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Tips from the journals of the American Society for Microbiology

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American Society for Microbiology

Human Norovirus In Groundwater Remains Infective After Two Months

Researchers from Emory University have discovered that norovirus in groundwater can remain infectious for at least 61 days. The research is published in the October Applied and Environmental Microbiology.

Human norovirus is the most common cause of acute gastroenteritis. The disease it causes tends to be one of the more unpleasant of those that leave healthy people unscathed in the long run, with diarrhea and vomiting that typically last for 48 hours. Norovirus sickens one in 15 Americans annually, causing 70,000 hospitalizations, and more than 500 deaths annually, according to the Centers for Disease Control and Prevention.

The results answer a question of great importance to public health, which had driven researcher Christine Moe and her colleagues to conduct this research: If well water becomes contaminated with noroviruses--perhaps from leaking sewer lines or a septic tank—how long do these noroviruses survive in water, and when would it be safe to drink from that well?

To answer that question, they prepared a safety-tested virus stock solution. They then put a known amount of this solution into a container of groundwater from an Atlanta well, which had met Environmental Protection Agency drinking water standards.

The researchers then tested the virus infectivity at days naught, 4, 14, 21, 27, and 61, by having volunteers drink the water on those days. The durability of the virus' infectivity was unexpected, says Moe. Most of the 13 volunteers became infected at various time points, exhibiting among them the complete range of norovirus symptoms, which endured for as long as five days post challenge. "We were surprised to observe that even the volunteers that drank the water 61 days after we had added the virus still got infected with the norovirus," says Moe.

Norovirus may remain infective far longer than 61 days. The researchers stored the groundwater at room temperature in the dark, using reverse transcription polymerase chain reaction to determine how much viral RNA remained after 622 days, and again after 1,266 days. They found no reduction after the first interval, and very little at the end of the second interval. Unfortunately, funding was insufficient to test infectivity in human volunteers beyond day 61.

"This study provides further evidence of the need to treat groundwater used for drinking water," says Moe, adding that the Environmental Protection Agency and other decision-makers who regulate drinking water need to take these findings into account, particularly since roughly half the US population relies upon groundwater for drinking.

To ensure that the volunteers' health would not be compromised, the investigators conducted the study in a special research unit of Emory University Hospital, while taking a variety of other precautionary measures.

Anticipating a question about who would volunteer to participate in a study with such potentially unpleasant consequences, Moe says that some volunteers have said that "they want to see how good their immune system is, and whether they will actually get sick." Three of the 13 volunteers did not become sick. One volunteer was the local librarian "who came to the research unit with a huge bag of books that she wanted to read while she was in the study," says Moe.

(S.R. Seitz, J.S. Leon, K.J. Schwab, G.M. Lyon, M. Dowd, M. McDaniels, G. Abdulhafid, M.L. Fernandez, L.C. Lindesmith, R.S. Baric, and C.L. Moe, 2011. Norovirus infectivity in humans and persistence in water. Appl. Environ. Microbiol. 77:6884-6888.)


Women Can Self-Test for HPV, Easily and Accurately

A team of German researchers has shown that women can accurately test themselves for human papillomavirus (HPV) infection, the most common cause of cervical cancer. The research is published in the October Journal of Clinical Microbiology.

"The high sensitivity of this self-sampling method guarantees to identify nearly all HPV-infected women," says first author Yvonne Delere, of the Robert Koch Institute of the Ministry of Health, Berlin.

Worldwide, cervical cancer is the second most common cancer in women, with half a million new cases and a quarter million deaths, annually, according to the World Health Organization. Virtually all cases are linked to certain strains HPV.

In the study, the researchers compared self sampling with conventional endocervical brush samples obtained by gynecologists in two groups of women 20-30 years of age, with (55 women) and without (101 women) a recent suspicious cytological smear. The two sampling methods were in accord in the two groups 84 and 91 percent of the time, respectively. Overall, the women rated the self-sampling method easy, at 12 on a scale of 0 (easy) to 100 (difficult).

The Netherlands has already introduced the new technique into cervical cancer screening programs, and Delere hopes to see the method become widespread in developing countries, where women frequently lack easy access to medical personnel and testing.

The researchers note that concordance between the conventional and the self-sampling methods is good despite the fact that the techniques sample different areas. The cervical brush sampling is directed towards the transformation zone, the area on the cervix where abnormal cells most commonly develop, while the lavage includes the whole cervical area.

"The higher prevalence of HPV, hr-HPV, and HPV16 in cervicovaginal lavage samples may be explained by additional infections at extracervical sites," according to the paper. "Since these infections may be a reservoir for virus infecting the cervical epithelium at the transformational zone, they are probably epidemiologically relevant. Therefore, cervicovaginal lavage sampling may be superior to cervix-directed sampling for future HPV prevalence studies."

Among teenaged girls, the transformation zone lies on the cervix's outer surface, where it is more vulnerable to infection than it is in adult women.

The self-sampling device, the Delphi Screener, is a sterile, syringe-like device containing five milliliters of buffered saline. One operates it by plunging the handle, releasing the saline into the vagina, holding it down for five seconds, then releasing the handle, so that the device retrieves the fluid. Next, one plunges the lavage specimens into prelabeled coded tubes, and mails it to the laboratory.

