Prevalence of common carbapenemase genes and multidrug resistance among uropathogenic Escherichia coli phylogroup B2 isolates from outpatients in Wasit Province/ Iraq

Carbapenems are the last resort antimicrobials for the treatment of extended spectrum β-lactamases (ESBLs) producing Enterobacteriaceae. Emergence of carbapenems resistant group B2 uropathogenic E. coli (UPEC) is a major concern because of their high virulence. Prevalence of these enzymes and multidrug resistance (MDR) among B2 UPEC isolates from Iraqi outpatients with acute urinary tract infection (UTI) was evaluated in this research. Urine cultures were performed and the isolates were identified biochemically. Escherichia coli isolates were tested for phylogroup reference by quadraplex PCR, then B2 isolates were detected for antimicrobial resistance by disc diffusion test and carbapenemase genes by PCR. Escherichia coli was the most prevalent among Gram-negative isolates (66.6%) and B2 was the most detected phylogroup among E. coli isolates (33.9%). Most of B2 isolates showed high resistance rates to tested antimicrobials, especially β-lactams with MDR revealed in 100% of them. Whereas, low resistance rates were noted against carbapenems, aminoglycosides and nitrofurantoin. Carbapenemase genes were detected in 76.3% of B2 isolates. Of which, blaOXA-48 was the most frequent (57.8%), followed by blaPER (47.3%), blaKPC (15.7%), blaVEB and blaVIM (10.5%, for each). Whereas, blaGES and blaIMP genes were not found. Coproduction of these genes occurred among 17 isolates. The combination of blaOXA-48 and blaPER was the most frequent (41.1%). All carbapenemase producing isolates were MDR. These results revealed high prevalence of carbapenemase genes and MDR among B2 UPEC recovered in this study. In the study area. it is strongly advised to use aminoglycosides and nitrofurantoin for empirical treatment of UPEC.


Introduction
Escherichia coli (E. coli) is the most versatile known microorganism [1]. It is a common commensal of gastrointestinal tract of human and animal. Also, it comprises pathogenic strains a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 their high virulence. Strains with high antimicrobial resistance accompanied by high virulence may emerge and this may lead to treatment failure and loss of effective treatment [10]. So that, awareness should be taken to prevent such strains from reaching our patients especially those who are immunocompromised. One of the effective measurements to achieve this aim is to do periodic surveillance of antimicrobial resistance of UPEC by both phenotypic and genotypic procedures. Selection of appropriate prevention and containment options requires good knowledge of the prevalence and incidence of carbapenemases. Strains-producing these enzymes are not limited to hospital environment, but also occur among hospitals, long-term care facilities, community, animals, and the environment [11]. As it is difficult to develop novel antimicrobial agents, efforts should be concentrated on the prevention of the spread of carbapenemase producers by early detection and reinforced hygiene measures [18] and careful monitoring of use of antimicrobials for UTI treatment is necessary [19]. In addition, predominance of carbapenemases among B2 E. coli isolates was reported from different parts of the world [20][21][22][23]. Hence, this research was designed to evaluate the prevalence of carbapenemase genes and MDR among B2 UPEC isolates from Iraqi outpatients in Wasit Province with acute UTI by multiplex PCR protocols.

Specimen collection and processing
Midstream urine samples were collected from outpatients attending AlKarama hospital and Al-Kut hospital for Gynecology and Obstetrics and Pediatrics in Al-Kut/Wasit Province/Iraq, during the period from July, 2018 to January, 2019. The urine samples were collected into sterile screw capped test tubes and streaked immediately on MacConkey agar (HIMEDIA, India) and Blood agar (HIMEDIA, India) plates for bacterial isolation [24].

