SOP104 General information about the different strains of ESKAPE pathogens and E. coli - Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

Standard Operating Procedure (SOP) – Biosafety level 2

Title: General information about the different strains of ESKAPE pathogens and E. coli - Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

ID: SOP104
Section: Section for Research
Department: Department of Microbiology
Division: Division of Laboratory Medicine (KLM), Oslo University Hospital (OUH)
Written by: Mari Kaarbø
Date: 01.06.2019
Approved by: Hilde Loge Nilsen
Date: 15.02.2022
Updated: 16.09.2025

 

Enterococcus faecalis (E. faecalis), not antibiotic resistant

Gram-positive, commensal bacterium. Found in healthy humans and has been used as a probiotic. E. faecalis can act as an opportunistic pathogen and cause life-threatening infections, especially in the nosocomial environment. Antibiotic resistance can contribute to pathogenicity. Disease caused by E. faecalis include endocarditis, sepsis, urinary tract infections and meningitis. Various virulence agents contribute to pathogenicity including, hemolysin, cytolysin and adhesion.  

Infection is typically caused by the fecal-oral transmission.

Generally patients suffering from comorbidities are at greater risk of infection such as cancer patients, particularly those with hematologic malignancy and those with structural abnormalities of the urinary tract and those who have undergone solid organ transplants. Long term antibiotic usage can also increase the risk of infection.

 

Enterococcus faecium (E. faecium), not antibiotic resistant

E. faecium can be a commensal bacterium within the gastrointestinal tract of humans, but it can also be pathogenic and cause serious infections. Infection can lead to neonatal meningitis, endocarditis, urinary tract infections, prostatitis, cellulitis, wound infection as well as concurrent bacteremia. Various virulence factors such as adhesins, invasins and haemolysin have been identified. However, E. faecium generally harbours few recognised virulence determinants (in comparison to E. faecalis). The presence of these factors varies between strains. In the US a high percentage of device-associated infections in intensive care units was due to vancomycin- and ampicillin-resistant E. faecium.

Infection is normally caused by the fecal-oral transmission route and cannot be transmitted by coughing or sneezing.

Most at risk are those who have been treated long term with antibiotics (as this can lead to antibiotic resistance development), people who are hospitalized or are immunocompromised (the elderly, sufferers of HIV/AIDS), have undergone surgical procedures, or have medical devices inserted into their bodies and patients with chronic medical conditions such as diabetes, cancer, or kidney disease.

Enterococci form part of the lactic acid bacteria and as such are important in the development of aroma in certain traditional Mediterranean cheeses and sausages. Enterococci are also used as human probiotics.

 

Staphylococcus aureus (S. aureus), not antibiotic resistant

1. aureus is commensal and frequently found as part of the microbiota in the upper respiratory tract and on the skin. Despite this S. aureus can become an opportunistic pathogen and is a common cause of skin infections, abscesses, respiratory infections, sinusitis and food poisoning. S. aureus is one of the five most common causes of nosocomial (hospital-acquired) infections and is particularly associated with wound infections following surgery.

People at risk include those who are immunocompromised, such as those with HIV/AIDS or cancer, hospital patients who have undergone surgical procedures or who have invasive medical devices such as catheters or ventilators. Additionally, those who inject drugs, those with skin injuries or conditions, athletes who share equipment or use communal showers and people living in crowded or unsanitary conditions are also at risk.

Virulence factors found in S. aureus include cell wall components that resist host immune defences, such as peptidoglycan, teichoic acids, and lipoteichoic acids. Enzymes such as coagulase so as to form a protective clot around the bacterium and hyaluronidase which breaks down connective tissue to allow the bacterium to spread. Toxins such as alpha-hemolysin which can lyse cells include erythrocytes and leukocidins which target white blood cells. Adhesion molecules which allows S. aureus to attach to host tissue and cells and S. aureus can also form biofilms protecting the bacteria from host immune defenses and antibiotics. The presence of these factors varies between strains.

 

Klebsiella pneumoniae (K. pneumoniae), not antibiotic resistant

K. pneumoniae is commensal in the mouth, skin and intestines, however if aspirated in large enough numbers it can cause damage to the lungs. K. pneumoniae is also an environmental bacteria found naturally in soil, water and on plants.

Immunocompromised people, particularly those hospitalized and those with chronic diseases are particularly at risk from infections. Infections can lead to pneumonia, urinary tract infections, bloodstream infections and wound infections. K. pneumoniae is a common cause of nosocomial infections especially in intensive care units (ICUs).

