Molecular evaluation of aminoglycosides resistance and biofilm formation in Klebsiella pneumoniae clinical isolates: A cross‐sectional study

Abstract Background and Aims Resistance to antibiotics and the capability to develop biofilm as two main virulent determinants of Klebsiella pneumoniae have important role in infection persistence. The aim of the study was to evaluate the association between the prevalence of aminoglycoside resistance and virulence genes and biofilm formation capacity in K. pneumoniae strains isolated from hospitalized patients in South‐West of Iran. Methods A total of 114 non‐duplicate clinical isolates of K. pneumoniae collected from Ahvaz teaching hospitals. Identification of species was performed by biochemical tests and then confirmed by polymerase chain reaction (PCR) of rpoB gene. The susceptibility to antibiotics was determined by Kirby−Bauer disk diffusion method. Biofilm formation was assessed by microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants including fimbrial genes, aminoglycoside modifying enzymes‐ and 16S rRNA methylase (RMTase) genes. Results Totally, all collected strains were carbapenem resistant and showed multidrug‐ and extensively drug‐resistance phenotype (75% and 25%, respectively). Seventy‐one percent (n = 81) of isolates were non‐susceptible to aminoglycosides. Among aminoglycoside antibiotics, K. pneumoniae isolates showed the highest and lowest resistance rates to tobramycin (71%) and the amikacin (25%), respectively. All biofilm producer strains were positive for the presence virulence determinants including ecpA, fimA, mrkD, and mrkA. Of 81 aminoglycosides non‐susceptible isolates 33% were positive for the presence ant (2″)‐Ia as the most prevalent gene followed by aac (3′)‐IIa and armA (27%), aac (6′)‐Ib (18%), and aph (3′)‐Ia (15%). Conclusion K. pneumoniae isolates showed the highest and the lowest aminoglycoside resistance rates to tobramycin and amikacin, respectively. Majority of isolates were biofilm producers and there was significant association between antibiotic resistance pattern and the strength of biofilm production. The ant(2″)‐Ia, aac (3′)‐IIa, and armA genes in aminoglycoside‐resistant isolates.

gastrointestinal tract of patients and the hands of medical personnel are the primary reservoirs for K. pneumoniae transmission. This microorganism is especially dangerous in immunocompromised patients and those who have indwelling medical devices on which biofilm could developed. 3,4 The occurrence and increase in multidrugresistant (MDR) bacteria have become a global public health issues.
The development of antimicrobial resistance in K. pneumoniae isolates poses a serious problem to public health, mainly with the rise of carbapenem-resistant K. pneumoniae (CRKP) isolates. The irrational use of antibiotics in clinical practice has fostered the spread of resistance to "old class antibacterials." 5 Clonal complex 258 (CC258) is one of the CRKP clones disseminating all over the world. 6 In particular, CRKP exhibiting resistance to colistin and tigecycline have raised important clinical concern, as these are the last-line drugs for the treatment of these isolates. 7 The worldwide prevalence of colistin-resistant K. pneumoniae simplified by chromosomal and plasmid-mediated LPS changes have steadily increased with increased colistin usage for treating CRKP isolates. 8 Colistin-resistant K. pneumoniae infections are increasing in intensive care units, particularly in patients who ultimately depend on vascular catheters for long-term or shortterm use. 9 CRKP isolates can cause serious infections in association with prolonged hospital stays, narrow therapeutic choices, and increased mortality. Several investigations indicated that MDR bacteria isolated from hosts with persistent infections are often biofilm formers. Also, it was noted that intrinsic resistance to antibacterial drugs increases when K. pneumoniae isolates grow as biofilms. 10 Biofilm enable the K.
pneumonia isolates to survive in the rough environmental conditions such as the antibiotic pressure and the host immune response.
Biofilm formation in therapeutic devices, including urinary catheters, as their important stage in their disease pathogenesis can result in biofilm-associated drug tolerance in these isolates. 11 In K. pneumoniae, biofilm development is an important virulence characteristic, and managing biofilms is very challenging as they confer significant tolerance to drug effects and host defense responses. 8 Biofilm can be considered as a reservoir of genetic diversity. In biofilm, the emergence and spread of antibiotic resistance genes increase through horizontal gene transfer. The horizontal transmission of antibiotic resistance genes increase between bacterial cells in an extracellular polymeric substance matrix. 12 In addition to biofilm, the ability to acquire antibiotics resistance is considered as another important virulence determinant which plays a significant role in the successful pathogenicity. Aminoglycosides are frequently used to treat infections caused by Enterobacteriaceae. 13 These antibiotics are one of the important groups of the broadspectrum antibiotics inhibiting protein synthesis by binding to the 30S ribosomal subunit in a wide range of gram-negative bacteria, especially Enterobacteriaceae. However, the improper use of them could lead to the emergence of resistant strains, leading to limitation in the choice of treatment options for life threating infectious diseases. Acquired resistance to aminoglycosides has been reported in these bacteria. The three mechanisms of resistance include the reduction of antibiotic affinity in binding to the ribosomal site, 16S rRNA, reduction in antibiotic permeability, and enzymatic inactivation of the antibiotic are responsible for resistance to aminoglycosides. 14 Enzymatic inactivation of aminoglycosides through three main known classes of aminoglycosides modifying enzymes (AMEs) including N-acetyltransferases (AACs), O-nucleotidyltransferases (ANTs), and O-phosphotransferases (APHs) is the most considerable mechanism of bacterial resistance to aminoglycosides. AACs cause resistance to aminoglycosides by transferring acetyl groups from acetyl-coenzyme A to amino group of these antibiotics, while ANTs by transferring adenyl group from ATP, and APHs by transferring γ-phosphate to hydroxyl groups of these antibiotics confer the resistance, respectively. [15][16][17] Another recently emerged mechanism of aminoglycosides resistance is methylation of ribosomal binding site of the antibiotic by 16S RMTase. The six important genes encoding 16S RMTase including armA, rtmA, rtmB, rtmC and rtmD, and npmA, are located on transposons within transferable plasmids which render high level of resistance to all clinically relevant aminoglycosides. 14

