Clonal Distribution of Monophasic Salmonella enterica Subsp. enterica Serotype 4,[5],12:i:- in the Czech Republic

Abstract

Salmonella 4,[5],12:i:- has become the third most common serotype in Europe, including the Czech Republic. In this study, phenotypic and genotypic methods for a more detailed description of this serotype were used. Analysis of a limited number of isolates revealed that 76% of them belonged to phage type DT193. Also, rare phage types DT208 and U311 were identified. In total, 88.6% of the isolates were resistant to at least four antimicrobial agents. In this study, 24 multilocus variable-number tandem-repeat analysis profiles were detected, and some of them matched with the profiles recently described in Europe.

Introduction

Recently, a monophasic serotype Salmonella enterica subsp. enterica serotype 4,[5],12:i:- has been listed as the third most common in the whole European Union. Many outbreaks caused by this serotype have been reported in Europe as well as in numerous countries worldwide (Bone et al., 2010; CDC, 2011). Studies focused on Salmonella 4,[5],12:i:- indicated that this serotype is usually linked to pigs (Bone et al., 2010). Our laboratory data show that Salmonella 4,[5],12:i:- is the third most common serotype among human isolates in the Czech Republic. Nevertheless, prevalence of this serotype in foodstuffs and food-producing animals with the exception of pigs is rare in the Czech Republic. Therefore, environmental sources have also been observed. The aim of our study was to perform further characterization of current and some archival monophasic isolates using phenotypic and genotypic methods, as well as to broaden the knowledge of the characteristics of Salmonella 4,[5],12:i:- in the Czech Republic.

Materials and Methods

In total, we collected 79 isolates of the serotype Salmonella serotype 4,[5],12:i:-. Altogether, we acquired 60 human (none of them being reported as a part of an outbreak), 10 animal, and 9 foodstuff isolates. Among 10 animal isolates, 5 seagull isolates were integrated because seagulls are typical environment indicator species.

Serotyping

The antigenic structure was obtained by slide agglutination with commercial antisera using the Kauffmann-White-LeMinor scheme (Grimont and Weill, 2007).

Detection of fljB gene by polymerase chain reaction (PCR)

The chromosomal DNA was obtained using the method of simple heat lysis. The primers used for the PCR were described by McQuiston (McQuiston et al., 2004).

Phage typing

Phage typing was performed according to the HPA Colindale protocol (Anderson et al., 1977). Salmonella enterica subsp. enterica serotype 4,[5],12:i:- DT193, DT208, and U302 and Salmonella enterica subsp. enterica serotype Typhimurium DT1 and DT27 were used as the control strains.

Antimicrobial susceptibility testing

Susceptibility to a panel of 17 antimicrobial agents was determined by the disk-diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) protocol (CLSI, 2006). The spectrum of the agents used was as follows: ampicillin, ampicillin/clavulanic acid, cefotaxime, chloramphenicol, streptomycin, kanamycin, gentamicin, neomycin, apramycin, sulfonamides, sulfamethoxazole/trimethoprim, trimethoprim, tetracycline, nalidixic acid, ciprofloxacin, enrofloxacin, and colistin. Escherichia coli CCM 3954 was used as the control strain.

MLVA typing

For Salmonella 4,[5],12:i:- MLVA typing, technical documents published by ECDC (2011) and Larsson et al. (2009) were used. Fluorescently labeled products were separated using capillary electrophoresis on ABI3100 Avant (Applied Systems, USA). The final profiles were assigned based on the fragment size from each locus; “NA” was used to indicate a locus not present. Salmonella Typhimurium SSI 1–SSI 33 (Statens Serum Institute, Denmark) were used as the control strains.

Results

All 79 isolates showed the antigenic formula O:4,12 H1:i H2:-. The presence of the gene of the second flagellar phase fljB was not detected. Phage types revealed in this study are summarized in Table 1. Our research resulted in 70 (88.6%) of all isolates being resistant to at least 4 antimicrobial agents, which leaves just 11.4% being resistant to less than 3 antimicrobials. Nineteen isolates (24%) were resistant to some additional agents, including resistance to quinolones. In our set of isolates, we differentiated 24 MLVA profiles. The most prevalent profile was 3-13-10-NA-211 and a very similar profile 3-12-10-NA-211. We detected 12 (15.2%) and 11 (13.9%) isolates, respectively. The profiles were shared by isolates of all origins (Table 1).

Table 1.

