Welcome to the Microbiology Information

In response to the need for an accurate, easy-to-use and cost effective identification system for medically important Staphylococci, Microgen
Bioproducts announces the release of Microgenâ„¢ Staph ID.

The Microgenâ„¢ Staph ID identifies all species of Staphylococcus related to humans, both as commensals, opportunistic pathogens, and
established pathogens. The kit may also be used to identify species of environmental and animal health significance. The Microgenâ„¢ Staph ID test panel consists of 13 biochemical tests specifically formulated to identify the target group of organisms. When used in conjunction with a coagulase or Staphylococcal latex agglutination test, these test panels provide identification of Staphylococci within 24 hours of inoculation. The interpretation of results is provided with an easy-to-use computer database employing up-to-date taxonomy. Regular updates to the software are available at no charge for customers registered as software users. Each kit contains sufficient test panels for 20 identifications.

The Microgenâ„¢ Staph ID is quick to set up and simple to read, providing a very effective means of identifying these organisms which are often simply classified as Coagulase Negative Staphylococci because existing methods are both laborious and expensive.

This is the latest addition to the family of innovative Microgen ID systems including Gram Negative Bacilli, Listeria spp and Bacillus spp. Staphylococci can cause many forms of infection from superficial skin lesions to deep seated infections such as endocarditis. Staphylococcus species are a major cause of nosocomial infections and coagulase negative Staphylococci are increasingly associated with indwelling medical devices.

For more information contact Vital Diagnostics. Email info@vitaldiagnostics.com.
Website: www.vitaldiagnostics.com.

Background
E. coli and coliform bacteria have been used for a century as indicators of faecal pollution in water since they occur in large numbers in the gut of many mammals. While total coliforms are being removed from water quality guidelines, except as a measure of treatment efficacy, anecdotal reports from around the world and a handful of publications in the last ten years have suggested that even some strains of E. coli can grow to high density (> 10000 per 100 mL) in large lakes and storages. This provides a challenge for the manager’s of such water bodies to differentiate these “blooms” from a sudden input of faecal pollution.

The ecology of E. coli
Culture methods allow us to identify a bacterial species as a “dominant” member of the gut microbial consortium if it represents more than 1% of the cells capable of growth on MacConkey agar. In fish, frogs and reptiles E. coli only reaches this threshold in about 10% of animals. In native Australian birds it can be detected at this level in around 20% of individuals, and in about 50 % of native mammal hosts. In humans, E. coli can be detected in 95 to 100% of people.

Factors known to affect the prevalence of E. coli in host animals include climate (being more prevalent in grassland and temperate climes than the tropics or deserts), host diet, body size and gut morphology. Omnivores are more likely to carry E. coli in substantial numbers than herbivores with carnivores the least likely. Prevalence also increases with body mass and, interestingly, with increased human association. The ecological niche of E. coli can be summarized as the gut of warm-blooded animals that have a microbial fermentation chamber in their hindgut or have a body mass of greater than one kilogram.

E. coli genogroups or subspecies
E. coli are divided into four subgroups: A, B1, B2 and D based on a range of factors including genetics, phenotype and ecology. Group A and B1 strains occur in all vertebrate hosts and in water; Group B2 strains are found in warm-blooded vertebrates with hindgut fermentation; Group D strains are found in warm-blooded vertebrates. In general, A and B1 strains appear to be generalists, acquired by their hosts from the environment, and which colonize well but persist poorly. B2 and D strains, which encode the most virulence factors, appear to be acquired from other host animals, colonize poorly but persist well. The distribution of the subgroups in human populations varies in different parts of the world. The prevalence of subgroups in humans may change with age in population and there appears to be a gender effect. In water, B1 strains are by far the most dominant, with B2 and D strains rarely found. Interestingly, B1 strains have similar sugar utilization patterns and optimal growth temperatures whether isolated from water or faeces, while A strains differ in both these characteristics depending on their origin. B1 strains appear to survive the transition to the aquatic environment best, with B2 and D strains surviving relatively poorly in water. Exposure to the aquatic environment appears to select for a subset of group A strains.

