study of Plasmid-Mediated Quinolone Resistance in Klebsiella pneumoniae : Relation to Extended-spectrum Beta-lactamases

Klebsiella pneumoniae ( K. pneumoniae ) is an important pathogen associated with various infections. The emergence of antibiotic resistances, such as quinolone resistance and those due to extended-spectrum beta-lactamases (ESBL), reduces the available choices for treatment. The objectives of the current study include the evaluation of the prevalence of the plasmid-mediated quinolone resistance genes qepA, acrA, acrB


iNtROduCtiON
Klebsiella pneumoniae (K.pneumoniae) are non-motile, encapsulated gram-negative bacilli that may be found in the environment, such as soil and water, and on medical devices. 1,2. pneumoniae is associated with various human infections, such as urinary tract, respiratory tract, wound, and blood stream infections.Antibiotic therapies against K. pneumoniae include cephalosporins, aminoglycosides, and quinolones.However, resistance to commonly used antibiotics has emerged. 3,4luoroquinolone resistance is among the antibiotic resistances identified in K. pneumoniae in the last few years.Fluoroquinolone resistance can be mediated via two different mechanisms: through mutations in the chromosomally encoded genes for gyrase and topoisomerase IV enzymes or through plasmid-mediated quinolone resistance (PMQR) determinants, namely, Qnr determinants, QepA and AcrAB efflux pumps, and aminoglycosideacetyltransferase Ib-cr enzymes. 5QepA efflux pumps extrude antibiotics to the extracellular environment, leading to decreased antibiotic activity.The other efflux system, AcrAB, acts as a chemical transporter that transports various antibiotics to the extracellular environment, leading to antibiotic resistance. 6,7AcrAB consists of an outer membrane component TolC, an inner membrane transporter AcrB, and a periplasmic protein AcrA that bridges the two integrated membrane components.aac(6')-Ib-cr encodes an acetyltransferase AAC(6')-Ib variant with substitutions in tryptophan 102 to arginine and in aspartic acid 179 to tyrosine.These substitutions lead to fluoroquinolone acetylation and to a reduction in susceptibility to fluoroquinolones and aminoglycosides, such as tobramycin, kanamycin, and amikacin. 8Moreover, there are reports about the association between the presence of quinolone resistance and extended-spectrum beta-lactamases (ESBL) in K. pneumoniae. 9,10he objectives of the current study were to evaluate the prevalence of the PMQR genes qepA, acrA, acrB, and aac(6')-Ib-cr by polymerase chain reaction (PCR) in K. pneumoniae and to determine their relationship with the ESBL phenotype and with other antibiotic resistances.

Bacterial strains
The analysis included 300 isolates of K. pneumoniae obtained from different clinical samples (100 blood, 80 urine, 70 wound, and 50 sputum cultures) from King Fahd Hospital, Jazan, Saudi Arabia, from January 2018 to May 2020.K. pneumoniae isolates were identified using standard microbiological techniques, including Gram staining and manual biochemical identification methods (according to the Clinical and Laboratory Standards Institute [CLSI] guidelines). 11Biochemical identification included the use of triple sugar iron agar, a motility test, the Simmons citrate test, and ornithine and lysine decarboxylation tests (Oxoid-Thermo Fisher Scientific, USA).Escherichia coli (E.coli) ATCC 25922 was used as the positive control for all biochemical reactions.

Antibiotic susceptibility tests by the disc Diffusion Method
Antibiotic susceptibility tests were performed according to the CLSI guidelines using the disc diffusion method. 11Briefly, pure colonies of K. pneumoniae were used for the preparation of cultures with a turbidity equivalent of 0.5 McFarland standards; cultures were spread over Mueller-Hinton agar.Discs were either impregnated with 5 μg of cefoxitin, 10 μg of imipenem, 20 or 10 μg of amoxicillin/clavulanate, or 30 μg of ceftazidime, tetracycline, cefotaxime, trimethoprim/sulfamethoxazole, gentamicin, amikacin, or cefepime (Oxoid-Thermo Fisher Scientific,).For antibiotic susceptibility testing, Escherichia coli strain ATCC 25922 was used as the quality control strain.

Minimum Inhibitory Concentrations (MICs) of the Quinolone Ciprofloxacin
The microdilution method (according to the CLSI guidelines) was used to determine the MIC of ciprofloxacin for the isolates. 11The results were interpreted according to the CLSI guidelines; isolates were classified as resistant to ciprofloxacin at MICs ≥ 4.0 μg/mL, as ciprofloxacin sensitive at MICs ≤ 1.0 μg/mL, and as ciprofloxacin intermediates at MICs between 1 and 2.0 μg/mL.E. coli ATCC 25922 was used as the quality control strain.

Determination of Extended-Spectrum Beta-Lactamase (ESBL) activity in K. pneumoniae
The double disc diffusion method was used to determine the ESBL activity for isolates of K. pneumoniae resistant to ceftazidime and cefotaxime according to the CLSI guidelines.The double disc method was performed using discs impregnated with 30 μg of cefotaxime and 30 μg of ceftazidime alone or in combination with 10 μg of clavulanic acid.An increase of 5 mm or more in the inhibition zone around the discs after combining the beta-lactam antibiotic with clavulanic acid was considered positive for ESBL activity. 11The ESBLpositive and negative control strains used in this study were E. coli ATCC 25922 and K. pneumoniae ATCC 700603, respectively.

Determination of PMQR genes in K. pneumoniae DNA Extraction
DNA from isolated colonies of K. pneumoniae was extracted using a Qiagen DNA extraction kit (Qiagen, Hampshire, United Kingdom).Extracted DNA was kept frozen at -20°C till amplification.

