|Year : 2020 | Volume
| Issue : 2 | Page : 50-54
Identification of Uropathogens: A Journey from Conventional to Molecular Level
Trupti Bajpai1, Maneesha Pandey2, Meena Varma3, Neelesh Gagrani4, Ganesh Bhatambare1
1 Department of Microbiology, Sri Aurobindo Medical College and PG Institute, Indore, MP, India
2 Discipline of Biochemistry, SOS, IGNOU, New Delhi, India
3 Department of Biochemistry, Sri Aurobindo Medical College and PG Institute, Indore, MP, India
4 Gagrani Hospital, Dewas, MP, India
|Date of Submission||09-May-2020|
|Date of Decision||17-Oct-2020|
|Date of Acceptance||12-Dec-2020|
|Date of Web Publication||31-Dec-2020|
Dr. Trupti Bajpai
Department of Microbiology, Sri Aurobindo Medical College and PG Institute, MR-10 Crossing, Indore-Ujjain Road, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: During the course of bacterial infection, the rapid and accurate identification of the causative agent is essential to determine the effective treatment option. Now, the question arises, is it necessary to identify the microbial pathogens up to the species level? Objective: The present prospective study involving uropathogens has been designed to highlight the journey from well-adapted, inexpensive but time-consuming and labor-intensive gold-standard conventional (biochemical) diagnostic methods to the rapid and specific upcoming rival in the form of molecular methods (16s rRNA sequencing). Materials and Methods: The study was carried out for a period of 12 months. Clean catch, midstream urine samples from 1101 admitted patients clinically suspected of urinary tract infection (UTI) were subjected to microscopy and culture on blood agar, MacConkey agar, and UTI chromogenic media (HiMedia, Mumbai). The uropathogens isolated from the culture-positive samples were identified up to the species level by the conventional method (Biochemical testing). The isolates were further confirmed by automated method (Vitek 2-Compact System, BioMérieux Inc., France). If required, then, further confirmation was done by molecular method (16S rRNA sequencing) (Yaazh Xenomics, Mumbai and Chennai). Results: A total of 463 (42%) urine samples were found to be culture positive out of 1101 patient samples processed. Four hundred and eighty-nine uropathogens were isolated from 463 culture-positive samples (26 samples had mixed flora i.e., two pathogens per sample). Conclusions: Although genotypic characterization of bacterial pathogen is advantageous when compared to phenotypic method, it is recommendable to use the combination of a traditional culture-based assays and rapid molecular diagnostic tool.
Keywords: 16s rRNA sequencing, biochemical tests, uropathogens, Vitek-2 automated system
|How to cite this article:|
Bajpai T, Pandey M, Varma M, Gagrani N, Bhatambare G. Identification of Uropathogens: A Journey from Conventional to Molecular Level. Biomed Res J 2020;7:50-4
|How to cite this URL:|
Bajpai T, Pandey M, Varma M, Gagrani N, Bhatambare G. Identification of Uropathogens: A Journey from Conventional to Molecular Level. Biomed Res J [serial online] 2020 [cited 2021 Apr 15];7:50-4. Available from: https://www.brjnmims.org/text.asp?2020/7/2/50/305771
| Introduction|| |
The prompt and accurate identification (ID) of the causative pathogen has always been essential for determining the effective treatment options, during the course of bacterial infection. Most of the microbiological laboratories continue to rely upon phenotypic methods of diagnosis involving conventional ID techniques. Such traditional approach presents some inherent problems because conventional databases mostly lack unusual or newly described organisms. It is well known that the delay between specimen submission and diagnosis may result in empiric and frequently inappropriate antimicrobial therapy against urinary tract infection (UTI).,,, A continuous progress is being made through the development of miniaturized ID systems, followed by modern automated ID systems that provide accurate and innovative IDs. In spite of their special features, they lack the reproducibility of molecular methods. The genotypic approaches have proved useful for slow-growing, unusual or fastidious bacteria and bacteria with ambiguous profiles which are poorly differentiated by conventional and automated methods. This is because of their speed, sensitivity, and ease of specimen processing. An excellent example of these molecular methods is MicroSeq 500 (Applied Biosystems) Foster City, California, USA, used to carry out 16s rRNA sequencing.,
Now, the question arises; is it necessary to identify the microbial pathogens up to the species level? The present prospective study involving uropathogens has been designed to highlight the journey from well-adapted, inexpensive but time-consuming and labor-intensive gold standard conventional (Biochemical) diagnostic methods through automated ID system to the rapid and specific upcoming rival in the form of molecular methods (16s rRNA sequencing).
| Materials and Methods|| |
The present prospective study was conducted for a period of 12 months in the department of microbiology of a teaching tertiary care hospital located in the central India. It was approved by the institutional ethical committee. Clean catch, midstream urine samples from 1101 admitted patients clinically suspected of UTI were subjected to microscopy and culture on blood agar, MacConkey agar, and UTI chromogenic media (HiMedia, Mumbai).
