Detection of Escherichia albertii in urinary and gastrointestinal infections in Kermanshah, Iran

Escherichia albertii (E. albertii) is a Gram-negative and facultative anaerobe bacterium. In recent years, the bacterium has been isolated from the feces of people with gastroenteritis as a pathogen that causes diarrhea. Due to insucient information on the phenotypic and biochemical characteristics of E. albertii, it is dicult to distinguish it from other species of the Enterobacteriaceae family. This is especially prevalent in the pathotypes of Escherichia coli (E. coli). Moreover, in clinical laboratories, it is mistakenly identied as E. coli or even Hafnia alvei (H. alvei). This study was performed for the rst time in Iran to identify E. albertii by PCR method from a sample of urinary and gastrointestinal infections obtained from clinical laboratories in Kermanshah, which were distinguished as E. coli.


Conclusion
The ndings of this study show that E. albertii can be considered as one of the causes of urinary and gastrointestinal infections that are mistakenly identi ed in clinical laboratories as E. coli.Therefore, the use of molecular methods for accurate and de nitive diagnosis of bacteria can be useful.

Background
Infectious diseases are always a threat to health and life, and they impose huge costs on families and the health-care system of the country every year. Infections related to gastrointestinal and urinary tract infections are of particular importance. A high proportion of these infections are attributed to the genus Escherichia from the Enterobacteriaceae family (1)(2)(3)(4).
Escherichia albertii is a relatively new member of this bacterial family, and there is strong evidence that the bacterium plays a role in many global con icts. Its misdiagnosis as Enteropathogenic Escherichia Coli (EPEC), Enterohemorrhagic Escherichia coli (EHEC), or other pathogenic bacteria has continued from the past to the present. Recently, its pathogenic potential has been strengthened by identifying strains with multiple antibiotic resistance (5)(6)(7).
Epidemiology, transmissibility, prevalence, pathogenicity, virulence factors, and antibiotic resistance in this emerging bacterium remain a challenge, and information about it is very poor. Its misdiagnosis as E. coli and the lack of a reliable diagnostic method to differentiate it are important points that have led to further investigation of E. albertii to identify its unique behaviors and characteristics. Comprehensive knowledge of the features of E. albertii, as a type of intestinal pathogen in humans and awareness of its prevalence, requires more analysis from different sources, different hosts, and the use of more accurate identi cation methods.
The uidA gene is one of the speci c housekeeping genes in E. coli that encodes beta-di-glucuronidase and is considered one of the virulence markers in E. coli. Numerous studies have used this gene to identify E. coli, and PCR methods based on the diagnosis of this gene have been repeatedly used to diagnose E.coli (8)(9)(10)(11). On the other hand, to identify E. albertii, tracking of two genes of mdh (Encoding Malate dehydrogenase) and lysP (Encoding Lysine Permease) as speci c and protected genes in E. albertii is performed in the form of a multiplex PCR method (8-10, 12, 13). Also rpoB gene sequencebased identi cation, Multilocus sequence typing (MLST), or Whole Genome Sequencing (WGS) methods have been proposed, one of the limitations of which is being time-consuming to complete the results (10,(13)(14)(15).
As can be seen, the species of E. albertii is considered a human pathogen that may be ignored due to weakness in diagnosis. Moreover, the prevalence of this species in Iran is unknown. Therefore, the present study aimed to investigate the prevalence of E. albertii and its differentiation from E. coli in clinical samples of patients with diarrhea and urinary tract infections in Kermanshah using molecular methods. Methods This is a cross-sectional-analytical study. The study population was the cultures of urine and fecal clinical samples collected from laboratories in Kermanshah. 180 culture samples belonging to 180 clinical samples were collected during 6 months (From February 2018 to July 2018), which after initial tests, a total of 100 samples infected with E. coli were identi ed. Finally, these 100 samples were examined to track study objectives. Samples of urine and fecal culture included 60 and 40 cases, respectively.

Culturing of samples
In the research laboratory, subcultures were prepared from cultures suspected of containing E. coli obtained from fecal and urinary specimens (Positive lactose and negative lactose) and biochemical tests including TSI, SIM, MRVP, Simon citrate, Urease, lysine, phenylalanine, and culture on the MacConkey agar were performed to identify E. coli (16).

Preparation of microbial stock
To make the isolates usable in later stages, including molecular tests, the microbial stock was prepared from pure isolates that were detected E. coli using biochemical tests. A preservation medium (Including glycerol and liquid BHI medium) was used for this purpose. The pure bacterial isolates were then placed in tubes and after two hours of heating stored in a 20˚C freezer (17).