(Y. Delere, M. Schuster, E. Vartazarowa, T. Hansel, I. Hagemann, S. Borchardt, H. Perlitz, A. Schneider, S. Reiter, and A.M. Kaufmann, 2011. Cervicovaginal self-sampling is a reliable method for determination of prevalence of human papillomavirus genotypes in women aged 20 to 30 years. J. Clin. Microbiol. 49:3519-3522.)


Newly Discovered Reservoir of Antibiotic Resistance Genes

Waters polluted by the ordure of pigs, poultry, or cattle represent a reservoir of antibiotic resistance genes, both known and potentially novel. These resistance genes can be spread among different bacterial species by bacteriophage, bacteria-infecting viruses, according to a paper in the October Antimicrobial Agents and Chemotherapy.

"We found great quantities of bacteriophages carrying different antibiotic resistance genes in waters with fecal pollution from pigs, cattle, and poultry," says Maite Muniesa of the University of Barcelona, Spain, an author on the study. "We demonstrated that the genes carried by the phages were able to generate resistance to a given antibiotic when introduced into other bacteria in laboratory conditions," says Muniesa.

Although we often think of antibiotic resistance genes as evolving into existence in response to the antibiotics that doctors use to fight human disease and that agribusiness uses to fatten farm animals, microbes had undoubtedly been using both antibiotics and resistance genes to compete with each other for millions of years before antibiotics revolutionized human medicine and resistance genes threatened their efficacy to the point where the World Health Organization considers them to be one of the biggest risks to human health.

Thus, the Spanish researchers suspect, based on their study, that these resistance gene reservoirs are the product of microbial competition, rather than pressure from human use of antibiotics. They note that the pasture-fed cattle in their study are not fed antibiotics, and they suggest that even if antibiotic feed additives were banned, new resistance genes might emerge while old ones spread from these reservoirs into bacteria that infect humans.

And if resistance genes are being mobilized from these reservoirs, it becomes important to understand how the resistance genes are transmitted from phage to new bacterial species, in order to develop strategies that could hinder this transmission, limiting the emergence of new resistance genes, says Muniesa.

(M. Colomer-Lluch, L.Imamamovic, J. Jofre, and M. Muniesa, 2011. Bacteriophages carrying antibiotic resistance genes in fecal waste from cattle, pigs, and poultry. Antim. Agents Chemother. 55:4908-4911.)


New Insights Into Insulin Resistance Could Lead to Better Drugs for Diabetics

Research published in the October Molecular and Cellular Biology moves us closer to developing drugs that could mitigate diabetes.

Diabetes afflicts an estimated 26 million Americans, while 79 million have prediabetes. In other words, one in three Americans confronts this disease. Diabetes raises the risk of heart disease and stroke by as much as fourfold, and it is the leading cause of blindness among adults 20-74. It is also the leading cause of kidney failure.

In earlier research, four years ago another team of researchers showed that they could boost insulin sensitivity in experimental rodents by giving the animals a drug called myriocin. People with diabetes have a condition called insulin resistance, which renders them poorly able to process sugar. That results in high blood sugar, which damages the blood vessels, leading to many of diabetes' ills. In their study, that team, led by Johannes M. Aerts of the University of Amsterdam, observed a decrease in a compound called ceramide, which sits on cell membranes in the circulatory system, which they postulated was responsible for the rise in insulin sensitivity.

In the new study, Xian-Cheng Jiang of Downstate Medical Center, Brooklyn, NY, and his collaborators set out to confirm this earlier work, using a genetic approach.

The new research provides strong evidence that ceramide was not causing insulin sensitivity, but that another membrane-bound compound, sphingomyelin, might be doing so.

Ceramide is the substrate for the last step in a five step cascade that produces sphingomyelin. In that step an enzyme called sphingomyline synthase 2 (SMS2) cleaves ceramide to produce sphingomyelin. The first enzyme in this pathway is called serine palmitoyltransferase (SPT).

To test the hypothesis that ceramide is involved in modulating insulin resistance the researchers used knockout mice for each of these enzymes. They postulated that (partially) knocking out the first enzyme in the cascade would decrease ceramide levels while knocking out the last enzyme in the sphingomyelin pathway would boost ceramide levels, since that enzyme uses ceramide to produce sphingomyelin. Thus, SPT knockout mice would have greater insulin sensitivity, while SMS knockout mice would have reduced insulin sensitivity.

Surprisingly, while ceramide levels changed as predicted, that change did not influence insulin sensitivity, which was higher in both groups.

The research has important implications for drug development for mitigating diabetes. Myriocin proved highly toxic and major efforts to modify the drug to reduce that toxicity have been fruitless. Myriocin's toxicity probably stems from the fact that it inhibits the first step of the sphingomyelin biosynthetic pathway, affecting all the downstream biology, says Jiang. The discovery that knocking out the last step in the biosynthetic pathway improves insulin sensitivity means that drug treatments could target that last enzyme, SMS, leaving the rest of that biosynthetic pathway to function normally.

(Z. Li, H. Shang, J. Liu, C.-P. Liang, Y. Li, Y. Li, G. Teitelman, T. Beyer, H.H. Bui, D.A. Peake, Y. Zhang, P.E. Sanders, M.-S. Kuo, T.-S. Park, G. Cao, and X.-C. Jiang, 2011. Reducing plasma membrane sphingomyelin increases insulin sensitivity. Mol. Cell. Biol. 31:4205-4218.)

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