Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil.

The study of carrier state phages challenged the canonical lytic-lysogenic binary, and carrier state appears to be ubiquitous and ecologically important. However, the mechanisms of the carrier state are not well elucidated due to the limited phage models. Herein, we reported phage HQ103, similar to Escherichia coli phage P2. In contrast to the temperate P2 phage, the HQ103 phage does not insert its genome into the bacterial chromosome and displays a dual behavior depending on the temperature. At 37°C, HQ103 lyses the host and forms clear plaques due to the truncation of repressor CI and mutation of promoter Pc. In contrast, HQ103 maintains a carrier state lifestyle with Y. pestis at an environmental temperature (21°C). Mechanistically, we found that the host-encoded histone-like nucleoid-structuring protein H-NS, which is highly expressed at 21°C to silence the Cox promoter Pe and inhibits the phage lytic cycle. Subsequently, the HQ103 carrier state Y. pestis could grow and co-exist with the phage in the soil at 21°C for one month. Thus, this study reveals a novel carrier state lifestyle of phage HQ103 due to the H-NS mediated xenogeneic silencing and demonstrates that the carrier state lifestyle could promote long-term phage-host coexist in nature.

Identification and characterization of two Bacillus anthracis bacteriophages.

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.

Direct Blood Culturing of Candida spp. on Solid Medium by a Rapid Enrichment Method with Magnetic Beads Coated with Recombinant Human Mannan-Binding Lectin.

A rapid and accurate method to identify the species and antibiotic resistance of Candida spp. in blood is vital to increase the survival rates of patients with bloodstream infections. However, the extremely low levels of Candida spp. in blood make rapid diagnosis by standard blood culture difficult. In this study, we constructed a direct blood culturing method (i.e., the M1 method) by a rapid enrichment method with magnetic beads coated with a recombined human mannan-binding lectin (rhMBL; i.e., M1 protein), which demonstrated much higher Candida sp.-binding capacity than that of full-length MBL expressed in vitro (i.e., M2). With the M1 method, individual colonies were obtained before the standard blood culture method for each species of Candida spp. tested at <1 CFU/ml (an average of 29 h earlier). Additionally, the clinical sensitivity of the M1 method was 90.5% compared with that of the standard blood culture method when detecting frozen plasma from patients. More significantly, the turnaround time of the M1 method for blood culture could be reduced by approximately 37 to 43 h compared with that of the standard blood culture method in clinical sample identification.

Characteristics of a pseudolysogenic phage vB_YpM_HQ103 infecting Yersinia pestis.

The plague, caused by Yersinia pestis, is a natural focal disease and the presence of Y. pestis in the environment is a critical ecological concern worldwide. The role of Y. pestis phages in the ecological life cycle of the plague is crucial. Previously, a temperature-sensitive phage named vB_YpM_HQ103 was isolated from plague foci in Yunnan province, China. Upon infecting the EV76 strain of Y. pestis, vB_YpM_HQ103 exhibits lysogenic behavior at 21 °C and lytic behavior at 37 °C. Various methods including continuous passage lysogenic tests, in vitro lysis tests, comparative genomic assays, fluorescence quantitative PCR and receptor identification tests were employed to demonstrate that the lysogenic life cycle of this phage is applicable to wild Y. pestis strains; its lysogeny is pseudolysogenic (carrying but not integrating), allowing it to replicate and proliferate within Y. pestis. Furthermore, we have identified the outer membrane protein OmpA of Y. pestis as the receptor for phage infection. In conclusion, our research provides insight into the characteristics and receptors of a novel Y. pestis phage infection with a pseudolysogenic cycle. The findings of this study enhance our understanding of Y. pestis phages and plague microecology, offering valuable insights for future studies on the conservation and genetic evolution of Y. pestis in nature.

Phylogenetic Relationships and Evolution of the Genus Eganvirus (186-Type) Yersinia pestis Bacteriophages.

