Abstract
The current study aimed to investigate the antimicrobial susceptibility profiles, biofilm production capabilities, and the prevalence of efflux pump and biofilm-associated genes among Klebsiella pneumoniae clinical isolates. One hundred sixty-seven K. pneumoniae isolates were collected from microbiology laboratories in Northern Jordan hospitals. Antimicrobial susceptibility was tested using the Kirby-Bauer method. The double-disk synergy test was used to detect the extended-spectrum beta-lactamase (ESBL) phenotype. PCR was used to detect the frequency of acrAB,tolC, and mdtk efflux pump genes and fimH-1,mrkA, and mrkD biofilm-associated genes among the isolates. The highest nonsusceptibility was observed against azithromycin (87.4 %) and nitrofurantoin (85.0 %). Among the isolates, 75.4 % and 92.2 % were multidrug resistant and produced biofilms, respectively. Efflux pump genes acrAB, tolC, and mdtK were found in 96.4 %, 95.2 %, and 90.4 % of the isolates, respectively. Biofilm-associated genes mrkD, mrkA, and fimH-1 were found in 92.2 %, 89.2 %, and 88.6 % of the isolates, respectively. The presence of the mrkA was significantly associated with biofilm formation. Overall, high percentages of multi-drug resistance, efflux pump, and biofilm-associated genes were observed among the isolates. Subsequent studies are recommended to monitor changes in the prevalence of resistance phenotypes and genotypes of isolates.
Keywords: Jordan, Klebsiella pneumoniae, Antibiotic, Efflux pump, Biofilm, Genotype, Phenotype
1. Introduction
Klebsiella pneumoniae is a Gram-negative opportunistic pathogen that is responsible for a wide range of community-acquired and healthcare-associated infections including urinary tract infections, bacteremia, and pneumonia [1]. The misuse and overuse of antimicrobial led to increased dissemination of multidrug resistant (MDR) K. pneumoniae strains. Infections by MDR K. pneumoniae strains are associated with high mortality rates (up to 40–50 %), particularly in immunocompromised patients [2]. Several factors are involved in the development of MDR K. pneumoniae strains, such as efflux pumps and biofilm formation [3,4].
Bacterial efflux pumps are transport proteins that can lead to nonsusceptibility to antimicrobial agents in many bacterial pathogens. These pumps confer resistance by extruding various types of antimicrobials out of the bacterial cells, thereby reducing their intracellular concentration [5,6]. Efflux pump systems in K. pneumoniae include AcrAB-TolC and MdtK, which are members of the resistance nodulation-division (RND) and the multidrug and toxic compound extrusion (MATE) families. Several studies showed that AcrAB-TolC and MdtK efflux pumps are involved in nonsusceptibility to different types of antimicrobials such as quinolones, tetracycline, and chloramphenicol in MDR K. pneumoniae [7,8].
Biofilms are complex associations of microorganisms embedded in a self-generated matrix of extracellular polymeric substances that grow attached to abiotic or biotic surfaces [9]. Bacteria within biofilms can resist higher concentrations of antimicrobials than planktonic bacteria, thus biofilm-associated infections are very difficult to eradicate [10]. Biofilms protect K. pneumoniae from environmental stresses such as host defense mechanisms. It also promotes bacterial persistence on host tissues and indwelling devices [[11], [12], [13]]. Biofilm formation in K. pneumoniae is an important factor in the development of MDR strains [14,15]. In Gram-negative bacteria, attachment to abiotic surfaces and host cell surfaces is mediated by fimbrial adhesins [16]. Type 1 and 3 fimbriae are the two major fimbrial adhesins in K. pneumoniae that are essential for adhesion and biofilm formation [17]. In K. pneumoniae, different biofilm-related genes including fimH-1 (encoding for the type 1 fimbrial adhesin), mrkA (encoding for the major structural subunit of type 3 fimbriae), and mrkD (encoding for the type 3 fimbrial adhesin) are involved biofilm formation and MDR phenotypes [[18], [19], [20]].
K. pneumoniae accounts for a considerable proportion of healthcare-associated infections. The increasing rate of K. pneumoniae strains resistant to antimicrobials is a global public health concern. The screening of efflux pump genes and biofilm genes among (MDR) K. pneumoniae isolates and their associations with their corresponding phenotypes provides a better understanding of the prevalent strains in the healthcare setting. This information also enables better management of infections caused by this pathogen. Several studies demonstrated the correlations between resistance to antimicrobials, efflux pumps, and biofilm formation, in K. pneumoniae [[21], [22], [23], [24], [25], [26]]. To date, the prevalence of efflux pump and biofilm genes and their association with biofilm formation and resistance to antimicrobials in K. pneumoniae has not been previously reported among clinical isolates from Jordan. Hence, in this study we aimed to determine the antimicrobial susceptibility profile and biofilm formation capacity of K. pneumoniae isolates obtained from Northern Jordan, to determine the frequency of acrAB, tolC, and mdtk efflux pump genes and fimH-1, mrkA, and mrkD biofilm-associated genes among the isolates, and to investigate possible associations between efflux pump genes and biofilm-associated genes, with biofilm formation and nonsusceptibility phenotypes in K. pneumoniae isolates.
2. Materials and methods
2.1. Bacterial isolates and identification
Approval for the study was granted by the IRBcommittee of Jordan University of Science and Technology (approval ID 2023/158/38; date 2023/3/23). One hundred and sixty-seven non-replicate previously collected K. pneumoniae isolates were used in this study. The isolates were from the microbiology laboratories of governmental and teaching hospitals in Northern Jordan. The isolates were collected over a period of 10 months (July 2021 - April 2022). The isolates were identified using the VITEK 2 system at the respective hospitals at the time of collection. The isolates were from various types of clinical samples and both genders. Supplementary Table S1 provides full details of the isolates and all isolates’ results.
2.2. Antimicrobial susceptibility testing
The antimicrobial susceptibility of K. pneumoniae isolates was tested using the Kirby-Bauer method according to the Clinical and Laboratory Standards Institute (CLSI 2023) guidelines. The antimicrobials disks tested were cefepime (30 μg), gentamicin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), levofloxacin [5 μg], imipenem (10 μg), ertapenem (10 μg), azithromycin (15 μg), chloramphenicol (30 μg), and nitrofurantoin (300 μg) disks. For each isolate, a bacterial suspension with a turbidity equivalent to 0.5 McFarland was prepared to inoculate MHA plates, the disks were placed, and the plates were incubated overnight at 37 °C. The diameters of the inhibition zones were recorded in millimeters and the results were interpreted according to CLSI guidelines (2023). MDR isolates were identified based on their nonsusceptibility (resistance or intermediate susceptibility) to at least one agent in three or more antimicrobial classes [27].
2.3. Detection of ESBL production by double disk synergy test
To detect ESBL production, the double disk synergy test was performed according to CLSI (2023) guidelines. Amoxicillin-clavulanate (20/10 μg) disk, with cefpodoxime (10 μg), cefotaxime (30 μg), and aztreonam (30 μg) disks were used. MHA plates were inoculated with each isolate as described above. An amoxicillin-clavulanate disk was placed in the center of the plate, and cefpodoxime (10 μg), cefotaxime (30 μg), and aztreonam (30 μg) disks were placed 25 mm (center to center) from the amoxicillin-clavulanate disk. An increase in the zone of inhibition (augmentation pattern) of any of the disks towards the central amoxicillin-clavulanate disk was interpreted as ESBL production. Escherichia coli ATCC 25922, K. pneumoniae BAA-2146, K. pneumoniae BAA-1705, K. pneumoniae BAA-1706, were used as controls for antimicrobial susceptibility and ESBL testing.
2.4. Quantitative biofilm formation assay
The biofilm assay was done as described previously with modifications [28]. The clinical isolates from frozen stock were cultured on LB broth at 37 °C for 24 h. Wells of a sterile 96-well plate were filled with 198 μL of Trypticase Soy Broth (TSB) supplemented with 1 % glucose and 2 μL of the overnight LB culture. Negative control wells contained sterile TSB broth. The plate was incubated for 24 h at 37 °C. The contents of each well were discarded by inversion and gentle tapping on absorbent paper towels, and the wells were washed four times with 200 μL of phosphate buffer saline (PBS) (pH 7.2) to remove free-floating planktonic bacteria. Wells were stained with 200 μL of 0.1 % crystal violet solution in water for 15 min at room temperature, the plate was rinsed with distilled water four times to remove any excess stain, and 200 μL of 95 % ethanol was added to each well to dissolve the stain (10–15 min at room temp). The absorbance for each well was measured at 550 nm using an ELISA plate reader (BioTek Epoch). The blank wells contained 95 % ethanol.
The test was done in duplicates and the average was calculated for each bacterial isolate. The cut-off optical density (ODc) for biofilm formation was defined as three standard deviations above the mean OD of the negative control. Optical density data was interpreted into negative, weak, moderate, or strong biofilm formation as previously described [29], per Supplementary Table S2.
