Eur J Wildl Res (2007) 53: 55–60 DOI 10.1007/s10344-006-0054-2
ORIGINA L PA PER
A. Čížek . M. Dolejská . R. Karpíšková . D. Dědičová . I. Literák
Wild black-headed gulls (Larus ridibundus) as an environmental reservoir of Salmonella strains resistant to antimicrobial drugs Received: 15 February 2006 / Accepted: 19 May 2006 / Published online: 28 September 2006 # Springer-Verlag 2006
Abstract Salmonella were isolated from black-headed gulls (Larus ridibundus) in six locations in the Czech Republic from 1984 to 2005 (Chropyně and Nymburk in 1984–1986; Nové Mlýny, Bartošovice, and Hodonín in 1991–1994; and Nové Mlýny, Bartošovice, and Ostrava in 2005). Antimicrobial susceptibility was determined in 12 antimicrobial drugs using disk diffusion. Although 95% of Salmonella isolates (197 out of 207) were pansusceptible, the prevalences of resistance increased significantly from 1 (2%) out of 59 isolates in 1984–1986 and 3 (3%) out of 100 isolates in 1991–1994 to 6 (13%) out of 48 isolates in 2005. Furthermore, in 2005, two isolates were nalidixic acid-resistant and one isolate was multidrug-resistant Salmonella Typhimurium DT 104. These findings suggest that the occurrence of salmonellae in black-headed gulls depends to a large extent on the contamination where the gulls feed and possibly reflects the dissemination of these strains among farm animals and humans. Black-headed gulls may also become infected with resistant Salmonella and thus pose a potential risk of Salmonella contamination of surface water and animal feeds, and consequently dissemination.
A. Čížek . M. Dolejská Department of Microbiology and Immunology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic R. Karpíšková . D. Dědičová Centre for Food Chain Hygiene, National Institute of Public Health, Brno, Czech Republic I. Literák (*) Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Palackého 1-3, 612 42 Brno, Czech Republic e-mail:
[email protected] Tel.: +420-54-1562630 Fax: +420-54-1562642
Keywords Gulls . Salmonella . Cloacal swab . Feces . Czech Republic . Drug resistance
Introduction The black-headed gull (Larus ridibundus) is an abundant, migrating species that nests in colonies on water reservoirs in inland Eurasia (Hudec and Št’astný 2005). In the Czech Republic, the gulls arrive in March and nest from April to June, usually in the same location each year. In July, the birds leave their nesting areas and spread throughout the country until November when they migrate over Germany, The Netherlands, and Belgium to the Atlantic coast of Western Europe or to the coast of Mediterranean Sea in Southern Europe to Italy, Croatia, and Greece. The diet of black-headed gulls consists of invertebrates, small vertebrates, and waste from municipal landfills and farms. During the nesting period, their diet consists mostly of insects, plant components, and earthworms and sometimes consists of vertebrates or cherries; the birds collect food within 10 km from their nesting colony (Honza and Modrý 1994). Nesting colonies may be large with thousands of pairs. In the Czech Republic; black-headed gulls often nest on islands in artificial water reservoirs in agricultural or industrial areas that commonly receive agricultural, municipal, and/or industrial wastewater that was insufficiently treated to kill Salmonella. In the Czech Republic, a high prevalence of Salmonella shedding was recorded among young black-headed gulls who may have become infected in the nest from the food brought to them, contaminated environment of the nesting colony, or contaminated water source for the reservoir (Literák et al. 1992; Čížek et al. 1994). It was also demonstrated that salmonellae survive in the environment of nesting colonies after the colony was abandoned and until the next nesting season and thereby infect gulls upon their return to the nesting colony (Literák et al. 1996). Although black-headed gulls are a well described reservoir of Salmonella, the public health consequence of this reservoir is not well known. Given their scavenging
56
diet and the large distances traveled, it may be postulated that black-headed gulls might be important vectors for the dissemination of Salmonella. Furthermore, although Salmonella was isolated from black-headed gulls, the antimicrobial resistance patterns of such isolates were not described. We therefore evaluated the antimicrobial resistance patterns of Salmonella isolated from black-headed gulls in the Czech Republic during the past two decades.
