Dissemination of multidrug resistant Acinetobacter baumannii in various hospitals of Antananarivo Madagascar
Annals of Clinical Microbiology and Antimicrobials volume 9, Article number: 17 (2010)
This study reports the dissemination of multidrug-resistant (MDR) OXA-23-producing Acinetobacter baumannii clones in hospitals in Antananarivo, Madagascar. A total of 53 carbapenem-resistant A. baumannii isolates were obtained from September 2006 to March 2009 in five hospitals. These resistant strains represent 44% of all A. baumannii isolates. The double disk synergy test was performed to screen for production of metallo-beta-lactamases. Polymerase chain reaction (PCR) and DNA sequencing were performed for the detection of bla(AmpC), bla(OXA-51),bla(OXA-23), bla(OXA-24), bla(IMP), bla(VIM). The presence of the insertion sequence ISAba1 relative to bla OXA-23 and bla OXA-51 was assessed by PCR. Isolates were typed by Rep-PCR. All the isolates were MDR and produced the OXA-23 carbapenemase, which was confirmed by sequencing. PCR analysis for AmpC and OXA-51 gave positive results for all strains studied. No isolates produced metallo-beta-lactamases. In all isolates ISAba1 laid upstream of bla OXA-23. The A. baumannii isolates were separated into two genotypes; genotype A had a higher prevalence (41 strains) than genotype B (12 strains). Genotype A was present in four hospitals, whilst genotype B had spread in two hospitals. The high frequency of MDR OXA-23-producing A. baumannii in various hospitals in Antananarivo is curious since carbapenems are not available in Madagascar, but it emphasises the need for infection control procedures and strict adherence to them to prevent the spread of these resistant organisms in Antananarivo and also the need to control the use of carbapenems in the future.
Acinetobacter baumannii is an agent of nosocomial infections, especially pneumonia. It is frequently associated with nosocomial outbreaks worldwide [1, 2]. Immunocompromised patients have a high risk of being infected with this organism. A. baumannii has become particularly problematic worldwide because of its resistance patterns. Hospital strains of A. baumannii are usually multidrug resistant. The problem is aggravated by increasing resistance to broad-spectrum antibiotics including carbapenems, the drugs of choice for hospital-acquired A. baumannii infections [3–5].
Indeed, carbapenems are generally the last resort in the treatment of lifethreatening infections caused by Acinetobacter spp. because they are not affected by most β-lactamases, including extended-spectrum β-lactamases. However their efficacy is increasingly compromised by the emergence of carbapenem-hydrolysing β -lactamase enzymes of Ambler molecular class B (metallo- β-lactamases) and D (oxacillinases) [3, 6]. Metalloenzymes are prevalent in East Asia but the OXA-type carbapenemases have emerged as the main mechanism responsible for this resistance worldwide. Recently, the number of OXA-type carbapenemases has increased and they have been divided into eight subgroups of enzymes; four of them have been identified in A. baumannii: OXA-23-like (OXA-23, OXA-27 and OXA-49); OXA-24-like (OXA-24, OXA-25, OXA-26, OXA-40 and OXA-72); OXA-58; and OXA-51-like. The last group constitutes a family of chromosomal enzymes typically present in A. baumannii.
Outbreaks of OXA-23-producing Acinetobacter have been reported from various regions of the world [8–13]. During a study on hospital-acquired infections conducted in various surgical units located in two hospitals of Antananarivo, the capital city of Madagascar, A. baumannii represented 9.4% of all pathogens isolated. Among them, 44% were resistant to imipenem although carbapenems are not available in Madagascar . We determined the resistance mechanisms of A. baumannii to imipenem in Antananarivo and attempted to explain the dissemination of these strains.