2.5. DNA extraction
The phenol-chloroform procedure was used with modifications to extract DNA from the isolates [30,31]. The isolates from the frozen stock were cultured into MHA plates and incubated overnight at 37 °C to obtain pure colonies. Next, Muller Hinton broth tubes were inoculated with a single pure colony and incubated in a shaking incubator (200 rpm) for 3–4 h at 37 °C. Then, bacterial cultures were transferred into sterile Eppendorf tubes. The cells were centrifuged at 18,000 rpm for 3 min and sediment was obtained. To break down the cell membrane, the sediment was suspended by adding 500 μL of lysis buffer (20 mM Tris pH 7.5 with HCl, 50 mM EDTA pH 8, 100 mM NaCl). To breakdown the cell wall, 100 μL of 0.04 g/mL lysozyme was added per sample, and the samples were incubated for 1 h at 37 °C.
To break down proteins, 60 μL of 10 % sarkosyl was added, followed by vortexing. To separate DNA from other components, 600 μL of phenol was added to each sample, and the tubes vigorously vortexed. Following centrifugation at 18,000 rpm for 5 min, the supernatant layer was transferred into a new Eppendorf tube. Next, 300 μL of phenol and 300 μL of chloroform were added, followed by centrifugation at 18,000 rpm for 5 min, and the supernatant layer was transferred into a new Eppendorf tube.
Next, 50 μL NaOAc (300 mM) was added and vortexed, followed by addition of 900 μL of 96 % ethanol to precipitate DNA. After centrifugation at 18,000 rpm for 2 min, the supernatant was discarded immediately to prevent the pellet from dissolving, and the tube was dried by tapping on absorbent tissue paper.
For the washing step, 150 μL of 70 % ethanol was added followed by vortexing and centrifugation at 18,000 rpm for 1 min. The supernatant containing 70 % ethanol was poured out and the tube was dried by tapping on absorbent tissue paper. Finally, for DNA elution, 100 μL of TE buffer (20 mM Tris pH 7.5 with HCl, 50 mM EDTA, pH 8) was added to each sample, and the DNA solution was stored at −80 °C for future use in PCR.
2.6. Species identity confirmation
To confirm the identity of K. pneumoniae isolates, amplification of the species-specific Phosphoporin E (phoE) gene was done using PCR. The reaction mix consisted of 1 μL of extracted DNA (10 ng/mL), 1 μL of each primer (each at 10 μmol/L), 5 μL 5x master mix (Ready-to load 5X Master Mix, myPOLS Biotech, Germany), and 17 μL nuclease-free water. The total PCR volume was 25 μL. The PCR conditions were initial denaturation at 95 °C for 5 min, followed by 30 cycles each consisting of denaturation (at 94 °C for 1 min), annealing (at 55 °C for 1 min), and extension (at 72 °C for 1 min), and one cycle of final extension at 72 °C for 5 min. Primer sequences are indicated in Supplementary Table S3.
2.7. Genotypic detection of efflux and biofilm genes
The frequency of acrAB, tolC, and mdtk efflux pump genes in addition to fimH-1, mrkA, and mrkD biofilm-associated genes in K. pneumoniae isolates was determined using 6 classical PCRs followed by gel electrophoresis to identify the bands. Representative bands for each of the genes were subjected to DNA sequencing to verify amplified sequences. The primers for target genes are listed in Supplementary Table S3. Each PCR was performed at a final reaction volume of 25 μL consisting of 1 μL of extracted DNA (10 ng/mL), 5 μL of 5x master (Ready-to load 5X Master Mix, myPOLS Biotech, Germany), 1 μL of each primer (each at 10 μmol/L), and 17 μL of nuclease-free water. PCR conditions were initial denaturation at 95 C for 5 min, followed by 25 cycles each consisting of denaturation (at 94 C for 1 min), 45 s of annealing (at the temperature listed in the table below), and extension (at 72 C for 1 min), and one cycle of final extension at 72 C for 10 min.
2.8. Agarose gel electrophoresis
PCR products were separated on 1.5 % agarose having ethidium bromide (0.5 μg/mL). Five microliters of PCR products were loaded per well of the gel. Electrophoresis was done at 140 V for 45 min. DNA was visualized using a UV transilluminator provided with a gel documentation system. Fragment sizes of each PCR were determined by comparison with a 100 bp DNA ladder. Identity of amplified genes was confirmed using DNA sequencing. Representative gel electrophoresis results are shown in Supplementary Figs. S1–S7.
2.9. Statistical analyses
The statistical package for social sciences (SPSS ver. 26, IBM, Armonk, NY, USA) was used to generate tables and cross-tabulations. Univariate analysis using the Chi-squared test was utilized to test associations between parameters. Resistance and intermediate susceptibility results in the Kirby-Bauer method were classified as nonsusceptible during statistical analysis. Furthermore, where indicated, biofilm results were analyzed as four groups (strong, moderate, weak, and negative) or two groups (strong or moderate, and weak or negative). Overall, the following associations were tested: MDR phenotype with ESBL production; ESBL and MDR phenotypes with biofilm formation; efflux and biofilm genes with biofilm formation; efflux and biofilm genes with antimicrobials’ susceptibility phenotypes; co-associations between efflux and biofilm genes. A p value < 0.05 was considered significant.
3. Results
3.1. Study isolates
One hundred sixty-seven K. pneumoniae isolates were collected from different types of clinical samples and several hospitals in Northern Jordan. Most isolates were from urine (n = 133) and blood (n = 21). While few isolates were from pus (n = 4), high vaginal swab (n = 3), wound (n = 2), sputum (n = 2), cerebrospinal fluid (n = 1), and tip of central line (n = 1). Females were the most frequent source for the isolates (65.9 %; 110/167).
3.2. Antimicrobial susceptibility profile
The antimicrobial susceptibility of K. pneumoniae isolates is presented in Table 1. The results are classified into susceptible and non-susceptible groups (intermediate susceptibility + resistant). The highest nonsusceptibility was against azithromycin (87.4 %), nitrofurantoin (85.0 %), and gentamicin (71.3 %). The highest susceptibility was against ertapenem (85.0 %), chloramphenicol (81.4 %), and imipenem (80.8 %). The MDR phenotype was observed among 126 isolates (75.4 %). The ESBL phenotype was observed among 55 isolates (32.9 %). Among the ESBL isolates, 85.5 % were MDR. A significant association was observed between the MDR phenotype and ESBL production (P = 0.035; Supplementary Table S4).
Table 1.
Antimicrobial susceptibility profile of the isolates.
| Antibiotic\ AST result | I |
R |
NS (I + R) |
S |
||||
|---|---|---|---|---|---|---|---|---|
| n | % | n | % | n | % | n | % | |
| Amoxicillin clavulanate | 33 | 19.8 | 43 | 25.7 | 76 | 45.5 | 91 | 54.5 |
| Cefpodoxime | 1 | 0.6 | 87 | 52.1 | 88 | 52.7 | 79 | 47.3 |
| Cefotaxime | 11 | 6.6 | 89 | 53.3 | 100 | 59.9 | 67 | 40.1 |
| Aztreonam | 12 | 7.2 | 59 | 35.3 | 71 | 42.5 | 96 | 57.5 |
| Cefepime | 7 | 4.2 | 75 | 44.9 | 82 | 49.1 | 85 | 50.9 |
| Gentamicin | 77 | 46.1 | 42 | 25.1 | 119 | 71.3 | 48 | 28.7 |
| Amikacin | 90 | 53.9 | 26 | 15.6 | 116 | 69.5 | 51 | 30.5 |
| Ciprofloxacin | 15 | 9.0 | 77 | 46.1 | 92 | 55.1 | 75 | 44.9 |
| Levofloxacin | 38 | 22.8 | 39 | 23.4 | 77 | 46.1 | 90 | 53.9 |
| Imipenem | 10 | 6.0 | 22 | 13.2 | 32 | 19.2 | 135 | 80.8 |
| Ertapenem | 3 | 1.8 | 22 | 13.2 | 25 | 15.0 | 142 | 85.0 |
| Azithromycin | 0 | 0 | 146 | 87.4 | 146 | 87.4 | 21 | 12.6 |
| Chloramphenicol | 5 | 3.0 | 26 | 15.6 | 31 | 18.6 | 136 | 81.4 |
| Nitrofurantoin | 29 | 17.4 | 113 | 67.7 | 142 | 85.0 | 25 | 15.0 |
AST: antimicrobial susceptibility test. I: intermediate susceptibility. n: count. NS: non-susceptible. R: resistant. S: susceptible.