The urine samples from patients already on antibiotics or those suspected of renal tuberculosis and leptospirosis were not included in the study. Furthermore, the samples from patients with the same episode of UTI were not repeated again. The uropathogens isolated from the culture-positive samples were identified up to species level by conventional method (biochemical tests) (HiMedia, Mumbai).,
The isolates were further confirmed by automated method (Vitek 2-Compact System, BioMérieux Inc., France). If required, then, further confirmation was done by molecular method (16S rRNA sequencing) (Yaazh Xenomics, Mumbai and Chennai). In 16s rRNA sequencing, genomic DNA was extracted, followed by its amplification (polymerase chain reaction). The amplified fragments were purified before sequencing. Bidirectional sequencing was performed for each amplified product by an automated sequencer., The analysis of sequencing data was performed by MicroSeq 500 software. The consensus sequences were compared (online) with the published sequences available in GenBank at the website (http://www.ncbi.nlm.nih.gov/) using nucleotide Basic Local Alignment Search Tool of National Center for Biotechnology Information. The phylogenetic tree of the identified bacterial isolates was analyzed. Data for the phylogenetic analysis were obtained from sequences contained in the GenBank nucleotide sequences database., The data were entered into MS Excel sheet, and simple percentages were used for the analysis of the data. The mycological work up for speciation of Candida isolates was simultaneously done through conventional and automated methods.,,
| Results|| |
A total of 463 (42%) urine samples were found to be culture positive out of 1101 patient samples processed. Four hundred and eighty-nine uropathogens were isolated from 463 culture-positive samples (26 samples had mixed flora i.e., two pathogens per sample). Among these 489 isolates, 315 (64.4%) were Gram-negative bacilli, 101 (20.6%) were Gram-positive cocci and 73 (14.9%) were Candida species.
Among the 315 Gram-negative isolates, 281 (89.2%) were members of Enterobacteriaceae, 27 (8.5%) were Pseudomonas aeruginosa, 06 (1.9%) were Acinetobacter species and 01 (0.3%) was a member of nonfermenter. Out of 101 Gram-positive isolates, 46 (45.5%) were Staphylococcus species and 55 (54.4%) were Enterococcus species. The detailed list of uropathogens isolated during the study period and the method by which they were identified up to the species level is mentioned in [Table 1].
|Table 1: List of uropathogen isolates confirmed through different methods of identification|
Click here to view
The ID of isolates up to the species level by means of automated and molecular methods is represented in [Figure 1] and [Figure 2], respectively.
In the present prospective study, 442 out of 489 microbial isolates were identified and confirmed up to the species level by conventional method alone or by the combination of both conventional and automated method. A total of 37 isolates identified as Coagulase Negative Staphylococcus (CoNS) by traditional method were not confirmed to the species level by automated system since it would not affect the treatment options. A total of ten isolates were confirmed through 16s rRNA sequencing.
| Discussion|| |
There exist certain “difficult” strains with ambiguous biochemical features that the automated system fails to characterize either by furnishing an inconclusive ID or by exhibiting implausible profile. All such partially identified or completely unidentified uropathogenic bacterial isolates that could not be confirmed by conventional or automated methods were subjected to molecular methods like 16s rRNA sequencing during our study. Therefore, gene sequencing is an accurate and reproducible method to identify such isolates thereby increasing our ability to capture the diversity of microbial taxa. One of the most attractive potential use of this new technology-based 16s rRNA sequence information is to provide genus and species ID for isolates that do not fit into any recognized biochemical profiles. It helps in the ID of unusual, novel, and “difficult to cultivate” organisms.,
During our study, six bacterial isolates that could not be identified by conventional methods were confirmed as Escherichia coli by the automated method. They were actually the “inactive” variants of E. coli that are nonmotile, anaerogenic, nonlactose fermenters and share several biochemical features with Shigella. Sequencing could not differentiate these two closely related members of Enterobacteriaceae because 16s rRNA sequences of these two genera contain highly conserved regions.
