ANTIMICROBIAL RESISTANCE OF SALMONELLA ISOLATED FROM ANIMALS AND FEED IN POLAND

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1 Bull Vet Inst Pulawy 48, 233-2, 4 ANTIMICROBIAL RESISTANCE OF SALMONELLA ISOLATED FROM ANIMALS AND FEED IN POLAND DARIUSZ WASYL AND ANDRZEJ HOSZOWSKI Department of Microbiology, National Veterinary Research Institute, 24-1 Puławy, Poland ahosz@piwet.pulawy.pl Received for publication April 6, 4. Abstract Five hundred and sixty Salmonella isolates belonging to 42 serovars were tested for antimicrobial resistance with ATBVET. The strains were collected from poultry, pigs, animal feed, and several other sources during two-year period (-1). The survey showed the recent trends in Salmonella antimicrobial resistance. A frequency of the isolation of resistant strains has been increasing. A high number of tested Salmonella strains were resistant to nitrofurantoin, doxycycline, streptomycin, tetracycline, flumequine, and oxolinic acid. Of the antimicrobials used only colistin was active against all tested isolates. Few strains revealed apramycin, gentamycin, kanamycin and ciprofloxacin resistance. A decrease in quinolone efficacy was observed. No Salmonella showing extended-spectrum β-lactamase activity was found. Differences were noted in antimicrobial resistance depending on Salmonella serovar and the source of isolation. Multiresistance was observed in the majority of S. Hadar and S. Gallinarum strains. However, S. Typhimurium revealed the most complex resistance patterns. Salmonellas originating from swine were more resistant than those from poultry and feed. It is concluded that Salmonella antibiotic resistance should be evaluated continuously because of serious human and animal health implications. Key words: Salmonella, antibiotic resistance, resistance patterns. Salmonella infections in animals have serious world-wide implications for public health. They result in the emergence of antimicrobial resistance and the risk of transfer to human population either resistant salmonellas or resistance genes into commensal flora or pathogens affecting man. To reduce such threats, Salmonella prevalence and resistance monitoring, surveillance, and control programs are introduced at national and international levels (1, 13, 14). There are two major objectives of antimicrobial resistance testing in bacteria. The first is to give a laboratory support for the therapy of infected humans and animals. The second is monitoring of the resistance and its trends for epidemiological purposes. Diffusion and dilution methods are used for antimicrobial resistance testing of microorganisms. ATBVET (biomerieux) is one of the commercial diagnostic kits that guarantees easy performance and interpretation of the results. The kit permits examining both Gram-negative and Gram-positive bacteria of animal origin against a wide range of antimicrobials in semisolid medium under conditions similar to those used in the reference agar dilution method. The results can be interpreted both qualitatively (resistant or sensitive) and quantitatively (MIC breakpoints). The general trends for resistance development in Salmonella are as follows: (i) the increase in number of resistant strains within isolates of animal origin, (ii) the occurrence of simultaneous resistance to a wide range of antimicrobials (multiresistance), and (iii) limited