3.3. Biofilm production among K. pneumoniae isolates
Among 167 K. pneumoniae isolates, 154 (92.2 %) produced biofilms. Among the biofilm producers, 94 isolates (56.3 %) were strong biofilm producers, 43 (25.7 %) moderate biofilm producers, and 17 isolates (10.2 %) weak biofilm producers. Table 2 indicates the association between biofilm formation and ESBL and MDR phenotypes. No significant association was observed between biofilm phenotypes with ESBL and MDR phenotypes. Furthermore, no significant association was observed between isolates’ source (urine vs non-urine) and the biofilm phenotype (85.0 % for strong or moderate vs 70.6 % for weak or none; P = 0.051; Chi-squared test).
Table 2.
Association of biofilms with ESBL and MDR phenotypes.
| Phenotype | ESBL phenotype |
MDR phenotype |
||||||
|---|---|---|---|---|---|---|---|---|
| Total |
– |
+ |
No |
Yes |
||||
| n (%) | n (%) | n (%) | P value | n (%) | n (%) | P value | ||
| Biofilm | Strong | 94 (56.3) | 63 (56.3) | 31 (56.4) | 0.786 | 22 (53.7) | 72 (57.1) | 0.584 |
| Moderate | 43 (25.7) | 27 (24.1) | 16 (29.1) | 9 (22.0) | 34 (27.0) | |||
| Weak | 17 (10.2) | 13 (11.6) | 4 (7.3) | 5 (12.2 %) | 12 (9.5) | |||
| Negative | 13 (7.8) | 9 (8.0) | 4 (7.3) | 5 (12.2) | 8 (6.3) | |||
| Biofilm (2 categories) | Strong or moderate | 137 (82.0) | 90 (80.4) | 47 (85.5) | 0.420 | 31 (75.6) | 106 (84.1) | 0.217 |
| Weak or negative | 30 (18.0) | 22 (19.6) | 8 (14.5) | 10 (19.1) | 20 (15.9) | |||
ESBL: extended spectrum beta-lactamase. MDR: multidrug resistant. “-”: absent. “+”: present. P value calculated using Chi-squared test. P < 0.05 is considered statistically significant.
3.4. Frequency of efflux pumps and biofilm-associated genes
All isolates demonstrated the K. pneumoniae specific phoE gene. The efflux pump genes acrAB, tolC, and mdtK were observed in 96.4 % (n = 161), 95.2 % (n = 159), and 90.4 % (n = 151) of the isolates, respectively. While the biofilm-associated genes mrkD, mrkA, and fimH-1 were observed in 92.2 % (n = 154), 89.2 % (n = 149), and 88.6 % (n = 148) of the isolates, respectively.
3.5. Association of efflux and biofilm genes with biofilm formation, ESBL, and MDR phenotypes
Table 3 shows the association between efflux and biofilm genes with biofilm formation, and ESBL and MDR phenotypes. Among the three efflux pump genes, tolC was significantly associated with the MDR phenotype. Among the three biofilm genes, mrkA was significantly associated with biofilm formation, and fimH-1 was significantly associated with the MDR phenotype.
Table 3.
Associations between efflux and biofilm genes with biofilm formation, ESBL, and MDR phenotypes.
| Gene\phenotype | ESBL phenotype |
MDR phenotype |
Biofilm (2 categories) |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| – |
+ |
– |
+ |
Strong or moderate |
Weak or negative |
||||||||
| n | % | n | % | n | % | n | % | n | % | n | % | ||
| acrAB | – | 3 | 50.0 | 3 | 50.0 | 3 | 50.0 | 3 | 50.0 | 4 | 66.7 | 2 | 33.3 |
| + | 109 | 67.7 | 52 | 32.3 | 38 | 23.6 | 123 | 76.4 | 133 | 82.6 | 28 | 17.4 | |
| P value | 0.365 | 0.140 | 0.318 | ||||||||||
| tolC | – | 4 | 50.0 | 4 | 50.0 | 5 | 62.5 | 3 | 37.5 | 6 | 75.0 | 2 | 25.0 |
| + | 108 | 67.9 | 51 | 32.1 | 36 | 22.6 | 123 | 77.4 | 131 | 82.4 | 28 | 17.6 | |
| P value | 0.293 | 0.011 | 0.595 | ||||||||||
| mdtK | – | 8 | 50.0 | 8 | 50.0 | 4 | 25.0 | 12 | 75.0 | 12 | 75.0 | 4 | 25.0 |
| + | 104 | 68.9 | 47 | 31.1 | 37 | 24.5 | 114 | 75.5 | 125 | 82.8 | 26 | 17.2 | |
| P value | 0.127 | 0.965 | 0.441 | ||||||||||
| mrkA | – | 12 | 66.7 | 6 | 33.3 | 3 | 16.7 | 15 | 83.3 | 11 | 61.1 | 7 | 38.9 |
| + | 100 | 67.1 | 49 | 32.9 | 38 | 25.5 | 111 | 74.5 | 126 | 84.6 | 23 | 15.4 | |
| P value | 0.970 | 0.411 | 0.014 | ||||||||||
| mrkD | – | 8 | 61.5 | 5 | 38.5 | 3 | 23.1 | 10 | 76.9 | 11 | 84.6 | 2 | 15.4 |
| + | 104 | 67.5 | 50 | 32.5 | 38 | 24.7 | 116 | 75.3 | 126 | 81.8 | 28 | 18.2 | |
| P value | 0.659 | 0.898 | 0.801 | ||||||||||
| fimH-1 | – | 13 | 68.4 | 6 | 31.6 | 9 | 47.4 | 10 | 52.6 | 15 | 78.9 | 4 | 21.1 |
| + | 99 | 66.9 | 49 | 33.1 | 32 | 21.6 | 116 | 78.4 | 122 | 82.4 | 26 | 17.6 | |
| P value | 0.894 | 0.014 | 0.710 | ||||||||||
ESBL: extended spectrum beta-lactamase. MDR: multidrug resistant. N: count. “-”: absent. “+”: present. P value calculated using Chi-squared test. P values in bold are statistically significant.
3.6. Association of genes encoding efflux pumps with nonsusceptibility phenotypes
As shown in Table 4, acrAB was significantly associated with nonsusceptibility to nitrofurantoin and susceptibility to cefpodoxime and cefotaxime, tolC was significantly associated with nonsusceptibility to azithromycin and nitrofurantoin, and mdtk was significantly associated with susceptibility to cefotaxime.
Table 4.
Association of efflux pump genes with antimicrobials’ susceptibility phenotypes.
| Antimicrobial\Gene |
acrAB |
tolC |
mdtK |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| – |
+ |
– |
+ |
– |
+ |
|||||||||||
| n | % | n | % | P value | n | % | n | % | P value | n | % | n | % | P value | ||
| Amoxicillin clavulanate | NS | 4 | 66.7 | 72 | 44.7 | 0.289 | 5 | 62.5 | 71 | 44.7 | 0.323 | 9 | 56.3 | 67 | 44.4 | 0.364 |
| S | 2 | 33.3 | 89 | 55.3 | 3 | 37.5 | 88 | 55.3 | 7 | 43.8 | 84 | 55.6 | ||||
| Cefpodoxime | NS | 6 | 100.0 | 82 | 50.9 | 0.018*I | 6 | 75.0 | 82 | 51.6 | 0.195 | 12 | 75.0 | 76 | 50.3 | 0.060 |
| S | 0 | 0.0 | 79 | 49.1 | 2 | 25.0 | 77 | 48.4 | 4 | 25.0 | 75 | 49.7 | ||||
| Cefotaxime | NS | 6 | 100.0 | 94 | 58.4 | 0.041*I | 6 | 75.0 | 94 | 59.1 | 0.371 | 14 | 87.5 | 86 | 57.0 | 0.018*I |
| S | 0 | 0.0 | 67 | 41.6 | 2 | 25.0 | 65 | 40.9 | 2 | 12.5 | 65 | 43.0 | ||||
| Aztreonam | NS | 4 | 66.7 | 67 | 41.6 | 0.223 | 4 | 50.0 | 67 | 42.1 | 0.661 | 8 | 50.0 | 63 | 41.7 | 0.524 |
| S | 2 | 33.3 | 94 | 58.4 | 4 | 50.0 | 92 | 57.9 | 8 | 50.0 | 88 | 58.3 | ||||
