Background
Inflammatory bowel disease (IBD), which consists of Crohn’s disease (CD) and ulcerative colitis (UC), share a variety of different genetic factors, environmental triggers, and treatment plans [
1‐
5]. Multiple recurring reports from our laboratory and others provided evidence that environmental triggers including enteric pathogens may be associated with IBD. Specifically
Mycobacterium avium subspecies
paratuberculosis (MAP), adherent-invasive
Escherichia coli (AIEC) strain LF82, and
Klebsiella pneumoniae have been implicated as possible causative agents in CD [
5‐
12].
MAP was first isolated as the causative agent for Johne’s disease, a CD-like enteritis in cattle [
8,
9,
13,
14]. MAP is an intracellular acid-fast pathogen that infects macrophages and dendritic cells and inhibits phagosome–lysosome fusion [
15,
16]. Interestingly, MAP was detected in the blood, milk and intestinal biopsies from patients with CD and most recently from the blood of Rheumatoid Arthritis (RA) [
5,
6,
8,
9]. On the other hand, AIEC is a gram-negative bacillus pathogen that has also been isolated from intestinal tissue from patients with CD [
10,
12,
17,
18]. AIEC strain LF82 has been studied intensely due to its ability to infiltrate intestinal tissue and increase pro-inflammatory cytokines level in CD patients [
10,
12,
17,
18]. Like MAP, AIEC strain LF82 resists phagosome–lysosome fusion and acidification [
10,
12,
17,
18]. Recent studies also showed that
K. pneumoniae might be associated with IBD pathogenesis [
11,
19].
K. pneumoniae is a gram-negative, facultative anaerobic bacillus pathogen that causes pneumonia in immunocompromised patients [
11,
19]. It colonizes the intestinal epithelial with an imbalance in gut flora leading to elevated humoral immune response [
11,
19].
Although there are numerous studies investigating the role of these pathogens in CD pathogenesis, none of the studies examined the co-occurrence or co-roles of these pathogens in CD. Despite of the rationale why these pathogens were investigated individually for their role in CD, the outcome of each study may contributed to the controversy as to which microorganism really causes inflammation in CD, or which bacterium play a more important role in CD. For sure, it is challenging to use standard experimental techniques and less specialized skills to study diverse microorganisms together in individual clinical samples. This study is designed to address these challenges in an attempt to develop the necessary protocols to evaluate the role of co-multiple pathogens in gastrointestinal tract inflammation. Consequently, we focused on development of a multiplex PCR based on rapid DNA extraction, and multi-color fluorescent in situ hybridization (m-FISH) on based specific oligonucleotide sequences.
Discussion
Recent studies have strongly supported the role of microbial infection in IBD development [
7‐
12]. It is still unclear which pathogen is found more readily in either UC or CD patients, where recent studies are more focused on detecting one pathogen at a time [
8,
10,
11,
18,
19]. In this study, the investigation of the presence of multiple pathogens including MAP,
E. coli strains, and
K. pneumoniae, was done to fully understand the role of possible co-infection or co-occurrence of multiple pathogens in individual IBD patients.
The development of a multiplex PCR protocol coupled with an
m-FISH detection protocol was successful, which together can detect multiple bacterial pathogens in a single sample. Along with this, a newly modified DNA extraction protocol was used in order to process the samples faster (~ 1 h) than previously used techniques (2–3 days) [
5,
6,
8]. Overall, combining all three protocols has produced a faster, more efficient way into detecting multiple bacterial species in one test sample.
The effectiveness of the two DNA extractions techniques that were used in this study was established, where the modified DNAzol
® technique showed similar specificity and sensitivity to the “traditional” phenol/chloroform/isoamyl-alcohol DNA extraction technique (Fig.
1). The modified DNAzol
® DNA extraction technique however has the advantage over the “traditional” DNA extraction due to the simplistic and the less time consuming protocol, thus could potentially lead to more samples being processed at a time. Also, due to the cost-effectiveness of the modified DNAzol
® DNA extraction protocol, which has a 1.5-fold lower cost than the “traditional” DNA extraction protocol, the modified protocol could be a better DNA extraction method to use in the clinical setting [
20].
