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Characterisation and clonal dissemination of OXA-23-producing Acinetobacter baumannii in Tabriz, northwest Iran
International Journal of Antimicrobial Agents 39 (2012) 526–528
Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Characterisation and clonal dissemination of OXA-23-producing Acinetobacter baumannii in Tabriz, northwest Iran Amir Peymani a , Paul G. Higgins b,∗ , Mohammad-Reza Nahaei c , Safar Farajnia d , Harald Seifert b a
Department of Microbiology, Qazvin University of Medical Sciences, Qazvin, Iran Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany Department of Microbiology, Tabriz University of Medical Sciences, Tabriz, Iran d Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 21 October 2011 Accepted 22 February 2012 Keywords: DiversiLab International clones Carbapenem resistance ISAba1
a b s t r a c t The characteristics and molecular epidemiology of carbapenemase genes amongst 68 imipenem-resistant Acinetobacter baumannii isolated from Imam Reza Hospital (Tabriz, Iran) during a 17-month period were studied. All 68 isolates were typed using sequence group-based multiplex polymerase chain reaction (PCR) to compare the clonal relationship of isolates with known international clonal lineages. Repetitive sequence-based PCR was further performed with representative isolates of each clone. PCR and sequencing were performed to detect OXA-type carbapenemases and class 1, 2 and 3 integron genes as well as to confirm the presence of insertion sequence ISAba1 upstream of blaOXA-23 and blaOXA-51-like genes. Sixty-four isolates (94%) belonged to international clone (IC) II, two isolates (3%) belonged to IC I and two isolates (3%) did not belong to known international clones. All isolates carried blaOXA-51-like , blaOXA-23 and class 1 integron genes. No other acquired blaOXA genes or class 2 or 3 integron genes were detected. Sequence analysis confirmed the presence of blaOXA-23 as well as the blaOXA-51-like variants blaOXA-66 , blaOXA-69 and blaOXA-88 . ISAba1 was present upstream of the blaOXA-23 gene in all of the isolates. Clonal spread of OXA23-producing A. baumannii emphasises the need for appropriate infection control measures to prevent further spread of these multidrug-resistant organisms. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Acinetobacter baumannii has become a major nosocomial pathogen that causes severe infections, especially in patients admitted to Intensive Care Units (ICUs) [1]. In recent years, the emergence of multidrug-resistant A. baumannii complicates the therapy of A. baumannii infections and may lead to treatment failure [1,2]. Carbapenems are the drugs of choice for serious A. baumannii infections in many medical centres, however resistance to these antibiotics is increasing owing to the production of carbapenem-hydrolysing -lactamases [2]. Carbapenemases found in A. baumannii include metallo--lactamases such as IMP and VIM and the recently described NDM-1 and NDM-2; however, the predominant carbapenemases belong to the class D oxacillinases (OXA enzymes) [3]. In A. baumannii, the OXA-type carbapenemases are classified into at least five subgroups corresponding to the intrinsic OXA-51-like and the acquired OXA-23-like, OXA-40-like, OXA-58like and OXA-143 [4]. The insertion sequence ISAba1 has been found
upstream of blaOXA-23-like and blaOXA-51-like genes in A. baumannii and leads to overexpression of blaOXA genes [5]. Several studies have shown the clonal dissemination of carbapenem-resistant A. baumannii strains in different geographic regions, including Pakistan and military hospitals in Iraq and Kuwait in the Middle East [6,7]. Three international clones (ICs) of A. baumannii (ICs I, II and III, previously designated European clones I, II and III, respectively) associated with multidrug resistance have been identified [8] and there is evidence of five other worldwide carbapenem-resistant clonal lineages [9]. Few reports exist regarding the molecular epidemiology of carbapenem-resistant A. baumannii in Iran. The aims of this study were to identify and characterise the OXA-type carbapenemase genes, ISAba1 and integrons as well as the clonal relationship amongst the imipenem-resistant A. baumannii isolates. 2. Materials and methods 2.1. Bacterial isolates
∗ Corresponding author. Tel.: +49 221 4783 2011; fax: +49 221 4783 2002. E-mail address: [email protected] (P.G. Higgins).
