INTRODUCTION:

Worldwide, one of the major bacterial diseases influencing the existences of 150-million- man yearly is the UTI, which can be complicated or uncomplicated. Typically, the latter affect people who are healthy and have no neurological or structural urinary tract abnormalities^1,2. Upper UTIs pyelonephritis) and lower UTIs cystitis) are the two types of such infections. On the other hand, urinary retention resulted from neurological diseases, urinary obstruction, renal failure, pregnancy, renal transplantation, immunosuppression, and the existence of foreign bodies like indwelling catheters, calculi, or other drainage devices are all examples of the former^3,4.

According to Bao 2020⁵, a 63-year-old female patient developed a recurrent UTI with extensive drug resistance K. pneumoniae ERkp). The pathogen responsible for UTIs was identified as ERKp, which was resistant to all antibiotics tested apart from polymyxin B and tigecycline. The patient's clinical history incorporated a long history of hypertension and type-2 diabetes. The major facilitator superfamily MFS), ATP binding cassette ABC) superfamily, the small multidrug resistance SMR) family, the Multidrug and toxic- compound extrusion MATE) family, and the resistance nodulation division RND) family are the five families of efflux pump systems. The arrangement of biofilms, joined with possessing of efflux siphons, lessens bacterial anti-toxin affectability to antibiotics^6,7,8.

The goal of this study is to look into the most common efflux pump genes from five different classes in K. Pneumoniae.

METHODOLOGY:

Bacterial Isolates Identification:

Fifty K. pneumoniae isolates were collected from patient with cystitis. First screening on MacConkey and EMB agar. Confirmation of E. coli was done via PCR using primer pairs of ptyr B: Forward primer: (GGCTGTACTACAACGATGAC), Reverse primer: (TTGAGCAGGTAATCCACTTTG) to give 931bp product at annealing temperature that was 56.5°C⁹.

Antibiotic Susceptibility Test:

Susceptibility of 50 UPEC isolates to 26 antibiotics was determined DDM based on the laboratory and clinical standards institute CLSI, 2019¹⁰. In addition, the activation of isolates has been carried out with the use of nutrient broth at a temperature of 37 Celsius for 18 hrs and the growth was adjusted to 0.5 McFarland’s standard 1.5×108 CFU/mL), after that spread on Mueller Hinton agar MHA) with a cotton swab. Antibiotic discs have been placed on MHA, pressed down gently for ensuring complete contact with the agar inoculated with bacteria, and after that incubated for 18–20 h at 37°C and then inhibition zone diameter in mm was recorded. Interpretation of results as a sensitive or resist was achieved according to the CLSI, 2019¹⁰.

DNA Extraction and PCR:

G-Spin Genomic DNA Extraction Kit for bacteria) are designed isolation of genomic DNA from various sample sources, such as frozen or fresh animal cells and Gram-negative bacteria. Conventional PCR was used to amplify the 19 genes of efflux pumps using specific primer pairs Table 1). The PCR condition is illustrated in Table 2 for the PCR mixture of 20μl consisted of 5μl of Maxime PCR Premix kit i-Taq) Intronbio/Korea), 2.5 μl of forward primer 10pmole/μl), 2.5μl of reverse primer 10pmole/μl), 5μl) of target DNA, and 5μl of nuclease-free water New Biolabs/US.

