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Tips from the journals of the American Society for Microbiology

March 2012

American Society for Microbiology

High Pressure Kills Pathogens, Maintains Green Onions' Taste and Color

Green onions cause about five percent of outbreaks of food poisoning from produce, worldwide. Now a team of researchers from the University of Delaware, Newark, shows that high pressure treatment of green onions can kill various strains of Escherichia coli O157:H7, and Salmonella enterica, two major sources of food poisoning. Unlike heating, the pressure treatment preserves the produce's gustatory attributes. The research is published in the March Applied and Environmental Microbiology.

In the study, the researchers cultivated green onions both in soil and hydroponically, irrigating each with different mixtures of the pathogen strains. The researchers verified that the microbes were taken up by the plants, into their roots, bulbs, stems, and leaves, says corresponding author Haiqiang Chen.

The researchers then grew green onions hydroponically, in water contaminated with the pathogens, for 15 days. At the end of this period, the plants were harvested and placed in a laboratory version of a commercial pressurizer for two minutes, at up to 5,000 times atmospheric pressure, at 20 or 40 degrees centigrade. In most cases, the pathogens were eradicated by this treatment. "To our knowledge, this is the first research to demonstrate that high pressure processing can kill foodborne pathogens internalized in green onions," says Chen.

In 2003, an outbreak of hepatitis associated with green onions consumed at a restaurant in Monaca, PA, sickened more than 550 people, killing at least three, according to the Centers for Disease Control and Prevention, and numerous outbreaks of Salmonella and E. coli O157:H7 have been linked to fresh produce.

(H. Neetoo, Y. Lu, C. Wu, and H. Chen. Use of high hydrostatic pressure to inactivate Escherichia coli 0157:H7 and Salmonella enterica internalized within and adhered to preharvest contaminated green onions. Appl. Environ. Microbiol. 78:2063-2065.)

Download a PDF of the journal article at: http://bit.ly/asm0312a


Antibiotic Resistant Bacteria Proliferate in Agricultural Soils

Infectious diseases kill roughly 13 million people worldwide, annually, a toll that continues to rise, aided and abetted by resistance genes. Now a study, published in the March Antimicrobial Agents and Chemotherapy finds reservoirs of resistance in agricultural soils. These contained more diverse populations of drug resistant bacteria, with greater levels of resistance, than composted and forest soils. Vegetable garden soil alone harbored multi-drug resistant bacteria, and also had the highest level of resistance to three major antibiotic classes.

"The observations of this study point to the widespread presence of high level antibiotic-resistant bacteria in agricultural soils," says first author Magdalena Popowska of the University of Warsaw, Poland.

Antibiotics, and resistance genes thereto, occur naturally in soil due to the arms race between microbial species competing for territory. "Almost 50 percent of Actinomycetes isolated from soil are capable of synthesizing antibiotics, which provide a natural antibiotic residue in soils," says Popowska. But the use of antibiotics to promote livestock growth boosts the resistance to a whole new level, as demonstrated by the differences in resistance level in agricultural and forested soils, she says. Manure from antibiotic-fed animals exacerbates the resistance spread, as demonstrated by the high levels in the manure-amended vegetable garden soils.

The spread of resistance and multi-resistant strains of pathogens and opportunistic bacteria that can infect humans and animals is aided and abetted by the fact that they are frequently carried on mobile genetic elements, notably plasmids and transposons, that can be transferred not only among bacteria of the same species, but among different species, says Popowska.

The results of this study "should assist in the development of regulations regarding the use of antibiotics in the broader environment e.g. in plant protection products fish farming, and industry," says Popowska. "We think they will also help optimize methods allowing the combating of emerging bacterial infections, as well as in the development and application of new chemotherapeutic agents."

The use of antibiotics "should be restricted to dangerous bacterial infections, and to strict medical supervision," says Popowska. "This cannot be emphasized strongly enough."

(M. Popowska, M. Rzeczycka, A. Miernik, A. Krawczyk-Balska, F. Walsh, and B. Duffy, 2012. Influence of soil use on prevalence of tetracycline, streptomycin, and erythromycin resistance and associated resistance genes. Antimicrobial Agents and Chemotherapy 56:1434-1443.)

Download a PDF of the journal article at: http://bit.ly/asm0312b


Cross-Reactive Antibodies Vanquish H5N1 in Preclinical Study

The H5N1 influenza has proven extraordinarily deadly. More than 50 percent of the 500 cases that have been documented since the virus first emerged in 1997 have been fatal. Thus, H5N1 is viewed as a serious threat to world public health. A major difficulty in developing antibodies to combat this virus is that ten different antigenic types have evolved since the virus first emerged. But now a team of researchers has produced a so-called cross-reactive antibody that can bind to nine of the ten H5N1 groups. They showed further that it could protect mice from infection, and that it could be used to treat established infections in the mice. The research is published in the March Journal of Virology.

