Subtyping of Salmonella enterica Serovar Typhimurium Outbreak Strains Isolated from Humans and Animals in Iceland

Abstract

A total of 75 Salmonella enterica serovar Typhimurium strains of various (mainly human and animal) origins were typed by pulsed-field gel electrophoresis (PFGE) and phage typing. These strains were collected during an outbreak in Iceland in 1999 and 2000. The typing revealed that 84% of the strains belonged to the same PFGE and phage type (PT), namely, PFGE type 1Aa and PT 1.

In most developed countries, the frequency of infections due to Salmonella enterica has been increasing steadily (1, 7) and is now considered second only to Campylobacter spp. as a cause of bacterial gastroenteritis (5).

Since 1990, the Department of Clinical Microbiology at Landspitali University Hospital in Reykjavik, Iceland, has kept records of all culture-confirmed Salmonella cases among humans diagnosed in Iceland. During this time period, the incidence of infections due to Salmonella has been relatively low (Fig. 1). With the exception of infections in the year 1995 and in two epidemic years, 1996 and 2000 (9, 13), the majority of Salmonella infections diagnosed in Iceland are acquired abroad. In the 12-year period from 1990 to 2001, 628 of the total 1,691 culture-confirmed human salmonellosis cases were recorded as being domestically acquired. S. enterica serovar Typhimurium and Salmonella serovar Enteritidis are by far the most common domestic serotypes, constituting 290 (46.2%) and 195 (31.1%) of the cases, respectively.

FIG. 1.

Incidence of human salmonellosis infection in Iceland from 1990 to 2001.

The present study deals with an outbreak of S. enterica serovar Typhimurium infections among animals and humans in the south and southwestern parts of Iceland. During the autumn of 1999 and the following winter, Salmonella serovar Typhimurium was diagnosed as a cause of death or disease in horses and cattle on five different farms in the southern part of Iceland, and in a subsequent survey (11), Salmonella serovar Typhimurium was also isolated from horses, cattle, sheep, and dogs on two additional farms in the area. On two of those seven farms, some members of the household got sick. During the winter, Salmonella serovar Typhimurium was also isolated from a poultry farm, stabled horses, and a dog in the southwestern part of the country. At the same time, an increase in salmonellosis was observed in humans, and many isolates were identified as Salmonella serovar Typhimurium. No strains of this serovar were isolated from food in Iceland at this time. It was suspected that the transmission was from animals to humans.

The application of pulsed-field gel electrophoresis (PFGE) provides precise information that can be used to accept or reject epidemiological associations with a high degree of confidence and has therefore been proven to be highly useful and reliable in outbreak situations (14).

Our study included 75 strains of S. enterica serovar Typhimurium, including 37 strains of human origin and 35 strains of animal origin, 2 strains from a slaughterhouse's drain pipes, and 1 strain from imported food (leg of duck) (Table 1).

TABLE 1.

Characteristics of the 75 strains typed by PFGE, phage typing and antimicrobial susceptibility analysis

No. of strains	Origin	Antimicrobial resistance	PTa	PFGE profileb
				XbaI	AvrII	SpeI
30	Human		1	1	A	a
10	Cattle		1	1	A	a
16	Horses		1	1	A	a
1	Sheep		1	1	A	a
1	Poultry		1	1	A	a
2	Cat and dog		1	1	A	a
2	Environmental swab		1	1	A	a
1	Human	AMP, SXT	1	1	A	a
2	Horses		92	1	A	a
1	Horse		206	1	NT	a
1	Human		1	1	G	a
1	Human		1	5	B	b
1	Horse		1	5	NT	b
1	Human		170	2	C	c
1	Food		NK	4	D	d
2	Human	SXT	208	6	E	f
1	Horse		193	6	F	f
1	Human		104	3	1	e

^aNK, not known.

^bNT, nontypeable.

All samples were cultured by standard procedures for Salmonella (10, 11). All isolates were serologically typed with antisera to O and H antigens (Dade Behring Marburg GmbH, Marburg, Germany) (8). Phage typing was performed at the Laboratory of Enteric Pathogens, Health Protection Agency, London, United Kingdom, with a scheme first described by Callow (4), further developed by Anderson (2), and still further developed at the Laboratory of Enteric Pathogens. Today, the Salmonella serovar Typhimurium phage-typing scheme can define over 300 phage types (PTs) by using 38 typing phages (L. Ward, personal communication).

PFGE typing was performed according to the method of Maslow et al. (15) with the following modifications. The agarose mix was placed in a syringe. The plugs were incubated overnight at 56°C in ESP buffer (0.5 EDTA [pH 8.0], 10% sodium larolyl sarcosine, 100 μg of proteinase K per ml) and then washed four times in Tris-EDTA for 1 h at 37°C. Chromosomal DNA was digested with 10 U of the restriction enzymes XbaI, AvrII (BlnI), and SpeI (New England Biolabs, Inc., Beverly, Mass.) at 37°C overnight. Electrophoresis was performed at 210 V on 1% SeaKem GTG agarose gels (FMC Bioproducts) with a CHEF-DR III system (Bio-Rad Laboratories, Richmond, Calif.). Running conditions were as follows: for XbaI digests, the running time was 5 to 70 s for 24 h; for AvrII digests, the running time was 5 to 50 s for 24 h; and for SpeI digests, the running time was 5 to 40 s for 24 h. A lambda ladder PFG marker (New England Biolabs, Inc.) was used as a molecular weight standard. The gels were stained with ethidium bromide and photographed under UV transillumination with the GelDoc 2000 documentation system (Bio-Rad Laboratories). The photos were saved as TIFF files.