| Antimicrobial susceptibility testing
The antibiotic susceptibility test was done by the disk diffusion method

| Quantification of the biofilm mass
The capacity of isolates to develop biofilm was measured using the microtiter plate (MTP) method. The isolates were classified into four categories using the previously described criteria by Haney et al. 19 as follows: no biofilm producers (A ≤ Ac), weak biofilm producers (Ac < A ≤ 2Ac), moderate biofilm producers (2Ac < A ≤ 4 Ac), and strong biofilm producers (A > 4Ac). The PAO1 strain of Pseudomonas aeruginosa was also used as a positive control for the biofilm assay. All samples were tested three times.

| Biofilm eradication assay
The MTP method was used to evaluate the capacity of amikacin and gentamicin to eradicate biofilms, as described previously. 20 Following biofilm formation for 48 h, the medium was discarded, and the wells were washed with PBS to remove nonattached bacteria. Then, the plates were filled with 100 μL of MHB in the presence of 2% glucose and 100 μL of antibiotics (0.25−512 μg/mL). After incubation at 37°C for 24 h, the plates were treated for the biofilm formation assay as described above. The minimal biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotics leading to a decrease in the metabolic activity of preformed biofilm by 100%.
The experiment was done in triplicate and repeated three times.  Table 1. [21][22][23][24] Sequencing of both sense and antisense strands of purified PCR product was performed to verify detected genes (Macrogen Company). Sequence confirmation was followed by alignment and analysis using the BLAST program of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast).

| Statistical analysis
Descriptive data were analyzed using SPSS version 22.0 statistics software (IBM Corporation). The results were presented as descriptive statistics in terms of relative frequency (mean ± standard deviation, percentile). Fisher's exact test was employed to analyze the significant differences between variables. In addition, p-value < 0.05 was considered as significance level.

| Bacterial characterization
Between October 2020 and March 2021, a total of 114 clinical isolates of K. pneumoniae were obtained from several clinical specimens which distribution of them in terms of sample type shown in Table 2. These isolates were recovered from 59 (52%) male and 55 (4%) female hospitalized patients which their mean age was 52 years    Table 3). It is noteworthy that only 11.5% (n = 3/26) of nonbiofilm producer isolates were resistant to aminoglycosides.