However, the organism was confirmed as E. coli instead of Shigella because based on our knowledge and different studies, extra-intestinal manifestations of Shigella are uncommon and in spite of the genotypic similarities E. coli and Shigella revealed variable sensitivity patterns and presenting treatments against UTI due to these two genera would be challenging.,,,,
One of the Gram-negative isolates that remained unidentified by the conventional method was identified as Moraxella More Details lacunata by automated system. This isolate was finally confirmed as Providencia rettgeri by 16s rRNA sequencing.,
Another genus, Acinetobacter was the center of attraction during our study. Several nosocomial infections and hospital outbreaks associated with Acinetobacter species have been reported in Intensive Care Units worldwide. The taxonomy of the genus Acinetobacter has a long history of debate. Where, biochemical method could not detect it, automated method nominated one of the species as Acinetobacter junni which was later on confirmed as the same through sequencing method while another isolate that was unidentified by conventional method and was identified as Acinetobacter baumannii ex (ABC) (mentioning it as one of the four species among A. baumannii, A calcoaceticus, A. nosocomialis, and A. pitti) by automated system. This was finally confirmed as Acinetobacter baumannii by 16s rRNA sequencing. ABC is a cluster of four different species of Acinetobacter that can hardly be distinguished on the basis of phenotypic and chemotaxonomic criteria. Clinically, they cannot be considered similar due to possible differences in the biology and pathology of the individual species. A total of 1.9% Acinetobacter spp detected as uropathogens in our study is a matter of concern for clinicians because few species of this genus are highly resistant and have the enormous potential to spread their notorious characteristics among other strains through horizontal transmission. Some of the species of Acinetobacter are generally more susceptible to antimicrobials and are usually considered to be of minor virulence.
These infections usually run a benign clinical course and their associated mortality is low. It is known that Acinetobacter species like A. calcoaceticus is a soil organism and A. lwoffii is a part of bacterial flora of the skin, while at the other extreme lies A. baumannii with the potential to inhabit patients as well as hospital environment.
Colonization in susceptible patients, carriage by medical staff, prolonged survival in the hospital environment, and resistance to common antibiotics and aseptic agents results in frequent outbreak of A. baumannii.
A. junni is an emerging and potential nosocomial human pathogen and the true incidence of this species might be underestimated since the phenotypic ID is usually difficult.,,,,,,,
One of the isolates in our study that could not be identified by the conventional method was identified and confirmed as Citrobacter koseri by both automated and molecular methods. The genus Citrobacter consists of facultative anaerobic, motile, Gram-negative bacilli, a common inhabitant of soil, water, food, and intestinal tracts of animals and humans. Species related differences have been noted within different species of Citrobacter. C. koseri is comparatively more sensitive isolate as compared to the species like C. freundii which belongs to C. freundii complex. Thus at this stage, it becomes necessary to confirm the species of Citrobacter through various available methods.
In the present prospective study, 73 Candida species were detected, among which Candida other than albicans (75.3%) predominated Candida albicans (24.6%). Although Candida albicans is frequently reported as the most prevalent species infecting the urinary tract, nonalbicans Candida species appear better adapted to the urinary tract environment. They are known to be increasing due to varying predisposing factors.,, At this stage, it is very important to identify Candida up to the species level because different species reveal different levels of resistance towards antimycotic therapy.
| Conclusions|| |
To conclude, a fast and reliable ID of pathogens causing acute infection is highly important issue in microbiological diagnosis. It is required that a perfect diagnostic method must be sensitive, specific, rapid, easy to perform and interpret but there exists no perfect method of diagnosis. On one hand, where, traditional diagnostics are low-priced reliable tools providing qualitative and quantitative results on the bacterial population; on the other hand, molecular diagnostics has revolutionized in form of rapid, more specific, sensitive, precise tools of diagnosis. On considering the cost–benefit ratio, the automated and molecular methods are less better choices but should be preferred if conventional methods could not provide any clues, and uropathogen identity plays a greater role in benefiting the health of the patient. Our study with urinary isolates has proved that proper integration of all the three methods can help us to identify the pathogenic isolates, which will be further helpful in administering appropriate antibiotics and in understanding the trend of pathogens in various clinical samples. Therefore, based on our results, it is recommendable to use a combination of a widely accepted culture-based assays involving biochemical testing, automated method, and molecular methods for confirmation.
The authors wish to thank the management, technical & clinical staff of Sri Aurobindo Medical College & PG Institute, Indore (MP) for their kind support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sun CP, Liao JC, Zhang YH, Gau V, Mastali M, Babbitt JT, et al.
Rapid, speciesspecific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dotblot hybridization and an electrochemical sensor array. Mol Genet Metab 2005;84:90-9.
Bakkali ME, Chaoui I, Zouhdi M, Melloul M, Arakrak A, Mzibri ME, et al
. Comparison of the conventional technique and 16s rDNA gene sequencing method in identification of clinical and hospital environmental isolates in Morocco. Afr J Microbiol Res 2013;7:5637=44.
de Melo Oliveira MG, Abels S, Zbinden R, Bloemberg GV, Zbinden A. Accurate identification of fastidious Gram-negative rods: Integration of both conventional phenotypic methods and 16S rRNA gene analysis. BMC Microbiol 2013;13:162.
Elgaml A, Hassan R, Barwa R, Shokralla S, Naggar WE. Analysis of 16s ribosomal RNA gene segments for the diagnosis of Gram negative pathogenic bacteria isolated from urinary tract infections. Afr J Microbiol Res 2013;7:2862-9.