Any difference between two PFGE profiles was considered sufficient to distinguish one from the other. XbaI digestion PFGE profiles were named with numbers starting with 1, AvrII digestion PFGE profiles were named with uppercase letters starting with A, and SpeI digestion PFGE profiles were named with lowercase letters starting with a.

The antimicrobial susceptibilities of the strains were determined by the Kirby-Bauer agar diffusion method according to NCCLS guidelines for the following antimicrobial agents: ampicillin (AMP), trimethoprim-sulfamethoxazole (SXT), ceftriaxone, ciprofloxacin, and chloramphenicol (antimicrobial susceptibility disks; Oxoid, Ltd., Basingstoke, Hampshire, England). Inhibition zones were interpreted according to the recommendations of NCCLS guidelines (17).

Conclusions.

A set of 75 S. enterica serovar Typhimurium isolates from various sources was analyzed by PFGE and phage typing to gain knowledge of the distribution of Salmonella serovar Typhimurium subtypes in humans and animals in Iceland. Such extensive subtyping of Salmonella isolates has never been carried out before in Iceland, and existing knowledge on the subject is very limited. Compiling the results of PFGE (all enzymes) and PT analysis enabled us to identify a total of 17 different subtypes among the 75 strains (Table 1). One dominating PFGE pattern and PT was identified and was applicable to 84% (63 of 75) of the strains of both human and animal origin. One of these strains, however, was resistant to AMP and SXT.

Out of the 74 strains submitted for phage typing, 66 strains were of PT 1, 2 strains were of PT 92, 2 strains were of PT 208, and 1 strain each belonged to PTs 170, 104, 193, and 206 (Table 1). The strain isolated from imported food was not submitted for phage typing.

PFGE analysis and identification of profile types were as follows. PFGE permitted the resolution of XbaI macrorestriction fragments of the 75 strains into six distinct patterns (Fig. 2). The profiles comprised 11 to 16 bands. Sixty-seven (89.3%) of the strains shared the most common pattern of 11 bands. Macrorestriction patterns generated by AvrII were resolved into eight distinct patterns. The profiles comprised 6 to 11 bands. Sixty-five (89.0%) of the strains shared the most common pattern of eight bands. This enzyme failed to digest two of the strains. Macrorestriction patterns generated by SpeI were resolved into six distinct patterns. The profiles comprised from 12 to 16 bands. Sixty-seven (89.0%) of the strains shared the most common pattern of 15 bands.

FIG. 2.

PFGE profiles determined by XbaI digestion. Lane St, lambda ladder molecular weight marker; lanes 1 through 6, PFGE profiles 1, 3, 5, 2, 4, and 6, respectively. Numbers at the left are molecular sizes (in base pairs).

All strains were sensitive to AMP, SXT, ceftriaxone, ciprofloxacin, and chloramphenicol, except for two strains that were resistant to SXT and one strain that was resistant to AMP and SXT (Table 1).

The results of the PFGE and phage typing supported our hypothesis of transmission of Salmonella from animals to humans. The discriminatory powers of the three restriction enzymes proved very similar. Each enzyme discriminated the strains at the same level, except on two occasions when AvrII discriminated between strains that showed the same banding pattern with XbaI and SpeI. The PTs were also consistent with the PFGE results, except that two strains of PFGE profile 1Aa were PT 92 while all others were PT 1, a PT that has not been widely reported. The discriminatory powers of XbaI and SpeI were the same but slightly less than that of AvrII. This finding is consistent with the results of work on Salmonella serovar Typhimurium, Salmonella serovar Enteritidis, and Salmonella serovar Typhi (3, 6, 16), whereas Ridley et al. found that XbaI and SpeI gave better discrimination than AvrII, NotI, and NheI for Salmonella serovar Enteritidis (18).

Some of the isolates with different PFGE subtypes had a difference of only one or two fragments in their PFGE banding patterns when they were digested with XbaI, AvrII, and SpeI, especially SpeI, which showed only two banding differentiations in the profiles. These results show the importance of using both PFGE and phage typing and more than one restriction enzyme to increase the discriminatory power. It has been reported previously that different PTs can give the same PFGE pattern and that the same PT can give different PFGE patterns (12, 18).

These data show the importance of subtyping strains related to outbreaks. Out of the 74 strains isolated (1 strain was not domestic), 63 strains were identified as an outbreak strain by PFGE and phage typing. This outbreak strain was spread over the whole outbreak area. Eleven strains differed from the outbreak strain; two of these strains had the same PFGE pattern but different PTs, and three of them were of PT 1 but had different PFGE patterns. Six human nonoutbreak strains may be traced to imported food.

In conclusion, although we identified an epidemic strain, there is considerable diversity in the endemic Salmonella serovar Typhimurium strains in Iceland.