Serratia marcescens, Pseudomonas spp. and other bacteria contaminating antiseptic and disinfectant solutions: Detection by conventional cultures; Molecular detection (PCR); Identification (sequencing); Molecular epidemiological comparison by MLST (Multilocus Sequence Typing).

Information 2019-02-28.

Serratia marcescens and Pseudomonas spp. are gram-negative bacilli that can be part of the human microbiota at the intestinal level, but which is also found in humid and nutrients-poor environments, such as soils and water, even in hospitals. Its affinity for wet environments means that they can be found in sinks and pipes. They usually form biofilms, which allows them to persist, even in unfavourable conditions, for days to months, in faucets for handwashing, intravenous fluids, instrumental equipment (mechanical ventilation equipment), antiseptic or disinfectant solutions, hand soaps, and even in baby shampoo. In addition, they can be found colonizing the gastrointestinal tract and the skin of health personnel.

Antiseptics are antimicrobial substances that are used on the skin and mucosa to reduce bacterial colonization. Currently, the main use of antiseptics in health institutions is intended for hand cleaning, preparing the skin of patients before surgery, removing microorganisms from the hands and forearms of the surgical team and preparing skin in some invasive procedures. such as central and arterial venous puncture, vascular and bladder catheterization, among others, where the normal cutaneous-mucous defense barriers of the individual are broken.

The information collected in the scientific literature on antiseptic products infected by microbiological contamination are a cause of concern for the control of infections related to medical care, because these contaminations have been the cause of outbreaks of hospital infections, sometimes serious. Despite the uncertainties regarding the resistance of some species of bacteria to biocides, the concentrations currently used in health services are considerably higher than the minimum inhibitory concentration (MIC) of bacteria capable of growing in antiseptic solutions.

Contamination of antiseptics can occur by two different mechanisms: 1) intrinsic when it occurs during the manufacturing process, and 2) extrinsic, when microorganisms are introduced into the antiseptic during use. Extrinsic contamination is the most frequent form and can happen when the product is diluted with contaminated tap water to prepare the disinfectant solutions, in the same way as when dilutions are not prepared with the appropriate aseptic techniques or when filling previously contaminated bottles. In addition, in the case of S. marcescens or Pseudomonas spp., its ability to produce biofilms means that it can survive and persist in the presence of high concentrations of some antiseptics such as chlorhexidine.

The most frequent errors include the use of dilute solutions, the use of obsolete products, the use of tap water to dilute the antiseptic, the filling of small volume dispensers from large volume containers and the inadequate selection of a product appropriate (for example, the use of a low level disinfectant instead of a high level disinfectant to disinfect an endoscope). Because there have been multiple outbreaks due to the filling of small volume dispensers from large volume containers, small volume containers should be used until they are completely empty, that is, small containers should not be refilled without prior care, but rather they must be previously rinsed with sterile water, and allowed to air dry before being refilled.

A critical element of skin disinfection is pre-cleaning to remove the protein material and biofilms to allow the antiseptic to achieve adequate microbial inactivation since otherwise the thickness of the cellular and extracellular material that accumulates on the surfaces, that is, biofilms, can protect microorganisms from the microbicidal actions of antiseptics and disinfectants. As an example, bacteria that grow in a biofilm can be up to 1,500 times more resistant to germicides than the bacteria grown in liquid cultures, so improper pre-cleaning of medical devices can make it difficult for microbial inactivation by antiseptics or disinfectants.

Clinical manifestations 

These reservoirs can be the source of outbreaks of nosocomial infections, such as bacteraemia, pneumonia, surgical wound infections and urinary tract infections. These infections frequently affect patients admitted to intensive care units and neonatal units. Clinically, bacteraemias occur more frequently in immunocompromised patients due to immunosuppressive treatments or systemic diseases such as diabetes, malignancies and chronic renal failure.

The clinical manifestations of the outbreaks caused by the use of contaminated antiseptics vary from infections located at the point of application of the antiseptic solution to serious systemic infections due to the penetration and spread of the contaminating bacteria from the point of entry where the antiseptic was applied.

