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ISOLATION AND CHARACTERISATION OF BACTERIOPHAGES INFECTING BORRELIA BURGDORFERI SENSU LATO
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Lysogens are bacterial cells that have survived after genomically incorporating the DNA of temperate bacteriophages infecting them. If an infection results in lysogeny, the lysogen continues to grow and divide normally, seemingly unaffected by the integrated viral genome known as a prophage. However, the prophage can still have an impact on the host's phenotype and overall fitness in certain environments. Additionally, the prophage within the lysogen can activate the lytic pathway via spontaneous prophage induction (SPI), killing the lysogen and releasing new progeny phages. These new phages can then lyse or lysogenize other susceptible nonlysogens, thereby impacting the competition between lysogens and nonlysogens. In a scenario with differing growth rates, it is not clear whether SPI would be beneficial or detrimental to the lysogens since it kills the host cell but also attacks nonlysogenic competitors, either lysing or lysogenizing them. Here we study the evolutionary dynamics of a mixture of lysogens and nonlysogens and derive general conditions on SPI rates for lysogens to displace nonlysogens. We show that there exists an optimal SPI rate for bacteriophage λ and explain why it is so low. We also investigate the impact of stochasticity and conclude that even at low cell numbers SPI can still provide an advantage to the lysogens. These results corroborate recent experimental studies showing that lower SPI rates are advantageous for phage-phage competition, and establish theoretical bounds on the SPI rate in terms of ecological and environmental variables associated with lysogens having a competitive advantage over their nonlysogenic counterparts.
Many vector-borne diseases are transmitted through complex pathogen-vector-host networks, which makes it challenging to identify the role of specific host groups in disease emergence. Lyme borreliosis in humans is now the most common vector-borne zoonosis in the Northern Hemisphere. The disease is caused by multiple genospecies of Borrelia burgdorferi sensu lato bacteria transmitted by ixodid (hard) ticks, and the major host groups transmit Borrelia genospecies with different pathogenicity, causing variable clinical symptoms in humans. The health impact of a given host group is a function of the number of ticks it infects as well as the pathogenicity of the genospecies it carries. Borrelia afzelii, with mainly small mammals as reservoirs, is the most common pathogen causing Lyme borreliosis, and it is often responsible for the largest proportion of infected host-seeking tick nymphs in Europe. The bird-borne Borrelia garinii, though less prevalent in nymphal ticks, is more likely to cause Lyme neuroborreliosis, but whether B. garinii causes disseminated disease more frequently has not been documented. Based on extensive data of annual disease incidence across Norway from 1995 to 2017, we show here that 69% of disseminated Lyme borreliosis cases were neuroborreliosis, which is three times higher than predicted from the infection prevalence of B. garinii in host-seeking ticks (21%). The population estimate of migratory birds, mainly of thrushes, explained part of the annual variation in cases of neuroborreliosis, with a one-year time lag. We highlight the important role of the genospecies' pathogenicity and the host associations for understanding the epidemiology of disseminated Lyme borreliosis.
Lyme disease, initially described as Lyme arthritis, was reported before nucleic-acid based detection technologies were available. The most widely used diagnostic tests for Lyme disease are based on the serologic detection of antibodies produced against antigens derived from a single strain of Borrelia burgdorferi. The poor diagnostic accuracy of serological tests early in the infection process has been noted most recently in the 2018 Report to Congress issued by the U.S. Department of Health and Human Services Tick-Borne Disease Working Group. Clinical Lyme disease may be caused by a diversity of borreliae, including those classified as relapsing fever species, in the United States and in Europe. It is widely accepted that antibiotic treatment of Lyme disease is most successful during this critical early stage of infection. While genomic sequencing is recognized as an irrefutable direct detection method for laboratory diagnosis of Lyme borreliosis, development of a molecular diagnostic tool for all clinical forms of borreliosis is challenging because a “core genome” shared by all pathogenic borreliae has not yet been identified. After a diligent search of the GenBank database, we identified two highly conserved segments of DNA sequence among the borrelial 16S rRNA genes. We further developed a pair of Borrelia genus-specific PCR primers for amplification of a segment of borrelial 16S rRNA gene as a “core genome” to be used as the template for routine Sanger sequencing-based metagenomic direct detection test. This study presented examples of base-calling DNA sequencing electropherograms routinely generated in a clinical diagnostic laboratory on DNA extracts of human blood specimens and ticks collected from human skin bites and from the environment. Since some of the tick samples tested were collected in Ireland, borrelial species or strains not known to exist in the United States were also detected by analysis of this 16S rRNA “core genome”. We recommend that hospital laboratories located in Lyme disease endemic areas begin to use a “core genome” sequencing test to routinely diagnose spirochetemia caused by various species of borreliae for timely management of patients at the early stage of infection.
Purpose:
We collected data from an online survey of 200 of our patients, which evaluated the efficacy of dapsone (diaminodiphenyl sulfone, ie, DDS) combined with other antibiotics and agents that disrupt biofilms for the treatment of chronic Lyme disease/post-treatment Lyme disease syndrome (PTLDS). We also collected aggregate data from direct retrospective chart review, including laboratory testing for Lyme, other infections, and associated tick-borne coinfections. This helped us to determine the frequency of exposure to other infections/coinfections among a cohort of chronically ill Lyme patients, evaluate the efficacy of newer "persister" drug regimens like DDS, and determine how other infections and tick-borne coinfections may be contributing to the burden of chronic illness leading to resistant symptomatology.