Isolation, identification and phylogenetic grouping of E. coli
Collected urine samples were cultured immediately after collection on MacConkey agar and Blood agar plates and incubated aerobically at 37˚C for 24 h. Positive urine cultures were defined by a growth of single morphotype of colony with counts >10 5 CFU/ml [24]. From each plate single colony (with the appropriate color and morphology, that is, characteristics of E. coli) was selected and subcultured onto MacConkey agar plate again, incubated, a single colony was selected and subcultured onto tryptic soy agar (TSA) (HIMEDIA, India) plate, and then kept in refrigerator for further work.
The isolates were identified biochemically by API20E rapid test system depending on the manufacturer's instructions (BioMerieux, France).
Thereafter, E. coli isolates were classified into eight phylogenetic groups (A, B1, B2, C, D, E, F, and clade I) according to the presence of chuA, yjaA, and arpA genes and TspE4.C2 DNA fragment, depending on methods provided by Clermont et al. [17] At first the DNA was extracted from the isolates by boiling method described by Yamamoto et al. [25] with modification which included suspending 24 hr. old bacterial culture (3 loopfuls) on TSA in 1 ml of sterile 1X TE buffer (pH 8.0) (Bio Basic, Canada) instead of sterile D.W. The cell suspension was boiled in water bath at 95˚C for 10 minutes. The suspension was centrifuged at 10,000 rpm for 5 minutes. The supernatant which contains purified DNA was dispensed in 100 μl aliquots and stored at -20˚C till use. The phylogroups were determined by quadraplex PCR (Tables 1 and 2). This work was approved by the Scientific Committee of the College of Science/ Wasit University/ Wasit Province/ Iraq (Scientific project discussing committee) and also it was performed after obtaining permission from Health Administration of Wasit, Wasit Province/Iraq. Oral consent was obtained from each patient for collecting specimens and publication of this report. The reason for just obtaining oral consent without the need for written consent is that collection of urine specimens is part of routine clinical laboratory work for diagnosis of these infections. All patients' data were anonymous.

Antimicrobial susceptibility of the isolates
Antimicrobial susceptibility of the isolates was performed by the disk diffusion method according to the instructions of CLSI [30] using Mueller-Hinton agar (HIMEDIA, India). The used antimicrobials were obtained from Bioanalyse/ Turkey and included: ampicillin (AMP:

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Carbapenemases production by B2 E. coli

Statistical analysis
Differences in the distributions of the studied determinants were tested by Chi square (SPSS software, version 2.1, IBM, NC, USA). A P value of � 0.05 was considered to indicate statistical significance.

Prevalence of E. coli among uropathogens
Of 1003 urine samples (one sample per patient) only 359 (35.7%) were positive for bacterial culture, where 168 (46.7%) of them were positive for Gram-negative bacteria. All of these Gram-negative isolates were identified biochemically by API20E rapid test according to index provided by the manufacturer (BioMerieux, France). Among all positive bacterial cultures, E. coli was one of the commonly isolated bacteria (112/359: 31.9%). Escherichia coli also was the most commonly isolated species among Gram-negative bacteria (112/168: 66.6%), followed by K. pneumoniae (28: 16.6%), Proteus mirabilis (P. mirabilis) and Pseudomonas spp. (11: 6.5%, for each) and Enterobacter spp. (6: 3.5%). These results were similar to those obtained by other Iraqi researchers such as that which was carried out in Kirkuk city by Alsamarai and Ali [32] where they showed 41.6% of urine samples were culture positive and E. coli was the predominantly isolated bacteria (57.7%), followed by K. pneumoniae (14.5%), and Proteus spp. (10.3%). In Baghdad, Ghaima et al. [33] reported that 57.9% specimens were positive for bacterial culture and E. coli was the most common bacteria (34.0%) followed by Klebsiella spp. (14.6%); Proteus spp. (4.5%); Pseudomonas spp. (3.7%)and Enterobacter sp. (1.4%). Also, similar results were reported from other countries as those achieved by Rafalskiy et al. [34] in the Russian Federation where they found that 64.2% of the isolated uropathogens were Gramnegative and E. coli was the most prevalent (49.1%), followed by K. pneumoniae (9.5%), P. mirabilis (2.9%), P. aeruginosa (1.7%) and Enterobacter spp. (1.0%). In Grenada, Sharma et al. [35] showed that 65.4% of the isolates were Gram-negative bacteria with E. coli being the frequently isolated species (51.5%), followed by K. pneumoniae (20.0%) and P. mirabilis (10.0%). The high predominance of E. coli in patients with UTI is expected and it is well known by physicians and researchers all over the world as this bacterium represents a normal component of the intestinal microbiota of humans and animals and has strains with the potential of causing UTI and other extraintestinal infections. These UPEC strains have virulence traits that allow their successful colonization. So that, these virulence traits are considered the most important features differentiating the ExPEC from commensal and enteric E. coli [36]. Uropathogenic E. coli exhibit various virulence-associated factors (VFs) that assist them in attaching to, invading, and injuring the host. Among these virulence factors are adhesins (e.g. fmbriae), siderophores, iron-acquisition systems, capsules, toxins (e.g. haemolysin), invasins, and serum resistance associated proteins [36, 37].

