Antibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936)

Caroline J. Henwooda, Tess Gatwarda, Marina Warnera, Dorothy Jamesa, Mark W. Stockdalea, Richard P. Spenceb, Kevin J. Townerb, David M. Livermorea and Neil Woodforda,*

a Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT; b Public Health Laboratory, University Hospital, Nottingham NG7 2UH, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A survey was conducted of the antimicrobial susceptibilities of 595 Acinetobacter spp. isolated from routine clinical specimens in 54 sentinel laboratories throughout the UK during 2000. Isolates of the Acinetobacter baumannii complex (genomic groups 2, 3 and 13TU; n = 443) were distinguished from other genomic groups (n = 152) by PCR fingerprinting of tDNA spacer regions. MICs of amikacin, cefotaxime, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, minocycline, piperacillin, piperacillin/tazobactam, rifampicin, sulbactam and tetracycline were determined on IsoSensitest agar and interpreted, wherever possible, using BSAC breakpoints. Tigecycline (GAR-936), a new glycylcycline, was also tested. Resistance to cephalosporins, aminoglycosides and ciprofloxacin was widespread, but carbapenems, colistin, sulbactam, minocycline and tigecycline were each active against >80% of the isolates. Isolates of A. baumannii were more often resistant to cefotaxime, ceftazidime, piperacillin, piperacillin/tazobactam, ciprofloxacin, gentamicin and tetracyclines than those belonging to other genomic groups, but were less often resistant to colistin; no significant differences between genomic groups were noted in the susceptibilities to amikacin, carbapenems, rifampicin or sulbactam. The relative activities of the tetracyclines were minocycline > tigecycline > tetracycline. Thirteen carbapenem-resistant isolates (MICs 8 mg/L; 2.2%) were received from six centres; four centres sent single isolates; one sent three and one sent six. An allele of bla IMP was detected in one of these isolates, but the other 12 isolates either had carbapenemase-independent resistance, or undetectable carbapenemase activity combined with other resistance mechanisms. In conclusion, carbapenems, colistin and minocycline retained greatest activity against the Acinetobacter isolates collected. Tigecycline was less active than minocycline, but both agents overcame most tetracycline resistance.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Acinetobacter spp., particularly A. baumannii, are important nosocomial pathogens, especially in intensive care and burns units,1 where they are frequent causes of ventilator-associated pneumonia and of bacteraemias.2 They are often multiresistant to antibiotics, meaning that therapy and infection control are complicated. Carbapenems have retained anti-Acinetobacter activity better than most other antimicrobial classes, but carbapenemases belonging to ß-lactamase classes B and D have begun to emerge in the genus, often in isolates already resistant to all other therapeutic antibiotics. Although carbapenemases are not always associated with high levels of phenotypic resistance in Acinetobacter spp.,3 major outbreaks of resistant carbapenemase producers have occurred in a few centres worldwide,4–6 and the International Network for the Study and Prevention of Emerging Antimicrobial Resistance (INSPEAR) has defined the emergence of carbapenem resistance in Acinetobacter as a ‘global sentinel event’.7

To assess the prevalence of antibiotic resistance in the UK, we examined 595 Acinetobacter spp. isolated during 2000 from routine clinical specimens at 54 sentinel laboratories. Each isolate was tested with a panel of 14 established antimicrobial agents, including representatives of all the major antibiotic classes. There is a paucity of new drugs active against Acinetobacter—or any other Gram-negative pathogens—but tigecycline (glycylcycline GAR-936)8 was included as a novel tetracycline known to evade both ribosomal- and efflux-mediated resistance to established analogues. Also included were the established carbapenems and colistin, which have been used against infections caused by multiresistant Acinetobacter strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study design

Fifty-four diagnostic laboratories with a good geographical spread across the UK (see Acknowledgements for a full listing) were each asked to collect up to 25 consecutive, non-replicate isolates of Acinetobacter from clinical specimens and to send these to the Antibiotic Resistance Monitoring & Reference Laboratory, CPHL, where susceptibility testing was performed. Species identification was confirmed centrally at Nottingham Public Health Laboratory. Data collected included the patient's age, sex, ward type, the site of isolation of organism and the susceptibility data generated by the source laboratory.

Identification

Genomic groups were assigned by amplification of the tDNA spacer regions,9 followed by separation of products on 1.5% agarose gels.