Table 1: Oligonucleotide sequence and PCR conditions

Efflux pump Class	Gene	Sequence	Product bp	Annealing Temp
RND	AcrA-F	ATCACCTTTCGCACTGTCGT	256	58.3 °C
	AcrA-R	CGACAAACAGGCCCAACAAG
	AcrB-F	CATAAACACGCCCTGGTCCT
	AcrB-R	GCTACCCGTAAGTCGATGGG	432	60.3 ºC
	AcrF-F	ATCCTCGCCGCTTTTGGTTA	432	60.3 ºC
	AcrF-R	AACACTTTTTGCGTCCGCTC	626	57.2ºC
	AcrE-F	CGCTGCAATTCTCCGATGTG	626	57.2ºC
	AcrE-R	GCAGTATCTGCGGGGGTATC	376	60.3 ºC
	AcrD-F	GCCGTGCAGCAAGTACAAAA	376	60.3 ºC
	AcrD-R	CTGGTGTTTGCAGCAGTGAC	424	58.3 °C
MFS	mdfA-F	TTCGATGACCGCGTATCTGG	206	59.3 °C
	mdfA-R	CAGCGCCAATGAAACAGAGG
	EmrD-F	ACGTTAATGTGGCAGTCGGT
	EmrD-R	ACGCCGGAACCAATGTTTTG	233	57.2 °C
	emrA-F	CGCTGGAGCGTACTCGTATT	233	57.2 °C
	emrA-R	ATTTTGCGCTGGAAGCAGTG	287	58.3 °C
	emrB-F	CTGCGCCGGTAGGGATTATT	287	58.3 °C
	emrB-R	ATCCCAAGCCCTTCCAGTTG	436	59.3 °C
	EmrE-F	ACACGGTTATGGCCATCTGT	436	59.3 °C
	EmrE-R	ATGTGGTGTGCTTCGTGACA	249	57.2 °C
	YnfA-F	CGCACCCGTCCAGTCATAAA	249	57.2 °C
	YnfA-F	ATGCTTTCTGCCCTGGTTGT	220	58.3 °C
	TehA-F	TTGCCCGACTCATACGCTTT	220	58.3 °C
SMR	TehA-R	CGCCATAATCCCGCAGTTTG	208	58.3 °C
	MacA-F	GCACAACAAGCACCGAACAT	208	58.3 °C
	MacA-R	CATATCCAGCCGCAGCAAAC	255	58.3 °C
	MacB-F	GCGTGAGCGGCGATTATTTT	255	58.3 °C
	MacB-R	AAACGCGTGAGTTGCTGTTC	364	57.2 °C
	MdlA-F	GCGCGTATTGATGCTCGTTT	364	57.2 °C
	MdlA-R	AGCATCTGACCGGGTTTCAG	383	58.3 °C
	MdlB-F	GTTACGCCAGCCATTAAGCG	383	58.3 °C
	MdlB-R	TCACCATTACCACCAGCACC	727	59.3 °C
ABC	MdtK-F	CTCTTTGGTCACGGACTGCT	350	59.3 °C
	MdtK-R	TTCACCGGGATGTTCACCAG
	DinF-F	TTGGTCTGCTAATGGTGCGT
MATE	DinF-R	ATGATGTGTTCCCCAGCCAG	400	58.3 °C
MATE	TolC-F	CGATCGTGATGCTGCCTTTG	400	58.3 °C
OM adapter	TolC-R	GGTTGCGTTTTTCGGCTTCT	596	58.3 °C

Table 2: Phenotype of coexisted antibiotic resistance among K. pneumoniae isolates

