study. S. Pullorum strain S06004 was used to infect 4 different avian cell lines (HD-11, DF-1, LMH, DT-40), and the results showed that the infection of S06004 in both LMH and HD-11 cells was more stable than in DF-1 and DT-40 cells. Therefore, the HD-11 and LMH cell lines were used as the appropriate macrophage and epithelial cell models, respectively, to study the infection of S. Pullorum. Fifty strains isolated during the years 1962 to 2010 were then analyzed to compare their infection rates in HD-11 and LMH cells. The result showed that the infection rates of most strains were very similar to that of S06004, except for S9876 which displayed the highest infection rate among these strains. Based on the cell infection results, 10 strains were selected to be used in the chicken embryo infection model. Sixteen-day-old SPF chicken embryos were infected with the pathogen at a dose of 103 CFU/100 µL via allantoic cavity inoculation. The strains C79-13, 7101, and S06013 caused death of more than 80% embryos, whereas S09C12 and 6703 resulted less than 20% death. Thus, this study established cell-based infection models to screen S. Pullorum strains in vitro, and a chick embryo model to evaluate their in vivo virulence.

Induction of arthritis in chickens by infection with novel virulent Salmonella Pullorum strains.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) is a host-specific serovar causing systemic infection with high mortality in young chicks. Pullorum disease is characterized by white diarrhea. However, arthritis has become increasingly frequent recently, particularly in southern China. The aim of the present study was to determine the pathogenesis and arthritis induction of new Salmonella Pullorum isolates. We isolated and identified five Salmonella Pullorum strains from broilers with bacterial arthritis and lameness in a commercial poultry farm. Four of five isolates were resistant to at least three classes of antibiotics and were defined as multidrug-resistant Salmonella Pullorum. All isolates had the same CRISPR sequence type and belonged to a single major cluster. The isolates exhibited high capability of biofilm formation, which may facilitate their dispersal and survival in hostile habitats, and showed high virulence based on embryo lethality and inoculation of newly hatched chicks. Tissue distribution analysis confirmed that SP1621 was more adapted to colonize the joint when compared to the white diarrhoea-causing Salmonella Pullorum reference strain S06004. Reproducible arthritis and typical joint lesions were observed in SP1621-infected chicks, and histopathological examination showed necrotic synovitis and cartilage tissue hyperplasia of the joint. Koch's postulates were confirmed when the novel Salmonella Pullorum strain was re-isolated from the joint tissues of experimentally inoculated chicks. These novel Salmonella Pullorum isolates have unique ability to induce arthritis in chickens, representing expanded pathogenic diversity in China. These results suggest the need for strict control strategies and new vaccines to prevent the disease.

A new PCR assay based on the new gene-SPUL_2693 for rapid detection of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are gram-negative bacteria, members of the most important infectious pathogens, and have caused common problems in the poultry industry, especially in the developing countries. O- and H-antigen specific anti-sera are commonly for slide and tube agglutination tests to identify Salmonella serovars. However, it is both labor intensive and time consuming, so there is an urgent need for a new technique for the rapid detection of the major Salmonella serovars. In this study, we developed a 1-step PCR assay to identify the serovar Gallinarum. This PCR-based assay was based on the SPUL_2693 gene, which was located in SPI-19 and found by comparing the genomes of the S. Pullorum and S. Gallinarum in the whole data of NCBI. The specificity of this gene was evaluated by bioinformatics analysis, and the results showed that the SPUL_2693 gene exists in all serovar Gallinarum. The specificity and sensitivity of this PCR assay were evaluated in our study. The developed PCR assay was able to distinguish the serovar Gallinarum from 27 different Salmonella serovars and 5 different non-Salmonella pathogens. The minimum limit of genomic DNA of S. Pullorum for PCR detection was 2.143 pg/µL, and the minimum limit number of cells was 6 CFU. This PCR assay was also applied to analyze Salmonella strains isolated from a chicken farm in this study. The PCR assay properly identified the serovar Gallinarum from other Salmonella serovars, and the results were in agreement with the results of a traditional serotyping assay. In general, the newly developed PCR-based assay can be used to accurately judge the presence of the serovar Gallinarum and can be combined with traditional serotyping assays, especially in the case of large quantities of samples.