Between May 2008 and September 2009, 68 non-repetitive imipenem-resistant isolates of A. baumannii were collected from Imam Reza Hospital (Tabriz, Iran). Bacterial isolates were recovered
0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2012.02.014
A. Peymani et al. / International Journal of Antimicrobial Agents 39 (2012) 526–528
527
Table 1 Molecular epidemiology and characterisation of 68 imipenem-resistant Acinetobacter baumannii. Epidemiological group
Collection date (No. of isolates)
Source (No. of isolates)
Ward (No. of isolates)
OXA-type carbapenemase
IC II
May–Aug. 2008 (9)
Tracheal aspirate (4), urine (2), bronchial washing (1), wound (1), ascites (1) Tracheal aspirate (7), urine (3), bronchial washing (2), pleural effusion (1) Tracheal aspirate (8), blood (1), urine (2), bronchial washing (2), sputum (2), wound (2) Tracheal aspirate (11), blood (2), urine (4), bronchial washing (3), sputum (1), CSF (1), ascites (1), wound (2) Urine (1), sputum (1)
ICU (5), Internal (1), Neurosurgery (1), Neurology (1), Infectious Diseases (1) ICU (7), Internal (3), Neurosurgery (2), Neurology (1)
OXA-66 and OXA-23
Sept.–Dec. 2008 (13)
Jan.–Apr. 2009 (17)
May–Sept. 2009 (25)
IC I
Feb. 2009, Mar. 2009 (2)
Clone C
June 2009, Aug. 2009 (2)
Urine (1), tracheal aspirate (1)
ICU (10), Internal (3), Neurosurgery (2), Neurology (1), Infectious Diseases (1) ICU (15), Internal (3), Neurosurgery (3), Neurology (2), Infectious Diseases (2) Internal (1), Infectious Diseases (1) Internal (1), Infectious Diseases (1)
OXA-69 and OXA-23 OXA-88 and OXA-23
IC, international clone; ICU, Intensive Care Unit; CSF, cerebrospinal fluid.
from different clinical specimens such as tracheal aspirate, urine, sputum, blood, bronchial washings, wound, cerebrospinal fluid, ascites and pleural effusion. Forty-six patients (68%) were male and 22 patients (32%) were female. The mean age of the patients was 49 ± 17.5 years (range 17–86 years). Acinetobacter baumannii is endemic in the hospital, causing 50–70 cases of A. baumannii colonisation or infection annually. However, precise epidemiological data are not available. Of note, there was no obvious outbreak situation at Imam Reza Hospital and the number of patients colonised or infected with A. baumannii per 1000 hospital admissions during the study period was not different from the previous 12-month period. Isolates were obtained from patients admitted to ICUs as well as internal, infectious diseases, neurosurgery and neurology wards (Table 1). Isolates were initially identified using standard laboratory methods. Species identification was confirmed by detection of blaOXA-51-like [5] and gyrB by multiplex polymerase chain reaction (PCR) [10]. 2.2. Antimicrobial susceptibility Antimicrobial susceptibility testing was performed by the standard agar disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [11]. The antimicrobial agents tested were amoxicillin/clavulanic acid, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/ clavulanic acid, cefpodoxime, ceftriaxone, ampicillin, cefepime, ceftazidime, cefotaxime, aztreonam, piperacillin, gentamicin, ciprofloxacin, levofloxacin, amikacin, imipenem and trimethoprim/ sulfamethoxazole (Mast Diagnostics Co., Bootle, UK). Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 reference strains were used as controls in the susceptibility testing. Resistance to imipenem was confirmed by Etest (AB BIODISK, Solna, Sweden). 2.3. Presence of carbapenem resistance genes and integrons A multiplex PCR assay was carried out for the detection of OXAtype carbapenemase genes [12]. The PCR products of representative isolates of each clone were purified using a QIAquick® PCR Purification Kit (QIAGEN, Hilden, Germany) followed by sequencing of both strands through direct sequencing at LGC Genomics GmbH (Berlin, Germany). PCR mapping experiments were performed using the
ISAba1 forward primer in combination with blaOXA-23-like and blaOXA-51-like reverse primers according to Turton et al. [5]. To determine whether the blaOXA-23-like gene was plasmid-borne, plasmid DNA was extracted from representative isolates using a QIAGEN Midi Kit and was transformed into carbapenem-susceptible A. baumannii ATCC 19606 by electroporation. Transformants were selected on Mueller–Hinton agar containing ticarcillin (100 g/mL) and the presence of the blaOXA-23 gene was confirmed by PCR. All isolates were tested for the presence of class 1, 2 and 3 integrons using specific primer pairs as described previously [13]. 2.4. Molecular typing Identification of PCR-based sequence groups was carried out using multiplex PCR assays to evaluate the possible epidemiological relationship of the isolates as previously described by Turton et al. [8]. Acinetobacter baumannii strains RUH 2036, RUH 3372 and LUH 5875 representing A. baumannii ICs I, II and III were used as controls. Based on the sequence types, 14 representative clinical isolates were selected for further epidemiological investigation using the DiversiLab® Microbial Typing System (bioMérieux, Nürtingen, Germany) [9]. 3. Results 3.1. International clonal lineages Multiplex PCR for identification of sequence groups revealed that 64 isolates (94%) belonged to IC II (previously European clone II), which were predominantly recovered from ICU patients (54%). These isolates were mostly obtained from tracheal aspirates (44%) and urine (16%). Two isolates (3%) belonged to the IC I (previously European clone I). Repetitive element sequence-based PCR (repPCR) confirmed clustering of isolates with the international clones. In addition, DiversiLab showed that the IC II isolates were highly related, clustering together with a similarity of ≥98.3%, and were regarded as evidence of clonal spread. Two isolates were identical (herein named clone C) and did not cluster with high similarity (≥95%) with any other isolate in our DiversiLab database, with the highest match to WW7 at 85% similarity, and are therefore regarded as unrelated. The two clone C isolates amplified only csuE and ompA from sequence group 2 by PCR.