| Cefepime | NS | 5 | 83.3 | 77 | 47.8 | 0.088 | 5 | 62.5 | 77 | 48.4 | 0.437 | 10 | 62.5 | 72 | 47.7 | 0.260 |
| S | 1 | 16.7 | 84 | 52.2 | 3 | 37.5 | 82 | 51.6 | 6 | 37.5 | 79 | 52.3 | ||||
| Gentamicin | NS | 4 | 66.7 | 115 | 71.4 | 0.800 | 7 | 87.5 | 112 | 70.4 | 0.298 | 11 | 68.8 | 108 | 71.5 | 0.816 |
| S | 2 | 33.3 | 46 | 28.6 | 1 | 12.5 | 47 | 29.6 | 5 | 31.3 | 43 | 28.5 | ||||
| Amikacin | NS | 6 | 100.0 | 110 | 68.3 | 0.098 | 7 | 87.5 | 109 | 68.6 | 0.256 | 12 | 75.0 | 104 | 68.9 | 0.613 |
| S | 0 | 0.0 | 51 | 31.7 | 1 | 12.5 | 50 | 31.4 | 4 | 25.0 | 47 | 31.1 | ||||
| Ciprofloxacin | NS | 3 | 50.0 | 89 | 55.3 | 0.799 | 5 | 62.5 | 87 | 54.7 | 0.666 | 8 | 50.0 | 84 | 55.6 | 0.667 |
| S | 3 | 50.0 | 72 | 44.7 | 3 | 37.5 | 72 | 45.3 | 8 | 50.0 | 67 | 44.4 | ||||
| Levofloxacin | NS | 3 | 50.0 | 74 | 46.0 | 0.846 | 4 | 50.0 | 73 | 45.9 | 0.821 | 7 | 43.8 | 70 | 46.4 | 0.842 |
| S | 3 | 50.0 | 87 | 54.0 | 4 | 50.0 | 86 | 54.1 | 9 | 56.3 | 81 | 53.6 | ||||
| Imipenem | NS | 3 | 50.0 | 29 | 18.0 | 0.051 | 2 | 25.0 | 30 | 18.9 | 0.667 | 3 | 18.8 | 29 | 19.2 | 0.965 |
| S | 3 | 50.0 | 132 | 82.0 | 6 | 75.0 | 129 | 81.1 | 13 | 81.3 | 122 | 80.8 | ||||
| Ertapenem | NS | 2 | 33.3 | 23 | 14.3 | 0.199 | 1 | 12.5 | 24 | 15.1 | 0.841 | 3 | 18.8 | 22 | 14.6 | 0.656 |
| S | 4 | 66.7 | 138 | 85.7 | 7 | 87.5 | 135 | 84.9 | 13 | 81.3 | 129 | 85.4 | ||||
| Azithromycin | NS | 4 | 66.7 | 142 | 88.2 | 0.118 | 5 | 62.5 | 141 | 88.7 | 0.029 | 13 | 81.3 | 133 | 88.1 | 0.433 |
| S | 2 | 33.3 | 19 | 11.8 | 3 | 37.5 | 18 | 11.3 | 3 | 18.8 | 18 | 11.9 | ||||
| Chloramphenicol | NS | 1 | 16.7 | 30 | 18.6 | 0.903 | 2 | 25.0 | 29 | 18.2 | 0.631 | 3 | 18.8 | 28 | 18.5 | 0.984 |
| S | 5 | 83.3 | 131 | 81.4 | 6 | 75.0 | 130 | 81.8 | 13 | 81.3 | 123 | 81.5 | ||||
| Nitrofurantoin | NS | 3 | 50.0 | 139 | 86.3 | 0.014 | 3 | 37.5 | 139 | 87.4 | 0.001 | 13 | 81.3 | 129 | 85.4 | 0.656 |
| S | 3 | 50.0 | 22 | 13.7 | 5 | 62.5 | 20 | 12.6 | 3 | 18.8 | 22 | 14.6 | ||||
N: count. NS: nonsusceptible. S: susceptible. “-”: absent. “+”: present. *I: statistically significant independence. P value calculated using Chi-squared test. P values in bold are statistically significant.
3.7. Association of biofilm-associated genes with nonsusceptibility phenotype
The association between biofilm-associated genes and nonsusceptibility phenotypes is indicated in Table 5. MrkD was significantly associated with nonsusceptibility to ciprofloxacin. FimH-1 was significantly associated with nonsusceptibility to nitrofurantoin. Furthermore, a significant association was observed between mrkA and susceptibility to several antimicrobials.
Table 5.
Association of biofilm genes with antimicrobials’ susceptibility phenotypes.
| Antimicrobial\Gene |
mrkA |
mrkD |
fimH-1 |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| – |
+ |
– |
+ |
– |
+ |
|||||||||||
| n | % | n | % | P value | n | % | n | % | P value | n | % | n | % | P value | ||
| Amoxicillin clavulanate | NS | 15 | 83.3 | 61 | 40.9 | 0.001*I | 6 | 46.2 | 70 | 45.5 | 0.961 | 9 | 47.4 | 67 | 45.3 | 0.863 |
| S | 3 | 16.7 | 88 | 59.1 | 7 | 53.8 | 84 | 54.5 | 10 | 52.6 | 81 | 54.7 | ||||
| Cefpodoxime | NS | 17 | 94.4 | 71 | 47.7 | 0.001*I | 7 | 53.8 | 81 | 52.6 | 0.931 | 10 | 52.6 | 78 | 52.7 | 0.995 |
| S | 1 | 5.6 | 78 | 52.3 | 6 | 46.2 | 73 | 47.4 | 9 | 47.4 | 70 | 47.3 | ||||
| Cefotaxime | NS | 17 | 94.4 | 83 | 55.7 | 0.002*I | 7 | 53.8 | 93 | 60.4 | 0.644 | 12 | 63.2 | 88 | 59.5 | 0.757 |
| S | 1 | 5.6 | 66 | 44.3 | 6 | 46.2 | 61 | 39.6 | 7 | 36.8 | 60 | 40.5 | ||||
| Aztreonam | NS | 13 | 72.2 | 58 | 38.9 | 0.007*I | 5 | 38.5 | 66 | 42.9 | 0.758 | 5 | 26.3 | 66 | 44.6 | 0.129 |
| S | 5 | 27.8 | 91 | 61.1 | 8 | 61.5 | 88 | 57.1 | 14 | 73.7 | 82 | 55.4 | ||||
| Cefepime | NS | 16 | 88.9 | 66 | 44.3 | 0.001*I | 6 | 46.2 | 76 | 49.4 | 0.825 | 7 | 36.8 | 75 | 50.7 | 0.256 |
| S | 2 | 11.1 | 83 | 55.7 | 7 | 53.8 | 78 | 50.6 | 12 | 63.2 | 73 | 49.3 | ||||
| Gentamicin | NS | 15 | 83.3 | 104 | 69.8 | 0.231 | 8 | 61.5 | 111 | 72.1 | 0.420 | 11 | 57.9 | 108 | 73.0 | 0.172 |
| S | 3 | 16.7 | 45 | 30.2 | 5 | 38.5 | 43 | 27.9 | 8 | 42.1 | 40 | 27.0 | ||||
| Amikacin | NS | 17 | 94.4 | 99 | 66.4 | 0.015*I | 11 | 84.6 | 105 | 68.2 | 0.217 | 15 | 78.9 | 101 | 68.2 | 0.340 |
| S | 1 | 5.6 | 50 | 33.6 | 2 | 15.4 | 49 | 31.8 | 4 | 21.1 | 47 | 31.8 | ||||
| Ciprofloxacin | NS | 14 | 77.8 | 78 | 52.3 | 0.040*I | 3 | 23.1 | 89 | 57.8 | 0.016 | 7 | 36.8 | 85 | 57.4 | 0.089 |
| S | 4 | 22.2 | 71 | 47.7 | 10 | 76.9 | 65 | 42.2 | 12 | 63.2 | 63 | 42.6 | ||||
| Levofloxacin | NS | 14 | 77.8 | 63 | 42.3 | 0.004*I | 3 | 23.1 | 74 | 48.1 | 0.083 | 5 | 26.3 | 72 | 48.6 | 0.066 |
| S | 4 | 22.2 | 86 | 57.7 | 10 | 76.9 | 80 | 51.9 | 14 | 73.7 | 76 | 51.4 | ||||
| Imipenem | NS | 12 | 66.7 | 20 | 13.4 | 0.001*I | 2 | 15.4 | 30 | 19.5 | 0.719 | 4 | 21.1 | 28 | 18.9 | 0.824 |
| S | 6 | 33.3 | 129 | 86.6 | 11 | 84.6 | 124 | 80.5 | 15 | 78.9 | 120 | 81.1 | ||||
| Ertapenem | NS | 9 | 50.0 | 16 | 10.7 | 0.001*I | 1 | 7.7 | 24 | 15.6 | 0.444 | 1 | 5.3 | 24 | 16.2 | 0.208 |
| S | 9 | 50.0 | 133 | 89.3 | 12 | 92.3 | 130 | 84.4 | 18 | 94.7 | 124 | 83.8 | ||||
| Azithromycin | NS | 15 | 83.3 | 131 | 87.9 | 0.579 | 10 | 76.9 | 136 | 88.3 | 0.234 | 15 | 78.9 | 131 | 88.5 | 0.236 |
| S | 3 | 16.7 | 18 | 12.1 | 3 | 23.1 | 18 | 11.7 | 4 | 21.1 | 17 | 11.5 | ||||
| Chloramphenicol | NS | 1 | 5.6 | 30 | 20.1 | 0.133 | 1 | 7.7 | 30 | 19.5 | 0.294 | 3 | 15.8 | 28 | 18.9 | 0.741 |
| S | 17 | 94.4 | 119 | 79.9 | 12 | 92.3 | 124 | 80.5 | 16 | 84.2 | 120 | 81.1 | ||||
| Nitrofurantoin | NS | 15 | 83.3 | 127 | 85.2 | 0.831 | 10 | 76.9 | 132 | 85.7 | 0.394 | 11 | 57.9 | 131 | 88.5 | 0.001 |
| S | 3 | 16.7 | 22 | 14.8 | 3 | 23.1 | 22 | 14.3 | 8 | 42.1 | 17 | 11.5 | ||||
N: count. NS: nonsusceptible. S: susceptible. “-”: absent. “+”: present. *I: statistically significant independence. P value calculated using Chi-squared test. P values in bold are statistically significant.
