Comparison of aminoglycoside resistance mechanisms in Pseudomonas aeruginosa isolates from Slovakia and Greece

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1 Biologia, Bratislava, 56/6: , 2001 Comparison of aminoglycoside resistance mechanisms in Pseudomonas aeruginosa isolates from Slovakia and Greece Andrea Žáková 1, Emília Talábová 1, Helen Giamarellou 2, Tatiana Mačičková 3, Dana Michálková-Papajová 1,MáriaBagová 4, Silvia Benczeová 1,ArpádJakab 5 & Milan Kettner 1 * 1 Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B-2, SK Bratislava, Slovakia; tel.: , fax: , kettner@fns.uniba.sk 2 4 th Department of Internal Medicine, Athens School of Medicine, GR Athens, Greece 3 Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, SK Bratislava, Slovakia 4 State Public Health Institute of the Slovak Republic, Trnavská 52, SK Bratislava, Slovakia 5 Bristol-Myers Squibb Slovakia, Kapucínska 5, SK Bratislava, Slovakia * Corresponding author áková, A., Talábová, E., Giamarellou, H., Maèièková, T., Michálková-Papajová, D., Bagová, M., Benczeová, S., Jakab, A. & Kettner, M., Comparison of aminoglycoside resistance mechanisms in Pseudomonas aeruginosa isolates from Slovakia and Greece. Biologia, Bratislava, 56: , 2001; ISSN (Biologia). ISSN (Biologia. Section Cellular and Molecular Biology). The most common mechanism of resistance to aminoglycoside antibiotics is the production of aminoglycoside-modifying enzymes (AGME). In our study we wanted to compare production of AGME, transferability of resistance and the occurrence of plasmid DNA in two series of Pseudomonas aeruginosa isolates, originating from hospitals in Athens (Greece) and Bratislava (Slovakia). Twenty-two P. aeruginosa isolates from Athens were nearly all highly resistant to gentamicin (GEN), tobramycin (TOB), netilmicin (NET), amikacin (AMI), and isepamicin (ISE). About 50% of isolates produced AAC(6 )-III enzyme causing resistance to TOB, NET, AMI; 18% of isolates produced APH(3 )-VI enzyme causing resistance to ISE, and 14% produced APH(2 ) enzyme, causing resistance to GEN and TOB. In 22 Slovak isolates presence of AAC(6 )-III was observed in 68%, APH(2 ) in 64% and APH(3 )-VI in 55% of isolates. In both series aminoglycoside resistance was transferable by bacterial conjugation with exception of ISE. In Greek donors and s the plasmid DNA in the range of MDal was observed, whereas the plasmid DNA from Slovak isolates ranged from 55 to 75 MDal. Interesting is the presence of ISE resistance in Slovak isolates as ISE has not been introduced into the therapy in Slovakia yet. Key words: aminoglycoside antibiotics, Pseudomonas aeruginosa, resistance mechanisms, R-plasmid. 617

2 Introduction Pseudomonas aeruginosa canbeconsideredasone of the most important bacterial causes of hospital acquired infections (Spencer, 1996). Nosocomial strains, in sharp contrast to communityacquired strains, exhibit high rates of resistance to antibiotics and are frequently multi-drug resistant (Archibald et al., 1997; Paraskaki et al., 1996), a fact probably related to the ease with which they can develop resistance in a hospital environment (Manian et al., 1996). Among nosocomial bacterial infections those caused by P. aeruginosa are associated with the highest mortality rate (Korvick & Yu, 1991; Bert & Lambert-Zechovsky, 1999). P. aeruginosa is difficult to eradicate from infected tissues and blood. Another problem is the increasing resistance against antimicrobial agents. Aminoglycoside antibiotics, especially tobramycin and amikacin, are suitable antimicrobials against drug-resistant