Patients and methods:
A total of 200 adult patients recruited from a specialized Lyme disease medical practice had been ill for at least 1 year. We regularly monitored laboratory values and participants' symptom severity, and the patients completed the online symptom questionnaire both before beginning treatment and after 6 months on DDS combination therapy (DDS CT). Paired-samples t-tests and Wilcoxon signed-rank nonparametric test were performed on each of eight major Lyme symptoms, both before DDS CT and after 6 months of therapy.
Results:
DDS CT statistically improved the eight major Lyme symptoms. We found multiple species of intracellular bacteria including rickettsia, Bartonella, Mycoplasma, Chlamydia, Tularemia, and Brucella contributing to the burden of illness and a high prevalence of Babesia complicating management with probable geographic spread of Babesia WA1/duncani to the Northeast. Borrelia, Bartonella, and Mycoplasma species, as well as Babesia microti had variable manifestations and diverse seroreactivity, with evidence of persistence despite commonly prescribed courses of anti-infective therapies. Occasional reactivation of viral infections including human herpes virus 6 was also seen in immunocompromised individuals.
Conclusion:
DDS CT decreased eight major Lyme symptoms severity and improved treatment outcomes among patients with chronic Lyme disease/PTLDS and associated coinfections.
Bacteriophage (phage) therapy is a promising alternative to antibiotics for the treatment of bacterial pathogens, including Clostridium difficile. However, as for many species, in C. difficile the physical interactions between phages and bacterial cells have not been studied in detail. The initial interaction, known as phage adsorption, is initiated by the reversible attachment of phage tail fibers to bacterial cell surface receptors followed by an irreversible binding step. Therefore binding can dictate which strains are infected by the phage. In this study, we investigated the adsorption rates and irreversible binding of three C. difficile myoviruses: CDHM1, CDHM3 and CDHM6 to ten strains that represent ten prevalent C. difficile ribotypes, regardless of their ability to infect. CDHM1 and CDHM3 phage particles adsorbed by ~75% to some strains that they infected. The infection dynamics for CDHM6 are less clear and ~30% of the phage particles bound to all strains, irrespective of whether a successful infection was established. The data highlighted adsorption is phage-host specific. However, it was consistently observed that irreversible binding had to be above 80% for successful infection, which was also noted for another two C. difficile myoviruses. Furthermore, to understand if there is a relationship between infection, adsorption and phage tail fibers, the putative tail fiber protein sequences of CDHM1, CDHM3 and CDHM6 were compared. The putative tail fiber protein sequence of CDHM1 shares 45% homology at the amino acid level to CDHM3 and CDHM6, which are identical to each other. However, CDHM3 and CDHM6 display differences in adsorption, which highlights that there is no obvious relationship between putative tail fiber sequence and adsorption. The importance of adsorption and binding to successful infection is often overlooked, and this study provides useful insights into host-pathogen interactions within this phage-pathogen system.
Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira.
As the importance of bacteriophages as novel antimicrobials and potential diagnostics comes increasingly into focus, there is a heightened interest in understanding the mechanisms of how they interact with their bacterial hosts. The first step of a bacteriophage (phage) infection is the recognition of specific moieties on the bacterial cell surface as determined by their phage receptor binding proteins (RBPs). Knowledge of RBPs and how they interact with bacteria has been driven by studies of model phages and of industrially important phages, such as those that impact the dairy industry. Therefore, data from these phage groups constitute the majority of this review. We start with a brief introduction to phages, their life cycles and known receptors. We then review the state-of-the-art knowledge of phage RBPs of Gram-positive bacteria in the context of the better understood Gram-negative bacterial RBPs. In general, more is known about the RBPs of siphoviruses than myoviruses, which is reflected here, but for both virus families, where possible, we show what RBPs are, how they are arranged within phage genomes and what is known about their structures. As RBPs are the key determinant of phage specificity, studying and characterising them is important, for downstream applications such as diagnostic and therapeutic purposes.
Phages are complex biomolecular machineries that have to survive in a bacterial world. Phage genomes show many adaptations to their lifestyle such as shorter genes, reduced capacity for redundant DNA sequences, and the inclusion of tRNAs in their genomes. In addition, phages are not free-living, they require a host for replication and survival. These unique adaptations provide challenges for the bioinformatics analysis of phage genomes. In particular, ORF calling, genome annotation, noncoding RNA (ncRNA) identification, and the identification of transposons and insertions are all complicated in phage genome analysis. We provide a road map through the phage genome annotation pipeline, and discuss the challenges and solutions for phage genome annotation as we have implemented in the rapid annotation using subsystems (RAST) pipeline.
Background
Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. ResultsWe have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). Conclusions
Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.
