Efflux Pumps among Urinary *E. coli* and *K. pneumoniae* Local Isolates in Hilla City, Iraq

*Hussein Al-Dahmoshi, Sahar A. Ali and Noor Al-Khafaji*

## **Abstract**

Urinary tract infections (UTI) are the most common bacterial infections affecting humans. *Escherichia coli* and *Klebsiella pneumoniae* were common enterobacteria engaged with community-acquired UTIs. Efflux pumps were vital resistance mechanisms for antibiotics, especially among enterobacteria. Overexpression of an efflux system, which results in a decrease in antibiotic accumulation, is an effective mechanism for drug resistance. The ATP-binding cassette (ABC) transporters, small multidrug resistance (SMR), and multidrug and toxic compound extrusion (MATE) families, the major facilitator superfamily (MFS), and the resistance-nodulation- cell division (RND) family are the five superfamilies of efflux systems linked to drug resistance. This chapter highlights the results of studying the prevalence of efflux pump genes among local isolates of *E. coli* and *K. pneumoniae* in Hilla City, Iraq. class RND AcrAB-TolC, AcrAD-TolC, and AcrFE-TolC genes detected by conventional PCR of *E. coli* and *K. pneumoniae* respectively. The result revealed approximately all studied efflux transporter were found in both *E. coli* and *K. pneumoniae* in different percentages. Biofilm formation were observed in 50(100%) of *K. pneumoniae* and 49(98%) of *E. coli* isolates were biofilm former and follow: 30(60%), 20(40%) were weak, 12(24%), 22(44%) were moderate and 7(14%) and 8(16%) were Strong biofilm former for *E. coli* and *K. pneumoniae*, respectively.

**Keywords:** UTIs, AcrAB-TolC, AcrAD-TolC, AcrFE-TolC, EmrAB-TolC, EmrD, MdfA, EmrE, YnfA, MacAB-TolC, MdlAB-TolCTehA

## **1. Introduction**

Urinary tract infections (UTI) are the most common bacterial infections affecting humans (Zhanel et al. [1]). They may be simple or complicated urinary tract infections (cUTIs), with the latter occurring in patients with urinary tract anatomic or functional abnormalities or major comorbidities [2]. UTIs may be categorized as either population- or hospital-acquired. In community-acquired UTIs, *Escherichia coli, Klebsiella pneumoniae,* and *Staphylococcus saprophyticus* are the most common bacteria, while in hospital-acquired UTIs, bacteria like *Staphylococcus* 

*aureus, Enterococcus* spp*., Proteus* spp*., Pseudomonas aeruginosa, Acinetobacter* spp*.*, and *Candida* spp. are more common [1, 3], UTIs are disproportionately prevalent in women, approximately half of them experiencing a UTI during their lifetimes. Although UTIs are prevalent in all age groups, they tend to be more common in postmenopausal and elderly women, because it is more susceptible to infection under this situation [4]. Three-quarters of all UTIs in outpatients are caused by *E. coli* [5]. However, some *E. coli* lineages are more likely to induce UTI than others [6]. Antibiotic resistance is more common in uropathogenic *E. coli* than in commensal *E. coli*, and one uropathogenic lineage [7] sequence type131 (ST131), especially sublineage ST131:O25b:H30, is linked to multidrug resistance [8].

In addition to being a clinically significant pathogen, *K. pneumoniae* is a common cause of both hospital-acquired (HA) and community-acquired (CA) urinary tract infections (UTI). Limited therapeutic options have been created due to the enlarged resistance of this pathogen [9]. Enterobacteriaceae that produce extended-spectrum beta-lactamase (ESBL) and carbapenemase are frequently multidrug resistance, posing significant therapeutic challenges [10]. *K. pneumoniae* strains that produce ESBL/carbapenemase are widely recorded around the world, and their spread is critical [11]. Quinolone resistance among *K. pneumoniae* clinical isolates has become a serious problem in both developing and developed countries since quinolones are commonly prescribed as broad spectrum antimicrobial agents for the treatment of UTI induced by ESBL-producing *K. pneumonia*. Efflux pumps are present in all major types of bacterial membrane transporters, and increased efflux levels are thought to cause MDR. The most important types for the maintenance of *E. coli* in the human gut are AcrAB-TolC, EmrAB-TolC, and MdtM for extruding bile salts, mammalian steroids, and different antibiotics [12]. The influx of antimicrobial agents is reduced when the outer membrane's permeability is reduced. As a result, resistance develops in a number of essential clinical microorganisms. The first discovered efflux pump system was the tetracycline efflux pump by Stuart Levy et al. in *E. coli* [13].

