Esra’a Jebreel Ibrahim Abu-Shoura¹, Shuaibu Abdullahi Hudu^1* and Tasneem Farouq AL- Quadan²

¹Department of Basic Medical and Dental Sciences, Faculty of Dentistry, Zarqa University, Zarqa, Jordan.

²Department of…University of Louisville, School of Medicine, 500 S Preston St, Louisville, KY, United States.

Corresponding Author E-mail: shudu@zu.edu.jo

Abstract

Background The biofilm phenomenon represents a prevalent mode of microbial life in nature which is characterized by cells irreversibly attaching to surfaces or each other and getting embedded in a matrix of extracellular polymeric substances. Object This study aimed to identify and characterize the genes associated with the common bacterial species responsible for biofilm formation in the catheters of hospitalized patients. Method Different bacterial strains were collected from catheterised patients at three local Jordanian hospitals for biofilm formation. The isolates were identified using Gram stain and Remel Rapid test. Biofilm formation was detected using the Tube method and tissue culture plate method. Result The presence of fimA and csgD genes was detected by polymerase chain reaction (PCR). Gram-negative bacteria species were isolated on the urethral catheters and the result shows the majority of the isolates were E. coli (40%), followed by K. pneumonia (27%). In comparison, the least was Citrobacter sp (2.6%). Similarly, some Gram-positive bacteria were also identified such as Staphylococcus aureus (4%) and Staphylococcus epidermidis (2.6%). K. pneumonia is strongly associated with biofilm formation (45%) followed by E. coli (33%). Conclusion Biofilm-associated genes, fimA and csgD were detected in all biofilm-positive samples containing the F plasmid, while csgD was detected in all biofilm-negative samples. Biofilm formation tends to be a prevalent process in patients included in this study and may cause dangerous complications in the patients especially in the elderly due to prolonged catheterization periods.

Keywords

Biofilm; Catheter; Genes; Jordan; Urinary infection

Copy the following to cite this article: Abu-Shoura E. J. I, Hudu S. A, AL- Quadan T. F. Exploring Genetic and Phenotypic Factors Contributing to Urethral Catheter Biofilm Formation in Hospitalised Patients in Jordan. Biomed Pharmacol J 2024;17(2).

Copy the following to cite this URL: Abu-Shoura E. J. I, Hudu S. A, AL- Quadan T. F. Exploring Genetic and Phenotypic Factors Contributing to Urethral Catheter Biofilm Formation in Hospitalised Patients in Jordan. Biomed Pharmacol J 2024;17(2). Available from: https://bit.ly/3VRTnTE

Introduction

Biofilm is a complex microbial system consisting of microorganisms attaching to surfaces; these microorganisms are embedded in a protective matrix characterized as a polysaccharide matrix produced by alginate polymerases ¹. Bacteria form microcolonies and surround themself with an extracellular polymeric substance (EPS), between these colonies water-filled channels control the influx and efflux of nutrients and waste ². In nature, bacteria live in polymicrobial ecosystems either as free planktonic floating cells suspended in a liquid medium or as a sessile community attached to surfaces³. Most bacteria tend to live in the attachment sessile mode attached to either biotic or abiotic surfaces such as aquatic systems living tissues, indwelling medical devices and industrial water system piping under certain circumstances such as pH, nutrient level, ionic strength and temperature⁴. Biofilm diseases are mainly associated with implant medical devices like catheters, valves, and joint prostheses⁵. However sometimes bacteria don’t need a device to form a biofilm layer; for example, lung infections, oral cavity infections, fibrosis and contact lenses could be associated with biofilm formation⁶. Usually, biofilm is associated with nosocomial infection which originates from the skin of the patients, health care workers and tap water. More than 35 % of nosocomial infections in the USA are urinary tract infections ⁷. Urinary Tract Infection (UTI) is the most common acquired infection by microbial invasion of the genitourinary tract ⁸. The infection extends from the urethra to the bladder until it arrives in the renal cortex of the kidneys. The risk of this type of infection increases with catheter usage; since catheters get intensively colonized with bacteria, this allows the bacteria to reach the bladder causing the infection more than the ordinary UTI ⁹. The bacteria can cause severe damage to the bladder mucosal tissue the enhanced invasion of the bacteria over time causes kidney failure ¹⁰. Catheter-acquired infection is classified according to the duration of catheterization in situ from long-term which is assumed to be more than 30 days, while the short-term is less than 30 days ¹¹. UTI Patients with short-term indwelling catheters usually acquire asymptomatic infection, however, the infection develops dramatically once catheterization lasts more than a month. Many diseases are associated with long-term catheterization term such as kidney renal inflammation, acute prostatitis and renal stones ^11,12. Whenever the catheter could be removed the physician should make this decision, the catheter must be the last choice.