Susceptibility testing

MICs for each isolate were determined on IsoSensitest agar with an inoculum of 104 to 105 cfu. End-points were read after overnight incubation at 37°C. Antimicrobial agents and ranges tested were: amikacin (0.125–64 mg/L); cefotaxime (0.25–32 mg/L); ceftazidime (0.25–32 mg/L); ciprofloxacin (0.064–8 mg/L); colistin (0.064–32 mg/L); gentamicin (0.25–32 mg/L); imipenem (0.032–32 mg/L); meropenem (0.032–32 mg/L); minocycline (0.125–8 mg/L); piperacillin (2–64 mg/L); piperacillin/tazobactam (2–64 mg/L with tazobactam at a fixed concentration of 4 mg/L); rifampicin (2–64 mg/L); sulbactam (1–32 mg/L); tetracycline (0.5–32 mg/L); tigecycline (formerly GAR-936, 0.032–32 mg/L). All powders were obtained from the Sigma Chemical Company (Poole, Dorset, UK), except ceftazidime (GlaxoSmithKline, Uxbridge, UK), colistin (Alpharma, Copenhagen, Denmark), imipenem (Merck, Hoddesdon, UK), meropenem (AstraZeneca, Macclesfield, UK), sulbactam (Pfizer, Sandwich, UK), tazobactam (Wyeth, Taplow, UK) and tigecycline (Wyeth-Ayerst, St Davids, PA, USA). Confirmatory tests, where needed, were performed using Etest strips (Cambridge Diagnostics, Cambridge, UK).

Susceptibilities were interpreted, where possible, using the breakpoints recommended for Acinetobacter spp. by the BSAC Working Party.10,11 Exceptions were sulbactam, with resistance defined as MIC >= 16 mg/L;12 and tigecycline, with resistance provisionally defined by an MIC >= 8 mg/L and susceptibility by an MIC <= 2 mg/L (R. Testa, Wyeth-Ayerst, personal communication). No breakpoint is recommended for rifampicin versus Acinetobacter either by the BSAC10,11 or by the NCCLS.13

Investigation for carbapenemases

Carbapenem-resistant isolates were screened by PCR for alleles of the gene families encoding known acquired carbapenemases. Published primers and amplification conditions were used for blaIMP,14 blaVIM,15 blaOXA-235 and blaOXA-24.5 The isolates were examined for their ability to hydrolyse 0.1 mM imipenem by spectrophotometry at 297 nm as described previously,16 except that the crude enzymes were released from the Acinetobacter cells by six alternate cycles of freezing and thawing.

Data handling and statistical analyses

All data were stored and analysed using Microsoft Access and Excel, WHONET 5.1 and the Statcalc component of EpiInfo 2000. The {chi}2 test was used with Yates' correction; P < 0.05 was used to indicate significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
General

A total of 595 of 649 isolates referred by the 54 sentinel laboratories were confirmed as Acinetobacter spp.; 443 of these isolates belonged to genomic groups comprising the clinically important members of the A. baumannii complex (genomic groups 2 = A. baumannii, 3 and 13TU), and 152 isolates belonged to other genomic groups, including Acinetobacter calcoaceticus (genomic group 1). Nineteen (35%) of the 54 participating hospitals referred 444 (75%) isolates (range 15–29), comprising 343 (77%) isolates of the A. baumannii complex and 101 (66%) isolates of other genomic groups. These ‘major referral sites’ were used to assess the geographical distribution of resistance across the UK.

Isolates of the A. baumannii complex and non-A. baumannii isolates were similarly distributed with regard to patient age or sex (not shown). A greater proportion of A. baumannii complex isolates were from intensive care units (ICUs): 164 isolates (37%), compared with 15 non-A. baumannii isolates (10%) (P < 0.0000001). A greater proportion of A. baumannii complex isolates were from sputum: 85 isolates (19%), compared with nine non-A. baumannii isolates (6%) (P = 0.00018). Non-A. baumannii isolates were more likely to be from blood (82 isolates, 54%) than were isolates of the A. baumannii complex (109 isolates, 25%) (P < 0.0000001). It should be noted that many UK laboratories only identify Gram-negative bacteria to species level if they have been isolated from blood, meaning that the collection as a whole probably overrepresented bloodstream isolates.

Antimicrobial susceptibilities

Susceptibility distributions are shown in Tables 1 and 2GoGo. Resistance to ciprofloxacin, gentamicin, rifampicin, sulbactam, tetracycline and, to a lesser extent, amikacin was unequivocal, with bimodal MIC distributions divided by the BSAC breakpoints, whereas resistance to other drugs was defined by the BSAC breakpoint dividing a ‘tail’ of resistant or susceptible organisms from a majority population with the converse phenotype.