3.8. Co-association of efflux pump and biofilm-associated genes
The co-association of efflux pump genes and biofilm genes among the isolates is demonstrated in Table 6. All investigated genes demonstrated significant co-associations.
Table 6.
Co-association of efflux pump genes and biofilm genes.
| Gene |
acrAB |
tolC |
mdtK |
mrkA |
mrkD |
fimH-1 |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| – | + | – | + | – | + | – | + | – | + | – | + | ||
| acrAB | – | 6 | 0 | 4 | 2 | 3 | 3 | 5 | 1 | 3 | 3 | 5 | 1 |
| + | 0 | 161 | 4 | 157 | 13 | 148 | 13 | 148 | 10 | 151 | 14 | 147 | |
| P value | NA | < 0.001 | 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||||
| tolC | – | 4 | 4 | 8 | 0 | 4 | 4 | 6 | 2 | 3 | 5 | 6 | 2 |
| + | 2 | 157 | 0 | 159 | 12 | 147 | 12 | 147 | 10 | 149 | 13 | 146 | |
| P value | < 0.001 | NA | < 0.001 | < 0.001 | 0.001 | < 0.001 | |||||||
| mdtK | – | 3 | 13 | 4 | 12 | 16 | 0 | 5 | 11 | 4 | 12 | 5 | 11 |
| + | 3 | 148 | 4 | 147 | 0 | 151 | 13 | 138 | 9 | 142 | 14 | 137 | |
| P value | 0.001 | < 0.001 | NA | 0.005 | 0.007 | 0.008 | |||||||
| mrkA | – | 5 | 13 | 6 | 12 | 5 | 13 | 18 | 0 | 4 | 14 | 6 | 12 |
| + | 1 | 148 | 2 | 147 | 11 | 138 | 0 | 149 | 9 | 140 | 13 | 136 | |
| P value | < 0.001 | < 0.001 | 0.005 | NA | 0.016 | 0.002 | |||||||
| mrkD | – | 3 | 10 | 3 | 10 | 4 | 9 | 4 | 9 | 13 | 0 | 8 | 5 |
| + | 3 | 151 | 5 | 149 | 12 | 142 | 14 | 140 | 0 | 154 | 11 | 143 | |
| P value | < 0.001 | 0.001 | 0.007 | 0.016 | NA | < 0.001 | |||||||
| fimh-1 | – | 5 | 14 | 6 | 13 | 5 | 14 | 6 | 13 | 8 | 11 | 19 | 0 |
| + | 1 | 147 | 2 | 146 | 11 | 137 | 12 | 136 | 5 | 143 | 0 | 148 | |
| P value | < 0.001 | < 0.001 | 0.008 | 0.002 | < 0.001 | NA | |||||||
“-”: absent. “+”: present. NA: not applicable. P value calculated using Chi-squared test. P values in bold are statistically significant.
4. Discussion
K. pneumoniae is an opportunistic pathogen that causes community-acquired along with healthcare-associated infections including bacteremia, pneumonia, and UTIs [32]. Efflux pump systems and biofilm formation are two major mechanisms contributing to resistance to antimicrobials among MDR K. pneumoniae [33]. Therefore, identifying the prevalence of MDR and biofilm-formation among K. pneumoniae isolates could enable better management of infections and the development of new preventive strategies. Moreover, findings of this study provide essential data for Jordan, a country in the middle east and north Africa (MENA) region and enable comparison with other countries and regions worldwide.
Among the study isolates, the highest nonsusceptibility was for azithromycin (87.4 %) and nitrofurantoin (85.0 %). While the highest susceptibility was for ertapenem (85.0 %) and chloramphenicol (81.4 %). Similar nonsusceptibility percentages to nitrofurantoin were previously reported in Iran (80 %) [34], India (73.1 %) [35], and Sudan (97.3 %) [36]. While, lower nitrofurantoin nonsusceptibility was observed in Pakistan (28.5 %) [37]. Consistent with our findings, a study from Sudan showed very high resistance to azithromycin (100 % of isolates were resistant) [38]. While, a study from Iran reported low resistance to azithromycin (20.2 %) [39]. The prevalence of MDR K. pneumoniae isolates was (75.4 %). Studies from other countries also showed a high incidence of MDR isolates; e.g., India at 90.2 % [35] and Egypt at 77.7 % [40]. In the present study, 32.9 % of isolates produced ESBLs, and 85.5 % of the ESBL positive isolates were MDR. Furthermore, a statistically significant association was observed between the MDR and ESBL phenotypes. Our MDR percentage was higher than that of India (52.4 %) [41] and Pakistan (58 %) [42], but lower than that of Brazil (84 %) [43] and China (89.5 %) [44]. There are several factors that potentially contribute to differences in the prevalence of ESBL and MDR phenotypes between countries worldwide. These include variations in the prevalent K. pneumoniae strains, differences in antibiotic prescribing practices among healthcare professionals, the range of available antimicrobials, variations in infection control practices, the socioeconomic characteristics of populations, the availability of high-quality health services, and possible variations in surveillance systems and detection methods, among others [[45], [46], [47]].
K. pneumoniae is known for its ability to form biofilms. In our study, 92.2 % of the isolates were biofilm producers. Among the biofilm producers, 56.3 %, 25.7 %, and 10.2 % were strong, moderate, and weak biofilm producers, respectively. Comparable percentages have been reported from Iran (33 % strong, 52.1 % moderate, and 8.5 % weak biofilm producers) [48], and Pakistan (64.7 % strong or moderate biofilm producers and 35.3 % weak biofilm producers) [49]. The urinary isolates demonstrated higher biofilm production than non-urinary isolates. However, the difference was not statistically significant. Likely due to the moderate sample size. In a study from China, 62.5 % of K. pneumoniae isolates recovered from urine, sputum, wound swabs, and blood were biofilm producers [50]. Differences in biofilm capacity between the isolates can be attributed to several factors including the sample source and the prevalence of different strains worldwide, among others [51].
Our results revealed no significant association between biofilm formation and ESBL production or the MDR phenotype. These results are in line with previous reports which indicated no statistically significant association between MDR phenotypes, biofilm formation, and ESBL production [52,53]. On the other hand, our results are in disagreement with Karimi et al. and Ostria-Hernandez et al. who showed a significant correlation between the MDR phenotype and the biofilm-forming ability of K. pneumoniae [54,55].
Efflux pumps are frequently involved in the development of MDR K. pneumoniae strains. These pumps can reduce the intracellular concentration of antimicrobials therefore enhancing bacterial survival. AcrAB and MdtK are two major efflux pumps in K. pneumoniae [56]. In our study, the most prevalent efflux pump gene was acrAB (96.4 %), followed by tolC (95.2 %), and mdtK (90.4 %). Similar percentages were reported in Saudi Arabia (acrAB at 93.3 % and tolC at 83.3 %) and Brazil (acrAB and tolC at 100 % each, and mdtK at 86 %) [43,57]. A study from Iran reported lower percentages for the efflux pump genes; acrAB (41 %), tolC (33 %), and mdtK (26 %) [58]. Our findings indicated a significant association between the MDR phenotype and tolC. Similarly, previous studies reported an association between tolC and MDR K. pneumoniae [43,59]. Furthermore, we showed that acrAB was significantly associated with nonsusceptibility to nitrofurantoin, and tolC was significantly associated with nonsusceptibility to azithromycin and nitrofurantoin. These results are in agreement with previous studies that showed an association between acrAB and tolC with nonsusceptibility to both azithromycin and nitrofurantoin [[60], [61], [62]]. None of the detected efflux pump genes in our study were associated with biofilm formation. In contrast to our findings, a study from Iraq reported a significant association between acrAB, tolC, and mdtK with biofilm formation in K. pneumoniae isolated from patients with cystitis [63].
