|Year : 2016 | Volume
| Issue : 8 | Page : 55-58
Species-level identification of Acinetobacter by 16s rRNA sequencing: Necessity today, essentiality tomorrow
Trupti Bajpai1, Ganesh S Bhatambare2, Meena Varma3, Maneesha Pandey4
1 Department of Microbiology, Sri Aurobindo Medical College and PG Institute, Indore, Madhya Pradesh; Department of Biochemistry, SOS, IGNOU, New Delhi, India
2 Department of Microbiology, Sri Aurobindo Medical College and PG Institute, Indore, Madhya Pradesh, India
3 Department of Biochemistry, Sri Aurobindo Medical College and PG Institute, Indore, Madhya Pradesh, India
4 Department of Biochemistry, SOS, IGNOU, New Delhi, India
|Date of Web Publication||3-Jan-2017|
Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, MR 10 Crossing, Indore Ujjain Road, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Purpose: When common organisms present with uncommon phenotypes, reliance on phenotype can compromise accurate identification. The use of 16s rRNA gene sequences to study bacterial phylogeny, and taxonomy has been the most common housekeeping genetic marker. The aim of our study was to identify "difficult" and notorious uropathogen such as Acinetobacter through multiple identification methods. Materials and Methods: The present prospective study was conducted for the period of 6 months in the year 2015 in the Department of Microbiology of a Teaching Tertiary Care Hospital. A total of 345 clean catch, midstream urine samples obtained from patients suspected of urinary tract infection were subjected to microscopy and culture. Uropathogens isolated from the culture-positive samples were identified to species level through conventional, automated, and molecular methods. Results: A total of 123 uropathogens were isolated from 118 culture-positive samples. Among them, 81.3% isolates were Gram-negative bacteria, 14.6% were Gram-positive bacteria, and 23.5% were Candida species. Four (3.2%) Acinetobacter isolates were detected among which three were confirmed as Acinetobacter baumannii, whereas one of them was confirmed as Acinetobacter junii by different methods of identification. Conclusion: Identification by gene sequencing is more objective, reliable, reproducible, and accurate and has the capability of defining taxonomical relations among bacteria.
Keywords: 16s rRNA sequencing, Acinetobacter spp., biochemical tests, Vitek-2 compact
|How to cite this article:|
Bajpai T, Bhatambare GS, Varma M, Pandey M. Species-level identification of Acinetobacter by 16s rRNA sequencing: Necessity today, essentiality tomorrow. N Niger J Clin Res 2016;5:55-8
|How to cite this URL:|
Bajpai T, Bhatambare GS, Varma M, Pandey M. Species-level identification of Acinetobacter by 16s rRNA sequencing: Necessity today, essentiality tomorrow. N Niger J Clin Res [serial online] 2016 [cited 2020 Aug 10];5:55-8. Available from: http://www.mdcan-uath.org/text.asp?2016/5/8/55/197438
| Introduction|| |
The genus Acinetobacter comprises a heterogeneous group of Gram-negative, nonfermentative, aerobic bacteria that have recently become the focus of attention for clinicians and scientists.  During the last 20 years, it has emerged as an important opportunistic and nosocomial pathogen, frequently reported to be the potential agents of serious hospital outbreaks. The notorious nosocomial Acinetobacter is usually pan-drug resistant and is capable of causing substantial morbidity and mortality in patients with severe underlying diseases both in community and hospital settings. They especially colonize in patients who are intubated, in those who have multiple intravenous lines or monitoring devices, surgical drains, or indwelling urinary catheters. ,
Identification of Acinetobacter at the species level is still erratic, and conventional phenotypic tests prove to be insufficient to identify Acinetobacter isolates to the species level which is especially essential for clinical reasons. However, in recent years, numerous molecular techniques have been successfully used for the description of Acinetobacter species. ,
Due to growing importance of Acinetobacter in hospital infections, the precise identification of the species is important to elucidate the taxonomy, epidemiology, ecology, genomics, and pathology of these species.  Our study aims at identification of a "difficult" uropathogen such as Acinetobacter through multiple identification methods.