This rebuttal Letter responds to a Letter in the IJSEM by Margos et al. challenging division of the genus Borrelia into two genera. We discuss here point-by-point the issues raised by Margos et al. and show that much of their criticism is unfounded and in several cases based on misreading of the presented results. We summarize here the extensive evidence based on genomic, genetic and phenotypic properties showing that the members of the family Borreliaceae (containing mainly the genus Borrelia) comprises two distinct and cohesive groups of microbes, differing in diseases they cause and other phenotypes. Prior to the proposed division, Borrelia spp. causing Lyme disease (LD) were already functionally treated as a distinct group, referred to as "B. burgdorferi sensu lato" to distinguish them from the other cluster of Borrelia spp. which includes all known species causing relapsing fever (RF). With the more explicit division of Borreliaceae species into two genus level groups, which are distinguishable from each other based on numerous unique genetic and molecular characteristics, the attention can now be focused on the biological significance of different molecular characteristics differentiating the two groups. The clear distinction of the LD and the RF groups of microbes based on numerous highly reliable markers, which are expected to be present even in uncharacterized members of these two groups, should aid in the improved diagnosis as well treatment of both these diseases, which is hindered by the conflation of a common name for agents causing two different types of diseases.
Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.
Due to the inherent limitations of conventional antibiotics for the treatment of C. difficile infection (CDI), there is a growing interest in the development of alternative treatment strategies. Both bacteriophages and R-type bacteriocins, also known as phage tail-like particles (PTLPs), show promise as potential antibacterial alternatives for treating CDI. Similar to bacteriophages, but lacking a viral capsid and genome, PTLPs remain capable of killing target bacteria. Here we describe our experience in the induction and purification of C. difficile PTLPs. These methods have been optimized to allow production of concentrated, non-contractile, and non-aggregated samples for both sensitivity testing and structural electron microscopy studies.
Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that are not killed by current Lyme antibiotics. To identify more effective drugs that are active against the round bodies of B. burgdorferi, we established a round body persister model induced by amoxicillin and screened the Food and Drug Administration (FDA) drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide (PI) viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven of these scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. While some drug candidates such as daptomycin and clofazimine overlapped with a previous screen against stationary phase B. burgdorferi persisters, additional drug candidates active against round bodies we identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even if pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.
PHASTER (PHAge Search Tool – Enhanced Release) is a significant upgrade to the popular PHAST web server for the rapid identification
and annotation of prophage sequences within bacterial genomes and plasmids. Although the steps in the phage identification
pipeline in PHASTER remain largely the same as in the original PHAST, numerous software improvements and significant hardware
enhancements have now made PHASTER faster, more efficient, more visually appealing and much more user friendly. In particular,
PHASTER is now 4.3× faster than PHAST when analyzing a typical bacterial genome. More specifically, software optimizations
have made the backend of PHASTER 2.7X faster than PHAST, while the addition of 80 CPUs to the PHASTER compute cluster are
responsible for the remaining speed-up. PHASTER can now process a typical bacterial genome in 3 min from the raw sequence
alone, or in 1.5 min when given a pre-annotated GenBank file. A number of other optimizations have also been implemented,
including automated algorithms to reduce the size and redundancy of PHASTER's databases, improvements in handling multiple
(metagenomic) queries and higher user traffic, along with the ability to perform automated look-ups against 14 000 previously
PHAST/PHASTER annotated bacterial genomes (which can lead to complete phage annotations in seconds as opposed to minutes).
PHASTER's web interface has also been entirely rewritten. A new graphical genome browser has been added, gene/genome visualization
tools have been improved, and the graphical interface is now more modern, robust and user-friendly. PHASTER is available online
at www.phaster.ca.
The potential for transport and dissemination of certain pathogenic microorganisms by migratory birds is of concern. Migratory birds might be involved in dispersal of microorganisms as their biological carriers. mechanical carriers. or as carriers of infected hematophagous ver-parasites (e.g., ixodid ticks). Many species of microorganisms pathogenic to homeothermic e vertebrates including humans have been associated with free-living migratory birds. Migratory birds of diverse species can play significant roles in the ecology and circulation of some arboviruses (e.g., eastern and western equine encephalomyelitis and Sindbis alphaviruses. West Nile and St., Louis encephalitis flaviviruses), influenza A virus. Newcastle disease virus, duck plague herpes-virus, Chlamydophila psittaci, Anaplasma phagocytophilum. Borrelia burgdorferi sensu lato. Campylobacter, jejuni, Salmonella enterica, Pasteurella multocida, Mycobacterium avium, Candida spp., and avian hematozoans. The efficiency of dispersal of pathogenic microorganisms depends on a wide variety of biotic and abiotic factors affecting the survival or the agent in. or disappearance from, a habitat or ecosystem in a new geographic area.
Borreliosis is highly prevalent in Xinjiang Uygur Autonomous Region, China. However, little is known about the presence of Borrelia pathogens in tick species in this region, in addition Borrelia pathogens have not been isolated from domestic animals.
We collected adult ticks from domestic animals at 19 sampling sites in 14 counties in northern Xinjiang from 2012 to 2014. Ticks were identified to species by morphology and were molecularly analysed by sequences of mitochondrial 16S rDNA gene; 4-8 ticks of each species at every sampling site were sequenced. 112 live adult ticks were selected for each species in every county, and were used to culture Borrelia pathogens; the genotypes were then determined by sequences of the 5S-23S rRNA intergenic spacer and the outer surface protein A (ospA) gene.