Analyses of prevalence and molecular typing reveal the spread of antimicrobial-resistant Salmonella infection across two breeder chicken farms.

In this study, Salmonella prevalence and antimicrobial resistance were evaluated at various production stages in 2 geographically separated breeder farms (referred to as G and F). Day-old chicks for the breeder flock at farm F were purchased from farm G. A total of 219 Salmonella isolates, all identified as Salmonella enterica subsp. enterica serovar Enteritidis, were recovered from 1,430 samples (sick chicken carcasses and/or dead embryos). The isolation rates at breeder farms G and F were 10.53% (56/532) and 18.15% (163/898), respectively. Resistance to 4-6 antimicrobial agents was the most frequent phenotype during the laying stage at both farms, suggesting that chicks are exposed to higher risk of antimicrobial-resistant Salmonella infection during this stage of the breeding process. Using clustered regularly interspaced short palindromic repeat (CRISPR) typing, 5 CRISPR patterns were identified, out of which one pattern was shared by the 2 farms. In addition, pulsed-field gel electrophoresis (PFGE) typing result indicated that 2 clusters (PF-1 and PF-2) were shared among the 2 breeder farms, suggesting that strains were transmitted from breeder farm G to farm F via the trade of day-old chicks. Our findings suggested that the trade of day-old breeder chicks could be one of the potential Salmonella transmission routes, and antibiotics should be administered with caution during the laying stage.

Genetic analysis and CRISPR typing of Salmonella enterica serovar Enteritidis from different sources revealed potential transmission from poultry and pig to human.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent serotypes in Salmonella isolated from poultry and the most commonly reported cause of human salmonellosis. In this study, we aimed to assess the genetic diversity of 329 S. Enteritidis strains isolated from different sources from 2009 to 2016 in China. Clustered regularly interspaced short palindromic repeat (CRISPR) typing was used to characterize these 262 chicken clinical isolates, 38 human isolates, 18 pig isolates, six duck isolates, three goose isolates and two isolates of unknown source. A total of 18 Enteritidis CRISPR types (ECTs) were identified, with ECT2, ECT8 and ECT4 as the top three ECTs. CRISPR typing identified ECT2 as the most prevalent ECT, which accounted for 41% of S. Enteritidis strains from all the sources except duck. ECT9 and ECT13 were identified in both pig and human isolates and revealed potential transmission from pig to human. A cluster analysis distributed 18 ECTs, including the top three ECTs, into four lineages with LI as the predominant lineage. Forty-eight out of 329 isolates were subjected to whole genome sequence typing, which divided them into four clusters, with Cluster I as the predominant cluster. Cluster I included 92% (34/37) of strains located in LI identified from the CRISPR typing, confirming the good correspondence between both typing methods. In addition, the CRISPR typing also revealed the close relationship between ECTs and isolated areas, confirming that CRISPR spacers might be obtained by bacteria from the unique phage or plasmid pools in the environment. However, further analysis is needed to determine the function of CRISPR-Cas systems in Salmonella and the relationship between spacers and the environment.

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ.

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90 fg/µl or 102 CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.

Genetic analysis of Salmonella enterica serovar Gallinarum biovar Pullorum based on characterization and evolution of CRISPR sequence.

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the cause of pullorum disease, characterized by white diarrhea, which leads to high mortality in poultry. In this study, we aimed to assess the genetic diversity of 655 S. Pullorum strains from 1962 to 2015 in China, Europe, and South America. A sequence typing scheme based on clustered regularly interspaced short palindromic repeats (CRISPR) was used to reveal the genetic relationships among these strains in this study. Overall, a total of 20 Pullorum sequence types (PSTs) of CRISPR were identified in the 655 isolates with PST7 (74%, 486/655) and PST3 (13%, 86/655) to be the most two frequent PSTs belonging to two different lineages, which confirmed the genetic conservation of S. Pullorum strains isolated from six provinces and two direct-controlled municipalities (Beijing and Shanghai) in China. However, the identification of seven new PSTs distributed in strains isolated since 2001 implied that genetic variation continues to develop in S. Pullorum. Interestingly, the whole-genome single-nucleotide polymorphism typing (WGST) of 96 strains out of the 655 isolates divided them into four lineages based on SNP analysis of core genomic sequence and exhibit good correspondence with the CRISPR subtyping method. Notably, 22 out of 26 isolates from Europe and South America were distributed in five distinctive PSTs (with no Chinese strains). Additionally, CRISPR data of spacers and their arrangement exhibit subtle but distinct specificity between different strains, and the dynamic adaptive nature of CRISPR loci provides critical insights into the evolution of S. Pullorum as the bacteria are influenced by their environment.