Plague is an endemic infectious disease caused by Yersinia pestis. In this study, we isolated fourteen phages with similar sequence arrangements to phage 186; these phages exhibited different lytic abilities in Enterobacteriaceae strains. To illustrate the phylogenetic relationships and evolutionary relationships between previously designated 186-type phages, we analysed the complete sequences and important genes of the phages, including whole-genome average nucleotide identity (ANI) and collinearity comparison, evolutionary analysis of four conserved structural genes (V, T, R, and Q genes), and analysis of the regulatory genes (cI, apl, and cII) and integrase gene (int). Phylogenetic analysis revealed that thirteen of the newly isolated phages belong to the genus Eganvirus and one belongs to the genus Felsduovirus in the family Peduoviridae, and these Eganvirus phages can be roughly clustered into three subgroups. The topological relationships exhibited by the whole-genome and structural genes seemed similar and stable, while the regulatory genes presented different topological relationships with the structural genes, and these results indicated that there was some homologous recombination in the regulatory genes. These newly isolated 186-type phages were mostly isolated from dogs, suggesting that the resistance of Canidae to Y. pestis infection may be related to the wide distribution of phages with lytic capability.

Genomic epidemiological analysis of county-scale Yersinia pestis spread pattern over 50 years in a Southwest Chinese prefecture.

Plague, one of the most devastating infectious diseases in human history, is caused by the bacterium Yersinia pestis. Since the 1950s, the Dehong Dai-Jingpo Autonomous Prefecture (DH) in Yunnan Province, China, has recorded plague outbreaks that have resulted in 1,153 human cases and 379 deaths. The genetic diversity and transmission characteristics of Y. pestis strains in this region remain unknown. Here, we performed high-resolution genomic epidemiological analysis of 175 Y. pestis strains isolated from five counties and 19 towns in DH between 1953 and 2007. Phylogenetic analysis revealed that most DH strains were located in lineage 1.ORI2, which could be further subdivided into seven sub-phylogroups (SPG1-SPG7). The dominant sub-phylogroups of Y. pestis in DH varied during different periods and presented a population shift. Genomic evidence showed that plague might have emerged from the southwest of DH (e.g., Longchuan or Ruili counties) or its bordering countries, and subsequently spread to the northeast in multiple waves between 1982 and 2007. Our study infers a fine-scale phylogeny and spread pattern of the DH Y. pestis population, which extends our knowledge regarding its genetic diversity and provides clues for the future prevention and control of plague in this region.

Two Novel Yersinia pestis Bacteriophages with a Broad Host Range: Potential as Biocontrol Agents in Plague Natural Foci.

Bacteriophages (phages) have been successfully used as disinfectors to kill bacteria in food and the environment and have been used medically for curing human diseases. The objective of this research was to elucidate the morphological and genomic characteristics of two novel Yersinia pestis phages, vB_YpeM_ MHS112 (MHS112) and vB_YpeM_GMS130 (GMS130), belonging to the genus Gaprivervirus, subfamily Tevenvirinae, family Myoviridae. Genome sequencing showed that the sizes of MHS112 and GMS130 were 170507 and 168552 bp, respectively. A total of 303 and 292 open reading frames with 2 tRNA and 3 tRNA were predicted in MHS112 and GMS130, respectively. The phylogenetic relationships were analysed among the two novel Y. pestis phages, phages in the genus Gaprivervirus, and several T4-like phages infecting the Yersinia genus. The bacteriophage MHS112 and GMS130 exhibited a wider lytic host spectrum and exhibited comparative temperature and pH stability. Such features signify that these phages do not need to rely on Y. pestis as their host bacteria in the ecological environment, while they could be based on more massive Enterobacteriales species to propagate and form ecological barriers against Y. pestis pathogens colonised in plague foci. Such characteristics indicated that the two phages have potential as biocontrol agents for eliminating the endemics of animal plague in natural plague foci.

New Genotype of Yersinia pestis Found in Live Rodents in Yunnan Province, China.

