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J. Med. Microbiol. Ð Vol. 50 (2001), 71±77 # 2001 The Pathological Society of Great Britain and Ireland Carriage of class 1 integrons and antibiotic resistance in clinical isolates of Acinetobacter Departamento de Immunologia, Microbiologia y Parasitologia, Facultad de Medicina y Odontologia, Universidad del Pais Vasco, 48080 Bilbao, Spain and ÃDepartment of Microbiology & PHLSLaboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH A collection of 70 clinical isolates of Acinetobacter baumannii from Bilbao in northern Spain was examined by PCR for the presence of class 1 integron structures. The organisms comprised 21 distinct RAPD genotypes, with 10 distinct antibiogram patterns.
Four different integron structures were detected in a total of 59 (84%) of the 70 isolates, with two predominant integron structures found in 20 and 30 isolates each. No clear antibiogram differences could be correlated with the presence or absence of integron structures, but sequence analysis of two of the internal integron regions indicated homology with genes encoding ANT(20) adenyltransferase activity and AAC(69)-Ib acetyltransferase activity. Phenotypic analysis of aminoglycoside resistance pro®les indicated that many isolates produced a combination of aminoglycoside-modifying enzymes, with most of the observed resistance to amikacin being associated with a gene encoding APH(39)-VI phosphotransferase, as detected by PCR. RAPD analysis indicated that all the Bilbao isolates producing APH(39)-VI were distinct from an epidemic integron-carrying and APH(39)-VI-producing Acinetobacter strain found in other regions of Spain. It is concluded that, although class 1 integrons are widely disseminated amongst clinical isolates of A. baumannii from the Bilbao region of Spain, at present they are not playing a major role in the dissemination of antibiotic resistance genes in As in other species, plasmids and transposons play an important role in the acquisition of antibiotic resistance Members of the genus Acinetobacter, predominantly A.
by Acinetobacter spp. In addition, antibiotic baumannii, are recognised as increasingly important resistance in Acinetobacter spp., particularly to ami- nosocomial pathogens, especially in intensive care noglycosides, has been associated increasingly with the units, where they are associated particularly with presence of integrons These elements possess a nosocomial pneumonia Such infections are often site-speci®c recombination system enabling the inser- dif®cult to treat because of the ability of acinetobacters tion, deletion and re-arrangement of discrete genetic to become rapidly resistant to multiple antibiotics, cassettes, often comprising antibiotic resistance genes, including aminoglycosides, expanded-spectrum cepha- within the integron structure Several classes of losporins, carbapenems and ¯uoroquinolones integrons have been described with class 1 Some strains are effectively untreatable, and the integrons being the most common and widely dis- severity and dif®culties associated with nosocomial tributed among gram-negative bacteria Inte- infection by A. baumannii are related directly to the grons have been found in isolates of Acinetobacter spp.
particularly high rates of resistance found from various locations worldwide and it has been suggested that multiresistant isolates of Acineto- bacter spp. may act as a reservoir of integron- associated antibiotic resistance genes, which could then spread to other pathogens in the hospital Received 31 Jan. 2000; revised version accepted 10 May Corresponding author: Dr K.J. Towner (email: Kevin.Towner The main aim of the present study was to determine the prevalence of class 1 integrons among nosocomial discrimination between genetically unrelated groups of isolates of A. baumannii from the city of Bilbao in Acinetobacter spp. is achieved at an SAB value of 0.7 northern Spain, and to examine whether there was any therefore, isolates that clustered together with an correlation between antibiotic resistance and carriage SAB value of .0.7, with both primers, were considered of class 1 integrons. The Hospital of Santa Marina, to be closely related and to form a single RAPD Bilbao, is a 200-bed institution specialising in respira- genotype. Comparison of isolates from Bilbao and tory illness. Infections caused by A. baumannii are a Barcelona was performed with M13 core primer, as particular problem. An epidemic strain of A. baumannii carrying a class 1 integron has been reported previously in Barcelona and other regions of Spain and the study also aimed to establish whether Detection and characterisation of class 1 this strain had spread from Barcelona to Bilbao.