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A. Peymani et al. / International Journal of Antimicrobial Agents 39 (2012) 526–528
3.2. Characterisation of carbapenem resistance genes and integrons The imipenem-resistant isolates were resistant or intermediate to all antimicrobials tested, and all were positive for blaOXA-51-like , blaOXA-23-like and class 1 integron genes. Isolates were negative for blaOXA-40-like , blaOXA-58-like , blaOXA-143 , and class 2 and 3 integron genes. ISAba1 was found upstream of the blaOXA-23-like gene in all of the isolates. No ISAba1 was detected upstream of the blaOXA-51-like gene. Sequencing of the entire blaOXA-51-like gene revealed blaOXA-66 in representative isolates belonging to IC II, blaOXA-69 in isolates belonging to IC I and blaOXA-88 in clone C. Presence of the blaOXA-23 gene was confirmed by sequencing of the representative isolates. Epidemiological characteristics of the 68 imipenem-resistant A. baumannii isolates are shown in Table 1. Transformation of plasmid preparations from the blaOXA-23 -positive isolates was successful only with the clone C isolates. Transfer of blaOXA-23 was confirmed by PCR. 4. Discussion In recent years, OXA carbapenemase-producing A. baumannii has increasingly been reported from Asian countries [6,7,14]. Previous studies have documented that clonal spread of carbapenem-resistant A. baumannii strains is often associated with the epidemic ICs I, II and III [9,15]. This is the first report indicating clonal dissemination of imipenem-resistant A. baumannii corresponding to known international clones in Iran. The results of this study revealed that 94% of imipenem-resistant A. baumannii isolates belonged to IC II, suggesting clonal spread and cross-transmission of this organism in different wards of Imam Reza Hospital, as well as presence of IC I isolates. The absence of appropriate infection control measures facilitated the spread of this organism in the different wards of the hospital. These findings are in agreement with other reports in which it was demonstrated that from a global perspective most carbapenem-resistant strains belong to IC II [9,15]. The results obtained in this study indicate that all isolates carried the blaOXA-23-like gene with an ISAba1 element upstream. These findings are in accordance with the results of other studies in which clonal outbreaks of OXA-23-producing A. baumannii strains are reported [2,6]. In a previous study from Tehran, OXA23 and OXA-40 were the most prevalent carbapenemases amongst imipenem-resistant isolates, however the epidemiological background of these isolates was not investigated [14]. Sequencing of the blaOXA-51-like gene revealed the presence of blaOXA-66 , blaOXA-69 and blaOXA-88 in the selected isolates belonging to ICs II and I and clone C, respectively. These findings are in good agreement with the results of Hamouda et al. who reported that OXA-66 and OXA-69 were the predominant members of the OXA51-like variants and were associated with particular clonal lineages [16]. In a study from Singapore, Koh et al. reported the presence of OXA-88 amongst OXA-23-producing A. baumannii isolates [17]. Given that blaOXA-51-like is a predominantly chromosomal gene, this highlights the international spread of this strain in this region. In two isolates belonging to clone C, the plasmids carrying the OXA-23 gene were successfully transferred by electroporation into A. baumannii ATCC 19606, making the infection control measures even more necessary because of the organism’s capability to transfer resistance to other strains. However, we did not succeed in transferring carbapenem resistance from blaOXA-23 -positive