The investigators approached the problem of finding cross-reactive antibodies by hypothesizing that H5N1 survivors might sometimes make small amounts of such versatile antibodies, thus accounting for their survival, says co-principal investigator John J. Skehel of the National Institute for Medical Research, London, UK. They then found such antibodies in an H5N1 survivor, which they expressed in insect cells, to produce sufficient quantities of antibody to conduct their medical experiments.

Skehel sees an eventual cross-reactive antibody product being used in conjunction with anti-Neuraminidase drugs as a more effective treatment for H5N1 than either alone, partly because the dual treatment could prevent development of resistance to the anti-Neuraminidase drugs, which is a problem when they are used as monotherapies.

An additional finding is that the cross-reactive antibody interacts with the virus' hemagglutinin, a protein that is responsible for binding the virus to the cell that it is invading. A clear understanding of this interaction might help researchers develop vaccines that would induce cross-reactive antibodies, thus overcoming the current need to make new influenza vaccines each year," says Skehel.

(H. Hu, J. Voss, G. Zhang, P. Buchy, T. Zuo, L. Wang, F. Wang, F. Zhou, G. Wang, C. Tsai, L. Calder, S.J. Gamblin, L. Zhang, V. Deubel, B. Zhou, J.J. Skehel, and P. Zhou, 2012. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses. J. Virol. 86:2978-2989.)

Download a PDF of the journal article at: http://bit.ly/asm0312c


Viruses Kill Pancreatic Tumors in Preclinical Model

An intra-tumor injection of a virus prevented further growth of some pancreatic tumors and eradicated others in mouse models of pancreatic ductal adenocarcinoma. However, some tumors continued growing despite this treatment, proving resistant to the viruses. The research is published in the March Journal of Virology.

About 95 percent of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAs). PDA is considered to be one of the most lethal malignancies, resulting in a five year survival rate of only 8-20 percent.

In this study, the researchers, led by Valery Z. Grdzelishvili of the University of North Carolina, Charlotte, tested several species of virus against pancreatic tumors, most notably vesicular stomatitis virus (VSV), a type of virus that is commonly used in the laboratory. Previous studies had demonstrated that some other viruses, including adenoviruses, herpesviruses, and reoviruses, could be used to kill pancreatic cancer cells in some animal models of pancreatic cancer.

VSV has several qualities which make it attractive as a potential oncolytic (cancer killing) agent. First, unlike some other viruses (including adenoviruses),VSV replication does not require the cancer cell to express a specific receptor in order to infect that cell, and therefore it can infect most any cancer cell. Second, replication occurs in the cytoplasm of host cells, which means that there is no risk that it will cause healthy host cells to become cancerous, says Grdzelishvili. Third, this virus's genome is easily manipulated, which would make it fairly practical to adjust levels of foreign gene expression to enhance the virus' specificity for particular cancers, and its ability to kill them. Fourth, unlike with some other viruses, humans have no preexisting immunity to VSV.

In the study, the cancer-killing potential of several VSV variants was tested against 13 clinically relevant cell lines of PDA, including both primary PDA tumors and PDA metastases to the liver and lymph nodes, all derived from human patients, and compared these to adenoviruses, Sendai virus, and respiratory syncytial virus.

"In general, VSV variants showed superior oncolytic abilities compared to other viruses, and some cell lines that exhibited resistance to other viruses were successfully eradicated by VSV," says Grdzelishvili. "However, we found that PDA cells were surprisingly heterogeneous in their susceptibility to virus-induced oncolysis and several cell lines were resistant to all tested viruses." In producing and responding to interferon, many pancreatic cancers seemed to retain the normal antiviral responses that normal, healthy cells have towards viruses, he says.

Grdzelishvili emphasizes that the VSV's ability to kill cancer cells in mouse models by no means guarantees that it would perform similarly in cancer patients due to complex tumor microenvironments and compromised immune responses. Most animal models involve simply inserting human cancer cells underneath the animal's skin, so that the cancers and their environments are both quite different from cancer growing naturally in a human.

However, cancer cells that are resistant to virus in laboratory dishes almost certainly would prove resistant in a human patient, which means that such virus-resistant cancers could be identified with simple laboratory tests prior to being applied to patients, says Grdzelishvili.

"Prescreening cells against an array of different viruses could identify the best option for treating a particular tumor," says Grdzelishvili. Combined virotherapy (analogous to combination drug therapy) could also potentially lead to enhanced cancer killing. "Understanding the mechanisms and identifying biomarkers of resistance is critical for the development of prescreening approaches and individualized oncolytic virotherapy against PDA," says Grdzelishvili.

(A.M. Murphy, D.M. Besmer, M. Moerdyk-Schauwecker, N. Moestl, D.A. Ornelles, P. Mukherjee, and V.Z. Grdzelishvili, 2012. Vesicular stomatitis virus as an oncolytic agent against pancreatic ductal adenocarcinoma. J. Virol. 86:3073-3087.)

Download a PDF of the journal article at: http://bit.ly/asm0312d

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