P. aeruginosa. The most frequently occurring aminoglycoside resistance mechanisms in P. aeruginosa are the production of modifying enzymes and reduced permeability (Miller et al., 1997). In our study we wanted to compare production of aminoglycoside-modifying enzymes (AGME), transferability of resistance by bacterial conjugation, and the occurrence of plasmid DNA in two series of P. aeruginosa isolates originating from hospitals in Athens and Bratislava. Many previous studies have shown certain similarities in the occurrence of aminoglycoside resistance mechanisms in the two countries mentioned (Dornbusch et al., 1990; Tsakris et al., 1992; Miller et al., 1995; Reshedko et al., 1997). The presented study is a contribution to the comparison of actual situation in aminoglycoside resistance mechanisms. Material and methods Bacterial strains Forty-four clinical isolates of P. aeruginosa (22 from hospitalized patients in Athens, Greece and 22 from hospitalized patients in Bratislava, Slovakia), resistant to several aminoglycoside antibiotics were studied. Only one isolate per patient was included in this study. Strains were preserved in Skim Milk (Merck) and kept in a freezer at C. When required, the suspensions were thawed and inoculated on McConkey agar (Merck). Antibiotic susceptibility testing Susceptibility to aminoglycoside antibiotics was performed using the agar dilution method according to NCCLS recommendations (NCCLS, 1990) on Mueller- Hinton agar (Merck) containing twofold dilutions of antibiotic solutions ranging in concentration from 64 to 0.5 mg/l. For evaluation of aminoglycoside resistance the following breakpoints were used: for GEN and TOB: MIC > 4 mg/l, for NET: MIC > 8 mg/l and for AMI and ISE: MIC > 16 mg/l. Antibiotics and chemicals The following aminoglycosides were included in this study: GEN, NET, ISE (Schering Plough, USA), TOB (Eli Lilly, USA) and AMI (Bristol-Myers-Squibb, USA). Radiolabelled chemicals were bought from Radiochemical Centre (Amersham, UK). Aminoglycoside resistance mechanisms The presence of AGME was assayed in cell-free preparations of P. aeruginosa isolates obtained by ultrasonic disruption. Enzymatic activities were measured as described previously (Kettner et al., 1981). Classification of enzymes was carried out according to the scheme by Shaw et al. (1993) (Tab. 1). Table 1. Substrate profiles of aminoglycoside-modifying enzymes. AGME Substrate AAC(3)-I GEN AAC(3)-II GEN TOB NET AAC(6 )-I TOB NET AMI AAC(6 )-II GEN TOB NET AAC(6 )-III TOB NET AMI ISE APH(2 ) GEN TOB APH(3 )-VI AMI ISE Transferability of aminoglycoside resistance Bacterial conjugation was performed using recipient P. aeruginosa 1008 rif r (obtained from S. Mitsuhashi, Japan) as described previously (Kettner et al., 1995). Transfer frequency was expressed as the number of CFU/mL divided by the number of input donor CFU/mL. Isolation of plasmid DNA Isolation of plasmid DNA from donor strains and P. aeruginosa 1008 s was prepared using the method of Takahashi & Nagano (1984). Plasmid DNA was studied by agarose gel electrophoresis with plasmid DNA standards. Results Susceptibility of 22 Greek isolates to five aminoglycoside antibiotics and production of different AGME is presented in Table 2. It is evident that all P. aeruginosa isolates from Athens were in vitro uniformly highly resistant to GEN, TOB, 618