isolates belonging to ICs I and II, indicating the probable chromosomal location of this gene. In conclusion, these results are indicative of the clonal spread of OXA-23-producing A. baumannii belonging to the international clones in northwest Iran. This reinforces the near ubiquity of these clonal lineages worldwide, particularly amongst carbapenemresistant isolates. It should be noted that carbapenem resistance is not only found in these international clones, but it is puzzling as to why the majority of carbapenem-resistant isolates belong to these lineages. Given that the natural reservoir is still unknown, as is the exact mechanism of dissemination, greater effort must be made to control this organism better within the hospital in an effort to contain its spread. Funding: PGH and HS were supported by a grant from Bundesministerium für Bildung und Forschung (BMBF), Germany, Klinische Forschergruppe Infektiologie (grant no. 01 KI 0771). Competing interests: None declared. Ethical approval: Not required. References [1] Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538–82. [2] Dalla-Costa LM, Coelho JM, Souza HA, Castro ME, Stier CJ, Bragagnolo KL, et al. Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. J Clin Microbiol 2003;41:3403–6. [3] Pfeifer Y, Wilharm G, Zander E, Wichelhaus TA, Göttig S, Hunfeld KP, et al. Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007. J Antimicrob Chemother 2011;66:1998–2001. [4] Higgins PG, Poirel L, Lehmann M, Nordmann P, Seifert H. OXA-143, a novel carbapenem-hydrolyzing class D -lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 2009;53:5035–8. [5] Turton JF, Ward ME, Woodford N, Kaufmann ME, Pike R, Livermore DM, et al. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett 2006;258:72–7. [6] Irfan S, Turton JF, Mehraj J, Siddiqui SZ, Haider S, Zafar A, et al. Molecular and epidemiological characterisation of clinical isolates of carbapenem-resistant Acinetobacter baumannii from public and private sector intensive care units in Karachi, Pakistan. J Hosp Infect 2011;78:143–8. [7] Scott P, Deye G, Srinivasan A, Murray C, Moran K, Hulten E, et al. Outbreak of multidrug-resistant Acinetobacter baumannii–calcoaceticus complex infection in the US military health care system associated with military operations in Iraq. Clin Infect Dis 2007;44:1577–84. [8] Turton JF, Gabriel SN, Valderrey C, Kaufmann ME, Pitt TL. Use of sequencebased typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii. Clin Microbiol Infect 2007;13:807–15. [9] Higgins PG, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenemresistant Acinetobacter baumannii. J Antimicrob Chemother 2010;65:233–8. [10] Higgins PG, Lehmann M, Wisplinghoff H, Seifert H. gyrB multiplex PCR to differentiate between Acinetobacter calcoaceticus and Acinetobacter genomic species 3. J Clin Microbiol 2010;48:4592–4. [11] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. Document M100-S18. Wayne, PA: CLSI; 2006. [12] Higgins PG, Lehmann M, Seifert H. Inclusion of OXA-143 primers in a multiplex polymerase chain reaction (PCR) for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 2010;35:305. [13] Shibata N, Doi Y, Yamane K, Yagi T, Kurokawa H, Shibayama K, et al. PCR typing of genetic determinants for metallo--lactamases and integrases carried by Gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol 2003;41:5407–13. [14] Feizabadi MM, Fathollahzadeh B, Taherikalani M, Rasoolinejad M, Sadeghifard N, Aligholi M, et al. Antimicrobial susceptibility patterns and distribution of blaOXA genes among Acinetobacter spp. isolated from patients at Tehran hospitals. Jpn J Infect Dis 2008;61:274–8. [15] Towner KJ, Levi K, Vlassiadi M; ARPAC Steering Group. Genetic diversity of carbapenem-resistant isolates of Acinetobacter baumannii in Europe. Clin Microbiol Infect 2008;14:161–7. [16] Hamouda A, Evans BA, Towner KJ, Amyes SG. Characterization of epidemiologically unrelated Acinetobacter baumannii isolates from four continents by use of multilocus sequence typing, pulsed-field gel electrophoresis, and sequencebased typing of blaOXA-51-like genes. J Clin Microbiol 2010;48:2476–83. [17] Koh TH, Sng LH, Wang GC, Hsu LY, Zhao Y. IMP-4 and OXA -lactamases in Acinetobacter baumannii from Singapore. J Antimicrob Chemother 2007;59:627–32.
Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Characterisation and clonal dissemination of OXA-23-producing Acinetobacter baumannii in Tabriz, northwest Iran Amir Peymani a , Paul G. Higgins b,∗ , Mohammad-Reza Nahaei c , Safar Farajnia d , Harald Seifert b a
Department of Microbiology, Qazvin University of Medical Sciences, Qazvin, Iran Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany Department of Microbiology, Tabriz University of Medical Sciences, Tabriz, Iran d Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 21 October 2011 Accepted 22 February 2012 Keywords: DiversiLab International clones Carbapenem resistance ISAba1
a b s t r a c t The characteristics and molecular epidemiology of carbapenemase genes amongst 68 imipenem-resistant Acinetobacter baumannii isolated from Imam Reza Hospital (Tabriz, Iran) during a 17-month period were studied. All 68 isolates were typed using sequence group-based multiplex polymerase chain reaction (PCR) to compare the clonal relationship of isolates with known international clonal lineages. Repetitive sequence-based PCR was further performed with representative isolates of each clone. PCR and sequencing were performed to detect OXA-type carbapenemases and class 1, 2 and 3 integron genes as well as to confirm the presence of insertion sequence ISAba1 upstream of blaOXA-23 and blaOXA-51-like genes. Sixty-four isolates (94%) belonged to international clone (IC) II, two isolates (3%) belonged to IC I and two isolates (3%) did not belong to known international clones. All isolates carried blaOXA-51-like , blaOXA-23 and class 1 integron genes. No other acquired blaOXA genes or class 2 or 3 integron genes were detected. Sequence analysis confirmed the presence of blaOXA-23 as well as the blaOXA-51-like variants blaOXA-66 , blaOXA-69 and blaOXA-88 . ISAba1 was present upstream of the blaOXA-23 gene in all of the isolates. Clonal spread of OXA23-producing A. baumannii emphasises the need for appropriate infection control measures to prevent further spread of these multidrug-resistant organisms. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Acinetobacter baumannii has become a major nosocomial pathogen that causes severe infections, especially in patients admitted to Intensive Care Units (ICUs) [1]. In recent years, the emergence of multidrug-resistant A. baumannii complicates the therapy of A. baumannii infections and may lead to treatment failure [1,2]. Carbapenems are the drugs of choice for serious A. baumannii infections in many medical centres, however resistance to these antibiotics is increasing owing to the production of carbapenem-hydrolysing -lactamases [2]. Carbapenemases found in A. baumannii include metallo--lactamases such as IMP and VIM and the recently described NDM-1 and NDM-2; however, the predominant carbapenemases belong to the class D oxacillinases (OXA enzymes) [3]. In A. baumannii, the OXA-type carbapenemases are classified into at least five subgroups corresponding to the intrinsic OXA-51-like and the acquired OXA-23-like, OXA-40-like, OXA-58like and OXA-143 [4]. The insertion sequence ISAba1 has been found
upstream of blaOXA-23-like and blaOXA-51-like genes in A. baumannii and leads to overexpression of blaOXA genes [5]. Several studies have shown the clonal dissemination of carbapenem-resistant A. baumannii strains in different geographic regions, including Pakistan and military hospitals in Iraq and Kuwait in the Middle East [6,7]. Three international clones (ICs) of A. baumannii (ICs I, II and III, previously designated European clones I, II and III, respectively) associated with multidrug resistance have been identified [8] and there is evidence of five other worldwide carbapenem-resistant clonal lineages [9]. Few reports exist regarding the molecular epidemiology of carbapenem-resistant A. baumannii in Iran. The aims of this study were to identify and characterise the OXA-type carbapenemase genes, ISAba1 and integrons as well as the clonal relationship amongst the imipenem-resistant A. baumannii isolates. 2. Materials and methods 2.1. Bacterial isolates
∗ Corresponding author. Tel.: +49 221 4783 2011; fax: +49 221 4783 2002. E-mail address: [email protected] (P.G. Higgins).
