Abstract
Acinetobacter spp. have been increasing in significance in hospitals as colonizers and pathogens, especially of immunocompromised hosts, patients in intensive care units and those with serious underlying diseases (Bergogne-Berezin & Towner, 1996). Within the genus, organisms within the Acinetobacter baumannii complex are particularly significant as opportunistic pathogens. The A. baumannii complex includes genospecies 1, 2, 3 and 13, which can only be identified by amplified rDNA restriction analysis (Vaneechoutte et al., 1995), and of which genospecies 2 most commonly causes hospital infections (Bergogne-Berezin & Towner, 1996). Treatment of acinetobacter infections is often complicated by their resistance to multiple antibiotics. Although carbapenems have remained more active than other antimicrobial agents and resistance to this class of antibiotics is still rare, carbapenem-resistant Acinetobacter spp. have increased in prevalence during the past few years (Afzal-Shah & Livermore, 1998). These strains were initially reported to be carbapenem resistant due to a mechanism other than production of β-lactamases, but β-lactamase-mediated resistance is commonly detected in recent strains (Clark, 1996; Héritier et al., 2005). The first β-lactamase reported in carbapenem-resistant A. baumannii was ARI-1, now renamed OXA-23 (Donald et al., 2000).
The carbapenem-hydrolysing OXA enzymes sequenced so far belong to three clusters. The first cluster comprises the OXA-23 and OXA-27 enzymes, with 99 % amino acid homology; the second cluster includes the OXA-24, OXA-25 and OXA-26 enzymes, which share 98 % homology; the homology between these two clusters is 60 % (Héritier et al., 2005). The third cluster, with OXA-58 as the only member, shares <50 % homology with the other two clusters.
In this study, we aimed to determine the prevalence of carbapenem-resistant Acinetobacter baumannii (IMRAB) in Shanghai and Hong Kong and to detect genes encoding carbapenem-hydrolysing β-lactamases by PCR in IMRAB isolates.
A total of 214 non-duplicate A. baumannii clinical isolates were collected from Renji Hospital, Shanghai (n=175), and the Prince of Wales Hospital, Hong Kong (n=39), from August 2002 to August 2003. Strains from Shanghai were isolated from clinical specimens that included sputum, wound, abscess, bile, urine and blood, and strains from Hong Kong were isolated from blood cultures only. All isolates were identified by the API 20NE system (API systems) together with other standard biochemical tests. They were further identified to genospecies by amplified rDNA restriction analysis (ARDRA) (Vaneechoutte et al., 1995).
MICs of antibiotics (Table 1⇓) were determined by an agar dilution method using Mueller–Hinton agar (Oxoid) according to the recommendation of the National Commmittee for Clinical Laboratory Standards (2003).
Characteristics and in vitro antimicrobial susceptibilities of 12 clinical isolates of A. baumannii
Abbreviations: TIC, ticarcillin; T+C, ticarcillin/clavulanic acid (clavulanic acid at a fixed concentration of 2 μg ml−1); PIP, piperacillin; P+T, piperacillin/tazobactam (tazobactam at a fixed concentration of 4 μg ml−1); CFP, cefoperazone; C+S, cefoperazone/sulbactam (1 : 1); A+S, ampicillin/sulbactam (2 : 1); SUL, sulbactam; CAZ, ceftazidime; CPE, cefepime; IMP, imipenem; MEM, meropenem; GM, gentamicin; AMK, amikacin; CIP, ciprofloxacin; LEV, levofloxacin; SXT, cotrimoxazole.
Strains were typed by macro-restriction analysis of chromosomal DNA with ApaI (Amersham Biosciences) using PFGE on a CHEF-DR apparatus (Bio-Rad) with pulses ranging from 5 to 35 s at a voltage of 6 V cm−1 at 14 °C for 32 h. DNA bands were photographed with a Gel Documentation System (Pharmacia Biotech) and analysed using Bionumerics version 2.5 software. Following conversion, normalization with molecular mass markers, and background subtraction, comparison matrices of the Dice coefficient were generated and dendrograms were constructed using the unweighted pair group method by arithmetic means (UPGMA).
