Identification of clinical isolates ofAcinetobacter baumanniifrom Iran and study of their heterogeneity : Journal of the Chinese Medical Association

Secondary Logo

Journal Logo

Original Article

Identification of clinical isolates ofAcinetobacter baumanniifrom Iran and study of their heterogeneity

Sadeghi, Parisaa,b; Khosravi, Azar Dokhtb,c,*; Shahraki, Abdolrazagh Hashemic,d; Beiranvand, Maryamb

Author Information
Journal of the Chinese Medical Association: July 2016 - Volume 79 - Issue 7 - p 382-386
doi: 10.1016/j.jcma.2016.01.012

    Abstract

    1. Introduction

    Although acinetobacters are strictly aerobic Gram-negative coccobacilli that are widely distributed in soil and water, they are also commonly found in the hospital environment. Over the past 20 years, Acinetobacter species have emerged as opportunistic pathogens that are associated with severe hospital-acquired infections.1,2 In particular, among the various species of this genus, Acinetobacter baumannii is responsible for a significant proportion of nosocomial infections.3 The management of infections caused by Acinetobacter baumannii is greatly hindered by its intrinsic and acquired resistance to a wide variety of antimicrobial agents. In addition to this, the number of multidrug-resistant strains has increased during the past two decades.4 Therefore, A. baumannii has emerged as one of the most troublesome pathogens for health-care institutions globally. To control the spread of A. baumannii in the hospital setting, it is necessary to identify potential reservoirs of the organism and the modes of transmission. In addition, to distinguish A. baumannii strains involved in the outbreaks from epidemiologically unrelated strains, a comparison of isolates at the subspecies level is required by application of molecular typing methods.2,5

    There are several typing methods for Acinetobacter spp., including ribotyping, polymerase chain reaction (PCR) hybridization with species-specific probes, pulse field gel electrophoresis, and random amplified polymorphic DNA typing.6,7 The main disadvantage of these typing methods is low reproducibility, especially in terms of global molecular epidemiology. Using sequence-based typing such as single or multilocus or whole-genome sequencing provides more reliable data to compare strain information from a local point of view or globally. However, high cost is the main disadvantage of sequence-based typing in developing countries.6,8 From this point of view, definitive identification of A. baumannii strains and investigation of their heterogeneity will be of value for both clinical studies and molecular epidemiology purposes. In this study, rpoB-based PCR sequencing was evaluated for definitive identification of A. baumannii strains and investigation of their heterogeneity. The presumptive relationship between genotypes and clinical presentation of the patients was also investigated.

    2. Methods

    2.1. Bacterial strains and phenotypic tests

    A total of 197 nonduplicate isolates from a wide range of clinical samples from laboratories of the university teaching hospitals in Ahvaz and Tehran, Iran, were collected from November 2011 to January 2013. The preliminary proposal of the work was reviewed and approved by the Institutional Review and Ethics Board of the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. We also received necessary permission for sample collection and starting the work. The sources of clinical samples and patients’ medical histories are summarized in Table 1. The isolates were all those identified as Acinetobacter spp. based on the results of preliminary conventional phenotypic tests including growth on MacConkey agar, sugar fermentation, motility, catalase and oxidase tests, and other standard recommended tests.9,10 For definitive identification of these isolates to the species level, molecular methods were used in the next step.

    T1-7
    Table 1:
    Characteristics of 50 clinical isolates of Acinetobacter baumannii from Ahvaz and Tehran, Iran.

    2.2. Molecular methods

    The isolates were identified to the species level using species-specific rpoB gene-based PCR as previously described.11 In brief, a 350-bp fragment of the rpoB gene was amplified from each isolate using two primers of 696F (5′-TAY CGY AAA GAY TTG AAA GAA G-3′) and 1093R (5′-CMA CAC CYT TGT TMC CRT CA-3′). To investigate the heterogeneity of A. baumannii isolates, restriction fragment length polymorphism (RFLP) was performed as described by other investigators, using TagI and HaeIII restriction endonucleases.12

    2.3. Data analysis of rpoB gene sequences

    The obtained sequences of the rpoB region of each strain were aligned with the published rpoB region sequences of A. baumannii strains retrieved from GenBank database using the JPhydit software package according to the primary structure.13 Comparative analyses of the rpoB region were performed with the distance matrix, maximum parsimony, and maximum likelihood methods as implemented in the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 (MEGA4) program.14 Tree topologies were tested by bootstrap analysis on 1000 replicates.