Studying biofilm formation is very important, especially in the medical field; the importance stems from the necessity to avoid the complications of biofilm formation. In the Middle East, more than 8%   of infections are nosocomial infections according to the World Health Organization which are related to the decline in health workers’ hygiene or other contamination sources¹³. More than 40% of these infections are acquired urinary tract infections associated with catheterization^13,14. Many diseases are related to biofilm formation such as cystic fibrosis, otitis media and urinary tract infection. Biofilm is formed by several types of microorganisms such as Escherichia coli, Klebsiella pneumoniae, Candida albicans and Staphylococcus aureus ¹⁵. This study aimed to identify the common bacterial species responsible for biofilm formation in the catheters of hospitalized patients and determine which genes in these organisms are partially responsible for biofilm formation.  

Methodology

To investigate the biofilm formation, urine catheters were collected from three governmental hospitals in Jordan; AL-Basher hospital, Prince Faysal Hospital and Zarka Governmental Hospital, the catheterisation period was at least four days.

Sample Identification

The urine catheters were collected directly from patients; the tip of the catheter was cut with a sterile sharp blade and scissors and placed directly in a sterile urine cup. The interior of the collected catheters was scraped with cotton swabs and cultured in enrichment media such as Luria-Bertani (LB) broth and was incubated at 37ºC for 24 hr. The suspensions were routinely cultured on Mac Conkey agar and blood incubated at 37ºC for 24h. Bacteria were classified into Gram-positive or Gram-negative using the Gram stain method as described previously¹⁶. Gram-negative bacteria were identified using a special kit (RemelRapID one) which consists of 18 biochemical reaction wells; the bacterial diluted sample reacts with each reagent. All bacteria were preserved in 20% glycerol at – 20 ºC until further use.

Biofilm Formation Assay.

The biofilm assay was performed in two methods. Tube Method and Tissue Culture Plate and the following steps explain both procedures as described previously ^17-19.

Growing a Film

Bacteria were growing in enrichment media (LB) overnight.The overnight suspension was diluted 1:100 into fresh media, 100 µl of the suspension was added to 96 well plate and 1ml of the suspension was added to the test tube; both the tubes and plate were incubated for 24 hrs. at 37 ºC.

Staining the Biofilm

After incubation, the plate and the tubes were washed with water two times to discard the planktonic bacteria. 125 µl of 0.1% of crystal violet (CV) was added to the microtiter plate for staining, and 1ml+50µl 1% of CV was added to test tubes for tube staining both were incubated for 15 min. After that, the microtiter plate and the tubes were rinsed 3 times with water to rid the plate of excess cells and dye.The plate and tubes were dried for a few hours or overnight.

Quantifying the biofilm

 To quantify the biofilm adhesion rate, 30% acetic acid was added to each tube for 10 min to solubilize the CV staining. The optical density of the adherent stained biofilm was measured at 570 nm. 

Genomic DNA Extraction  

Bacteria were grown in 5 ml of LB media at 37°C for one night. After centrifuging the culture at 14,000 rpm for one minute, the supernatant was thrown away and the pellet was kept. It was mixed with 500 µl of processing buffer (10 µM Tris-HCL, 10 µM EDTA, 50M NaCl, and 2% SDS) and 4 µL of proteinase K. After that, this blend was kept in a water bath at 56°C for two hours. The samples were spun at 14000 rpm for 3 minutes after being left to sit for a while. Another mix of 500 µl of processing buffer, 4 µl of proteinase K, and 7 µl of dithiothenitol (DTT) was added to the pellet. The mixture was then left to sit overnight at 56°C.

After being left to sit overnight, 500 µl of phenol was added to each tube. The tubes were then left at room temperature for 10 minutes before being centrifuged at 300 rpm for 5 minutes. After the supernatant was made, it was put in a new tube and 50 µl of 3M sodium acetate and 1 ml of cold isopropanol were added. After being kept at -20°C for 20 minutes, this mixture was spun at 3000 rpm for 10 minutes. Carefully, the supernatant was taken away, and 500 µl of 70% ethanol was used to wash the pellet. The ethanol was thrown away after another 3 minutes of spinning at 3000 rpm, and the pellet was left to dry in the air. Finally, the pellet was mixed again in 50 µl of Tris/EDTA buffer (TE buffer), and 0.8% agarose gel electrophoresis was used to look at the DNA that had been recovered.       

Plasmid DNA Extraction.  

