MicrobiologyBytes

Methicillin-resistant Staphylococcus aureus (MRSA), first identified in the 1960s, was initially considered to be a nosocomial pathogen (hospital acquired infection). Beginning in the late 20th century, a specific clone of MRSA known as USA300 emerged as a leading cause of community-acquired infection, but doubts remain as to where many cases of MRSA infection originate, and how to break the transmission of this dangerous strain.

A new study finds that 8% of hospital outpatients carrying methicillin-resistant MRSA lived with an MRSA-positive pet. When faced with chronic and or recurrent MRSA cases, physicians should consider the possibility of household pets as MRSA source. Patients should be informed of this possibility. Unnecessary close contact should be avoided and heightened hygiene practices should be instituted. Sampling/swabbing of all the human and animals in a household seems appropriate to identify unrecognized sources and break potential cycles of reinfection especially in cases involving immunocompromised patients.

Transmission of MRSA between Companion Animals and Infected Human Patients Presenting to Outpatient Medical Care Facilities. PLoS ONE 6(11): e26978. doi:10.1371/journal.pone.0026978
Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen in both human and veterinary medicine. The importance of companion animals as reservoirs of human infections is currently unknown. The companion animals of 49 MRSA-infected outpatients (cases) were screened for MRSA carriage, and their bacterial isolates were compared with those of the infected patients using Pulsed-Field Gel Electrophoresis (PFGE). Rates of MRSA among the companion animals of MRSA-infected patients were compared to rates of MRSA among companion animals of pet guardians attending a “veterinary wellness clinic” (controls). MRSA was isolated from at least one companion animal in 4/49 (8.2%) households of MRSA-infected outpatients vs. none of the pets of the 50 uninfected human controls. Using PFGE, patient-pets MRSA isolates were identical for three pairs and discordant for one pair (suggested MRSA inter-specie transmission p-value = 0.1175). These results suggest that companion animals of MRSA-infected patients can be culture-positive for MRSA, representing a potential source of infection or re-infection for humans. Further studies are required to better understand the epidemiology of MRSA human-animal inter-specie transmission.

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As the incidence of type 1 diabetes in developed countries has been increasing at a rate far beyond the rate of population growth, environmental factors have been considered as likely candidates responsible for this change in disease incidence in recent decades. Of those factors, the gut microbiota have come under recent interest; supported in part by observations in both non-obese diabetic (NOD) mice and BioBreeding Diabetes Prone (BB-DP) rats where antibiotic use prevents the onset of diabetes.

To explore specific differences in the microbial communities responsible for T1D modulation, a metagenomic analysis of bacteria from susceptible rodents was performed. This revealed bacteria whose members were either positively or negatively correlated with diabetes. Lactobacillus and Bifidobacterium were more abundant in BB-DR (Diabetes Resistant) rats while Bacteroides and Clostridium were more abundant in BB-DP (Diabetes Prone) rats. Both Lactobacillus and Bifidobacterium are well known to have members with probiotic characteristics. These data suggest a model for the role of bacteria in a healthy gut. The total number of lactic acid producing and butyrate producing bacteria is higher in controls than in diabetic animals. This suggest that microbial-induced butyrate production, and subsequent mucin synthesis, with a corresponding enhancement of tight junctions may contribute to the development of autoimmunity for type 1 diabetes in humans.

Gut Microbiome Metagenomics Analysis Suggests a Functional Model for the Development of Autoimmunity for Type 1 Diabetes. (2011) PLoS ONE 6(10): e25792. doi:10.1371/journal.pone.0025792
Recent studies have suggested a bacterial role in the development of autoimmune disorders including type 1 diabetes (T1D). Over 30 billion nucleotide bases of Illumina shotgun metagenomic data were analyzed from stool samples collected from four pairs of matched T1D case-control subjects collected at the time of the development of T1D associated autoimmunity (i.e., autoantibodies). From these, approximately one million open reading frames were predicted and compared to the SEED protein database. Of the 3,849 functions identified in these samples, 144 and 797 were statistically more prevalent in cases and controls, respectively. Genes involved in carbohydrate metabolism, adhesions, motility, phages, prophages, sulfur metabolism, and stress responses were more abundant in cases while genes with roles in DNA and protein metabolism, aerobic respiration, and amino acid synthesis were more common in controls. These data suggest that increased adhesion and flagella synthesis in autoimmune subjects may be involved in triggering a T1D associated autoimmune response. Extensive differences in metabolic potential indicate that autoimmune subjects have a functionally aberrant microbiome. Mining 16S rRNA data from these datasets showed a higher proportion of butyrate-producing and mucin-degrading bacteria in controls compared to cases, while those bacteria that produce short chain fatty acids other than butyrate were higher in cases. Thus, a key rate-limiting step in butyrate synthesis is more abundant in controls. These data suggest that a consortium of lactate- and butyrate-producing bacteria in a healthy gut induce a sufficient amount of mucin synthesis to maintain gut integrity. In contrast, non-butyrate-producing lactate-utilizing bacteria prevent optimal mucin synthesis, as identified in autoimmune subjects.