Several studies indicate that efflux pumps can play at least four roles in biofilm formation efflux of EPSs and/or QS and quorum quenching (QQ ) molecules to promote biofilm environment creation and regulate QS, correspondingly; indirect regulation of genes engagement in biofilm formation; abolition of harmful particles such as antibiotics and metabolic intermediates; and efflux of harmful molecules such as antibiotics and metabolic intermediates and by encouraging or preventing adhesion to surfaces and other cells, aggregation can be influenced [14]. Overexpression of an efflux system, which results in a decrease in antibiotic accumulation, is an effective mechanism for drug resistance [15, 16].

### **1.1 Efflux pumps**

Efflux pumps are membrane proteins that are involved in the export of noxious substances from within the bacterial cell into the external environment. Efflux proteins are found in both Gram-negative and Gram-positive bacteria as well as in eukaryotic organisms [17]. The ATP-binding cassette (ABC) superfamily [18], the resistance-nodulation-division (RND) family [19], the small multidrug resistance (SMR) family [20], the major facilitator superfamily (MFS) [21], and the multidrug and toxic compound extrusion (MATE) [22] family. The ABC family uses ATP hydrolysis to power substrate export, while the other families depend on the proton motive force for energy. The MFS, ABC, SMR, and MATE families are

found in Gram-positive and Gram-negative bacteria, respectively, while the RND superfamily is only found in Gram-negative bacteria. Members of the RND family are often found as part of a three-part complex that spans Gram-negative bacteria's two membranes [23].

## *1.1.1 Resistance-nodulation-division (RND) efflux pump*

This type of pump is occupied as a tripartite complex constituting the RND protein (the inner membrane component), membrane fusion protein (the periplasmic compartment), and the outer membrane protein. These three proteins form a constitute channel crossways the Gram-negative cell envelope guaranteeing that the molecule, taken from the outer leaflet of the inner membrane bilayer, is replied directly transversely to the periplasm and the outer membrane into the exterior medium with the assistance of the proton-gradient as an energy source. RND family shows a significant role in the intrinsic resistance of Gram-negative bacteria [24, 25]. The most frequent member of RND family in enterobacteria include AcrAB-TolC, AcrAD-TolC, and AcrFE-TolC.

AcrAB-TolC, one of the efflux systems, constitutively expressed in *E. coli*, is composed of the outer membrane protein TolC, the inner membrane transporter AcrB, and the periplasmic adaptor protein AcrA [26]. Overexpression of the AcrABTolC efflux pump is an intrinsic mechanism of multidrug resistance in Gram-negative bacteria [27]. AcrA, a highly elongated protein, is thought to bring the outer and inner membranes closer. It composed a trimer that interacts with a monomeric AcrB, which was shown by in vitro reconstitution to be a proton antiporter [28]. Efflux pumps, such as AcrAB-TolC and MexAB-OprM, are essential for bacteria survival and virulence/colonization, especially during the course of infection when the pathogen is attacked by toxic substances or adherence to the host [29]. The resistance mechanism by efflux pumps is the most important antibiotic resistance type because the efflux pump is able to remove more than one antibiotic such as B-lactam, erythromycin fluoroquinolones, and chloramphenicol [30]. Ruzin et al. [31] showed up that the resistance to quinolones related to the overexpression of the AcrAB efflux pump of *K. pneumoniae* was associated with resistance to other antibiotics, including erythromycin, chloramphenicol, tetracycline, and also tigecycline, a recently commercialized molecule.

AcrD is a component of an efflux pump that mediates the export of aminoglycosides and a few amphiphilic compounds such as sodium dodecyl sulfate (SDS), deoxycholate, and novobiocin, AcrA is a periplasmic fusion protein that also exports aminoglycosides in association with the cytoplasmic membrane protein AcrD [30]. Despite increased acrA expression, the mutant strain showed no increase in resistance, suggesting that the deletion of acrD contributes to adaptive resistance directly, rather than indirectly via mechanisms like control of other efflux components. As a result, the previously reported adaptive cross-resistance to non-aminoglycoside antibiotics cannot be due to the development of the AcrAB-TolC complex as shown by an acrD mutant that showed significantly reduced biofilm formation and expression of key biofilm proteins encoded by csgBD, the AcrD efflux pump has an effect on biofilm formation, and appears to play a special biological function, according to Buckner et al. [32] findings. The transcriptome showed major changes, which supported this theory. The transcriptomes of the acrD mutant were compared to the transcriptome of the acrB mutant, which had previously been released AcrD is not a "backup" efflux pump, but serves a