| Materials And Methods|| |
The present prospective study was conducted from January 2015 to June 2015 in the Department of Microbiology of a teaching Tertiary Care Hospital located in the Central India. Clean catch, midstream urine samples from 345 patients clinically suspected of urinary tract infection (UTI) were subjected to microscopy and culture. The uropathogens isolated from culture-positive samples were identified by conventional methods on the basis of microscopy, culture characteristics, and colony characteristics on blood agar, MacConkey agar, and UTI chromogenic media followed by biochemical testing methods (HiMedia, Mumbai). , The isolates that were identified as Acinetobacter by conventional methods were identified and confirmed to species level by automated system (Vitek-2 Compact, BioMérieux Inc., France) and if required they were further confirmed by molecular method (16s rRNA sequencing, Yaazh Xenomics, Chennai). The 16s rRNA sequencing involved genomic DNA extraction 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. , This was followed by analysis of sequencing data 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 trees of the identified Acinetobacter species were analyzed. Data for the phylogenetic analysis were obtained from sequences contained in the GenBank nucleotide sequences database. ,
| Results|| |
A total of 118 (34.2%) out of 345 urine samples from patients clinically suspected of UTI were found to be culture positive. One hundred and twenty-three uropathogens were isolated from 118 culture-positive samples (five samples had mixed flora, i.e., two pathogens per urine sample). Among these, 123 uropathogens, 76 (81.3%) were Gram-negative bacteria, 18 (14.6%) were Gram-positive bacteria, and 29 (23.5%) were Candida species. Among 76 Gram-negative bacteria, 65 (85.5%) were members of Enterobacteriaceae, 07 (9.2%) were Pseudomonas aeruginosa, and 04 (5.2%) were Acinetobacter species. Out of the four Acinetobacter, two (50%) isolates were identified and confirmed as Acinetobacter baumannii by conventional and automated method, respectively. One (25%) isolate was identified and confirmed as Acinetobacter junii by automated method and 16s rRNA sequencing [Figure 1], respectively. One (25%) isolate that was identified as Acinetobacter spp. by a conventional method and A. baumannii complex (any of the four species among A. baumannii, Acinetobacter calcoaceticus, Acinetobacter Pittii, or Acinetobacter nosocomialis was suspected) by Vitek-2 Compact was confirmed as A. baumannii by 16s rRNA sequencing [Figure 2].
|Figure 1: Phylogenetic tree of Acinetobacter junii based on the nucleotide sequences of 16s rRNA genes|
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|Figure 2: Phylogenetic tree of Acinetobacter baumannii complex (confirmed as Acinetobacter baumannii) based on the nucleotide sequences of 16s rRNA genes|
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| Discussion|| |
Prompt and accurate detection of bacterial pathogens is essential for improving the management of infectious diseases. Most of the current clinical microbiology laboratory practices in developing countries still continue to rely on the bacterial identification based on the use of conventional techniques including Gram-staining, colony morphology, growth requirements, and enzymatic and/or metabolic activities. This phenotypic approach presents some inherent problems such that they are laborious and time consuming, fail to identify some bacteria because of the variability of characteristics generated with repeated testing, stress or evolution, and the test results are mostly based on an individual and subjective judgment and interpretation. Such techniques also cannot distinguish between strains belonging to the same species. Moreover, the characteristic database also lacks newly described species and unusual microorganisms. Moreover, it is well known that in the case of UTI, the delay between specimen submission and diagnosis results in empiric and frequently inappropriate antimicrobial therapy. ,,,
Much progress has been made through the development of miniaturized ID systems followed by innovative automatic ID systems such as Vitek 2 (Biomerieux, France) and Phoenix (BD Microbiology systems, Cockeysville, MD) that provide accurate and reproducible IDs as well as antibiotic sensitivity tests. In spite of the undoubtedly innovative results obtained with the widespread use of these automated systems, they do have some drawbacks, particularly when microbiologists need to identify microorganisms exhibiting biochemical features that do not fit into any known patterns of genus and species. Such unusual isolates are commonly isolated from patients in Intensive Care Units (ICUs), especially those who have undergone long-term antimicrobial therapy. Such isolates can lose their typical biochemical characteristics and become extremely difficult to cultivate. 
The application of molecular approaches to clinical diagnostics has a number of advantages over standard microbiological techniques including sensitivity, speed, and ease of specimen processing. This has proved useful not only for slow-growing, unusual, or fastidious bacteria but also for identification of rare bacteria having ambiguous profiles, which are poorly differentiated by conventional methods. , An excellent example of these molecular methods is MicroSeq 500 (Applied Biosystems, California, USA) 16s rRNA sequencing that helps in identifying some "difficult" strains that conventional and automated systems have failed to characterize either by furnishing an inconclusive ID or by exhibiting an unlikely (implausible) profile. 
The taxonomy of the genus Acinetobacter has a long history of debate. Based on the most recent taxonomic studies, Acinetobacter belongs to subclass Gammaproteobacteria, family Moraxellaceae comprising catalase positive and oxidase negative nonmotile coccobacilli.  Recent molecular studies have shown 31 distinct Acinetobacter species with valid names among the genus Acinetobacter. , Besides, the genus comprises a number of taxa including species with published names. Of these, A. calcoaceticus, A. baumannii, A. pittii, and A. nosocomialis (formerly referred to as genomic species 1, 2, 3, and 13 TU) are phenotypically and genotypically similar. 
Only a few phenotypic techniques (based on 19 biochemical test pattern developed by Bouvet and Grimont)  have been validated to identify 12 clinically important Acinetobacter species. Therefore, in our study, it was possible to identify two isolates as A. baumannii by conventional method with further confirmation by automated system (Vitek-2 Compact). However, these techniques are unable to identify rest of the Acinetobacter species that have already been identified by DNA-DNA hybridization.
