Susceptibility of catheter-related Klebsiella pneumoniae strains to quaternary ammonium compounds under biofilm and planktonic conditions Fatemeh Monirzadeh;1 Mohammad Reza Shakibaie;1,2,3 Melika Gholamrezazadeh;1 Shalaleh Masoumi1 1 Department of Microbiology and Virology, Kerman University of Medical Sciences, Kerman, Iran 2 Research Center for Tropical Medicine and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran 3 Environmental Health Engineering Research Center, Kerman University of Medical Sciences, Kerman, Iran Corresponding author: Mohammad Reza Shakibaie Department of Microbiology and Virology Kerman University of Medical Sciences Kerman, Iran Tel.: +98 3403221660 Fax: +98 3403221671 mr_shakibaei@kmu.ac.ir ORIGINAL ARTICLE Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 INTRODUCTION Klebsiella pneumoniae is a gram-negative opportunistic pathogen, which is responsible for 10% of all hospital-acquired infections [1, 2]. This bacterium causes important diseases such as pneumonia, septicemia, urinary tract infections, wounds and intensive care unit infections in immunocompromised patients, diabetic patients, and still-born infants [3]. In recent years, the emergence of K. pneumoniae resistant to both antibiotics as well as biocides (disinfectants) has caused serious concern for infectious diseases specialists around the world [4]. It has been reported that about 60% to 70% of Klebsiella infections in hospitals are resistant to more than three classes of antibiotics [5]. Studies of pneumonia in Chinese hospitals revealed a pathogen unlike any the investigators had previously seen in healthcare settings. It was hyper-virulent K. pneumoniae harbouring various antibiotic resistance genes, a combination that causes severe, quick-developing, and deadly infections that are nearly impossible to treat with currently available drugs [6]. Both infections and antibiotic resistances in K. pneumoniae are often associated with the formation of biofilm. A biofilm is broadly considered a population of microorganisms grown on a surface or interface and embedded in a matrix of extracellular polymeric substances [7]. This will provide effective resistance ABSTRACT Background: The aim of this study was to evaluate the susceptibility of catheter-related Klebsiella pneumoniae isolates to two biocides (benzalkonium chloride and Deconex) under biofilm and planktonic conditions. Methods: A total of 85 strains of K. pneumoniae were isolated from catheters of inpatients hospitalized in four hospitals in Kerman, Iran. Susceptibility to antibiotics and biocides under biofilm and planktonic growths was performed using the microdilution method. Antibiofilm activity of the biocides was determined by microtiter assay. Biofilm eradication was carried out at different periods of time. The presence of cepA and qacEΔ1 genes were detected by polymerase chain reaction (PCR). Results: We found that 15% (n = 12) of the isolates showed strong biofilm activity, 40% (n = 35) displayed moderate activity, 30% (n = 26) demonstrated weak activity, and 15% (n = 12) showed no attachment to microtiter wells. Both the biocides had profound inhibitory activities on planktonic cells (average minimum inhibitory concentration [MIC] 0.06 ± 0.2 mg/ml for Deconex and 0.03 ± 0.1 mg/ml for benzalkonium chloride). They exerted the least antibiofilm activity at a sub-MIC concentration of 0.015 mg/ml. The isolates that formed high biofilm also harboured the cepA gene. Furthermore, a considerable increase in MIC to piperacillin/tazobactam, tetracycline, and cefotaxime was observed for cells grown in biofilm conditions for 24 hours, but all the isolates were sensitive to colistin and tigecycline. These differences were statistically significant, with a p-value of < 0.05. Most of the biofilms were eradicated from the microtiter plate within 30 minutes' exposure to these biocides. Conclusions: As the data indicates, benzalkonium chloride and Deconex have good potential as hospital disinfectants for catheter-related