efficacy of some newly licensed antimicrobials representing, e.g. fluoroquinolones or β-lactams (5). The aims of the paper were to evaluate those trends in Salmonella strains isolated from animals and feed in Poland and to get a general overview on antibiotic resistance levels by Salmonella serovars and sources of isolation. Material and Methods The study included 56 Salmonella strains isolated in and 1. They were obtained from poultry, poultry farms and hatcheries (n = 391), swine (n = 63), animal feed (n = 72), and other sources (n = 34) such as food, exotic animals and sewage sludge. The following serovars were examined: S. Enteritidis (n = 287), S. Typhimurium (n = 54), S. Choleraesuis (n = 35), S. Agona (n = 28), S. Hadar (n = 23) and S. Gallinarum (n = 13). The remaining strains (n = 1) belonged to one of the remaining 36 serovars, some of them showed autoagglutination or unspecific antigenic properties. ATBVET (biomerieux) was applied to determine antibiotic resistance of the isolates. Twentyeight antimicrobials in single concentrations were tested: apramycin, gentamycin, kanamycin, spectinomycin,

2 234 streptomycin, amoxicillin/clavulanic acid, amoxicillin, cefoperazone, cefalotine, oxolinic acid, flumequine, enrofloxacin, chloramphenicol, sulfamethizol, cotrimoxazol, tetracycline, doxycycline, colistin, nitrofurantoin. Ciprofloxacin in two concentrations was used additionally to examine 25 strains. The test was performed according to the manufacturer s instructions and the results were read and interpreted with miniapi (biomerieux). Because of the natural Salmonella resistance to penicillin G, erythromycin, lincomycin, pristinamycin, oxacillin, tylosin, fusidic acid, rifampicin and metronidazole those results were not analysed. Independence test at.5 significance was used for statistical analysis of the results. Results The number and percentage of the resistant Salmonella strains by source of isolation are shown in Table 1. Very few strains were resistant to apramycin, gentamycin and kanamycin. Spectinomycin, streptomycin and amoxicillin resistance were found in the range of 7.1% to 28.8% and mostly in swine isolates (P.5; P.1 and P.1, respectively). Amoxicillin/clavulanic acid and cephalosporins resistance varied from 3.6% to 8.2% of tested strains. Resistance to all quinolones except for ciprofloxacin ranged from 15.7% to 22.1% and was seldom in feed isolates (1.4%; P.1). Only a few strains isolated from poultry and swine were resistant to ciprofloxacin. Chloramphenicol resistance was observed in 8.% of the tested strains mostly isolated from swine (27.%; P.1). Sulfamethizol showed lack of activity with respect to 16.8 in contrast to 3.% noted in the case of cotrimoxazol. Swine isolates were more often resistant to the two above mentioned drugs than those obtained from other sources (P.1 and P.5, respectively). The percentage of tetracycline resistant swine isolates was higher in comparison with salmonellas from the remaining sources (47.6% versus 23.4%, P.1). Doxycycline was the second less efficient agent with 45.5% of resistant isolates. None of the strains was resistant to colistin. Nitrofurantoin resistance (48.2%) resulted mostly from a high number of resistant poultry isolates (62.7%, P.1). Antibiotic resistance by the most prevalent Salmonella serovars and selected antimicrobials (at least 3% of resistant