Canberra: The lake, the bug, the issue
Lake Burley Griffin experiences coliform blooms on occasional basis, from late summer to early autumn, where confirmed coliform counts exceed acceptable levels and necessitate the closure of the lake to a range of recreational activities, often at substantial cost and inconvenience. These bloom events prompted an investigation of the lake’s coliform microbiology funded by the National Capital Authority. The research program has three main objectives:

Sampling program
Over the past two years Lake Burley Griffin has been regularly monitored during both dry and wet weather. During the summer months Lake Tuggeranong and Lake Ginninderra, two other recreational lakes in Canberra, were monitored weekly. To gain an understanding of the frequency and characteristics of Escherichia coli in the external environment soil, sediment and water samples were also collected from localities across Australia.

E. coli strain characteristics tested
All environmental samples were screened for the presence of E. coli in order to develop a strain database. The phenotypic characteristics of the strains were determined, e.g. their growth rates at a range of temperatures, as well as their genotypic characteristics, e.g. virulence factor profile.

Findings
The study’s major finding has been the clear identification of three strains of E. coli capable of survival in the external environment and which do not appear to require a host population. Two of the three strains type as Group A (strains 000 and 010) while the third types in Group B1 (strain 001). Two would be described as phenotypically “typical” E. coli while the B1 strain would be “very atypical”. Of interest is the fact that this strains 001 (B Group) gives a negative indole test, meaning that it may not confirm as an E. coli but rather be classified as a total or thermotolerant (“faecal”) coliform, depending on the method being used. These three strains have been responsible for all bloom events in
Lake Burley Griffin since April 2000. The bloom strains have also been found in Lake Burragorang, Sydney’s primary drinking water reservoir, and detected in other water bodies in the ACT. These strains can be detected in these water bodies outside of bloom events and the evidence suggests these strains have been responsible for blooms over the past 30 years in Australia. The main distinguishing feature of these bloom strains is their mucoid colony morphology (Figure 1).

Figure 1: Colony appearance of Klebsiella pneumoniae (top left) showing a typical mucoid appearance due to their Type 1 capsule, typical unencapsulated Escherichia coli (top right) and the three bloom strains (bottom).

Examination of the genes responsible for the mucoid appearance demonstrates that the bloom strains are unlikely to be detected in a human or animal host. These bloom E. coli also have none of the genes thought to enable bacteria to cause disease. A simple Restriction Fragment Length Polymorphism assay is able to readily distinguish the three strains (Figure 2). A more sophisticated Real-time PCR assay has been developed based on unique genome fragments in each strain to allow rapid, definitive confirmation of the bloom strains should they be suspected when
elevated counts occur.

Figure 2. RFLP analysis of the bloom strains from different sampling locations.

Use of the research findings
The Real-time PCR assay will now be used to determine whether elevated coliform counts in Lake Burley Griffin are due to one of the identified environmental E. coli strains enabling more informed decisions to be made concerning the extent to which recreational activities in the lake should be curtailed.

When is it likely to be a bloom rather than faecal pollution?
If faecal material is deposited in a water body it would be expected that a range of E. coli genotypes would be detected in the polluted water. The genotypes would have a heterogeneous spatial distribution and most of the genotypes isolated would be rarely identified. There should also be other faecal indicators present in elevated numbers, such as enterococci. On the other hand, if there is a bloom of coliform bacteria one would expect to see a limited range of genotypes amongst the isolates and the range of isolates would be consistent across the area of the bloom. One would not expect to find other faecal indicators.

Related research
This study demonstrates that in some cases elevated coliform counts may not be a result of faecal contamination. This outcome suggests that future research is required to identify more appropriate indicator organisms and that water quality standards need to be modified to include such exceptions to the indicator assumption. The coliform group is still used extensively as a water quality indicator. Many water bodies throughout the world are considered to have coliform counts above acceptable levels. In an effort to manage this problem, programmes are underway to develop methods that will allow the source of faecal contamination to be identified (Microbial Source Tracking, MST). The success of these efforts critically depend on a number of assumptions being valid and these assumptions need to be carefully articulated and worked through in such programs. There is some evidence that wastewater treatment processes may select for specific strains of E. coli. For example, one study showed that the dominant E. coli in a septic tank was not a dominant strain in the householders. A similar process may be taking place in large wastewater systems.

Water Testing Laboratory
Independent Monitoring Consultants
www.imclive.com

Authors:
Jane Littlefield-Wyer is a PhD candidate in the School of Botany and Zoology at the Australian National University. Jane has presented at the Australian Society of Microbiology meetings in September 2004 and 2005 and at the 2006 annual meeting of the Society for General Microbiology.

