Molecular Study of Escherichia albertii in Pediatric Urinary Tract Infections

E. albertii was identified by PCR in 7% of the isolates and E.coli was identified in 93% of the isolates. Two mdh and lysP genes were detected for E. albertii and the uidA gene for E. coli. E. albertii isolates had marked resistance to gentamicin (71.4%), followed by resistance to ciprofloxacin (57.1%), meropenem and imipenem (42.9% each) and ESBL activity by double discs method was reported in 57.1% of the isolates. However, none of the isolates had shown resistance to nalidixic acid and only one isolate had resistance to norfloxacin. There was a statistically insignificant difference between resistance to the used antibiotics such as aztreonam (P=0.083), ampicillin/clavulanate (P=0.5), ciprofloxacin (P=0.69), gentamicin (P=0.3) and ceftazidime (P=1.00).


INTRODUCTION
Urinary tract infections (UTI) represent a major infection in children with high incidence and tendency to relapse [1,2]. This infection is associated with marked morbidity due to difficulty in diagnosis based upon clinical symptoms only and requires careful laboratory diagnosis based upon accurate urine collection and culture [3,4]. The accurate diagnosis and appro-animals. The Escherichia fergusonii is classified as opportunistic infections in animals, birds and human with no reported intestinal infections [12]. The Escherichia albertii(E. albertii) has been recognized to be associated with entrotpathogenic effects and fewer studies have described its association with extraintestinal infections [13,14]. The main enteropathogenic strains of E. coli have been identified to be E. albertii [15]. Pathogenesis of E. albertii depends mainly on its ability to adhere to epithelial cells with formation of attachingeffacing lesions causing formation of dense microfilaments beneath the adherent strains which helps in the invasion process, the intracellular presence of E.albertii protect them from intestinal clearance and immune system causing prolongation of diarrheal disease, those isolates and their products also have the ability to translocation from the intestinal lumen to the mesenteric lymph nodes [16]. E.albertii strains are capable of production of Shiga toxins Stx2a and Stx2f) and causes outbreaks of gastroenteritis [17,18]. The complete genomic structure and the virulence factors of E. albertii are not fully investigated [19]. E. albertii is associated with different intestinal and extrintestinal infections and even there are strains with multiple antibiotics resistance [3 -5].
The diagnosis of infections associated with this species remains a challenge due to poor discrimination between E. albertii and E. coli based upon biochemical identification. The accurate method for identification of E. albertii depends mainly on identification of specific genes namely of mdh that produces malate dehydrogenase and lysP that produces Lysine permease. These genes are preserved genes in E. albertii and can be detected by a multiplex PCR method [20,21]. The other molecular identification method depends on identification of rpoB gene by the use of multilocus sequence typing and the method of whole genome sequencing. However, these methods are tedious and time consuming and therefore cannot be used for routine diagnosis of infections [20,21].
The molecular identification of E.coli depends on the detection of the uidA gene, housekeeping and virulence gene. Molecular identification of E.coli used this gene in laboratory diagnosis [20,21].
There are insufficient data about the presence of E. albertii as a causative organism in urinary tract infection in pediatric patients. Hence, the present study aimed to detect E. albertii by polymerase chain reaction (PCR) for detection of uidA, mdh, and lysP genes among isolated E.coli from children with urinary tract infection.

MATERIALS AND METHODS
The present study was a cross-sectional retrograde study which was carried out on 100 isolates of phenotypically confirmed E. coli detected in urine samples of children suffering from urinary tract infection recruited from Mansoura University Children Hospital, Egypt. The isolates are obtained from March 2019 till May 2020. The study was approved by Mansoura Faculty of Medicine Ethical committee (R. 20.12.1110) and approval was taken from parents of the patients.
Urine specimens obtained from children by clean catch method and from children trained to use toilet clean catch midstream samples were obtained. The urine is collected in a sterile container and transported within 30 minutes to the laboratory for microscopic examination, urine culture and colony counts [22]. Isolated E. coli was identified by Gram stain and manual biochemical reactions that include the use of triple sugar iron agar, Simmon citrate, urease, lysine, phenylalanine, Methyl red voges-proskauer test and subculture on MacConkey agar [23]. For molecular studies, pure suspension of the isolated E. coli was prepared in brain heart infusion broth and glycerol kept frozen at -20ºC.

Determination of the Isolates Producing Extended Spectrum Beta Lactamase (ESBL)
The isolated E. coli which was resistant to ceftazidime and /or cefotaxime were evaluated for production of ESBL by the double discs method (CLSI). The prepared dilution of 0.5 McFarland was spread over Mueller-Hinton plates and cefotaxime + cefotaxime/clavulanic acid and ceftazidime + ceftazidime/clavulanic acid discs were added and the plates were incubated at 37ºC for 24 hours. Positive ESBL production was considered if there was an increase of inhibition zone diameter around antibiotics ≥5 mm after combination of antibiotic with clavulanic acid [24]. Klebsiella pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as the ESBL-negative and positive control strains, respectively.

Extraction of Genomic DNA
DNA was extracted by the use of QIA amp DNA Mini Kit (Qiagen-Wade RoadBasingstokeHampshireRG24 8PWUnited Kingdom).