Between May 2008 and September 2009, 68 non-repetitive imipenem-resistant isolates of A. baumannii were collected from Imam Reza Hospital (Tabriz, Iran). Bacterial isolates were recovered
0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2012.02.014
A. Peymani et al. / International Journal of Antimicrobial Agents 39 (2012) 526–528
527
Table 1 Molecular epidemiology and characterisation of 68 imipenem-resistant Acinetobacter baumannii. Epidemiological group
Collection date (No. of isolates)
Source (No. of isolates)
Ward (No. of isolates)
OXA-type carbapenemase
IC II
May–Aug. 2008 (9)
Tracheal aspirate (4), urine (2), bronchial washing (1), wound (1), ascites (1) Tracheal aspirate (7), urine (3), bronchial washing (2), pleural effusion (1) Tracheal aspirate (8), blood (1), urine (2), bronchial washing (2), sputum (2), wound (2) Tracheal aspirate (11), blood (2), urine (4), bronchial washing (3), sputum (1), CSF (1), ascites (1), wound (2) Urine (1), sputum (1)
ICU (5), Internal (1), Neurosurgery (1), Neurology (1), Infectious Diseases (1) ICU (7), Internal (3), Neurosurgery (2), Neurology (1)
OXA-66 and OXA-23
Sept.–Dec. 2008 (13)
Jan.–Apr. 2009 (17)
May–Sept. 2009 (25)
IC I
Feb. 2009, Mar. 2009 (2)
Clone C
June 2009, Aug. 2009 (2)
Urine (1), tracheal aspirate (1)
ICU (10), Internal (3), Neurosurgery (2), Neurology (1), Infectious Diseases (1) ICU (15), Internal (3), Neurosurgery (3), Neurology (2), Infectious Diseases (2) Internal (1), Infectious Diseases (1) Internal (1), Infectious Diseases (1)
OXA-69 and OXA-23 OXA-88 and OXA-23
IC, international clone; ICU, Intensive Care Unit; CSF, cerebrospinal fluid.
from different clinical specimens such as tracheal aspirate, urine, sputum, blood, bronchial washings, wound, cerebrospinal fluid, ascites and pleural effusion. Forty-six patients (68%) were male and 22 patients (32%) were female. The mean age of the patients was 49 ± 17.5 years (range 17–86 years). Acinetobacter baumannii is endemic in the hospital, causing 50–70 cases of A. baumannii colonisation or infection annually. However, precise epidemiological data are not available. Of note, there was no obvious outbreak situation at Imam Reza Hospital and the number of patients colonised or infected with A. baumannii per 1000 hospital admissions during the study period was not different from the previous 12-month period. Isolates were obtained from patients admitted to ICUs as well as internal, infectious diseases, neurosurgery and neurology wards (Table 1). Isolates were initially identified using standard laboratory methods. Species identification was confirmed by detection of blaOXA-51-like [5] and gyrB by multiplex polymerase chain reaction (PCR) [10]. 2.2. Antimicrobial susceptibility Antimicrobial susceptibility testing was performed by the standard agar disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [11]. The antimicrobial agents tested were amoxicillin/clavulanic acid, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/ clavulanic acid, cefpodoxime, ceftriaxone, ampicillin, cefepime, ceftazidime, cefotaxime, aztreonam, piperacillin, gentamicin, ciprofloxacin, levofloxacin, amikacin, imipenem and trimethoprim/ sulfamethoxazole (Mast Diagnostics Co., Bootle, UK). Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 reference strains were used as controls in the susceptibility testing. Resistance to imipenem was confirmed by Etest (AB BIODISK, Solna, Sweden). 2.3. Presence of carbapenem resistance genes and integrons A multiplex PCR assay was carried out for the detection of OXAtype carbapenemase genes [12]. The PCR products of representative isolates of each clone were purified using a QIAquick® PCR Purification Kit (QIAGEN, Hilden, Germany) followed by sequencing of both strands through direct sequencing at LGC Genomics GmbH (Berlin, Germany). PCR mapping experiments were performed using the
ISAba1 forward primer in combination with blaOXA-23-like and blaOXA-51-like reverse primers according to Turton et al. [5]. To determine whether the blaOXA-23-like gene was plasmid-borne, plasmid DNA was extracted from representative isolates using a QIAGEN Midi Kit and was transformed into carbapenem-susceptible A. baumannii ATCC 19606 by electroporation. Transformants were selected on Mueller–Hinton agar containing ticarcillin (100 g/mL) and the presence of the blaOXA-23 gene was confirmed by PCR. All isolates were tested for the presence of class 1, 2 and 3 integrons using specific primer pairs as described previously [13]. 2.4. Molecular typing Identification of PCR-based sequence groups was carried out using multiplex PCR assays to evaluate the possible epidemiological relationship of the isolates as previously described by Turton et al. [8]. Acinetobacter baumannii strains RUH 2036, RUH 3372 and LUH 5875 representing A. baumannii ICs I, II and III were used as controls. Based on the sequence types, 14 representative clinical isolates were selected for further epidemiological investigation using the DiversiLab® Microbial Typing System (bioMérieux, Nürtingen, Germany) [9]. 3. Results 3.1. International clonal lineages Multiplex PCR for identification of sequence groups revealed that 64 isolates (94%) belonged to IC II (previously European clone II), which were predominantly recovered from ICU patients (54%). These isolates were mostly obtained from tracheal aspirates (44%) and urine (16%). Two isolates (3%) belonged to the IC I (previously European clone I). Repetitive element sequence-based PCR (repPCR) confirmed clustering of isolates with the international clones. In addition, DiversiLab showed that the IC II isolates were highly related, clustering together with a similarity of ≥98.3%, and were regarded as evidence of clonal spread. Two isolates were identical (herein named clone C) and did not cluster with high similarity (≥95%) with any other isolate in our DiversiLab database, with the highest match to WW7 at 85% similarity, and are therefore regarded as unrelated. The two clone C isolates amplified only csuE and ompA from sequence group 2 by PCR.
