Cats as a risk for transmission of antimicrobial drug-resistant Salmonella.
Salmonella infections are still a leading cause of human odborne infections in the world (1,2). These infections primarily originate from eating contaminated food, especially chicken eggs and egg products, and also meat products from pigs and chickens (3,4). Considering the high frequency of food contamination and the emergence of multidrug-resistant Salmonella strains, control of Salmonella in food-producing animals has become a worldwide challenge. Other environmental sources can lead to accidental human infections with Salmonella as well. The role of pet animals as a source of Salmonella has not been fully investigated, but severe human infections originating from reptiles, especially pet turtles, have been reported (5).
Cats and dogs are the most widely kept pet animals, yet the incidence of Salmonella in these animals is largely unknown, and the risk that these animals pose for transmission of Salmonella to humans is unclear. In particular, cats that can freely roam outside, and are therefore able to scavenge or hunt food of unknown quality, are potential candidates for Salmonella carriage. Most reports concerning Salmonella and cats are case studies of clinical salmonellosis, which resulted in septicemia and death (6,7). Subclinical infections and carrier animals, however, are much more important with respect to transmission to humans. In this study, rectal swabs from cats of different origin (house cats, group-housed cats, diseased cats) were cultured for Salmonella. The serotype and phage type of the Salmonella isolates were determined, and the isolates were characterized with respect to their antimicrobial drug resistance pattern and interaction with human intestinal epithelial cells.
Collection of Fecal Samples
A total of 278 rectal swab samples from house cats of different age, sex, and breed were taken between July and November 2003. All house cats came from different owners. The animals came from all over the Dutch-speaking part of Belgium, i.e., north of Brussels. Rectal swab specimens were also taken from 58 cats that were submitted for autopsy to the Faculty of Veterinary Medicine, Ghent University. The latter died or were euthanized because of incurable disease. All cats came from different owners, except three cats that had feline immunodeficiency virus (FIV), which came from one owner. Finally, rectal samples of 35 kittens (all <4 months of age) were taken at a facility where the animals were group-housed, waiting to be adopted. These animals came from 16 different owners.
Bacteriologic analysis was performed by enrichment of the rectal swabs. The samples were first pre-enriched in buffered peptone water (BPW) (Oxoid, Basingstoke, Hampshire, UK) overnight at 37[degrees]C, after which 1 mL of this suspension was added to 9 mL oftetrathionate brilliant green broth (Oxoid) (enrichment). After incubation overnight at 37[degrees]C, a drop of this suspension was spread on brilliant green agar (BGA) (Oxoid). Both the serotype and phage type of positive isolates were determined.
Antimicrobial Susceptibility Testing
Resistance to antimicrobial agents was tested by using the disk difliision assay on Mueller-Hinton agar with commercial antimicrobial susceptibility disks (Oxoid) according to the international standards of the National Council for Clinical Laboratory Standards (NCCLS) (8). The following antimicrobial agents were tested: ampicillin (A, 10 [micro]g), chloramphenicol (C, 30 [micro]g), streptomycin (S, 10 [micro]g), sulfonamide (Su, 300 [micro]g), tetracycline (T, 30 [micro]g), ciprofloxacin (Cip, 5 [micro]g), kanamycin (K, 30 [micro]g), gentamicin (Gn, 10 [micro]g), sulfamethoxazole-trimethoprim (Sxt, 25 [micro]g), cefotaxime (Cxt, 30 [micro]g), nalidixic acid (Na, 30 [micro]g), and amoxicillin-clavulanic acid (Amc, 30 [micro]g). Salmonella enterica serovar Typhimurium 8420 (resistance type ACSSuT), 6237 (sensitive), 3520 (resistance type T), 2200 (resistance type ASSuT), and 5833 (sensitive) isolates from human patients in Belgium were used as control strains in antimicrobial susceptibility testing.