In K. pneumoniae, mrkD, mrkA, and fimH-1 encode for Type 1 and 3 fimbrial adhesins which mediate binding to various biotic and abiotic surfaces and play an important role in biofilm formation [64]. In the present study, mrkD, mrkA, and fimH-1 were detected in most isolates (92.2 %, 89.2 %, and 88.6 %, respectively). For comparison, a high prevalence of biofilm-related genes was reported in Iran (mrkA at 100 %), mrkD at 100 %, and fimH-1 at 93.2 %) [39], Turkey (mrkD at 83.0 % and fimH-1 at 64.2 %) [65], China (mrkD at 95.8 % and fimH-1 at 91.6 %) [66], and Algeria (fimH-1 at 100 % and mrkD at 96.3 %) [23]. While, a low prevalence was reported in Pakistan (fimH-1 at 19 % and mrkD at 18 %) [67]. Murphy and colleagues confirmed that type 3 fimbria is a major colonization factor in biofilm-associated infections [68]. Our results revealed that mrkA was significantly associated with biofilm formation. Similar to our results, previous studies showed that mrkA was significantly associated with biofilm formation in K. pneumoniae [[69], [70], [71]]. In addition, fimH-1 was significantly associated with the MDR phenotype. A previous study from Iran reported that out of the 102 K. pneumoniae isolates, 50 (49 %) were MDR, and among the MDR isolates, 48 (96 %) carried fimH-1 [72]. We also found a significant association between mrkD and fimH-1 with nonsusceptibility to different antimicrobials such as ciprofloxacin and nitrofurantoin. Similarly, Ranjbar et al. reported high prevalence of mrkD and fimH-1 among K. pneumoniae isolates and an association with resistance to antimicrobials including ciprofloxacin, tobramycin, and aztreonam [73]. In our investigation, each of the efflux pump genes and biofilm-associated genes was significantly co-associated with each other. In-line with our findings, a report from Iran showed an association between mrkA and mrkD with antimicrobials’ resistance genes [22].
In the present study, several reliable methods were used. The antimicrobial susceptibility profiles of K. pneumoniae isolates were determined using the Kirby-Bauer method and the double disk synergy test. The quantitative biofilm formation assay was used to evaluate biofilm-formation among the isolates. Furthermore, conventional PCR was used to confirm the isolates’ species identity and to assess the distribution of efflux pump and biofilm-associated genes. However, our study had some limitations. The study had a modest research budget. The number of clinical isolates included in this study was moderate and the isolates were mostly from urine. The study involved only specific regions and hospitals in Northern Jordan which potentially limits the generalizability of the findings to other regions or healthcare settings. In our study, due to budget limitations we were not able to use techniques such as Real-Time PCR which allows detection of gene expression among the isolates. Additionally, we only investigated the presence of three efflux pump genes and three biofilm-associated genes and their association with biofilm formation and MDR phenotypes. However, other genes in K. pneumoniae might be related to biofilm formation and MDR phenotypes. Therefore, future studies could include additional virulence genes, a larger sample size from a variety of clinical sample sources, a wider array of analyses, and possibly encompass other species from different settings.
In conclusion, among 167 K. pneumoniae isolates, high percentages of antimicrobial nonsusceptibility and multidrug resistance were observed. Approximately one third of the isolates were ESBL producers and a significant association was observed between the MDR phenotype and ESBL production. The majority of the isolates were biofilm producers. All investigated efflux and biofilm genes were detected at high frequencies. The presence of the mrkA was significantly associated with biofilm formation. The presence of efflux pump genes and biofilm-associated genes was significantly associated with nonsusceptibility to nitrofurantoin and ciprofloxacin. The tolC efflux pump gene was significantly associated with nonsusceptibility to azithromycin. Additionally, strong co-associations of efflux pump genes were observed with biofilm-associated genes.
Data availability statement
Data included in article/supp. material/referenced in article.
Ethics statement
Approval to conduct the study was granted by the IRBcommittee of Jordan University of Science and Technology (Approval ID 38/158/2023).
CRediT authorship contribution statement
Samer F. Swedan: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Dima B. Aldakhily: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment/Funding Statement
This study was supported by the deanship of research at Jordan University of Science and Technology (Grant no. 20230262)
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e34370.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
References
- 1.Chiu S.K., Ma L., Chan M.C., Lin Y.T., Fung C.P., Wu T.L., et al. Carbapenem nonsusceptible Klebsiella pneumoniae in Taiwan: dissemination and increasing resistance of carbapenemase producers during 2012-2015. Sci. Rep. 2018;8(1):8468. doi: 10.1038/s41598-018-26691-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Xu L., Sun X., Ma X. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae. Ann. Clin. Microbiol. Antimicrob. 2017;16(1):18. doi: 10.1186/s12941-017-0191-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Anderl J.N., Franklin M.J., Stewart P.S. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 2000;44(7):1818–1824. doi: 10.1128/AAC.44.7.1818-1824.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Srinivasan V.B., Singh B.B., Priyadarshi N., Chauhan N.K., Rajamohan G. Role of novel multidrug efflux pump involved in drug resistance in Klebsiella pneumoniae. PLoS One. 2014;9(5) doi: 10.1371/journal.pone.0096288. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann. Med. 2007;39(3):162–176. doi: 10.1080/07853890701195262. [DOI] [PubMed] [Google Scholar]
- 6.Pages J.M., Lavigne J.P., Leflon-Guibout V., Marcon E., Bert F., Noussair L., et al. Efflux pump, the masked side of beta-lactam resistance in Klebsiella pneumoniae clinical isolates. PLoS One. 2009;4(3) doi: 10.1371/journal.pone.0004817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Pakzad I., Zayyen Karin M., Taherikalani M., Boustanshenas M., Lari A.R. Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients. GMS Hyg Infect Control. 2013;8(2) doi: 10.3205/dgkh000215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Muhsin E.A., Sajid Al-Jubori S., Abdulhemid Said L. Prevalence of efflux pump and porin-related antimicrobial resistance in clinical Klebsiella pneumoniae in baghdad, Iraq. Arch Razi Inst. 2022;77(2):785–798. doi: 10.22092/ARI.2022.356976.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Flemming H.C., Wingender J., Szewzyk U., Steinberg P., Rice S.A., Kjelleberg S. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 2016;14(9):563–575. doi: 10.1038/nrmicro.2016.94. [DOI] [PubMed] [Google Scholar]
- 10.Van Acker H., Van Dijck P., Coenye T. Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends Microbiol. 2014;22(6):326–333. doi: 10.1016/j.tim.2014.02.001. [DOI] [PubMed] [Google Scholar]
- 11.Piperaki E.T., Syrogiannopoulos G.A., Tzouvelekis L.S., Daikos G.L. Klebsiella pneumoniae: virulence, biofilm and antimicrobial resistance. Pediatr. Infect. Dis. J. 2017;36(10):1002–1005. doi: 10.1097/INF.0000000000001675. [DOI] [PubMed] [Google Scholar]
- 12.Bandeira M., Carvalho P.A., Duarte A., Jordao L. Exploring dangerous connections between Klebsiella pneumoniae biofilms and healthcare-associated infections. Pathogens. 2014;3(3):720–731. doi: 10.3390/pathogens3030720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Chung P.Y. The emerging problems of Klebsiella pneumoniae infections: carbapenem resistance and biofilm formation. FEMS Microbiol. Lett. 2016;363(20) doi: 10.1093/femsle/fnw219. [DOI] [PubMed] [Google Scholar]