A commercial kit from OMEGA bio-tek was used to get plasmid DNA from E. coli. To begin, E. coli samples were grown in 5 ml of LB broth in 10 ml tubes to make sure they had enough air. The tubes were then heated to 37oC and shaken for 12 to 16 hours. After the incubation time, the cultured broth was centrifuged at 10,000 rpm for 10 minutes to separate the bacterial cells in the pellet from the supernatant. The pellet was kept and the liquid was thrown away. After that, 250 µl of solution I (RNase A solution) was added to the pellets. Then, 250 µl of solution II was added. 350 µl of solution III was added to this mixture, and it was mixed well until a white residue formed. After that, the mixture was spun at 13,000 rpm for 10 minutes. After getting the clear residue, it was put on a DNA mini-column and spun at 13,000 rpm for 10 minutes. The column was then turned on and 700 µl of alcohol was added. It was then spun at 13,000 rpm for 2 minutes. The last step was to add 50 µl of elution solution to the column. It was then spun at 13,000 rpm for 2 minutes. After the plasmid DNA was removed, it was tested using 1% agarose gel electrophoresis.

Polymerase Chain Reaction (PCR) for Gene Detection

Polymerase Chain Reaction (PCR) was performed to determine the presence or absence of certain genes known to be responsible for biofilm formation. The bacterial genera selected for PCR were E. coli and K. pneumonia. Two genes were selected for E. coli (fimA and csgD), and one (mrkD) for K. pneumonia based on previous reports ^20,21. E. coli PCR condition and protocol; 5µL of 1.9 µg/µl DNA plasmid extract or 5.08 µg/µl  DNA genomic extract of  E. coli isolated were added to 12.5 µL of 2_× master mix, 1 µL of forward primer (100µM) and 1 µL of reverse primer (100 µM ) (Table 1), 5.5 µL H₂O in a total volume of 25 µL. DNA amplification was performed according to the following conditions in Bio-Rad thermocycler; 94 ºC for 2 min, followed by 35 cycles of 20s at 94 ºC, 30s at 56ºC, 45s at 72 ºC, and final extension for 5 min at 72 ºC. The PCR amplicons as shown in Table 1 were examined on 2% agarose gel electrophoresis. DNA amplification was performed according to the following conditions in the (Bio-Rad) thermocycler. 94 ºC for 2 min, 10 initial cycles at 94 ºC for 10s, 30s at 63 ºC and 15 min at 68 ºC, followed by 20 cycles of 10s at 94 ºC, 30s at 63 ºC and 15 min plus 20s for each new cycle at 72 ºC, the final elongation step was 7 min at 72 ºC. PCR were amplicons examined on 2% agarose gel electrophoresis.

Table 1: Primes nucleotides sequences used in PCR amplification of E. coli fim A and csgD gene^22,23

Organism	Primers	Amplicon Size	Reference
E. coli	fim A F: 5′-GTTAGGACAGGTTCGTACCGCAT-‘3 fimA R: 5′-AAATAACGCGCCTGGAACGAATG-‘3	315bp	22
	csgD F: 5′-CGCGAATTCTCGCTGGCAATTACAGG-‘3 csgD R: 5-‘CGCGGATCCGCTGATGAACAACGAAC-‘3	480bp	23

 

Ethical Approval

The study was approved by the Hashemite University Institutional Review Board (IRB) on 20/05/2014, session number (2014/2013/7/36) with reference No. 1404123/440/1

Results

Urine Catheter culture

 One hundred and thirty-five (135) samples were collected from patients’ urinary catheters. Bacterial growth was observed in 99 (73%) out of the 135 isolates. The 99 isolates were classified into Gram-positive and Gram-negative based on Gram stain and biochemical tests, 24 samples out of 99 showed mixed growth as such they were excluded from the study. The (RemelRapID ONE) kit was used to identify the different genera. Nine different Gram-negative and Gram-positive bacterial genera were isolated from the tips of the catheters. E. coli was the major pathogen in the three Jordanian hospitals 30/75 (40%), followed by K. pneumonia 20/75 (27%), Pseudomonas 7/75 (9%), Enterobactersp 6/75 (8%), Proteus sp 3/75 (4%), S. aureus 3/75 (4%), Shigellasp 2/75 (2.6%), Citrobactersp 2/75 (2.6%), and S. epidermidis 2/75 (2.6%), 43/75 (55%) of isolated samples were isolated from female patients.