infections caused by K. pneumoniae in planktonic conditions. Antimicrobial stewardship programs must be performed weekly in our hospitals to improve the quality of antimicrobial use, reduce the use of antibiotics, and shorten the length of hospital stay without increasing mortality rates. KEYWORDS Klebsiella pneumoniae; biocides; biofilm; PCR; hospital hygiene Acknowledgements: We thank staff members of the Department of Microbiology at Kerman University of Medical Sciences for helping us during this research. Conflicts of interest: None. Funding: None. 209 Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 against large molecules such as antimicrobial agents, lysozyme, and biocides [8]. It has been estimated that biofilms can tolerate antimicrobial agents (disinfectants, antibiotics, surfactants) at concentrations of ten to 1,000 times that needed to inactivate genetically equivalent planktonic bacteria [8, 9]. The presence of catheters favours biofilm formation by providing an inert surface for the attachment of bacterial cells, thereby enhancing microbial colonization and the development of biofilm [10, 11]. At present, there are few studies describing K. pneumoniae isolates resistant to benzalkonium chloride and Deconex through biofilm formation [12]. Reports suggest that, in the presence of benzalkonium chloride, biofilm development of K. pneumoniae was inhibited fourfold and the compound's MIC value was reduced by one-eighth [13]. The primary aim of this study was to evaluate the susceptibility of K. pneumoniae to antibiotics, benzalkonium chloride, and Deconex under biofilm and planktonic conditions. Furthermore, we studied the prevalence of quaternary ammonium compound-resistant genes (qacEΔ1 and cepA) in these isolates. METHODS Bacterial samples and identification A total of 85 non-duplicate strains of K. pneumoniae were isolated from catheter-associated specimens of 345 patients hospitalized in four referral hospitals in Kerman (southeastern Iran). Sampling was carried out from February to November 2015. For each isolate, patient demographic data and culture site were obtained. The samples were collected from intravascular and urinary tract central catheters peripherally inserted by a trained laboratory technician that were inoculated into 5 ml Stuart transport medium (Merck Group, Darmstadt, Germany) and transferred to our lab within 24 hours of collection. The identification process was carried out based on biochemical and conventional diagnostic tests for Enterobacteriaceae, as described by Kilian and Bülow (1979) [14]. Compound sources and analysis Biocides, benzalkonium chloride as standard concentrate 50% w/v solution, under trade name BC50, and Deconex were purchased from NIPCO chemical group (Tehran, Iran). Determination of susceptibility to biocides under planktonic conditions The MIC and minimum bactericidal concentration (MBC) of Deconex and benzalkonium chloride against isolates were determined with a broth microdilution test, explained in detail in a previous report [15]. In brief, 10 ml of 32 mg/ml stock solutions of benzalkonium chloride and Deconex were prepared and diluted to 0.015 mg/ml. 50 μl of each dilution was added to 96-well microtiter plates containing 150 μl Luria-Bertani broth. To this preparation, 50 μl of the bacterial sample at 1 × 106 colony-forming units (CFU)/ml was added. To prevent bacterial aggregations, the microtiter plates were incubated under shaking conditions (100 RPM) for 24 hours at 37° C. MIC was defined as the lowest concentration of the above compounds that inhibits bacterial growth (no visible growth). A loopful of MIC was then streaked into Muller-Hinton agar and checked for grown colonies after 24 hours' incubation at 37° C. The number of colonies formed was considered the MBC. The MBC was defined as the lowest concentration of biocides killing at least 99.9% of the initial inoculums. Simultaneously, E. coli ATTC 25922 was used as a control strain. Biofilm formation under static conditions Biofilm