David Gordon is an Associate Professor in the School of Botany and Zoology at the Australian
National University. David presented a seminar for the US Environmental Protection Agency in 2003 and attended a three-day workshop held by the Water Environment Research Foundation on Microbial Source Tracking in Texas 2005. The finding of this research have been published in the primary scientific literature and presented at both national and international conferences.

Acknowledgments
The Australian National University (ANU) would like to thank the Australian Government, represented by the National Capital Authority (NCA) for requesting the ANU to carry out this research based on samples from Lake Burley Griffin, Canberra. The NCA’s initiative in providing funding support and professional assistance to this research has been invaluable in this critical work. The Sydney Catchment Authority sponsored David and Jane to visit Sydney and present their findings on the 19th April 2006. That evening the NSW Branch of the Australian Society for Microbiology sponsored an evening seminar on the ecology of E. coli and the blooming strains at Macquarie University, hosted by Dr Martin Slade.0

Did you know that according to researchers in Hong Kong more than 10% of imported oysters screened using a reverse transcription polymerase chain reaction (RT-PCR) assay showed evidence of norovirus (Norwalk-like virus) contamination. Senior investigator Dr. Wilina W. L. Lim explains that although the approach is of limited use in demonstrating an epidemiological link with human cases, “it appears that oysters may be an important vehicle for introducing novel strains of norovirus.”

Outbreaks of norovirus gastroenteritis are often associated with consumption of oysters and contamination appears to be widespread, Dr. Lim of the Public Health Laboratory Centre, Kowloon, and colleagues note in the August issue of the Journal of Medical Virology. They found that 10.5% of 507 samples of oysters from 11 countries tested on arrival showed evidence of norovirus contamination. In particular, oysters from six countries were contaminated; those from the remaining five countries were not. A wide variety of strains was found, including two novel genetic clusters.

Norovirus screening was also conducted following 13 outbreaks of oyster-associated gastroenteritis in hotels or restaurants in Hong Kong. Norovirus RNA sequences were detected in at least one oyster in six outbreaks. However, only in one outbreak was there a match between the strains isolated from patients and those found in the oysters.

Dr Lim concludes with “Given the popularity of consuming raw oysters in many countries, oysters may serve (as) a vehicle for the dissemination of new norovirus strains,”

Journal of Medical Virology 2005;76:593-597.

Did you know that approximately four million cases of food borne infectious disease occur annually in Australia; new food borne pathogens, such as enterohaemorrhagic Escherichia coli, are emerging. Climate change, combined with changes in how we produce and distribute food and how we behave as consumers, have the potential to affect food borne disease in the coming century. Food borne disease outbreaks are now more far-reaching (and sometimes global) due to modern mass food production and widespread food distribution. There are strong seasonal patterns for Salmonella and Campylobacter infection in Australia. Global warming may increase the incidence of infections, such as salmonellosis, and diseases caused by toxins, such as ciguatera.

Let’s hope all the world leaders take climate change seriously.

This biofilm-producing terrorist is the bane of all industrial microbiologists. Industry can be humming along quite happily and then up pops Listeria and its panic stations. The micro response team rushes to the site armed with gauzes, swabs, sampling and HACCP (Hazard Analysis and Control of Critical Points) plans to do combat.

As we all know the genus Listeria is a gram positive rod, psychrotrophic, and displays a peculiar tumbling motility caused by a low number of peritrichous flagella which beat in a clockwise motion due to a defective CheY gene (Dons et al, 2004). This organism is ubiquitous and is found primarily in soil (Sutherland et al, 2003). The only species that is truly a human opportunistic infector is Listeria monocytogenes, public enemy number one. Its sibling Listeria ivanovii is attempting to cause confusion in the ranks of those over-worked industrial microbiologists. L. ivanovii has shown similar pathogenicity as seen by L. monocytogenes, in mice and other animals, but is rarely seen in humans (FDA/CFSAM, 2003). Are these two species protected or masked by Listeria innocula the harmless one? With the perceived threat of Listeriosis, the government bodies are debating the move towards zero tolerance for the genus. The federal government food body FSANZ standard only states that L. monocytogenes absence is required in ready-to-eat products and the FAO/WHO risk assessment concluded that levels of L. monocytogenes <100 cells per gram has the same risk as zero cells per gram (FAO/WHO, 2001). To complicate matters, Dussarget (2004) stated that of the 13 known serovars of L. monocytogenes, only 1/2a, 1/2b and 4b are responsible for 98% of reported human Listeriosis cases. The serovar 4b is associated with the majority of food borne outbreaks and sporadic cases. This single genus has been responsible for more product recalls and media hype than any other micro-organism. We all have heard of Conroy’s and the two deaths from ham in Adelaide in the last few months. Industries that produce ready-to-eat products all have great concern for this ubiquitous terrorist.