physiological function in the cell, as shown by the fact that the effect was quite distinct. This comparison found 232 major gene expression changes that were only caused by the inactivation of acrD and not by the disruption of acrB. Both the acrB and acrD mutant transcriptomes had 169 genes that were differentially expressed as compared to the acrB mutant transcriptome. Experiments have shown that the AcrB and AcrD efflux pumps have different substrate profiles when it refers to aminoglycoside antibiotics [33]. AcrEF-TolC pump is known to exhibit higher expression levels in quinolone-resistant *E. coli*. AcrEF shares high homology (65–77%) with AcrAB and so AcrEF-TolC expression complement the activity of AcAB-TolC. Overexpression of acrEF restores resistance to acriflavine, erythromycin, novobiocin, and crystal violet. Additionally, AcrEF is responsible for increased resistance for compounds such as dyes, detergents, and antibiotic substrates like that of AcrAB especially levofloxacin [34].

## *1.1.2 ATP-binding cassette (ABC) efflux pump*

There is a variety of transport systems in *E. coli*, including ABC-type transporters as well as substrate-binding proteins, outer membrane receptors, and a number of transporters with various functions. We have gained a better understanding of the molecular basis of transport through recent structures of ATPases, substrate-binding proteins, and full-length transporters [35]. Specialized ABC transporter types transport a diverse range of substrates, ranging from small molecules such as ions, sugars, or amino acids to larger compounds such as antibiotics, drugs, lipids, and oligopeptides [36]. The most important ABC family efflux pumps in *E. coli* include MAcAB-TolC and MdlAB-TolC.

The MacA-MacB-TolC assembly of *E. coli* is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism [37].

In a macrolide-susceptible AcrAB deficient *E. coli* strain, only overexpression of MacAB may increase resistance to macrolide antibiotics. MacAB, on the other hand, has recently been linked to the secretion of an *E. coli* heat-stable enterotoxin [38]. MacA is the MFP in the MacA–MacB–TolC pump, and due to high sequence similarity, it is predicted to share structural similarities with AcrA (44%) [39]. The C-terminal periplasmic membrane-proximal domain of MacA is required for these MacA–MacB interactions [40]. By modifying the conformation of MacA's membrane-proximal domain and disrupting the proper assembly of the MacA–MacB complex, a single G353A substitution in this domain impaired MacAB–TolC function [41]. In the *E. coli* genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters. MdlAb-TolC is multidrug efflux transporter with few studies concerned it is a function [42].

*Efflux Pumps among Urinary* E. coli *and* K. pneumoniae *Local Isolates in Hilla City, Iraq DOI: http://dx.doi.org/10.5772/intechopen.104408*

#### *1.1.3 Small multidrug resistance (SMR) efflux pump*

Small multidrug resistance transporters (SMR) systems provide to study the minimal requirements for active transport [43]. They are also small multidrug transporters, with four transmembrane helices and no significant extra membrane domain, although they function as dimers the minimum functional unit is a bundle of eight α-helices [44]. SMR transporter exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. Genes encoding SMR proteins (variously annotated emrE, ynfA and tehA) are frequently found in mobile drug resistance gene arrays, and provide a broad selective advantage by conferring resistance to ubiquitous environmental pollutants with low-grade toxicity to microbes [45]. The SMR family consists of small hydrophobic proteins of about 100 amino acid residues with four transmembrane α-helical spanners [46].

SMR family includes more than 40 proteins in eubacteria, a few of which have been studied in detail. One of them, EmrE, is an *E. coli* multidrug transporter (MDT), that utilizes proton gradients as an energy source to drive substrate translocation and confers resistance to a wide variety of toxicants by actively exchanging them with hydrogen ions [47]. EmrE is the smallest ion-coupled transporter known; it functions as an oligomer and each monomer comprises four transmembrane segments [48]. EmrE is a tetramer comprised of two conformational heterodimers related by a pseudo-two-fold symmetry axis perpendicular to the cell membrane. Based on the structure and biochemical evidence, we propose a mechanism by which EmrE accomplishes multidrug efflux by coupling conformational changes between two heterodimers with proton gradient [49]. The overexpression of EmrE causes bacteria to become resistant to a wide variety of toxic cationic hydrophobic compounds such as ethidium bromide, methyl viologen, tetracycline, and tetraphenylphosphonium, as well as other antiseptics and intercalating dyes [50].

