Catheter-associated biofilms play a crucial role in the development and persistence of infections. Considering this, studying biofilms, especially, formed by pathogenic bacteria, is highly important not only for the understanding of the physiology of biofilms and their regulation but for Medicine and healthcare to identify new strategies for infection treatment. In our study, we focused on one of the most clinically important pathogens P. aeruginosa associated with urinary catheters, and aimed to analyze its antibiotic resistance.
Identification of bacterial biofilms associated with the surface of urological catheters
Urinary tract indwelling catheters were analyzed for the presence of biofilms. 8 samples of catheters were obtained from patients with urinary tract diseases including bladder stones, prostatic hyperplasia, urinary retention, acute tubulointerstitial nephritis. Catheter samples were stained with CV to determine biofilm presence. Bacterial biofilms were detected on 6 out of the 8 studied catheter samples. Three samples had low-density biofilms, two samples were characterized as having medium-density biofilms, and only one sample exhibited a high-density biofilm (Table 1).
Table 1
– An analysis of biofilms on the catheter surfaces taken from patients with urological diseases.
Catheter samples |
| №1 | №2 | №3 | №4 | №5 | №6 | №7 | №9 |
Biofilm OD | 0 | 0.060 | 0 | 0.232 | 0.770 | 0.233 | 0.522 | 1.991 |
Interpretation | NI | Weak | NI | Weak | Moderate | Weak | Moderate | Strong |
*Biofilm OD = average OD-ODc; ODc = negative control OD-(3xSD negative control). |
NI – not identified
The morphology and biofilm composition of the catheter sample with the high-density biofilm (catheter No. 9, Table 1) was analyzed using scanning electron microscopy (SEM). Scanning electron microscopy revealed the presence of a heterogeneous matrix on the inner and outer surfaces of the catheter (Figs. 1, 2). Rod-shaped and coccoid bacterial cells, matrix fragments, inclusions of blood cells, and salt crystals were observed both on the inner and the outer layer of the catheter (Fig. 1A, B; Fig. 2).
As far as urological catheters directly contact with the patient’s internal tissues, host blood cells and salt crystals derived from the urea and other inclusions can be detected in bacterial biofilm structure [21]. Overall, urinary catheterization induces fibrinogen release into the bladder as part of the inflammatory response. This fibrinogen can accumulate both on the bladder surface and catheter surface. For instance, it was shown that Enterococcus faecalis cells attach to fibrinogen-coated catheters more efficiently than to the fibrinogen-free surfaces and use it for growth as the source of nutrients, enhancing biofilm development on the catheter [22].
Isolation and identification of catheter-associated biofilm-forming microorganisms.
We focused on the isolation of Pseudomonas spp., especially P. aeruginosa due to its vital significance in the CAUTI. Among 25 obtained bacterial isolates several strains had a blue-green pigmentation after 3 days of cultivation at RT which allowed us to consider these strains as candidate representatives of Pseudomonas genus. MALDI-TOF MS protein profile analysis confirmed that three of the isolated strains belong to P. aeruginosa (strains numbers 96345, 96347, and 96349) (Score value > 2.4) (Table 2). All strains had a rod-shaped cell morphology and 3–4 µm long cells, which is usual for Pseudomonas genus (Fig. 3) [23]. All three P. aeruginosa strains were isolated from different catheter samples. Also, P. aeruginosa 96345 was isolated from a catheter on which biofilm formation was not detected (Table 1).
Table 2
– A catheter-associated bacterial isolates identified by Biotyper MALDI-TOF MS.
Strain number | Bacterial species |
96345 | Pseudomonas aeruginosa |
96347 | Pseudomonas aeruginosa |
96349 | Pseudomonas aeruginosa |
Although the majority of bacteria isolated from urinary catheters belongs to the Enterobacteriaceae family (the most common group associated with the human urogenital tract), P. aeruginosa has an extremely high value in urinary tract infections [24]. Overall, P. aeruginosa is involved in the development of different human diseases including dental caries, otitis media, cystic fibrosis pneumonia, chronic wound infections, musculoskeletal infections, biliary tract infection, bacterial prostatitis, urinary tract infection, and a wide range of medical device-related infections. Additionally, chronic bacterial prostatitis and medical device-related infections are prominently caused by P. aeruginosa [8]. Pseudomonas aeruginosa is one of the most environmentally significant species among the genus Pseudomonas, widespread in nature. However, the increasing importance of P. aeruginosa strains acquired in medicine in nature, due to its high antimicrobial and broad spectrum resistance virulence factors [25]. P. aeruginosa is an opportunistic pathogen causing severe nosocomial infections in people with weakened immunity, it causes several types of infections, including dermatitis, endocarditis and infections of the urinary tract, eyes, ears, bones, joints, respiratory tract. Patients with cystic fibrosis who extremely susceptible to Pseudomonas infections, have poor prognosis and high mortality. Urinary tract infections (UTIs) are among the most common bacterial infections which annually affect about 150 million people in all the world. UTIs are a significant cause of morbidity in boys, older men and women of all ages.
