a Unit of Antibiotic Research, Pasteur Institute Brussels, Engelandstraat 642, B-1180, Brussels, Belgium
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Abstract |
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Introduction |
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The aim of this study was to evaluate the prevalence of aminoglycoside resistance in Gram-negative bacterial isolates from patients with septicaemia. Furthermore, we wanted to evaluate possible geographic differences. For this purpose recently isolated strains were obtained from various hospitals located in different parts of Belgium and the Grand Duchy of Luxembourg. The level of resistance to aminoglycosides was determined and the presence of resistance mechanisms in isolates with resistance or intermediate sensitivity was evaluated.
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Materials and methods |
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A total of 1102 consecutive clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa and other non-fermenters were prospectively collected in 13 hospitals from blood cultures over a 6 month period from October 1996 until March 1997. Multiple isolates from the same patient were omitted from the study and no attempt was made to differentiate between nosocomial and community-acquired isolates. The isolates were routinely identified by conventional methods by the participating laboratories and suspensions were stored at 70°C. When required the suspensions were thawed and sub-cultured on to MuellerHinton agar (Merck Belgium, Overijse, Belgium).
Participating hospitals
A total of eight university hospitals and five university-affiliated hospitals participated in the study. These hospitals were divided into a Northern and a Southern group. Based on the predominant linguistic register, two hospitals located in Brussels were allocated to the Southern group, which included the following centres: Cliniques Universitaires Saint-Luc, UCL, Brussels; Hôpital ErasmeULB, Brussels; Hôpital de Jolimont, Haine St Paul; C. H. R. de la Citadelle, Liège; C. H. U. Sart Tilman, Liège; C. H. Luxembourg, Luxembourg; C. H. U. Vésale, Montigny-le-Tilleul and Cliniques Universitaires de Mont-Godinne, UCL, Yvoir. The Northern group comprised the following centres: Universitair Ziekenhuis Antwerpen, Edegem; St Jan ziekenhuis, Brugge; Akademisch ZiekenhuisVUB, Brussels; Universitair Ziekenhuis Ghent, Ghent and St Raphaël ZiekenhuisKUL, Leuven.
Antimicrobial agents
The following aminoglycosides are included in the study: amikacin (A) (Bristol-Myers Squibb, Princeton, NJ, USA), gentamicin (G), isepamicin (I) and netilmicin (N) (Schering Plough, Kenilworth, NJ, USA) and tobramycin (T) (Eli Lilly, Indianapolis, IN, USA).
Sensitivity testing
MICs of the antibiotics were determined for all isolates according to NCCLS procedures13 by microdilution using a Dynatech Laboratories Quick Spense dispenser and a Cooke Dynatech MIC2000 inoculator (Alexandria, VA, USA) with a final inoculum of approximately 1.5 x 105 cfu/mL.
Reference strains Escherichia coli ATCC 25922 and P. aeruginosaATCC
27853 were included as controls. Based on NCCLS interpretive criteria14 the isolates were categorized as aminoglycoside non-susceptible (ANS
= Intermediate and resistant; MIC: gentamicin and tobramycin 8 mg/L, netilmicin
16 mg/L, amikacin and isepamicin
32 mg/L) or aminoglycoside resistant (AR, MIC:
gentamicin and tobramycin
16 mg/L, netilmicin
32 mg/L, amikacin and isepamicin
64 mg/L). Evaluation of the results was done with the Chi-squared test with Yates
correction if necessary.
