Thursday, July 21, 2011

The Need for Rapid Methods Continues to Grow Within the Food Industry

I recently came across an interesting article from a produce industry online trade journal (The Packer). The article discusses a new mandate from Costco that requires pathogen testing. But produce suppliers have been testing for pathogens for some time, and they routinely use diagnostic assays for this purpose. However, there is some debate on the accuracy of rapid tests that detect a broad range of pathogens as compared with PCR assays that target specific strains. Here is the full story from

New pathogen testing requirements for produce companies supplying Costco Wholesale Corp. may be the first of their kind, but large suppliers say they were already conducting the tests.
Craig Wilson, Costco’s vice president of quality assurance and food safety, said July 18 that the company began requiring its produce suppliers to test finished products for the “Big 6” E. coli strains “a couple of months ago.”
He said the company added the rare strain O104:H4 that recently sickened more than 3,900 people in Europe to its mandatory test list “in the past two or three weeks.”

“The tests don’t make the food safer, but they do tell us if the vendors’ food safety programs are working,” Wilson said.

Representatives of two of Costco’s produce suppliers agreed with Wilson’s assessment of the role of testing. They also said none of their other customers require finished product testing.

Earthbound Farm, San Juan Bautista, Calif., began testing raw products in 2006 and finished products in 2007 in response to the E. coli outbreak in 2006. Consequently, the Costco requirement won’t add any costs or disrupt their supply chain, said Will Daniels, senior vice president of operations and organic integrity.

“We test 100% of raw and 100% of our finished products, unlike many companies that just do spot checks,” Daniels said, adding that the company’s cost for the aggressive testing program is only 3 cents per retail unit, “which we do not pass along to our customers.”

At Ready Pac Foods Inc. in Irwindale, Calif., tests for salmonella and various E. coli strains have been standard procedure for several years, said Brian Zomorodi, senior vice president of technology and quality.

Both Earthbound Farm and Ready Pac work with IEH Laboratories & Consulting Group, Lake Forest Park, Wash., which has 76 labs across the country.
More than three years ago Ready Pac and IEH partnered to open a lab onsite at one of Ready Pac’s locations in California. The lab is staffed 24 hours a day, seven days a week to help reduce delays that finished product testing can cause.

The PCR (polymerase chain reaction) testing used by both produce companies takes about 12 hours, including sample preparation time and an enrichment process — or growth period — for each sample. If tests come up positive an additional four hours are needed for confirmation.

Zomorodi and Daniels both said that so-called rapid tests may offer results in as little as 15 minutes, but the quality of the results and their accuracy are far less reliable than the PCR method.

“With the rapid tests you are looking for a broader category of organisms,” Daniels said. “There is a large class of E. coli out there and many of them don’t make you sick. So if you are using a rapid test and get a positive result you could be throwing away good product because you don’t know which strain is present.”

Mansour Samadpour, president of IEH, echoed Daniels comments about the rapid tests. Companies have to follow the rapid test with the 12-hour PCR method for strain-specific results.

DuPont’s Qualicon division markets PCR test kits to the produce industry; the company warns that rapid tests are not reliable for specific pathogen testing.
Amy Smith, technical applications and regulatory support specialist for Qualicon, said a positive from a rapid test still leaves uncertainty.
“Diagnostic tests are only useful if they find the organisms you are looking for,” Smith said. “The enrichment step is the key in any of these kinds of tests.”

Smith said Qualicon is working on a test for the rare E. coli strain that hit Europe, but because of the evolutionary nature of the pathogen the company is waiting to see how it develops to create the most accurate test possible.
Similarly, BioControl Systems, a multinational company specializing in food safety testing with offices in Bellevue, Wash., and seven other countries, also has a test for the O104:H4 strain in the works. Anita Kressner, vice president for marketing, said the company hopes to have the new test available by the fourth quarter this year.

Samadpour said July 19 that IEH has already developed a test for O104:H4, which it made available to customers beginning two weeks ago.

Friday, July 8, 2011

Direct Identification of Bacteria in Blood Culture by MALDI TOF Mass Spec

A recent publication highlights the use of MALDI TOF Mass Spectrometry for the identification of microbial contaminants to blood cultures. The full reference and abstract are provided below.

Direct identification of bacteria in blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new methodological approach. Kroumova V, Gobbato E, Basso E, Mucedola L, Giani T, Fortina G. Laboratory of Microbiology and Virology, Azienda Ospedaliero Universitaria 'Maggiore della Carità', Corso Mazzini 18, Novara, 28100, Italy. In Rapid Commun Mass Spectrom. 2011 Aug 15;25(15):2247-9.