Strains were also typed by repetitive extragenic palindromic PCR (REP-PCR) using primer pair REP1 and REP2 according to Martín-Lozano et al. (2002). Strains with similar profiles (up to two-band difference) were assigned to the same DNA group. A Dice coefficient of ⩾80 % was used to denote closely related isolates (Tenover et al., 1995).
Crude β-lactamases were extracted from bacteria by sonication, and pIs were determined by isoelectric focusing on PAGplates (Pharmacia Biotech, pH range 3.5–9.5) in a Multiphor II System (Pharmacia-LKB) (Matthew et al., 1975). The following β-lactamases of known pI were used as markers: TEM-1 (pI 5.4), TEM-2 (pI 5.6), TEM-3 (pI 6.3), K1 (pI 6.5), OXA-1 (pI 7.4), SHV-1 (pI 7.6), OXA-2 (pI 7.7), SHV-4 (pI 7.8) and P99 (pI 8.2).
The extracted DNA was screened by PCR for the presence of blaOXA-23-specific and blaOXA-24-related sequences (Afzal-Shah et al., 2001), and blaTEM- and blaPER-related sequences (primer pairs 5′-GAGTATTCAACATTTCCGTGTC-3′ and 5′-TAATCAGTGAGGCACCTATCTC-3′, and 5′-ATGAATGTCATTATAAAAGC-3′ and 5′-AATTTGGGCTTAGGGCAGAA-3′, respectively, were used) (Jiang et al., 2002).
Of the 214 acinetobacters tested, 12 were resistant to imipenem and meropenem (IMRAB) (MICs 8 to >64 μg ml−1). They were identified to be A. baumannii; 10 belonged to genospecies 3 and two to genospecies 2. They were also resistant to ampicillin-sulbactam (MIC ⩾16/8 μg ml−1); piperacillin and ticarcillin (MIC ⩾128 μg ml−1); piperacillin-tazobactam (MIC 64/4 to >128/4 μg ml−1); ticarcillin-clavulanate (MIC >128/2 μg ml−1); cefoperazone, ceftazidime and cefepime (MIC ⩾32 μg ml−1); gentamicin (>64 μg ml−1) and cotrimoxazole (MIC 16 to >64 μg ml−1) (Table 1⇑). The MIC of sulbactam alone was the same as that of sulbactam in combination with ampicillin or cefoperazone. When piperacillin-tazobactam was tested in the ratio of 2 : 1, the MICs for all isolates were 32/16 μg ml−1 (results not shown). Two isolates were susceptible to 16 μg amikacin ml−1. Only cefoperazone-sulbactam, ciprofloxacin and levofloxacin could inhibit more than 50 % of the isolates (Table 1⇑).
The isolates were shown to belong to two clusters by PFGE analysis (Fig. 1a⇓). One cluster, consisting of nine isolates, all belonging to genospecies 3, was referred to as type A (Table 1⇑). PFGE patterns within this type could be divided into four subtypes: A1, A2, A3 and A4. Subtypes A1, A2 and A3 shared >90 % similarity. There was only one isolate with PFGE subtype A4, and it shared 84 % similarity with the other A subtypes. The other cluster, designated type B, consisted of three isolates, of which two belonged to genospecies 2 (subtypes B1 and B2) and one to genospecies 3 (subtype B3) (Table 1⇑). Agarose gel electrophoresis of the amplified fragments obtained after REP-PCR of these isolates is shown in Fig. 1(b)⇓. The patterns of five isolates were assigned to PCR group 1 and those of four to PCR group 2. The band pattern of strains 190, 193 and 108132 (all with PFGE type B) was assigned to PCR group 3 (Table 1⇑).