Polymerase Chain Reaction (PCR)
For PCR, a loop of bacterial culture was resuspended in 50 [micro]k of water, and DNA was released from bacterial cells by boiling for 20 min. After the mixture was spun for 1 min in a microlilge at 14,000 x g, 2 [micro]L of the supematant was taken as a template DNA for PCR. PCR was carried out in 20-[micro]L volumes by using PCR Master Mix from Qiagen (Hilden, Germany), according to the manufacturer's instructions. All the resistant strains were tested for the presence of the genes typical for particular resistance. The genes determined and primers used are listed in Table 1. Cycling consisted of 50-s incubations at 92[degrees]C, 55[degrees]C, and 72[degrees]C, which were repeated 25 times. After PCR, amplification products were detected by electrophoresis in 2% agarose gel, stained with ethidium bromide, and visualized under UV light. Antimicrobial drug-sensitive strain S. Typhimurium F98 was used as a negative control in all the amplifications. S. Typhimurium strains 8420, 6237, 3520, 2200, and 5833 were used as positive controls.
All Salmonella strains were tested for the presence of the SopB gene. The primers were GATAGGAAAGATTGAGCACCTCTG and TACAGAGCTTCTATCACTCAGCTTC, and the PCR cycle consisted of 30 cycles of(30 s 95[degrees]C, 1 min 58[degrees]C, 1 min 72[degrees]C).
Pulsed-Field Gel Electrophoresis (PFGE)
The bacteria were grown while being shaken overnight at 37[degrees]C in Luria-Bertani broth (LB). The Xbal PFGE patterns were determined for all 21 S. Typhimurium strains by using previously described PFGE methods (16,17)with some slight modifications. The patterns were grouped in a dendrogram with GelCompar II software (Applied Maths, St.-Martens-Latem, Belgium) by using the Dice coefficient and the unweighted pair group method with an arithmetic averages clustering algorithm.
Invasion of the Human intestinal Epithelial Cell Line T84
The capacity of all cat Salmonella isolates and the human S. Typhimurium isolates 8420, 6237, 3520, 2200, and 5833 to invade human intestinal epithelial cells was determined. Cells of the human colon carcinoma cell line T84 were seeded in 96-well cell culture plates (Greiner, Frickenhausen, Germany) at a density of 5.[10.sup.5] cells/mL culture lnediuln (DMEM + 10% fetal calf serum + 2% L-glutamine, without antimicrobial drugs) and grown for 24 h. Bacteria were grown for 20 h in LB-lnedium, after which the suspension was diluted 1:50 in fresh LB-medium. After 4 h of incubation at 37[degrees]C, suspensions were centrifuged and resuspended in DMEM with 10% fetal calf seruln (FCS). The number of colony-forming units (CFU)/mL was determined by plating 10-fold dilutions on BGA. The suspensions were stored overnight at 4[degrees]C. The next day, [10.sup.6] CFU in 200 [micro]L were added to the T84 cell cultures, which were then centrifuged for 10 min at 1,500 rpm to make close contact between the bacteria and the colon cells. The plates were incubated ibr 1 h at 37[degrees]C and 5% C[O.sub.2]. The cells were then rinsed three times with Hanks' Balanced Salt Solution (HBSS, Life Technologies, Paisley, Scotland). Cell culture medium with gentamicin (50 [micro]g/mL) was added, and plates were incubated for 1 h at 37[degrees]C and 5% C[O.sub.2]. Hereafter, the cells were rinsed three times with PBS and analyzed with 1% Triton X-100 (Sigma, St. Louis, MO) in distilled water. From this lysate, 10-fold dilution series were made. From each dilution, 6 x 20 [micro]L was added to BGA, to determine the number of CFU Salmonella per mL The assays were performed in triplicate. The percentage of intracellular bacteria, relative to the number of Salmonella bacteria, initially incubated with the cells, was calculated. The previously mentioned human isolates of S. Typlmnurium were used for comparison between the cat isolates and human isolates. Statistical analysis was performed by analysis of variance methods using the SPSS 11.0 software.
