Spotlight on Enterococci

Enterococci are a subgroup of fecal streptococci and are characterized by their ability to grow at low and elevated temperatures (10°C and 45°C) and at elevated pH (9.5). These microorganisms have been used as indicators of fecal pollution for many years especially in water samples.

Enterococci are benign bacteria when they reside in their normal habitat such as the gastrointestinal tracts of humans or animals. Outside of their normal habitat, enterococci are pathogenic causing urinary tract and wound infections, and life-threatening diseases such as meningitis. Enterococci easily colonize open wounds.

Compounding their pathogenesis, enterococci are also some of the most resistant bacteria, particularly from human sources. Studies have shown that certain strains of enterococci are resistant to expensive and potent antibiotics such as vancomycin. Several intrinsic features of the enterococcus genus allow it to survive for extended periods of time. These bacteria have been shown to survive for 30 minutes at 60°C and persist in the presence of detergents. Enterococci may remain viable on surfaces for days or weeks because the organisms are resistant to desiccation.

Fortunately the bacteria are easily killed by disinfectants used in the bottling plant, such as peracetic acid and hydrogen peroxide. However, the main reason for enterococci to be mentioned in quality standards for water is the link with possible fecal contamination. Sources of enterococci can be from toilet flushes, animal faeces brought in on shoes or boots or poor hand hygiene.

Enterococcus faecalis is a Gram-positive bacterium and it is interesting to note that Gram-positive strains are very common in indoor environments and have been measured in one study to represent 40% of the species recovered from airborne bacteria. 

Weekly testing for enteroccoci is recommended while bottling water.  An easy-to-use test from IDEXX called Enterolert makes measurement of presence/absence possible in the bottling plant in 24hrs. 

Food Grade Detergents and Disinfectants

I am often asked about the requirements for food grade approvals when using cleaners and disinfectants in the bottling plant, and elsewhere.

The regulations concerning the washing of polycarbonate bottles and other indirect food contact materials are paraphrased as follows: Detergent compositions may be used for washing surfaces that afterwards come into contact with food or beverage, provided there is a thorough rinse with potable water after the washing process. Washing compositions with components such as alkalis, water softening agents and small amounts of anionic, non-ionic or amphoteric detergents, may be employed for this purpose.

Peracetic acid is a terminal disinfectant (or sanitizer in American English) and regulations are somewhat stricter. The material is usually supplied as either a 5% or 15% peracetic acid composition, which is generic and well-known in the bottled water industry. Federal Regulations of the FDA describe peracetic acid and confirm its allowed use in food and beverage applications.

Disinfectant/sanitising products containing a single quaternary ammonium (cationic) compound, on the other hand, should not be used on surfaces which come into contact with potable water.  This is because cationics are fairly tenacious in sticking to surfaces, and while the residual effect can be beneficial for non-water-contact surfaces, such as floors and walls, retention of small amounts on water-contact surfaces can lead to bacterial acclimatisation.  If cationics are your preferred disinfectant (and there are several advantages), it is better to use a product containing a mixture of cationic types, which will offset the acclimatisation and often provide a synergistic disinfectant effect.
Always read the label or safety data sheet.

Bulk Chemicals and the Environment

The use of large containers (IBCs) for storage of bottle wash chemicals can be both convenient and cost effective.  However, the volume of chemical will be large, up to 1100 litres, and it is important to have environmental safety measures in place.

The best solution is to have a stand for the IBC which is also an effective bund, in other words, if an accident occurs where the IBC is fractured, the contents will flow into the bund and not over the ground.  Several types of bunding are available, but the one that I would recommend is a single moulded unit with integral drip  catchment.

Spills easily can  occur when, for example, 25L containers are being refilled from the IBC. The dispensing area will catch all spills which then flow back into the bund. A typical bunded stand with dispensing area is shown in the photo.

 A stand such as this is can cost about  £500.  Of course, an alternative arrangement is to have automatic dosing into the machine directly from the IBC. This reduces manual operations and improves safety.

Final safety note, the bunded stands can be moved around with a fork-lift truck but should not be moved with a full IBC on top.

Peracetic Acid Test Strips

I have been asked by several customers if there is an alternative way to determine peracetic acid concentration without using the expensive test strips.

The answer is yes, there is an alternative method, but it is complicated and the savings would be limited.  The alternative method uses a titration procedure involving 5 different chemicals. You may ask “why is it so complicated?”. The reason is that peracetic acid, or peroxyacetic acid, to give it its correct name, is not a single substance, it is a mixture of hydrogen peroxide, peracetic acid and acetic acid in an equilibrium.

The balance of the three components may change at different temperatures or at different concentrations of the product.  The hydrogen peroxide and peracetic acid components mutually interfere with simple titration procedures, hence the requirement for the complex procedure. The chemicals are expensive because they are analytical grade materials. The test strips cover 100 determinations and are more cost effective and less prone to operator error.

ATP Meters

Over the past ten years or so, a great deal of emphasis has been placed on the use of bioluminescence technology in the detection of micro-organisms.

The mechanism by which fireflies produce a flash of light was first analyzed and identified by William McElroy in 1947 . McElroy found that central to the light emission process was a specific enzyme reaction catalyzing the consumption of adenosine triphosphate (ATP).  In microbes, ATP can only be detected when living cells are present.

It has since been established that the amount of light emitted from this reaction is directly proportional to the amount of ATP present. A high reading of relative light units (RLUs) indicates that the sample contains a high number of micro-organisms, provided that the background ATP level is low. Unlike traditional testing methods, results from a bioluminescent reaction can be obtained quickly. Light is produced within seconds and can be measured with a luminometer (ATP Meter).

I have reviewed several ATP Meters on the market, for example, those from Celsis International and Biotrace.  However, for application in the bottled water industry I would recommend SystemSURE II from Hygiena.  This palm-sized instrument brings together state-of-the-art photodiode technology with simple user-friendly design to produce an affordable hygiene-monitoring system. Used with Ultrasnap ATP swabs, levels of contamination can be determined in just 15 seconds.

 Because bacterial counts in water are very low, a sample enrichment process is necessary. The process will be described in a later Blog. Key features of the luminometer include, low cost, high sensitivity, compactness, simple to use,self-calibrating with background check. Readings may be down-loaded onto a PC or, as an optional extra, analysis software will provide spreadsheet-compatible data.

Sulphite-Reducing Clostridia

These are mentioned in the water microbiology check, but what on earth are they?

They are Gram-positive anaerobic spore-forming bacteria in the shape of rods. In the method of detection, sulphite is reduced by the bacteria to sulphide at 37 deg C within 24 hours.  The organisms form spores which are environmentally resistant and could be an indication of ground water contamination of drinking water. The important species within the group is Clostridium perfringens, often associated with fecal contamination.

Clostridia are everywhere, in soil, decaying vegetation, marine sediment, and the intestinal tract of humans and other vertebrates.  Virtually every soil sample ever examined, with the exception of the sands of the Sahara desert, has been shown to contain C. Perfringens.