Polymerase Chain Reaction (PCR)
The identified E. coli, according to biochemical reaction, was subjected to PCR intended for detection of uidA, mdh, and lysP genes to differentiate E. albertii from E. coli.  The used sequences of the used genes primers of lysP, uidA and mdh are listed in Table 1 [25,26]. The used amplification mixture ready to use kit was supplied from Qiagen (Wade RoadBasingstokeHampshireRG24 8PWUnited Kingdom). The amplification procedures were carried out for each gene separately with the following amplification conditions: an initial denaturation for 5 minutes at 95°C, then 35 cycles with denaturation for 30 seconds at 95°C followed by primer annealing for 30 seconds using different temperatures for each primer, the annealing temperatures were 67°C, 65°C and 64°C for uidA, mdh, and lysP, respectively and the extension step was done for 60 seconds at 72°C. Final extension was performed for 5 minutes at 72°C. Then electrophoresis was performed for 30 minutes Electrophoresis in 2% agarose gel stained with ethidium bromide was performed to visualize DNA.

Statistical Analysis
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 22 software (SPSS Inc., Chicago, IL). Chi-square was run to verify the P-value. Statistically significant P-value was considered if it was less than 0.05.

RESULTS
The study included 100 isolates that were identified by biochemical methods as E. coli. The isolates were separated from urine samples of children complaining of urinary tract infection. The children were 46 males and 54 females with an age range from 0.7 up to 14 years old. The most common symptoms were fever (46%), dysurea (39%), abdominal pain (36%) and vomiting (35%) ( Table 2).
E. albertii was identified by PCR in 7% of the isolates and E.coli was identified in 93% of the isolates. E. albertii isolates had marked resistance to gentamicin (71.4%), followed by resistance to ciprofloxacin (57.1%), meropenem and imipenem (42.9% each). ESBL activity by double discs method was reported in 57.1% of the isolates. However, none of the isolates had shown resistance to nalidixic acid and only one isolate had resistance to norfloxacin. There was statistically insignificant difference between resistance to the used antibiotics such as azteronam (P=0.083), ampicillin /clavulanate (P=0.5), ciprofloxacine (P=0.69), gentamicin (P=0.3) and ceftazidime (P=1.00) ( Table 3).
There was a statistically insignificant difference in clinical symptoms between children infected with E. albertii and those infected with E. coli (Table 4).

DISCUSSION
E. albertii is a species derived from E.coli with difficulty to differentiate with biochemical or phenotypic characterization. Some reports identify E. albertii on the base of negative lactose fermentation, however, some strains can ferment lactose [27]. From genetic studies conducted on E. albertii, two genes of lysP and mdh have been identified as diagnostic markers for the identification of E. albertii. E. albertii has been reported to be associated with mortality in animals such as cats, pigs and birds. Moreover, environmental and food contamination may be associated with this bacterium [28].
In the present study, by using mdh and lysP genes, seven isolates had been identified as E. albertii in 100 isolates from urinary tract infections. A previous study had reported identification of 5 isolates as E. albertii among 60 isolates from urinary tract infections [6]. Higher prevalence of E. albertii was reported in previous studies from patients with diarrhea by the use of these two genes among isolates recognized as E.coli by biochemical reactions and phenotypic methods. Nimri reported 48 isolates as E. albertii from total of 250 isolates with the absence of uidA gene [29], and Aoshima identified six isolates of E. albertii detected from 20 E. coli recognized phenotypically in specimens from a gastrointestinal tract infection of food origin [30]. These studies highlight the emergence of E. albertii as a causative organism in human infections with the requirement of application of specific genetic studies for identification of E. albertii [19].
Specific identification of E. albertii is an important issue for determination of its resistance to antibiotics [31]. Until now, little data is known about antibiotic resistance of E. albertii. In the present research, E. albertii isolates had marked resistance to gentamicin, followed by resistance to ciprofloxacin, meropenem and imipenem. Another study from China reported different resistance pattern of 51 isolates of E. albertii, as those isolates were found to be either sensitive or exhibited intermediate susceptibility to amoxicillin-clavulanic acid, levofloxacin, imipenem and meropenem. The difference in the resistance pattern can be attributed to the difference in the number of isolates and the difference in the protocol of antibiotics prescription between different countries. ESBL activity detected in E. albertii by double discs method was reported in 57.1% of the isolates. This is higher than that reported in a previous study from China, as only 15 isolates from 51 E.albertii isolates had ESBL activity [31].
There was statistically insignificant difference in resistance to the used antibiotics between E.coli and E. albertii isolates.
There was insufficient data about the resistance difference between E.coli and E. albertii. Therefore, there is a need for more epidemiological studies on the patterns of drug resistance of E. albertii and to determine the mechanisms of molecular resistance for the management of infections caused by those isolates [19].
Urinary tract infection signs in children are highly heterogeneous, and the symptoms can be relatively unclear. The gold standard diagnostic test for urinary tract infection is still urine culture [32]. The management of UTI in children has been changed from the use of aggressive radiological exploration and prophylactic antimicrobial therapy to a more controlled approach [33]. In the present study, the common symptoms were fever, dysuria, abdominal pain and vomiting. However, these symptoms are widely shared by many clinical conditions in children.

CONCLUSION
The present study highlights the emergence of E. albertii as a pathogen associated with urinary tract infections in children. There is marked antibiotic resistance of this pathogen, especially toward extended spectrum beta-lactams antibiotics. The identification method depends mainly on genetic studies. Further longitudinal studies with a large number of patients are required to verify the accurate prevalence of this bacterium.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This study was approved by the ethics committee of Mansoura Faculty of Medicine, Egypt (R. 20.12.1110).

HUMAN AND ANIMAL RIGHTS
No animals were used for studies that are base of this research. All the humans used were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013 (http://ethics.iit.edu/ecodes/node/3931).

CONSENT FOR PUBLICATION
The approval was taken from the parents of the patients.

STANDARDS OF REPORTING
STROBE guidelines and methodologies were followed for this study.