Resistance of the B2 E. coli isolates to antimicrobials
Susceptibility to 17 antimicrobials was performed for all of the B2 UPEC isolates (n = 38). Resistance to β-lactams was the most common except carbapenems (imipenem and meropenem) to which all of the isolates were sensitive. High resistance rates were also found against tetracycline, trimethoprim-sulfamethoxazole, and nalidixic acid. Whereas, the isolates showed high sensitivity to ciprofloxacin, gentamicin, nitrofurantoin and amikacin. (Table 6). Recently, similar resistance patterns were published by Iraqi researchers [23].
Antimicrobial resistance in UPEC is a major concern in both humans and animals at a worldwide scale due to its increased resistance to several antibiotics [51], and expanding resistance to different classes of antimicrobial agents generally [52]. Indiscriminate and widespread use of antibiotics in addition to the practice of prescribing antibiotics to treat UTI without bacterial characterization led to increased resistance among uropathogens and to decreased effectiveness of oral therapies [5,53], which gave an alarming level of antimicrobial resistance developing in UTI pathogens. Thus, rapid initiation of appropriate empirical treatment requires a good knowledge of epidemiological data concerning the sensitivity of uropathogens to antibacterial agents [54]. Furthermore, plasmids harboring resistance determinants can be transferred between bacteria, even between species, leading to the acquisition of resistance to new antibiotics via the emergence of mutant strains [55]. Also, some bacteria produce multiple β-lactamases, which may reduce the efficiency of β-lactam/β-lactamases inhibitor combination [5]. The rapid development of resistance to β-lactam antibiotics attributed to the emergence of ESBLs in the enteric bacteria [56]. This may be due to the excessive use of expanded spectrum cephalosporins (ESC) during clinical practice, where several studies have found a relationship between third-generation cephalosporins use and acquisition of ESBL-producing strains [57].

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Therefore, the limited use of these antibiotics might be helpful to inhibit/avoid the emerging or spreading of multidrug-resistant Gram-negative bacteria [58]. One hundred percent sensitivity to imipenem and meropenem was observed among all B2 UPEC isolates investigated in this work. For bacterial infections, these antibacterials are the most reliable last-resort treatment [59]. Furthermore, the low resistance rates against aminoglycosides (gentamicin: 18.4% and amikacin: 2.6%) reported in the present study may be attributed to the rare use of these antibiotics in AL-Kut hospitals which may be due to their high costs in comparison with β-lactams [57]. Nitrofurantoin resistance was also noted in 7.8% of E. coli clinical isolates, and this may be due to the lower frequency use of this drug. In addition, 100% of this study included isolates were MDR, while none of them were XDR or PDR. This may be due to the fact that E. coli pathogens have developed resistance to every class of antibiotics introduced to treat human and animal infections [60], and these infections are particularly challenging to treat [50]. This high prevalence of MDR among the isolates of the present study is alarming and necessitate the need for the clinicians to ensure the use of appropriate antibiotics for recommended periods in adequate doses in order to prevent emergence of multidrug resistant organisms. Many factors may have contributed to such high rates of resistance including misuse of antibiotics by health care professionals or non-skilled practitioners, misuse of antibiotics by the general public and inadequate surveillance due to lack of information arising from routine antimicrobial susceptibility testing, like reports from other developing countries. Careless usage of antibiotics is the most important factor that facilitates the development of MDR, which triggers the selection and distribution of antibiotic-resistant pathogens in clinical practice [56,61]. Iraq is one of the developing countries where antibiotics are sold over the counter, an attitude that encourages self-medication. On the other hand, it is remarked that during period, a group of antibiotics become more used than others without susceptibility tests, which may lead to variability in their resistance [62]. Other factors known to influence the evolution and transfer of MDR among microorganisms are incomplete doses, . Antibiotics resistance arises quickly and spreads rapidly, especially when resistance genes are horizontally transferred via plasmids and integrons among individuals, among species, and even among bacterial kingdom [63].
Much of the problem of antimicrobial resistance has been shown to be due to the presence of transferable plasmids encoding MDR and their dissemination among different enterobacterial species and it is common for a single plasmid to simultaneously mediate resistance to multiple antimicrobials and to be shared among different bacterial genera [64]. These MDR isolates showed resistance to most antimicrobials tested in this study except carbapenems (all isolates were sensitive), amikacin (2.6%) and nitrofurantoin (7.8%). These results ensure that continuous and inappropriate use of antimicrobials is a major risk factor for development of resistance. It was realized that the inappropriate use of antimicrobials has been shown to play a pivotal role in the emergence of MDR organisms [65,66]. Also, MDR is largely associated with ESBLs' production as mentioned by several researchers [64,65,[67][68][69].
Also, coproduction of these enzymes occurred among 17 isolates. The combination of bla OXA-48 and bla PER was the most frequent (41.1%) ( Table 8). Antimicrobial susceptibility of these carbapenemase producers was depicted in Table 9.
From these results, it seems likely that the situation is more complicated than it is expected as the isolates: (1) belonged to the highly virulent phylogroup B2 and (2) carried not only one