3 Table 2. Susceptibility of Pseudomonas aeruginosa isolates originating from Greece to aminoglycoside antibiotics and production of aminoglycoside-modifying enzymes (AGME). No Microorganism Acetyltransferases Phosphotransferases 1 Pseudomonas aeruginosa 1G >64 >64 >64 >64 >64 AAC(3)-II 2 Pseudomonas aeruginosa 2G >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 3 Pseudomonas aeruginosa 3G >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 4 Pseudomonas aeruginosa 4G >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 5 Pseudomonas aeruginosa 5G >64 >64 >64 64 >64 AAC(3)-II AAC(6 )-III APH(2 ) 6 Pseudomonas aeruginosa 6G >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III 7 Pseudomonas aeruginosa 7G >64 > >64 AAC(3)-II AAC(6 )-III 8 Pseudomonas aeruginosa 8G >64 >64 >64 >64 >64 AAC(6 )-II 9 Pseudomonas aeruginosa 9G >64 >64 > Pseudomonas aeruginosa 10G 64 >64 >64 >64 >64 AAC(6 )-III 11 Pseudomonas aeruginosa 11G 64 >64 >64 > Pseudomonas aeruginosa 12G 64 >64 >64 >64 64 AAC(6 )-II 13 Pseudomonas aeruginosa 13G >64 >64 >64 >64 >64 AAC(3)-II 14 Pseudomonas aeruginosa 14G 64 >64 >64 >64 64 AAC(6 )-III 15 Pseudomonas aeruginosa 15G >64 >64 > AAC(3)-I AAC(6 )-III APH(2 ) APH(3 )-VI 16 Pseudomonas aeruginosa 16G 64 >64 >64 >64 >64 AAC(3)-I AAC(6 )-III 17 Pseudomonas aeruginosa 17G >64 >64 >64 >64 >64 AAC(3)-II APH(3 )-VI 18 Pseudomonas aeruginosa 18G 64 >64 >64 >64 >64 19 Pseudomonas aeruginosa 19G 64 >64 >64 >64 >64 AAC(6 )-III 20 Pseudomonas aeruginosa 20G 64 >64 >64 >64 64 AAC(6 )-I 21 Pseudomonas aeruginosa 21G 32 >64 64 >64 >64 AAC(3)-II 22 Pseudomonas aeruginosa 22G 32 >64 >64 >64 64 Resistance (% ) Table 3. Susceptibility of Pseudomonas aeruginosa isolates originating from Slovakia to aminoglycoside antibiotics and production of aminoglycoside-modifying enzymes (AGME). No Microorganism Acetyltransferases Phosphotransferases 1 Pseudomonas aeruginosa 1S >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 2 Pseudomonas aeruginosa 2S >64 64 > AAC(6 )-III APH(2 ) 3 Pseudomonas aeruginosa 3S >64 >64 >64 32 >64 APH(2 ) APH(3 )-VI 4 Pseudomonas aeruginosa 4S 64 >64 >64 >64 >64 AAC(6 )-III 5 Pseudomonas aeruginosa 5S >64 32 >64 32 >64 AAC(6 )-III APH(2 ) APH(3 )-VI 6 Pseudomonas aeruginosa 6S >64 >64 >64 > Pseudomonas aeruginosa 7S >64 >64 >64 >64 >64 AAC(6 )-III APH(2 ) APH(3 )-VI 8 Pseudomonas aeruginosa 8S >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III 9 Pseudomonas aeruginosa 9S 64 >64 >64 >64 >64 AAC(6 )-III APH(2 ) APH(3 )-VI 10 Pseudomonas aeruginosa 10S >64 >64 >64 >64 >64 AAC(6 )-III 11 Pseudomonas aeruginosa 11S >64 >64 > AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 12 Pseudomonas aeruginosa 12S AAC(3)-II AAC(6 )-III 13 Pseudomonas aeruginosa 13S >64 >64 >64 >64 >64 14 Pseudomonas aeruginosa 14S >64 >64 >64 64 >64 AAC(6 )-III APH(2 ) APH(3 )-VI 15 Pseudomonas aeruginosa 15S >64 >64 >64 >64 >64 AAC(6 )-III 16 Pseudomonas aeruginosa 16S AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 17 Pseudomonas aeruginosa 17S APH(2 ) APH(3 )-VI 18 Pseudomonas aeruginosa 18S APH(2 ) APH(3 )-VI 19 Pseudomonas aeruginosa 19S >64 >64 > AAC(3)-II AAC(6 )-III APH(2 ) 20 Pseudomonas aeruginosa 20S AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 21 Pseudomonas aeruginosa 21S >64 >64 >64 >64 >64 AAC(6 )-III 22 Pseudomonas aeruginosa 22S >64 >64 >64 >64 >64 AAC(6 )-III APH(2 ) APH(3 )-VI Resistance (%)