Materials and methods Cloacal swabs were collected from young, not flying, black-headed gulls captured in six colonies in the Czech Republic: Chropyně and Nymburk in 1984–1986; Nové Mlýny, Bartošovice, and Hodonín in 1991–1994; and Nové Mlýny, Bartošovice, and Ostrava in 2005 (Fig. 1). After collecting the specimen, the birds were released. The Nové Mlýny colony is the largest in the Czech Republic with approximately 7,500 gull pairs (Hudec and Št’astný 2005). Each of the nesting colonies, except for the Ostrava colony, were situated in intensively used agricultural areas with cattle, pig,` and chicken farms. The Ostrava colony was on a pond next to a town with about 350 thousand inhabitants. The health status of the birds examined appeared good. Cloacal swabs were placed overnight in buffered peptone water (Oxoid) at 37°C, and then selectively propagated in semisolid Rappaport–Vassiliadis agar (Oxoid) at 41.5°C for 24–48 h. Selected colonies were then subcultivated on xylose lysine deoxycholate (XLD) agar (Oxoid). Suspect Salmonella colonies were confirmed biochemically using triple sugar iron agar (Oxoid) and the API 10S test (BioMérieux, France). Salmonella isolates collected before 2005 were stored on Dorset’s egg medium at laboratory temperature or kept in meat peptone water (Oxoid) at −80°C before being cultivated in 2005 on XLD agar for this study. Confirmed Salmonella were serotyped using Salmonella antisera (Denka Seiken, Japan). Phage typing of Salmonella (S.) Enteritidis and S. Typhimurium isolates was performed using the schemes and phages provided by the Laboratory of Enteric Pathogens, HPA (Colindale, UK) (Ward et al. 1987,
1994). Disk diffusion according to Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) standards was used to determine antibiotic susceptibility to the following drugs and concentrations per disc: ampicillin (A, 10 μg), amoxicillin-clavulanic acid (30 μg), cefalothin (CF, 30 μg), ceftazidime (30 μg), chloramphenicol (C, 30 μg), streptomycin (S, 10 μg), gentamicin (10 μg), tetracycline (T, 30 μg), sulfonamide (Su, 300 μg), sulfamethoxazoletrimethoprim (25 μg), nalidixic acid (Na, 30 μg), and ciprofloxacin (5 μg). Escherichia coli strain ATCC 25922 was used as a control. Isolates resistant to ≥1 antimicrobial drugs where tested using polymerase chain reaction (PCR) to detect specific antimicrobial resistance genes and the integrase-coding gene int1, which is a part of class 1 integrons (Ng et al. 1999; Zhao et al. 2001; Briñas et al. 2002; Faldynová et al. 2003; Ferrari and Turnidge 2003). An overview of tested genes, used primers, PCR conditions, and product sizes is given in Table 1. Several colonies cultivated on meat peptone agar (Oxoid) were resuspended in 300 μl of sterile distilled water. The cells were lysed by boiling in a water bath at 95°C for 10 min. The mixture was then centrifuged at 16,000×g for 2 min. The supernatant was used as template DNA for PCR. To prepare 25 μl of PCR mixture, Taq-Purple DNA polymerase PCR Master Mix with 2.5 mM MgCl2 was used (Top-Bio, Czech Republic). DNA amplification was performed in accordance with the following steps of PCR reaction: initial denaturation at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing for 1 min, extension at 72°C for 1.5 min, and final extension at 72°C for 10 min. Multidrug-resistant (MDR) S. Typhimurium DT 104 type strain 110/2 obtained from Veterinary Research Institute (Brno, Czech Republic) was used as positive control for the integron and genes blaPSE-1, floR, aadA, sul1, tetG, and int1. PCR products were detected by electrophoresis at 1.5% agarose gel with ethidium bromide and were visualized in UV light. The χ2 test was used for statistic evaluation of the difference in prevalences of resistant isolates.