Industry has spent millions on the combat, control and the eradication of this organism. As with all terrorist organizations, the sleeper cells are very hard to find and the fact that Listeria produce a fatty acid biofilm on solid surfaces makes it very difficult to treat with standard chloride based surface sanitizers. This biofilm aids the survival of Listeria due to its lipid composition which is hydrophobic and thus prevents the entrance of water-based sanitizers; it also acts as a food reserve and selects for the survival of other symbiotic organisms that aid in the survival and proliferation of Listeria (Sutherland et al, 2003) (Somers & Wong, 2004). The destruction of one biofilm may lead to the establishment of others from that original source and to product contamination. Biofilms are living entities and thus, when critical mass is achieved, cells detach and contaminate the product. This is known to the industrial microbiologist as ‘spitting’. There is reported resistance developing in the standard chemicals used in the eradication of biofilms (Chavant, 2004). The only effective way to clean down contaminated areas is by high-pressure (area needs to be sealed) acid washes as well as physical scrubbing followed by contact sanitization (quats, chlorine, acid and peroxide sanitizer) - the chemical equivalent of hunting down terrorist cells with thermonuclear warheads. Listeria has also displayed an ability to survive and thrive in some of the most extreme environments found in industry such as saturated brine. Listeria has been associated with many of our most loved and highly consumed foods. These include: ice cream, soft cheeses, smoked salmon, pate, fermented meats, cooked further processed chicken meats and fresh leaf produce (Sutherland et al, 2003). This cowardly bacterium attack the elderly, infirmed and the defenseless fetus with relatively low infective doses, 2 to 3 log less than is required to infect healthy adults (CFSAN, 2003). To complicate matters further, this organism presents to the treating clinician as flu -like systems and initial diagnosis may be difficult.

The total number of victims recorded in Australia is 3 cases in 1,000,000 and is steadily decreasing as the industrial microbiologist is slowly eradicating all known niches. The consumers demand for ‘fresh’ products with minimal preservatives and additives results in additional pressures on the industrial microbiologist to discover strategies to meet the consumer demand without endangering the public. This has resulted in the steady development of non-thermal treatments such as microwave and radio frequency, ohmic and inductive heating, high pressure processing, pulsed electrical field and pulsed light, just to name a few that are in development or have been used in commercial food manufacturing (FDA/CFSAN, 2000). These intervention strategies amplify nature’s only controls in controlling these terrorists. For example, high pressure processing uses water pressures to burst the cell. There is a plethora of methods available for the industrial microbiologist to screen and identify this organism. The selection of methods is primarily based on quality and turnaround time. The longer a company’s product takes to reach the market the more it costs the company. Therefore, there is always pressure to find faster methods to screen out negatives. Some of the most common rapid methods are based either on ELISA type tests (BioMerieux VIDAS, TECRA Unique) or PCR (Oxoid’s BAX and Roche’s real-time PCR protocol). These methods are all automated and have the required regulatory approvals. The covert battle between the industrial microbiologist and Listeria is ongoing with no definite exit time. As long as the consumer enjoys the convenience of ready to eat food, Listeria will be waiting to strike; however, the industrial microbiologist will be there to contain, prevent and eliminate any danger to the public.

While antibiotics eliminate disease-causing pathogens they also disrupt the protective micro flora of the intestinal tract. This micro flora normally prevents opportunistic pathogens from establishing in the intestinal tract via competitive inhibition. However, after long-term antibiotic exposure this protective barrier is disrupted, leaving the host vulnerable to subsequent infection by organisms such as Clostridium difficile. Almost all antimicrobials have been implicated in the process, however, clindamycin, second and third generation cephalosporins, and fluoroquinolones are the major antibiotics involved (Gerding, 2004). The next step leading to Clostridium difficile associated diseases (CDAD) involves exposure to toxigenic Clostridium difficile. This can be facilitated via asymptomatic carriers, hospital staff for example or symptomatic hospital patients who shed Clostridium difficile cells and spores into the hospital environment. Consequently, infected fomites also become vehicles for transmission.