Thus, the study of infections caused by this microorganism is the highest priority task of modern medicine.
Urease activity of catheter-associated P. aeruginosa.
Salt crystals from the host’s internal environment can often be found in the biofilm matrix [23]. Presumably, the formation of salt crystals in the urogenital tract can be initiated by bacterial ureases. The pH value of human urine under normal conditions is usually 6.0–7.5 with a tendency to slightly acidic values, with a normal range from 4.5 to 8.0. Higher urine pH values of 8.5 or 9.0 indicate infections induced by urea-splitting microorganisms, such as Proteus, Klebsiella, and Ureaplasma urealyticum. Therefore, an asymptomatic patient with a high urine pH means UTI regardless of the other urine test results. Alkaline pH can also lead to the formation of struvite kidney stones, which are also known as “infection stones” [26]. Urease-producing pathogens hydrolyze host urea with the formation of ammonium (NH4+) and hydrogen (H+) ions, which leads to pH increasing and changes in the urine composition. These reactions result in the sedimentation of struvite (ammonium magnesium phosphate) and apatite (calcium phosphate) on the surface of urinary tract epithelium, as well as on the surface of catheters and stents.
As mentioned above, the presence of salt crystals was identified in biofilm samples using SEM (Fig. 2). Hence, a urease activity of the isolated Pseudomonas strains was studied to confirm the role of biofilm-associated bacteria in salt crystals formation. However, high urease activity was determined for only one strain, P. aeruginosa 96349. We suppose that the high urease activity causes the formation of salt crystals associated with the biofilm formed by this strain, which also correlates with the density of the biolfilm.
Antibiotic resistance of catheter-associated biofilm-forming P. aeruginosa strains.
Antibiotic resistance of P. aeruginosa strains 96345, 96347, and 96349 were investigated using both microbiological and molecular methods. The growth of P. aeruginosa 96345 was significantly inhibited by both imipenem and meropenem. Diameters of growth inhibition zones induced by imipenem and meropenem were 33 and 11 mm, respectively (Fig. 4A). In contrast, it was shown that two P. aeruginosa strains (96347 and 96349) exhibit high resistance to β-lactam antibiotics, particularly carbapenems (imipenem and meropenem). Growth inhibition zones were not observed for these strains (Fig. 4B, C).
The DD-test is usually recommended for the qualitative analysis of antibiotic resistance. Moreover, this method provides no detailed information on the mechanisms of resistance. Instead, qPCR analysis is commonly adopted for the detection of wide-spread beta-lactamase genes such as KPC, NDM, OXA-48, VIM, IMP, and OXA-23 carbapenemases. Generally, antibiotic resistance of many Gram-negative bacteria can be associated with other mechanisms including efflux pumps and reducing of porin number.
However, according to many studies on antibiotic resistance [8; 27; 28], P. aeruginosa usually shows high β-lactamase activity. Thus, we performed a qPCR analysis to detect genes of the most widespread types of these enzymes, metallo-β-lactamase (NDM - metallo-β-lactamase of New Delhi, VIM- and IMP-types). The VIM-type metallo-β-lactamase gene was detected in P. aeruginosa 96347 and 96349 strains but not in the strain 96345, which correlated with the DD-test results (Fig. 5).
VIM- metallo-β-lactamase is extensively widespread in different bacteria including Pseudomonas spp., Acinetobacter spp., and members of the Enterobacteriaceae family [28]. Moreover, carbapenemase activity is one of the most common resistance mechanisms in the family Pseudomonadaceae. While carbapenemases belonging to class A (NMC/IMI, SME, and KPC) and class D (several types of OXA) are mainly detected in bacteria of the genus Acinetobacter, class B which includes zinc-depended metalloenzymes are often found in Pseudomonas spp. [29, 30]. All types of carbapenemases pose a serious threat to the therapeutic value of all β-lactam antibiotics. Particularly, the distribution of metallo-β-lactamase among the major Gram-negative human pathogens (P. aeruginosa, Acinetobacter spp., Enterobacteriaceae) represents a high clinical and epidemiological importance. Therefore, the presence of the VIM-type metallo-β-lactamase genes in the genomes of studied isolates confirmed the distribution of these enzymes among Pseudomonas sp. Moreover, this fact could allow us to suppose that the VIM metallo-β-lactamases play the leading role in the high antibiotic resistance and pathogenesis of studied CAUTI isolates.