Characterization of Salmonella Isolates from Cats
Of 278 healthy house cats, 1 Salmonella strain was isolated, an S. Enteritidis phage type 21 strain, sensitive to all tested antimicrobial drugs. Five strains were isolated from cats that died from or were euthanized because of incurable disease. Feline AIDS (caused by feline immunodeficiency virus [FIV]) was diagnosed in three cats, one died due to feline panleukopenia parvovirus infection, and one was poisoned. Three isolates were identified as being ampicillin-resistant S. Typhimurium phage type 193, harboring the [bla.sub.TEM] gene. They had the same pulsed-field gel electrophoresis (XbaI) pattern, indicating that the isolates were of clonal origin (Figure 1). The three cats came from the same owner. One isolate was an antimicrobial drug-sensitive Salmonella Bovismorbificans strain. One isolate was Salmonella 4:i:-, which was resistant to ampicillin, chloramphenicol, sulfonamides, tetracycline and sulfamethoxazole-trimethoprim (ACSuTSxt), harboring the [bla.sub.TEM], cat, sul2, tet(A), and dfrA1 antimicrobial drug resistance genes. Eighteen strains were isolated from the group-housed cats. All of these were S. Typhimurium phage type 120/ad. Fourteen of these strains showed acquired resistance to ampicillin, chloramphenicol and tetracycline and harbored the [bla.sub.TEM], cat, and tel(A) antimicrobial drug-resistance genes, while four isolates were resistant to chloramphenicol only and only harbored the cat gene (Table 2). Pulsed-field gel electrophoresis showed that the isolates from the group-housed cats were of the same XbaI PFGE type, and that three subtypes within this type were present, indicating a clonal origin (Figure 1). One subtype contained the 14 strains that were resistant to the three mentioned antimicrobial drugs. All Salmonella strains harbored the SopB gene.
[FIGURE 1 OMITTED]
Invasion of the Human Intestinal Epithelial Cell Line T84
All isolates invaded T84 cells, with the cat isolates of S. Typhimurium PT193 (strains 1147, 1145, and 55, which belong to the same clone) and the human isolate X Typhimurium strain 2200, the most invasive, yielding a percentage of invasion of 8% to 10%. The multidrug-resistant cat isolate Salmonella 4:i:- (strain 11) was the least invasive strain, having an invasion percentage of about 0.5%. Invasion percentages of the different isolates are shown in Figure 2. Of the strains of the same PFGE type, only one was shown in Figure 2, since no significant differences were detected between the invasion percentages of these strains. Statistically significant differences are shown in the figure.
[FIGURE 2 OMITTED]
This study concluded that, although cats can transmit Salmonella strains, healthy house cats are generally safe. Earlier reports regarding isolation of pathogens from healthy cats showed low percentages (mostly around 1%) of Salmonella-positive rectal swabs (18,19). In our study, 1 of 278 healthy cats was found to be positive. Immunodeficiency and nonhygienic housing can be predisposing factors for cats to shed Salmonella in the feces, resulting in contamination of the environment. Rectal swabs from 18 of 35 group-housed kittens were Salmonella-positive in our study. The fact that the 35 kittens were derived from more than 10 different owners before being group-housed and that one PFGE type (three subtypes) of S. Typhimurium 120/ad was isolated, indicates spread of the Salmonella strain between the cats or a common source. The age of these animals may also play a role, since all animals in this group were <4 months. Young animals are more susceptible to Salmonella infection. Also immunodeficiency can result in Salmonella excretion. One outbreak of fatal salmonellosis in cats has been reported after mild immunosuppression induced by live panleukopenia virus vaccination (7). In our study, animals infected with FIV and one animal that had panleukopenia shed Salmonella. Three animals that were infected with FIV were derived from the same owner, which indicates that the animals were infected with Salmonella from the same source or that one animal contaminated the others.
In our study, serotypes Typhimurium, Enteritidis, Bovismorbificans, and 4:i:- were isolated from cats. The isolated serotypes indicate that the cats were infected from the same sources compared with other animals and man. Indeed, serotypes Typhimurium and Enteritidis are the most widespread serotypes and the serotype Bovismorbificans is not uncommon in other animals, including humans (2,20).
Generally, invasion in the human intestinal epithelial cell line T84 was comparable between the cat isolates and isolates from humans. Invasion in intestinal epithelial cells is the primary step in the pathogenesis of Salmonella that causes gastrointestinal problems (21). This finding implies that the cat isolates are potentially pathogenic for humans. Moreover, all cat isolates harbored the SopB gene, which is involved in blocking the closure of chloride channels in gut epithelium and thus in inducing diarrhea. As in most other animal species, the cat isolates of the serotype Typhimurium harbored antimicrobial drug-resistant genes, raising concerns about spreading antimicrobial drug-resistant strains to humans.