Once Clostridium difficile establishes in the colon, it can produce toxin A (an enterotoxin), toxin B (a cytotoxin) or a binary toxin. Individually or in combination these toxins cause changes to the cytoskeletal organization of enterocytes, fluid secretion, leukocyte chemotaxis and the up regulation of cytokines, all subsequently leading to mucosal damage, which manifests as diarrhea or PMC.

Data from the National Nosocomial Infections Surveillance system, the Centers for Disease Control and Prevention, and the Emerging Infection Network of the Infectious Diseases Society of America, all show that the disease rates and severity of CDAD have increased over the past few years (McDonald et al., 2005). Reasons for this rise may include the emergence of hypervirulent strains, strains with a functional binary toxin gene or deletions in the tcdC gene, or those that have emerged with resistance to common antibiotic therapy. Whatever the reasons may be, it is paramount that we reassess current laboratory diagnostic techniques so that all possible strains including epidemic strains can be detected.

One of the problems with current laboratory techniques is the failure to quickly detect hypervirulent strains of toxinotype III ribotype 027 – a strain that has been reported to be present and active in Canada, the United States, the United Kingdom, and the Netherlands. This hyper-producer of toxins produces 16 to 23 times more toxins A and B than the reference strain VPI 10463 (Pépin et al., 2005); it is therefore capable of inducing very severe diarrhea. To complicate matters, hyper producing strains (those similar to that of ribotype 027) cause what is known as a prozone effect. This is a phenomenon in which mixtures of specific antigen and antibody do not agglutinate or precipitate visibly because of an excess of either antigen or antibody, and in this case, it is the antigen (Clostridium difficile toxins) that is in excess. As a result, commercial enzyme immunoassays often fail to detect these strains because false negative results are often instigated by this prozone effect.

A similar problem applies to strains with functional binary toxin genes and those with partial deletions. Studies by McDonald et al. (2005) and Loo et al. (2005) have detected epidemic strains, which contained binary toxin genes and a partial deletion in the tcdC gene. Because the tcdC protein is thought to function as a negative regulator of the production of toxins A and B, the authors have proposed that this 18- base pair deletion could lead to increased toxin production, and subsequently induce problems clinically as well as posing challenges for laboratory detection as discussed above. In addition, because many laboratories persist with kits that only detect toxins A or B (despite a decade of literature outlining issues with single antigen testing), those strains without functional toxin A or B genes will not be detected by this approach. Increasing literature reporting clinically significant binary toxin-producing isolates of Clostridium difficile will pose problems to labs not performing sophisticated molecular testing. However, it must be noted that molecular techniques are not economically viable, require technical laboratory expertise, and demand discovery of relevant primers for particular strains. Furthermore, very few laboratories have adopted PCR as routine practice to diagnose CDAD.

Laboratory diagnosis of CDAD in many institutions consists of a positive faecal toxin test. Laboratories have been moving more towards kits testing for both toxin A and B and away from a single antigen test strategy, albeit slowly.

Some laboratories are also incorporating faecal isolation of Clostridium difficile from stool specimens as a safety net approach to detect these newly described strains. Studies by Delmée et al. (2005) have shown that the direct faecal cytotoxin assay (the “gold standard” technique) missed almost as many cases as it detected, while the toxigenic culture was successful in detecting those that were missed by the former technique. Toxigenic culture involves a stool culture for Clostridium difficile followed by a toxin assay of Clostridium difficile colonies. This method, although time-consuming, is highly sensitive and vital for correct diagnosis and early recognition of possible outbreaks.

In addition to toxigenic culture, presumptive cell counts of leukocytes and erythrocytes should also be included in routine practices. Even though this is not a definitive marker of CDAD, knowledge of the leukocyte numbers in combination with positive culture isolation and toxin detection, provides a much stronger basis for correct diagnosis. In retrospect, there is an urgent need for laboratories to reassess their diagnostic techniques because globally, CDAD cases are increasing in numbers and severity, and emerging aberrant strains are appearing undetected by out-dated laboratory practices.

Well-known Clostridium difficile expert Prof. Thomas Riley from Western Australia has recently published a succinct article in the Medical Journal of Australia outlining the sentiment of this commentary. It is a good read for those laboratories performing Clostridium difficile detection to further enforce a review of Clostridium difficile detection methodology.

By Valerie Nguyen Department of Microbiology, School of Molecular and Microbial
Biosciences, University of Sydney Department of Microbiology, Concord Hospital, NSW Australia.