Genomic DNA extraction
In the present study, DNA extraction was performed by the Phenol-Chloroform-Isoamyl method (16).

Determination of quality and quantity of extracted genomic DNA
To determine the quantity and quality of DNA, electrophoresis was used on 0.8% agarose gel, and the quality of the band was assessed using Ethidium bromide and placing the gel inside the Gel Document device.

Polymerase chain reaction (PCR)
Isolates that belonged to E. coli (Negative lactose and positive lactose) based on biochemical tests were nominated to track down E. albertii. For this purpose, three genes of uid A, mdh, and lysP were evaluated using the PCR method.
In the present study, the polymerase chain reaction was performed using a thermocycler device in a volume of 20 μl. The materials required for each reaction are shown in Table 1.

Primer selection
The primers of the three required genes (uidA, mdh, and lysP) were purchased from CinnaGen Company (Iran). The selected primer pairs were diluted (1:10) according to the de ned standard by adding speci c amounts of sterile distilled water and a working solution was prepared from them ( Table 2).

Thermal cycling of PCR reaction
The thermal cycle was rst performed with an initial denaturation at 95℃ for 5 min, followed by 35 cycles. During these 35 cycles, denaturation at 95℃ for 30 s, the primer annealing at different temperatures for each primer (Annealing temperatures in this study for uidA, mdh, and lysP primers were 67, 65, and 64℃, respectively) was performed for 30 s and the extension step was performed at 72°C for 60 s. Finally, after the cycles were completed, the nal extension of the samples was performed at 72℃ for 5 min (Table 3). After the cycles were completed, the samples were removed from the device and stored at 4℃ until electrophoresis was performed (10 and 11 with minor modi cations).
Electrophoresis of PCR products on agarose gel DNA electrophoresis was performed horizontally in 2% agarose gel. The gel was placed inside the gel documentation device and the proliferated DNA bands were compared by the indicator DNA to view the desired bands.

Results
As mentioned earlier, during the initial processing, 100 samples (Negative lactose and positive lactose samples suspected of E. coli), including 60 urine samples and 40 fecal samples, were sent to the research laboratory for phenotypic and complementary biochemical diagnosis and molecular testing to identify E. albertii.

Results from phenotypic and biochemical tests
Biochemical tests performed on 100 samples, including TSI, SIM, MRVP, Simon citrate, urease, lysine, phenylalanine, and culture on MacConkey agar indicated that they belonged to E. coli.
The PCR results of uidA, mdh, and lysP genes The results of the polymerase chain reaction of 100 samples showed that 94% of them were E. coli (Positive uidA gene) (Figs. 1) and 6% of them were positive in terms of tracking two speci c genes of E. albertii (mdh and lysP) ( Fig. 2 and 3). On the other hand, these 6 isolates were negative for the uidA gene. Five isolates of these 6 cases were related to the urinary tract and one to gastrointestinal infections.

