Significance of the bacteriophage treatment schedule in reducing salmonella colonization of poultry

Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time.     

Restriction of bacteriophage plaque formation in Streptomyces spp.

Several Streptomyces species that produce restriction endonucleases were characterized for their ability to propagate 10 different broad host range bacteriophages. Each species displayed a different pattern of plaque formation. A restrictionless mutant of S. albus G allowed plaque formation by all 10 phages, whereas the wild-type strain showed plaques with only 2 phages. DNA isolated from three of the phages was analyzed for the presence of restriction sites for Streptomyces species-encoded enzymes, and a very strong correlation was established between the failure to form plaques on Streptomyces species that produced particular restriction enzymes and the presence of the corresponding restriction sites in the phage DNA. Also, the phages that lacked restriction sites in their DNA generally formed plaques on the corresponding restriction endonuclease-producing hosts at high efficiency. The DNAs from the three phages analyzed also generally contained either many or no restriction sites for the Streptomyces species-produced enzymes, suggesting a strong evolutionary trend to either eliminate all or tolerate many restriction sites. The data indicate that restriction plays a major role in host range determination for Streptomyces phages. Analysis of bacteriophage host ranges of many other uncharacterized Streptomyces hosts has identified four relatively nonrestricting hosts, at least two of which may be suitable hosts for gene cloning. The data also suggest that several restriction systems remain to be identified in the genus Streptomyces.     

基于智能计算噬菌体的细菌宿主范围预测

针对噬菌体的细菌宿主范围预测对于深入理解噬菌体及将其作为抗生素替代用于生物疗法具有重要意义。传统生物实验方法确定噬菌体的细菌宿主范围受到极有限的噬菌体可培养性和严苛的培养条件限制,而高通量测序技术所提供的海量基因组或宏基因组序列提供了噬菌体及细菌重要的序列信息,因此智能计算为预测噬菌体的细菌宿主范围提供了可行方法。本文从智能计算的角度对噬菌体的细菌宿主范围预测研究进行系统梳理,从噬菌体感染细菌的过程入手,描述配对预测模型所依赖的特征及其生物合理性,归纳宿主范围预测的智能模型、建模原理及预测策略,总结建模训练和评估所依赖的参考数据集与真实数据及评价指标。本文特别注意挖掘和分析各信息手段、模型、方法其背后的生物合理性及其依赖的生物机理。本综述期望推动基于智能算法的噬菌体的细菌宿主范围预测研究发展,并探索将生物先验结合人工智能实现噬菌体侵袭细菌宿主的本质机理推断,同时也为基于噬菌体的临床应用提供参考与借鉴。     

Host range of chlamydiaphages phiCPAR39 and Chp3

The host range of phiCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that phiCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.     

A guard-killer phage cocktail effectively lyses the host and inhibits the development of phage-resistant strains of Escherichia coli

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

     

Adsorption of bacteriophages on bacterial cells

The biological functions of bacteriophage virions come down to the solution of three basic problems: to provide protection of viral nucleic acid from the factors of extracellular environment, to recognize a host suitable for phage replication, and to provide the delivery of nucleic acid through bacterial cell envelopes. This review considers the main regularities of phage–cell interaction at the initial stages of infection of tailed bacteriophages, from the reversible binding with receptors on the surface to the beginning of phage DNA entry. Data on the structure and functions of the phage adsorption apparatus, the main quantitative characteristics of the adsorption process, and the mechanisms of adaptation of phages and their hosts to each other effective at the stage of adsorption are presented.     

An ORFan no more: the bacteriophage T4 39.2 gene product, NwgI, modulates GroEL chaperone function

Bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (ORFans) encoded by their immense genetic repertoire. These genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. To test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host Escherichia coli GroEL/GroES chaperone machine, which is essential for the folding of certain host and bacteriophage proteins. As a result, we identified the bacteriophage RB69 gene 39.2, of previously unknown function and showed that homologs of 39.2 in bacteriophages T4, RB43, and RB49 similarly modulate GroEL/GroES. Production of wild-type bacteriophage T4 Gp39.2, a 58-amino-acid protein, (a) enables diverse bacteriophages to plaque on the otherwise nonpermissive groES or groEL mutant hosts in an allele-specific manner, (b) suppresses the temperature-sensitive phenotype of both groES and groEL mutants, (c) suppresses the defective UV-induced PolV function (UmuCD) of the groEL44 mutant, and (d) is lethal to the host when overproduced. Finally, as proof of principle that Gp39.2 is essential for bacteriophage growth on certain bacterial hosts, we constructed a T4 39.2 deletion strain and showed that, unlike the isogenic wild-type parent, it is incapable of propagating on certain groEL mutant hosts. We propose a model of how Gp39.2 modulates GroES/GroEL function.     

Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus

Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage φIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, φIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.     

6 (p-Hydroxyphenylazo)-Uracil: a Reversible, Selective Inhibitor of the Replication of Deoxyribonucleic Acid of Staphylococcal Bacteriophage P11-M15

6(p-Hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of the semiconservative replication of deoxyribonucleic acid (DNA) of gram-positive bacteria, was found to inhibit the replication of DNA of bacteriophage P11-M15, a virulent derivative of the temperate Staphylococcus aureus bacteriophage P11. At appropriate concentration, HPUra inhibited DNA synthesis by P11-M15-infected S. aureus immediately and completely, regardless of the stage of the lytic cycle at which infected cells were exposed to drug. The effect of HPUra was reversible since the capacity of inhibited, infected cells to replicate phage DNA and produce mature phage could be restored by removal of HPUra from incubation media. Concentrations of HPUra which completely inhibited the replication of P11-M15 in its drug-sensitive host did not inhibit the replication of this phage or its DNA in several drug-resistant host mutants. HPUra also did not inhibit the replication of two other serologically distinct, virulent staphylococcal bacteriophages, P1 and 44AHJD, in drug-sensitive hosts.     

Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

工业微生物的噬菌体感染与防治策略

以细菌为基础的生物技术在蓬勃发展的同时也不断受到噬菌体感染的威胁,噬菌体感染已成为微生物发酵过程中的一个顽疾,其实质是噬菌体与细菌之间复杂的共进化关系。在漫长的进化过程中,噬菌体已经形成了多种针对细菌抗性系统的逃逸机制。合理的工厂设计、菌株的轮换策略和传统的基因工程方法能在一定程度上降低噬菌体感染的风险,但仍然无法避免。基于CRISPR-Cas系统的防治策略仅需噬菌体的序列信息就可以理性设计噬菌体抗性菌株,且可以通过叠加效应不断增强菌种抗性,从而避免噬菌体的逃逸;群体感应信号分子则可以从整体水平上调节细菌的噬菌体抗性。这些新发现为噬菌体感染问题的解决带了新的希望,而噬菌体基因组编辑技术和合成生物学的快速发展则将进一步加深人们对噬菌体感染防治领域的认识。     

Bacterial host strains that support replication of somatic coliphages

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log₁₀ fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.     

Longitudinal Study of Campylobacter jejuni Bacteriophages and Their Hosts from Broiler Chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

Bacteriophage cocktail and multi-strain probiotics in the feed for weanling pigs: effects on intestine morphology and targeted intestinal coliforms and Clostridium

Two experiments were conducted to investigate the effects of dietary supplementation of bacteriophage cocktail, probiotics and a combination of these two supplements on performance and gut health of weanling pigs. In Experiment 1, 150 weaned piglets were randomly allotted to three treatments on the basis of BW. The dietary treatments included a basal diet supplemented with 0 (control), 1.0 and 1.5 g/kg bacteriophage cocktail. Pigs fed 1.0 and 1.5 g/kg bacteriophage product had greater (P<0.05) average daily gain (ADG), apparent total tract digestibility of dry matter from day 22 to 35, ileal Lactobacillus spp., villus height (duodenum and jejunum), and fewer coliforms (ileum) and Clostridium spp. (ileum). In Experiment 2, 200 weaned piglets were randomly allotted to four treatments. Dietary treatments included basal diet, basal diet supplemented with 3.0 g/kg fermented probiotic product (P), 1.0 g/kg bacteriophage cocktail (B) and combination of 1.0 g/kg bacteriophage cocktail and 3.0 g/kg fermented probiotic product. Pigs fed bacteriophage cocktail diets had greater (P<0.05) overall ADG, gain to feed ratio (G:F), fecal score from day 8 to day 21, and pigs fed bacteriophage cocktail diets had fewer coliforms (ileum) Clostridium spp. (ileum and cecum). Probiotics significantly increased G:F, colonization of Lactobacillus spp. in ileum. At day 35, bacteriophage treatment group showed greater (P<0.05) villus height of the duodenum, but a deeper crypt in duodenum. The present results indicate that the bacteriophage cocktail had a potential to enhance the performance and gut health of weanling pigs, however their combination with probiotics did not show an interaction.     