- 14.Galdiero E., Di Onofrio V., Maione A., Gambino E., Gesuele R., Menale B., et al. Allium ursinum and allium oschaninii against Klebsiella pneumoniae and Candida albicans mono- and polymicrobic biofilms in in vitro static and dynamic models. Microorganisms. 2020;8(3) doi: 10.3390/microorganisms8030336. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Subramanian P., Shanmugam N., Sivaraman U., Kumar S., Selvaraj S. Antiobiotic resistance pattern of biofilm-forming uropathogens isolated from catheterised patients in Pondicherry, India. Australas. Med. J. 2012;5(7):344–348. doi: 10.4066/AMJ.2012.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Livrelli V., De Champs C., Di Martino P., Darfeuille-Michaud A., Forestier C., Joly B. Adhesive properties and antibiotic resistance of Klebsiella, Enterobacter, and Serratia clinical isolates involved in nosocomial infections. J. Clin. Microbiol. 1996;34(8):1963–1969. doi: 10.1128/jcm.34.8.1963-1969.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Vuotto C., Longo F., Balice M.P., Donelli G., Varaldo P.E. Antibiotic resistance related to biofilm formation in Klebsiella pneumoniae. Pathogens. 2014;3(3):743–758. doi: 10.3390/pathogens3030743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Sahoo R.K., Das A., Gaur M., Pattanayak A., Sahoo S., Debata N.K., et al. Genotypic validation of extended-spectrum beta-lactamase and virulence factors in multidrug resistance Klebsiella pneumoniae in an Indian hospital. Pathog. Glob. Health. 2019;113(7):315–321. doi: 10.1080/20477724.2019.1705020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Shao L., Yao B., Yang J., Li X., Ye K., Zhang Y., et al. Characterization of a multidrug-resistant Klebsiella pneumoniae ST3330 clone responsible for a nosocomial outbreak in a neonatal intensive care unit. Ann. Palliat. Med. 2020;9(3):1092–1102. doi: 10.21037/apm-20-958. [DOI] [PubMed] [Google Scholar]
- 20.Khosravi M., Mirzaie A., Kashtali A.B., Noorbazargan H. Antibacterial, anti-efflux, anti-biofilm, anti-slime (exopolysaccharide) production and urease inhibitory efficacies of novel synthesized gold nanoparticles coated Anthemis atropatana extract against multidrug- resistant Klebsiella pneumoniae strains. Arch. Microbiol. 2020;202(8):2105–2115. doi: 10.1007/s00203-020-01930-y. [DOI] [PubMed] [Google Scholar]
- 21.Lv F., Cai J., He Q., Wang W., Luo Y., Wang X., et al. Overexpression of efflux pumps mediate Pan resistance of Klebsiella pneumoniae sequence type 11. Microb. Drug Resist. 2021;27(10):1405–1411. doi: 10.1089/mdr.2020.0395. [DOI] [PubMed] [Google Scholar]
- 22.Bakhtiari R., Javadi A., Aminzadeh M., Molaee-Aghaee E., Shaffaghat Z. Association between presence of RmpA, MrkA and MrkD genes and antibiotic resistance in clinical Klebsiella pneumoniae isolates from hospitals in tehran, Iran. Iran. J. Public Health. 2021;50(5):1009–1016. doi: 10.18502/ijph.v50i5.6118. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.El Fertas-Aissani R., Messai Y., Alouache S., Bakour R. Virulence profiles and antibiotic susceptibility patterns of Klebsiella pneumoniae strains isolated from different clinical specimens. Pathol. Biol. 2013;61(5):209–216. doi: 10.1016/j.patbio.2012.10.004. [DOI] [PubMed] [Google Scholar]
- 24.Dan B., Dai H., Zhou D., Tong H., Zhu M. Relationship between drug resistance characteristics and biofilm formation in Klebsiella pneumoniae strains. Infect. Drug Resist. 2023;16(null):985–998. doi: 10.2147/IDR.S396609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Ashwath P., Deekshit V.K., Rohit A., Dhinakaran I., Karunasagar I., Karunasagar I., et al. Biofilm Formation and associated gene expression in multidrug-resistant Klebsiella pneumoniae isolated from clinical specimens. Curr. Microbiol. 2022;79(3):73. doi: 10.1007/s00284-022-02766-z. [DOI] [PubMed] [Google Scholar]
- 26.Yazgan B., Turkel I., Guckan R., Kilinc K., Yildirim T. Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae. J Infect Dev Ctries. 2018;12(3):156–163. doi: 10.3855/jidc.9677. [DOI] [PubMed] [Google Scholar]
- 27.Magiorakos A.P., Srinivasan A., Carey R.B., Carmeli Y., Falagas M.E., Giske C.G., et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012;18(3):268–281. doi: 10.1111/j.1469-0691.2011.03570.x. [DOI] [PubMed] [Google Scholar]
- 28.O'Toole G.A. Microtiter dish biofilm formation assay. J. Vis. Exp. 2011;(47) doi: 10.3791/2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Leoney A., Karthigeyan S., Asharaf A.S., Felix A.J.W. Detection and categorization of biofilm-forming Staphylococcus aureus, viridans streptococcus, Klebsiella pneumoniae, and Escherichia coli isolated from complete denture patients and visualization using scanning electron microscopy. J. Int. Soc. Prev. Community Dent. 2020;10(5):627–633. doi: 10.4103/jispcd.JISPCD_256_20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Green M.R., Sambrook J. fourth ed. Cold Spring Harbor Laboratory Press; 2012. Molecular Cloning: A Laboratory Manual. [Google Scholar]
- 31.Sambrook J., Russell D.W. Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc. 2006;(1) doi: 10.1101/pdb.prot4455. 2006. [DOI] [PubMed] [Google Scholar]
- 32.Marques C., Menezes J., Belas A., Aboim C., Cavaco-Silva P., Trigueiro G., et al. Klebsiella pneumoniae causing urinary tract infections in companion animals and humans: population structure, antimicrobial resistance and virulence genes. J. Antimicrob. Chemother. 2019;74(3):594–602. doi: 10.1093/jac/dky499. [DOI] [PubMed] [Google Scholar]
- 33.Tang M., Wei X., Wan X., Ding Z., Ding Y., Liu J. The role and relationship with efflux pump of biofilm formation in Klebsiella pneumoniae. Microb. Pathog. 2020;147 doi: 10.1016/j.micpath.2020.104244. [DOI] [PubMed] [Google Scholar]
- 34.Babakhani S., Derikvand S.S., Nazer M.R., Kazemi M.J. Comparison frequency and determination antibiotic resistance pattern of Klebsiella spp. isolated from Nosocomial infection in Khorramabad Shohadaye Ashayer hospital. Bull Env Pharmacol Life Sci. 2014;3(12):149–154. [Google Scholar]
- 35.N G.M., G C.M., Nagshetty K., Patil S.A., Gaddad S.M., Shivannavar C.T. Antibiotic susceptibility pattern of ESbetaL producing Klebsiella pneumoniae isolated from urine samples of pregnant women in Karnataka. J. Clin. Diagn. Res. 2014;8(10):DC08–11. doi: 10.7860/JCDR/2014/9594.5048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Ibrahim M.E., Bilal N.E., Magzoub M.A., Hamid M.E. Prevalence of extended-spectrum beta-Lactamases-producing Escherichia coli from hospitals in khartoum state, Sudan. Oman Med. J. 2013;28(2):116–120. doi: 10.5001/omj.2013.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Ejaz H., Zafa A., Mahmood S., Javed M.M. Urinary tract infections caused by extended spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae. Afr. J. Biotechnol. 2011;10(73):16661–16666. [Google Scholar]
- 38.Abdellatif A O., Ali I N., Mohamed M A., Ibrahim M A., Mohamed W A. Evaluation of the antibiotic resistance pattern at the medical services administration hospital in khartoum, Sudan, 2021. Journal for Research in Applied Sciences and Biotechnology. 2022;1(4):50–56. doi: 10.55544/jrasb.1.4.6. [DOI] [Google Scholar]
- 39.Khoshnood S., Akrami S., Saki M., Motahar M., Masihzadeh S., Daneshfar S., et al. Molecular evaluation of aminoglycosides resistance and biofilm formation in Klebsiella pneumoniae clinical isolates: a cross-sectional study. Health Sci Rep. 2023;6(5) doi: 10.1002/hsr2.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Wasfi R., Elkhatib W.F., Ashour H.M. Molecular typing and virulence analysis of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from Egyptian hospitals. Sci. Rep. 2016;6(1) doi: 10.1038/srep38929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Navaneeth B.V. Extended-spectrum and AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae from a rural South Indian tertiary care hospital. Int. J. Antimicrob. Agents. 2007;29(5):602–603. doi: 10.1016/j.ijantimicag.2006.12.004. [DOI] [PubMed] [Google Scholar]
- 42.Ullah F., Malik S.A., Ahmed J. Antimicrobial susceptibility pattern and ESBL prevalence in Klebsiella pneumoniae from urinary tract infections in the North-West of Pakistan. Afr. J. Microbiol. Res. 2009;3(11):676–680. [Google Scholar]
- 43.Ferreira R.L., da Silva B.C.M., Rezende G.S., Nakamura-Silva R., Pitondo-Silva A., Campanini E.B., et al. High prevalence of multidrug-resistant Klebsiella pneumoniae harboring several virulence and beta-lactamase encoding genes in a Brazilian intensive care unit. Front. Microbiol. 2018;9:3198. doi: 10.3389/fmicb.2018.03198. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Hou X.H., Song X.Y., Ma X.B., Zhang S.Y., Zhang J.Q. Molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates. Braz. J. Microbiol. 2015;46(3):759–768. doi: 10.1590/S1517-838246320140138. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Quan J., Zhao D., Liu L., Chen Y., Zhou J., Jiang Y., et al. High prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae in community-onset bloodstream infections in China. J. Antimicrob. Chemother. 2017;72(1):273–280. doi: 10.1093/jac/dkw372. [DOI] [PubMed] [Google Scholar]
