Genotypic diversity and antibiotic susceptibility of Acinetobacter ...

Research Notes 1135

RESEARCH NOTE Genotypic diversity and antibiotic susceptibility of Acinetobacter baumannii isolates in a Bulgarian hospital R. Dobrewski1, E. Savov1, A. T. Bernards2, M. van den Barselaar3, P. Nordmann4, P. J. van den Broek3 and L. Dijkshoorn3 1

Department of Clinical Microbiology, Military Medical Academy, Sophia, Bulgaria, 2Department of Medical Microbiology, 3Department of Infectious Diseases, Centre of Infectious Diseases, Leiden University Medical Centre, Leiden, The Netherlands and 4Department of Bacteriology-Virology, Hoˆpital de Biceˆtre, Le-KremlinBiceˆtre, France

ABSTRACT A set of 18 Acinetobacter baumannii isolates, collected prospectively in a Bulgarian hospital during episodes of increased A. baumannii occurrence during 2000–2002, was investigated for genotypic diversity and antibiotic susceptibility. Four genotypes were identified by amplified fragment length polymorphism genomic fingerprinting, one of which (type 1) accounted for 13 isolates, indicating that a specific strain was predominant. The single isolate allocated to type 2 was identified to European clone I. All isolates were resistant to multiple antibiotics, but most retained susceptibility to tobramycin and colistin, and all except one were susceptible to imipenem. Keywords Acinetobacter baumannii, AFLP typing, antibiotic susceptibility, Bulgaria, genotypes, nosocomal infection Original Submission: 9 December 2005; Revised Submission: 1 March 2006; Accepted: 9 April 2006

Clin Microbiol Infect 2006; 12: 1135–1137 10.1111/j.1469-0691.2006.01530.x

Corresponding author and reprint requests: L. Dijkshoorn, Department of Infectious Diseases, Leiden University Medical Centre, Albinusdreef 2, PO Box 9600, 2300 RC Leiden, The Netherlands E-mail: L. [email protected]

Most reports of multiresistant endemic or epidemic Acinetobacter baumannii are from western European countries, and information concerning this organism in central and eastern Europe is scarce. A. baumannii has been endemic in the hospital of the Military Medical Academy (MMA), Sophia, Bulgaria since the early 1990s. The present report investigated a set of A. baumannii isolates, collected prospectively in the MMA hospital during 2000–2002, for genotypic relatedness and susceptibility to antibiotics. The aims of the study were to determine whether a single strain predominated in the hospital during the collection period, and whether the isolates were related to clones I–III found in hospital outbreaks in several other European countries [1–3]. The MMA is a 500-bed university hospital for military personnel and civilians. It is a referral hospital that provides acute medical and surgical care, but not for paediatrics, obstetrics and burns patients. The hospital has two intensive care units, i.e., a 20-bed medical unit and a 16-bed surgical unit. Many patients admitted to the surgical intensive care unit are emergency cases with poly-trauma following car accidents or gunshot wounds. The hospital employs an infection control doctor and an infection control nurse, who both participate in the infection control committee of the hospital. The occurrence of multidrug-resistant Acinetobacter has increased steadily since the early 1990s, with recurrent clusters of cases, especially in the two intensive care units [4]. Control measures during such epidemic episodes usually include isolation of patients, as well as disinfection of the environment and surgical instruments on wards. Antibiotics for treatment of patients with infections caused by Acinetobacter spp. include imipenem, and amikacin plus ampicillin–sulbactam. In total, 18 multidrug-resistant isolates of Acinetobacter spp. from 18 patients were investigated (Table 1). These isolates were selected from among 65 multidrug-resistant Acinetobacter isolates recovered during documented episodes of increased occurrence during 2000–2002. The isolates were selected to represent presumptive cases of cross-infection, based on their antibiotic susceptibility profile and their origin in time and space. Most isolates were from multi-trauma patients, i.e., a group susceptible to colonisation with A. baumannii [5]. The organisms were iden-

2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 1131–1146

1136 Clinical Microbiology and Infection, Volume 12 Number 11, November 2006

Isolate

Patient

Date of isolation (day/month/year)