integronsTotal genomic DNA extracts of A. baumannii isolates were used as templates in PCR experiments to detect the hot-spot insertion regions of class 1 integrons. Each PCR was performed with a Ready-To-Go PCR bead (Amersham Pharmacia Biotech, Little Chalfont, Bucks) In total, 70 isolates of A. baumannii were obtained in a ®nal 25-ìl volume containing 5 ìl of DNA from different patients attending the Hospital of Santa extract and 2.5 pmol each of primers 59CS and 39CS, Marina, Bilbao, Spain, from June 1998 until April as described previously Initial comparison of the 1999. The specimens from which A. baumannii were inserted cassettes in the class 1 integron structures isolated comprised 58 sputum samples, three blood detected was performed by taking 5-ìl portions of the cultures, one broncho-alveolar sample, one urine, three primary PCR products and digesting them with HinfI, wound swabs, three pleural aspirates and one stool. All the isolates were identi®ed initially with the API 32 system (bioMeÂrieux, Marcy l'Etoile, France), with Further PCR analysis of possible inserted cassettes was identi®cation to the level of genomic species being performed by taking total genomic DNA or initial con®rmed by tDNA ®ngerprinting The isolates integron PCR products and using the primers and were stored at À208C in nutrient broth containing methodology described by Vila et al. to seek the glycerol 50% v/v before inclusion in this study. An aph(39)-VIa gene, which is reported to be responsible integron-carrying strain of A. baumannii from Barce- for much of the amikacin resistance found in A.
lona (generously donated by Professor J. Vila), designated previously as strain Ab41 or strain ESP41 was included in the study, as was the sensitive type strain of A. baumannii, ATCC 19606.
Preliminary sequence dataCycle sequencing of PCR products, puri®ed with a GFX PCR DNA Gel Band Puri®cation Kit (Amer- sham), was performed with the 59CS primer and a Cy5 MICs for the isolates were determined by the broth Thermo Sequenase Dye Terminator Kit (Amersham), micro-dilution method described by the National with both kits used as recommended by the manufac- Committee for Clinical Laboratory Standards turer. DNA sequencing reactions were analysed on with Pseudomonas aeruginosa strain ATCC 27853 as a acrylamide (Premix Long Ranger Gel Solution; Flow- control. The antibiotics tested were aztreonam, cefe- gen, Ashby de la Zouch, Leics) 6% w/v gels in an pime, cefotaxime, ceftazidime, imipenem, meropenem, automated ALFexpress DNA Sequencer (Amersham).
ticarcillin, amikacin, gentamicin, cipro¯oxacin, levo- The DNA sequences obtained were screened against ¯oxacin, nor¯oxacin and o¯oxacin. Similarity coef®- known sequences contained in nucleic acid sequence cients (SAB values) were calculated and subjected to databases by the BLAST (Basic Local Alignment cluster analysis as described previously except Search Tool) on-line method (www.ncbi.nlm.nih.gov/ that MIC values rather than inhibition zone sizes were used. Aminoglycoside resistance pro®les were analysed further by a disk method, with 12 different drug ResultsDNA ®ngerprinting experiments and antibiograms Twenty-one distinct RAPD genotypes of A. baumannii Total genomic DNA extracts of A. baumannii isolates were identi®ed among the 70 isolates included in the were prepared and then compared by analysis of randomly ampli®ed polymorphic DNA (RAPD) ®nger- antibiogram data for one isolate of each type. Three prints, obtained with primers AP3 and ERIC2 Previous investigations have determined that good collectively accounting for 44 (63%) of the 70 isolates.
Table 1. Antibiograms for A. baumannii isolates representing the 21 RAPD genotypes delineated during the study Spt, sputum; B/c, blood culture; Wnd, wound; Plp, pleural aspirate; TIC, ticarcillin; CTX, cefotaxime; CAZ, ceftazidime; CFM, cefepime; IMP, imipenem; MEM, meropenem; AZT, aztreonam; AMI, amikacin; GEN, gentamicin; NOR, nor¯oxacin; CIP, cipro¯oxacin; LEV, levo¯oxacin; OFL, o¯oxacin.
Minor variations in antibiograms were observed within For screening purposes, preliminary DNA sequences each genotype, and the isolates listed are the most were generated from the 59 end of puri®ed PCR resistant representatives of each type. Antibiograms products corresponding to the inserted cassette regions with an SAB value of .0.9 were considered to be in each of the four integron structures. Isolate SM36 indistinguishable. On this basis, 10 distinct antibio- (structure a) yielded a 550-base sequence that showed grams were distinguishable among the 21 different 95% base homology with the aadB gene cassette genotypes, of which the most common was shared by encoding ANT(20) adenyltransferase activity (accession 54 (77%) isolates belonging to 10 genotypes (A, B, E, no. U14415 Similarly, isolate SM49 (structure c) L, M, N, P, Q, R and T). None of the other yielded a 650-base sequence that showed 93% base antibiograms was shared by more than two genotypes.
homology with an aacA4 gene encoding AAC(69)-Ib acetyltransferase activity (accession no. AF043381).