Since the 1990s, concerns have arisen about the emergence and spread of multidrug-resistant Typhimurium strains, especially the multidrug-resistant ACSSuT type, which is resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (2). In our study, some S. Typhimurium isolates from cats were resistant to a single drug such as ampicillin or chloramphenicol, while most isolates from the group-housed cats (same clone) were resistant to ampicillin, chloramphenicol, and tetracycline. Resistance genes were found to be [bla.sub.TEM] (ampicillin), cat (chloramphenicol), and tet(A) (tetracycline). The genes in the class 1 integron of the multidrug-resistant genomic island in ACSSuT type S. Typhimurium, required for the resistances to the above three mentioned antimicrobial drugs, are [bla.sub.PSE1],floR, and tet(G) (22). This illustrates that these isolates did not acquire their resistance genes from horizontal transfer from pentadrug-resistant ACSSuT type strains. The isolate Salmonella 4:i:- was resistant to ampicillin, chloramphenicol, sulfonamides, tetracycline, and sulfamethoxazole/trimethoprim (ACSuTSxt-type), encoded by [bla.sub.TEM] (ampicillin), cat (chloramphenicol), sul2 (sulfonamides), tet(A) (tetracycline), and dfrA1 (trimethoprim). Also the resistance shown by this example had no relationship to the typical S. Typhimurium DT104 multidrug-resistant genomic island.
In conclusion, healthy house cats are generally safe with regard to excretion of Salmonella in the environment. Cats that are sick or are receiving medication resulting in immune deficiencies can potentially pose a threat to public health. Young children, the elderly, and immunocompromised persons are at risk because of their high sensitivity for the infection. All persons should follow good hygiene practices when keeping cats as pets.
Table 1. List of primers used in the PCR reactions for detection of resistance genes Sequence Size Refer- Resistance Gene Primer (5'-3') (bp) ence Ampicillin blaPSE1 PSEF TAG CCA TAT TAT 321 AF261825 GGA GCC TC PSER TTA ACT TTT CCT TGC TCA GC [bla.sup. TEMF GCA CGA GTG GGT 310 9 TEM] TAC ATC GA TEMR GGT CCT CCG ATC GTT GTC AG blaoxa1 oxa1F AGC AGC GCC AGT 708 10 GCA TCA oxa1R ATT CGA CCC CAA GTT TCC Chloram- floR floRF GCG ATA TTC ATT 425 11 phenicol ACT TTG GC floRR TAG GAT GAA GGT GAG GAA TG Cat catF CCT GCC ACT CAT 623 10 CGC AGT catR CCA CCG TTG ATA TAT CCC Streptomy- aadA1 aad1For CGA CTC AAC TAT 384 AY534545 cin CAG AGG TA aad1Rev CTT TTG TCA GCA AGA TAG CC aadA2 aadA2F CGG TGA CCA TCG 249 12 AAA TTT CG aadA2R CTA TAG CGC GGA GCG TCT CGC strA strAF CCT ATC GGT TGA 250 11 TCA ATG TC strAR GAA GAG TTT TAG GGT CCA CC Tetracy- tetA tetAF GCT ACA TCC TGC 210 13 cline TTG CCT TC tetAR CAT AGA TCG CCG TGA AGA GG tetB tetBF TTG GTT AGG GGC 659 13 AAG TTT TG tetBR GTA ATG GGC CAA TAA CAC CG tetC tetCF GCG GGA TAT CGT 207 14 CCA TTC CG tetCR GCG TAG AGG ATC CAC AGG ACG tetG tetGF GCT CGG TGG TAT 468 13 CTC TGC TC tetGR AGC AAC AGA ATC GGG AAC AC Sulfona- sul1 sul1F ATG GTG ACG GTG 841 15 mide TTC GGC ATT CTG sul1 R GCT AGG CAT GAT CTA ACC CTC GG sul2 sul2F AGG GGG CAG ATG 249 11 TGA TCG AC sul2R GCA GAT GAT TTC GCC AAT TG Trimetho- dfrA1 dfrA1F