Molecular identification of K. pneumoniae
Table 1 shows the results from the molecular identification of K. pneumoniae isolates by PCR using universal 16S rRNA bacterial primers (16S forward and reverse primers).The PCR was carried out in a total volume of 50 μL using Dream Tag Green Master Mix (Thermo Fisher Scientific) containing 25 μL of master mix ready to use, 1 μL (10 pmol) of each reverse and forward primers, 5 μL of DNA template, and 18 μL of nuclease-free water.The mixture was subjected to an initial denaturation process at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 56°C, and primer extension at 72°C for 1 min, and a final extension cycle at 72°C for 5 min.Negative and positive controls were included in each experiment.PCR products were analyzed by gel electrophoresis using 1.5% (wt/vol) agarose gels in Tris-Acetate EDTA buffer stained with ethidium bromide and visualized using a Gel Doc XR imaging system (Bio-Rad).

Standard Sequencing and Data Analysis
The PCR products were subjected to a high purification process.An ABI PRISM® 3730XL Analyzer with 96 capillary type using a BigDyeTM Terminator Cycle Sequencing Kit with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems) was used to perform sequencing reactions according to the manufacturer's protocols.Single-pass sequencing was performed on each template using 16S F primers.Gel elution was performed using the MG Gel Extraction SV (MD007) kit (MGmed), following the manufacturer's instructions.
The sequence similarity was examined and compared to the reference sequences through the Basic local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST/) and GenBank (www.ncbi.nlm.nih.gov/genbank/). 12n evolutionary tree was constructed for the sequenced samples using molecular evolutionary genetic analysis (MEGA 5).

PCR for PMQR gene detection in K. pneumoniae
The amplification of qepA, acrA, aac(6')-Ib-cr, and acrB was carried out using the primers listed in Table 1.A Qiagen (ready to use) amplification mixture (25 µL) was used for the entire process.
The amplification process was performed as follows: an initial denaturation step at 94°C for 5 min, 36 amplification cycles consisting of 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C, and a final extension step at 72°C for 5 min. 8el electrophoresis of the amplification products was carried out on a 1% agarose gel stained with ethidium bromide.
Positive PCR products were purified, and direct sequencing was performed to confirm the positive results (Thermo Fisher Scientific).

Statistical Analysis
The data were analyzed using the Statistical Package for the Social Sciences (SPPS) 22 and were stated as number and percentage.The results were compared using the chi-square test, P values < 0.05 were considered as statistically significant.

Molecular identification
To confirm the biochemical identification results, approximately 1500 bp of the 16S gene of each isolate were amplified (Fig. 1) and partial sequencing of the PCR products using the 16S forward primer was performed.BLAST search results showed that the 16S sequences from the isolates from the Jazan region aligned with many K. pneumoniae sequences published in GenBank (Table 2).Sequences were also used to determine the evolutionary relationship between K. pneumoniae isolates from the Jazan region and other isolates from GenBank (Fig. 2).
A PCR study searching for PMQR genes was positive in 233 of the isolates.acrA was found in 74.3% of the isolates, whereas aac(6')-Ib-cr, acrB, and qepA were found in 73.7%, 71%, and 6.7% of the K. pneumoniae isolates, respectively (Table 4).K. pneumoniae positive for PMQR genes represented 82.7% of the isolates that were also positive for ESBL, as determined by the double disc method (Fig. 3).
Antibiotic resistance was compared between PMQR-positive and negative K. pneumoniae isolates (Table 5).The results showed that the isolates of K. pneumoniae positive for PMQR genes had significantly increased resistance to amikacin (P = 0.0001), amoxicillin/clavulanate (P = 0.0001), gentamicin (P = 0.001), and cefoxitin (P = 0.002).disCussiON Recently, there has been an increase in worldwide reports on PMQR genes in K. pneumoniae.Most of these studies report a higher prevalence of PMQR genes in K. pneumoniae compared than in E. coli. 13,14However, the rates of prevalence vary considerably with the geographic area. 13,15n the present study, 80% of the K. pneumoniae isolates were found to be resistant to quinolones, according to the MIC study.7][18] Resistance to fluoroquinolone may be attributed to improper use of quinolone, especially in urinary tract infections, leading to the transference of resistance genes to other susceptible Enterobacteriaceae by horizontal plasmid transfer.
A significant association between ESBL production and the presence of PMQR genes was found (P = 0.01).][21][22] Moreover, a recent study reported that all K. pneumoniae strains with quinolone resistance genes were also ESBL strains. 10The co-resistance to fluoroquinolones and beta-lactams was explained by the presence of quinolone-resistant genes, including ESBL determinants, in identical mobile genetic elements. 10he molecular investigation done in the present study revealed that the acrA and acrB frequencies in K. pneumoniae were 74.3% and 71%, respectively.][25] The second most prevalent gene was aac(6')-Ib-cr.Previous studies also reported a high frequency of aac(6')-Ib-cr associated with quinolone resistance. 87][28] Therefore, in addition to quinolone resistance, PMQR determinants can play a significant role in resistance to other antibiotics, particularly β-lactams and aminoglycosides.This hypothesis supports the finding of increased resistance to amikacin and gentamicin among isolates positive for quinolone resistance genes.
The least frequently determined gene was qepA.][31] The current study highlights the emergence of the quinolone resistance plasmid-

Table 1 .
Genes, primers and the amplified base pair (bp)

Table 2 .
Similarity to some K. pneumoniae isolates from Gen Bank

Table 3 .
Prevalence of ESBL and quinolone resistance among isolated K. pneumoniae

table 5 .
Comparison of antibiotics resistance between positive versus negative K. pneumoniae for the studied genes