Discussion
The share of Escherichia, especially E. coli, is signi cant in urinary and gastrointestinal infections (3, 4, and 18). In recent years, E. albertii has been reported to cause gastrointestinal infections in some cases of epidemics, and the results indicated that E. albertii resembles E. coli pathotypes (19,20). This has challenged a range of studies to verify the diagnosis of E. coli pathotypes, especially EPEC, in recent years. Thus, it was proved that E. albertii was mistakenly identi ed as EPEC due to its unknown features and similar phenotypic and biochemical characteristics to E. coli (12, 21, and 22). The reliable classi cation of diarrhea-causing bacteria into distinct pathotypes requires molecular tools, and the unavailability of this equipment in clinical laboratories has led to misdiagnosis and neglect of some pathogens, including E. albertii (22). Recently, researchers, have examined this unknown species in terms of prevalence, biochemical characteristics, pathogenicity, and virulence factors. Although studies in this area are increasing day by day, there is still insu cient information about these indicators in E. albertii.
The study of this bacterial species is of particular importance since some ndings have obscured the information from previous research. Therefore, given the importance of E. albertii as a lesser-known bacterial species and its importance in gastrointestinal and urinary tract infections and its neglect in diagnosis, for the rst time in Iran (As far as the authors know) the present study identi ed E. albertii among urinary and fecal samples collected from clinical laboratories in Kermanshah province. These specimens were previously identi ed as E. coli.
Of the 100 clinical samples, 94 were positive for the uidA gene (Speci c for E. coli) and using the speci c genes of E. albertii (lysP and mdh), 6 cases (6%) of E. albertii were nally identi ed. The uidA gene was not found in these six samples. Of the 6 cases of E. albertii, 5 cases belonged to urinary tract infections. It is believed that the origin of E. coli, which causes urinary tract infections, is intestinal ora (23). As a result, the gastrointestinal tract can be the origin of E. albertii identi ed from urinary samples in the present study.
To identify E. albertii, similar studies have traced two genes of lysP, and mdh. Nimri reported that out of a total of 250 isolates obtained in about ten years from the feces of people with diarrhea, 48 cases of E. albertii were identi ed using the lysP and mdh genes. The uidA gene was not found in these 48 isolates. It should be noted that these specimens had previously been identi ed as E. coli (8).
Aoshima also reported 6 E. albertii isolates from 20 phenotypically recognized E. coli samples by identifying the lysP and mdh genes in a population with a gastrointestinal infection of food origin (12).
By tracking the eae gene and sequencing it, Ooka identi ed 21 out of 31 samples related to the gastrointestinal infection as E. albertii. They were initially diagnosed as E. coli (27). In another study, Ooka reported that out of a total of 278 samples from the human, animal, and environmental sources previously identi ed as E. coli using common diagnostic methods, 26 were identi ed as E. albertii using MLST analysis of the eae gene. 14 cases of them belonged to human samples (21).
Hinenoya re-examined 20 strains of E. coli isolated from diarrheal infections. He identi ed all 20 isolates as E. albertii by MLST analysis of housekeeping genes of E. albertii (24).
Ori et al., in a 6-year care program re-examined diarrhea-causing E. coli isolates. They identi ed 10 E. albertii cases out of a total of 693 isolates by tracking speci c genes including cdgR, DNA-binding transcription activating gene in cysteine biosynthesis, and palmitoyl-acyl carrier protein-depended acetyltransferase gene (22).
In a research conducted by Lindsey, out of a total of 1,644 samples of chicken carcasses over one year at the slaughterhouse, lysP and mdh genes were positive in 61 isolates, which were identi ed as possible E. albertii species. However, the sequencing of the rpoB gene reduced the number of E. albertii to 27 (14).
In general, due to the lack of a speci c diagnostic protocol for E. albertii, different studies have used different methods and speci c genes to identify it, and this information is constantly changing. It can be argued that although most studies have identi ed the two genes of lysP and mdh as speci c genes in the diagnosis of E. albertii, in several studies these two genes have not been able to identify all E. albertii. Therefore, efforts have been made to design more speci c areas of the genome (10,(13)(14)(15).
Also, there is a discrepancy in the ndings of the phenotypic and biochemical characteristics of E. albertii. For example, E. albertii was previously considered as a type of negative lactose, but a recent study found that strains of E. albertii could ferment the lactose (26). One of the reasons for the limited recognition of the phenotypic and biochemical properties of E. albertii is the small number of known strains to date (14).
In general, there is no su cient information about the features of E. albertii to isolate and diagnose it optimally. As a result, it is di cult to identify the true prevalence of infections associated with E. albertii (24).
In several studies, the role of E. albertii as a potential and related pathogen in cases of gastroenteritis and diarrhea in humans has been con rmed (14 and 25-30). In countries such as Japan and Norway, the number of infections caused by E. albertii is increasing, which is a warning sign that the bacterium is causing problems around the world (24). E. albertii is important not only for its pathogenicity and its role in gastroenteritis in the world but also for its resistance to certain antibiotics (6,7). Therefore, it is important to continuously examine patients with diarrhea and urinary tract infections to detect E. albertii. Deaths among birds are another reason for global attention to E. albertii. The bacterium has also been isolated from animals such as pigs, cats, and in some cases from environmental and food contamination (21,(31)(32)(33)(98)(99)(100). As a result, epidemiological studies should include not only the clinical level but also animal and environmental patterns such as water and food.
In the rst step, the study of the frequency of E. albertii in the world can help further identify it. Epidemiological studies also help identify pathogenic strains, biochemical characteristics, and virulence genes. Finally, the integration of data and results leads to a comprehensive and accurate de nition of the characteristics of E. albertii. In general, it can be said that in the future, diagnostic tests for E. albertii will be routinely performed in clinical laboratories to differentiate it from other members of the Enterobacteriaceae family.

Conclusions
As far as the authors know, the present study is the rst report on the identi cation and separation of E. albertii from gastrointestinal and urinary tract infections in Iran. The results show that E. albertii is one of the possible causes of diarrhea, gastroenteritis, and urinary tract infections. Therefore, it is necessary to study and recognize this bacterial species as much as possible. Extensive and comprehensive epidemiological studies in Iran and other parts of the world are recommended using well-known molecular methods to achieve preventive and therapeutic goals before major con icts caused by E. albertii.