Results
GERMANY POLAND NYMBURK (1985)
CZECH REPUBLIC
OSTRAVA (2005) BARTOŠOVICE (1991, 2005) Š (1984-1986) CHROPYNE
NOVÉ MLÝNY (1991, 2005)
AUSTRIA
HODONÍN (1993-1994)
SLOVAKIA
Fig. 1 A map of the Czech Republic with locations from where black-headed gulls were examined. Years of sampling are in parentheses
Cloacal swabs were collected from 1,095 young blackheaded gulls from 1984 to 2005, of which 207 (19%) yielded Salmonella; 12% (59 out of 473) in 1984–1986, 30% (100 out of 331) in 1991–1994, and 17% (48 out of 291) in 2005 (Table 2). Sixteen different serotypes of Salmonella were isolated. The most common serotypes were Typhimurium (34%) and Enteritidis (12%). Phage typing revealed types DT 1, 2, 30, 36, 41, 46, 66, 86, 93, 101, 104, 132, 135, 141, and 194 in S. Typhimurium isolates and PT 1b, 4, 6, 6a, 8, reaction did not conform (RDNC), and untypable (U) in S. Enteritidis isolates (Table 3). One out of 59 (2%) isolates from 1984–1986 was resistant; it was S. Typhimurium resistance (R)-type S
57 Table 1 Primers used in the study Resistance
Primer
Ampicillin
TEMF TEMR PSEF PSER Tetracycline tetAF tetAR tetBF tetBR tetCF tetCR tetGF tetGR Streptomycin/spectinomycin aadAF aadAR strAF strAR Chloramphenicol catF catR floRF floRR Sulfonamide sul1F sul1R sul2F sul2R int1F int1R 5CS 3CS
Sequence (5′–3′)
Target gene Product size (bp) Ref.
ATT CTT GAA GAC GAA AGG GC ACG CTC AGT GGA ACG AAA AC TAG CCA TAT TAT GGA GCC TC TTA ACT CCT TGC TCA GC GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG TTG GTT AGG GGC AAG TT1T TG GTA ATG GGC CAA TAA CAC CG CTT GAG AGC CTT CAA CCC AG ATG GTC GTC ATC TAC CTG CC CAG CTT TCG GAT TCT TAC GG GAT TGG TGA GGC TCG TTA GC TGA TTT GCT GGT TAC GGT GAC CGC TAT GTT CTC TTG CTT TTG CCT ATC GGT TGA TCA ATG TC GAA GAG TTT TAG GGT CCA CC CCT GCC ACT CAT CGC AGT CCA CCG TTG ATA TAT CCC GCG ATA TTC ATT ACT TTG GC TAG GAT GAA GGT GAG GAA TG CTT CGA TGA GAG CCG GCG GC GCA AGG CGG AAA CCC GCG CC AGG GGG CAG ATG TGA TCG AC GCA GAT GAT TTC GCC AAT TG CCT CCC GCA CGA TGA TC TCC ACG CAT CGT CAG GC GGC ATC CAA GCA GCA AG AAG CAG ACT TGA CCT GA
(Table 4). Three out of 100 (3%) isolates from 1991–1994 were resistant; they were S. Typhimurium (DT 104) R-type Su and S. Berta R-type AST (two isolates). Six out of 48 (13%) isolates from 2005 were resistant: they were S.