LUH8587 LUH8588 LUH8589 LUH8590 LUH8591 LUH8592 LUH8595 LUH8597 LUH8598 LUH8599 LUH8600 LUH8601 LUH8602 LUH8603 LUH8604 LUH8605 LUH8606 LUH8607

LP AP MA KR VS LI NV LD RN AD VT PP SS EA PJ AD BB AA

30 ⁄ 06 ⁄ 00 02 ⁄ 11 ⁄ 00 24 ⁄ 11 ⁄ 00 25 ⁄ 11 ⁄ 00 04 ⁄ 09 ⁄ 01 24 ⁄ 09 ⁄ 01 15 ⁄ 11 ⁄ 01 23 ⁄ 11 ⁄ 01 23 ⁄ 11 ⁄ 01 27 ⁄ 12 ⁄ 01 08 ⁄ 01 ⁄ 02 18 ⁄ 01 ⁄ 02 31 ⁄ 01 ⁄ 02 16 ⁄ 08 ⁄ 02 23 ⁄ 08 ⁄ 02 23 ⁄ 08 ⁄ 02 02 ⁄ 09 ⁄ 02 02 ⁄ 09 ⁄ 02

Warda Surg. ICU Surg. ICU Surg. ICU Surg. ICU Traum. Surg. ICU Surg. ICU Surg. ICU Surg. ICU Surg. ICU Uro. Haem. Surg. ICU Surg. ICU Surg. ICU Surg. ICU Med. ICU Med. ICU

Specimenb

Underlying conditionc

AFLP typed

SW CC BA BA SW UR BL BL BA BA UR UR BA UR.CATH. SW SW BA BA

Abd. abscess Poly-trauma Poly-trauma Poly-trauma Trauma Poly-trauma Poly-trauma Poly-trauma Poly-trauma Poly-trauma URP Myeloma Contusio cerebri Poly-trauma Poly-trauma Poly-trauma Insultus cerebri Insultus cerebri

1 1 1 1 1 2 1 1 1 4 4 1 1 1 1 3 4 3

Table 1. Data for Acinetobacter baumannii isolates from recurrent epidemic episodes in the Military Medical Academy, Sofia, Bulgaria during 2000–2002

a

Surg., surgical; Traum., traumatology; Uro., urology; Haem., haematology, Med., medical. SW, surgical wound; CC, central catheter; BA, bronchial aspirate; UR, urine; BL, blood; UR.CATH., urine catheter. Abd., abdominal; URP, trans-urethral resection of prostate gland. d Amplified fragment length polymorphism (AFLP) type as shown in the dendrogram of Fig. 1. b c

tified initially to the genus Acinetobacter using the automated Mini API System with ID32 strips (bioMe´rieux, Marcy l’Etoile, France), and were then identified to the species level by amplified ribosomal DNA restriction analysis [6] and amplified fragment length polymorphism (AFLP) fingerprint analysis [7]. Electrophoretic fragment separation and data capture were performed with the ALF express instrument (Amersham Biosciences, Roosendaal, The Netherlands). For final species identification, the AFLP profiles were grouped with those of strains of described genomic species in the Leiden AFLP library with BioNumerics software v.3.5 (Applied Maths, St Martens-Latem, Belgium). Isolates grouping at ‡50% similarity were considered to belong to the same species [7], while those that clustered at ‡82% and ‡90% similarity were considered to belong to the same clone or strain, respectively [2,8]. All 18 isolates were identified as A. baumannii by both amplified ribosomal DNA restriction analysis and AFLP analysis. At a clustering level of 90%, the dendrogram of the AFLP fingerprints comprised one cluster with 13 isolates (type 1), including two isolates with one extra fragment of c. 250 bp, one cluster with three isolates (type 4), and two single isolates (type 2 and type 3) (Fig. 1, Table 1). The linkage of the 13 type 1 isolates at 94% in one main cluster indicated that one particular strain predominated among the isolates. Isolate LUH8592 (type 2) was identified as European clone I, as it clustered at >82% with