We developed a multiplex PCR that is specific to detect pathogens’ targets when DNA or bacteria were used purely or in a mixture (Figs.
2,
3). This technique was validated using DNA extracts from intestinal tissue from IBD subjects (Tables
1,
3). The labeling of oligonucleotide primers used in multiplex PCR with fluorophores in our
m-FISH confirmed the identity of the microorganisms used in this study and the results obtained by multiplex PCR from intestinal tissue samples (Figs.
4,
5).
m-FISH illustrations validated the efficiency of the multiplex PCR in detecting multiple pathogens within hours using one protocol and limited reagents and steps. The significance of this technique will be tremendous for those investigating the role of multiple pathogens in tissue sections from patients with idiopathic or multifactorial diseases. Since CD is considered a syndrome with multifactorial etiology, the outcome of this study will provide new tools toward understanding which microorganism(s) is/are play significant role in disease pathogenies. As expected, both RS1 (UC) and RS2 (CD) were positive for non-pathogenic
E. coli strain K-12 and
K. pneumoniae (Fig.
6). However, detection of MAP in RS2 tissue (CD) and not in RS1 (UC) by multiplex PCR and validated with images by
m-FISH support published reports of association of MAP with CD [
5,
6,
8,
9,
13,
21]. Astonishing, both multiplex PCR and
m-FISH did not detected the AIEC strain LF82 in the two IBD patients samples, where previous studies have shown that this bacterium has been associated with IBD pathogenesis [
7,
10,
12,
18]. This could be due to the small sample size in this study, but also could be due to the possibility that the
gipA gene being amplified in the AIEC strain LF82 could be a low copy gene target [
22,
23]. The virulent gene
gipA was chosen as the target gene for amplification in AIEC strain LF82 is due to its association with AIEC infection in Peyer’s patches of CD patients [
22,
23].
When comparing the intensities of the multiplex PCR DNA bands and the
m-FISH signaling of non-pathogenic
E. coli strain K-12 with the signaling of
K. pneumoniae in both patient samples, it is evident that the dysregulation of commensal
E. coli could potentially play a role in a higher rate of
K. pneumoniae infection in CD patients (Figs.
3,
5a, d). The data suggests that the more
K. pneumoniae signaling is present in the CD patient (RS2) sample, the less non-pathogenic
E. coli strain K-12 signaling is present. This could suggest and confirm that an opportunistic infection of
K. pneumoniae can occur if there is a dysfunction of the microbiome in IBD patients due to the dysregulation of commensal bacteria, such as
E. coli strain K-12 [
11,
24‐
28]. With the increasing influx of
K. pneumoniae in the CD patient more so in the UC patient, it could be a potential pathogen to investigate for CD pathogenesis studies.
The
m-FISH assay on tissue from additional four other CD patients (RS3–RS6) showed that we able to detect and visualize multiple bacterial species in a single biopsy sample (Fig.
6). Further testing of more tissue from other diseases using the
m-FISH probes and multiplex PCR for different microorganisms, such as other commensal bacteria species like
Bacteroidetes fragilis and other opportunistic bacterial species like
Pseudomonas aeruginosa, would be helpful to further validate these techniques to help elucidate idiopathic diseases with multiple etiology [
29,
30].
Overall, this study was done as a pilot study in order to verify and examine these newly developed protocols, and as such, more IBD patient samples are required for future examination.
Authors’ contributions
RS and EN has performed the experiments, collected and analyzed all data, and were the primary writers in this study. KA, AQ, and LA contributed to the experimentations and helped create the protocols used in this study. SN is the corresponding author and primary investigator of this study. He managed the entire study, supervised all experiments, helped analyze and interpret the data, and the writing of the manuscript. All authors read and approved the final manuscript.