Biofilm Detection Assay

All samples were applied to the biofilm colourimetric assay, and classified as non-adherent, weakly, moderately and strongly. The cut-off OD was the negative control (ODc = 0.129 nm), the OD of the non-adherent ≤ ODc. Weakly adherent sample OD should be ODc ˂ OD ≤ 2×ODc (0.258). The moderate adherent samples OD should be 2× ODc ˂ OD ≤ 4× ODc (0.516). The strongly adherent sample OD should be 4×ODc ˂ OD (0.516), and E. coli DH5α used as a negative control. The OD values using tissues culture plate (TCP) showed that E. coli 11/30 (33%) tend to form biofilm. In K. pneumonia 12/20 (60%) of samples have the strong to moderate ability to form the biofilm.

Table 2: Shows the different isolates’ biofilm formation ability using the tube method and Tissue Culture Plate method.

Biofilm formation	Strongly adherent (+++)		Moderate adherent (++)		Weakly adherent (+)		Non-adherent (0)		Total
	TM	TCP	TM	TCP	TM	TCP	TM	TCP
E. coli	5	5	10	6	4	4	11	15	30
K. pneumonia	9	10	7	2	1	4	3	4	20
Pseudomonas sp	–	4	–	0	–	0	–	3	7
Proteus sp	–	2	–	0	–	0	–	1	3
Shigella sp	–	0	–	0	–	0	–	2	2
Cirtrobacter sp	–	0	–	0	–	0	–	2	2
Enterobacter sp	–	2	–	0	–	0	–	4	6
S. aureus	–	2	–	0	–	1	–	0	3
S. epidermidis	–	1	–	0	–	0	–	1	2

TM: tube method TCP: tissue culture plate

PCR for Biofilm Formation Genes

In this study, two genes are directly involved in biofilm formation (fimA and csgD). All E. coli samples that were phenotypically positive for biofilm formation contained (fimA, csgD) genes; fimA was detected in the plasmid, as shown in Figure 1, while csgD was detected in the genomic DNA, as shown in Figure 2. A positive control confirmed the gene amplification for fimA and csgD genes of E. coli ATCC 25922. The products were analyzed using electrophoresis, and the gel results are shown in Figure 3. The phenotypic and genotypic characteristics of   E. coli and K. pneumonia are summarized in Table 3.

Figure 1: PCR Amplification products of fimA gene. Amplified products were analyzed by electrophoresis on 2% ethidium bromide-stained agarose gel. Lane L:   Click here to view Figure

 

Figure 2: PCR Amplification products of csgD. Amplified products were analyzed by electrophoresis on 2% ethidium bromide-stained agarose gel. Lane L:   Click here to view Figure

 

Figure 3: PCR Amplification products of fim A and csgD. Amplified products were analyzed by electrophoresis on 1% ethidium bromide-stained agarose gel. Lane L:   Click here to view Figure

 

Table 3: E. coli and K. pneumonia phenotypic and genotypic characteristics

Types of E. coli Serotype	Biofilm (TM)	Biofilm (TCP)	F-Plasmid	fimA	csgD
01	+++	+++	+	+	+
02	++	++	+	+	+
03	++	++	+	+	+
04	++	+	+	+	+
05	++	++	+	+	+
06	+++	+++	+	+	+
07	+++	+++	+	+	+
08	+++	+++	+	+	+
09	++	+	+	+	+
10	++	++	+	+	+
11	++	++	+	+	+
12	++	++	+	+	+
13	++	+	+	+	+
14	+++	+++	+	+	+
15	++	+	+	+	+
16	+	–	+	+	+
17	+	–	+	+	+
18	+	–	+	+	+
19	+	–	+	+	+
20	–	–	+	+	+
21	–	–	–	–	+
22	–	–	–	–	+
23	–	–	–	–	+
24	–	–	–	–	+
25	–	–	–	–	+
26	–	–	–	–	+
27	–	–	–	–	+
28	–	–	–	–	+
29	–	–	–	–	+
30	–	–	–	–	+
Types of K. pneumonia Strain	Biofilm (TM)	Biofilm (TCP)	F-Plasmid	fimA	csgD
Kp1	++	++	–	–	–
Kp 2	++	+	–	–	–
Kp 3	++	+	–	–	–
Kp 4	+++	+++	–	–	–
Kp 5	++	++	–	–	–
Kp 6	++	+	–	–	–
Kp 7	++	+++	–	–	–
Kp 8	+++	+++	–	–	–
Kp 9	+++	+++	–	–	–
Kp 10	++	+	–	–	–
Kp 11	+++	+++	–	–	–
Kp 13	+++	+++	–	–	–
Kp 14	+++	+++	–	–	–
Kp 15	+++	+++	–	–	–
Kp 16	+++	+++	–	–	–
Kp 17	+	–	–	–	–
Kp 18	–	–	–	–	–
Kp 19	–	–	–	–	–
Kp 20	–	–	–	–	–