A total of 5257 adult ticks, belonging to four genera and seven species, were collected. Compared with three decades ago, the abundance of the five common tick species during the peak ixodid tick season has changed. Certain tick species, such as Rhipicephalus turanicus (Rh. turanicus), was found at Jimusaer, Yining, Fukang, and Chabuchaer Counties for the first time. Additionally, the sequence analyses showed that the Hyalomma asiaticum (Hy. asiaticum), Haemaphysalis punctata (Ha. punctata), and Dermacentor marginatus (D. marginatus) that were collected from different sampling sites (≥3 sites) shared identical 16S rDNA sequences respectively. For the tick species that were collected from the same county, such as Hy. asiaticum from Shihezi County and Rh. turanicus from Yining County, their 16S rDNA sequences showed genetic diversity. In addition, sixteen Borrelia isolates were found in Hy. asiaticum, Ha. punctata, D. marginatus and Rh. turanicus, which infested cattle, sheep, horse and camel in Yining, Chabuchaer, Shihezi and Shawan Counties. All of the isolates were genetically identified as B. Burgdorferi sensu stricto.
Warmer and wetter climate may have contributed to the altered distribution and abundance of the five most common ticks in northern Xinjiang. The genetic analyses showed that certain tick species, such as Hy. asiaticum or Rh. turanicus, exhibit genetic commonness or diversity. Additionally, this study is the first to isolate B. burgdorferi sensu stricto in Hy. asiaticum asiaticum, H. punctata, D. nuttalli and D. marginatus ticks from domestic animals. These ticks may transmit borreliosis among livestock.
Diagnostic methods in erythema migrans are still not standardized.
To evaluate the frequency of Borrelia burgdorferi s.l. DNA presence in patients with erythema migrans (EM); to assess the polymerase chain reaction (PCR) procedure for detecting B. burgdorferi s.l. DNA in patients with the skin form of Lyme borreliosis; and to compare the results of the PCR-based method with the traditional ELISA method.
Skin biopsy and blood samples from 93 patients with EM were examined for B. burgdorferi s.l. DNA detection (PCR). Seventy-one of these patients were examined for the presence of anti-B. burgdorferi s.l. antibodies (ELISA).
Borrelia burgdorferi s.l. DNA was detected in 48% of the skin biopsy specimens and in 2% of blood samples. Only 1 patient was PCR positive in both blood and skin samples. Seventy percent of patients whose PCR results were positive were bitten by a tick less than 14 days before. IgM anti-B. burgdorferi s.l - specific antibodies were present in the serum of 35% of patients and IgG antibodies - in 30% of patients. Seventeen percent were positive in both IgM and IgG.
Polymerase chain reaction of skin biopsy specimens seems to be currently the most sensitive and specific test for the diagnosis of patients with EM, especially in patients with a short duration of the disease (< 14 days) but still its effectiveness is much lower than expected. Polymerase chain reaction of blood samples cannot be recommended at the present time for the routine diagnostic of patients with EM.
Spirochete bacteria Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common tick-borne infection in the Northern hemisphere. There is a long-standing debate regarding the role of pleomorphic forms in Lyme disease pathogenesis, while very little is known about the characteristics of these morphological variants. Here, we present a comprehensive analysis of B. burgdorferi pleomorphic formation in different culturing conditions at physiological temperature. Interestingly, human serum induced the bacteria to change its morphology to round bodies. In addition, biofilm-like colonies in suspension were found to be part of B. burgdorferi's normal in vitro growth. Further studies provided evidence that spherical round bodies had an intact and flexible cell envelope demonstrating that they are not cell wall deficient, or degenerative as previously implied. However, the round bodies displayed lower metabolic activity compared to spirochetes. Furthermore, our results indicated that the different pleomorphic variants were distinguishable by having unique biochemical signatures. Consequently, pleomorphic B. burgdorferi should be taken into consideration as being clinically relevant and influence the development of novel diagnostics and treatment protocols.
Lyme borreliosis is increasing rapidly in many parts of the world and is the most commonly occurring vector-borne disease in Europe and the USA. The disease is transmitted by ticks of the genus Ixodes. They require a blood meal at each stage of their life cycle and feed on a wide variety of wild and domestic animals as well as birds and reptiles. Transmission to humans is incidental and can occur during visits to a vector habitat, when host mammals and their associated ticks migrate into the urban environment, or when companion animals bring ticks into areas of human habitation. It is frequently stated that the risk of infection is very low if the tick is removed within 24-48 hours, with some claims that there is no risk if an attached tick is removed within 24 hours or 48 hours. A literature review has determined that in animal models, transmission can occur in <16 hours, and the minimum attachment time for transmission of infection has never been established. Mechanisms for early transmission of spirochetes have been proposed based on their presence in different organs of the tick. Studies have found systemic infection and the presence of spirochetes in the tick salivary glands prior to feeding, which could result in cases of rapid transmission. Also, there is evidence that spirochete transmission times and virulence depend upon the tick and Borrelia species. These factors support anecdotal evidence that Borrelia infection can occur in humans within a short time after tick attachment.