- 46.Calbo E., Garau J. The changing epidemiology of hospital outbreaks due to ESBL-producing Klebsiella pneumoniae: the CTX-M-15 type consolidation. Future Microbiol. 2015;10(6):1063–1075. doi: 10.2217/fmb.15.22. [DOI] [PubMed] [Google Scholar]
- 47.Bassetti M., Righi E., Carnelutti A., Graziano E., Russo A. Multidrug-resistant Klebsiella pneumoniae: challenges for treatment, prevention and infection control. Expert Rev. Anti Infect. Ther. 2018;16(10):749–761. doi: 10.1080/14787210.2018.1522249. [DOI] [PubMed] [Google Scholar]
- 48.Seifi K., Kazemian H., Heidari H., Rezagholizadeh F., Saee Y., Shirvani F., et al. Evaluation of biofilm formation among Klebsiella pneumoniae isolates and molecular characterization by ERIC-PCR. Jundishapur J. Microbiol. 2016;9(1) doi: 10.5812/jjm.30682. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Hassan A., Usman J., Kaleem F., Omair M., Khalid A., Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz. J. Infect. Dis. 2011;15(4):305–311. [PubMed] [Google Scholar]
- 50.Yang D., Zhang Z. Biofilm-forming Klebsiella pneumoniae strains have greater likelihood of producing extended-spectrum beta-lactamases. J. Hosp. Infect. 2008;68(4):369–371. doi: 10.1016/j.jhin.2008.02.001. [DOI] [PubMed] [Google Scholar]
- 51.Cherif-Antar A., Moussa–Boudjemâa B., Didouh N., Medjahdi K., Mayo B., Flórez A.B. Diversity and biofilm-forming capability of bacteria recovered from stainless steel pipes of a milk-processing dairy plant. Dairy Sci. Technol. 2015;96(1):27–38. doi: 10.1007/s13594-015-0235-4. [DOI] [Google Scholar]
- 52.Cepas V., Lopez Y., Munoz E., Rolo D., Ardanuy C., Marti S., et al. Relationship between biofilm formation and antimicrobial resistance in gram-negative bacteria. Microb. Drug Resist. 2019;25(1):72–79. doi: 10.1089/mdr.2018.0027. [DOI] [PubMed] [Google Scholar]
- 53.Dumaru R., Baral R., Shrestha L.B. Study of biofilm formation and antibiotic resistance pattern of gram-negative Bacilli among the clinical isolates at BPKIHS, Dharan. BMC Res. Notes. 2019;12(1):38. doi: 10.1186/s13104-019-4084-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Karimi K., Zarei O., Sedighi P., Taheri M., Doosti-Irani A., Shokoohizadeh L. Investigation of antibiotic resistance and biofilm formation in clinical isolates of Klebsiella pneumoniae. International journal of microbiology. 2021;2021 doi: 10.1155/2021/5573388. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Ostria-Hernandez M.L., Juarez-de la Rosa K.C., Arzate-Barbosa P., Lara-Hernandez A., Sakai F., Ibarra J.A., et al. Nosocomial, multidrug-resistant Klebsiella pneumoniae strains isolated from Mexico City produce robust biofilms on abiotic surfaces but not on human lung cells. Microb. Drug Resist. 2018;24(4):422–433. doi: 10.1089/mdr.2017.0073. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56.Maurya N., Jangra M., Tambat R., Nandanwar H. Alliance of efflux pumps with beta-lactamases in multidrug-resistant Klebsiella pneumoniae isolates. Microb. Drug Resist. 2019;25(8):1155–1163. doi: 10.1089/mdr.2018.0414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Lagha R., Ben Abdallah F., Aah A.L., Amor N., Hassan M.M., Mabrouk I., et al. Molecular characterization of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from king abdulaziz specialist hospital at taif city, Saudi Arabia. J Infect Public Health. 2021;14(1):143–151. doi: 10.1016/j.jiph.2020.12.001. [DOI] [PubMed] [Google Scholar]
- 58.Mirzaie A., Ranjbar R. Antibiotic resistance, virulence-associated genes analysis and molecular typing of Klebsiella pneumoniae strains recovered from clinical samples. Amb. Express. 2021;11(1):122. doi: 10.1186/s13568-021-01282-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Mohammed N., Samad A.B.A., Hussien A. Detection of some virulence gene in multidrug-resistant Klebsiella pneumoniae isolated from urethritis patients in wasit province, Iraq. Journal of Survey in Fisheries Sciences. 2023:503–517. doi: 10.17762/sfs.v10i1.619. [DOI] [Google Scholar]
- 60.Jabar R.M.A., Hassoon A.H. The expression of efflux pump AcrAB in MDR Klebsiella pneumoniae isolated from Iraqi patients. J. Pharmaceut. Sci. Res. 2019;11(2):423–428. [Google Scholar]
- 61.Xu Q., Jiang J., Zhu Z., Xu T., Sheng Z.K., Ye M., et al. Efflux pumps AcrAB and OqxAB contribute to nitrofurantoin resistance in an uropathogenic Klebsiella pneumoniae isolate. Int. J. Antimicrob. Agents. 2019;54(2):223–227. doi: 10.1016/j.ijantimicag.2019.06.004. [DOI] [PubMed] [Google Scholar]
- 62.Xu Q., Sheng Z., Hao M., Jiang J., Ye M., Chen Y., et al. RamA upregulates multidrug resistance efflux pumps AcrAB and OqxAB in Klebsiella pneumoniae. Int. J. Antimicrob. Agents. 2021;57(2) doi: 10.1016/j.ijantimicag.2020.106251. [DOI] [PubMed] [Google Scholar]
- 63.Amer Ali S., Al-Dahmoshi H.O.M. Molecular study on Efflux pumps of Klebsiella pneumonia Isolated from patients with Cystitis. Res. J. Pharm. Technol. 2022;15(10):4559–4564. doi: 10.52711/0974-360x.2022.00765. [DOI] [Google Scholar]
- 64.Paczosa M.K., Mecsas J. Klebsiella pneumoniae: going on the offense with a strong defense. Microbiol. Mol. Biol. Rev. : MMBR (Microbiol. Mol. Biol. Rev.) 2016;80(3):629–661. doi: 10.1128/MMBR.00078-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65.Kus H., Arslan U., Turk Dagi H., Findik D. [Investigation of various virulence factors of Klebsiella pneumoniae strains isolated from nosocomial infections] Mikrobiyoloji Bulteni. 2017;51(4):329–339. doi: 10.5578/mb.59716. [DOI] [PubMed] [Google Scholar]
- 66.Liu X., Zhang J., Li Y., Shen Q., Jiang W., Zhao K., et al. Diversity and frequency of resistance and virulence genes in bla (KPC) and bla (NDM) co-producing Klebsiella pneumoniae strains from China. Infect. Drug Resist. 2019;12(null):2819–2826. doi: 10.2147/IDR.S214960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.Imtiaz W., Syed Z., Rafaque Z., Andrews S.C., Dasti J.I. Analysis of antibiotic resistance and virulence traits (Genetic and phenotypic) in Klebsiella pneumoniae clinical isolates from Pakistan: identification of significant levels of carbapenem and colistin resistance. Infect. Drug Resist. 2021;14:227–236. doi: 10.2147/IDR.S293290. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 68.Murphy C.N., Mortensen M.S., Krogfelt K.A., Clegg S. Role of Klebsiella pneumoniae type 1 and type 3 fimbriae in colonizing silicone tubes implanted into the bladders of mice as a model of catheter-associated urinary tract infections. Infect. Immun. 2013;81(8):3009–3017. doi: 10.1128/IAI.00348-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 69.Fang R., Liu H., Zhang X., Dong G., Li J., Tian X., et al. Difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution. Microb. Pathog. 2021;152 doi: 10.1016/j.micpath.2021.104743. [DOI] [PubMed] [Google Scholar]
- 70.Vuotto C., Longo F., Pascolini C., Donelli G., Balice M.P., Libori M.F., et al. Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains. J. Appl. Microbiol. 2017;123(4):1003–1018. doi: 10.1111/jam.13533. [DOI] [PubMed] [Google Scholar]
- 71.Gual-de-Torrella A., Delgado-Valverde M., Perez-Palacios P., Oteo-Iglesias J., Rojo-Molinero E., Macia M.D., et al. Prevalence of the fimbrial operon mrkABCD, mrkA expression, biofilm formation and effect of biocides on biofilm formation in carbapenemase-producing Klebsiella pneumoniae isolates belonging or not belonging to high-risk clones. Int. J. Antimicrob. Agents. 2022;60(4) doi: 10.1016/j.ijantimicag.2022.106663. [DOI] [PubMed] [Google Scholar]
- 72.Pourmohammad-Hosseini G., Ghandehari F., Hoveida L. The abundance of capsule (wabG) and fimbria (fimH) coding genes in multidrug-resistant Klebsiella pneumoniae strains isolated from patients admitted to Isfahan hospitals. Microbiology, Metabolites and Biotechnology. 2023;6(1):27–34. doi: 10.22104/MMB.2023.6333.1111. [DOI] [Google Scholar]
- 73.Ranjbar R., Fatahian Kelishadrokhi A., Chehelgerdi M. Molecular characterization, serotypes and phenotypic and genotypic evaluation of antibiotic resistance of the Klebsiella pneumoniae strains isolated from different types of hospital-acquired infections. Infect. Drug Resist. 2019;12(null):603–611. doi: 10.2147/IDR.S199639. [DOI] [PMC free article] [PubMed] [Google Scholar]
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