Phenotype	Resistance Pattern	No.	%
B-Lactam/FLOURO/CARBA/MONO/AMINO/NITRO/MACRO	MDR	1	2
B-Lactam/FLOURO/AMINO/TETRA/SULFA/NITRO/MACRO	MDR	1	2
B-Lactam/FLOURO/MONO/AMINO/TETRA/NITRO	MDR	1	2
B-Lactam/FLOURO/MONO/AMINO/TETRA/SULFA	MDR	1	2
B-Lactam/FLOURO/CARBA/MONO/AMINO/SULFA	MDR	2	4
B-Lactam/FLOURO/MONO/AMINO/SULFA/MACRO	MDR	1	2
B-Lactam/FLOURO/MONO/AMINO/SULFA/NITRO	MDR	1	2
B-Lactam/FLOURO/AMINO/TETRA/SULFA	MDR	1	2
B-Lactam/AMINO/NITRO/MACRO	MDR	1	2
B-Lactam/FLOURO/AMINO/NITRO	MDR	1	2
B-Lactam/CARBA/AMINO/MACRO	MDR	1	2
B-Lactam/CARBA/AMINO/NITRO	MDR	1	2
B-Lactam/FLOURO/MONO/AMINO	MDR	1	2
B-Lactam/FLOURO/AMINO/SULFA	MDR	1	2
B-Lactam/AMINO/SULFA/NITRO	MDR	3	6
B-Lactam/MONO/AMINO/NITRO	MDR	2	4
B-Lactam/FLOURO/AMINO/NITRO	MDR	1	2
B-Lactam/AMINO/SULFA	MDR	2	4
B-Lactam/FLOURO/AMINO	MDR	1	2
B-Lactam/AMINO/NITRO	MDR	5	10
B-Lactam/SULFA/NITRO	MDR	1	2
B-Lactam/AMINO/SULFA	MDR	1	2
B-Lactam/AMINO	MDR	8	16
B-Lactam/FLOURO	non-MDR	1	2
B-Lactam/Tetracyclin	non-MDR	1	2
B-Lactam	non-MDR	9	18
		50	100

Molecular Detection of Efflux Pumps for K. pneumoniae:

Results of molecular investigation of efflux pumps in K pneumoniae revealed that class RND AcrAB-TolC, AcrAD-TolC and AcrFE-TolC genes were distributed as follow: acrA 4896%), acrB 4488%), acrD 46 92%), acrF 3264%), acrE 4692%) and tolC 50100%). Three class MFS pumps EmrAB-TolC, EmrD and MdfA) were also investigated for K. pneumoniae and the results: emrA 4896%), emrB 4998%), emrD 50100%) and mdfA 50100%). Three class SMR pumps EmrE, YnfA and TehA) genes were distributed as follow: emrE 3570%), ynfA 3366%) and tehA 3876%). Two class ABC pumps MacAB-TolC and MdlAB-TolC) genes were investigated for K. pneumoniae and the results: macA 3876%), macB 4896%), mdlA 3876%) and mdlB 50100%). Two MATE pumps MdtK and DinF) genes were studies and the results revealed that: mdtK and dinF genes were present 50100%) and 4386%) respectively in K. pneumoniae isolates (Figure 3)

Figure 3: Distribution of efflux pumps genes among K. pneumonia isolates A) AcrABTolC, B) AcrAD-TolC, C) EmrAB-TolC, D) AcrFE-TolC, E) EmrE,YnfA, TehA, F) MdfA and EmrD, G) MacAB-TolC, H) MdlAB-TolC, I) Mdtk and DinF

Coexisted Genotypes of Efflux Pumps for UPEC:

Concern results of coexisted pumps in same K. pneumoniae isolates the results showed that, the highest genotypes were: 816%) for genotype AcrAB-TolC/ AcrAD-TolC/AcrFE-TolC/MdfA/EmrD/EmrAB- TolC/ EmrE/YnfA/TehA/MacAB-TolC/MdlAB-TolC/ Mdtk/ DinF, 816%) for genotype AcrAB-TolC/AcrAD-TolC/ MdfA/EmrD/EmrAB-TolC/EmrE/YnfA/TehA/ MacAB-TolC/ MdlAB-TolC/ Mdtk/ DinF (Table3)

Table 3: Genotypes of efflux pump among K. pneumoniae

Genotype

No.

AcrAB TolC/ AcrAD TolC/ AcrFE TolC/ MdfA/ EmrD/ EmrAB TolC/

EmrE/ YnfA/ TehA/ MacAB TolC/ MdlAB TolC/ Mdtk/ DinF

AcrAB TolC/ AcrAD TolC/ AcrFE TolC/ MdfA/ EmrD/ EmrAB TolC/

YnfA/ TehA/ MacAB TolC/ MdlAB TolC/ Mdtk/ DinF

AcrAB TolC/ AcrAD TolC/ MdfA/ EmrD/ EmrAB TolC/ EmrE/ YnfA/TehA/ MacAB TolC/ MdlAB TolC/ Mdtk/ DinF

AcrAB TolC/ AcrAD TolC/ MdfA/ EmrD/ EmrAB TolC/ EmrE/ YnfA/MacAB TolC/ MdlAB TolC/ Mdtk/ DinF

Biofilm Formation for K. pneumonia:

Results of biofilm formation revealed that 100% of isolates were biofilm former while 0% were non-biofilm. 40% was weak biofilm, 44% was moderate and 16% was strong biofilm former (figure 4).