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently been demonstrated to be a powerful tool for the rapid identification of bacteria from growing colonies. In order to speed up the identification of bacteria, several authors have evaluated the usefulness of this MALDI-TOF MS technology for the direct and quick identification bacteria from positive blood cultures. The results obtained so far have been encouraging but have also shown some limitations, mainly related to the bacterial growth and to the presence of interference substances belonging to the blood cultures. In this paper, we present a new methodological approach that we have developed to overcome these limitations, based mainly on an enrichment of the sample into a growing medium before the extraction process, prior to mass spectrometric analysis. The proposed method shows important advantages for the identification of bacterial strains, yielding an increased identification score, which gives higher confidence in the results.

Wednesday, July 6, 2011

PDA Journal Paper Outlines Molecular Applications to Pharmaceutical Processes and Cleanroom Environments

In the May/June issue of the PDA Journal of Pharmaceutical Science and Technology, Luis Jimenex provides an outstanding overview of genetic sequencing applications as well as a case study in using molecular technologies to rapidly detect microbial contamination, provide a more accurate identification of microorganisms, understand the types of microbes in pharmaceutical laboratories, and make pharmaceutical processes more efficient. The full reference and abstract are provided below.

Molecular Applications to Pharmaceutical Processes and Cleanroom Environments. LUIS JIMENEZ. PDA J Pharm Sci and Tech 2011, 65 242-253.


This review article discusses several technologies using the power of genetic analysis that were used to rapidly detect microbial contamination, provide a more accurate identification of microorganisms, understand the microbial diversity in cleanroom environments, and optimize pharmaceutical processes. Nucleic acids were extracted and purified from enrichment cultures of product suspensions, growing cultures, microbial isolates, and directly from water and cleanroom surfaces. The extracted microbial DNA was either amplified using different polymerase chain reaction (PCR) assays or analyzed using DNA microarray technology. PCR assays were targeting genes coding for structural proteins, ribosomal subunits, or catabolic enzymes. Different PCR formats were tested ranging from single gene amplification to multiplexing. The detection of the amplified fragments was performed by qualitative or quantitative analysis. In some studies the amplified products were cloned/sequenced or sequenced to accurately determine the identity and diversity of the microbes present in the samples. DNA microarray analyses provided a higher multiplexing capability than PCR assays, as more than 3000 genes were analyzed in a single reaction, providing a higher resolution and specificity of the microorganisms present in cleanroom environments and pharmaceutical samples.

PDA Journal Paper Describes Identification Strategy Following Rapid Method Sterility Test Positive

In the May/June issue of the PDA Journal of Pharmaceutical Science and Technology, Jennifer Gray and colleagues describe a unique strategy for obtaining microbial identifications from positive sterility tests when using the Milliflex Rapid system. The full reference and abstract are provided below.

Identification of Micro-Organisms after Milliflex Rapid Detection—A Possibility To Identify Nonsterile Findings in the Milliflex Rapid Sterility Test. JENNIFER C. GRAY, DIETER MORANDELL, GUNTHER GAPP, NATHALIE LE GOFF, GUNTHER NEUHAUS, and ALEXANDRA STAERK. PDA J Pharm Sci and Tech 2011, 65 42-54.


The Milliflex Rapid System is used as a rapid microbiological method based on adenosine triphosphate (ATP) bioluminescence in the pharmaceutical industry to quantify the amount of micro-organisms present in water and in bioburden samples. The system can also be used for qualitative analyses, for example, to perform a rapid sterility test. This rapid sterility test has been successfully validated and implemented at Novartis and Sandoz. As the reagents used for the ATP bioluminescence reaction, which are directly sprayed on a micro-colony, disrupt the walls/membranes of the present cells to release ATP and therefore no intact cells for subsequent identification were believed to be present, the identification was supposed to be impossible until now.

During development and validation of a rapid sterility test with the Milliflex Rapid System, a possibility to identify contaminants was found. A method based on regrowth of the Milliflex Rapid-treated microbial cells and consecutive 
genotypic identification reproduced feasible and robust results. The data presented here show that sufficient recovery of the micro-colonies detected with the Milliflex Rapid System was reached with the test strains, except with 
Penicillium spec. The chosen micro-organisms represent the full spectrum of environmental isolates and ATCC strains, and it was shown that they are not destroyed after application of the reagents for the ATP bioluminescence reaction. 

Overall, 22 stressed microbial strains were examined during the study.