(a) Dendrogram showing results of Dice coefficient based on PFGE of restricted DNA fragments of all 12 isolates. Nine isolates were assigned to type A, with three isolates within subtype A1 (166, 176 and 191), one each within subtypes A2 and A4 (182 and 192, respectively) and four within subtype A3 (3, 16, 25 and 126). Three isolates were assigned to subtypes B1, B2 and B3 (190, 193 and 108132, respectively) within type B. (b) Dendrogram showing results of Dice coefficient based on REP-PCR of all 12 isolates. Five isolates were assigned to PCR group 1 (3, 16, 25, 126 and 166), four to group 2 (176, 182, 191 and 192) and three to group 3 (190, 193 and 108132).
The sonicated extract of all 12 strains showed three β-lactamase bands at pI 5.3, 6.9 and 7.2 by isoelectric focusing. These strains harboured the OXA-23 β-lactamase gene (1062 bp), and all 11 isolates from Shanghai also harboured a PER-1-type gene (∼600 bp). Three of these isolates harboured a TEM-like gene (1079 bp) (strains 190, 193 and 180132) (Table 1⇑). An OXA-24-type gene was not detected in any of the isolates.
Carbapenem resistance in Acinetobacter spp. is an emerging problem because many strains are already resistant to most other antibiotics (Bergogne-Berezin & Towner, 1996; Afzal-Shah et al., 2001). This study revealed that all 12 carbapenem-resistant A. baumannii isolates were resistant to ticarcillin, piperacillin and ticarcillin-clavulanic acid (MIC ⩾128 μg ml−1). Our results also showed that the combination of β-lactam and β-lactamase inhibitor, e.g. piperacillin-tazobactam and cefoperazone-sulbactam, was more active than the β-lactam alone. Sulbactam alone has an antibacterial effect on Acinetobacter strains, and β-lactams in combination with sulbactam were found to be effective against this group of organisms.
In order to investigate the source of infection and the route of spread of the infecting organism, several phenotypic and molecular typing methods have been used. Although an antibiogram may alert us to the emergence of a multiply resistant A. baumannii outbreak, distinguishing between strains with slight differences in their resistance profiles may be difficult. Therefore, PFGE of chromosomal DNA restriction fragments and PCR fingerprinting have been used to investigate nosocomial A. baumannii outbreaks (Dimopoulou et al., 2003). By using PFGE and REP-PCR, we demonstrated the spread of IMRAB strains in a Shanghai hospital during a period of 1 year. These isolates belonged to two different clones, as evidenced by PFGE analysis, and all except one belonged to the same genospecies.
Several class B and D β-lactamases have been reported to be responsible for carbapenem resistance in acinetobacters (Héritier et al., 2005). Three different β-lactamases were produced by our IMRAB strains: a TEM-like enzyme, a PER-1-type β-lactamase, and an OXA-derived enzyme.
All our 12 IMRAB isolates harboured an OXA-23 gene, 11 of which, all from Shanghai, also harboured a PER-1-type gene as demonstrated by PCR. Two isolates from Shanghai (strains 190 and 193) and one from Hong Kong (strain 108132) harboured a TEM-type gene as well. These three IMRAB isolates belonged to PFGE type B, and were different from the other nine isolates, which were of PFGE type A and did not harbour the TEM-type gene. The MICs of imipenem and meropenem (MIC 32 to >64 μg ml−1) for the three strains that harboured an OXA-23, TEM-like and/or PER-1-type gene were higher than those for other isolates.
The results of this study showed that both OXA-23- and PER-1-type β-lactamases were prevalent among IMRAB in Shanghai and had already spread among different clones. The PER-1-type β-lactamase has also been shown to be prevalent in Turkey (Vahaboglu et al., 1997). Whether the presence of a TEM-like gene augments the hydrolytic activity of OXA-23 remains to be determined. Interestingly, the strain isolated from Hong Kong that did not have the PER-1-type gene (strain 108132) was more resistant to meropenem than strains with this gene.
Since carbapenem-resistant A. baumannii strains are highly resistant to β-lactams, aminoglycosides and fluoroquinolones, their emergence and dissemination deserve great attention. Continuous surveillance should be carried out to monitor the spread of these strains.