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Researchers have discovered a toxin – SElX – released by methicillin-resistant Staphylococcus aureus (MRSA) which leads the body’s immune system to go into overdrive and damage healthy cells. SElX is made by 95 per cent of S. aureus strains, making it a potential drug target to fight this hospital superbug. SElX belongs to a family of toxins known as superantigens that can invoke an extreme immune response. When it is released it triggers an over multiplication of immune cells, which can lead to high fever, toxic shock and potentially fatal lung infections looked at a strain of MRSA known as USA300 that can cause severe infections in otherwise healthy individuals. If we can find ways to target this toxin, we may be able to stop it from triggering an over-reaction of the body’s immune system and prevent severe infections.

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia. (2011) PLoS Pathog 7(10): e1002271. doi:10.1371/journal.ppat.1002271

Other research has linked a naturally occurring mutation in the bacterium Clostridium difficile to severe and debilitating diarrhoea in hospital patients undergoing antibiotic therapy. These antibiotics destroy the “good” bacteria in the gut, which allows this “bad” bacterium to colonise the colon, where it causes bowel infections that are difficult to treat. The mutation wipes out an inbuilt disease regulator, called anti-sigma factor TcdC, producing hypervirulent strains of C. difficile that are resistant to antibiotics and which have been found to circulate in Canada, the US, UK, Europe and Australia. The results suggest that bacterial strains carrying this mutation have the potential to produce more of the harmful toxins that cause disease in susceptible individuals – commonly patients aged 65 years or over. As we now have a better understanding of these strains, we can design new strategies to prevent, control and treat these infections.

The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of Clostridium difficile. (2011) PLoS Pathog 7(10): e1002317. doi:10.1371/journal.ppat.1002317

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Two days ago I wrote about monkeys as an animal model for smallpox. Animal models certainly have a place in microbiology research, but here’s a rather less controversial one:

Pseudomonas aeruginosa causes serious infections in people with compromised immune systems. Individuals with cystic fibrosis and hospital patients are particularly vulnerable to P. aeruginosa infections. This bacterium does not respond to many antibiotics, making these infections difficult to treat. P. aeruginosa can grow as free-floating planktonic cells or as microcolonies known as biofilms. The ability of P. aeruginosa to form biofilms is thought to contribute to their ability to cause chronic infections. The aim of this research was to develop a simple biofilm model of infection using the fruit fly (Drosophila melanogaster). The immune system of the fruit fly has similarities with the vertebrate innate immune system. Understanding how P. aeruginosa causes infections in Drosophila will aid in understanding virulence mechanisms in mammals. This study shows that feeding P. aeruginosa to Drosophila results in a biofilm infection and biofilm infections induced expression of antimicrobial peptide immune response genes in the fly. Using fly survival as a measure of virulence it shows that biofilm infections were less virulent than non-biofilm infections. These results provide novel insight into host-pathogens interactions during P. aeruginosa infection.

Drosophila melanogaster as an Animal Model for the Study of Pseudomonas aeruginosa Biofilm Infections In Vivo. (2010) PLoS Pathog 7(10): e1002299. doi:10.1371/journal.ppat.1002299
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

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Stomach ulcers are caused by chronic infection with the bacterium Helicobacter pylori, which is also the leading risk factor for stomach cancer. One reason for the cancer risk could be that the pathogen creates breaks in the DNA molecules of infected cells:

Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells. PNAS USA 108: 14944–14949 (2011)
The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors—p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)—and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.

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One of the great advances in medical technology has unwittingly spawned a serious threat to public health. Implanted medical devices, from cardiac stents to artificial hip joints, are commonly infected with biofilms, complex microbial communities that can prove remarkably resistant to host defenses and treatment. It appears, however, that biofilms, even those arising from the same microbe species, may harbor innate differences in their response to treatments like antifungal agents. Understanding how these microbe colonies might produce structures that appear similar but have very different physical properties and functions is a critical step in figuring out how to overcome antimicrobial resistance.

In humans, Candida albicans can cause problems like oral thrush and yeast infections. Far more serious is its increasing tendency to colonize catheters, heart valves, and other medical devices, where it serves as a seeding source for potentially deadly bloodstream infections. The finding that C. albicans can form two different types of biofilm is interesting not only because it is an example of how similar structures can have very different functions, but also because it provides information about how signaling pathways may evolve by modifying preexisting signaling modules for entirely new purposes. This phenomenon may be widespread, extending beyond slimy fungal biofilms to a variety of organisms. On a practical note, when researchers are designing new methods to discourage fungal biofilms it will be useful for them to keep in mind that there are two types of biofilms being formed by C. albicans.

A Tale of Two Biofilms. 2011 PLoS Biol 9(8): e1001119. doi:10.1371/journal.pbio.1001119

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Sudan is a large country with a diverse population and history of civil conflict. Poverty levels are high with a gross national income per capita of less than two thousand dollars. The country has a high burden of tuberculosis (TB) with an estimated 50,000 incident cases during 2009, when the estimated prevalence was 209 cases per 100,000 of the population. Few studies have been undertaken on TB in Sudan and the prevalence of drug resistant disease is not known.

