Diagnosis of biofilm- associated infections in medical devices
4.5 Examples of methods for the detection of biofilms associated with infections
4.5.1 Culture- based methods
Intra-vascular and urinary catheterization and endotracheal intubation may cause device- associated infection. Confirmation of catheter- associated infection requires isolation of the same organism from the patient’s body fluids (blood, urine, tracheal and broncho alveolar aspiration) and from the catheter tips submitted for culture.
The three common device- associated infections by biofilm- producing organisms are catheter-associated urinary tract infection (CAUTI), catheter related bloodstream infection (CRBSI) and ventilator associated pneumonia (VAP) (Maki et al. , 1977;
Mermel et al. , 2001; Sheretz et al. , 1990; Siegman-Igra et al. , 1997; Singh et al. , 2010). Central catheter infection may show infection at the site of surgical insertion, or perhaps as bacteremia. Various methods can be employed to diagnose CRBSI, such as semi- quantitative (direct inoculation by pressing device tip on to plate, or culture of catheter fluid), quantitative culture (sonication) and qualitative culture. In the case of CRBSI, two blood samples are collected, one through the catheter itself, and other from a peripheral site. This should be done at the time when the catheter is sent for culture. Following appropriate aseptic and antiseptic procedures, the catheter is removed from the patient with the use of sterile forceps. Two to three inches of catheter tip is cut with sterile scissors and put in a sterile capped container, which should be sent to the laboratory as soon as possible. For confirmation of CAUTI, urine is collected from the sampling port of the urinary catheter using a sterile syringe and needle (Singh et al. , 2010). Clinically, a patient is considered to be suffering from VAP if they are on a mechanical ventilator and have developed a fever and cough with purulent expectoration. For VAP, the sample collected is broncho- alveolar lav- age (BAL) through endotracheal intubation. (Maki et al. , 1977; Mermel et al. , 2001;
Sheretz et al. , 1990; Siegman-Igra et al. , 1997; Singh et al. , 2010). A majority of microbiology laboratories perform the semi- quantitative method because it is easy to perform, and is usually associated with decreased costs to the laboratories in terms of equipment and personnel training. The requirement for the infection to be diagnosed clinically as a case of CAUTI, is if the patient is catheterized and has developed one or more of the following conditions, such as, fever, supra- pubic pain frequency and urgency of micturition. Urine and/or device samples are cultured on blood and MacConkey’s agar and incubated for 24 hours. More than or equal to 10 5 CFU/ml is considered as significant (Singh et al. , 2010). For catheter-associated VAP, the samples (BAL and tracheal aspirate) are inoculated on blood and MacConkey’s agar and incubated for 24 to 48 hours. For BAL, 10 3 CFU/ml and for tracheal aspirates 10 5 CFU /ml are considered as significant. All the isolates thus obtained are identi- fied by standard microbiological culture techniques and tested for biofilm produc- tion. Various culture- based techniques for detection of biofilm are in use, such as the Congo red agar method, tube method and tissue culture plate method (Bose et al. , 2009; Mathur et al. , 2006).
Diagnosis of biofilm- associated infections in medical devices 75
4.5.2 Microscopy 4.5.2.1 Light microscopy
The biofilm is removed from the device, such as a catheter, by swab or scraping, and a culture is inoculated in to a tube containing nutrient medium. The diluted culture is poured in to a flat- bottom 12-well plate, where the uppermost surface of the liquid should reach the center of a cover slip placed over the top. The plate is then incubated at 37 °C for 18 hours, after which time the cover slip is taken out and dipped in 0.1%
crystal violet to visualize the cells using a standard light microscope (Taj et al. , 2012).