2. Material and methods
2.1. Bacterial isolates
From September 2006 to March 2009, 53 non repetitive clinical isolates of imipenem non-susceptible A. baumannii, based on the results of disk diffusion tests, were collected from 53 patients from four hospitals (three public and one private) in Antananarivo, Madagascar. Bacterial isolates were obtained from skin wounds (54.6%), urine (18.9%), respiratory tract secretions (15.1%), blood (5.7%) and other sites (5.7%). Most of the isolates were obtained from patients admitted to the Soavinandriana (HOMI) (45.3%) or Ravoangy Andrianavaolona (HJRA) (39.6%) hospitals. Five control strains obtained at the same time from other patients at two of the hospitals studied and susceptible to carbapenems, with minimum inhibitory concentrations (MICs) of 0.5-2 μg/ml for imipenem were used.
Isolates were identified by conventional techniques. They were kept frozen at -20°C in skimmed milk until further testing.
2.2. Antimicrobial susceptibility testing
The disk diffusion method was used to assess susceptibility to the following antimicrobial agents: amikacin; tobramicin; gentamycin; ticarcillin/clavulanic acid; piperacillin/tazobactam; ceftazidime; ampicillin/sulbactam; ciprofloxacin; sulfamethoxazole/trimethoprim, imipenem and colistin. MIC values of imipenem were determined using agar plate dilution. Two-fold serial dilutions of imipenem were added to molten Mueller-Hinton agar base (Oxoid) at a temperature of 45°C. The resulting plates were seeded with 104 cfu/spot of bacteria by means of a multipoint inoculator and incubated at 37°C for 24 h. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as controls and included in each run. The imipenem concentration range was from 0.5 to 512 μg/ml. MIC values of meropenem were determined by Etest (BioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. The meropenem concentration range from 0.02 to 32 μg/ml. Antimicrobial susceptibility was scored using breakpoint criteria shown in table 1 as defined by the Antibiogram Committee of the French Microbiology Society (CASFM) , except for ampicilin/sulbactam for which we used the criteria of the Clinical and Laboratory Standards Institute .
Carbapenem-resistant A. baumannii (CRAB) isolates were screened by the modified Hodge test, to evaluate the inactivation of imipenem by carbapenemases, .
All strains were tested for the presence of metallo-enzymes using the imipenem-EDTA double-disk synergy test .
2.3. PCR amplification and sequencing
DNA was extracted from the isolates by boiling five colonies in 250 μl of sterile ultrapure water for 10 minutes, followed by cooling in ice for 10 minutes and centrifugation for 1 min at 14,000 rpm. Supernatants were conserved at -20°C until amplification. Genes coding for class B and D carbapenemases were sought by PCR using Taq polymerase with specific primers. Attempts were made to detect and map the presence of ISAba1 relative to bla OXA-23 and bla OXA-51 using the primer pairs ISAba1 F/OXA-23-seqR (expected size 1456 bp) and ISAba1 F/OXA-51-R (expected size 1223 bp), respectively. All primers used are listed in table 2.
The amplified products were observed after electrophoresis on a 1% agarose gel with ethidium bromide staining; purified, amplified products were then sequenced with the dye termination cycle sequencing technique (Genome Express Company, Meylan, France). Searches and alignments for the nucleotide sequences were performed with the BLAST program http://www.ncbi.nlm.nih.gov/BLAST.
REP-PCR, which uses consensus primers for the REP sequences found in many bacterial chromosomes, was used in the genotyping of A. baumannii clones [23, 24]. This highly conserved REP sequence is approximately 35 nucleotides long, includes an inverted repeat, and can occur in the genome singly or as multiple adjacent copies. The primer pair REP 1 (5'-IIIGCGCCGICATCAGGC-3') and REP 2 (5'-ACGTCTTATCAGGCCTAC-3') was used to amplify putative REP-like elements in the bacterial DNA. Amplification PCRs were performed as described previously [23, 24]. A negative control to detect reagent contamination was included in each PCR, containing all components except the DNA extract, which was replaced by 5 μl of sterile distilled H2O. Aliquots (12 μl) of each sample were subjected to electrophoresis in a 1.5% agarose gel. Amplified products were detected by staining with ethidium bromide and photographed with Polaroid type 665 film.