Taken together with the ampli®ed external non-coding regions, the known sizes of these aadB and aacA4 gene Ampli®cation with primers speci®c for the 59 and 39 cassettes (591 bp and 637 bp, respectively were conserved regions of class 1 integrons yielded PCR suf®cient to account for the entire ampli®ed regions products from 59 of the 70 isolates. Four predominant integron structures a and c. No signi®cant PCR products of 760, 550, 800 and 600 bp were database homology was detected with a 400-base visualised Additional faint PCR products of sequence generated from isolate SM80 (structure b) various sizes were visualised occasionally, but were not or a 650-base sequence generated from isolate SM52 reproducible and were not investigated further. Four different HinfI ®ngerprint patterns, corresponding to the four predominant PCR products, were obtained and designated a±d The most common structures were a and b, found in 20 and 30 isolates, respectively.
Structures c and d were detected in eight isolates and Members of the genus Acinetobacter frequently have one isolate, respectively. Based on the sizes of the complex combinations of aminoglycoside resistance inserted cassette regions detected and the reported sizes of previously characterised gene cassettes it identi®ed here, aadB, encoding ANT(20), confers seemed likely that each of the four integron structures resistance to gentamicin and tobramycin, whereas contained one, or at most two, inserted gene cassettes.
aacA4, encoding one of the AAC(69)-I family of Integrons of the same internal structure were found in enzymes, confers resistance to tobramycin, amikacin more than one Acinetobacter genotype, but no single and netilmicin. Amikacin resistance in Acinetobacter genotype contained more than one type of integron spp. can be associated with production of either 69-N- acetyltransferase type I (AAC(69)-I) enzymes or 39-O-phosphotransferase type VI (APH(39)-VI) To distinguish these possibilities, total DNA and integron Correlation of antibiotic resistance pro®les with PCR products from the representative isolates listed in responding to the 21 different genotypes, were screened by PCR for the presence of the aph(39)- Attempts were made to correlate the observed minor VIa gene. Of the 10 isolates that expressed resistance variations in antibiograms within each genotype with (MIC . 16 mg=L) to amikacin, eight (SM10, 16, 28, the presence or absence of integron structures, but no 29, 30, 36, 49 and 80) yielded a 234-bp PCR product clear distinctions could be made. For example, only 10 characteristic of the aph(39)-VIa gene when total, of the 14 isolates that belonged to genotype P yielded a but not integron, DNA was used as a template. Thus, type a integron structure, and these 10 isolates varied the aph(39)-VIa gene was widely distributed amongst slightly in antibiotic susceptibilities among themselves the different genotypes, but did not form part of the and from the four isolates that lacked an integron integron structures identi®ed. Isolate SM49 possessed both an aph(39)-VIa gene and an integron-associated Table 2. Characteristics of the inserted cassette regions of the class 1 integron CLASS 1 INTEGRONS AND ANTIBIOTIC RESISTANCE IN A. BAUMANNII aacA4 gene. Two isolates (SM69 and 77) were resistant but all eight Bilbao genotypes with the aph(39)-VIa to amikacin, but lacked an aph(39)-VIa gene; these gene were clearly distinguishable from strain ESP41.
isolates were assumed to express an AAC(69)-I enzyme, but lacked the type c integron structure found in SM49. In contrast, isolate SM15 contained the type c integron structure, but was susceptible to amikacin Class 1 integron structures were detected by PCR in 59 (84%) of 70 clinical isolates of A. baumannii from Bilbao. Although these clinical isolates were geneti- side disks for four representative isolates carrying the cally diverse, with 21 distinct genotypes identi®ed, only four different integron structures. Isolate SM52 (in- four integron structures ± recognised on the basis of tegron structure d) did not differ signi®cantly in their HinfI restriction endonuclease ®ngerprint pro®les ± behaviour from the sensitive type strain of A.
were identi®ed. These structures were disseminated baumannii. Therefore, it was concluded that the type amongst 18 genotypes, with types a and b being found d integron structure did not encode aminoglycoside in 20 and 30 isolates, respectively. These ®ndings resistance. After taking account of the resistance clearly indicate the spread of integron structures among phenotype associated with aph(39)-VIa (already demon- different A. baumannii genotypes found in Bilbao.
strated by PCR in isolates SM36, SM49 and SM80), However, and in contrast to many integrons found in A.
the pro®les obtained also suggested production of baumannii elsewhere in the world the Bilbao ANT(20) and an AAC(3) enzyme by isolate SM36, the structures seemed to have acquired only one (or at most production of AAC(3)-II by isolate SM80, and the two) inserted resistance gene cassettes.