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Table 1. Distributions of antibiotic susceptibilities for isolates of the A. baumannii complex (n = 443)
 

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Table 2. Distributions of antibiotic susceptibilities for isolates of Acinetobacter spp. not belonging to the A. baumannii complex (n = 152)
 
Over 75% of the isolates were resistant to cefotaxime and ceftazidime, and over 30% to ciprofloxacin, gentamicin, piperacillin and piperacillin/tazobactam. The only established drugs active against over 90% of the isolates at the BSAC breakpoints were the two carbapenems, colistin and sulbactam. Minocycline should perhaps be added to this list: although only 82% of isolates were susceptible at the BSAC breakpoint of 0.5 mg/L, 97% were susceptible at the NCCLS breakpoint of 4 mg/L (see below).

Statistically, isolates of the A. baumannii complex were more often resistant than non-A. baumannii isolates to cephalosporins, ciprofloxacin, gentamicin, piperacillin and piperacillin/tazobactam. However, A. baumannii complex isolates were less often resistant to colistin. Isolates of the A. baumannii complex were neither more nor less often resistant than non-A. baumannii isolates to amikacin, carbapenems, rifampicin or sulbactam.

Antimicrobial resistance was geographically scattered across the UK; 18 of the 19 ‘major referral sites’ submitted gentamicin-resistant isolates; 17 submitted isolates resistant to amikacin; 17 submitted isolates resistant to ciprofloxacin; four submitted isolates resistant to imipenem; and two submitted isolates resistant to meropenem. The extent of multiresistance among Acinetobacter spp. was investigated by analysing resistance to eight antimicrobial agents: amikacin, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, minocycline and sulbactam (Table 3Go). Multiresistance was more frequently associated with isolates of the A. baumannii complex than with other genomic groups; nevertheless, 206 (47%) A. baumannii complex isolates and 85 (56%) other isolates were susceptible to all of the eight selected agents. Isolates with each of the most frequently observed multiresistance patterns were referred from multiple sentinel laboratories (Table 3Go).


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Table 3. Prevalence and distribution of common resistance and multiresistance patterns among Acinetobacter spp. in the UK
 
Relative activity of tigecycline and established tetracycline analogues

On a weight-for-weight basis, the relative activities of the tetracyclines were minocycline > tigecycline > tetracycline. For the entire collection, the MIC50 and MIC90 of tigecycline were 0.5 and 2 mg/L, compared with 0.125 and 1 mg/L of minocycline, and 4 and >32 mg/L of tetracycline. Calculation of resistance prevalence to tetracyclines, however, presents some difficulty. The BSAC adopts a single breakpoint of 1 mg/L for most established analogues except minocycline, to which it attributes a breakpoint of 0.5 mg/L.10 The NCCLS recommends identical criteria for all licensed tetracycline analogues, with susceptible and resistant categories defined by MIC <= 4 and >=16 mg/L, respectively.13 These differences, together with the manufacturer's provisional breakpoints of <=2 and >=8 mg/L of tigecycline, distort attempts to compare susceptibilities, so uninterpreted distributions are shown in Tables 1 and 2GoGo. Using BSAC criteria, 84% of isolates were resistant to tetracycline, although this fell to 30% resistant and 10% intermediate if the NCCLS breakpoints were used. It should be noted, however, that tests reported here were not performed in accordance with NCCLS recommended methodology. Similarly, 18% of total isolates were resistant to minocycline according to BSAC criteria, but none was considered resistant and just 3% were intermediate using the NCCLS breakpoints. Sixteen isolates (2.7%) from 10 centres were resistant to tigecycline (at the provisional breakpoint of MIC >= 8 mg/L); these all belonged to the A. baumannii complex. Twenty-six isolates (4.4%) showed intermediate susceptibility to tigecycline (MIC 4 mg/L); 22 belonged to the A. baumannii complex and four to other genomic groups. Irrespective of breakpoints, A. baumannii complex isolates were more often resistant to tetracyclines than non-A. baumannii isolates.

