Whole Genome Sequencing for Outbreak Detection of Salmonella enterica

Salmonella typhimurium Salmonella bacteria are a common cause of infectious disease in human and animals. Salmonella is classically divided into species S. bongori and S. enterica – which is in turn further divided into more than 2,500 different serotypes. However, only a limited number of serovars that are responsible for most infections. In Europe, the most prevalent S. enterica serovars isolated from humans are Enteritidis and Typhimurium, responsible for over 75% of the human cases of salmonellosis.

In order to understand the epidemiology and implement control programs, reliable and rapid sub-typing is essential. Different typing methods are commonly used as a central part of the detection and investigation of Salmonella outbreaks, for instance, serotyping, phage typing, pulse-field gel electrophoresis (PFGE) and multilocus variable number of tandem repeat analysis (MLVA). PFGE has become the standard for epidemiological investigations of foodborne bacterial pathogens including Salmonella. A drawback of PFGE is that it is unable to separate very closely related strains because the low rate of genetic variation does not significantly impact the electrophoretic mobility of a restriction fragment.

During recent years the cost of whole genome sequencing (WGS) has decreased dramatically and the technology becomes increasingly available for routine use around the world. The speed of sequencing is decreasing from several days or weeks to perhaps hours for a bacterial genome in the near future. This combination of low cost and high speed opens an opportunity for WGS to become very useful and practical in bacterial infection studies including the routine use in diagnostic and public health microbiology.

A new study evaluates WGS for outbreak typing of S. enterica and compares it to results obtained using the conventional typing method, PFGE. The results show that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

Evaluation of Whole Genome Sequencing for Outbreak Detection of Salmonella enterica. (2014) PLoS ONE 9(2): e87991. doi: 10.1371/journal.pone.0087991
Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly ‘real-time’ monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE reveling that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

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Where will Ebola strike next?

Ebola risk Since the first outbreaks of Ebola virus disease in 1976, there have been numerous other outbreaks in humans across Africa with fatality rates ranging from 50% to 90%. Humans can become infected with the Ebola virus after direct contact with blood or bodily fluids from an infected person or animal. The virus also infects and kills other primates, though fruit bats are suspected to be the most likely carriers of the virus in the wild.

The largest recorded outbreak of Ebola virus disease is ongoing in West Africa: more people have been infected in this current outbreak than in all previous outbreaks combined. The current outbreak is also the first to occur in West Africa – which is outside the previously known range of the Ebola virus. A new paper in eLife updates predictions about where in Africa wild animals may harbour the virus and where the transmission of the virus from these animals to humans is possible.

Mapping the zoonotic niche of Ebola virus disease in Africa. (2014) eLife 3:e04395 doi: 10.7554/eLife.04395
Ebola virus disease (EVD) is a complex zoonosis that is highly virulent in humans. The largest recorded outbreak of EVD is ongoing in West Africa, outside of its previously reported and predicted niche. We assembled location data on all recorded zoonotic transmission to humans and Ebola virus infection in bats and primates (1976–2014). Using species distribution models, these occurrence data were paired with environmental covariates to predict a zoonotic transmission niche covering 22 countries across Central and West Africa. Vegetation, elevation, temperature, evapotranspiration, and suspected reservoir bat distributions define this relationship. At-risk areas are inhabited by 22 million people; however, the rarity of human outbreaks emphasises the very low probability of transmission to humans. Increasing population sizes and international connectivity by air since the first detection of EVD in 1976 suggest that the dynamics of human-to-human secondary transmission in contemporary outbreaks will be very different to those of the past.

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Recent developments linking retroviruses to human breast cancer

Retrovirus replication We have known for decades that viruses =can couse breast cancer in animals (Mouse Mammary Tumour Virus – MMTV). Evidence has been accumulating that retroviruses may play a part in human breast cancers. This short review summarizes iur prsent state fo knowledge.


