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Detection of bacterial growth and Biofilm formation in pipelines made easy

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By Giovanni Pavanello

The Biofilm Problems

On the surfaces that are in contact with water, a “biofilm” layer of microorganisms that are contained in a matrix (slime layer) can be formed. It is as a complex community of microorganisms, embedded in self-created extracellular polymeric substances (EPS). The biofilm is a microbial population adherent to each other and to the surfaces or interfaces enclosed in the matrix. Their presence in manufacturing pipelines can be responsible for a wide range of water quality and operational problems.
A Biofilm layer can form on any surface, even in extreme conditions (e.g., very high and very low pH values and temperature, in radioactive waters, even in space stations), and it causes a variety of problems:
  • Increase in the risk of pathogens – biofilm is the ideal environment for the proliferation of harmful bacteria by offering shelter and food;
  • Decrease in thermal efficiency of heat exchangers – for example, the biofilm is more insulating than calcium carbonate;
  • Microbiologically influenced corrosion (MIC) – also known as bacterial corrosion, it accounts for billions of dollars of industrial damages all over the world.
Large amounts of chemical substances (sanitizers, biocides, and disinfectants) are usually employed to prevent biofilm development in industrial piping systems. However, often they do not provide the expected results. In most cases, Clean-In-Place (CIP) and sanitation treatments are applied out on a regular basis (daily, or weekly) or when visible problems arise. The real need for neither the treatment nor its effectiveness is verified.
It is important to note that it is usually hard to take representative samples (“swabs”) from internal pipe surfaces. Standard plate counts applied to water samples are of limited use. Indeed, it is well known that bacterial growth on pipe surfaces is mostly unrelated to the presence/number of bacteria in the water. Planktonic (free-floating) bacteria attach to the pipes by the production of ESP; they multiply and form a mature biofilm, stabilizing the microcolony from environmental stress. Therefore, most bacteria live in biofilms, not free in the water.
Bacteria embedded in the biofilm are more difficult and expensive to eliminate, than free-floating planktonic bacteria. The extracellular substances (EPS) increase the bacterial resistance to external agents, by up to 1000x. As the biofilm matures, the ESP matrix becomes thicker. Thus it is much more resistant than an early-stage one. Sanitation and cleaning treatments should be applied on early-phase biofilm, to obtain the best results.
Moreover, when the biofilm is mature, its outermost layers tend to detach and float away. In these cases, it increases the likelihood of biofilm formation in other areas of the piping system.

The ALVIM Biofilm Monitoring System

Innovative technology for the detection of biofilms was developed by ALVIM, an Italian Company, in collaboration with the Italian National Research Council.
By measuring bacteria bio-electrochemical activity (a phenomenon known as “ennoblement” or “cathodic depolarization”), the ALVIM technology detects biofilm formation since its first phases, online and in real time. The technology allows checking the effectiveness of CIP and sanitation treatments, without the need for water sampling and lab analyses.   An example, from a reverse-osmosis (RO) system, is illustrated in the graph on the right. As it can be seen in the figure, the user can decide when to apply CIP, checking in real time if biofilm was actually removed.
The system helps in keeping track of bacterial settlement and growth inside water lines, tanks, and other surfaces in contact with the water and other liquids allowing checking, simultaneously, the efficacy of sanitation treatments.
The ALVIM probes are more specific than other sensors since they detect bacterial fouling (biofilm formation), not other kinds of deposit, such as minerals, proteins, fats, etc. This is extremely important since different fouling types require different treatments. It is important to emphasize that the ALVIM sensors are very sensitive to the detection of biofilm in its initial colonization phase, the best time to apply CIP and sanitation treatments.
ALVIM biofilm probes are currently used worldwide, in applications ranging from Cooling Towers to Food and Beverage, Paper Production, Oil and Gas and others, including large international Companies like Total, GDF Suez (ENGIE) and Danone.
The Massachusetts Institute of Technology (MIT) used ALVIM probes, in a Project regarding water distribution systems, and the European Defence Agency (EDA) indicated ALVIM Technology as a promising solution to the cleaning of biofilms. Overall, the ALVIM Technology allows for a simple and flexible approach to biofilm monitoring, considering different applications such as:
  • Analysis and characterization of biofilm growth;
  • Assessment and comparative evaluation of different chemical biocides, CIP and sanitation treatments;
  • Real-time, continuous monitoring of water treatment systems;
  • Automatic and/or remote control and optimization of sanitation treatment;
  • Effective prevention of pathogens, like Legionella, Listeria and Staphylococcus
 
More info available at: http://www.alvimcleantech.com/cms/en/biofilmsensors 
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Precedence-setting Food Safety Case: Foster Chicken Farm found Liable in Salmonella Case