4 Table 4. Transferability of aminoglycoside resistance from Pseudomonas aeruginosa donors from Greece and Slovakia to Pseudomonas aeruginosa 1008 recipient. GREECE Frequency Microorganism Resistance determinants Transfered determinants Selection on of transfer P. aeruginosa 2G GEN TOB NET GEN P. aeruginosa 3G GEN TOB NET GEN P. aeruginosa 4G GEN TOB NET AMI GEN P. aeruginosa 5G GEN TOB NET GEN SLOVAKIA Frequency Microorganism Resistance determinants Transfered determinants Selection on of transfer P. aeruginosa 11S GEN TOB NET ISE GEN TOB NET GEN P. aeruginosa 14S GEN TOB NET GEN P. aeruginosa 15S GEN TOB NET GEN P. aeruginosa 19S GEN TOB NET ISE GEN TOB NET GEN P. aeruginosa 20S GEN TOB NET ISE GEN TOB NET GEN Table 5. Susceptibility of Pseudomonas aeruginosa s originating from Pseudomonas aeruginosa donors from Greece and Slovakia to aminoglycoside antibiotics and production of aminoglycoside-modifying enzymes (AGME). GREECE Microorganism Acetyltransferases Phosphotransferases Pseudomonas aeruginosa 2G >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI P. aeruginosa 2GT1 32 > AAC(6 )-III APH(2 ) APH(3 )-VI Pseudomonas aeruginosa 4G >64 >64 >64 >64 >64 AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI P. aeruginosa 4GT AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI P. aeruginosa 4GT AAC(6 )-III APH(2 ) SLOVAKIA Microorganism Acetyltransferases Phosphotransferases Pseudomonas aeruginosa 14S >64 >64 >64 64 >64 AAC(6 )-III APH(2 ) APH(3 )-VI P. aeruginosa 14ST AAC(6 )-III APH(2 ) APH(3 )- VI Pseudomonas aeruginosa 20S AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI P. aeruginosa 20ST AAC(3)-II AAC(6 )-III APH(2 ) APH(3 )-VI 620

5 Table 6. Molecular weight of plasmid DNA isolated from Greek and Slovak Pseudomonas aeruginosa donors and their s. GREECE Microorganism Molecular weight (MDal) Pseudomonas aeruginosa 2G 65 Pseudomonas aeruginosa 2GT1 65 Pseudomonas aeruginosa 3G 64 Pseudomonas aeruginosa 3GT1 64 Pseudomonas aeruginosa 4G 70 Pseudomonas aeruginosa 4GT1 70 Pseudomonas aeruginosa 4GT2 67 Pseudomonas aeruginosa 5G 60 Pseudomonas aeruginosa 5GT1 58 Pseudomonas aeruginosa 5GT2 60 SLOVAKIA Microorganism Molecular weight (MDal) Pseudomonas aeruginosa 11S 50 Pseudomonas aeruginosa 11ST1 50 Pseudomonas aeruginosa 14S 65 Pseudomonas aeruginosa 14ST1 65 Pseudomonas aeruginosa 15S 60 Pseudomonas aeruginosa 15ST1 60 Pseudomonas aeruginosa 19S 60 Pseudomonas aeruginosa 19ST1 60 Pseudomonas aeruginosa 20S 48 Pseudomonas aeruginosa 20ST1 45 NET and AMI and nearly all to ISE, too. Majority of Greek isolates produced two acetyltransferases and some of them (4 isolates) produced even two phosphotransferases. Three isolates produced 4 different enzymes inactivating all 5 aminoglycosides. Susceptibility of 22 Slovak isolates and their production of AGME is presented in Table 3. Eighteen percent of Slovak isolates were susceptible to AMI, but the majority of them was highly resistant to all aminoglycosides tested. Transferability of aminoglycoside resistance by bacterial conjugation in selected isolates and the frequency of transfer are shown in Table 4. Susceptibility of s to aminoglycosides and the transferred AGME in selected Greek and Slovak isolates are shown in Table 5. Molecular weight of plasmid DNA from Slovak and Greek donors and their s is presented in Table 6. Comparison of the occurrence of produced AGME in P. aeruginosa isolates from Athens and Bratislava is shown in Figure 1. Discussion Pseudomonas aeruginosa is recognized as a major opportunistic pathogen in the hospital environment. Its role in hospitalized patients has markedly increased in the past decade (Vanhoof et al., 1993; Kresken et al., 1996; Cercenado- Mansilla & García-Garrote, 1998; Bert & Lambert-Zechovsky, 1999). It is possible, that indiscriminate use of antibiotics can encourage the spread of certain multiresistant phenotypes (Kettner et al., 1995; Schmitz et al., 1999). The com- 621