In this study Mycobacterium tuberculosis isolates from 235 patients attending three treatment centers in Sudan were screened for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin by the proportion method on Lowenstein Jensen media. 232 isolates were also genotyped by spoligotyping. Demographic details of patients were recorded using a structured questionnaire. Statistical analyses were conducted to examine the associations between drug resistance with risk ratios computed for a set of risk factors (gender, age, case status – new or relapse, geographic origin of the patient, spoligotype, number of people per room, marital status and type of housing).

Multi drug-resistant tuberculosis (MDR-TB), being resistance to at least rifampicin and isoniazid, was found in 5% of new cases and 24% of previously treated patients. Drug resistance was associated with previous treatment with risk ratios of 3.51 for resistance to any drug and 5.23 for MDR-TB. Resistance was also associated with the geographic region of origin of the patient, being most frequently observed in patients from the Northern region and least in the Eastern region with risk ratios of 7.43 and 14.09 for resistance to any drug and MDR-TB.

“We conclude that emergence of drug resistant tuberculosis has the potential to be a serious public health problem in Sudan and that strengthened tuberculosis control and improved monitoring of therapy is needed. Further surveillance is required to fully ascertain the extent of the problem.”

Tuberculosis in Sudan: a study of Mycobacterium tuberculosis strain genotype and susceptibility to anti-tuberculosis drugs. BMC Infectious Diseases 11:219 2011

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The most amazing thing about the “common cold”, or at least, the proportion of this spectrum of diseases which is caused by rhinoviruses (as opposed to adenoviruses, coronaviruses, or something else), is how little we still know about it. A few years ago I was involved in a proposal for a large project which would look at some of the issues adressed by a new research paper which has just appeared.  That proposal eventually collapsed under its own weight, so it’s fascinating to read these new results and see what we might have found.

The new study describes a phylogenetic analysis to compare the relative distribution of HRV species or serotypes according to the respiratory site (upper respiratory tract (URT) versus lower respiratory tract (LRT) ) and in protracted infection in hospital patients and immunosuppressed lung transplant recipients. In one case, rhinovirus genome variation was followed in the URT and LRT over a period of 27 months using both classical and ultra-deep sequencing methods.

Based on phylogenetic analysis, the frequency distribution of strains infecting the URT and LRT did not reveal any apparent correlation between a given HRV serotype or species and their ability to infect the LRT. Five lung transplant recipients were chronically infected with HRV during periods of time ranging from three to 27 months. Mutation mapping along the HRV genome showed that synonymous changes were roughly spread along the entire ORF, whereas non-synonymous changes clustered mostly in the capsid VP2, VP3, and VP1 genes. The capsid genes are also the most variable during acute infections in immunocompetent hosts.

As expected, the data suggests that immunocompromised patients cannot clear virus infections as well as immunocompetent individuals, and represent a potential reservoir for the emergence of new variants and inter-host transmission due to chronic virus infection. In addition, these patients may be co-infected by two viruses, thus opening the door to recombination, another putative driving force of rhinovirus evolution. With the emergence of new therapies and progress in transplantation, the population of immunocompromised patients is constantly increasing. Our results suggest that this could accelerate the ability of viruses to adapt to the host, evolve, and propagate and may favor a more rapid emergence of new viral variants.

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections. (2011) PLoS ONE 6(6): e21163. doi:10.1371/journal.pone.0021163
Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5′ UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

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Despite the availability of antibiotics that rapidly kill bacteria in vitro, the treatment of chronic bacterial infections, such as tuberculosis, requires long-term drug therapy. The reasons for this are unclear, but many have hypothesized that the slow replication and concomitantly low metabolic rate of bacteria in the host environment produce an “antibiotic-tolerant” state. Researchers tested this hypothesis by identifying the bacterial pathways responsible for slowing the growth and metabolism of Mycobacterium tuberculosis in response to stress. They found that diverse growth-limiting stresses trigger a common signal transduction pathway that slows bacterial growth by redirecting cellular carbon fluxes away from central metabolic pathways and towards storage. Disruption of this metabolic switch increased the antibiotic sensitivity of the bacterium during infection, verifying that this response significantly contributes to antibiotic tolerance and suggesting new strategies for accelerating therapy.

Metabolic Regulation of Mycobacterial Growth and Antibiotic Sensitivity. (2011) PLoS Biol 9(5): e1001065. doi:10.1371/journal.pbio.1001065
Treatment of chronic bacterial infections, such as tuberculosis (TB), requires a remarkably long course of therapy, despite the availability of drugs that are rapidly bacteriocidal in vitro. This observation has long been attributed to the presence of bacterial populations in the host that are “drug-tolerant” because of their slow replication and low rate of metabolism. However, both the physiologic state of these hypothetical drug-tolerant populations and the bacterial pathways that regulate growth and metabolism in vivo remain obscure. Here we demonstrate that diverse growth-limiting stresses trigger a common signal transduction pathway in Mycobacterium tuberculosis that leads to the induction of triglyceride synthesis. This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle. Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection. Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

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