Characterization of CRISPR-Cas system in clinical Staphylococcus epidermidis strains revealed its potential association with bacterial infection sites.

Staphylococcus epidermidis is considered as a major cause of nosocomial infections, bringing an immense burden to healthcare systems. Virulent phages have been confirmed to be efficient in combating the pathogen, but the prensence of CRISPR-Cas system, which is a bacterial immune system eliminating phages was reported in few S. epidermidis strains. In this study, the CRISPR-Cas system was detected in 12 from almost 300 published genomes in GenBank and by PCR of cas6 gene in 18 strains out of 130 clinical isolates obtained in Copenhagen. Four strains isolated in 1965-1966 harboured CRISPR elements confirming that this immunity system was not recently acquired by S. epidermidis. In these CRISPR-positive strains, 44 and 12 spacers were found to belong to CRISPR1 and CRISPR2 elements, respectively. However, only 15 spacers displayed homology to reported phages and plasmids DNA. Interestingly, 5 different spacers located in the CRISPR1 locus with homolgy to virulent phage 6ec DNA sequences, and 19 strains each carrying 2 or 3 different spacers recognizing this phage, implied that the CRISPR-Cas immunity could be abrogated by nucleotide mismatch between the spacer and its target phage sequence, while new spacers obtained from the evolved phage could recover the CRISPR interference. In addition, phylogenetic analysis of the 29 CRISPR-positive isolates divided them into four lineages, with 81% human blood isolates as a distinct sub-lineage, suggesting that the CRISPR difference is closely related to diverse habitats. Knowledge of CRISPR and its prevalence may ultimately be applied in the understanding of origin and evolution of CRISPR-positive S. epidermidis strains.

Complete Genome Sequence of Salmonella enterica Serovar Pullorum Multidrug Resistance Strain S06004 from China.

As Salmonella enterica serovar Pullorum remains a major economic problem for the poultry industries of countries with no efficient control measures, we presented a multidrug resistance strain S06004 (isolated from a clinically sick chicken in China in 2006) for genome sequencing. The genome comparison showed that the strain contained two prophages, the ST104 and prophage-4 (Fels2) of E. coli LF82, which were not detected in the only published genomes of S. Pullorum RKS5078 and CDC1983-67. In addition, the GyrA Ser83 point mutation, drugresistant genes, and many antibiotic pump systems that are present in S06004 may be contributing to the multidrug resistance of this strain.

[Construction and characterization of Salmonella Pathogenicity Island 2 (SPI-2) deletion mutant of Salmonella Pullorum S06004].

OBJECTIVE: To research the pathogenicity of Salmonella Pathogenicity Island 2 (SPI-2) deletion mutant of Salmonella Pullorum and preliminary explore the feasibility of developing safe attenuated Salmonella Pullorum candidate vaccine strain. METHODS: The SPI-2 (-40 kb) deletion mutant of Salmonella Pullorum S06004 was constructed using the λ-red recombinant system. Then the biological characteristics such as growth rate, biochemical properties, genetic stability and virulence were evaluated between the deletion mutant strain S06004ΔSPI2 and its parent strain S06004. RESULTS: S06004ΔSPI2 was successfully constructed. The growth rate and biochemical properties of S06004ΔSPI2 were consistent with those of its parent strain S06004. The mutant was stable with the deletion of SPI-2. Chicken lethal test showed that the LD50 of S06004ΔSPI2 was 252 times higher than the parent strain S06004. CONCLUSION: The virulence of S06004ΔSPI2 was obviously attenuated. This study provided basic data for further study of the functions of SPI-2, and implied its potential to develop attenuated Salmonella vaccine.