Light microscopy can be enhanced by the use of fluorescent dyes, which have shown to be effective on catheter surfaces (Zufferey et al. , 1988). Two typical dyes are 4,6 diamidino-2 phenylindone (DAPI) or acridine orange (AO), which stain the nucleic acid of both dead and live microbial cells. Propidium iodide can also be used to stain cells with a damaged cytoplasmic membrane, and 5-cyano- 2,3-ditolyltetrazo- lium chloride is used to stain the cells with a functional cytochrome system. Live cells reduce this dye into 5-cyano-2,3-ditolyltetrazolium chloride formazan, which can be visualized due to the evolution of a red fluorescent precipitate. Using this dye, it is possible to detect whether cells present in the biofilm are viable or not, and reveal the total number and distribution of cells. This has the advantage of being able to inform on how effective a treatment was on the biofilm, and how the cells are developing within the matrix. One significant disadvantage of this method is that thick biofilms cannot be analyzed due to the scattering of light associated with increased layers of exopolymers and cells (Donlan and Costerton, 2002; Schaule et al. , 1999). This sug- gests that the technique is inappropriate for the analysis of advanced- stage biofilm device infections.
4.5.2.2 Scanning Electron Microscopy (SEM)
This technique can be used to stain devices themselves, as well as laboratory- cultured samples in infection models. The biofilm is sputter coated with a gold or palladium film to facilitate visualization of the matrix. By using SEM, it is possible to detect surface attached cells, and the extent of biofilm growth. One notable disadvantage of this technique is that complete dehydration of the samples is required, which can result in sample shrinkage and artificial images being recorded. To prevent excessive shrinkage of samples, biofilms can be treated with ruthenium red prior to dehydra- tion. A modified SEM method has been developed to overcome this drawback, known as Environmental Scanning Electron Microscopy (ESEM). But the problem with ESEM is that the magnification is much lower than conventional SEM (Schaule et al. , 1999; Taj et al. , 2012).
4.5.3 Genotypic methods
One problem associated with the detection of biofilms and assigning treatment is the presence of viable but non- culturable (VBNC) cells within patient samples (Anderson et al. , 2004), which can hamper the identification and treatment processes. For the
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detection of non- culturable atypical or fastidious microbes within biofilms, molecu- lar diagnostic techniques have been developed and are becoming more mainstream in their utilization. These techniques can also identify other microbes that might not additionally be suspected of causing infection, adding a greater depth of scru- tiny where cells, which might be undetected by conventional, culture methods. The molecular techniques are highly sensitive but costlier than the conventional meth- ods, making a significant detraction to their adoption globally, alongside the need for implementation of an appropriate training program. It has been reported that by adopting molecular diagnostic techniques, bacterial colonization could be detected in more than 60% of arthroplasty samples, whereas by using conventional methods, infection could be diagnosed in less than one quarter of those patients (Arciola et al. , 2001; O’ Gara and Humphreys, 2001; Patrick and Rocky, 2011). This could represent a significant saving in terms of morbidity and mortality, as well as representing a significant advance in terms of reducing overall healthcare costs.
4.5.4 Polymerase chain reaction
Polymerase chain reaction (PCR) may be used to amplify gene sequences specific to target organisms suspected of causing infections, and also to determine whether genes associated with biofilm infections are switched on at the time of infection. This allows researchers to identify stages of the infection, and also to determine epidemio- logical trends in pathogen virulence. There are three main steps in the PCR process:
● Nucleic acid extraction
● PCR amplification
● Visualization of amplified products by gel electrophoresis
For formation of biofilm in coagulase- negative staphylococci, the expression of the Polysaccharide Intercellular Adhesin (PIA) virulence factor appears to be an impor- tant feature, however, PIA-negative/biofilm- positive strains have also been reported (Agarwal and Jain, 2013). The synthesis of PIA is coded by ica genes, which are regu- lated by an operon, consisting of ica ABDC and ica R genes. By using PCR, detection of gene expression is relatively straightforward, and can elucidate information critical to the disease pathogenesis specific to the patient, particularly when considering hos- pital- and community- acquired staphylococcal infections. Amplification of bacterial DNA can be performed relatively easily, however there is a chance of false positive results. This problem can be solved by detection of messenger RNA (mRNA) and ribosomal RNA (rRNA) as a marker of infection in biofilm (O’ Gara and Humphreys, 2001; Patrick and Rocky, 2011), and could have the potential to solve many current such issues. Detection of mRNA can be problematic, however, as it can quickly degrade after bacterial cell death and is present only in active infection. Another limitation of using mRNA is the low copy numbers and lack of a universal target sequence in all bacteria (Patrick and Rocky, 2011), suggesting that multiplex PCR setups might be required. It should be noted, however, that viable cells only produce mRNA, hence detection of mRNA in a biofilm is evidence of bacterial cell viability within a biofilm (Birmingham et al. , 2008; Roberts et al. , 2006). rRNA is a structural
Diagnosis of biofilm- associated infections in medical devices 77
subunit of ribosomes, and is abundant in active cells. It is a highly conserved marker, however molecules do not last for a significant period of time after cell death. Some segments of rRNA are unique to specific species, meaning that individual species can be identified by this technique, as well as groups of bacteria. Investigating incidents of fungal keratitis, a novel rRNA PCR technique demonstrated a sensitivity of 90.9%
and specificity of 94.7% (Embong et al. , 2008). Such results suggest that PCR-based technology platforms could represent a relatively easy to operate and cost effective strategy for pathogen diagnosis, if brought in to mainstream use.