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3.2. Characterisation of carbapenem resistance genes and integrons The imipenem-resistant isolates were resistant or intermediate to all antimicrobials tested, and all were positive for blaOXA-51-like , blaOXA-23-like and class 1 integron genes. Isolates were negative for blaOXA-40-like , blaOXA-58-like , blaOXA-143 , and class 2 and 3 integron genes. ISAba1 was found upstream of the blaOXA-23-like gene in all of the isolates. No ISAba1 was detected upstream of the blaOXA-51-like gene. Sequencing of the entire blaOXA-51-like gene revealed blaOXA-66 in representative isolates belonging to IC II, blaOXA-69 in isolates belonging to IC I and blaOXA-88 in clone C. Presence of the blaOXA-23 gene was confirmed by sequencing of the representative isolates. Epidemiological characteristics of the 68 imipenem-resistant A. baumannii isolates are shown in Table 1. Transformation of plasmid preparations from the blaOXA-23 -positive isolates was successful only with the clone C isolates. Transfer of blaOXA-23 was confirmed by PCR. 4. Discussion In recent years, OXA carbapenemase-producing A. baumannii has increasingly been reported from Asian countries [6,7,14]. Previous studies have documented that clonal spread of carbapenem-resistant A. baumannii strains is often associated with the epidemic ICs I, II and III [9,15]. This is the first report indicating clonal dissemination of imipenem-resistant A. baumannii corresponding to known international clones in Iran. The results of this study revealed that 94% of imipenem-resistant A. baumannii isolates belonged to IC II, suggesting clonal spread and cross-transmission of this organism in different wards of Imam Reza Hospital, as well as presence of IC I isolates. The absence of appropriate infection control measures facilitated the spread of this organism in the different wards of the hospital. These findings are in agreement with other reports in which it was demonstrated that from a global perspective most carbapenem-resistant strains belong to IC II [9,15]. The results obtained in this study indicate that all isolates carried the blaOXA-23-like gene with an ISAba1 element upstream. These findings are in accordance with the results of other studies in which clonal outbreaks of OXA-23-producing A. baumannii strains are reported [2,6]. In a previous study from Tehran, OXA23 and OXA-40 were the most prevalent carbapenemases amongst imipenem-resistant isolates, however the epidemiological background of these isolates was not investigated [14]. Sequencing of the blaOXA-51-like gene revealed the presence of blaOXA-66 , blaOXA-69 and blaOXA-88 in the selected isolates belonging to ICs II and I and clone C, respectively. These findings are in good agreement with the results of Hamouda et al. who reported that OXA-66 and OXA-69 were the predominant members of the OXA51-like variants and were associated with particular clonal lineages [16]. In a study from Singapore, Koh et al. reported the presence of OXA-88 amongst OXA-23-producing A. baumannii isolates [17]. Given that blaOXA-51-like is a predominantly chromosomal gene, this highlights the international spread of this strain in this region. In two isolates belonging to clone C, the plasmids carrying the OXA-23 gene were successfully transferred by electroporation into A. baumannii ATCC 19606, making the infection control measures even more necessary because of the organism’s capability to transfer resistance to other strains. However, we did not succeed in transferring carbapenem resistance from blaOXA-23 -positive