Resistance to carbapenems

Thirteen carbapenem-resistant isolates (MICs >= 8 mg/L; 2.2%) were received from six centres (Table 4Go); eight belonged to the A. baumannii complex and five to other genomic groups. Single resistant isolates were referred from four centres; one centre sent three isolates (all non-A. baumannii); and one sent six (five A. baumannii and one of another genomic group). Based on BSAC breakpoints, nine isolates were resistant to imipenem only, two to meropenem only and two to both carbapenems. The imipenem-resistant, meropenem-susceptible isolates all required raised meropenem MICs of 1 to 4 mg/L, indicating reduced susceptibility; the two meropenem-resistant, imipenem-susceptible isolates required elevated imipenem MICs (2 to 4 mg/L) as compared with typical isolates (Table 4Go). The carbapenem-resistant isolates were all multiresistant, but mostly remained susceptible to colistin at <=4 mg/L (11/13 isolates), tigecycline at <=2 mg/L (11/13), minocycline at <=4 mg/L (13/13) and sulbactam at <=8 mg/L (9/13). A blaIMP PCR product was obtained from one meropenem-resistant, imipenem-susceptible isolate (Table 4Go). None of the other 12 isolates yielded products with primers for genes known to encode carbapenemases, or detectably hydrolysed imipenem in spectrophotometric assays.


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Table 4. Isolation of carbapenem-resistant Acinetobacter spp.
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All of the Acinetobacter included in this survey were isolated from clinical specimens and were considered to be significant by the referring laboratory. The A. baumannii complex accounted for 75% (443/595) of those collected and we confirmed the association between isolates belonging to this complex and ICUs. Furthermore, the high isolation rate of A. baumannii complex isolates from sputa is consistent with their association with lower respiratory tract infections.1,2 A small majority of the isolates belonging to other genomic groups were recovered from blood, indicating that they also may cause serious infections, albeit less frequently than isolates of the A. baumannii complex; however, this association with blood could also reflect the fact that non-A. baumannii isolates are ubiquitous members of the normal human skin flora and are therefore prone to cause contamination of blood cultures. The isolates represented a wide distribution across the UK, but 75% were referred from only 35% of the sentinel laboratories. This bias may reflect the incidence of clonal outbreaks. Molecular epidemiological typing of the isolates is ongoing to investigate this possibility, to determine the extent of any intra- and inter-hospital spread of strains, and to compare resistant and susceptible isolates from individual sentinel laboratories. These studies will be reported separately.

The BSAC Working Party on Susceptibility Testing has advocated identical interpretative breakpoints for Acinetobacter spp. and the Enterobacteriaceae.11 We used these values wherever possible, although the criteria for sulbactam and tigecycline were taken from other sources (see Materials and methods). Resistance and multiresistance to many agents, including aminoglycosides, cephalosporins and ciprofloxacin, were frequent and geographically scattered, especially among A. baumannii isolates. Among the established agents tested, the carbapenems, colistin, minocycline and sulbactam retained greatest activity. Carbapenems are increasingly the drugs of choice against infections caused by the genus and colistin has been perceived as a drug of last resort, generally active in vitro, though with variable efficacy and significant toxicity in vivo. Uniquely, colistin retained better activity versus A. baumannii isolates than those of other genomic groups. The potential role of rifampicin as a synergist in the treatment of Acinetobacter infections is interesting.17–19 Hogg et al.17 reported synergy between colistin and rifampicin against 11/13 multiresistant A. baumannii isolates, even though nine isolates were categorized as resistant to rifampicin (MIC >= 4 mg/L). The BSAC Working Party does not recommend interpretative criteria for rifampicin versus Gram-negative bacteria other than Neisseria spp., for which resistance is defined as MIC >= 2 mg/L.11 Similarly, N. meningitidis and Haemophilus spp. are the only Gram-negative bacteria for which the NCCLS gives interpretative criteria for rifampicin; these being MIC <= 1 mg/L (susceptible) and >=4 mg/L (resistant).13 How such values relate to the potential for rifampicin to act as an adjunct in anti-Acinetobacter treatment is unknown. Tentatively, 76% of isolates belonging to the A. baumannii complex and 52% of isolates belonging to other genomic groups were considered resistant (MICs >= 4 mg/L) to rifampicin.