    3. Results

    A total of 50 isolates (25.4%) were identified as A. baumannii using conventional phenotypic methods with subsequent confirmation by rpoB sequencing. These 50 isolates were recovered from 20 male adults (40%), 14 female adults (28%), and 16 infants (32%). RFLP analysis demonstrated five different restriction enzyme patterns, designated as A–E clusters (designated as “isolate sequence type or seqtype”). These clusters had 98.2–100% similarity with the A. baumannii type/reference strain in GenBank (CIP70.34). A majority of A. baumannii isolates were categorized as Cluster A (32%; Table 2). Twenty-eight isolates (56%) were recovered from patients with pulmonary disease (Table 1). About half of the isolates that originated from pulmonary diseases were classified under Clusters A and B (53.57%), and all the isolates from meningitis cases, though the number was low, were classified under Clusters C and D. However, significant association between A. baumannii seqtype and age and sex of patients, pulmonary disease, and other clinical presentations was not seen.

    T2-7
    Table 2:
    Restriction patterns and clustering of Iranian clinical isolates of Acinetobacter baumannii by rpoB RFLP and their similarity with type strain.

    A dendrogram based on maximum parsimony analysis reflecting the rpoB sequence-based clustering of all test strains of A. baumannii is shown in Fig. 1. Within the consensus tree, five clusters with distinct branches among the A. baumannii reference strains could be defined. The branches were supported with the highest bootstrap value (100%).

    F1-7
    Fig. 1:
    Distance matrix tree showing the divergence of rpoB sequences of the clinical isolates of Acinetobacter baumannii. All alignment positions that are occupied by residues were used for the calculation of binary distance values. The topology of the tree was evaluated and corrected according to the results of maximum parsimony and maximum-likelihood analyses. The bar represents 0.1 estimated sequence divergence.

    4. Discussion

    In recent times, A. baumannii has emerged as a main opportunistic pathogen, and there is a high incidence of morbidity and mortality related to A. baumannii infection among immunocompromised hospitalized patients.15 This organism is known for its involvement in hospital outbreaks and has sometimes caused interinstitutional spread.5 Several studies have demonstrated the usefulness of rpoB gene sequencing for the identification and taxonomic classification of various bacterial species.16,17 This study showed that the rpoB region has high discriminatory power to identify the clinical isolates of this pathogen to the species level and is of value for heterogeneity analysis. Our findings also suggest that a large variety of A. baumannii seqtypes may be responsible for causing diseases in humans, of which certain seqtypes seem to be more predominant. In our study, we identified and constructed phylogenetic trees incorporating 50 genospecies of A. baumannii based on the partial rpoB sequences following the RFLP analysis. RFLP demonstrated five different restriction enzyme patterns, designated as A–E clusters (seqtypes), and showed homogeneous grouping of A. baumannii.

    From the total tested isolates that were initially identified as Acinetobacter spp. by phenotypic tests, only a small number was actually confirmed to be A. baumannii by a subsequent molecular examination. Although, in general, phenotypic tests are not reliable enough for the identification of Acinetobacter spp. isolates to the species level,18 these tests exceptionally offer high sensitivity in identification of A. baumannii strains. This study showed that it is possible to achieve 100% confirmation of the isolates recovered (25.4%) as belonging to A. baumannii by application of confirmatory rpoB-based PCR followed by subsequent sequencing. The Acinetobacter spp. isolation rate in Iran varies from one setting to another. One significant report, conducted by Rahbar et al19 from the Reference Health Laboratory of Iran, demonstrated the isolation of 88 isolates of A. baumannii in a 1-year survey in a reference hospital in Tehran. In another similar study, Rahbar and Hajia20 reported the isolation rate of Acinetobacter spp. from nosocomial infections to be 3–6%. Moreover, in other similar studies, 78% and 8.8% of isolates recovered were, respectively, confirmed as A. baumannii by application of molecular techniques,21,22 but these rates were not similar to that of our study. The difference in the obtained rates among these studies may be explained by the type of clinical sample, the methods adopted for sample processing and DNA extraction, and other factors that affect the rate of positivity.

    In this work, the A. baumannii strains were categorized into five different clusters by application of the RFLP technique. Our study result was concordant with that of Karah et al,22 who also reported five clusters for the A. baumannii isolates recovered on the basis of rpoB PCR and RFLP analysis. However, in the study by Gundi et al,16 among the 32 rpoB seqtypes identified for the Acinetobacter spp., A. baumannii strains were grouped into 14 distinct clusters, which was more discriminative than our study or the clustering presented by Karah et al.22 By contrast, in the study by Turton et al,21 only three clusters were generated, showing less heterogeneity among their A. baumannii isolates. The confirmation of A. baumannii identification and clustering in our study and other mentioned works were achieved by comparing similarities of up to 97–100% with the A. baumannii reference strain. However, this similarity was reported to be wider, 77–100%, in the study by Turton et al.21

    The species variation among A. baumannii strains revealed by RFLP enabled us to investigate the correlation between different seqtypes with demographic information and clinical presentation of the patients. Although the majority of patients who entered the study had some pulmonary complications and, from ˜56% of these patients, strains belonging to A. baumannii seqtypes A and B were isolated, we could not find a significant correlation between the A. baumannii seqtype and clinical complication or age and sex of the patients.