Venous catheters are used in many types of patients with infusion of intravenous solutions or undergoing hemodialysis. The application of an antiseptic solution for the management of tunneled catheters is recommended to reduce risks in patients with this type of catheter. The morbidity and mortality associated with bacteraemia secondary to these infections is high and, therefore, care measures for tunneled catheters must be extreme in hemodialysis units. 

Preventive measures 

To prevent outbreaks associated with antiseptics/disinfectants, the following recommendations should be followed:

  1. Use only antiseptics or disinfectants registered and recommended by Official Medicines and Medical Devices Agencies;
  2. Use all antiseptics or disinfectants at the recommended use dilution and do not excessively dilute the products;
  3.      Use sterile water to dilute products and not use tap water;
  4. Use all products during recommended contact times;
  5. Do not use products labeled as antiseptics for disinfection of medical devices or surface disinfection;
  6. Follow the recommended procedures in the preparation of products to prevent extrinsic contamination;
  7. Small volume dispensers that are refilled from large volume containers should be used until they are completely empty, and then must be rinsed with running water, or better sterile water, and air dried before refilling;
  8. Keep the stock solutions of the products as indicated on the product label.

Some ancient scientific published descriptions of bacteria present in antiseptic or disinfectant solutions 

  • Alcohols: 

Bacillus cereus causing bacteraemia due to intrinsic contamination (1999). 

Burkholderia cepacia (formerly Pseudomonas cepacia) causing catheter bacteraemia due to contamination from tap water to dilute alcohol for cutaneous antisepsis (2004). 

  • Chlorhexidine: 

Pseudomonas spp. by refilling use containers for use of low concentration (0.05%) (167).

Burkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia, urinary tract and wounds infections (1971).

Flavobacterium meningosepticum causing bacteraemia, meningeal infection, wounds, skin, possibly from contaminated water or use of low concentration (1:1,000–1:5,000) (1976).

Pseudomonas spp., Serratia marcescens, Flavobacterium spp. possibly due to over-dilution or refilling of reused bottles (1981).

Pseudomonas aeruginosa causing wounds infections by using tap water to dilute stock solutions at low concentration (0.05%) (1982).

Bulkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia, wound infection, urinary, oral, or vaginal infections, by contamination of deionized water pipes during dilution at low concentration (1982).

Ralstonia pickettii (formerly Pseudomonas pickettii) causing bacteraemia due to contamination of double-distilled water for dilution at low concentration (0.05%) (1983).

Ralstonia pickettii (formerly Pseudomonas pickettii) causing bacteraemia due to deionized water contamination to dilute at a low concentration (0.05%) (1987).

Ralstonia pickettii (formerly Pseudomonas pickettii) causing bacteraemia due to contamination of distilled water to dilute at a low concentration (0.05%) (1985).

Ralstonia pickettii (formerly Pseudomonas pickettii) causing bacteraemia due to contamination of distilled water used for dilution at low concentration (0.05%) (2000).

Achromobacter xylosoxidans causing bacteraemia, or injuries infections due to low concentration spray contamination (600 mg/L) (1988).

Achromobacter xylosoxidans causing bacteraemia due to spray contamination (2005).

Serratia marcescens causing bacteraemia, urinary infections, wounds or sputum infections due to the use of non-sterile water to dilute 2% and distribution in non-sterile reused containers (1998).

  • Chlorhexidine + Cetrimide:

Pseudomonas multivorans causing wound infections due to tap water used to prepare stock solutions, at low concentrations of 0.05% chlorhexidine and 0.5% cetrimide (1970).

Stenotrophomonas maltophilia (formerly Pseudomonas maltophilia) causing urinary, umbilical infections, catheter tips and others, due to the use of deionized water to prepare solutions, and the use of contaminated containers not disinfected between uses (1976).

  • Chloroxylenol:

Serratia marcescens causing infections of multiple locations due to extrinsic contamination of soaps of 1% chloroxylenol (1997).

  • Benzalkonium Chloride:

Pseudomonas spp. causing bacteremia due to contamination during storage or solutions of benzalkonium chloride (0.1%) with cotton or gauze (1958).

Pseudomonas spp. and Achromobacteriaceae group that causes bacteraemia and urinary infections, due to the preservation of benzalkonium chloride solutions (0.1%) with cotton or gauze, or to dilution with non-sterile water (1961).