isolates) is presented in Fig. 1. S. Typhimurium and S. Hadar were more resistant to spectinomycin and streptomycin in comparison with the remaining serovars (P.1). No resistance to the above mentioned drugs were found in S. Agona and S. Gallinarum. Additionally, streptomycin resistance was rarely observed in S. Enteritidis strains (P.1). A similar resistance among the predominant serovars was noted in β-lactames, their combination with clavulanic acid and cephalosporins. The highest resistance was demonstrated in S. Typhimurium and S. Hadar, but no or only a limited number of resistant strains were noted in S. Agona, S. Gallinarum, and S. Enteritidis. The exceptions were a low resistance to cefalotine in S. Typhimurium and a high one to amoxycillin in S. Choleraesuis. Quinolone resistance was frequent in S. Hadar, S. Gallinarum (P.1), and S. Typhimurium (P.5). Resistance to chloramphenicol was mostly found in S. Typhimurium and S. Agona and never noted in S. Hadar and S. Gallinarum (P.1). A high level of sulfamethizol resistance (P.1) occurred in S. Typhimurium and S. Choleraesuis, but S. Enteritidis was mostly susceptible to the mentioned antimicrobial (P.1). Cotrimoxazole was less effective against S. Choleraesuis and other serovars (P.1). S. Hadar, S. Typhimurium and S. Agona showed a high resistance to tetracyclines and no resistance was found in S. Gallinarum. S. Enteritidis strains were also more susceptible to tetracycline than the isolates of the remaining serovars (9.%; P.1). Nitrofurantoin resistance was noted in 78.7% of S. Enteritidis strains (P.1). S. Typhimurium was more susceptible in comparison with S. Enteritidis but also demonstrated a high nitrofurantoin resistance (38.%; P.1). Only 12.9% of the isolates were susceptible to all applied antimicrobials (Table 2). The remaining strains showed resistance to few antimicrobials (37.3%) or were multiresistant (49.8%). Both S. Choleraesuis and S. Gallinarum strains were not susceptible to all tested antimicrobials. Moreover, S. Enteritidis, S. Typhimurium and S. Hadar strains were more frequently resistant than S. Agona and other strains (P.1). A high level of multiresistance was observed among the predominant serovars. Statistical differences in the frequency of multiresistance were noted (P.1). Common and the most complex of 96 resistance patterns (data not shown) found in the study are presented in Table 2. Discussion The purpose of the present report was to estimate the resistance of Salmonella strains isolated from different sources and representing different serovars in relation to possible wide range of antimicrobials. Some of the drugs have not been used in food producing animals in Poland and therefore the report should be considered as an epidemiological survey of the resistance rather than a support for therapeutic recommendation. Moreover, ATBVET defines the tested strains as resistant or susceptible and therefore isolates with MIC close to the breakpoint value may be misdiagnosed and classified as resistant. Nevertheless, the method is reproducible and comparable with reference testing techniques (data not shown). The ATBVET results provided evidence that the general trends towards the development of antimicrobial resistance seen among Salmonella strains isolated from animals and feed in Poland were in agreement with those observed in agar dilution method (7, 8).