production of AAC(69)-I and AAC(3)-II by isolate SM49. These results were consistent with the pre- Ten different antibiograms were seen amongst the 21 liminary integron sequencing data in the two cases genotypes, but there were no clear phenotypic differ- (isolates SM36 and SM49) where signi®cant homology ences between isolates of the same genotype with and without an integron. Preliminary sequence analysis indicated the presence of cassettes encoding ANT(20) adenyltransferase and AAC(69)-Ib acetyltransferase Dissemination of the aph(39)-VI gene in Spain activity for two of the integron structures (a and c, respectively). Aminoglycoside resistance determinants Amikacin resistance in Acinetobacter spp. from differ- are the predominant gene cassettes found in integrons ent regions of Spain has previously been associated from gram-negative bacteria including Acineto- with the spread of an epidemic strain carrying the bacter isolates collected worldwide However, many multiply-resistant acinetobacters produce complex com- amikacin resistance found in Bilbao was associated, binations of aminoglycoside-modifying enzymes, many at least in part, with spread of the same epidemic of which are not encoded by integrons and strain, representatives (SM10, 16, 28, 29, 30, 36, 49 analysis of disk inhibition tests indicated that this was and 80) of the eight genotypes found to carry the aph(39)-VIa gene were directly compared in RAPD experiments with the epidemic strain ESP41 found in As a strain of A. baumannii carrying a class 1 integron Barcelona and other regions of Spain. The RAPD was reported to be widely disseminated in Barcelona pro®les obtained with M13 core primer (Fig. and other regions of Spain it was interesting indicated that isolates SM28 and SM29 were indis- to determine whether this strain had spread to the tinguishable from each other with this primer (although Bilbao region. The Barcelona strain encodes APH(39)- they were distinguished with primers AP3 and ERIC2), VI, a 39-O-phosphotransferase that inactivates amikacin Table 3. Aminoglycoside disk zone sizes and deduced aminoglycoside-modifying enzymes for representative isolates of A. baumannii carrying the four integron structures AMK, amikacin; APR, apramycin; EPI, episisomicin; FOR, fortimicin; GEN, gentamicin; ISP, isepamicin; KAN, kanamycin; NEO, neomycin; NET, netilmicin; 69-NET, 69-netilmicin; 29Net, 29-netilmicin; TOB, tobramycin; NZ, no zone.
ÃThe presence of the gene encoding APH(39)-VI was con®rmed by PCR. ySensitive type strain of A. baumannii.
Fig. 1. Comparative RAPD pro®les obtained with M13 primer for A. baumannii isolates carrying the aph(39)-VI gene.
Lanes: 1, isolate SM10; 2, SM16; 3, SM28; 4, SM29; 5, SM30; 6, SM36; 7, SM49; 8, SM80; 9, ESP41 (epidemic This gene was detected by PCR in eight of the 10 L.G. is indebted to the Basque Government and the British Council genotypes found in Bilbao that expressed resistance to for ®nancial support, and to M.J. Canduela and F. LoÂpez-Otsoa for amikacin. However, all these genotypes were clearly distinguished from the Barcelona strain. As in France it seems that aph(39)-VIa has spread to several different genotypes, including two predominant types in Bilbao, and that spread of a single resistant strain is 1. Bergogne-BeÂreÂzin E, Towner KJ. Acinetobacter spp. as not implicated, as in other areas in Spain Work in nosocomial pathogens: microbiological, clinical, and epidemio- logical features. Clin Microbiol Rev 1996; 9: 148±165.
France suggests that aph(39)-VIa may be transposable 2. Bergogne-BeÂreÂzin E. Resistance of Acinetobacter spp. to or associated with plasmids that are self- antimicrobials ± overview of clinical resistance patterns and transmissible among some Acinetobacter strains, or therapeutic problems. In: Bergogne-BeÂreÂzin E, Joly-Guillou ML, Towner KJ (eds) Acinetobacter ± microbiology, epidemi- both Preliminary experiments indicate that ology, infections, management. Boca Raton, CRC Press. 1996: many of the Bilbao isolates carry diverse collections of plasmids of various molecular sizes (unpublished 3. Wolff M. Nosocomial infections caused by Acinetobacter spp.