formation was quantified by the microtiter method, as described by O'Toole et al. (2000) [9]. In this method, 1:100 diluted K. pneumoniae isolates at a concentration of 1 × 106 CFU/ml were inoculated into a 96-well microtiter plate containing 100 μl of fresh Tryptic Soy Broth (TSB) (BioMérieux, Marcy-I'Étoile, France). Growths were monitored after 24 hours' incubation at 37° C. Non-adherent cells were aseptically aspirated with pasture pipettes and washed with 300 μL of phosphate-buffered saline (PBS) solution (pH 7.2) to remove any remaining suspended cells. The biofilm was subsequently stained with the addition of 150 μL of 0.2% safranin (Merck Group, Darmstadt, Germany) for 30 minutes. The stain was removed by thorough washing, once with PBS at a pH of 7.2 and then with distilled water. The wells containing biofilm matrix were kept at room temperature or at 60° C until dry. Quantification of cells in the biofilms was carried out by solubilization of dye with 300 μL of glacial acetic acid. Optical density at 480 nm was then measured for each well [15]. All mentioned experiments were performed in duplicate. Pseudomonas aeruginosa PAO1 was used as positive control for biofilm formation study. MIC of antibiotics under planktonic and biofilm conditions Susceptibility of 12 strong biofilm-forming K. pneumoniae isolates to different antibiotics was determined under planktonic and biofilm conditions using the microdilution method [16]. In the case of planktonic conditions, the microtiter plates were incubated under shaking at 100 RPM for 18 hours. In case of biofilm, cells attached to the wall of the microtiter wells after 24 hours were scraped in 1 ml of phosphate buffer (pH 7.2). The suspensions were then diluted to 1 × 106 CFU/ml using 5 ml of D/W. 10 μl of bacterial suspensions was added to each microtiter well containing 100 μl of sterile TSB medium and 50 μL different concentrations of antibiotics (0.5-516 μg/ml). The following antibiotics were used in this study: tigecycline, colistin, ceftazidime, imipenem, amikacin, cefotaxime, cefoxitin, gentamicin, ciprofloxacin, trimethoprim/sulfamethoxazole, piperacillin/tazobactam, tetracycline, and chloramphenicol (Mast Group Ltd., Bootle, Merseyside, UK). E. coli ATCC 25922 was selected as a quality control strain in this antimicrobial susceptibility study. Biofilm inhibitory activities of Deconex and benzalkonium chloride The antibiofilm activities of Deconex and benzalkonium chloride were evaluated as described here. Briefly, microtiter plates containing 180 μl TSB medium were prepared. 10 210 Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 μL of bacterial suspension at 10 × 106 CFU/ml and 10 μl Deconex and benzalkonium chloride preparation at a subMIC concentration of 0.03 mg/ml were added to each well. The sub-MIC was defined as the highest concentration of the antimicrobial compounds that bacterial cells can grow and showing visible turbidity. The microtiter plates were kept at 37° C for 24 hours and the amount of biofilm formed in each well was measured as described above. Prior to the experiments, we measured the growth rate and viable count of each isolate in the absence and presence of biocides. The adherence efficiency was calculated as described by Stepanovíc et al. (2007) [17]. Determination of biofilm eradication time To determine biofilm eradication time, we decanted the contents of a microtiter plate with 24-hour biofilm treated with biocides for contact times of five, 15, 30, and 60 minutes. Microplate wells were then rinsed gently with sterile water and stained. The bound dye was re-solubilized in 33% glacial acetic acid. Absorbance was read as described by Leary et al. (2017) [18]. The combination of benzalkonium chloride at a concentration of 0.3 mg/ml and Deconex at a concentration of 0.06 mg/ml was also used for biofilm eradication. Detection of qacEΔ1 and cepA genes by PCR For PCR assays, the genomic DNA of each isolate was extracted using the boiling method and subjected to phenol/chloroform