Bacteriophages and genetic elements, such as prophage-like elements, pathogenicity islands, and phage morons, make up a considerable amount of the bacterial genome. Their transfer and subsequent activity within the host's genetic circuitry have had a significant impact on bacterial evolution. In what follows, it is considered what underlying mechanisms might cause the spontaneous activity of lysogenic phages in single bacterial cells, how the spontaneous induction of prophages can lead to competitive advantages for and influence the life style of bacterial populations or the virulence of pathogenic strains.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium sized mammals, birds, companion animals (dogs, cats) and larger mammals (roe deer, wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, 'Candidatus Neoehrlichia mikurensis', Rickettsia helvetica, R. monacensis), Borrelia miyamatoi and protozoans (Babesia divergens, B. venatorum and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.
Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.
In recent years, the use of lytic bacteriophages as antimicrobial agents controlling pathogenic bacteria has appeared as a promising new alternative strategy in the face of growing antibiotic resistance which has caused problems in many fields including medicine, veterinary medicine, and aquaculture. The use of bacteriophages has numerous advantages over traditional antimicrobials. The effectiveness of phage applications in fighting against pathogenic bacteria depends on several factors such as the bacteriophages/target bacteria ratio, the mode and moment of treatment, environmental conditions (pH, temperature...), the neutralization of phage and accessibility to target bacteria, amongst others. This report presents these factors and the challenges involved in developing phage therapy applications.
Ticks are highly susceptible to global environmental and socio-economical changes. Several tick-borne pathogens have been reported in new geographical regions while new species, strains or genetic variants of tick-borne microorganisms are continually being detected. However, tick-borne pathogens are still poorly understood, and it is estimated that half of all human tick-borne disease has an unknown origin. Therefore in order to prevent these diseases, more effort is required to identify unknown or unexpected tick-borne pathogens. Ixodes ricinus is the vector for a broad range of bacterial pathogens and the most prevalent tick in Europe. The aim of the present study was to evaluate the capability of Next Generation Sequencing (NGS) to extend the inventory of pathogenic bacteria carried by this species of tick in France.
RNA and DNA were extracted from 1450 I. ricinus questing nymphs collected by flagging in Alsace, France. RNA was pooled and used for NGS. Following de novo assembly, bacterial contigs were assigned to the closest known taxonomy. DNA was used for real time PCR to confirm taxonomic species assignment of NGS-derived contigs for the doubtful cases, and for determination of prevalence.
We have generated a global in-depth picture of tick-borne bacteria. We identified RNA from the main pathogenic bacterial species known to be transmitted by I. ricinus. In addition we also identified unanticipated bacterial species for which we have estimated the prevalence within those ticks inhabiting the studied areas.
The data obtained from this study has proven that NGS has an enormous potential to detect the unexpected and provides the means to monitor pathogen occurrence.
In the 1960s-1980s, determination of bacterial growth rates was an important tool in microbial genetics, biochemistry, molecular biology, and microbial physiology. The exciting technical developments of the 1990s and the 2000s eclipsed that tool; as a result, many investigators today lack experience with growth rate measurements. Recently, investigators in a number of areas have started to use measurements of bacterial growth rates for a variety of purposes. Those measurements have been greatly facilitated by the availability of microwell plate readers that permit the simultaneous measurements on up to 384 different cultures. Only the exponential (logarithmic) portions of the resulting growth curves are useful for determining growth rates, and manual determination of that portion and calculation of growth rates can be tedious for high-throughput purposes. Here, we introduce the program GrowthRates that uses plate reader output files to automatically determine the exponential portion of the curve and to automatically calculate the growth rate, the maximum culture density, and the duration of the growth lag phase. GrowthRates is freely available for Macintosh, Windows, and Linux.We discuss the effects of culture volume, the classical bacterial growth curve, and the differences between determinations in rich media and minimal (mineral salts) media. This protocol covers calibration of the plate reader, growth of culture inocula for both rich and minimal media, and experimental setup. As a guide to reliability, we report typical day-to-day variation in growth rates and variation within experiments with respect to position of wells within the plates. © 2013 The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected]
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Patients with central nervous system (CNS) infection experience very high levels of morbidity and mortality, in part because of the many challenges inherent to the diagnosis of CNS infection and identification of a causative pathogen. The clinical presentation of CNS infection is nonspecific, so clinicians must often order and interpret many diagnostic tests in parallel. This can be a daunting task given the large number of potential pathogens and the availability of different testing modalities. Here, we review traditional diagnostic techniques including Gram stain and culture, serology, and polymerase chain reaction (PCR). We highlight which of these are recommended for the pathogens most commonly tested among U.S. patients with suspected CNS infection. Finally, we describe the newer broad-range diagnostic approaches, multiplex PCR and metagenomic sequencing, which are increasingly used in clinical practice.
ABSTRACT
In the majority of cases, Lyme disease responds well to antibiotic treatment. Nevertheless, some patients may suffer from chronic nonspecific symptoms such as musculoskeletal pain and fatigue, termed as a post-treatment Lyme disease syndrome (PTLDS).
Proper understanding and management of PTLDS may prevent from the incorrect diagnosis of patients with autoimmune, neuromuscular or other somatic disorders, where identical symptoms are equally prevalent.
We report a 37-year-old female with systemic lupus erythematosus (SLE) who was repeatedly misdiagnosed for a chronic Lyme disease and treated with tetracycline antibiotics before she developed an acute exacerbation of SLE and finally died due to multi-organ failure.
The internet encourages patients to ask physicians for serology testing for Lyme disease and to demand further antibiotic treatment. However, there is clear evidence that positive serology does not indicate infection with B.burgdorferi and most importantly, antibiotic therapy for PTLDS is potentially harmful for patients.