| Ability of amikacin and gentamicin for biofilm eradication
To investigate the ability of gentamicin and amikacin in eradication of preformed biofilm, we determined MBEC. So those, after treatment with antibiotics at the different concentrations of amikacin and gentamicin, living bacteria were counted in all tested isolates. Table 5 shows the capacity of mentioned antibiotics to disperse preformed biofilm in K. pneumonia isolates. Gentamicin eradicated the pre-

| Distribution of AMEs and biofilm related genes in K. pneumonia
The results of our study showed that among 85 isolates that were resistant to at least one of the aminoglycoside antibiotics, 82.4% (n = 70/85) had AME genes. Some isolates had only one gene and some others harbored a combination of two or more genes ( Table 4).
The presence of these genes was not confirmed in remaining

| DISCUSSION
This study evaluated the aminoglycosides resistance and biofilm formation potency in K. pneumoniae strains collected from hospitalized patients in Ahvaz, southwest Iran. The isolates showed different resistance rates against aminoglycoside antibiotics with the highest and the lowest resistance to tobramycin and amikacin, respectively.
In a previous study from China, the lowest aminoglycoside resistance was found to amikacin and streptomycin. 26 Also, Nasiri et al. 27 from Iran reported the lowest resistance rate to netilmicin and amikacin.
Despite the fact that tobramycin is not available in the pharmaceutical markets of Iran, there was a high resistance to this antibiotic (71%) in this study, which was consistent with a previous study by Latifi et al. 28 from Iran. This could be explained by the cross-resistance phenomenon among aminoglycoside antibiotics. In a previous study by Jansen et al. 29 from Germany, significant cross-resistance among three aminoglycosides gentamicin, tobramycin, and streptomycin was observed in Pseudomonas isolates collected from cystic fibrosis patients.
Despite the emergence of resistant bacteria to various antibiotics, aminoglycosides are still one of the best options in the treatment of carbapenem-resistant gram-negative bacteria. However, the results of the current study did not confirm this fact. In this study, the majority of K. pneumoniae strains were resistant to at least one aminoglycoside, making them not proper treatment option especially in case of carbapenem-resistant and MDR isolates. This observation was in contrast to a previous study by Galani et al. 30 from Greece.
Indiscriminate and uncontrolled use of antibiotics, especially in third world countries like Iran, is one of the reasons for increasing antibiotic resistance. Two of the most principal mechanisms of resistance to aminoglycosides are AME and RMTase genes, which are known to be present in gram-positive and gram-negative bacteria.
The existences of these coding genes has been reported in several studies from Iran. 28,31 In the current research, the backbone gene of RMTase, armA, was detected in 27% of the amikacin resistant strains that was higher than a previous report by Nasiri et al. 27  However, in contrast with our findings, in several studies the combination of aac(6′)-Ib and aac(3′)-IIa was reported as the most common genotype. 28,34 One of the notable findings in this study was the 100% rate of carbapenems resistance of K. pneumoniae isolates. Moreover, the majority of isolates were biofilm producers. These findings were in line with the previous studies that revealed significant correlation between biofilm formation capacity and antibiotic resistance among K. pneumonia isolates. [35][36][37] Carbapenems are extremely successful against infections produced by MDR K. pneumoniae strains. MDR K.
pneumoniae is a significant source of concern because it not only causes severe and fatal infections, but it also prolongs hospitalization, resulting in higher treatment costs. 36 Our findings revealed that biofilm producer strains were significantly aminoglycosides non-susceptible isolates that was in agreement with a previous study by Karimi et al. 38  Abbreviation: AME, aminoglycoside modifying enzymes.
T A B L E 5 Demographic data, prevalence of fimbrial and AME genes, and biofilm production in Klebsiella pneumoniae isolates. biofilm-producing K. pneumoniae isolates were more resistant to gentamicin and amikacin than those of non-biofilm producers. In this study, the majority of XDR isolates were strong biofilm producers. In line with our results, Vuotto et al. 39 showed a positive relationship between antibiotic resistance profile and biofilm-forming ability in XDR K. pneumoniae strains. However, the presence of association between antibiotic resistance and biofilm formation capacity is still debated by some researchers. 40,41 This study showed that ecpA, fimA, mrkD, and mrkA genes were harbored by all biofilm producing K. pneumoniae strains, In conclusion, this study showed that all K. pneumoniae isolates were carbapenem-resistant. Moreover, K. pneumoniae isolates showed the highest and the lowest aminoglycoside resistance rates to tobramycin and amikacin, respectively. Majority (more than 70%) of isolates were biofilm producers and there was significant association between antibiotic resistance pattern and the strength of biofilm production. Also, ant(2")-Ia and aph(3′)-Ia were the most and the least frequent AME genes, respectively. Finally, most AMEs harboring isolates were also positive for presence of the biofilm related genes including ecpA, mrkA, and fimbrial genes. investigation; supervision.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

TRANSPARENCY STATEMENT
The lead author Zahra Farshadzadeh affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article. All data is in article.