treatment. The cell lysate was centrifuged at 8.000 RPM for ten minutes and used to detect quaternary ammonium compounds (QACs) (qacEΔ1, cepA) genes with the PCR technique using a set of primer sequences F: 5-GCCCTACACAAATTGGGAGA-3 and R: 5-CTGCGGTACCACTGCCACAA-3 specific for the qacEΔ1 gene; and primer pairs F: 5-CAACTCCTTCGCCTATCCCG-3 and R: 5-TCAGGTCAGACCAAACGGCG-3 specific for the cepA gene [19]. The PCR assay was carried out using a temperature gradient thermal cycler (Biometra-T300, Göottingen, Germany). The amplification program consisted of an initial denaturation at 95° C for 300 seconds, 95° C for 30 cycles for 30 seconds, annealing 60° C for 35 seconds, extension at 72° C for 40 seconds, and final extension at 72° C for 300 seconds. The 100 bp DNA Ladder was used as a molecular weight standard. Statistical analysis The SPSS program version 17.0 for Windows (SPSS, Chicago, IL, U.S.A.) was used for statistical analysis. Data are presented as the mean ± standard error of the mean. For all tests, a twosided p-value < 0.05 was considered significant. RESULTS Biofilm quantification revealed that 15% of the isolates showed strong biofilm activity, 40% showed moderate activity, 30% showed weak activity, and 15% showed no biofilm activity. However, no significant antibiofilm activity of benzalkonium chloride and Deconex were detected after exposure of the isolates to a sub-MIC concentration of 0.05 mg/ml (p ≤ 0.05), as shown in Figure 1. Further analysis of the susceptibility of K. pneumoniae to antibiotics grown under planktonic and FIGURE 1: Biofilm formation and the effect of benzalkonium chloride and Deconex on biofilm in K. pneumoniae multidrug-resistant isolates. No statistical significance in the amount of biofilm masses was observed in the presence and absence of these agents. 211 Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 biofilm conditions (Figure 2) revealed a significant increase in the MIC level for the piperacillin/tazobactam, tetracycline, chloramphenicol, trimethoprim/sulfamethoxazole, and cefotaxime antibiotics for cells taken from 24 hour biofilm. Resistance to imipenem and ceftazidime increased more than fourfold; however, in the case of tigecycline and colistin, the MIC did not differ under planktonic and biofilm growths (Figure 2). These differences were statistically significant, with a p-value of < 0.05. To assess the antimicrobial activity of benzalkonium chloride and Deconex, we performed MIC and MBC tests using different concentrations of the above biocides. More than 58.8% and 35.2% of the isolates exhibited an MIC of 0.015 mg/ ml to benzalkonium chloride and Deconex under planktonic conditions, while 17.6% and 18.8% of the isolates showed an MIC of 0.03 mg/ml to the above compounds. We detected one isolate with an MBC of 1 mg/ml to benzalkonium chloride (isolate 53) and one isolate with an MIC of 4 mg/ml to Deconex (isolate 43), respectively. The results of survival curves of the planktonic cells and biofilm eradication time in the presence of biocides are illustrated in Figures 3a and 3b. Here, benzalkonium chloride at a concentration of 0.03 mg/ml and Deconex at a concentration of 0.06 mg/ml reduced bacterial population to 3 log 10 units within four hours of exposure (Figure 3a). Further exposure of planktonic cells to biocides indicated a continuous drop of viable cells with no detectable CFUs after eight hours of incubation. Furthermore, we challenged biofilms with benzalkonium chloride at concentrations of 0.03 mg/ml and Deconex at concentrations of 0.06 mg/ml, respectively. A minimum biofilm eradication time assay revealed that both biocides were not effective against preformed biofilm at the recommended time of five minutes, but that they eradicated the biofilm masses from microplate wells after 30 minutes' exposure (Figure 3b). In addition, combinations of benzalkonium chloride at a concentration of 0.03 mg/ml and Deconex at a concentration of 0.06 mg/ml had a synergistic effect and removed most of the biofilm