GTG AAA CTA TCA 470 10 prim CTA ATG G dfrA1R CCC TTT TGC CAG ATT TGG dfrA10 dfrA10F TTA ATT ACC AGA 374 AY049746 GCA TTC GG dfrA10R TAC ACA TCA GCA TGA ACA GG dfrA12 dfrA12F ACT CGG AAT CAG 463 10 TAC GCA dfrA12R GTG TAC GGA ATT ACA GCT Kanamycin aadD aadD ATA TTG GAT AAA 161 12 [logical TAT GGG GAT not] F aadD TCC ACC TTC CAC [logical TCA CCG GTT not] R aphA/aph aphAaphIdF ATG GGC GCC TAT 257 12 (3') [lo- CAC AAT TGG gical not] Id aphAaphIdR TCG CCT CCA GCT CTT CGT AGA aphAI aphAI [lo- AAA CGT CTT GCT 461 12 [logical gical not] CGA GGC not] IAB IABF aphAI [lo- CAA ACC GTT ATT gical not] CAT TCG TGA IABR aph(3') KanAphF GAG AAA GTA TCC 465 L19385 [logical ATC ATG GC not] Ila KanAphR GCT CAG AAG AAC TCG TCA AG Table 2. Characteristics of Salmonella isolates from cats (a) PFGE Resist- pat- ance Isolate Phage tern pheno- Resistance no. Source Serotype type (b) type (b) genotype 11 Diseased 4:i:- -- ND ACSuTSxt [bla.sub. house TEM], cat, cat sul2, tet(A), dfrA1 40 Diseased Bovis- -- ND -- -- house morbifi- cat cans 109 House Enteri- 21 ND -- -- cat tidis 1145, Diseased Typhimu- 193 II A [bla.sub. 1147, 55 house rium TEM] cats 89, 165, Group- Typhimu- 120/ Ia ACT [bla.sub. 174, 198, housed rium ad TEM], cat, 320, 326, cats tet(A) 352, 355, 358, 359, 369, 380, 390, 392 161, 350 Group- Typhimu- 120/ Ib C cat housed rium ad cats 220, 339 Group- Typhimu- 120/ Ic C cat housed rium ad cats (a) A, ampicillin; C, chloramphenicol; Su, sulfonamides, T, tetracycline; Sxt, sulfamethoxazole-trimethoprim; ND, not determined; PFGE, pulsed-field gel electrophoresis. (b) Roman numerals indicate the major types of fragment patterns; lowercase letters indicate minor variations in the respective fragment pattern.
We thank V. Eeckhaut, M. Foubert, and L. Winters for their excellent technical assistance.
Ivan Rychlik was supported by grant 1B44019 from the Ministry of Agriculture of the Czech Republic.
Dr. Van Immerseel is a postdoctoral researcher at Ghent University, Faculty of Veterinary Medicine, Department of Pathology, Bacteriology and Avian Diseases, where the work described in this study was performed. His research interests include bacterial pathogenesis and host-pathogen interactions, with a focus on Salmonella.
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Filip Van Immerseel, * Frank Pasmans, * Jeroen De Buck, * Ivan Rychlik, ([dagger]) Helena Hradecka, ([dagger]) Jean-Marc Collard, ([double dagger]) Christa Wildemauwe, ([section]) Marc Heyndrickx, ([paragraph]) Richard Ducatelle, * and Freddy Haesebrouck *
* Ghent University, Merelbeke, Belgium; ([dagger]) Veterinary Research Institute, Brno, Czech Republic; ([double dagger]) Scientific Institute of Public Health, Brussels, Belgium; ([section]) Pasteur Institute of Brussels, Brussels, Belgium; and ([paragraph]) Center for Agricultural Research, Melle, Belgium.
Address for correspondence: F. Van Immerseel, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium; Fax: 09-264-74-94; email: filip.vanimmerseel@UGent.be
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|Publication:||Emerging Infectious Diseases|
|Date:||Dec 1, 2004|
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