Investigation of Antibiotic Resistance and Biofilm Formation in Clinical Isolates of Klebsiella pneumoniae

Aim Klebsiella pneumoniae (K. pneumoniae) is an encapsulated Gram-negative bacterium that can lead to 14–20% of nosocomial infections. The ability of biofilm formation in this bacterium decreases the host immune response and antibiotic efficacy. This may impose a huge impact on patients and healthcare settings. This study aimed to evaluate the antibiotic resistance pattern and biofilm formation in K. pneumoniae strains isolated from two major Hamadan hospitals, west of Iran. Methods A total of 83 K. pneumoniae strains were isolated from clinical samples of patients in different wards of Hamadan hospitals from September 2018 to March 2019. Determination of antimicrobial susceptibility was performed using the disk diffusion method. Biofilm formation was evaluated by the crystal violet method. Data were analyzed by the SPSS software and chi-square test. Results The results showed that clinical samples included 18 urinary tract samples (22%), 6 wound samples (7%), 6 blood samples (7%), 17 tracheal tube aspiration samples (20%), 32 throat cultures (38%), 2 sputum samples (2.5%), and 2 abscess drain cultures (2.5%). High-level resistance to cefotaxime was detected in 92%, and all of isolates were susceptible to colistin. Biofilm formation was seen in 62 (75%) isolates. Strong biofilm formation was observed in 17 (20%) strains. A significant correlation was seen between biofilm formation and antibiotic resistance (P value <0.05). Conclusion Our findings emphasize the need for proper diagnosis, control, and treatment of infections caused by K. pneumoniae especially in respiratory tract infections due to the strong biofilm formation and high antibiotic resistance in these strains.


Introduction
Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative, encapsulated, nonmotile, rod-shaped bacterium and an important member of Enterobacteriaceae which can lead to various infections including gastrointestinal, skin, nasopharyngeal, osteomyelitis, biliary and urinary tract infections, and bacteremia. K. pneumoniae virulence factors including polysaccharide capsule which protects against bactericidal serum factors, type I and III pili that adhere to the surfaces, adhesins, and siderophore, play a key role in its pathogenicity [1,2]. K. pneumoniae strain is more common in immunocompromised individuals such as diabetics and the elderly and children and widely colonized in hospitalized patients [3]. Urinary tract infection (UTI) is known to be the most prevalent type of nosocomial infection, and K. pneumonia is the second cause of urinary tract infections among other Gram-negative bacteria [4]. e increasing emergence of multidrug-resistant (MDR) bacterial pathogens is known as a major public health challenge worldwide [5]. e overuse of antibiotics has resulted in difficulties in the treatment of K. pneumoniae and limitations in our available options for effective management of this bacterial infection [4][5][6]. In Gram-negative bacteria, beta-lactam resistance has been detected through chromosomal or plasmid genes, but in clinical specimens, resistance is usually dependent on plasmid R genes [7,8]. Extended-spectrum β-lactamase (ESBLs), part of group A beta-lactamases, can lead to the hydrolysis of broad-spectrum cephalosporin and lead to resistance to penicillin and cephalosporins. However, they can be inhibited by beta-lactamase inhibitors such as clavulanic acid [9,10]. Previous studies have shown that K. pneumoniae strains that are resistant to a broad spectrum of antibiotics are rapidly expanding, especially when the bacteria are capable of forming biofilm [2]. ese bacteria can form a thick layer of extracellular biofilm which helps them attach to living and abiotic surfaces [11]. Treatment of infections caused by biofilm-forming K. pneumoniae strains is more difficult than other strains [12]. e antibiotic resistance and bacterial tendency to biofilm formation may play a key role in the emergence of MDR-K. pneumoniae strains [13]. Due to the antiphagocytic feature of biofilm, it is more challenging for the host immunity to eliminate this kind of bacterial pathogens [14]. e use of antibiotics in patients with bacterial infections can lead to bacterial elimination and accelerate the treatment process. Consequently, the increasing incidence of drug resistance causes complications in patients and higher medical costs [15]. is study was designed to investigate the biofilm formation and antibiotic resistance patterns in clinical isolates of K. pneumoniae isolated from Hamadan hospitals, west of Iran.

Methods
is study was performed on 83 K. pneumoniae strains isolated from clinical samples of patients with various infections in different wards of Hamadan hospitals from September 2018 to March 2019. e isolates were identified as K. pneumoniae, using conventional microbiological tests [16]. After recognition, the K. pneumoniae strains were stored in trypticase soy broth (TSB) containing 18% glycerol at −70°C.
SPSS software (version 22) was used for statistical analysis. Chi-square analysis was used for comparisons between the capacity of biofilm production and antibiotic resistance.
According to antibiogram results of 83 clinical isolates of K. pneumoniae, the highest antibiotic resistance was related to cefotaxime (92%), and all isolates were susceptible to colistin. After cefotaxime, the most resistance was to piperacillin-tazobactam (91%) and ampicillin-sulbactam (87%). Resistance to levofloxacin and tobramycin was detected in 85% of isolates. e prevalence of resistance to ceftazidime, trimethoprim-sulfamethoxazole, amikacin, ciprofloxacin, meropenem, imipenem, gentamicin, and ceftriaxone are shown in Figure 1. Different antibiotic resistance patterns were detected in various clinical samples, for example, the highest resistance to amikacin, ceftazidime, and gentamicin was found in wound and blood samples and the highest antibiotic resistance to trimethoprim-sulfamethoxazole, tobramycin, cefotaxime, piperacillin-tazobactam, ampicillin-sulbactam, and meropenem was observed in urine samples. Table 1 provides the prevalence of antibiotic resistance in different clinical specimens. A significant correlation was seen between the type of specimen and antibiotic resistance (P value <0.05). All K. pneumoniae isolates were susceptible to colistin, and the colistin MICs of 63 (75%), 11 (%13), and 9 (12%) of K. pneumoniae isolates were 0.125 µg/ml, 0.5 µg/ml, and 1 µg/ml, respectively.
Our findings indicate that 62 (74.5%) K. pneumoniae isolates formed biofilm. e strains were classified into four categories as described above. Based on the biofilm analysis, 27 (32.5%) K. pneumoniae isolates formed biofilm weakly, 18 (21.6%) isolates created moderately, and 17 (20.4%) isolates were strong producers of biofilms. Among different clinical specimens, the lowest biofilm formation was seen in drainage abscess, and sputum culture and endotracheal aspiration samples had the highest biofilm formation. Additionally, blood culture samples formed biofilm weakly more than others and aspiration of the endotracheal tube showed the highest moderate biofilm formation. A significant correlation was seen between the type of clinical sample and the rate of biofilm formation (P value � 0.000), and the results are given in Table 2. Statistical analysis showed that there is a meaningful correlation between biofilm formation and resistance to antibiotics (P value <0.05). e results are given in Table 3.