It is crucial to follow the recent guidelines for diagnosis and treatment of Lyme disease and PTLDS to avoid the repetition of antibiotic regimens and misdiagnosing patients.
Keywords
systemic lupus erythematosus
SLE
Lyme disease
post-treatment Lyme disease syndrome
Borrelia
autoimmunity
IVF
self-diagnosis
Background:
Non-guideline endorsed post-treatment courses of antibiotics for post-Lyme disease syndrome (PLDS) have been linked to adverse patient outcomes, but these findings have yet to be validated in large systematic evaluations.
Methods:
A retrospective cohort analysis of medical and pharmacy claims derived from the Truven Health Market Scan® Commercial Claims and Encounters Database assessed 90-day incidence rates of adverse events (AEs) associated with PLDS treatments (PLDS-TX). Patients were diagnosed with PLDS ≥6 months after initial diagnosis and standard antibiotic treatment for Lyme disease. Comparison cohorts included intravenous (IV) PLDS-TX with or without oral antibiotics; oral antibiotic-only PLDS-TX; or neither.
Results:
Composite AE incidence rates were higher for patients treated with IV or oral PLDS-TX than for patients not receiving either treatment (18.7%, 16.8%, and 13.4%, respectively; P=0.019). Significant between-group differences in AE incidence rates were noted for electrolyte imbalance (4.0%, 1.5%, and 0.7%, respectively; P=0.001) and infection (14.0%, 12.7%, and 9.3%; P=0.006). Infection prevalence increased by 22.0% in the IV treatment group and 17.7% in the oral group. Incidence rates for all-cause and AE-related hospital stays and emergency department (ED) visits were higher for treated than nontreated patients, particularly when treatment was IV (all P<0.01). Of IV-treated patients, 7.3% experienced an incident all-cause inpatient stay and 11.3% an incident all-cause ED visit, compared with, respectively, 2.2% and 3.4% of those treated with oral antibiotics and 0.9% and 1.9% of nontreated patients.
Conclusions:
Use of IV therapies or oral antibiotics for PLDS was associated with increased patient morbidity within 90 days.
Relapsing fever (RF) in North America is caused primarily by the spirochete Borrelia hermsii and is associated with the bite of its tick vector Ornithodoros hermsi. Although this spirochete was known long before the discovery of the Lyme disease (LD) spirochete, Borrelia burgdorferi, basic methods to facilitate the study of B. hermsii have lagged behind. One important technique to expedite the study of the molecular biology and pathogenesis of B. hermsii would be a reliable method to grow and clone these bacteria in solid medium, which we now describe. We have defined the solidifying agent, plating temperature, oxygen concentration, and pH for the efficient plating of two species of RF spirochetes, B. hermsii and Borrelia turicatae. Importantly, this technique allowed us to successfully isolate virulent, clonal cell lines of spirochetes, and to enumerate and isolate viable B. hermsii from infected mouse blood and tick tissues. Our results also demonstrate the value of testing a range of several environmental variables to increase the efficiency of bacterial isolation, which may be helpful for researchers working on other prokaryotes that are intractable for in vitro growth.
The microbiota of hard ticks has been an area of growing interest due to the potential role that the broader microbial community may play in pathogen carriage and transmission. In the last two decades, Ontario, Canada has experienced rapid changes in the risk of tick-borne disease, primarily due to the range expansion of Ixodes scapularis. Another human-biter, Dermacentor variabilis, is a longstanding resident of the province, but currently poses minimal risk of pathogen transmission. To examine the microbiota of these two species, we collected adult and nymphal I. scapularis and D. variabilis from the eastern and southern regions of the province via tick dragging, and conducted next generation sequencing of 19 samples (composed of 45 ticks) via Illumina MiSeq. A total of 1400469 sequences were detected (median 69118/sample; range 23350-155227). The most abundant families of bacteria were unclassified Clostridiales and Ruminococcaceae for both I. scapularis and D. variabilis. No significant differences in the relative abundances of any phylum, class, order, family or genus were detected between locations (east vs south), sex, life stage or tick species. There were no differences in community membership or structure based on unifrac and AMOVA analyses. Female and male ticks had lower microbial diversity when compared to nymphs, based on the Simpson's index and Shannon evenness index. The findings of our study differ from previous studies of these tick species conducted in other geographic areas, and highlight the potential role geography and related ecological factors may have in shaping the tick microbiota.
Borrelia miyamotoi is a spirochete bacterium related to Borrelia burgdorferi sensu lato, the cause of Lyme borreliosis, and vectored by ticks. In 2014, B. miyamotoi was identified in three questing Ixodes ricinus collected in the UK. We sought to confirm the presence of B. miyamotoi in the UK. Ticks were collected from four locations not previously investigated for B. miyamotoi or B. burgdorferi s.l. and of which two are considered as Lyme borreliosis "hotspots" based on hospital records of the disease. We independently confirm that B. miyamotoi is present in the UK and support the view that B. miyamotoi is likely to have a broad geographic distribution, at low levels. Our study also adds to the existing data on the distribution of B. burgdorferi s.l. in the UK and demonstrates that although the two "hotspots" had relatively high tick densities, they did not have the highest proportion of infected ticks.