isolates belonging to ICs I and II, indicating the probable chromosomal location of this gene. In conclusion, these results are indicative of the clonal spread of OXA-23-producing A. baumannii belonging to the international clones in northwest Iran. This reinforces the near ubiquity of these clonal lineages worldwide, particularly amongst carbapenemresistant isolates. It should be noted that carbapenem resistance is not only found in these international clones, but it is puzzling as to why the majority of carbapenem-resistant isolates belong to these lineages. Given that the natural reservoir is still unknown, as is the exact mechanism of dissemination, greater effort must be made to control this organism better within the hospital in an effort to contain its spread. Funding: PGH and HS were supported by a grant from Bundesministerium für Bildung und Forschung (BMBF), Germany, Klinische Forschergruppe Infektiologie (grant no. 01 KI 0771). Competing interests: None declared. Ethical approval: Not required. References [1] Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538–82. [2] Dalla-Costa LM, Coelho JM, Souza HA, Castro ME, Stier CJ, Bragagnolo KL, et al. Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. J Clin Microbiol 2003;41:3403–6. [3] Pfeifer Y, Wilharm G, Zander E, Wichelhaus TA, Göttig S, Hunfeld KP, et al. Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007. J Antimicrob Chemother 2011;66:1998–2001. [4] Higgins PG, Poirel L, Lehmann M, Nordmann P, Seifert H. OXA-143, a novel carbapenem-hydrolyzing class D -lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 2009;53:5035–8. [5] Turton JF, Ward ME, Woodford N, Kaufmann ME, Pike R, Livermore DM, et al. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett 2006;258:72–7. [6] Irfan S, Turton JF, Mehraj J, Siddiqui SZ, Haider S, Zafar A, et al. Molecular and epidemiological characterisation of clinical isolates of carbapenem-resistant Acinetobacter baumannii from public and private sector intensive care units in Karachi, Pakistan. J Hosp Infect 2011;78:143–8. [7] Scott P, Deye G, Srinivasan A, Murray C, Moran K, Hulten E, et al. Outbreak of multidrug-resistant Acinetobacter baumannii–calcoaceticus complex infection in the US military health care system associated with military operations in Iraq. Clin Infect Dis 2007;44:1577–84. [8] Turton JF, Gabriel SN, Valderrey C, Kaufmann ME, Pitt TL. Use of sequencebased typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii. Clin Microbiol Infect 2007;13:807–15. [9] Higgins PG, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenemresistant Acinetobacter baumannii. J Antimicrob Chemother 2010;65:233–8. [10] Higgins PG, Lehmann M, Wisplinghoff H, Seifert H. gyrB multiplex PCR to differentiate between Acinetobacter calcoaceticus and Acinetobacter genomic species 3. J Clin Microbiol 2010;48:4592–4. [11] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. Document M100-S18. Wayne, PA: CLSI; 2006. [12] Higgins PG, Lehmann M, Seifert H. Inclusion of OXA-143 primers in a multiplex polymerase chain reaction (PCR) for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 2010;35:305. [13] Shibata N, Doi Y, Yamane K, Yagi T, Kurokawa H, Shibayama K, et al. PCR typing of genetic determinants for metallo--lactamases and integrases carried by Gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol 2003;41:5407–13. [14] Feizabadi MM, Fathollahzadeh B, Taherikalani M, Rasoolinejad M, Sadeghifard N, Aligholi M, et al. Antimicrobial susceptibility patterns and distribution of blaOXA genes among Acinetobacter spp. isolated from patients at Tehran hospitals. Jpn J Infect Dis 2008;61:274–8. [15] Towner KJ, Levi K, Vlassiadi M; ARPAC Steering Group. Genetic diversity of carbapenem-resistant isolates of Acinetobacter baumannii in Europe. Clin Microbiol Infect 2008;14:161–7. [16] Hamouda A, Evans BA, Towner KJ, Amyes SG. Characterization of epidemiologically unrelated Acinetobacter baumannii isolates from four continents by use of multilocus sequence typing, pulsed-field gel electrophoresis, and sequencebased typing of blaOXA-51-like genes. J Clin Microbiol 2010;48:2476–83. [17] Koh TH, Sng LH, Wang GC, Hsu LY, Zhao Y. IMP-4 and OXA -lactamases in Acinetobacter baumannii from Singapore. J Antimicrob Chemother 2007;59:627–32.