To group isolates for photographic documentation, the REP-PCR fingerprints of strains were exposed to UV light, photographed, and compared by visual inspection. The molecular sizes of fragments generated by electrophoresis were estimated from comparisons with relative molecular mass standards run concurrently. Fingerprints were considered to be highly similar when all visible bands from the two isolates had the same apparent migration distance. Variations in the intensity or shape of bands were not taken into account. The absence of up to two bands from a fingerprint was allowed, when all other visible bands in the fingerprints matched, before isolates were considered to be different by visual inspection [23, 24]. Each isolate was run in duplicate. Fingerprint profiles were interpreted with no knowledge of the clinical data.
According to the results of the antimicrobial susceptibility testing by disk diffusion for 53 imipenem non-susceptible A. baumannii isolates, all isolates were resistant to most of the antimicrobials tested. Apart from colistin to which all isolates were susceptible, the highest rates of susceptibility were observed with ampicillin/sulbactam (79.2%; n = 42), tobramycin (67.9%; n = 36) and amikacin (47,2%; n = 25) (Table 1). According to the results of MICs, one isolate was intermediate and all isolates were resistant to imipenem and to meropenem. The MIC of imipenem varied slightly; for 13 isolates it was 16 μg/ml, for 39 isolates it was 32 μg/ml whereas for one strain it was 64 μg/ml. All strains had MICs ≥ 32 μg/ml to meropenem. No synergy was observed using the imipenem-EDTA double-disk synergy test. Using the modified Hodge test, the presence of carbapenemases was detected in 46 (86,8%) of the CRAB consistent with PCR results. Indeed, using PCR and DNA sequencing of the PCR product, all 53 CRAB isolates showed the presence of bla OXA-23 and bla OXA-51 but none had bla OXA-24-like, bla OXA-58, bla IMP or bla VIM. In susceptible isolates, only bla OXA-51 was detected.
ISAba1 was detected only upstream of bla OXA-23 in all isolates.
Isolates were analysed by REP-PCR to determine the genomic diversity of CRAB. Two genotypes were observed. The five carbapenem-susceptible control isolates belonged to different genotypes from the CRAB (figure 1). Genotype A (77.4% of isolates; n = 41) was the most prevalent and was found in four hospitals, whilst genotype B (22.6% of isolates; n = 12) had spread in two hospitals.
Our study revealed that OXA-23 carbapenemase was present in A. baumannii isolates in Antananarivo. OXA-23-type carbapenemase-producing A. baumannii are becoming increasingly widespread, with reports from Europe [25–27], South America [9, 13, 28], or Asia [29–31]. In 2002, 49 strains of imipenem resistant A. baumannii producing the carbapenenase OXA-23 were isolated in South Africa . Many strains of OXA-23 producing A. baumannii from the same clone were responsible for an epidemic of nocomial infection from 2005 to 2007 in Tunisia .
As in the present study, such isolates usually exhibit resistance to many antimicrobials, creating a serious problem for choice of therapy. In our study, patients with skin infections caused by carbapenem-resistant A. baumannii received local treatments and most survived without sequelae. On the contrary, among patients with urinary, pulmonary or blood stream infections who received only antibiotics ineffective on the A. baumannii strains isolated (third generation cephalosporins, ciprofloxacin or amoxicillin-clavulanic acid), the rate of mortality was high. Indeed 2 of 3 patients with bloodstream infection, 4 of 8 patients with pulmonary infection and 2 of 10 patients with urinary infection died.