Ticks were collected during March-July 2015 from dogs by veterinarians throughout the U.K. and used to estimate current prevalences and distributions of pathogens. DNA was extracted from 4750 ticks and subjected to polymerase chain reaction and sequence analysis to identify Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) and Babesia (Piroplasmida: Babesiidae) species. Of 4737 ticks [predominantly Ixodes ricinus Linneaus (Ixodida: Ixodidae)], B. burgdorferi s.l. was detected in 94 (2.0%). Four Borrelia genospecies were identified: Borrelia garinii (41.5%); Borrelia afzelli (31.9%); Borrelia burgdorferi sensu stricto (25.5%), and Borrelia spielmanii (1.1%). One Rhipicephalus sanguineus Latreille (Ixodida: Ixodidae), collected from a dog with a history of travel outside the U.K., was positive for B. garinii. Seventy ticks (1.5%) were positive for Babesia spp. Of these, 84.3% were positive for Babesia venatorum, 10.0% for Babesia vulpes sp. nov., 2.9% for Babesia divergens/Babesia capreoli and 1.4% for Babesia microti. One isolate of Babesia canis was detected in a Dermacentor reticulatus (Ixodida: Ixodidae) tick collected from a dog that had recently travelled to France. Prevalences of B. burgdorferi s.l. and Babesia spp. did not differ significantly between different regions of the U.K. The results map the widespread distribution of B. burgdorferi s.l. and Babesia spp. in ticks in the U.K. and highlight the potential for the introduction and establishment of exotic ticks and tick-borne pathogens.
Importance:
The small and exceptionally fragmented genome of the Lyme disease spirochete Borrelia burgdorferi encodes for over 120 lipoproteins. Studies in the field have predominantly focused on a relatively small number of surface lipoproteins that play important roles in the transmission and pathogenesis of this global human pathogen. Yet, a comprehensive spatial assessment of the entire borrelial lipoproteome has been missing. The current study newly identifies 52 surface and 23 periplasmic lipoproteins. Overall, two thirds of the B. burgdorferi lipoproteins localize to the surface, while outer membrane lipoproteins facing the periplasm are rare. This analysis underscores the dominant contribution of lipoproteins to the spirochete's rather complex and adaptable host-pathogen interface, and encourages further functional exploration of its lipoproteome.
Global temperatures continue to rise, reaching new records almost every year this decade. Although the causes are debated, climate change is a reality. Consequences of climate change include melting of the arctic ice cap, rising of sea levels, changes in precipitation patterns, and increased severe weather events. This article updates dermatologists about the effects of climate change on the epidemiology and geographic ranges of selected skin diseases in North America. Although globalization, travel, and trade are also important to changing disease and vector patterns, climate change creates favorable habitats and expanded access to immunologically naïve hosts. Endemic North American illnesses such as Lyme disease, leishmaniasis, and dimorphic fungal infections have recently expanded the geographic areas of risk. As temperatures increase, epidemic viral diseases such as hand-foot-and-mouth disease may develop transmission seasons that are longer and more intense. Chikungunya and dengue are now reported within the southern United States, with Zika on the horizon. Cutaneous injuries from aquatic and marine organisms that have expanding habitats and longer durations of peak activity include jellyfish envenomation, cercarial dermatitis, and seabather eruption, among others. Skin cancer rates may also be affected indirectly by changes in temperature and associated behaviors.
Background:
Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA.
Methods:
At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR.
Findings:
100 545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site.
Interpretation:
We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis.
Funding:
US Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) Cooperative Agreement and Mayo Clinic Small Grant programme.
This chapter discusses approaches to mass-balance-based modeling of aerobic and anaerobic waste treatment processes originating in IAWQ Activated Sludge Models 1, 2, and 3. Within this framework, three mathematical models of thermophilic treatment processes are described: two aerobic models for cheese whey and potato stillage/distiller’s slops and an anaerobic model for “dry” chicken waste and dehydrated waste-activated sludge. Additionally, a new model of an autothermal thermophilic aerobic digestion (ATAD) reactor based on material and energy balances is presented. Process sampling issues including methods for assuring representative samples from bioreactors treating highly heterogeneous solid food wastes are addressed. Applying the principles of Process Analytical Technology, the task of effective monitoring and controlling the biological processes is discussed. Finally, knowledge-based control strategies developed during our EC-funded thermophilic bioremediation project are outlined under three distinct categories: supervisory control, physiological state classification, and direct control strategies.
The majority of laboratory tests performed for the diagnosis of Lyme disease are based on detection of the antibody responses against B burgdorferi in serum. The sensitivity of antibody-based tests increases with the duration of the infection. Patients early in their illness are more likely to have a negative result. There is a need to simplify the testing algorithm for Lyme disease, improving sensitivity in early disease while still maintaining high specificity and providing information about the stage of infection. The development of a point of care assay and biomarkers for active infection would be major advances for the field.
Published by Elsevier Inc.