Discussion
In this current research, the ability of biofilm formation in K. pneumoniae isolates obtained from the clinical specimens and the correlation between the strength of biofilm formation and patterns of antibiotic resistance, sites of infection, or type of clinical samples were assayed. Our findings indicated that high percentages (74.5%) of K. pneumoniae isolates were able to form biofilms, and 20.4% of them formed biofilm strongly. It has already been determined that the formation of biofilms by bacteria is linked to the development of infections associated with implants and catheters which can even threaten the lives of patients with cystic fibrosis and chronic wound infections [22]. K. pneumoniae is now considered a biofilm-forming bacterium that can lead to nosocomial opportunistic infections and also affects the efficacy of antibiotic treatments [23,24].
ere are different findings of the correlation between biofilm formation and site of infection. Yang and colleagues conducted an investigation on biofilm formation by K. pneumoniae strains isolated from blood samples, wounds, swabs, urine, and sputum samples. eir study indicated that 62.5% of the isolates generated biofilms, which is less than our results [24]. Seifi et al. in Iran reported that 93.6% of K. pneumoniae isolates had the ability of biofilm formation and 33% of them could produce biofilm strongly which is more than the results of our study. ey also reported that strong biofilms were more common in wound and sputum specimens, while in our study, the samples collected from the tracheal tube were able to form stronger biofilms, and sputum specimens formed weaker biofilm compared to them [11]. Diversities in the results of different studies can be related to the geographical area, type, and number of samples or characteristics of bacterial isolates, including antibiotic resistance patterns.
In the present study, isolates related to the respiratory system were more than isolates extracted from urine culture and blood samples, consistent with the studies of Yang et al. from China and Nirwaty et al. from Indonesia [24,25]. However, according to research by Seifi et al. from Iran, the isolates from urine culture (61.7%) were higher than the wound, blood, and sputum samples [11].
Our findings reveal that over 90% of isolates were resistant to cefotaxime and piperacillin-tazobactam, and also, more than 80% of strains were resistant to ampicillinsulbactam, levofloxacin, tobramycin, ceftriaxone, and ciprofloxacin. Furthermore, more than 70% of isolates were resistant to ceftazidime and trimethoprim-sulfamethoxazole. Colistin was the only effective antibiotic. is is in contrast to the findings of Borges et al. who have identified meropenem and piperacillin-tazobactam as effective antibiotics against K. pneumoniae [23]. In our study, the resistance to ciprofloxacin is higher than the resistance reported by Madahiah et al. and Cepas et al. [28,29].   According to the results of a research from Iran, the highest antibiotic resistance in K. pneumoniae isolates belongs to ampicillin (91%), the lowest resistance is related to imipenem (5.5%), and more than 50% of the strains were resistant to ceftazidime and cefalotin [30]. However, our results indicate that 62% of isolates were resistant to imipenem, and almost 73% of specimens were resistant to ceftazidime. Another finding of the study was that the strength of biofilm formation in antibiotic-resistant strains was higher than the sensitive strains, and a remarkable correlation was observed between antibiotic resistance and biofilm formation. However, among sensitive isolates, biofilm formation has been observed, or in resistant strains, nonbiofilm producers and weak and moderate biofilm former have been observed. Different results have been reported in this regard. Zheng et al. have demonstrated no correlation between biofilm formation and the production of broad-spectrum beta-lactamase (ESBL) enzymes in the K. pneumoniae strains [14]. According to Zheng et al. and Yang's and Zhang study, about 80% percent of the K. pneumoniae strains, which were extracted from urine and sputum samples and tested positive for biofilm, were also able to produce ESBL [14,24]. It has also been claimed that there may be a correlation between the strength of biofilm formation in bacterial strains and different geographical areas. One of the most important limitations of this study was the almost insufficient number of K. pneumoniae isolates from patients, due to limited supply of materials for laboratory tests and limitations in financial support. More sampling and molecular epidemiological studies are recommended to obtain stronger results.
In conclusion, results of this study confirm the role of biofilm formation in resistance to clinical isolates of K. pneumoniae isolated from Hamadan hospitals, west of Iran. e strength of biofilm formation was different in clinical samples. So, characterization of nosocomial pathogens is very useful to control and treat infections caused by these pathogens.

Conflicts of Interest
e authors declare that there are no conflicts of interest.