Microbiology – Minimise the Microbial Risk

Minimise the Microbial Risk - Microbiology is the study of a range of microscopic organisms which will typically fit into one of the following categories;

Acellular microbes – such as viruses and prions

Unicellular microbes – such as bacteria and yeast (fungi)

Multicellular microbes – such as moulds (fungi)

The vast majority of microbes are completely harmless to us. Some microbes are essential to our survival and enhance our health and wellbeing, others are pathogenic and given the appropriate conditions, have the potential to cause clinically significant infections which in some cases can be life-threatening. Although infections caused by microbes such as moulds, yeasts, and prions are often clinically significant and can have major implications for patients and healthcare providers they tend to be less prevalent than infections caused by bacteria and viruses which are responsible for the majority of healthcare associated infections (HCAI).

Minimise the Microbial Risk- Introduction and overview of bacteria

Size: Typically 0.5-5.0 ?m (0.0005 – 0.005mm).

Morphology: The three main bacterial shapes are bacillus (rod), coccus (spherical) or spirillum (coiled, helical or spiral).

Genetics: Deoxyribonucleic acid (DNA) within the bacterial cell cytoplasm

Key structures: Important bacterial cell structures can include the following; Cell wall consisting of tough peptidoglycan layers which confer structural rigidity to the bacteria and prevent distortion and lysis of the bacterial cell; Plasma membrane, a lipid bilayer which performs multiple processes essential to the survival and reproduction of the bacteria; Flagella which enable the bacteria to move in order to find nutrients and penetrate host mucus; Slime, a polysaccharide material secreted by some bacteria during biofilm formation to help protect the bacteria from the host immune system and antibiotics; Lipopolysaccharide (LPS), a powerful endotoxin which is an integral part of the outer membrane of Gram-negative bacteria and often responsible for significant immunological reactions in the host; Pili which aid bacterial colonisation by securing the bacteria to a suitable surface; Capsule, a loose polysaccharide structure which protects the bacteria from drying out and phagocytosis; Spores; (see ‘Classification’ section below).

Classification: Gillespie and Bamford 2 have described several ways in which bacteria can be classified. These include; Cell shape; Reaction to the Gram Stain procedure. Differences in the structure of the bacterial cell wall will result in bacteria being classified as Gram-positive (several layers of peptidoglycan with no outer membrane) or Gram-negative (minimal peptidoglycan, encased in an outer membrane); Endospore (spore) formation. In times of environmental stress, some bacterial species can produce spores which are metabolically inert and adapted for long term survival. Under suitable environmental conditions, spores regrow into vegetative bacteria. Classification by endospore notes the presence, shape, and position of the spore within the cell. Atmospheric preference. Different bacteria require different conditions for growth, for example, some are ‘aerobic’ (requiring oxygen), others ‘anaerobic’ (require little or no oxygen), some can grow with or without oxygen (facultative aerobes), some prefer reduced oxygen levels (microaerophiles) and others prefer elevated levels of carbon dioxide (capnophiles). Although historically, bacterial classification has been based on physical characteristics DNA sequencing is now increasingly used as a key classification tool.

Determining bacterial classification based on genetic characteristics has identified wide variety within each species. This helps explain how different strains of the same organism can create significant variations in terms of the infections they cause. One example of this is the toxin producing O157 strain of E. coli which can cause serious illness while other strains of E. coli can be carried without any ill effects.

Replication: For a bacterial cell to survive and replicate it must find a suitable environment and a food source which it can metabolise. Unlike viruses, bacteria possess both the genetic material and all necessary components and mechanisms required for replication, therefore they do not require a host cell within which to multiply. Given the correct conditions bacterial numbers can increase significantly within a relatively short space of time. For example under perfect conditions and assuming a 20 minute replication time a single bacteria would multiply into 68,719,476,736 bacteria over a 12 hour period.

The growth cycle of a bacterial population can be split into the following four distinct phases

Lag phase - although there is no bacterial cell division the cells metabolic activity increases as the synthesis of RNA, enzymes, proteins etc. takes place and the volume / mass of the cell increases in readiness of reproduction;

Log phase - an exponential growth phase where the bacterial cells divide at a constant rate by binary fission. The rate of bacterial growth will be determined by the environmental conditions and availability of the food source;

The Stationary phase marks the end of exponential bacterial growth and typically occurs due to one (or more) of the following factors: 1) exhaustion of available nutrients; 2) accumulation of inhibitory metabolites or end products; 3) insufficient space for growth. It is during the stationary phase that bacteria produce secondary metabolites (defined as metabolites produced after the active growth phase) such as antibiotics, and spore-forming bacteria can switch on the mechanisms involved in the sporulation process.

During the Death phase, the number of viable bacterial cells declines exponentially.

Control: Within healthcare, it is essential to manage the bacterial bioburden as this will have a direct impact on HCAI risk reduction. Bacterial control can be achieved by numerous different methods and with evidence now linking environmental surfaces and equipment with nosocomial pathogen transmission and enhanced clinical environmental cleanliness reducing HCAI incidence, cleaning, disinfection, and sterilisation are all preventative measures which aim to reduce the HCAI risk by managing the bacterial bioburden. Sterilisation technologies such as gamma irradiation, ethylene trioxide (ETO), gas plasma or autoclaving (steam at high temperature and pressure) will eliminate bacteria and bacterial spores In high-risk situations such as surgery it is essential to eliminate all microbes, therefore all medical devices used for the procedure should be sterile. Disinfection using proven biocides and antimicrobials will typically reduce the bioburden by killing viable bacteria within the clinical environment which in turn will help reduce the HCAI risk. Cleaning will help reduce the risk of HCAI by physically removing some of the bioburden along with any organic matter that could be a potential bacterial food source.