Yunnan Province, China is thought to be the original source of biovar Orientalis of Yersinia pestis, the causative agent of the third plague pandemic that has spread globally since the end of the 19th century. Although encompassing a large area of natural plague foci, Y. pestis strains have rarely been found in live rodents during surveillance in Yunnan, and most isolates are from rodent corpses and their fleas. In 2017, 10 Y. pestis strains were isolated from seven live rodents and three fleas in Heqing County of Yunnan. These strains were supposed to have low virulence to local rodents Eothenomys miletus and Apodemus chevrieri because the rodents were healthy and no dead animals were found in surrounding areas, as had occurred in previous epizootic disease. We performed microscopic and biochemical examinations of the isolates, and compared their whole-genome sequences and transcriptome with those of 10 high virulence Y. pestis strains that were isolated from nine rodents and one parasitic flea in adjacent city (Lijiang). We analyzed the phenotypic, genomic, and transcriptomic characteristics of live rodent isolates. The isolates formed a previously undefined monophyletic branch of Y. pestis that was named 1.IN5. Six SNPs, two indels, and one copy number variation were detected between live rodent isolates and the high virulence neighbors. No obvious functional consequence of these variations was found according to the known annotation information. Among genes which expression differential in the live rodent isolates compared to their high virulent neighbors, we found five iron transfer related ones that were significant up-regulated (| log2 (FC) | > 1, p.adjust < 0.05), indicating these genes may be related to the low-virulence phenotype. The novel genotype of Y. pestis reported here provides further insights into the evolution and spread of plague as well as clues that may help to decipher the virulence mechanism of this notorious pathogen.

The characteristics and genome analysis of the novel Y. pestis phage JC221.

The pathogen of plague is Yersinia pestis (Y. pestis), one of the deadliest pathogens in the world and belonging to the family Enterobacteriaceae. In this work, the biological characteristics and complete genome sequence analysis of a novel lytic Y. pestis-specific phage JC221 isolated from Yunnan Province, China, was studied. JC221 belongs to the Myoviridae family and has a regular icosahedral head and a long contractile tail. The double-stranded DNA genome of JC221 contains 174,931 bp, and the G + C content is 41.23 %. There are 274 predicted genes, of which only 103 hits of genes or gene products are found in database searches, and there are no known virulence-related or antibiotic resistance genes. The genome sequence of JC221 showed <80 % identity to other phages, and evolutionary analysis revealed that bacteriophage JC221 belongs to the Yersinia phage cluster. Furthermore, the bacteriophage could completely lyse most of the tested Y. pestis strains (12/13) at 28 °C and 37 °C, and some Shigella strains could be lysed at 37°C. Morphological and genomic analysis indicated that JC221 is a new Y. pestis phage and a new member of the Tequatrovirus phages. The novel Y. pestis phage JC221 has important reference value for the study of environmental microecology and epidemiology of plague foci.

Predictors for presence and abundance of small mammals in households of villages endemic for commensal rodent plague in Yunnan Province, China.

BACKGROUND: Ninety-one rodent plague epidemics have occurred in Lianghe county, Yunnan Province, China, between 1990 and 2006. This study aimed to identify predictors for the presence and abundance of small mammals in households of villages endemic for rodent plague in Lianghe county. RESULTS: Rattus flavipectus and Suncus murinus were the two species captured in 110 households. Keeping cats decreased the number of captures of R. flavipectus by one to two thirds and the chance of reported small mammal sightings in houses by 60 to 80%. Food availability was associated with fewer captures. Keeping food in sacks decreased the small mammal captures, especially of S. murinus 4- to 8-fold. Vegetables grown around house and maize grown in the village reduced the captures of S. murinus and R. flavipectus by 73 and 45%, respectively. An outside toilet and garbage piles near the house each reduced R. flavipectus captures by 39 and 37%, respectively, while raising dogs and the presence of communal latrines in the village increased R. flavipectus captures by 76 and 110% but were without detectable effect on small mammal sightings. Location adjacent to other houses increased captures 2-fold but reduced the chance of sightings to about half. In addition, raising ducks increased the chance of sighting small mammals 2.7-fold. Even after adjusting for these variables, households of the Dai had higher captures than those of the Han and other ethnic groups. CONCLUSION: Both species captures were reduced by availability of species-specific foods in the environment, whereas other predictors for capture of the two species differed. Other than the beneficial effect of cats, there were also discrepancies between the effects on small mammal captures and those on sightings. These differences should be considered during the implementation and interpretation of small mammal surveys.