In this study, the OXA-23-producers originated from two clones. According to Rep-PCR patterns, it appears that OXA-23-producing CRAB belonging to two predominant genotypes spread between public and private hospitals in Antananarivo. Different studies have already shown that clones of CRAB may spread in a town  or even a country . The occurrence of common Rep-PCR types in OXA-23-producing A. baumannii from various hospitals in Antananarivo suggests that dissemination of isolates contributes to the increase in prevalence of CRAB. Dissemination of CRAB in the community could be favoured by poor health facilities and the transfer of infected or colonised patients from a hospital to another. However, this is rarely the case in Antananarivo. Sharing of common healthcare staff is more likely. Indeed staff from public hospitals also works together in the various clinics of Madagascar. This hypothesis has already been evoked to explain the spread of clonal strains between different hospitals [13, 35]. If poor sanitary conditions probably explain how these CRAB clones can spread, it does not explain how these clones appeared in Antananarivo. One can suppose that resistant clones were introduced by patients treated in a developed country (hospitalization in Reunion Island is rather frequent for Malagasy patients). However, since carbapenem are not available in Madagascar, resistance to these drugs should not confer any advantage to these clones. Therefore the selection of these clones is probably due to their resistance to most drugs used in Madagascar than to resistance to imipenem.
Whatever the reason for this spread, the high level of CRAB in the hospitals of Antananarivo is a cause for concern as carbapenems are not available in Madagascar. Thus, the presence of MDR OXA-23 producing A. baumannii genotypes emphasises the need for infection control procedures and strict adherence to them to prevent the spread of these resistant organisms in Antananarivo and also the need to control the use of carbapenems in the future.
Bergogne-Bérézin E, Towner KJ: Acinetobacter spp . as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 1996, 9: 148-165.
Villegas MV, Hartstein AI: Acinetobacter outbreaks, 1977-2000. Infect Control Hosp Epidemiol 2003, 24: 284-295. 10.1086/502205
Livermore DM: The impact of carbapenemases on antimicrobial development and therapy. Curr Opin Investig Drugs 2002, 3: 218-224.
Gaynes R, Edwards JR: National Nosocomial Infections Surveillance System. Overview of nosocomial infections caused by gram-negative bacilli. Clin Infect Dis 2005, 41: 848-854. 10.1086/432803
Abbo A, Navon-Venezia S, Hammer-Muntz O, Krishali T, Siegman-Igra Y, Carmeli Y: Multidrug - resistant Acinetobacter baumannii . Emerg Infect Dis 2005, 11: 22-29.
Poirel L, Nordmann P: Acquired carbapenem-hydrolyzing beta-lactamases and their genetic support. Curr Pharm Biotechnol 2002, 3: 117-127. 10.2174/1389201023378427
Walter-Rasmussen Hoiby N: OXA-type carbapenemases. J Antimicrob Chemother 2006, 57: 373-383. 10.1093/jac/dki482
Naas T, Levy M, Hirschauer C, Marchandin H, Nordmann P: Outbreak of carbapenem-resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in a tertiary care hospital of Papeete, French Polynesia. J Clin Microbiol 2005, 43: 4826-4829. 10.1128/JCM.43.9.4826-4829.2005
Dalla-Costa LM, Coelho JM, Souza HA, Castro ME, Stier CJ, Bragagnolo KL, Rea-Neto A, Penteado-Filho SR, Livermore DM, Woodford N: Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. J Clin Microbiol 2003, 41: 3403-3406. 10.1128/JCM.41.7.3403-3406.2003
Jeong SH, Bae IK, Park KO, An YJ, Sohn SG, Jang SJ, Sung KH, Yang KS, Lee K, Young D, Lee SH: Outbreaks of imipenem-resistant Acinetobacter baumannii producing carbapenemases in Korea. J Microbiol 2006, 44: 423-431.
Koh TH, Sng LH, Wang GC, Hsu LY, Zhao Y: IMP-4 and OXA β-lactamases in Acinetobacter baumannii from Singapore. J Antimicrob Chemother 2007, 59: 627-632. 10.1093/jac/dkl544
Villegas MV, Kattan JN, Correa A, Lolans K, Guzman AM, Woodford N, Livermore D, Quinn JP: Dissemination of Acinetobacter baumannii clones with OXA-23 carbapenemase in Colombian hospitals. Antimicrob Agents Chemother 2007, 51: 2001-2004. 10.1128/AAC.00226-07
Carvalho KR, Carvalho-Assef AP, Peirano G, Santos LC, Pereira MJ, Asensi MD: Dissemination of multidrug-resistant Acinetobacter baumannii genotypes carrying bla(OXA-23) collected from hospitals in Rio de Janeiro, Brazil. Int J Antimicrob Agents 2009, 34: 25-28. 10.1016/j.ijantimicag.2008.12.009
Randrianirina F, Vaillant L, Ramarokoto CE, Rakotoarijaona A, Andriamanarivo ML, Razafimahandry HC, Randrianomenjanahary J, Raveloson JR, Ratsima Hariniaina E, Carod JF, Talarmin A, Richard V: Antimicrobial resistance in pathogens causing nosocomial infections in two hospitals in Antananarivo, Madagascar. J Infect Dev Ctries 2010, 4: 74-82.