3 235 Table 1 Antibiotic resistance by source of Salmonella isolation Antimicrobials (concentraction) No (%) of resistant strains poultry (n=391) swine (n=63) feed (n=72) other (n=34) total (n=56) apramycin (16 mg/l) 1 (.3) 1 (.3) gentamycin (4 mg/l) 6 (1.5) 6 (1.1) kanamycin (8 mg/l) 4 (1.) 2 (3.2) 1 (2.9) 7 (1.3) spectinomycin (64 mg/l )* 25 (6.4) 1 (15.9) 4 (5.6) 1 (2.9) (7.1) streptomycin (8 mg/l)*** 85 (21.7) 49 (77.8) 16 (22.2) 11 (32.4) 161 (28.8) amoxicillin (4 mg/l)*** 45 (11.5) 22 (34.9) 4 (5.6) 71 (12.7) amoxicillin/clavulanic acid (4/2 mg/l) 33 (8.4) 9 (14.3) 4 (5.6) 46 (8.2) cefoperazone (4 mg/l) 32 (8.2) 9 (14.3) 4 (5.6) 45 (8.) cefalotine (8 mg/l) 17 (4.3) 1 (1.6) 1 (1.4) 1 (2.9) (3.6) oxolinic acid (2 mg/l)*** 99 (25.3) 13 (.6) 1 (1.4) 1 (29.4) 123 (22.) flumequine (4 mg/l )*** 1 (25.6) 13 (.6) 1 (1.4) 1 (29.4) 124 (22.1) enrofloxacin (.5 mg/l)*** 69 (17.6) 12 (19.) 7 (.6) 88 (15.7) ciprofloxacin (1 mg/l and 2 mg/l) 2 (1.1) 1 (5.) 3 (1.2) chloramphenicol (8 mg/l )*** 22 (5.6) 17 (27.) 4 (5.6) 2 (5.9) 45 (8.) sulfamethizol (1 mg/l )*** 51 (13.) 24 (38.1) 11 (15.3) 8 (23.5) 94 (16.8) cotrimoxazol (2/38 mg/l)* 12 (3.1) 5 (7.9) 17 (3.) tetracycline (4 mg/l)*** 79 (.2) 3 (47.6) 16 (22.2) 6 (17.6) 131 (23.4) doxycycline (4 mg/l) 172 (44.) 35 (55.6) 37 (51.4) 11 (32.4) 255 (45.5) colistin (4 mg/l) nitrofurantoin (25 mg/l)*** 245 (62.7) 8 (12.7) 8 (11.1) 9 (26.5) 27 (48.2) statistical significance: * P.5; ** P.1; *** P.1. Table 2 Character of antimicrobial resistance by Salmonella serovars Salmonella serovar No (%) of strains Resistance pattern c (No of strains) susceptible resistant a multiresistant b commonest multiresistant Enteritidis 31 (1.8) 126 (44.1) 129 (45.1) N (15) GSRTDMXLPHN (1) Typhimurium 4 (7.4) 14 (25.9) 36 (66.7) CSRTDMXPOFHN (8) CGSRTDMXLPOEFHN (1) Choleraesuis 12 (34.3) 23 (65.7) R (12) CSRTDMXPOEFHN (1) Agona 6 (21.4) 9 (32.1) 13 (46.4) D (14) TDLH (1) Hadar 1 (4.3) 22 (95.7) RTDOEF (1) RTDMXLPOEFH (1) Gallinarum 2 (15.4) 11 (84.6) OEF (9) OEFN (1) others 3 (24.8) 46 (38.) 45 (37.2) different different total 72 (12.9) 9 (37.3) 279 (49.8) Resistant + Multiresistant 488 (83.1) a resistance to 1-3 antimicrobials from the same class, b resistance to at least 2 antimicrobials from different antibiotic class, c amoxicillin (M), amoxicillin/clavulanic acid (X), cefalotine (L), cefoperazone (P), streptomycin (R), sulfamethizol (H), spectinomycin (S), flumequine (F), oxolinic acid (O), gentamicin (G), enrofloxacin (E), nitrofurantoin (N), chloramphenicol (C), tetracycline (T), doxycycline (D) 235

4 spectinomycin (7.1%) SE Tm*** Chs Ag Ha*** Gal other streptomycin (28.8%) SE*** Tm*** Chs Ag Ha*** Gal other amoxicillin (12.7%) SE*** Tm*** Chs*** Ag Ha*** Gal other amoxicillin/clavulanic acid (8.2%) SE*** Tm*** Chs Ag Ha*** Gal other cefoperazone (8.%) SE*** Tm*** Chs Ag Ha** Gal other cefalotine (3.6%) SE Tm Chs Ag Ha*** Gal other Fig. 1. Antibiotic resistance by Salmonella serovar (SE Enteritidis, Tm Typhimurium, Chs Choleraesuis, Ag Agona, Ha Hadar, Gal Gallinarum)

5 oxolinic acid (22.%) SE Tm* Chs Ag Ha*** Gal*** other flumequine (22.1%) SE Tm* Chs Ag Ha*** Gal*** other enrofloxacin (15.7%) SE Tm Chs Ag Ha*** Gal*** other chloramphenicol (8.%) SE Tm*** Chs Ag*** Ha Gal other sulfamethizol (16.8%) SE*** Tm*** Chs*** Ag Ha Gal other cotrimoxazol (3.%) SE Tm Chs*** Ag Ha Gal other*** Fig. 1. cont.