6 $*0( $$&, $$&,, *5((&( 6/29$.,$ $$&, (Q]\PHV $$&,,, $3+ $3+9, Fig. 1. Occurrence of aminoglycoside-modifying enzymes (AGME) in clinical isolates of Pseudomonas aeruginosa from Greece and Slovakia. mon mechanism of resistance to the aminoglycosides in Gram-negative bacilli is the production of AGME, which are capable of acetylating, adenylating or phosphorylating the target of aminoglycosides (Miller et al., 1995; Miller et al., 1997). The major mechanism of resistance to aminoglycosides formed mainly in P. aeruginosa are enzymatic modification of the drug and a penetration barrier or impermeability (AMINOGLYCO- SIDE RESISTANCE STUDY GROUPS, 1994). Several previous studies examined the occurrence of aminoglycoside resistance mechanisms in Pseudomonas spp. from different geographical regions (Dornbusch et al., 1990; Shaw et al., 1993; Miller et al., 1995; Reshedko et al., 1997). In these studies the overall incidence of combinations of aminoglycoside resistance mechanisms was much higher than had been observed previously and was even higher in individual geographical regions. Combinations of aminoglycoside resistance mechanisms were particularly common in isolates from Greece and Turkey, but were less common in northern Europe (Benelux, Germany and Scandinavia). In our previous studies we observed high complexity of AGME in clinical isolates of Gramnegative bacilli in Slovakia (Kallová et al., 1995; Kallová et al., 1997; Reshedko et al., 1997). The aim of this study was to compare the mechanisms of aminoglycoside resistance in P. aeruginosa isolates from hospitals in Slovakia and Greece, as on the basis of previous studies we expected certain similarities based on the similar aminoglycoside usage pattern in both countries. In the last decade, the usage of AMI in Slovakia has increased by more than 300%. This may also have contributed to higher aminoglycoside resistance rates in Slovakia when compared with West European countries (Germany, Switzerland, Benelux), where the spectrum of therapeutically used antimicrobials is much wider (Milo¹oviè et al., 1994). When comparing the production of AGME in Greek and Slovak isolates (Fig. 1), a higher occurrence rate of enzymes inactivating AMI and ISE in Slovakia is evident. This might be due to the mentioned increased use of AMI in Slovakia. ISE has not been introduced into therapy in Slovakia till now, but as AMI is also a substrate for the enzyme inactivating ISE [APH(3 )-VI], selection pressure of AMI may have contributed to the dissemination of ISE inactivating enzyme. Reshedko et al. (1997), using 22 aminoglycoside resistance gene probes, studied aminoglycoside resistance mechanisms in a series of Gram-negative bacilli from Slovakia. Sixteen of 19 P. aeruginosa isolates (84%) produced AAC(6 )-III enzyme inactivating GEN, TOB, NET, AMI, ISE. In our study 75% of Slovak isolates and 50% of Greek isolates produced this enzyme. Our results document the prevailing occurrence of P. aeruginosa isolates with multiple mechanisms of resistance in both series of strains originating from Greece and Slovakia. This suggests that the genes responsible for such resistance mechanisms have a greater than usual tendency for recombination into existing plasmids that encode resistance to aminoglycosides, and on the other hand it may help to explain the incredible variety of mechanisms observed. Acknowledgements This research was financially supported by the grant VEGA No. 1/5128/98. References AMINOGLYCOSIDE RESISTANCE STUDY GROUPS Resistance to aminoglycosides in Pseudomonas. Trends Microbiol. 2:

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