Determination of the survival of bacteriophage M13 from chemical and physical challenges to assist in its sustainable bioprocessing

Bacteriophages are naturally infectious particles that replicate extremely efficiently in their bacterial hosts. Consequently, a facility processing bioproducts from a bacterial strain would be typically expected to focus on avoiding bacteriophage contamination. However, bacteriophages themselves are now showing great promise as a whole new class of industrial agents, such as biologically based nano-materials, delivery vectors and antimicrobials. This therefore raises a new challenge for their large-scale manufacture, potentially in contracted facilities shared with the host organism. The key issue is that knowledge of individual bacteriophage behaviour in the face of physical and chemical challenges is frequently incomplete, complicating decision-making regarding their safe introduction to a facility. This study tackles this issue for the filamentous bacteriophage M13. It was found that experimentation to determine an effective decontamination agent was important: Two of the three tested were ineffective. Virkon was considered to be the disinfectant of choice. Bacteriophage M13 was confirmed to be highly desiccation resistant, exhibiting a half-life of up to 120 days. Conversely, it was completely inactivated by strongly acidic and alkaline conditions and by temperatures above 95°C. By understanding the response of a bacteriophage to these challenges, steps towards their sustainable manufacture can be achieved.     

F-RNA噬菌体YM1肠道宿主分析

【背景】F-RNA噬菌体近年来常被作为水环境中诺如病毒污染的指示物。本课题组前期以大肠杆菌ATCC700891^T为宿主,从人便样中筛选出一株F-RNA噬菌体YM1,其与大肠杆菌噬菌体MS2亲缘关系最近,MS2宿主通常为含有性菌毛的雄性大肠杆菌。【目的】探索F-RNA噬菌体与其肠道宿主及诺如病毒之间的互作关系,筛选YM1的肠道宿主。【方法】采用选择性培养基筛选YM1阳性便样中的大肠杆菌并进行YM1侵染验证,结合16S rRNA基因扩增子测序分析YM1接种前后便样中的差异性菌群种类,对YM1阳性便样中潜在的YM1肠道宿主进行分析。【结果】筛选到351个大肠杆菌菌株,YM1侵染结果表明这些大肠杆菌均不是YM1的宿主;16S rRNA基因扩增子测序分析差异性菌种显示,Enterobacter sp. (OTU144)和Enterobacter sp. (OTU11)这2株肠杆菌属细菌的相对丰度在YM1感染后发生显著性的降低,表明该2种细菌可能为YM1的潜在肠道宿主。【结论】YM1具有严格的宿主特异性,便样中大肠杆菌并非YM1的肠道宿主,同时发现了2种YM1的潜在宿主,为进一步筛选分离YM1的肠道宿主提供了方向和依据。     

Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards treatment and prevention

Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy--the use of specific viruses that infect bacteria--is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections.     

Bacteriophage attachment to the S-layer proteins of the mosquito-pathogenic strains ofBacillus sphaericus

The linearly arrayed surface layer proteins found on the mosquito-pathogenic strains ofBacillus sphaericus function as the site of bacteriophage attachment for the ten lytic bacteriophages used in a bacteriophage typing scheme. Attachment to the surface layer proteins was demonstrated by the ability to block bacteriophage binding with antisera and the ability of the purified proteins to neutralize bacteriophage. Bacteriophage-resistant mutants have modified surface proteins that are less able to neutralize bacteriophages than is the protein of the parent strain. No evidence was obtained that sugar residues play a part in bacteriophage attachment. Phage neutralization by surface proteins from strains that do not serve as host to the phage indicates that, although strains in each phage group have a unique surface protein, the proteins do not determine the phage groups.     

噬菌体与宿主菌间的角逐

噬菌体和它们的宿主菌组成了地球上教目最庞大的微生物种群,噬菌体靠寄生宿主菌来扩增繁衍。但在漫长的进化中,噬菌体与宿主菌间不单是捕食关系,它们间还形成了复杂的相互对抗机制,其中抵抗防御机制的抗防御机制也会促使抗．抗防御机制的产生。从生态学角度来看,噬菌体和宿主菌间的共进化保持着动态平衡。本文综述近年来这一领域的研究,为更清楚地了解噬菌体与宿主菌间的关系和应用提供了参考。