Detection of resistance genes
The genes encoding the aminoglycoside modifying enzymes were detected using PCR. The techniques used for DNA isolation and the PCR were described previously.15,16 Specific sequences of sense and antisense primers were chosen within the nucleotide sequence of the published regions of the various genes. Sets of primers for the following genes were included in the PCR tests: aac(6')-Ia, aac(6')-Ib, aac(6')-Ic, aac(6')-Id, aac(6')-If, aac(6')-Ig, aac(6')-Ih, aac(6')-Ij, aac(6')-Ik, aac(6')-Il, aac(6')-Im, aac(6')-IIa, aac(6')-IIb, aac(3)-Ia, aac(3)-IIc,aac(3)-IIIa,aac(3)-IVa,aac(3)-VIa, aac(2')-Ia, ant(2'')-Ia, ant(4' ,4'')-IIa, aph(3')-VIa. Genotypes were determined by PCR for isolates which appeared to be ANS by NCCLS interpretive criteria. Permeability resistance was deduced from the phenotype obtained with a set of 12 selected aminoglycosides for isolates which were found to lack any of the above mentioned resistance genes or isolates in which the presence of genes did not explain the resistance profile. An isolate resistant to all these compounds was considered as resistant by permeability. The following compounds were used: amikacin, apramycin (Eli Lilly), gentamicin, isepamicin, kanamycin (Bristol-Myers Squibb), lividomycin, neomycin (Sigma Chemicals, St Louis, MO, USA), netilmicin, paromomycin (Parke-Davis, Ann Arbor, Mi, USA), spectinomycin (Upjohn, Kalamazoo, MI, USA), streptomycin (Duchefa, Haarlem, The Netherlands), tobramycin.
The bacterial population
A total of 1102 isolates (81.4% Enterobacteriaceae, 18.6% non-fermenting bacilli) were included. In the Southern hospitals (632 isolates), 83.1% were Enterobacteriaceae and 16.9% were non-fermenters. The Northern population (470 isolates) was composed of 79.1% Enterobacteriaceae and 20.9% non-fermenters. The most frequently isolated species were E. coli (44.9%), P. aeruginosa (12.5%), Klebsiella spp. (11.5%) and Enterobacter spp. (8.8%). Enterobacter aerogenes was significantly more likely to be isolated in the Southern hospitals (0.05 > P> 0.02) while Klebsiella pneumoniae was more frequently found in the Northern hospitals (0.01 > P> 0.001).
Aminoglycoside resistance levels and patterns
The mean levels of resistance and susceptibility for the various aminoglycosides are summarized in Table I. The only significant difference between North and South concerned amikacin and isepamicin (0.01 > P> 0.001). In total, 157 isolates (14.2%) were considered as ANS, comprising 122 isolates (11.1%) with aminoglycoside resistance (AR) and 35 (3.1%) with intermediate susceptibility. The most frequently encountered resistance patterns were TN (20.4%), GTN (19.1%), GTNAI (12.7%), G (10.2%) and TNA (9.6%). The distributions were very comparable between the Southern and Northern isolates.
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Table II summarizes the presence of the aminoglycoside resistance mechanisms in the various bacterial species. Overall, 179 aminoglycoside resistance mechanisms (i.e. 150 genes encoding aminoglycoside modifying enzymes and 29 permeability mechanisms) were found in a total of 148 ANS isolates. The aac(6') genes were the most frequently found genes (8.3% of the total population), followed by the aac(3) genes (3.4% ) and the permeability resistance mechanism (2.6%). Furthermore, the isolated aac(6'')-Ib gene represented the most frequently encountered resistance mechanism (4.4% of the total population). Impermeability, as a single mechanism, was detected in 2.5% of the total population and was found significantly more frequently in Northern than in Southern isolates (3.8% versus 1.4%; 0.02 > P> 0.01). The third most important resistance mechanism was the isolated aac(3)-IIc gene which was found in 1.0% of the total population. A total of 29 ANS isolates (2.6% of the total population) harboured a combination of two genes while two isolates had a triple combination. There were only minor differences in the prevalence of certain resistance genes between the Southern and the Northern regions. Theaph(3')-VI gene was more prevalent in the Northern isolates (0.02 > P> 0.01) while the ant(2'') gene, 57.1% of which were found in P. aeruginosa, was present significantly more frequently in the Southern isolates (0.02 > P> 0.01).
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Discussion |
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The great variability in resistance rates between the different hospitals and regions may be due to clusters of isolates. This is demonstrated by the isepamicin resistance level in the Northern hospitals in which the overall isepamicin resistance rate of 2.3% is almost exclusively attributed to two centres. The isepamicin resistance rate of 3.9% in the first centre was exclusively due to permeability resistance in four E. coli isolates and three non-fermenters. The second hospital had an isepamicin resistance rate of 7.5% caused by the presence of four Acinetobacter baumannii isolates with the aac(3)IIc + aph(3')-VI gene combination, one non-fermenter showing impermeability and one E. aerogenes with an unknown mechanism.