from the microtiter wells within five minutes of incubation (Figure 3b). PCR analysis showed the presence of cepA in 15% of the isolates showing strong biofilm, while the qacEΔ1 gene was absent in this study. DISCUSSION The U.S. Centers for Disease Control and Prevention alone records nearly 560,000 catheter-related urinary tract infections in U.S. hospitals [20]. Catheterization increases the risk of developing bacteriuria by about 3% to 6% per day. Almost 50% of short-term catheterized patients acquire infections within this period, whereas the risk of infection from long-term catheters is 100% [21]. In the present study, we selectively isolated K. pneumoniae strains from catheters in four hospitals and exposed them FIGURE 2: Antibiotic susceptibility of K. pneumoniae under biofilm and planktonic conditions. MIC was defined as the lowest concentration of antibiotics that inhibits growth (visible turbidity) after 24 hours of incubation. Legend Abbreviations: CAZ: ceftazidime CTX: cefotaxime PZT: piperacillin/ tazobactam CIP: ciprofloxacin IMP: imipenem TIG: tigecycline CLS: colistin TE: tetracycline C: chloramphenicol Gm: gentamicin SXT: sulfamethoxazole/ trimethoprim 212 Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 FIGURE 3a: Survival of K. pneumoniae in the presence of benzalkonium chloride and Deconex. The above results are the average of two replicate experiments. FIGURE 3b: Eradication of biofilm in the presence of benzalkonium chloride and Deconex at different time intervals. Mean values and standard deviation of the compounds are indicated by the error bar. 213 Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 to different concentrations of Deconex and benzalkonium chloride. Both compounds act on the cell membrane, causing the dissociation of the bilayer membrane and the disruption of cell permeability. Our results show a high rate of sensitivity to these compounds by strains of K. pneumoniae (average MIC to Deconex of 0.06 ± 0.2 mg/ml and benzalkonium chloride of 0.03 ± 0.01 mg/ml). However, the compounds did not exert biofilm inhibitory activity. A recent study revealed that bacterial adherence to the benzalkonium chloride-impregnated catheters was significantly reduced compared with control catheters containing no antimicrobial agent (p < 0.01) [22]. Furthermore, we found a direct relationship between biofilm formation and high MIC values to antibiotics, which highlights how current planktonic-based antimicrobial susceptibility tests are often misleading [23]. This increased resistance occurs mainly in mature biofilms and is attributed to the formation of antibiotic-tolerant subpopulations in the deeper layers of biofilms in combination with impaired molecule diffusion [24]. A fourfold increase in MIC to imipenem in biofilm-grown cells was unexpected; Chen et al. (2014) [25] reported that imipenem displayed potent activity against established K. pneumoniae biofilms under both static and flow conditions in vitro, while Vuotto et al. (2017) [26] demonstrated that the association of biofilm production and imipenem resistance in K. pneumoniae was statistically significant. Recently, research has been directed at understanding why antibiotics do not effectively penetrate biofilms (specific antibiotics are required at higher dosages to treat biofilms) [27]. There was no significant relationship between antibiotic resistance and resistance to biocides in our study. On the contrary, many isolates were susceptible to biocides but resistant to antibiotics. We propose that hand hygiene, contact precautions, active patient screening, and disinfecting hospital equipment and catheter-related devices with these compounds at concentrations of 0.03 mg/ml can effectively reduce the transmission of infections caused by K. pneumoniae planktonic cells but not biofilm conditions. A population-based study on bloodstream infections caused by K. pneumoniae from 2000 to 2007 in Canada, a setting with low prevalence of antimicrobial resistance, showed an increase of