± therapeutic problems. In: Bergogne-BeÂreÂzin E, Joly-Guillou ML, Towner KJ (eds) Acinetobacter ± microbiology, epidemi- ology, infections, management. Boca Raton, CRC Press. 1996: It was concluded that although class 1 integrons appear 4. Towner KJ. Plasmid and transposon behaviour in Acinetobac- to be widely disseminated amongst clinical isolates of ter. In: Towner KJ, Bergogne-BeÂreÂzin E, Fewson CA (eds) The A. baumannii from the Bilbao region of Spain, these biology of Acinetobacter. Taxonomy, clinical importance, structures are not playing a major role in the molecular biology, physiology, industrial relevance. New York, dissemination of antibiotic resistance genes. Clinical 5. Young H-K, Gonzalez G, Zemelman R. Rearrangement of isolates of A. baumannii from diverse worldwide antibiotic resistance genes within a Tn7 integron structure in locations seem to share resistance mechanisms with clinical Acinetobacter isolates. Abstracts of the 7th European Congress of Clinical Microbiology and Infectious Diseases other genera, although many resistance plasmids from other genera are unstable in A. baumannii. Thus, 6. Vila J, Navia M, Ruiz J, Casals C. Cloning and nucleotide despite their limited role at present, the presence of sequence analysis of a gene encoding an OXA-derived â- lactamase in Acinetobacter baumannii. Antimicrob Agents integrons in 84% of the clinical isolates examined in Bilbao is a cause for concern, as these structures may 7. Gonzalez G, Sossa K, Bello H, Dominguez M, Mella S, enhance the ability of these organisms to respond Zemelman R. Presence of integrons in isolates of different biotypes of Acinetobacter baumannii from Chilean hospitals.
rapidly to the challenge of new antibiotics, enabling FEMS Microbiol Lett 1998; 161: 125±128.
otherwise unstable resistance genes to be incorporated 8. Seward RJ, Towner KJ. Detection of integrons in worldwide easily into the bacterial chromosome or into indigenous nosocomial isolates of Acinetobacter spp. Clin Microbiol Infect Acinetobacter plasmids. Isolates of A. baumannii from 9. Stokes HW, Hall RM. A novel family of potentially mobile some centres are already extremely dif®cult to combat DNA elements encoding site-speci®c gene-integration func- therapeutically and the widespread occurrence of tions: integrons. Mol Microbiol 1989; 3: 1669±1683.
10. Hall RM, Recchia GD, Collis CM, Brown H, Stokes HW. Gene integrons in this species can only add to the problem.
cassettes and integrons: moving antibiotic resistance genes in CLASS 1 INTEGRONS AND ANTIBIOTIC RESISTANCE IN A. BAUMANNII Gram-negative bacteria. In: AmaÂbile-Cuevas CF (ed) Antibiotic Acinetobacter isolates from two contrasting hospitals in the resistance: from molecular basics to therapeutic options.
United Kingdom and South Africa. Eur J Clin Microbiol Infect 11. Hall RM, Brookes DE, Stokes HW. Site-speci®c insertion of 20. Seward RJ, Lambert T, Towner KJ. Molecular epidemiology of genes into integrons: role of the 59-base element and aminoglycoside resistance in Acinetobacter spp. J Med determination of the recombination cross-over point. Mol 21. Grundmann HJ, Towner KJ, Dijkshoorn L et al. Multicenter 12. Collis CM, Hall RM. Gene cassettes from the insert region of study using standardized protocols and reagents for evaluation integrons are excised as covalently closed circles. Mol of reproducibility of PCR-based ®ngerprinting of Acinetobacter spp. J Clin Microbiol 1997; 35: 3071±3077.
13. Hall RM. Mobile gene cassettes and integrons: moving 22. Webster CA, Towner KJ, Humphreys H, Ehrenstein B, Hartung antibiotic resistance genes in Gram-negative bacteria. In: D, Grundmann H. Comparison of rapid automated laser Chadwick DJ, Goode J (eds) Antibiotic resistance: origins, ¯uorescence analysis of DNA ®ngerprints with four other evolution, selection and spread (Ciba Foundation Symposium computer-assisted approaches for studying relationships be- 207). Chichester, John Wiley & Sons. 1997: 192±202.
tween Acinetobacter baumannii isolates. J Med Microbiol 14. Sallen B, Rajoharison A, Desvarenne S, Mabilat C. Molecular epidemiology of integron-associated antibiotic resistance genes 23. Recchia GD, Hall RM. Gene cassettes: a new class of mobile in clinical isolates of Enterobacteriaceae. Microb Drug Resist element. Microbiology 1995; 141: 3015±3027.
24. Recchia GD, Hall RM. Plasmid evolution by acquisition of 15. Martinez-Freijo P, Fluit AC, Schmitz F-J, Grek VSC, Verhoef mobile gene cassettes: plasmid IE723 contains the aadB gene J, Jones ME. Class I integrons in Gram-negative isolates from cassette precisely inserted at a secondary site in the IncQ different European hospitals and association with decreased plasmid RSF1010. Mol Microbiol 1995; 15: 179±187.
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