Recent developments linking retroviruses to human breast cancer: infectious agent, enemy within or both? J Gen Virol. 12 Sep 2014. doi: 10.1099/vir.0.070631-0
Evidence is accumulating that one or more beta-retrovirus is associated with human breast cancer. Retroviruses can exist as an infectious (exogenous) virus or as a part of the genetic information of cells due to germline integration (endogenous). An exogenous virus with a genome that is highly homologous to mouse mammary tumor virus (MMTV) is gaining acceptance as possibly being associated with human breast cancer and recently furnished evidence is discussed in this article, as is the evidence for involvement of an endogenous human beta-retrovirus, HERV-K. Modes of interaction are also reviewed and linkage to APOBEC3 suggested.


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Streptococcus pneumoniae causes cardiac lesions

Streptococcus pneumoniae Hospitalization for community-acquired pneumonia carries a documented risk for adverse cardiac events. These occur during infection and contribute to elevated mortality rates in convalescent individuals up to a year after. A new paper describes a previously unrecognized pathogenic mechanism by which Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, causes direct cardiotoxicity and forms microscopic bacteria-filled lesions within the heart.

Cardiac microlesions were detected in experimentally infected mice and rhesus macaques, as well as in heart sections from humans who succumbed to invasive pneumococcal disease. Cardiac microlesion formation required interaction of the bacterial adhesin CbpA with host laminin receptor and bacterial cell wall with platelet-activating factor receptor and also involved the pore-forming toxin pneumolysin. When infected mice were rescued with antibiotics, collagen deposition occurs at former lesion sites. Thus, microlesions and the scarring that occurs thereafter may explain why adverse cardiac events occur during and following pneumococcal disease.

Streptococcus pneumoniae Translocates into the Myocardium and Forms Unique Microlesions That Disrupt Cardiac Function. (2014) PLoS Pathog 10(9): e1004383. doi:10.1371/journal.ppat.1004383
Hospitalization of the elderly for invasive pneumococcal disease is frequently accompanied by the occurrence of an adverse cardiac event; these are primarily new or worsened heart failure and cardiac arrhythmia. Herein, we describe previously unrecognized microscopic lesions (microlesions) formed within the myocardium of mice, rhesus macaques, and humans during bacteremic Streptococcus pneumoniae infection. In mice, invasive pneumococcal disease (IPD) severity correlated with levels of serum troponin, a marker for cardiac damage, the development of aberrant cardiac electrophysiology, and the number and size of cardiac microlesions. Microlesions were prominent in the ventricles, vacuolar in appearance with extracellular pneumococci, and remarkable due to the absence of infiltrating immune cells. The pore-forming toxin pneumolysin was required for microlesion formation but Interleukin-1b was not detected at the microlesion site ruling out pneumolysin-mediated pyroptosis as a cause of cell death. Antibiotic treatment resulted in maturing of the lesions over one week with robust immune cell infiltration and collagen deposition suggestive of long-term cardiac scarring. Bacterial translocation into the heart tissue required the pneumococcal adhesin CbpA and the host ligands Laminin receptor (LR) and Platelet-activating factor receptor. Immunization of mice with a fusion construct of CbpA or the LR binding domain of CbpA with the pneumolysin toxoid L460D protected against microlesion formation. We conclude that microlesion formation may contribute to the acute and long-term adverse cardiac events seen in humans with IPD.


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What’s the diference between cow TB and human TB?

Mycobacterium tuberculosis In 1901, when Robert Koch proposed that the bacilli causing human and bovine tuberculosis were not identical, this view caused much controversy. 113 years later we know that the bovine tuberculosis agent, Mycobacterium bovis, together with other animal strains, forms a separate phylogenetic lineage apart from the human Mycobacterium tuberculosis lineages, but the molecular reasons why bovine and animal strains only play minor roles in human tuberculosis epidemiology remain unknown. A recent paper in PNAS uses genetic transfer and virulence experiments to show that specific mutations in a virulence regulator contribute to lower fitness and virulence of M. bovis and related strains for the human host, likely obstructing the capacity of causing overt disease needed for efficient human-to-human transmission.

Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator. (2014) PNAS USA 111(31): 11491–11496, doi: 10.1073/pnas.1406693111
Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis–associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.

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Rabies Rapid Transit System

Rabies virus Negri bodies Rabies virus hijacks the transport systems of neurons, manipulating axonal transport machinery to get to the central nervous system at maximum speed. 

Rabies virus is a neurotropic negative-strand RNA rhabdovirus. It is transmitted mostly via bites from diseased animals and causes a fatal infection of the nervous system in both animals and humans. A key step in rabies virus pathogenesis is rapid transfer to the Central Nervous System (CNS) through the Peripheral Nervous System. Due to its extraordinary properties in directed axonal transport and trans-synaptic spread, rabies virus has also been used as a neuro-tracing agent to map neuronal circuitry. Understanding the mechanism of rabies virus neuronal transport is important for both basic and applied fields.

Rabies virus enters the peripheral nervous system and undergoes long-distance transport before arriving at the cell body and subsequently the CNS. Axonal transport is the cellular process of trafficking proteins, organelles, vesicles, RNA and other cellular factors to and from the neuronal cell body. The molecular motor kinesin drives transport from the cell body anterogradely, supplying proteins, lipids and other essential materials to the cell periphery. Dynein/dynactin complexes drive retrograde transport, moving damaged proteins for degradation and critical signaling molecules such as neurotrophins to the cell body. Although rabies virus phosphoprotein P, a component of the viral nucleocapsid of infecting virions, was shown to directly interact with a light chain of the dynein motor complex, axonal RABV transport and CNS infection are independent of that interaction and long distance transport of complete enveloped virions within internalized endosomes is more likely. The cellular and molecular mechanisms involved in rabies virus’ infection and retrograde trafficking are yet to be understood.

A new paper in PLOS Pathogens shows that rabies virus interacts with the p75 neurotrophin receptor (p75NTR) at peripheral neuron tips to enter the axon. Then the virus moves in acidic compartments, mostly with p75NTR. Rabies virus is transported faster than NGF, an endogenous p75NTR ligand. p75NTR-dependent transport of rabies virus is faster and more directed than p75NTR-independent rabies virus transport. Hence, rabies virus not only exploits the neurotrophin transport machinery, but also has a positive influence on transport kinetics, facilitating its own arrival at the CNS. These findings reveal how RABV can spread from the periphery, describe a novel role for the RABV-p75NTR interaction beyond ligand-receptor binding, and define a mechanism that allows proficient long-distance accelerated transport in axons.

Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery. (2014) PLoS Pathog 10(8): e1004348. doi:10.1371/journal.ppat.1004348
Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV- G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.

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HIV-Mediated Apoptosis: New Therapeutic Targets

Involvement of HIV proteins in apoptotic pathways Currently, there is no cure for HIV infection. Antiretroviral therapy against HIV has been successful, but now research is focusing on achieving the ultimate goal, a cure. Consequently, understanding the host factors that control disease progression will be fundamental to the development of new therapeutic strategies. This review discusses the apoptotic pathways that result in the demise of the immune system and possible apoptotic-mediated interventions for therapeutic purposes and looks at the drugs currently under development.


Human Immunodeficiency Virus-1 (HIV-1)-Mediated Apoptosis: New Therapeutic Targets. (2014) Viruses, 6(8): 3181-3227; doi:10.3390/v6083181
HIV has posed a significant challenge due to the ability of the virus to both impair and evade the host’s immune system. One of the most important mechanisms it has employed to do so is the modulation of the host’s native apoptotic pathways and mechanisms. Viral proteins alter normal apoptotic signaling resulting in increased viral load and the formation of viral reservoirs which ultimately increase infectivity. Both the host’s pro- and anti-apoptotic responses are regulated by the interactions of viral proteins with cell surface receptors or apoptotic pathway components. This dynamic has led to the development of therapies aimed at altering the ability of the virus to modulate apoptotic pathways. These therapies are aimed at preventing or inhibiting viral infection, or treating viral associated pathologies. These drugs target both the viral proteins and the apoptotic pathways of the host. This review will examine the cell types targeted by HIV, the surface receptors exploited by the virus and the mechanisms whereby HIV encoded proteins influence the apoptotic pathways. The viral manipulation of the hosts’ cell type to evade the immune system, establish viral reservoirs and enhance viral proliferation will be reviewed. The pathologies associated with the ability of HIV to alter apoptotic signaling and the drugs and therapies currently under development that target the ability of apoptotic signaling within HIV infection will also be discussed.