the burden of disease during the last decade and a case fatality rate of 19% [28]. Our data also suggest that for resistance to QACs, the presence of both qacEΔ1 and cepA genes is essential. The absence of the qacEΔ1 gene led to sensitivity to these biocides. One study performed in Tehran hospitals investigated the susceptibility of 26 isolates of K. pneumoniae to a hospital biocide in the presence of qacEΔ1 and cepA genes [19]. The qacEΔ1 and cepA genes were detected in 26 and 19 biocide-resistant isolates. The results suggest that susceptibility to biocide in clinical isolates of K. pneumoniae is directly related to the presence of both qacEΔ1 and cepA genes. Similarly, it has been shown that 50%, 49%, and 53% of K. pneumoniae strains had reduced susceptibility to chlorhexidine and benzalkonium chloride. The antiseptic-resistant genes cepA and qacEΔ1 were found in 56, 34, and one isolates, respectively [19]. CONCLUSION Based on the above data, we concluded that benzalkonium chloride and Deconex antiseptics are very good at controlling infections caused by K. pneumoniae planktonic cells and could be included in the antimicrobial stewardship program of our hospitals. REFERENCES 1. Itokazu, G. S., Quinn, J. P., Bell-Dixon, C., Kahan, F. M., & Weinstein, R. A. (1996). Antimicrobial resistance rates among aerobic gram-negative bacilli recovered from patients in intensive care units: Evaluation of a national post marketing surveillance program. Clinical Infectious Diseases, 23(4), 779-84. 2. Nordmann, P., Cuzon, G., & Naas, T. (2009). The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infectious Diseases, 9(4), 228-236. doi: 10.1016/S1473-3099(09)70054-4 3. Podschun, R., & Ullmann. (1998). Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clinical Microbiology Reviews, 11(4), 589-603. 4. Henson, S. P., Boinett, C. J., Ellington, M. J., Kagia, N., Mwarumba, S., Nyongesa, S., Mturi, N., Kariuki, S., Scott, J. A. G., Thomson, N. R., & Morpeth, S. C. (2017). Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya. International Journal of Medical Microbiology, 30(7), 422-429. doi: 10.1016/j.ijmm.2017.07.006 5. Bi, W., Liu, H., Dunstan, R. A., Li, B., Torres, V. V. L., Cao, J., Wilksch, J. J., Strugnell, R. A., Lithgow, T., & Zhou, T. (2017). Extensively drugresistant Klebsiella pneumoniae causing nosocomial bloodstream infections in China: Molecular investigation of antibiotic resistance determinants, informing therapy, and clinical outcomes. Frontiers in Microbiology, 30(8), 1230. doi: 10.3389/fmicb.2017.01230 6. Yao, H., Qin, S., Chen, S., Shen, J., & Du, X. D. (2018). Emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae. Lancet Infectious Diseases, 18(1), 25. doi: 10.1016/S1473-3099(17)30628-X 7. Shakibaie, M. R. (2018). Bacterial biofilm and its clinical implications. Annals of Microbiology and Research, 2(1), 45-50. 8. Ishida, H., Ishida, Y., Kurosaka, T., Otani, T., Sato, K., & Kobayashi, H. (1998). In vitro and in vivo activities of levofloxacin against biofilm-producing Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 42(7), 1641-1645. 9. O'Toole, G., Kaplan, H. B., & Kolter, R. (2000). Biofilm formation as microbial development. Annual Review of Microbiology, 54, 49-79. Retrieved from https://www.annualreviews.org/doi/10.1146/annurev. micro.54.1.49 10. Stahlhut, S. G., Struve, C., Krogfelt, K. A., & Reisner, A. (2012). Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunology and Medical Microbiology, 65(2), 350-359. 11. Jefferson, K. K. (2004). What drives bacteria to produce biofilm? FEMS Microbiology Letters, 236(2), 163-173. Retrieved from https://academic. oup.com/femsle/article/236/2/163/535288 12. Gainor, B. J., Hockman, D. E., Anglen, J. O., Christensen, G., & Simpson, W. A. (1997). Benzalkonium chloride: A potential disinfecting irrigation solution. Journal of Orthopaedic Trauma, 11(2), 121-125. 