The gene, ynfA of *E. coli* is the newest member of the small multidrug resistance (SMR) gene family, identified in both Gram-negative and Gram-positive bacterial species. It might be involved alone or with tolC or any other way by complex regulation in which the initial susceptible bacteria become resistant. The level of ynfA gene expression was observed between 2 and 6 folds equivalent to tolC gene [51].

### *1.1.4 Major facilitator superfamily (MFS) efflux pump*

The major facilitator superfamily (MFS) is the largest known superfamily of secondary active transporters. MFS transporters are responsible for transporting a broad spectrum of substrates, either down their concentration gradient or uphill using the energy stored in the electrochemical gradients. The major facilitator transporters form a superfamily that composed a number of subfamilies; of these subfamilies, transporters of sugars and drugs are by far the most numerous [52].

These MFS transporters are typically composed of approximately 400 amino acids that are putatively arranged in 12 membrane-spanning helices, with a large cytoplasmic loop among helices six and seven [43]. The MFS family of drug transporters is made up of two domains that are centered around a central pore and two domains that transfer conformations from the cytoplasmic to the periplasmic side of the membrane in response to a Na + or H+ ion gradient [53]. The MFS drug transporters are classified into subfamilies −12-helix and 14-helix transporters (e.g., TetA(B) and TetA(K), class two and class K tetracycline transporters from *E. coli* and S. aureus respectively [54]. *E. coli* have many MFS transporter-like EmrAB-TolC, EmrD, MdfA.

EmrAB–TolC from *E. coli* is such a tripartite system, comprised of EmrB an MFS transporter, EmrA, a membrane fusion protein, and TolC, an outer membrane channel. The whole complex is predicted to form a continuous channel allowing direct export from the cytoplasm to the exterior of the cell [55]. The components of EmrAB-TolC were identified in *E. coli* for the first time more than a decade ago resistance to hydrophobic toxins like carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Its overexpression causes increased resistance to nalidixic acid, thiolactomycin, nitroxoline, hydrophobic proton uncouplers [56].

EmrD is a multidrug transporter from the Major Facilitator Superfamily that expels amphipathic compounds across the inner membrane of *E. coli*. It can transport detergents such as benzalkonium and sodium dodecylsulfate [57]. EmrD may have vital role in biofilm formation via the efflux of arabinose, which promotes cell aggregation and biofilm formation [58]. MdfA is drug/proton antiporter consisting of 410 amino acid long membrane protein responsible for resistance to a diverse group of cationic or zwitterionic lipophilic compounds such as ethidium bromide, tetraphenylphosphonium, rhodamine, daunomycin, benzalkonium, rifampin, tetracycline, and puromycin. Surprisingly, however, MdfA also confers resistance to chemically unrelated, clinically important antibiotics such as chloramphenicol, erythromycin, and certain aminoglycosides and fluoroquinolones. Synergistic overexpression of *mdfA* along with *acrAB* leads to increases in quinolone resistance [59].

## *1.1.5 Multidrug and toxic compound extrusion (MATE) efflux pump*

Export of substrates and toxins by the cell is a fundamental life process and members of the MATE family represent the last class of multidrug resistance (MDR) transporters to be structurally characterized. MATE transporters involved a variety of important biological functions across all kingdoms of life [60]. MATE transporters are very similar in size to the MFS transporters and are typically composed of approx. 450 amino acids which are putatively arranged into 12 helices however, they do not have any sequence similarity to members of the MFS transporters [61]. MdtK is one of the important MATE inner membrane transporter in *E. coli* conferring resistance to quinolone and fluoroquinolone when overexpressed [62].