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From the dandruffome to deep sea vents

Malassezia globosa Malassezia fungi have been linked to skin diseases like dandruff and eczema. Until recently, they were assumed to have evolved to inhabit mammalian skin. More recently, however, they have been found in a much wider range of habitats, and this short review focuses on what researchers know and would like to know about marine Malassezia.

Discoveries of Malassezia representatives in fish, plankton, sponges, corals, lobster, etc., and the fact that marine Malassezia appear to dominate certain marine habitats, suggest that marine Malassezia should be the focus of future research into the diversity and distribution of this enigmatic group. The paper also discusses the challenges of such research: Malassezia fungi are notoriously difficult to grow in laboratory cultures, especially in “clean” cultures that consist only of a single species, and recreating marine environments with specialized pressure and saltiness are likely to further complicate cultivation efforts.

From Dandruff to Deep-Sea Vents: Malassezia-like Fungi Are Ecologically Hyper-diverse. (2014) PLoS Pathog 10(8): e1004277. doi: 10.1371/ journal.ppat.1004277
As the dominant component of the mycobiota on human skin – both healthy and diseased – the genus Malassezia has received a fair amount of attention. Since the middle of the 19th century, researchers have linked these fungi with skin maladies such as dandruff and eczema, but their difficulty to culture axenically long hampered studies of their systematics and diversity. Malassezia is the sole genus within the fungal order Malasseziales, contained within the proposed subphylum Malasseziomycetes (anonymous reviewer; personal communication). Although Malassezia is sister to the so-called ”smut” plant pathogens, they are markedly divergent in ecological terms. A hallmark of Malassezia species is their incomplete fatty acids synthesis metabolic pathway, and reliance, instead, on a suite of extracellular lipases, phospho-lipases, and acid sphingomyelinases. In fact, only a single species, M. pachydermatis, is able to survive in culture. Until recently, it was assumed that Malassezia evolved into a specialized and narrow niche associated with the skin of mammalian hosts. However, culture-independent studies of fungi from environmental samples show that Malassezia are exceedingly widespread and ecologically diverse. Recent studies in little- characterized marine environments point to extensive diversification of Malassezia-like organisms, providing exciting opportunities to explore the ecology, evolution and diversity of this enigmatic group.

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If I have seen further than others it is because I have a bloody big microscope

Atomic Force Microscopy Microbial cells sense and respond to their environment using their surface constituents. Therefore understanding the assembly and biophysical properties of cell surface molecules is an important research topic. With its ability to observe living microbial cells at nanometer resolution and to manipulate single-cell surface molecules, atomic force microscopy has emerged as a powerful tool in microbiology. This short review surveys major breakthroughs made in cell surface microbiology using AFM techniques, emphasizing the most recent structural and functional insights.

Atomic Force Microscopy in Microbiology: New Structural and Functional Insights into the Microbial Cell Surface. (2014) mBio 5(4) e01363-14 doi: 10.1128/mBio.01363-14


And while we’re on the subject of advances in microscopy:

Microscopy: Cryo-EM enters a new era. (2014) eLife 3: e03678 doi: http://dx.doi.org/10.7554/eLife.03678
The development of new detector hardware has led to a resolution revolution in electron cryo-microscopy (cryo-EM). This is evident from a series of striking new structures obtained by cryo-EM at near-atomic resolution: these include ribosomes from human pathogens, mitochondria, ribosomes in complex with a protein translocase, ion channels, or a key enzyme in the biogenesis of methane. The ability to solve such structures in atomic detail is an essential prerequisite for the development of novel antibiotics and drugs.


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