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gene but combination of genes in addition to ESBLs' production. This study results revealed that all carbapenemase producers (100%) were MDR. Patterns of antimicrobial resistance and carbapenemase genes of these isolates were presented in Table 10. Multidrug resistance is not attributed to a certain set of genes but in fact it is a result of possession of a combination of different genes especially ESBLs. Nikaido [70] concluded that MDR is due to accumulation of genes, each coding for resistance to a single antimicrobial agent, on R plasmids. This accumulation of these genes is carried out by transposons, integrons, and insertion sequences (ISCR). In UPEC, the mechanisms of antimicrobial resistance are diverse and comprise: production of inactivating enzymes either hydrolytic enzymes such as β-lactamases or no hydrolytic enzymes such as aminoglycoside acetyl transferase enzymes. Other mechanisms include: active efflux pumps, alteration of target site, horizontal gene transfer by insertion sequences, gene cassettes, integrons, and transposons [3,71]. Eshetie et al. [72] reported that carbapenemase producing Enterobacteriaceae had significant drug resistance rates compared to other MDR Enterobacteriaceae and that production of these enzymes is one of the main mechanisms in the occurrence of drug resistance in this family. So that, Jain et al. [73] concluded that antimicrobial resistance due to β-lactamases is emerging as a major problem in UTI; routine surveillance of these β -lactamases will help in control of treatment failures. In fact, one of the critical

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priorities of the World Health Organization is carbapenem-resistant ESBL-producing bacterial pathogens for which new antibiotics should be designed [4].
Although, all of the isolates in this study were phenotypically susceptible to imipenem and meropenem by disc diffusion method, genotypically, carbapenemase production was obvious among them ranging from 10.5% for both VEB and VIM to 57.8% for OXA-48. This discrepancy was noticed by other researchers [20,74]. It was explained that detection of

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carbapenemase-producing E. coli (CP-E. coli) isolates is often difficult due to low carbapenem MICs that may remain within the susceptibility range [83]. Also, OXA-48 enzymes are known to exhibit only low hydrolytic activity toward carbapenems [84]. Moreover, it seems better to use ertapenem disc diffusion for phenotypic detection of those especially producing OXA-48 rather than imipenem or meropenem [20]. The same researchers concluded that laboratory detection of CP-E. coli may be more difficult in comparison with CP-K. pneumoniae, particularly in the case of OXA-48, because: (i) the isolates may appear susceptible to imipenem and meropenem; and (ii) there is a high frequency of OXA-48-producing isolates without ESBL co-production.
The results of the present investigation showed high occurrence of carbapenemase genes among B2 UPEC isolates. This may be due to increased use of carbapenems by physicians for treatment of serious and even non-serious cases in the study area (personal communication).

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Also, in this work only B2 isolates were investigated for carbapememases among which these genes are concentrated. In spain [20], it was revealed that the CP-E. coli occurred after a continuous increase in resistance to third-generation cephalosporins and fluoroquinolones. The production of all kinds of carbapenemases by E. coli isolates represent a major issue with further problem in UTI treatment [60]. From a therapeutic perspective, CRE represent a threat as only a few antibiotics retain activity against them. This is due to the ability of carbapenemases to hydrolyze most other β-lactam antibiotics, and to frequent coexistence in CRE isolates of additional mechanisms of resistance against other antibiotics such as fluoroquinolones and aminoglycosides [85]. Furthermore, the indiscriminate use of carbapenems can select resistance to these main drugs and sow seeds for significant therapeutic problems that may occur in the future. Efficient infection-control methods for outbreak management are also needed; and prevention approaches, e.g., antibiotic rotation, are needed to reduce the selection and spread of these highly resistant pathogens [86]. Today, the extensive international movement and exchange has helped OXA-48 producing Enterobacteriaceae to spread from many Middle-Eastern countries into other parts of the world [11].

Conclusions
This study revealed high occurrence of carbapenemase genes and MDR among phylogroup B2 UPEC isolates from outpatients with acute UTI in Wasit Province, Iraq. These findings may indicate the concentration of these genes among E. coli phylogroup B2 members. Also, this study included isolates showed high resistance rates to most antimicrobials used in this work except aminoglycosides and nitrofurantoin, so that, it is strongly recommended to use aminoglycosides and nitrofurantoin for empirical treatment of UTIs in the study area.