Figure 4: Biofilm Formation patterns

DISCUSSION:

In immunocompromised patients, like patients with diabetes and cancer and in transplant recipients, UTI with extensively ERKp) is a difficult infectious complication. Antibiotics' efficacy in treating UTIs is decreasing. There strong association between resistance to those 3 antibiotics and presence of 5 classes’ efflux pump genes. Multidrug efflux pumps are of high importance in the resistance to various antibiotic classes ^11,12,13, the acridine resistance complex of E. coli, is the most well-studied RND transporter. An outer-membrane channel TolC, inner-membrane transporter AcrB, and a periplasmic protein AcrA make up this tripartite complex^14,15. The most effective drug efflux pump E. coli's AcrAB works with TolC to extrude a wide variety of antimicrobial compounds from the cell, including antibiotics, dyes, detergents, and organic solvents^16,17.

Chetri et al 2018¹⁸ showed Overexpression of AcrEFTolC was found in 13 E. coli isolates that were also resistant to quinolones and carbapenems ⁽¹⁸⁾. The first being that EmrAB is very active at effluxing these antibiotics, so its deletion resulted in measurable MIC reductions^19,20 EmrD belongs to the multidrug resistance exporter family, which is part of the main facilitator superfamily MFS EmrE^21,22, a member of the small multidrug resistance SMR) protein family, uses secondary active antiport to transport medicines across the PM in exchange for protons^23,24,25. Antibiotic resistance to penicillins and cephalosporins is also thought to grow in ynfA-expressing E. coli²⁶.

MacA is a periplasmic membrane fusion protein that activates the operation of the MacB transporter and connects it to the outer membrane channel TolC in this complex^27,28. The AcrAB and mdtK complexes are the most well-studied efflux pumps in K. pneumonia^29,30 This group of bacteria's biofilm-forming capacity leads them to stick together and get embedded in a matrix of extracellular polymeric substance known as exopolysaccharides^31,32,33. This generates a protective environment that makes antibiotic penetration more difficult and protects against insults like dehydration and food scarcity^34,35.

CONCLUSION:

The current study concludes that, all efflux pumps may be highly associated with resistance to ceftazidime, Amoxicillin, cefotaxime, ceftriaxone, cefixime, cefepime and moderately associated with to streptomycin, trimethoprime, nitrofuraniton, Aztreonam, Kanamycin and may be unrelated to resistance of other studied antibiotics or the concentration of theses antibiotics inadequate to induce the expression of these pumps. Additionally, biofilm formation was highly related to presence of studies pumps.

ACKNOWLEDGMENT:

It is my pleasure to thankful the head of Biology department and advanced microbiology laboratory at college of science, university of Babylon for their permission and facilitate the work at their labs. Also many thanks to Assistant Prof. Dr. Noor S.K. Al-Khafaji for their assistant in PCR work.

ETHICAL APPROVAL:

Informed consent was obtained from all human adult participants or parents or legal guardians of minors. The project was approved by scientific committee and Bioethics committee under project no. 325 at 29 December 2020.

CONFLICT OF INTEREST:

All authors declare that, the is no conflict of interest

FUNDING:

The project was funded by authors themselves

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Received on 03.12.2021 Modified on 30.01.2022

Research J. Pharm. and Tech 2022; 15(10):4559-4564.

DOI: 10.52711/0974-360X.2022.00765

Sahar Amer Ali, Hussein O. M. Al-Dahmoshi*