Additional control measures for bacteria include; Antibiotics, which can be given either as a prophylactic (typically as part of a surgical procedure) to help prevent an infection or for the treatment of localised or systemic infections; Topical antimicrobials such as iodine, chlorhexadine, silver etc. can often be used for removing microbes from the skin or for the treatment of localised infection such as wound infection.

Broadly speaking antimicrobials, disinfectants and sterilisation technologies physically disrupt and alter the bacteria in order to kill them. Antibiotics tend to work by blocking one or more steps in the bacterial replication process.

Minimise the Microbial Risk - Introduction and overview of viruses

Size: Typically 0.02-0.6 ?m (0.00002 – 0.0006mm). 14, 15

Morphology: A typical virus will consist of nucleic acid (DNA or RNA) surrounded by a protective protein coat or capsid with either icosahedral or helical symmetry (see Figure 5). The capsid of some viruses will also be encased within a bi-layered lipid “envelope” containing integral, virus-specific, proteins. The lipid envelope is derived from either the nuclear or cell membrane of the host and enables the virus particles to leave the host cell without disrupting the cell membrane and therefore without killing the cell.

Genetics: Deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA) located within the capsid

Key structures: viral structures include; Nucleic acid (see above); Nucleoproteins, proteins linked to the viral nucleic acid and which are essential for multiple steps in successful virus replication; Capsid, a protein shell consisting of multiple sub-units or capsomers; Envelope (see above) and Envelope virus proteins or glycoprotein which are specific to the virus and may act as receptors for other host cells.

Classification: Viral classification is determined according to the following factors: Genetic Material, which will either be DNA or RNA. The viral DNA and RNA is either single strand (ss) or double strand (ds). RNA viruses can also be further classified according to whether their RNA is ‘positive-strand’ (sense) or ‘negative strand’ (antisense); Capsid structure and symmetry i.e. whether the shape of the virus is icosahedral, helical or a combination of the two shapes; 17 The presence or absence of a lipid envelope. The capsid of some viruses will be surrounded by a lipid envelope; Mode of replication.

Replication: Viruses contain only the genetic material necessary for reproduction – they possess none of the necessary components or mechanisms required for replication. A virus can only replicate when it has infected a host cell. The virus effectively redirects the genetic mechanisms of the host cell to create new viruses. This process is often lethal to the host cell in which it occurs and the typical outcome is often cell lysis. Host cell lysis releases the newly created viruses which then come in to contact with neighbouring cells which then become infected.

There is significant variation between viruses with regard to the structure of the viral capsid or envelope, type of nucleic acid, method of entry into the host cell and mechanism of replication once inside.

Control: Cleaning, disinfection and sterilisation (see previous pages on bacteria): Proven virucidal biocides or antimicrobials will reduce the bioburden of viable viruses within the clinical environment which in turn will help reduce the HCAI risk posed by viruses. Not all disinfectants are virucidal. Vaccination provides human immunity to specific viruses. Vaccines introduce a suspension of killed or attenuated viruses (live viruses where pathogenicity has been removed) or virus products into the body, to enable the body to build up immunity to a particular virus; Antiviral drug therapy may help manage viral infection and reduce its overall severity. However, due to the intracellular location of viruses and their use of the host cell, antiviral therapy can be complex to develop. Antibiotics are only effective against bacteria and therefore antibiotics should never be taken for a viral infection.

Minimise the Microbial Risk - Introduction to biofilms

A simplistic definition of a biofilm is one where the bacteria are embedded in a thick, slimy barrier of polysaccharides (sugars). Biofilms can consist of a single bacterial or fungal species or they can be polymicrobial and consist of several different microbial species. The bacteria synthesise and secrete the protective extracellular polymeric substance (EPS) that attaches the biofilm firmly to a living or non-living surface.

Within the medical environment biofilms are known to occur over a wide range of invasive or implantable devices including but not limited to;

  1. Orthopedic implants (e.g. hip joints, knee joints etc.)
  2. Urinary catheters
  3. Contact lenses
  4. Hemodialysis systems
  5. Central venous catheters
  6. Endoscopes

In addition to the problems posed by biofilms on implantable devices, James et al detected biofilms in 60% of all chronic wounds sampled in their study.

The bacteria within biofilms can demonstrate up to 1,000 times more resistance to antimicrobials than the same bacteria within a suspension. This increased tolerance of disinfectants often arises due to a combination of several factors, including production of neutralising enzymes from microbes embedded within the biofilm, physiologic gradients within the biofilm (e.g., pH), genotypic variation of the bacteria and physical characteristics of older biofilms.

Within the healthcare, environment biofilms can have serious implications for immunocompromised patients and patients with indwelling medical devices such as catheters, prosthetic joints, stents, intra-uterine implants, heart valves and contact lenses.  Coagulase-negative staphylococci are the bacteria most frequently isolated from biofilm infections, however, Gram-negative bacilli and Candida have also been associated with implant infections.

Biofilm related infections often appear weeks or months after placement of the implant and in patients who appear to have a successful outcome. Successful treatment normally requires removal of the affected implant.

Biofilm development and formation on surfaces is well understood and has several well defined stages.

However, despite this understanding, the prevention of biofilm formation and the disruption of established biofilms is a significant problem in many industries including healthcare.

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