Monica Cheesbrough, District Laboratory Practice in Tropical Countries. Cambridgeshire, England: Cambridge University Press; 1984;2: p. 985.
Collee JG, Fraser AG, Marmian BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. Standard Edition. 14th
ed. London, UK: Churchill Livingstone; 2000.
Fontana C, Favaro M, Pelliccioni M, Pistoia ES, Favalli C. Use of the MicroSeq 500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J Clin Microbiol 2005;43:615-9.
Fukushima M, Kakinuma K, Kawaguchi R. Phylogenetic analysis of salmonella shigella and Escherichia coli
strains on the basis of the gyrb gene sequence. J Clin Microbiol 2002;40:2779-85.
Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, et al.
Phylogeny.fr: Robust phylogenetic analysis for the nonspecialist. Nucleic Acids Res 2008;36:W465-9.
Deorukhkar S, Saini S. Non albicans-Candida
species: Its isolation pattern, species distribution, virulence factors and antifungal sensitivity profile. Int J Med Sci Public Health 2013;2:533-8.
Chander J. Candidiasis in Text Book of Medical Mycology. 3rd
ed., Ch. 13. New Delhi: Mehta Publishers; 2002.
Ng KP, Kuan CS, Kaur H, Na SL, Atiya N, Velayuthan RD. Candida
species epidemiology 2000-2013: A laboratory-based report. Trop Med Int Health 2015;20:1447-53.
Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Med Microbiol 2007;44:1108-14.
Janda JM, Abbott SL. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls. J Clin Microbiol 2007;45:2761-4.
Raksha R, Srinivasa H, Macaden RS. Occurrence and characterisation of uropathogenic Escherichia coli
in urinary tract infections. Indian J Med Microbiol 2003;21:102-7.
] [Full text]
Khot PD, Fisher MA. Novel approach for differentiating Shigella
species and Escherichia coli
by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2013;51:3711-6.
Karakas A, Coskun O, Kilic A, Bedir O, Besirbellioglu BA. Urinary tract infections caused by Shigella
species. Travel Med Infect Dis 2016;14:1-3.
Shyamkuwar A, Deogade N, Wadher B, Roychoudhury K. Incidence of non-fermenter Moraxella
species in clinical isolates. Int J Pharm Sci Rev Res 2014;29:105-9.
Wie SH. Clinical significance of providencia bacteremia or bacteriuria. Korean J Intern Med 2015;30:167-9.
Visca P, Seifert H, Towner KJ. Acinetobacter infection – An emerging threat to human health. IUBMB Life 2011;63:1048-54.
Sundar SK, Kumari TP, Vijayalakshmi B, Murugan M. Isolation and 16s rRNA sequencing of clinical isolates of Acinetobacter baumannii
. Int J Curr Microbiol Appl Sci 2014;3:855-8.
Khosravi AD, Sadeghi P, Shahraki AH, Heidarieh P, Sheikhi N. Molecular methods for identification of Acinetobacter species by partial sequencing of the rpoB and 16S rRNA genes. J Clin Diagn Res 2015;9:DC09-13.
Smidt GP, Tjernberg I, Ursing J. Reliability of phenotypic tests for identification of Acinetobacter
species. J Clin Microbiol 1991;29:277-82.
Ahmed SS, Alp E. Genotyping methods for monitoring the epidemic evolution of A. baumannii
strains. J Infect Dev Ctries 2015;9:347-54.
Chang HC, Wei YF, Dijkshoorn L, Vaneechoutte M, Tang CT, Chang TC. Species-level identification of isolates of the Acinetobacter calcoaceticus
complex by sequence analysis of the 16S-23S rRNA gene spacer region. J Clin Microbiol 2005;43:1632-9.
Nemec A, Krizova L, Maixnerova M, Van der Reijden TJ, Deschaght P, Passet V, et al
. Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus
complex with the proposal of Acinetobacter pittii
sp. nov. (formerly Acinetobacter genomic
species 3) and Acinetobacter nosocomialis
sp. nov. (formerly Acinetobacter genomic
species 13TU). Res Microbiol 2011;162:393-404.
Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii
: Emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538-82.
Arens S, Verhaegen J, Verbist L. Differentiation and susceptibility of citrobacter isolates from patients in a university hospital. Clin Microbiol Infect 1997;3:53-7.
Deorukhkar SC, Saini S, Mathew S. Non-albicans Candida
infection: An emerging threat. Interdiscip Perspect Infect Dis 2014;2014:1-7.
Sobel JD, Fisher JF, Kauffman CA, Newman CA. Candida urinary tract infections-Epidemiology. Clin Infect Dis 2011;52 Suppl 6:S433-36.
[Figure 1], [Figure 2]