[Predictors for abundance of Rattus tanezumi in households of commensal rodent plague foci].

OBJECTIVE: To explore the predictors on the abundance of Rattus (R.) tanezumi in households of commensal rodent plague foci. METHODS: Thirty natural villages that experienced previous plague cases in Lianghe county, Yunnan province, were selected followed by random selection of 20 households in each village through computer technique. Live traps were set in households to capture small mammals which were then identified to species in the field according to their morphological features. Data on potential factors for abundance of R. tanezumi were collected through questionnaires and field observation and were coded and computerized using EpiData software and further analyzed by hurdle regression model under R software. RESULTS: A total of 166 rodents (133 R. tanezumi and 33 Suncus murinus) were captured. RESULTS: from final multilevel hurdle regression model showed that the likelihood of R. tanezumi captures increased by 1.67- to 2.76-fold in households belonged to Dai ethnic families that stored foodstuff in metal pail, often raising dogs, and having adjacent houses. The number of R. tanezumi captures increased by 2.18-fold in the villages where over 80% of the households would raise chickens. In the villages with communal latrine, the likelihood and the number of R. tanezumi capture increased 1.93-fold and 2.38-fold, respectively. While the likelihood of R. tanezumi captures would reduce by 45% - 61% in those households where there were cats and cattle being raised and maize grown in the village. The number of R. tanezumi captures would reduce by 63% in the households where there were outside toilet. CONCLUSION: The abundance of R. tanezumi seemed to be closely related to the ecological environment factors. Programs on plague control and prevention should relate to ecological factors that influencing the abundance of R. tanezumi.

Predictors for abundance of host flea and floor flea in households of villages with endemic commensal rodent plague, Yunnan Province, China.

BACKGROUND: From 1990 to 2006, fifty-five natural villages experienced at least one plague epidemic in Lianghe County, Yunnan Province, China. This study is aimed to document flea abundance and identify predictors in households of villages with endemic commensal rodent plague in Lianghe County. METHODS: Trappings were used to collect fleas and interviews were conducted to gather demography, environmental factors, and other relevant information. Multivariate hurdle negative binomial model was applied to identify predictors for flea abundance. RESULTS: A total of 344 fleas were collected on 101 small mammals (94 Rattus flavipectus and 7 Suncus murinus). R. flavipectus had higher flea prevalence and abundance than S. murinus, but the flea intensities did not differ significantly. A total of 315 floor fleas were captured in 104 households. Xenopsylla cheopis and Ctenocephalides felis felis were the predominant flea species on the host and the floor flea, respectively. The presence of small mammal faeces and R. flavipectus increased host flea prevalence odds 2.9- and 10-fold, respectively. Keeping a dog in the house increased floor flea prevalence odds 2-fold. Keeping cattle increased floor flea intensity by 153%. Villages with over 80% of houses raising chickens had increased prevalence odds and intensity of floor flea about 2.9- and 11.6-fold, respectively. The prevalence and intensity of floor flea in brick and wood houses were decreased by 60% and 90%, respectively. Flea prevalences of host and floor flea in the households that were adjacent to other houses were increased 7.4- and 2.2-fold, respectively. Houses with a paddy nearby decreased host flea intensity by 53%, while houses with an outside toilet increased host flea intensity by 125%. CONCLUSION: Rodent control alone may not be sufficient to control plague risk in these areas. In order to have successful results, plague control programs should pay attention to ecological and hygiene factors that influence flea populations.