4.5.5 Fluorescent in situ hybridization
Fluorescent in situ hybridization (FISH) is based upon the identification of DNA sequences that have been denatured in the sample by fluorescent labeling probes, and is used to determine the presence of specific pathogens due to its ability to dis- criminate between genetic sequences. However, the fading of fluorescent markers could mean that this technique is not suitable for all laboratories, as samples cannot be archived. This is a quick and reliable method, and is very useful for detection of infected joint prosthesis, and can also be helpful in culture- negative cases. FISH is a rapid method, only taking a few hours to obtain the result, meaning that diagnosis and the time taken to assign a particular treatment is vastly reduced. In situ PCR can be used to identify specific nucleic acid sequences, such as those of microbes in human tissue samples (Nakamura et al. , 2001; Taksukiko et al. , 2001). Nelson (2006) compared FISH and calcoflour white (CW) stain for the direct detection of filamentous fungi in water biofilms. By adopting this method, FISH demonstrated that the microbes were present in biofilm formations after five hours, whereas the CW staining method showed chitinous, filamentous structures in less than one hour. This suggests that a combination of techniques might be an appropriate approach to obtain a more complete picture of what is happening as diseases progress.
4.5.6 IBIS T5000 technology
This technique allows for the detection of a wide spectrum of microbes, and combines broad range PCR and high performance mass spectrometry. In broad range PCR, it targets the gene encoding 16s rRNA, also known as 16s PCR (Renovoise et al. , 2013). First, PCR is performed to obtain amplified genomes of microbes present in the biofilm. After that, mass spectrometry technology is used to obtain base com- position signatures, which facilitates the identification of microbes to the species level, and can detect genes coding for antibiotic resistance within their genomes (Ecker et al. , 2008). It is a highly sensitive PCR based technique and can detect infec- tion due to those microbes, which are difficult to culture, suggesting a potentially wide range of applications where initial culture- negative samples could be further investigated.
Traditional PCR requires the use of a specific primer for a specific pathogen.
However, IBIS T5000 uses mass spectrometry- derived base units of microbes obtained from the amplified PCR products of multiple genes present in the sample.
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Broad range PCR can produce products from many organisms at a time, rather than a single species, thus increasing its potential application, and the mass spectrometer accurately provides base composition data for PCR products in a short span of time, thus potentially reducing the time taken to prescribe an effective treatment. This avoids the requirement of sequence specific probes for a suspected pathogen, and the technique can be used for large- scale analysis of clinical or environmental sam- ples. The T5000 can analyze complex PCR amplicon at a rate of approximately one well/minute, because of highly automated software, meaning that a technician without specialized expertise can operate this, thus potentially reducing long- term costs (Paul et al. , 2011; Michael et al. , 2011; David et al. , 2006; Nistico et al. , 2011;
Costerton et al. , 2011).