13. Houari, A., & Di Martino, P. (2007). Effect of chlorhexidine and benzalkonium chloride on bacterial biofilm formation. Letters in Applied Microbiology, 45(6), 652-656. Retrieved from https://www.ncbi.nlm.nih. gov/pubmed/17944843 14. Kilian, M., & Bülow, P. (1979). Rapid identification of Enterobacteriaceae. II. Use of a beta-glucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples. Acta Pathologica et Microbiologica Scandinavica. Section B, Microbiology, 87(5), 271-276. 15. Gholamrezazadeh, M., Shakibaie, M. R., Monirzadeh F., Masoumi, S., & Hashemizadeh, Z. (2018). Effect of nano-silver, nano-copper, deconex 214 Canadian Journal of Infection Control | Winter 2018 | Volume 33 | Issue 4 | 219-215 and benzalkonium chloride on biofilm formation and expression of transcription regulatory quorum sensing gene (rh1R) in drug-resistance Pseudomonas aeruginosa burn isolates. Burns, 44(3), 700-708. doi: https://doi.org/10.1016/j.burns.2017.10.021 16. Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy, 48(Suppl S1), 5-16. 17. Stepanović, S., Vuković, D., Hola, V., Di Bonaventura, G., Djukić, S., Ćirković, I., & Ruzicka, F. (2007). Quantification of biofilm in microtiter plates: Overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS, 115(8), 891-899. doi: https://doi.org/10.1111/j.1600-0463.2007.apm_630.x 18. Leary, J. T., Werger, M. M., Broach, W. H., Shaw, L. N., Santoni, B.G., Bernasek, T.L., & Lyons, S. T. (2017). Complete eradication of biofilm from orthopedic materials. Journal of Arthroplasty, 32(8), 2513-2518. doi: 10.1016/j.arth.2017.03.050 19. Azadpour, M., Nowroozi, J., Goudarzi, G. R., & Mahmoudvand, H. (2015). Presence of qacEΔ1 and cepA genes and susceptibility to a hospital biocide in clinical isolates of Klebsiella pneumoniae in Iran. Tropical Biomedicine, 32(1), 109-115. 20. Desai, D. G., Liao, K. S., Cevallos, M. E., & Trautner, B. W. (2010). Silver or nitrofurazone impregnation of urinary catheters has a minimal effect on uropathogen adherence. Journal of Urology, 184(6), 2565-2571. doi: 10.1016/j.juro.2010.07.036 21. Ortega, M., Marco, F., Soriano, A., Almela, M., Martınez J. A., Pitart, C., & Mensa, J. (2013). Epidemiology and prognostic determinants of bacteraemic catheter-acquired urinary tract infection in a single institution from 1991 to 2010. Journal of Infection, 67(4), 282-287. doi: 10.1016/j.jinf.2013.06.003 22. Tebbs, S. E., & Elliott, T. S. (1993). A novel antimicrobial central venous catheter impregnated with benzalkonium chloride. Journal of Antimicrobial Chemotherapy, 31(2), 261-271. 23. Brady, A. J., Laverty, G., Gilpin, D. F., Kearney, P., & Tunney. M. (2017). Antibiotic susceptibility of planktonicand biofilm-grown staphylococci isolated from implant-associated infections: Should MBEC and nature of biofilm formation replace MIC? Journal of Medical Microbiology, 66(4), 461-469. doi: 10.1099/jmm.0.000466 24. Ito, A., Taniuchi, A., May, T., Kawata, K., & Okabe, S. (2009). Increased antibiotic resistance of Escherichia coli in mature biofilms. Applied and Environmental Microbiology, 75(12), 4093-4100. 25. Chen, P., Seth, A. K., Abercrombie, J. J., Mustoe, T. A., & Leung, K. P. (2014). Activity of Imipenem against Klebsiella pneumoniae biofilms in vitro and in vivo. Antimicrobial Agents and Chemotherapy, 58(2), 12081213. Retrieved from https://aac.asm.org/content/aac/58/2/1208.full.pdf 26. Vuotto, C., Longo, F., Pascolini, C., Donelli, G., Balice, M. P., Libori, M. F., Tiracchia, V., Salvia. A., & Varaldo, P. E. (2017). Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains. Journal of Applied Microbiology, 123(4), 1003-1018. Retrieved from https:// onlinelibrary.wiley.com/doi/abs/10.1111/jam.13533 27. Kostakioti, M., Hadjifrangiskou, M., & Hultgren, S. J. (2013). Bacterial biofilms: Development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harbor Perspectives in Medicine, 3(4), a010306. doi: 10.1101/cshperspect.a010306 28. Meatherall, B. L., Gregson, D., Ross, T., Pitout, J. D., & Laupland, K. B. (2009). Incidence, risk factors, and outcomes of Klebsiella pneumoniae bacteremia. American Journal of Medicine, 122(9), 866-873. doi: 10.1016/j.amjmed.2009.03.034