    In conclusion, this study showed that the rpoB region possesses high discriminatory power to identify the isolates to the species level. This marker also showed high interspecies variability when combined with the RFLP technique, which might be useful for strain typing. The results also suggest the possibility of the existence of predominant clones of A. baumannii affecting patients in Iran. Although some results in this study did not show statistical significance, it still provides groundwork for future studies.

    Acknowledgments

    This work has been approved by the Infectious and Tropical Diseases Research Center and was financially supported by Research Affairs (Grant No. 91125), Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

    References

    1. Sieniawski K, Kaczka K, Rucińska M, Gagis L, Pomorski L. Acinetobacter baumannii nosocomial infections. Pol Przegl Chir. 2013;85:483-490.
    2. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 2008;21:538-582.
    3. Gaynes R, Edwards JR. Overview of nosocomial infections caused by gram-negative bacilli. Clin Infect Dis. 2005;41:848-854.
    4. Poirel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect. 2006;12:826-836.
    5. Griffith ME, Ceremuga JM, Ellis MW, Guymon CH, Hospenthal DR, Murray CK. Acinetobacter skin colonization of U.S. Army soldiers. Infect Control Hosp Epidemiol. 2006;27:659-661.
    6. Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodriguez-Valera F. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol. 2005;43:4382-4390.
    7. Ahmed SS, Alp E. Genotyping methods for monitoring the epidemic evolution of A. baumannii strains. J Infect Dev Ctries. 2015;9:347-354.
    8. Rafei R, Pailhoriès H, Hamze M, Eveillard M, Mallat H, Dabboussi F, et al. Molecular epidemiology of Acinetobacter baumannii in different hospitals in Tripoli, Lebanon using bla OXA-51-like sequence-based typing. BMC Microbiol. 2015;15:103.
    9. Forbes BA, Sahm DF, Weissfeld AS. 2007. Bailey & Scott's diagnostic microbiology, 12th ed. Mosby, St Louis, MO.
    10. Golanbar GD, Lam CK, Chu YM, Cueva C, Tan SW, Silva I, et al. Phenotypic and molecular characterization of Acinetobacter clinical isolates obtained from inmates of California correctional facilities. J Clin Microbiol. 2011;49:2121-2131.
    11. La Scola B, Gundi VA, Khamis A, Raoult D. Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species. J Clin Microbiol. 2006;44:827-832.
    12. Gomila M, Gasco J, Busquets A, Gil J, Bernabeu R, Buades JM, et al. Identification of culturable bacteria present in haemodialysis water and fluid. FEMS Microbiol Ecol. 2005;52:10-14.
    13. Zhang W, Sun Z. Random local neighbor joining: a new method for reconstructing phylogenetic trees. Mol Phylogenetics Evol. 2008;47:117-128.
    14. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596-1599.
    15. Howard A, O’Donoghue M, Feeney A, Sleator RD. Acinetobacter baumannii: an emerging opportunistic pathogen. Virulence. 2012;3:243-250.
    16. Gundi VA, Dijkshoorn L, Burignat S, Raoult D, La Scola B. Validation of partial rpoB gene sequence analysis for the identification of clinically important and emerging Acinetobacter species. Microbiology. 2009;155:2333-2341.
    17. Zhan Y, Yan Y, Zhang W, Chen M, Lu W, Ping S, et al. Comparative analysis of the complete genome of an Acinetobacter calcoaceticus strain adapted to a phenol-polluted environment. Res Microbiol. 2012;163:36-43.
    18. Wang J, Ruan Z, Feng Y, Fu Y, Jiang Y, Wang H, et al. Species distribution of clinical Acinetobacter isolates revealed by different identification techniques. PLoS One. 2014;9:e104882.
    19. Rahbar M, Mehrgan H, Haji-Aliakbari N. Prevalence of antibiotic-resistant A. baumannii in a 100-bed tertiary care hospital in Tehran, Iran. Indian J Pathol Microbiol. 2010;53:290-293.
    20. Rahbar M, Hajia M. Detection and quantitation of the etiologic agents of ventilator-associated pneumonia in endotracheal tube aspirates from patients in Iran. Infect Control Hosp Epidemiol. 2006;27:884-885.
    21. Turton JF, Shah J, Ozongwu C, Pike R. Incidence of Acinetobacter species other than A. baumannii among clinical isolates of Acinetobacter: evidence for emerging species. J Clin Microbiol. 2010;48:1445-1449.
    22. Karah N, Haldorsen B, Hegstad K, Simonsen GS, Sundsfjord A, Samuelsen O. Species identification and molecular characterization of Acinetobacter spp. blood culture isolates from Norway. J Antimicrob Chemother. 2011;66:738-744.
    Keywords:

    Acinetobacter baumannii; heterogeneity; nosocomial infections; rpoB

    © 2016 by Lippincott Williams & Wilkins, Inc.