6 tetracycline (23.4%) SE*** Tm*** Chs Ag* Ha*** Gal other doxycycline (45.5%) SE Tm*** Chs Ag*** Ha*** Gal other nitrofurantoin (48.2%) SE*** Tm*** Chs Ag Ha Gal other Fig. 1. cont. Our study proved that nitrofurantoin was the least efficient drug (Table 1). Although it is not used for animal salmonellosis treatment in Poland, a surprisingly high resistance was observed (48.2%). A low nitrofurantoin efficacy in salmonellas was reported both in Poland (9) and the Netherlands (21). In the case of the resistance to tetracycline and streptomycin the results were similar to our earlier data, but we noted a distinguished increase in resistance in the case of quinolones mainly those used in animal husbandry (8). Those results are in accordance with observations of Madsen et al. (11) and Malorny et al. (12). Beta-lactamresistance reached several percent, but no extendedspectrum β-lactamase (ESBL) producers were found among the tested salmonellas (data not shown). Although ESBL-positive Salmonella occurrence was rare, such strains of human origin had been isolated in Poland (17) and they caused public health problems in some European countries (3, 18, 25). Therefore ESBL activity in bacteria, including salmonellas, should be monitored. Although chloramphenicol is banned in food animals in Poland for more than a decade, 8% of resistant isolates were found during the examination. It was probably a result of a co-resistance with other resistance phenotypes (3, 25). Sulphonamide-resistance is often integrated with multiresistance and encoded chromosomaly (2, 22). In the present study the highest number of sulfamethizol resistance was observed in multidrug resistant S. Typhimurium (data not shown). Other salmonellas revealed moderate sulfamethizol efficacy. It increased, however, when a combination with trimethoprim was used. Those results were in agreement with the observations of van Duijkeren et al. (21). The majority of tested salmonellas were susceptible to most aminoglycosides and colistin. The observations of Pedersen et al. (15) and van der Wolf et al. () were similar with respect to turkey and swine isolates. According to Threlfall et al. (19) the use of aminoglycosides was responsible for the appearance of multiresistant strains in cattle and further on in humans. The above mentioned tendencies were in agreement with our findings (7) and those of other authors supporting the opinion that the Salmonella resistance has been increasing, although some regional diversities could be recognised (3-5). It is well known that the selective pressure of antibiotics used in animal husbandry makes foodproducing animals the major reservoir of resistant salmonellas (5, 24). Isolates originating from different animal species, feed or environment show different resistance patterns (4, 6-8, 15, 21, 23). The present study proved that salmonellas of animal origin show higher resistance than the strains isolated from feed and quinolones can serve as an example. In addition, differences were also seen among strains obtained from various animal species. In general, swine isolates were

7 239 more often resistant to most of antimicrobials while poultry isolates showed high resistance to nitrofurantoin (Table 1). The study showed that the resistance of isolates linked with animal husbandry has been continuously rising (5). Resistance was observed in 83.1% of the 56 tested strains (Tables 1 and 2) and it rose several times compared to previous studies (8). However, as shown in Fig. 1, the differences in resistance were not only limited to production sector but they also depended on Salmonella serovar (4, 7, 12, 17, 24). No strain susceptible to all applied antimicrobials was found within species-specific serovars S. Choleraesuis and S. Gallinarum. It may indicate the antibiotic overuse in swine and poultry. The correlation between increasing resistance and antibiotic use in animal husbandry was also indicated by others, however, multiresistance was considered the biggest threat for public health and disease therapy (5, 12). For the purpose of present study we defined multiresistance as a state of resistance to several drugs belonging to at least two antibiotic classes. Therefore, almost every second tested strain was multiresistant. Three serovars: S. Hadar, S. Gallinarum, and S. Typhimurium showed high multiresistance (P.1). This finding was in agreement with the observations of others (12, 17, 24). Although S. Typhimurium showed a lower percentage of multiresistant strains than the two other serovars, its resistance patterns comprised markedly more antimicrobials. It was connected with the prevalence of multiresistant clones within this particular serovar (1, 16, 22). Multiresistance in S. Enteritidis was not so frequent as in other prevalent Salmonella serovars, but much higher than that previously observed (8). It may be concluded that world-wide observed trends of Salmonella antimicrobial resistance are reflected in Polish isolates. There is an increase in the percentage of isolates showing either resistance or multiresistance. Some resistance is serovar specific, e.g. multiresistant S. Typhimurium clones or nitrofurantoin resistance in S. Enteritidis. Quinolone resistance is also found among isolates originating from animals. No ESBL positive Salmonella isolates are noted in animals and their feed so far. Swine isolates are more often antibiotic resistant compared to poultry. An increase in antimicrobial resistance, alike in other countries, can cause problems during human and animal therapy and therefore the surveillance of antimicrobial resistance should be intensified in Poland. Acknowledgments: We thank Dr Roman Franiczek, Department of Microbiology, Wrocław Medical University for re-testing of the isolates suspected to be ESBL producers. References 1. Bager F.: DANMAP: monitoring antimicrobial resistance in Denmark. 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