Comité de l'Antibiogramme de la Société Française de Microbiologie: Communiqué 2009. Paris, France, SFM; 2009.
National Committee for Clinical Laboratory Standards: Performance standards for antimicrobial susceptibility testing: 13thinformational supplement. Document M100-S13. National Committee for Clinical Laboratory Standards, Wayne, Pa; 2003.
Lee K, Lim YS, Yong D, Yum JH, Chong Y: Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo- β-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2003, 41: 4623-4629. 10.1128/JCM.41.10.4623-4629.2003
Corvec S, Caroff N, Espaze E, Giraudeau C, Drugeon H, Reynaud A: AmpC cephalosporinase hyperproduction in Acinetobacter baumannii clinical strains. J Antimicrob Chemother 2003, 52: 629-635. 10.1093/jac/dkg407
Vahaboglu H, Budak F, Kasap M, Gacar G, Torol S, Karadenizli A, Kolayli F, Eroglu C: High prevalence of OXA-51-type class D beta-lactamases among ceftazidime-resistant clinical isolates of Acinetobacter spp.: co-existence with OXA-58 in multiple centres. J Antimicrob Chemother 2006, 58: 537-542. 10.1093/jac/dkl273
Poirel L, Figueiredo S, Cattoir V, Carattoli A, Nordmann P: Acinetobacter radioresistens as a silent source of carbapenem resistance for Acinetobacter spp. Antimicrob Agents Chemother 2008, 52: 1252-1256. 10.1128/AAC.01304-07
Yang HY, Lee HJ, Suh JT, Lee KM: Outbreaks of imipenem resistant Acinetobacter baumannii producing OXA-23 beta-lactamase in a tertiary care hospital in Korea. Yonsei Med J 2009, 50: 764-770. 10.3349/ymj.2009.50.6.764
Chu YW, Cheung TK, Chu MY, Lo JY, Dijkshoorn L: OXA-23-type imipenem resistance in Acinetobacter baumannii in Hong Kong. Int J Antimicrob Agents 2009, 34: 285-286. 10.1016/j.ijantimicag.2009.03.010
Martín-Lozano D, Cisneros JM, Becerril B, Cuberos L, Prados T, Ortíz-Leyba C, Cañas E, Pachón J: Comparison of a Repetitive Extragenic Palindromic sequence-based PCR method and Clinical and Microbiological methods for determining strain sources in cases of nosocomial Acinetobacter baumannii bacteremia. J Clin Microbiol 2002, 40: 4571-4575. 10.1128/JCM.40.12.4571-4575.2002
Snelling AM, Gerner-Smidt P, Hawkey PM, Heritage J, Parnell P, Porter C, Bodenham AR, Inglis T: Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak. J Clin Microbiol 1996, 34: 1193-1202.