The success of phage therapy is dependent on the development of strategies able to overcome the limitations of bacteriophages as therapeutic agents, the creation of an adequate regulatory framework, the implementation of safety protocols, and acceptance by the general public. Many approaches have been proposed to circumvent phages' intrinsic limitations but none have proved to be completely satisfactory. In this review we present the major hurdles of phage therapy and the solutions proposed to circumvent them. A thorough discussion of the advantages and drawbacks of these solutions is provided and special attention is given to the genetic modification of phages as an achievable strategy to shape bacteriophages to exhibit desirable biological properties.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Lyme disease was originally identified in Lyme, Connecticut, based upon an unusual cluster of what appeared to be patients with juvenile rheumatoid arthritis. It was subsequently identified as a new clinical entity originally called Lyme arthritis based on the observation that arthritis was a major clinical feature. However, Lyme arthritis is now called Lyme disease based upon the understanding that the clinical features include not only arthritis, but also potential cardiac, dermatologic and neurologic findings. Lyme disease typically begins with an erythematous rash called erythema migrans (EM). Approximately 4–8% of patients develop cardiac, 11% develop neurologic and 45–60% of patients manifest arthritis. The disease is transmitted following exposure to a tick bite containing a spirochete in a genetically susceptible host. There is considerable data on spirochetes, including Borrelia burgdorferi (Bb), the original bacteria identified in this disease. Lyme disease, if an organism had not been identified, would be considered as a classic autoimmune disease and indeed the effector mechanisms are similar to many human diseases manifest as loss of tolerance. The clinical diagnosis is highly likely based upon appropriate serology and clinical manifestations. However, the serologic features are often misinterpreted and may have false positives if confirmatory laboratory testing is not performed. Antibiotics are routinely and typically used to treat patients with Lyme disease, but there is no evidence that prolonged or recurrent treatment with antibiotics change the natural history of Lyme disease. Although there are animal models of Lyme disease, there is no system that faithfully recapitulates the human disease. Further research on the effector mechanisms that lead to pathology in some individuals should be further explored to develop more specific therapy.
Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens. Curr. Protoc. Microbiol. 34:12C.1-12C.31. © 2014 by John Wiley & Sons, Inc.
Background
Current Clostridium difficile infection (CDI) antibiotic regimens have become increasingly ineffective at achieving cure and preventing recurrence. A recently developed alternative to conventional antibiotics are phage tail-like particles (PTLPs), which are proteins that are morphologically similar to bacteriophages and are produced by C difficile. This study examines the in vitro killing spectrum of a previously unreported PTLP isolated from a clinical isolate of C difficile.
Methods
Using patient-derived samples from an institutional review board-approved C difficile tissue bank, a ribotype 078 C difficile isolate was anaerobically incubated on blood agar plates that were preswabbed with norfloxacin to induce the production of PTLPs. Concentrated PTLP populations were confirmed using transmission electron microscopy. Using a standard lawn spot approach, bactericidal activity was assessed as indicated by a clearing within the bacterial lawn. The PTLP genomic cluster was also fully sequenced and open reading frames were annotated according to predicted function.
Results
PTLPs were assessed using 64 patient-derived C difficile isolates of varying ribotypes. PTLPs demonstrated complete bactericidal activity in 21 of 25 ribotype 027 isolates with partial activity in 2 of the 25. Complete bactericidal activity was not demonstrated against any other ribotype or non-difficile bacteria, suggesting a species and ribotype specificity. Functional genes, which may be necessary for killing, were identified within the PTLP genetic locus.
Conclusion
PTLPs demonstrate capability in eradicating C difficile in vitro, and with further development, may represent an organism-specific, microbiome-sparing therapy for CDI.
Interstrand cross-linking of DNA is believed to account for the cytotoxicity of many bifunctional alkylating agents, some of which are useful in the treatment of human cancer. The nucleotide sequences at which these cross-links are formed have been defined at single nucleotide resolution in DNA fragments for several agents, including mechlorethamine, cisplatin, mitomycin C, and some structurally related agents. Taken together, the structure of duplex DNA, the sequences which are cross-linked, and the atomic sites on DNA which are linked, indicate that cross-linking occurs preferentially at locations which will result in minimal distortion of B-DNA. The proposal that this preference is primarily expressed by minimizing the energy of the transition state for conversion of monoadducts to cross-links is supported by experiments with mechlorethamine. It is suggested that extension of the modest sequence-recognizing capacity of these cross-linking agents by conjugation to highly sequence-selective “delivery vehicles” may yield second generation, targeted antitumor drugs.
About 300 000 cases of Lyme disease are diagnosed in the United States each year, according to data presented by scientists from the US Centers for Disease Control and Prevention (CDC) at the International Conference on Lyme Borreliosis in Boston in August. The new estimate is 10 times greater than the number of cases of the infection reported to the CDC annually. Each year, about 30 000 cases of Lyme disease are reported to the CDC, primarily from states in the upper Midwest and Northeast. But many cases may not come to the attention of state public health authorities, so the CDC embarked on an effort to better assess the prevalence of Lyme disease by looking at clinical records, laboratory reports, and a survey of the public. The new estimate is based on preliminary results from those efforts.
A review of reports on the genetic and phenotypic characteristics of strains of the spirochete which causes Lyme disease revealed that these organisms are representative of a new species of Borrelia. We propose the name Borrelia burgdorferi for this species. The type strain of B. burgdorferi is strain B31 (= ATCC 35210). In two separate studies the guanine-plus-cytosine content of the deoxyribonucleic acid of the type strain was determined to be 29.0 to 30.5 mol% (thermal denaturation method).