Associated virulence factors includes a thick capsule that helps it evade the host’s immune system by preventing phagocytosis. Lipopolysaccharide (LPS) is an endotoxin that can cause septic shock in severe cases. Fimbriae, hair-like structures that facilitate adherence to host cells and that can also aid in the formation of biofilms. Siderophores that scavenge iron from the host to enable faster growth of K. pneumoniae. Proteases that can degrade host proteins and hypermucoviscosity in which the bacterium produces excessive capsular polysaccharide. The presence of these factors varies between strains.

 

Acinetobacter baumannii (A.baumannii), not antibiotic resistant

A. baumannii can be commensal but is commonly found in soil and water. A. baumannii is an opportunistic pathogen typically only infecting immunocomrompised people or those who have other underlying health conditions. A. baumannii can survive in a variety of environments including hospital surfaces and medical equipment where it can cause nosocomial infections. Infections result in pneumonia, bloodstream and wound infections and urinary tract infections.

Virulence factors include a capsule and the ability to form biofilms. A. baumannii can also secrete a variety of factors that can damage host cells. The bacteria can also acquire iron from the host to improve growth and they gain resistance readily through horizontal gene transfer (HGT).

 

Pseudomonas aeruginosa (P. aeruginosa), not antibiotic resistant

P. aeruginosa is an environmental pathogen found in the soil, water and other moist environments. It is an opportunistic pathogen that particularly causes infections in individuals with weaken immune systems or those suffering from chronic lung conditions such as cystic fibrosis.

P. aeruginosa can contain many virulence factors. P. aeruginosa can form biofilm which can lead to nosocomial infection due to the colonization of catheters and ventilators, particularly with immunocompromised patients and those suffering with burns and wounds. P. aeruginosa can produce exotoxins that can damage host cells and tissue. Proteases that can degrade host cells and tissue. LPS which can trigger inflammation and an excessive immune response. Other factors include quorum sensing molecules and type III secretion systems.

 

Escherichia coli (E. coli), not antibiotic resistant

E. coli is a commensal found in the lower intestines of many warm-blooded animals including humans. Some strains are beneficial to humans, many are harmless and some can cause illness. Infections can results in diarrhea, urinary tract infections, meningitis and sepsis. Pathogenic strains are often transmitted through contaminated food or water or by direct contact with infected individuals or animals.

Strains such as O157:H7, ETEC, EIEC and EPEC are virulent. Such strains can contain adhesins, toxins, secretion systems, capsules, iron acquisition systems and can be motile.

E. coli strains are used in probiotics and in the production of fermented foods such as yoghurt and cheese.

 

When working with these strains:

1. Medical help
   1. For acute medical emergencies call 113
   2. For GP out-of-hours services (legevakt) call 116 117
   3. In the case of suspected poisoning call 22591300 for help, if acute help is needed call 113
2. Virkon is often referred to here for deactivation of the BSL2 organisms, you need to be aware that:
   1. The powder is an irritant to the skin (wash thoroughly with water in case of physical contact). Use gloves to avoid this.
   2. The powder can cause serious eye damage, in case of contact wash with water for 15 minutes and then contact a doctor. Use safety glasses to reduce the chances of contact.
   3. The powder is harmful to aquatic life, do not wash down the sink, if small amounts get into the sink dilute with copious amounts of water.
   4. The powder can cause respiratory irritation, use tablets to avoid inhalation
   5. If the powder or a 1% solution are ingested seek medical advice immediately (see 1)
   6. Use tissues to mop up small spills, place the tissues in the risk waste for thermal destruction
3. Transport – Movement of the strains to/from storage (freezer or fridge), to/from the incubator and the lab bench or the designated hood. The strains must be within a closed primary container (container (closed tubes or plates sealed with parafilm or cling wrap) within a suitable secondary container (e.g. plastic box with the lid closed).
   1. This is so that if there is an accident and the box containing the strains is dropped the strains will at least still be contained within the primary or secondary container.
   If transporting the cultures outside the primary lab areas (e.g. staircase), a new (unused) glove will be worn when transporting the double contained cultures (primary and secondary containers) following a one-glove rule, i.e. the gloved hand is used to hold the container, the ungloved hand is used for doors, keys etc.
4. Protective equipment – All laboratory staff who will come into contact with the BSL2 strains must wear the correct personal protective equipment (PPE). A lab coat must be used at all times, and disposable gloves when necessary. Preferably, a short-sleeved shirt should be worn under the lab coat. Closed shoes should also be worn. Only experienced personnel with knowledge in handling BSL2 microorganisms will be allowed to perform laboratory work with the Class II organisms.
   1. If a spillage occurs so that the strains comes into contact with the member of staff then the contaminant clothing can be removed and sterilized
5. Work with live Class II strains is reserved to the designated BSL2 areas, bench, hood, incubator and freezer.
6. Work with live BSL2 organisms is only allowed to occur in the presence of a ready-made virkon solution of sufficient volume to inactivate all of the organisms if required. Additionally, 70% ethanol should be available as a backup for the same purpose of organism deactivation.
7. All designated areas where BSL2 organisms will be handled will be clearly indicated with the biohazard symbol.
8. All equipment that comes into contact with BSL2 organisms must be thoroughly sterilized after handling is concluded. To as large extent as possible use separate equipment for BSL2 work.
9. All personal protective equipment must be correctly treated after the handling of the BSL2 strains is concluded. Disposal of gloves, washing of any contaminated clothing.
10. Face, and especially the mouth, nose and eyes, should not under any circumstances come into contact with Class II strains or aerosols during the work, neither should objects including pencils, pens and more.
11. In case of contact with eyes/face – flush any affected skin area with sterile water and wash with soap and then 70% ethanol. Rinse eyes using eye-wash bottle located in the room. Inform supervisor (James Booth) immediately (Tel. 41045852) and in the case of eye exposure, seek medical attention.
12. All workspaces should always be sprayed and wiped down with 70% EtOH before and after work.
13. In order to minimize the risk of spills a rack(s) should be used at all times, tube lids should remain closed when not in use. If the tubes/cuvettes are open use parafilm.
14. The volumes of liquids containing BSL2 organisms should be reduced to a minimum and only handled within the designated hood.
15. Avoid producing aerosols whilst pipetting, spreading or handling liquid bacterial cultures by working with calm and controlled hand movements. Sterilize pipettes before and after working with BSL2 organisms.
16. All experiments should be well planned in advance and with plenty of time allowed for any experiment.
17. Only approved and trained personnel are to work with BSL2 organisms.
18. All waste containing or contaminated by BSL2 organisms must be decontaminated before is it placed in a sealable plastic bag and then in the risk waste for destruction. Liquid waste should be placed in a water tight sealable container following deactivation and before being placed in the risk waste.
    1. 96 well plates and plates of a similar size. Place in two black plastic bags in a risk waste yellow plastic container. Decontaminate with virkon, after deactivation tie the black plastic bags and place in the risk waste for thermal destruction.
    2. Agar plates can be deactivated with either 70% EtOH (5 min) or virkon (20 min). The deactivated plates are then placed in the risk waste for thermal destruction.
19. Physical contact with bacterial lawns on solid media: Spray with 70% EtOH and keep the contaminated surfaces wet with 70% EtOH for at least 30 seconds but up to 5 minutes if possible. Place deactivated disposable material in the risk waste, lab coats should be packed into a sealed plastic bag and autoclaved.
20. Minor spills (less than 50 μl e.g. drops from pipette tip): Spray 70% EtOH onto the spill and leave for 5 minutes, and then spray 70% EtOH onto absorbent material and place over the effected work area and/or equipment. Wash hands, then put on new gloves and return to the hood.
21. Small spills (less than 1 ml): to disinfect spills, spray with 70% EtOH and leave for 10 min or mix with a generous amount of Virkon solution, incubate for at least 20 minutes. Spray and immediately remove and dispose of gloves inside the hood. Wash hands (soap and water, then 70% ethanol) and put on new gloves. Wipe spill area with absorbent material and dispose of in closed double plastic bags. Dispose of the bag in the risk waste. Further disinfect spill area and any equipment (e.g. pipettes) with 5% hypochlorite or Virkon, leave for 10 min and wipe off with 70% EtOH. Spray and immediately remove and dispose of gloves inside the hood. Wash hands and put on new gloves. Spills onto lab coat should be soaked in 70% EtOH and left for 10 min – the lab coat should be packed in a sealed plastic bag and autoclaved (NB. Virkon should not be used if the lab coat is to be autoclaved).
22. Work with volumes over 1 ml is not permitted.
23. Sharps (e.g. hypodermic needles, scalpels) must never be used with these organisms.
24. Following completion of work inside the hood it should be thoroughly sprayed with 70% EtOH.
25. On completion of work and transport, hands must be washed thoroughly in warm water with soap and dried. Once dry, hands may be cleansed using alcohol 70%.
26. If you suspect you have been exposed, infected or become unwell seek immediate medical attention and report the situation to the supervisor (JAB) immediately (Tel. 41045852).
27. The risks involved with other BSL2 bacteria, not specified here, must be thoroughly investigated and approved before work can be initiated.

Decontamination: Virkon (>20 minutes), 70% EtOH (>30 seconds) as long as the surfaces are wet. Times can vary between organisms.