Phage Types and Multilocus Variable-Number Tandem-Repeat Analysis Profiles Together with the Isolate Origin

Isolate origin	Phage type (number of isolates)	MLVA profile (number of isolates)
Human	DT193 (42)	3-12-10-NA-211 (9)
		3-13-10-NA-211 (4)
		3-12-9-NA-211 (3)
		3-15-10-NA-211 (3)
		3-11-9-NA-211 (3)
		3-13-8-NA-211 (3)
		3-13-9-NA-211 (2)
		3-15-9-NA-211 (2)
		3-11-10-NA-211 (2)
		3-14-11-NA-211 (2)
		1-6-NA-NA-511 (2)
		3-8-10-NA-211 (1)
		3-14-10-NA-211 (1)
		3-13-11-NA-211 (1)
		3-13-6-12-311 (1)
		3-14-14-NA-311 (1)
		3-16-8-NA-211 (1)
		3-21-10-NA-211 (1)
	DT208 (8)	3-12-9-NA-211 (3)
		3-13-10-NA-211 (2)
		3-12-10-NA-211 (1)
		3-13-9-NA-211 (1)
		3-21-11-NA-211 (1)
	U311 (6)	3-14-10-NA-211 (4)
		3-13-10-NA-211 (2)
	U (2)	3-13-7-NA-211 (1)
		3-13-8-NA-211 (1)
	U302 (1)	2-11-6-9-212 (1)
	DT193/10+ (1)	3-13-10-NA-211 (1)
Pork meat	DT193 (7)	3-8-10-NA-211 (1)
		3-11-9-NA-211 (1)
		3-11-10-NA-211 (1)
		3-12-10-NA-311 (1)
		3-12-11-NA-211 (1)
		3-13-9-NA-211 (1)
		3-15-9-NA-211 (1)
Cream cake	DT193 (1)	3-13-10-NA-211 (1)
Fish	DT193 (1)	3-13-10-NA-211 (1)
Seagull	DT193 (5)	3-14-9-NA-211 (4)
		3-13-8-NA-211 (1)
Piglet	DT193 (4)	3-8-10-NA-211 (1)
		3-12-10-NA-211 (1)
		3-13-10-NA-211 (1)
		3-15-9-NA-211 (1)
Pig	U302 (1)	3-16-3-15-311 (1)

U – untypable.

Discussion

Our study showed that 76% of our isolates belonged to the phage type DT193, which appears to be usual in European countries (Hopkins et al., 2012; Mandilara et al., 2013). We also detected some rather rare phage types such as DT208 (10.1%) and U311 (7.6%). Until now, DT208 has not been detected in Slovakia (a neighboring country of the Czech Republic). On the contrary, U311 was identified as the second most frequent phage type in Slovakia (Majtan et al., 2013).

The latest studies focusing on Salmonella 4,[5],12:i:- confirm that the typical antibiotic resistance pattern contains ampicillin, streptomycin, sulfonamides, and tetracycline, and strains are more often resistant to some additional agents (Dionisi et al., 2009; Hopkins et al., 2010). Like in the Slovak study, we also did not detect any fully susceptible isolate (Majtan et al., 2013).

Altogether, 24 MLVA profiles were described. Some of them originated from all three sources and showed different phage types. On the other hand, there were three profiles that did not occur in human isolates and were detected only in pork meat isolates. A possible explanation of this discrepancy might be due to the fact that pork meat and offal in some cases cannot be tracked back to the country of origin. Thus, it could have been a single batch that did not affect the epidemiological situation in the Czech Republic.

MLVA profiles of the environmental indicator species—seagulls – were taken in the same locality at different times, and thus they evinced the same or related profiles. Moreover, the profile 3-13-8-NA-211 also occurred in human isolates, which may indicate a possible role for environmental sources in epidemiology of this serotype. On the other hand, piglet isolates as well as foodstuff isolates showed significant heterogeneity due to the fact that they come from different farms and food business operators.

We also described some MLVA profiles occurring in other European countries. Profile 3-12-10-NA-211 was revealed as one of the common profiles in a survey performed in England (Hopkins et al., 2012). Three isolates from our set shared the profile 3-13-9-NA-211, which is identical to the profile of the strain confirmed to cause the outbreak linked to pork sausages in France in 2010 (Gossner et al., 2012). Our study suggests that some of the human isolates listed as separate cases might have been interrelated.

This very first study of this kind in the Czech Republic may help broaden knowledge of the characteristics of Salmonella 4,[5],12:i:-.

Footnotes

Acknowledgments

The authors wish to thank Mr. Paul Veater (Bristol, United Kingdom) for proofreading the translated manuscript. The results of the project LO1218 were obtained with a financial support from the MEYS of the CR under the NPU I program.

Disclosure Statement

No competing financial interests exist.

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