Enterobacter aerogenes causing bacteraemias, sinus infections, due to preservation of benzalkonium chloride solutions (0.13%) with cotton or gauze (1960).

Pseudomonas kingii causing urinary infections due to intrinsic contamination of antiseptics (1969).

Pseudomonas EO-1 causing urinary infections due to intrinsic contamination of cleaning or antiseptic solution (1970).

Pseudomonas spp. causing bacteraemia due to intrinsic contamination of 0.2% solutions (1976).

Bulkholderia cepacia (formerly Pseudomonas cepacia) and Enterobacter spp. causing bacteraemia by preservation of benzalkonium chloride solutions with cotton or gauze; by improper dilution; or by conservation in non-sterilized containers (1976).

Bulkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia due to the preservation of benzalkonium chloride with rayon balls; or for failures to disinfect use containers (1976).

Serratia marcescens joint infections due to inadequate preservation of benzalkonium chloride solutions with cotton or gauze (1987).

Serratia marcescens causing meningeal infections (CSF) due to extrinsic contamination of the stock container (1984).

Mycobacterium chelonae causing skin abscess due to the preservation of benzalkonium chloride with cotton or gauze; or at inadequate dilution (1990).

Mycobacterium abscessus causing joint infections due to preservation of benzalkonium chloride with cotton or gauze; or by dilution with probable contaminated tap water (2003).

  • Benzalkonium Chloride / Picloxidine:

Burkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia, urinary tract infections, wounds or sputum infections due to contaminated water to dilute the antiseptic (1976).

Burkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia, due to the water used to dilute the antiseptic (1976).

  • Povidone-iodine:

Burkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia due to intrinsic contamination of 10% solutions, and probable proliferation of B. cepacia in a deionizing resin in the water system (1981).

Burkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia and peritoneal infection, due to intrinsic contamination (1989; 1991; 1992).

Burkholderia cepacia (formerly Pseudomonas cepacia) causing bacteraemia, due to intrinsic contamination (1981).

Pseudomonas putida causing bacteraemia and catheter tip infection without a specific cause (2004).

  • Poloxamer-iodide:

Pseudomonas aeruginosa causing peritoneal and wound infection, due to intrinsic contamination (1982).

  • Triclosan: 

Serratia marcescens causing conjunctival infection due to intrinsic contamination (1995).

Tests performed in IVAMI: 

  • Conventional culture with prior enrichment in liquid medium followed by plaque isolation (recommended to avoid antiseptic inhibitory effect).
  • Molecular diagnosis (PCR) (recommended after previous enrichment broth culture).
  • Molecular identification (sequencing) (recommended to determine the species of causative bacteria).
  • Molecular comparison of strains in outbreaks (MLST: MultiLocus Sequencing Typing) (recommended to help determine focus of common origin).

Recommended samples: 

  • Sample of antiseptic or disinfectant.
  • Strains isolated by cultures for identification or for molecular epidemiological comparison of strains). 

Storage and shipment of the sample: 

  • For samples of antiseptics or disinfectants: usual storage temperature.
  • For bacterial strains on culture plates: refrigerated for less than 2 days. 

Delivery of results 

  • Conventional culture with prior enrichment in liquid medium followed by plaque isolation (recommended to avoid antiseptic inhibitory effect): 48 hours in working days.
  • Molecular diagnosis (PCR) (recommended after previous enrichment broth culture): 24 to 48 hours in working days.
  • Molecular identification (sequencing) (recommended to determine the species of causative bacteria): 3 to 4 working days
  • Molecular comparison of strains in outbreaks (MLST: MultiLocus Sequencing Typing) (recommended to help determine focus of common origin). 7 working days. 

Cost of the test: 

  • Conventional culture with prior enrichment in liquid medium followed by plaque isolation (recommended to avoid antiseptic inhibitory effect in Molecular methods): Consult to ivami@ivami.com.
  • Molecular diagnosis (PCR) (recommended after previous enrichment broth culture): Consult to ivami@ivami.com.
  • Molecular identification (sequencing) (recommended to determine the species of causative bacteria): Consult to ivami@ivami.com.
  • Molecular comparison of strains in outbreaks (MLST: MultiLocus Sequencing typing) (recommended to help determine focus of common origin) (each strain compared): Consult to ivami@ivami.com.