Kp: Klebsiella pneumonia‘ TM: tube method TCP: tissue culture plate (+): strength of adherence, (-): No adherence

Discussion

Bacterial adhesion has been considered a virulence factor contributing to infections associated with indwelling medical devices, particularly catheters. This ability to form the biofilm helps to resist the host’s immune response and is considered the main factor responsible for chronic infection. In this study, we investigated the ability of isolates to form biofilm. It included 75 specimens, both Gram-positive and Gram-negative bacteria. This study found that E. coli is the most prevalent organism (40%), followed by K. pneumonia (26%) associated with catheter-associated urinary tract infection, which is in agreement with the findings of other researchers ^24-26. More than 50% of the samples were isolated from the ICU, indicating that patients with serious diseases are more affected by nosocomial acquired urinary tract infections, as reported previously ^27-29.

In this study, the samples were collected from both males and females, and we found that more than 57% of the isolates were from female patients. This might not be unrelated to the fact that UTI is more common in females than males due to their short urethra ^30-32. Catheter insertion should be done under sterile conditions using iodine or specific insertion gel, and in the case of female patients positioning of the catheter needs to be checked to ensure that it is correctly positioned in the urethra. During the insertion procedure, the bacteria could get access to the urinary tract even with sterilization. Male patients are not subjected to this process; moreover, using condom catheters for males decreases the catheterization-associated infection^33,34. Studies showed that E. coli is still heading the urinary tract infection causative list ^35,36 which is in line with the findings of this study. On the other hand, the number of Gram-positive isolates was very low which indicates that the contamination sources were not related to the skin of health workers or patients, Staphylococcus epidermidis constituted only 2.5% the of isolates which concurs with previous studies ^37,38. Fifty-seven per cent of the isolates were biofilm producers while 43% of the isolates were non-biofilm producers, 45% of K. pneumonia isolates were strong biofilm producers and 33 % of E. coli isolates were strong biofilm producers.

The ability of isolates to form biofilm indicates that patients in Jordanian hospitals are exposed to biofilm formation complications just like patients in other health settings globally ^25,39,40. Since E. coli and K. pneumonia were the most frequently isolated species, we investigated the presence of certain genes which are known to be involved in biofilm formation. These genes fimA and csgD are regulatory genes for pili and curli production which are involved essentially with biofilm formation in E. coli ^41-43. In this study, the fimA gene and csgD genes were detected in all E. coli biofilm-producing isolates, which confirmed that the catheter-isolated organisms can form biofilm; and more than 50% of the patients were under antibiotic treatment but were not responding to it. Thus, patients would suffer from catheter-acquired infection complications which include fever, urine burning, back pain, and pyelonephritis and it could ultimately lead to kidney failure septicemia or even death^24,44,45. Antibiotics fight side by side with the immune system to overstep the bacterial infection and survive without any server damage, thus any paralysis of the antimicrobial agent job would weaken the body effort. Only 26% of the collected samples were free of bacterial growth which indicates that nosocomial infection shows a prevalent mode and this in turn increases the risk of indwelling urinary catheter-related disease^45-48.

Conclusion

In Jordanian hospitals, E. coli was the most common infectious organism followed by K. pneumonia. The biofilm formation process represents a prevalent mode in Jordanian patients included in this study. The conjugative bacteria which have an F plasmid tend to form a multilayer biofilm while non-conjugative can’t form the biofilm even if the genes are present in another plasmid. Thetime and csgD adhesion genes were detected in all phenotypically positive for biofilm in the presence of optimum environmental conditions. The key to controlling the biofilm formation phenomenon is applying a prevention system in hospitals by controlling the environmental conditions to protect patients by providing them with probable nutrition, vaccination, and antimicrobial agents. Educating the health workers including doctors, nurses, therapists and the cleaning crew is important to controlling the nosocomial infection. This education must include: what is meant by biofilm, its complications, what the preventive actions should be done, what is the corrective action if biofilm was detected and what is the personal and economical benefit that we can gain in preventing such process. However, this study is limited by its sample size and details of antibiotic resistance profiles, which could be crucial for understanding treatment challenges.

Acknowledgement

The researchers appreciate Zarqa University for supporting this research publication

Conflict of interest

Authors declare no conflict of interest

Funding Source

No funding was received for this study

Authors’ contributions

Data curation: EIA; Formal analysis: SAH; Methodology: EIA, SAH; Project administration: TFA; Writing original draft: EIA, TFA; Writing review & editing: SAH, TFA, EIA; All authors approved the final submission.

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