The detection of aminoglycoside resistance genes is a very useful tool in an epidemiological setting. Genes could not be detected in nine isolates while in a further 14 isolates the detected genes did not fully explain the resistance phenotype. These negative results can plausibly be explained by the presence of genes for which we did not include primers or genes that are as yet unknown. In this study, the aac(6')-I genes were found most frequently. These genes were not more prevalent in the Southern hospitals despite the apparently higher percentage of amikacin prescription (figures from Information Medical Statistics) and the subsequent possible higher selective pressure. Interestingly, the lowest rate of resistance to amikacin was found in the region with the highest percentage of prescription. However, this does not imply necessarily a causal relationship since other factors such as species distribution, sampling size, type of hospital and use of other classes of antibiotics can also have an influence. Miller et al.4 reported AAC(6')-I as a single aminoglycoside resistance mechanism to be present in 20.3% of the Enterobacteriaceae (versus 36.0% in our study) while combinations with AAC(6') were present in more than 18.4% of the Enterobacteriaceae and only 8.6% of the P. aeruginosa isolates. Moreover, from their data, it is clear that, both in the 1987 and the 19881993 surveillances, the AAC(3)s were the most frequently detected mechanisms in Enterobacteriaceae (41.9% and 49.8%, respectively). This finding was in general agreement with the results published by Shaw et al.3 A European collaborative study2 performed in the mid-eighties, reported ANT(2''), AAC(3) and AAC(6')-I as the predominant mechanisms. In our study, the AAC(3)s formed the second most important resistance mechanism while ANT(2'') was found only at a very low incidence.
Isepamicin was included in the study because it has been launched recently in Belgium. It is expected to be modified by the APH(3')-VI and ANT(4')-II enzyme from Gram-negative isolates and to be refractory to the activity of most AAC(6')-I enzymes.4,18 Mostly, isepamicin resistance was due to the presence of the aph(3')-VI gene or to impermeability. One E. cloacae isolate, intermediately resistant to isepamicin, harboured only the aac(6')-Ib gene while an isepamicin-resistant Serratia marcescens had the aac(6')-Ib + aac(6')-Ic + aac(6')-Im gene combination. The presence of the aac(6')-Ibgene may partly explain the resistance. Indeed, Caulin et al.19 concluded from an experimental endocarditis model with an aac(6')-Ib bearing K. pneumoniae that the AAC(6')-Ib enzyme, irrespective of the level of enzyme production, compromised the efficacy of both amikacin and isepamicin. This finding was confirmed by another experimental endocarditis study.20 Isepamicin resistance can also be due to the presence of an unknown resistance mechanism. Recently an AAC(6')-III enzyme reducing the activity of both amikacin and isepamicin has been detected and seems to be associated with the aac(6'>)-Il, aac(6')-Im and aac(6')-In genes,21 one of which was present in our S. marcescensisolate.
In general it can be said that the aminoglycosides remain useful drugs despite their intensive use. Antibiotic usage certainly is an incriminating factor in the development of resistance but other factors such as hospital hygiene, hospital organization, type of hospital, introduction and subsequent spread of bacterial strains or resistance determinants should also be taken into account. The control of antibiotic consumption is an important element in combating multidrug resistance while the significance of infection control programmes has also been documented.22 Eventually, rules governing antibiotic policies should not be based solely on international or national recommendations but should take into account local parameters.
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Notes |
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Participants. M. Carpentier (Liège), M. Delmee (brussels),
P. De Mol (Liège), M.-G. Garrino (mont Godinne), H. Gossens (antwerp), Y.
glupczynski (montigny-le-Tilleul), R. Hemmer (Luxembourg), S. Lauwers (brussels), F. Meunier
(Jolimont), M. Struelens (Brussels), H. Van Landuyt (Brugge), L. Verbist (Leuven) and G.
Verscyhraegen (Ghent).
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Received 21 January 1999; returned 19 March 1999; revised 28 April 1999; accepted 10 May 1999