reference strains of this clone (data not shown), but none of the other isolates corresponded to European clones I–III [1–3]. All isolates were tested routinely for antimicrobial susceptibility using the ATB-PSE 5 strip of the Mini API system (bioMe´rieux). Screening for extended-spectrum b-lactamases (ESBLs) was performed with the ATB-BLSE strip. Sixteen of the 18 isolates were resistant or intermediatelyresistant to piperacillin, piperacillin–clavulanate, ceftazidime, aztreonam, gentamicin, netilmycin, fosfomycin, pefloxacin and ciprofoxacin, but most isolates were susceptible to ticarcillin (n = 11), tobramycin (n = 11), colistin (n = 15) and imipenem (n = 17). Based on the MICs of expandedspectrum cephalosporins and use of the ATB-BLSE strip, the isolates were considered to be possible ESBL producers. To further investigate susceptibilities, a sub-set of six isolates that represented the different genotypes identified (LUH8604, LUH8605, LUH8600, LUH8599, LUH8592 and LUH8590; Table 1) was tested by the Vitek 2 system (bioMe´rieux) with the AST-N020 card. Vitek-derived MICs (mg ⁄ L) were 8 to ‡32 for ampicillin–clavulanic acid, 16 to ‡64 for ceftazidime, 0.5 to 2 for meropenem, £1 to ‡16 for tobramycin, and ‡4 for ciprofloxacin. None of the isolates produced a clavulanic acid-inhibited ESBL when tested by reference methods [9], or a metallo-enzyme, and the blaVEB and blaPER genes were not detected by PCR. The contradictory results concerning ESBL detection may be explained by the fact that the


Research Notes 1137

1

2 3

4

100

95

90

85

80

Pearson correlation (Opt:0.10%) [4.0%-97.0%] AFLP

LUH08595

15-11-01 Surg ICU

LUH08598

23-11-01 Surg ICU

LUH08601

18-01-02 UR

LUH08602

31-01-02 BA

LUH08603

16-08-02 Surg ICU

LUH08604

23-08-02 Surg ICU

LUH08589

24-11-00 Surg ICU

LUH08591

04-09-01 Traum

LUH08587

30-06-00 Surg ICU

LUH08588

02-11-00 Surg ICU

LUH08597

23-11-01 Surg ICU

LUH08590

25-11-00 Surg ICU

LUH08607

02-09-02 Med ICU

LUH08592

24-09-01 Surg ICU

LUH08605

23-08-02 Surg ICU

LUH08599

27-12-01 Surg ICU

LUH08600

08-01-02 Uro

LUH08606

02-09-02 BA

Fig. 1. Dendrogram showing cluster analysis of amplified fragment length polymorphism (AFLP) fingerprints for the 18 Acinetobacter baumannii isolates included in the study. The dotted line at 90% marks the level above which individual strains are delineated. Surg, surgical; ICU, intensive care unit; UR, urine; BA, bronchial aspirate; Traum, traumatology; Med, medical; Uro, urology.

ATB-BLSE strips contain sulbactam as an inhibitor, and this antibiotic has intrinsic activity against acinetobacters [10]. For this reason, the ATB-BLSE strip is not useful for detection of ESBL activity in A. baumannii. The present report indicates that several multidrug-resistant strains, as distinguished by AFLP analysis, were present in the MMA during the study period, with one strain being predominant. Except for one isolate identified to European clone I, none of the isolates belonged to the widespread European clones I–III [1–3]. This raises the question of whether the evolution to antibiotic resistance has occurred in these organisms independently from other A. baumannii strains in Europe. Further to the present report from Bulgaria, multidrug-resistant A. baumannii

isolates have been reported in other central ⁄ eastern European countries [2,8]. With the recent expansion of the European Union, and the expected increase in travel between eastern and western Europe, it is important to establish systems for monitoring the occurrence of multidrug-resistant A. baumannii in the new EU member states. ACKNOWLEDGEMENTS T. van der Reijden is gratefully acknowledged for excellent technical support.

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