blaTEM
1,150
Briñas et al. (2002)
blaPSE-1
321
Faldynová et al. (2003)
tetA
210
Ng et al. (1999)
tetB
659
Ng et al. (1999)
tetC
418
Ng et al. (1999)
tetG
468
Ng et al. (1999)
aadA
284
Clark et al. (1999)
strA
250
Faldynová et al. (2003)
cat
623
Faldynová et al. (2003)
floR
425
Faldynová et al. (2003)
sul1
417
Zhao et al. (2001)
sul2
249
Faldynová et al. (2003)
int1
280
Zhao et al. (2001)
5′–3′CS
variable
Faldynová et al. (2003)
Typhimurium (DT 104) R-type ACSSuTNa, S. Typhimurium (DT 1) R-type A, S. Enteritidis (PT 6) R-type Na, and S. Typhimurium (DT 194) R-type A (three isolates). The prevalences of resistant isolates in 1984–1986 (2%), 1991–
Table 2 Salmonellae in black-headed gulls from six locations in the Czech Republic examined from 1984 to 2005 Year (location)
Number of Salmonella Isolated serotypes (n) isolates/examined specimens (%)
1984–1986 (Chropyně) 57/377 (15.1) 1985 (Nymburk) 1991 (Nové Mlýny)
2/96 (2.1) 83/267 (31.1)
1993–1994 (Hodonín) 2005 (Ostrava) 2005 (Bartošovice) 2005 (Nové Mlýny)
17/64 (26.6) 35/132 (26.5) 4/54 (7.4) 9/105 (8.6)
Total
207/1,095 (18.9)
S. Typhimurium (38), S. Agona (11), S. Enteritidis (2), S. Panama (2), S. Isangi (2), S. Hadar (1), S. Schwarzengrund (1) S. Typhimurium (2) S. Typhimurium (48), S. Derby (13), S. Enteritidis (8), S. Agona (5), S. Montevideo (4), S. Berta (2), S. Abony (1), S. Hadar (1), S. Infantis (1) S. Typhimurium (14), S. Bareilly (2), S. Derby (1) S. Typhimurium (13), S. Enteritidis (13), S. Derby (8), S. Kentucky (1) S. Typhimurium (3), S. Derby (1) S. Enteritidis (4), S. Indiana (2), S. Typhimurium (1), S. Abony (1), S. Saintpaul (1) S. Typhimurium (119), S. Enteritidis (27), S. Derby (22), S. Agona (16), S. Panama (2), S. Isangi (2), S. Hadar (2), S. Schwarzengrund (1), S. Montevideo (4), S. Berta (2), S. Abony (1), S. Infantis (1), S. Bareilly (2), S. Kentucky (1), S. Indiana (2), S. Saintpaul (1)
58 Table 3 Phagotypes of S. Typhimurium and S. Enteritidis in black-headed gulls from six locations in the Czech Republic from 1991 to 2005 Year (location)
Serotype (n)
Phage type (n)
1984–1986 (Chropyně)
S. S. S. S.
Typhimurium (38) Enteritidis (2) Typhimurium (2) Typhimurium (48)
S. S. S. S. S. S. S.
Enteritidis (8) Typhimurium (6) Typhimurium (13) Enteritidis (13) Typhimurium (3) Typhimurium (1) Enteritidis (4)
DT 1 (1), DT 93 (4), DT 104 (4), DT 135 (2), ND (27) PT 6a (1), PT 4 (1) DT 141 (1), DT 86 (1) DT 1 (5), DT 2 (2), DT 30 (1), DT 41 (1), DT 46 (3), DT 66 (1), DT 104 (7), DT 132 (2), DT 141 (16), RDNC (5), U (5) PT 6 (1), PT 8 (7) DT 141 (5), U (1) DT 101 (10), DT 36 (2), DT 104 (1) PT 4 (1), PT 6 (6), PT 8 (3), ND (3) DT 194 (3) DT 104 (1) PT 8 (3), PT 1b (1)
1985 (Nymburk) 1991 (Nové Mlýny)
1993–1994 (Hodonín) 2005 (Ostrava) 2005 (Bartošovice) 2005 (Nové Mlýny) ND Not determined
1994 (3%), and 2005 (13%) were significantly (p
