Antimicrobial resistance is the increasing challenge in healthcare associated infections. K. pneumoniae is one of the main causes of nosocomial infections especially among immunocompromised individuals with increasing resistance rates. Drug resistance and virulence such as its biofilm forming ability are the key factors involved in the persistence of infections10. These factors found to have a significant association with the clinical outcomes. Currently, increasing AMR in K. pneumoniae have become a worldwide problem and there is still very limited data regarding biofilm producing K. pneumoniae in India. These biofilms forming K. pneumoniae are highly resistance to many commonly used antibiotics thus making the current treatment challenging.
The situation become more complicated when biofilm producing organisms are treated with inappropriate antibiotics and with insufficient concentrations. For instance, biofilms can resist empiric antibiotic therapy and contribute to bacterial persistence in chronic infections that result in high morbidity and mortality. Antibiotics such as piperacillin, piperacillin/tazobactam, cefoperazone, ceftazidime, cefepime, meropenem, ciprofloxacin, netilmicin and amikacin were reported to show reduced activity against adherent bacteria when compared to the planktonic counterparts11. In addition, several studies have shown that certain antibiotics induce biofilm formation when treated with sub-inhibitory concentration12. For this reason, early detection of biofilm forming nosocomial pathogens mainly from high dependency units is crucial. The present study was designed to inform the clinicians in identifying and choosing the appropriate antibiotic therapy for biofilm mediated infections.
The key aspect of this study was to develop a diagnostic assay that uses a set of virulence genes of K. pneumoniae as a marker. Currently, there are no rapid diagnostic methods available to identify K. pneumoniae biofilms from clinical isolates and direct samples. Conventional assays take 48–72 hrs for identification of K. pneumoniae and biofilm formation ability. This assay based on real-time (RT)-PCR provided an added advantage by offering information on the biofilm forming ability of K. pneumoniae as well as confirming the identity of the pathogen in a short span.
Among the various genes responsible for the biofilm production, mrk (Type 3 fimbriae) and pgaC (polysaccharide adhesion) are the candidate genes linked with biofilm formation in K. pneumoniae and has shown to promote strong biofilm formation, enabling surface adhesion13. Studies have indicated that mrkA gene contributes to rapid biofilm formation while mrkD responsible for dense K. pneumoniae biofilms14,15,16,17. MrkD, a fimbrial adhesin from Klebsiella pneumoniae, causes adherence to the basement membranes of tissues and the basolateral surfaces of renal and pulmonary epithelia. This adhesin, which is an extracellular matrix binding protein, has been demonstrated to bind to type V collagen. Even though all isolates containing the MrkD adhesin induce the agglutination of erythrocytes treated with tannic acid in vitro, the mrkD gene is not conserved across species. The ability of a plasmid-borne mrkD gene product to induce type V collagen binding is usually associated with K. oxytoca strains and seldom with K. pneumoniae strains. The MrkD adhesin is a chromosomally borne adhesin that mediates binding to collagen types IV and V in K. pneumoniae18,19.
Similarly, pgaC also reported to serves as an adhesion factor for the initiation and maintenance of biofilm structure20. pgaC is known to be closely associated with pgaB/D, a biofilm adhesin polysaccharide and luxR gene, an N-acyl homoserine lactone (AHL)-dependent transcriptional regulator. AHL is one of the most common quorum sensing (QS) mechanism utilized by Proteobacteria21. QS is a well-established mechanism in the process of biofilm formation22. LuxR protein plays a key role in QS mechanism in most of the Gram-negative bacteria by detecting the presence of signalling molecules which enable inter- and intra-species interaction in response to external stimuli according to population density23.
Furthermore, in-silico screening of the clinical K. pneumoniae genomes available in the public database revealed that 98%, 99.3% and 34.6% of the genomes carried mrkD, pgaC and wcaJ genes13. It was also found that mrkD and pgaC genes were present in all biofilm forming K. pneumoniae isolates in the present study. This indicates their conservation in K. pneumoniae strains and the specificity of the chosen targets in the detection of K. pneumoniae biofilms from clinical samples.
Occasionally, some strains of K. pneumoniae may lose their fimbriae during culturing or may lack this fimbriae gene17. This issue has been addressed by using more than one target as demonstrated in the present study to ensure the reliability of the assay. Interestingly, the PCR panel evaluated in this study identified K. pneumoniae from the samples that were even culture-negative, showing the high sensitivity of the assay. Further, the samples that were PCR positive and culture negative were confirmed by 16S metagenomics to have Klebsiella reads. This could be due to the limited sensitivity of the culture method, or lesser load of the pathogen. In such cases, treatment with broad spectrum antibiotics will be helpful since Klebsiella in this individual may or may not be associated with the infection due to its low bacterial load. However, this approach also has its limitations where the patient may be over-treated leading to the development of drug resistance. To avoid this difficulty, the RT-PCR results can be coupled with clinical diagnosis to make this an accurate diagnostic tool.
Further, wcaJ gene appears to act as a negative regulator, where its absence indicates the high potential of biofilm-forming capacity of the strain. The hypothesis was supported by earlier study by Pal et al, where they demonstrated that the inactivation of the wcaJ gene results in the disruption of colanic acid synthesis and enhances the biofilm formation in K. pneumoniae3. Based on the observed results for known positives and negatives, the cut off for Ct values to define negatives were ≥ 30 Ct for mrkD and > 30 for pgaC genes. However, this needs further standardization with a higher number of clinical samples.
Antimicrobial resistance rates among biofilm forming bacteria are higher compared to its planktonic forms and above the breakpoints proposed for therapeutic clinical use. This shows that treatment of biofilms with standard antimicrobial therapy would be unhelpful mainly among patients in high dependency units. This may also explain the treatment failure in some patients, despite susceptibility to antimicrobials in vitro, to result in clinical resistance. The PCR evaluated in this study in combination with clinical diagnosis will help in early detection of K. pneumoniae biofilms in critically ill patients and for their appropriate treatment either with high-dosage broad spectrum antimicrobials or with combinations.
In conclusion, the PCR assay standardized in this study is the first of its kind for rapid identification of biofilm forming K. pneumoniae from clinical samples. Considering the limited resource settings like primary health laboratories, the cost of the PCR test and maintenance of the sample integrity might be the limiting factors. Overall, the results of the study highlight that the rapid detection of K. pneumoniae biofilms based on the real time PCR results coupled with clinical conditions would be appropriate to treat emerging infections or to prevent re-infections in the clinical settings.