### **1.2 Antibiotic resistance and efflux pumps**

The study include antibiotic susceptibility profile (for 20 antibiotics) according to CLSI-2021 [63] and efflux pumps gene profile for (19 genes) for 50 isolates of *E. coli* and 50 isolates of *K. pneumoniae* isolated from patients with UTIs. The results revealed high level of resistance to β-lactams and cephalosporin and low level of resistance to piperacillin, aminoglycosides and carbapenem (**Table 1**). Multidrug resistance for more than 3 antibiotics (at least one for each class) were studied and the results revealed that 68% of *E. coli* and 90% of *K. pneumoniae* were MDR (**Table 2**). Results of biofilm formation shown that, approximately all isolates were biofilm former (**Table 3**). Concern presence of efflux pump genes, the results of polymerase chain reaction revealed that: acrA 50 (100%), 48 (96%)–acrB


## *Efflux Pumps among Urinary* E. coli *and* K. pneumoniae *Local Isolates in Hilla City, Iraq DOI: http://dx.doi.org/10.5772/intechopen.104408*

#### **Table 1.**

*Antibiotic resistance among* E. coli *and* Klebsiella pneumoniae*.*


### **Table 2.**

*Classes of MDR among* E. coli *and* Klebsiella pneumoniae.

43 (86%), 44 (88%)–acrD 48 (96%), 46(92%)–acrF 33 (66%), 32 (64%)–acrE 50 (100%), 46 (92%)–and tolC 50 (100%), 50 (100%), while class MFS pumps (EmrAB-TolC, EmrD and MdfA) were investigated for *E. coli* and *K. pneumonia* the


#### **Table 3.**

*Biofilm formation patterns among* E. coli *and* Klebsiella pneumoniae.


#### **Table 4.**

*Efflux pump genes among* E. coli *and* Klebsiella pneumoniae.

results emrA 50 (100%), 48 (96%)–emrB 50 (100%), 49 (98%)–emrD 50 (100%), 50 (100%)–and mdfA 49 (98%), 50 (100%), class SMR pumps (EmrE, YnfA and TehA) genes were distributed as follow:: emrE 48 (96%), 35 (70%)–ynfA 50 (100%), 33 (66%)–tehA 49 (98%), 38 (76%), Two class ABC pumps (MacAB-TolC and MdlAB-TolC) The result revealed that the: macA 50 (100%), 38 (76%), macB 49 (98%), 48 (96%)–mdlA 50 (100%), 38 (765%)–and mdlB 49 (98%), 50 (100%), Two MATE pumps (MdtK and DinF) genes were studies and the results revealed

that: mdtK and dinF genes were present in all *E. coli* isolates while *K. pneumoniae* revealed 50 (100%), 43 (86%) of MdtK and DinF respectively (**Table 4**).

## **1.3 Coexisted genotypes of efflux pumps**

Concern results of coexisted pumps in the same *E. coli* or *K. pneumoniae* isolate the results were shown in **Tables 5** and **6**.


#### **Table 5.**

*Co-existed efflux pump genes among* E. coli*.*



#### **Table 6.**

*Co-existed efflux pump genes among* Klebsiella pneumoniae*.*

## **2. Conclusion**

There is a strong correlation between antibiotic resistance, especially to β-lactams, and the presence of efflux pump genes, which may be reflected in biofilm formation in both *E. coli* and *K. pneumoniae.*

## **Conflict of interest**

There is no conflict of interest for this work.

*Efflux Pumps among Urinary* E. coli *and* K. pneumoniae *Local Isolates in Hilla City, Iraq DOI: http://dx.doi.org/10.5772/intechopen.104408*

## **Author details**

Hussein Al-Dahmoshi1 \*, Sahar A. Ali<sup>2</sup> and Noor Al-Khafaji1

1 Biology Department, College of Science, University of Babylon, Hilla, Iraq

2 General Directorate for the Education of Babylon, Hilla, Iraq

\*Address all correspondence to: dr.dahmoshi83@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## *Edited by Guillermo Tellez-Isaias*

Antibiotic resistance is a global health crisis. Misuse of antibiotics in humans, animals, food, and agriculture has compounded the situation. Bacterial infections have returned decades after medicines were first used. This book discusses antibiotic resistance and some of the organisms that pose immediate, serious, and alarming dangers. It highlights the need for a broader, more comprehensive approach to fighting bacterial infections, which may involve non-compound techniques (other than standard antibacterial drugs) that target bacteria or the host, such as antibodies, probiotics, phytobiotics, and vaccinations.

Published in London, UK © 2022 IntechOpen © Sinhyu / iStock

The Global Antimicrobial Resistance Epidemic - Innovative Approaches

and Cutting-Edge Solutions

The Global Antimicrobial

Resistance Epidemic

Innovative Approaches

and Cutting-Edge Solutions

*Edited by Guillermo Tellez-Isaias*