The MIC distribution for tetracycline was clearly bimodal, whereas those for minocycline and tigecycline were unimodal with tails of organisms with resistance or reduced susceptibility. Although we did not investigate the molecular basis of resistance, these distributions are consistent with the wide distribution of the Tet(A) and Tet(B) efflux proteins in clinical isolates of A. baumannii reported previously.20 Tet(A) confers resistance to tetracycline, but not to minocycline or glycylcyclines, while Tet(B) confers resistance to tetracycline and minocycline, but not to glycylcyclines.21 Comparison of resistance rates among the tetracyclines is, however, difficult. The BSAC breakpoints for established tetracyclines10 are four- to eight-fold lower than those recommended by the NCCLS,13 and only a provisional breakpoint value is available for tigecycline. If, however, the BSAC breakpoints were applied, 66% of isolates (390/595) collected in this survey were resistant to tetracycline, and a further 18% (110 isolates) were resistant to both tetracycline and minocycline. We found tigecycline (GAR-936) to be less active than minocycline, but both agents overcame most tetracycline resistance. The MIC50 and MIC90 of tigecycline determined in this study were 0.5 mg/L and 2 mg/L, agreeing with values for Acinetobacter spp. from a previous study.22 Using the provisional tigecycline breakpoint of 2 mg/L for susceptibility, we identified 42 insusceptible isolates (16 with full resistance, and 26 with intermediate susceptibility). The genetic basis of this resistance will be examined further; mutations in either Tet(A) or Tet(B) potentially may lead to glycylcycline resistance.21,23

Although increasing concern has been expressed about the emergence of class B and D carbapenemases in Acinetobacter spp.,3,7 carbapenem resistance evidently remains very rare among Acinetobacter in the UK, seen in only 13 (2.2%) of 595 isolates. A blaIMP allele was detected in one isolate with low-level meropenem resistance (MIC 8 mg/L), but susceptibility to imipenem (MIC 2 mg/L). This represents the first confirmed isolation in the UK of a bacterium with an IMP ß-lactamase. Detailed characterization of the particular allele is being presented separately.24 Genes encoding enzymes belonging to known carbapenemase families were not detected in any of the other 12 carbapenem-resistant isolates, and extracts prepared from them did not hydrolyse imipenem. Thus, they may have carbapenemase-independent resistance, or very weak carbapenemases linked with other resistance factors such as impermeability or efflux. The isolate with the IMP enzyme was barely resistant to meropenem (MIC 8 mg/L) and apparently susceptible to imipenem (MIC 2 mg/L) confirming international results that production of even a potent metallo-carbapenemase does not necessarily confer high levels of phenotypic resistance to carbapenems in Acinetobacter spp. or other Gram-negative bacteria.5

In conclusion, carbapenems, colistin and minocycline retained greatest activity against the Acinetobacter isolates collected. Tigecycline was less active than minocycline, but both agents overcame most tetracycline resistance.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We wish to thank Wyeth-Ayerst for tigecycline and for financial support of its inclusion in this study, to Hamish Beattie for his advice on using WHONET software, and to Marie-France Palepou and Luke Tysall for their technical assistance with the PCR and hydrolysis assays. We are grateful to colleagues in the following clinical microbiology laboratories who submitted isolates for inclusion in this study: Aberdeen Royal Infirmary; Ashford Hospital, Middlesex; Bangor PHL; Bath PHL; Birmingham City Hospital; Birmingham PHL; Brighton PHL; Bristol PHL; Cambridge PHL; Cardiff PHL; Chelmsford PHL; Chester PHL; Chelsea & Westminster Hospital; Crawley Hospital; Derbyshire Royal Infirmary; Dulwich PHL; Epsom PHL; Exeter PHL; Victoria Hospital, Fife; Gloucester PHL; Greenwich District Hospital; Hammersmith Hospital; Hereford PHL; Hope Hospital; Ipswich PHL; Leeds General Infirmary; Leicester PHL; Lincoln PHL; Manchester PHL; Middlesborough PHL; North Middlesex Hospital; Nottingham PHL; Plymouth PHL; Poole PHL; Portsmouth PHL; Reading PHL; Rhyl PHL; Royal Gwent Hospital; Royal Lancaster Infirmary; Salisbury PHL; Sheffield PHL; Southmead Hospital; Southampton PHL; St George's Hospital; St Mary's Hospital NHS Trust; Stoke Mandeville Hospital; Stoke-on-Trent PHL; Trafford General Hospital; Truro PHL; University College Hospital; Queen Elizabeth II Hospital, Welwyn Garden City; Worthing & Southlands Hospital; York District Hospital; Wrexham Maelor Hospital. This work was presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Chicago, IL, USA in December 2001.


    Notes
 
* Corresponding author. Tel: +44-20-8200-4400; Fax: +44-20-8358-3292; E-mail: nwoodford{at}phls.org.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 23 August 2001; returned 14 November 2001; accepted 4 December 2001