Therefore, in our study, one of the Acinetobacter isolates that could not be identified by conventional method up to species level was identified as A. junii by automated method and was further confirmed by molecular method (16s RNA sequencing). Apart from these, one of the isolates that was identified as Acinetobacter conventionally was identified as A. baumannii complex by our automated system. Studies have proved that differentiation of genetically closely related species in an A. calcoaceticus-baumannii (ACB) complex is not possible using conventional methods. In addition, commercially available platforms are also insufficient and inaccurate to reach the confirmation. 
Associated with the A. baumannii is a group of phenotypically and genotypically closely related Gram-negative bacteria that can hardly be distinguished by phenotypic and chemotaxonomic criteria. These particular species have been named as A. calcoaceticus, A. pittii, and A. nosocomialis. Collectively, they are now referred as ACB complex, having a 16s rRNA sequence identity value between 97% and 99%, and interspecies values between 65% and 75%. Although, ACB complex is considered to be an important nosocomial agent, the clustering of these species together in the ACB complex is unsatisfactory for clinical reasons because it obscures possible differences in biology and pathology of the individual species. For example, A. calcoaceticus is predominantly an environmental isolate while other three are clinically important species with A. baumannii, especially involved in infection and epidemic spread. ,,
A. baumannii is the most troublesome species associated with the significant proportion of nosocomial infections such as UTI, endocarditis, surgical-site infections, meningitis, soft tissue infections, blood stream infections, and nosocomial pneumonia, particularly ventilator-associated pneumonia in ICU patients. , They have more recently become a cause for major concern in clinical practice due to its high level of antimicrobial resistance, especially to carbapenems which have been accounted as the most effective antimicrobial agents for the treatment of infections caused by multidrug-resistant organisms, thereby leading to reduced therapeutic options. The circumstances of some outbreaks demonstrated the long survival of Acinetobacter in dry, inanimate environments.  Our uropathogen (ACB complex) was also confirmed as A. baumannii that needs a major concern in our clinical setting. Thus, 75% of isolates were confirmed as A. baumannii in our study.
Other Acinetobacter species including A. junii that was one of our isolates are exceedingly rare and their infections usually run a benign clinical course associated with low mortality rates. Acinetobacter species other than Baumannii and its close relatives are normal commensals, often colonizing the skin and mucous membranes of the humans and their recovery may be misinterpreted as the causative agents of infection. Small-sized outbreaks caused by Acinetobacter species other than Baumannii and its close relatives have been observed occasionally. 
Acinetobacters are occasionally known to cause UTIs, especially related to indwelling Foley's catheters. These infections are usually benign and can be either community acquired or hospital acquired.  In our study also, a very small percentage (3.2%) of uropathogens were detected as Acinetobacter spp. Even 3.2% Acinetobacter isolated as uropathogens in our clinical setting should attract the attention of clinicians and microbiologists because they are a matter of concern having the enormous potential to spread its notorious characteristics among other strains through horizontal transmission. Actually, several clonal lineages of A. baumannii have disseminated worldwide and seem to have a selective advantage over nonepidemic strains. The reasons for the success of these epidemic lineages remain to be elucidated but could be related to the potential of these organisms to achieve very dynamic reorganization and rapid evolution of their genome including acquisition and expression of additional antibiotic resistance determinants under fluently environmental and selective conditions.
Several genotypic methods have been developed for the identification of Acinetobacter species. Despite significant advances in genotypic methodologies, genotypic identification is not being used extensively these days. It should be introduced, especially because of faster turnaround time and high sensitivity as compared to phenotypic methods.
| Conclusion|| |
Microbial identification by gene sequencing plays an important role in the identification of isolates with ambiguous biochemical profiles, thereby proving to be an accurate, reliable, reproducible tool with excellent capability of defining taxonomical relations among bacteria. Currently, 16s rRNA sequencing plays a limited role in the identification of microorganisms in clinical laboratories, mainly due to high costs and requirements of great technical skill; however, multiple identification methods are highly desirable, especially for "difficult" pathogens.
The authors wish to thank the management, technical, and clinical staff of Sri Aurobindo Institute of Medical Science Medical College and PG Institute, for their kind support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
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.
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.
Cheesbrough M. Microbiology. Medical Laboratory Manual for Tropical Countries. Vol. 2. Cambridgeshire, England: 1984. p. 985.
Collee JG, Fraser AG, Marmian BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. (Reprinted 1999). 14 th
ed. (Reprinted 1999); 1996.
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 Mircobiol Res 2013;7:5637-44.
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
, 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 non-specialist. Nucleic Acids Res 2008;36:W465-9.
Sun CP, Liao JC, Zhang YH, Gau V, Mastali M, Babbitt JT, et al.
Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array. Mol Genet Metab 2005;84:90-9.
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.
Gerner-Smidt P, 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.
[Figure 1], [Figure 2]