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Meatingplace reported that a jury in Arizona returned a precedent-setting verdict in favor of 5 ½ years old boy (Noah Craten)   who had brain surgery in 2013, as a result of a massive brain abscess. The doctors determined that the abscess had been caused by bacterial meningitis due to Salmonella Heidelberg infection from what was believed to be chicken meat produced by Foster Poultry Farms.
Although the USDA does not consider Salmonella an adulterant in raw chicken, and even though cooking the chicken will kill the bacteria, the case establishes that chicken producers can be held liable for Salmonella contamination.
The jury on March 1 returned a verdict in the amount of $6.5 million in favor of Noah. The jury concluded that Foster Farm was negligent was based on microbiological and epidemiological evidence alone. Fluid removed from the abscess during surgery showed that the Salmonella bacteria present in the abscess matched one of the strains in the Foster Farm’s outbreak.
The jury seems to reject the argument that Salmonella contamination is natural to raw chicken. In a verdict that is a first of its kind, the jury found Foster Farm to be negligent in producing Salmonella Heidelberg contaminated product. It is a first to have the verdict based solely on epidemiological and microbiological evidence.
The jury attributed 30% of the fault to Foster Farms and 70% to family members for their preparation of the chicken. The net verdict against the family was $1.95 million.
Eric Hageman, the lead trial attorney for Noah Craten, claimed that the verdict established a precedent that could change the poultry industry. “Traditionally, Foster Farms and other poultry producers have argued that they are under absolutely no obligation to address even pervasive Salmonella contamination. The jury, in this case, said enough is enough. Clean up your act.” The jury’s verdict, Hageman said, “showed that Foster Farms could not simply hide behind USDA ‘approval’ of its chicken” and was “a rejection of the argument that poultry companies can produce contaminated product and then blame consumers who get sick from eating it.”
Noah Craten case was part of an extensive Salmonella Heidelberg outbreak investigated by the Centers for Disease Control (CDC) from March 1, 2013, to July 11, 2014. In this outbreak, 634 persons were infected with seven outbreak strains of Salmonella Heidelberg from 29 states and Puerto Rico, from March 1, 2013, to July 11, 2014. 38% of the sick persons were hospitalized, but no deaths were reported. The outbreak strains of Salmonella Heidelberg were resistant to several commonly prescribed antibiotics, as was the strain isolated from the abscess.
This case is groundbreaking and sets a precedent for food safety in the poultry industry.
According to Meatingplace  Foster Farms issued the following statement in response to the verdict: “Since 2013, Foster Farms has instituted a multi-hurdle Salmonella control program and committed to a company-wide Salmonella prevalence level of 5 percent in whole body chickens and parts. This compares to the USDA permissible level of 9.8 percent for whole body chickens and 15.4 percent for parts. Foster Farms’ current food safety performance record is recognized as being among the best in the US poultry industry, and the company is committed to advancing food safety for the benefit of consumers, customers, and the poultry industry.”
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A Single Genetic Code Change Makes African Salmonella More Deadly

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A new study has identified a single genetic code change that allows Salmonella Typhimurium (ST313) to play a significant role in human bloodstream infections in sub-Saharan Africa.  The study was published in Proceedings of the National Academy of Sciences, 2018 (PNAS) [Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella Disa L. Hammarlöf, Carsten Kröger, Siân V. Owen, Rocío Canals, Lizeth Lacharme-Lora, Nicolas Wenner, Anna E. Schager, Timothy J. Wells, Ian R. Henderson, Paul Wigley, Karsten Hokamp, Nicholas A. Feasey, Melita A. Gordon and Jay C. D. Hinton; PNAS 2018] 
Invasive non-typhoidal Salmonellosis (iNTS) is killing approximately 390,000 people annually in sub-Saharan Africa. iNTS is caused by Salmonella that enters the bloodstream and spreads through the body. The African iNST is caused by a variant of Salmonella Typhimurium (ST313) that is antibiotic resistant, and affect people with deficient immune system. This Salmonella strain seems to be very similar to the Salmonella Typhimurium that causes gastrointestinal illness.
The team of scientists, led by Professor Jay Hinton at the University of Liverpool, used advanced genetic techniques to switch various single-nucleotide polymorphism (SNPs) to find the one responsible for the difference in the Salmonella strain between the antibiotic-resistant strain that can enter the bloodstream, and the one causing gastroenteritis.
The team analyzed hundreds of Salmonella genomes and showed that the Salmonella Typhimurium ST313 is closely related to the ST19 strains that cause gastroenteritis. The core genome of the two strains share over 4,000 genes, varies by only ~1,000 SNPs. They hypothesized that one or more of these SNPs are accountable for the difference between the ST313 and ST19.  A single nucleotide difference that was unique to the African ST313, was identified as the cause of the virulence of the strain and its ability to grow in the bloodstream.
Using transcriptomics (a type of RNA analysis), the scientists identified SNPs that affected the level of expression of important Salmonella genes. After studying 1000 different SNPs, they found the single nucleotide difference that is unique to the African ST313 strain which causes high expression of a virulence factor called PgtE that prevents Salmonella being killed in the bloodstream.  The virulence factor PgtE is an outer membrane protease Salmonella that causes diseases ranging from gastroenteritis to severe enteric fever.
There are thousands of SNP differences between different types of Salmonella; therefore it is surprising that a change of just one letter in the DNA sequence will cause such a profound difference in the disease-causing ability of the organism. Until now it has been hard to connect an individual SNP to the bacterial ability to cause a disease.
The researchers used animal models (chicken) to infect with bacteria with altered SNP to show that by removing it the organism lost its ability to cause the disease.

Summary

The research team from the Universities of Birmingham and Liverpool has identified a single-nucleotide polymorphism (SNPs), which helps the African Salmonella to survive in the human bloodstream.  SNPs represent a change in a single DNA letter between the two Salmonella strains. It seems to be the first link between an individual SNP and a disease.
The single SNP responsible for high levels of expression of the PgtE in the outer membrane protease was linked to the virulence of African S. Typhimurium ST313. The study has implications for bacterial genome-wide association studies, which they claim should clearly include a focus on noncoding regions of the genome. The study findings also emphasize the value of identifying all gene promoters in bacterial pathogens, to allow nucleotide differences to be correlated with the process of transcriptional initiation.