Bogaerts P, Cuzon G, Naas T, Bauraing C, Deplano A, Lissoir B, Nordmann P, Glupczynski Y: Carbapenem-resistant Acinetobacter baumannii isolates expressing the blaOXA-23 gene associated with ISAba4 in Belgium. Antimicrob Agents Chemother 2008, 52: 4205-4206. 10.1128/AAC.01121-08
Gur D, Korten V, Unal S, Deshpande LM, Castanheira M: Increasing carbapenem resistance due to the clonal dissemination of oxacillinase (OXA-23 and OXA-58)-producing Acinetobacter baumannii : report from the Turkish SENTRY Program sites. J Med Microbiol 2008, 57: 1529-1532. 10.1099/jmm.0.2008/002469-0
Stoeva T, Higgins PG, Bojkova K, Seifert H: Clonal spread of carbapenem-resistant OXA-23-positive Acinetobacter baumannii in a Bulgarian university hospital. Clin Microbiol Infect 2008, 14: 723-727. 10.1111/j.1469-0691.2008.02018.x
Merkier AK, Catalano M, Ramírez MS, Quiroga C, Orman B, Ratier L, Famiglietti A, Vay C, Di Martino A, Kaufman S, Centrón D: Polyclonal spread of bla (OXA-23) and bla (OXA-58) in Acinetobacter baumannii isolates from Argentina. J Infect Dev Ctries 2008, 2: 235-240.
Jeon BC, Jeong SH, Bae IK, Kwon SB, Lee K, Young D, Lee JH, Song JS, Lee SH: Investigation of a nosocomial outbreak of imipenem-resistant Acinetobacter baumannii producing the OXA-23 beta-lactamase in Korea. J Clin Microbiol 2005, 43: 2241-2245. 10.1128/JCM.43.5.2241-2245.2005
Niumsup PR, Boonkerd N, Tansawai U, Tiloklurs M: Carbapenem-resistant Acinetobacter baumannii producing OXA-23 in Thailand. Jpn J Infect Dis 2009, 62: 152-154.
Zhou H, Pi BR, Yang Q, Yu YS, Chen YG, Li LJ, Zheng SS: Dissemination of imipenem-resistant Acinetobacter baumannii strains carrying the IS Aba1 bla OXA-23 genes in a Chinese hospital. J Med Microbiol 2007, 56: 1076-1080. 10.1099/jmm.0.47206-0
Segal H, Elisha BG: Use of Etest MBL strips for the detection of carbapenemases in Acinetobacter baumannii . J Antimicrob Chemother 2005, 56: 598. 10.1093/jac/dki265
Mansour W, Poirel L, Bettaieb D, Bouallegue O, Boujaafar N, Nordmann P: Dissemination of OXA-23-producing and carbapenem-resistant Acinetobacter baumannii in a University Hospital in Tunisia. Microb Drug Resist 2008, 14: 289-92. 10.1089/mdr.2008.0838
Fu Y, Zhou J, Zhou H, Yang Q, Wei Z, Yu Y, Li L: Wide dissemination of OXA-23-producing carbapenem-resistant Acinetobacter baumannii clonal complex 22 in multiple cities of China. J Antimicrob Chemother 2010, 65: 644-650. 10.1093/jac/dkq027
Randrianirina F, Vedy S, Rakotovao D, Ramarokoto CE, Ratsitohaina H, Carod JF, Ratsima E, Morillon M, Talarmin A: Role of contaminated aspiration tubes in nosocomial outbreak of Klebsiella pneumoniae producing SHV-2 and CTX-M-15 extended-spectrum beta-lactamases. J Hosp Infect 2009, 72: 23-29. 10.1016/j.jhin.2009.02.004
The authors thank all hospital participants who collected isolates for the study.
This work was supported by a grant from the Institut Pasteur de Madagascar.
The authors declare that they have no competing interests.
TSA and ER participated in the design of the study, performed the MICs, the genotyping and the characterization of betalactamase, HCR performed the genotyping and the characterization of betalactamase, FR, LR and JFC participated in the collection of the strains and the antimicrobial susceptibility testing, VR participated in the analysis of the results and AT participated in the design of the study and in the analysis of the results. All authors contributed in the writing of the article, read and approved its final version.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Andriamanantena, T.S., Ratsima, E., Rakotonirina, H.C. et al. Dissemination of multidrug resistant Acinetobacter baumannii in various hospitals of Antananarivo Madagascar. Ann Clin Microbiol Antimicrob 9, 17 (2010). https://doi.org/10.1186/1476-0711-9-17
- Acinetobacter Baumannii