Journal of Medical Microbiology (2014), 63, 831–834
DOI 10.1099/jmm.0.072702-0
Salmonella enterica subspecies II infections in England and Wales – the use of multilocus sequence typing to assist serovar identification Satheesh Nair,1 John Wain,2 Steve Connell,1 Elizabeth de Pinna1 and Tansy Peters1 Correspondence
1
Satheesh Nair
2
[email protected]
Received 31 December 2013 Accepted 30 March 2014
Salmonella Reference Service, Public Health England, Colindale, London NW9 5EQ, UK Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK
Identifying rare Salmonella serotypes by conventional serotyping can be a problem for diagnostic or reference microbiology laboratories. We report two cases of the seldom encountered serovar Salmonella Dubrovnik, which is known as Salmonella subspecies II 41 : z : 1,5, in an elderly man and a young child. Multilocus sequence typing, a technique that is being used more frequently in our laboratory, was used to assist serovar identification as serotyping proved to be inconclusive. To our knowledge, these cases were not linked geographically and there were no known associations between them although they occurred within a very short time of each other.
INTRODUCTION Classification and identification of Salmonella are complex, and are mainly based on serological detection of antigens. The complexity is often due to the presence of Salmonella strains that do not fully express their antigens and hence confuse the identification process (Grimont & Weill, 2007; Peters et al., 2010; Achtman et al., 2012). Therefore reference laboratories frequently receive isolates that are difficult to type and require a combination of microbiological and molecular methods for identification. Our laboratory screens Salmonella isolates using TaqMan realtime PCR for identification of the most common subspecies of Salmonella enterica (subsp. I and III) (Hopkins et al., 2009, 2011), as well as multilocus sequence typing (MLST) for problematic strains. Between July and August 2010, three unusual isolates were referred to the Public Health England Salmonella Reference Service (formerly the Health Protection Agency Salmonella Reference Unit) for confirmation and typing. Two isolates came from the same elderly male patient (83 years of age) admitted to hospital with fever. The first isolate was from a blood culture and had a presumptive identification from the sending laboratory of Salmonella sp., while the second isolate from a faecal culture had a presumptive identification of Salmonella arizonae. The third isolate was from faeces of a 3-year-old female hospitalized with severe diarrhoea. There was no known link between the cases, and they were hospitalized in geographically separated regions. Despite treatment with appropriate antibiotics the elderly male deteriorated and died following admission. Abbreviations: MLST, multilocus sequence typing; ST, sequence type.
072702 G 2014 Crown Copyright
Printed in Great Britain
In order to establish whether the isolates from these two cases belonged to the less common S. enterica subsp. salamae (subsp. II), S. enterica subsp. houtenae (subsp. IV) or Salmonella bongori (previously subsp. V), a range of biochemical tests were performed together with serum agglutination tests, MLST (Kidgell et al., 2002) and fljB and fliC genotyping (Mortimer et al., 2004; McQuiston et al., 2004).
METHODS Bacterial isolates and identification. The two isolates from the
elderly male were submitted on 9 and 22 July 2010, and the isolate from the child was submitted on 11 August 2010, to the Salmonella Reference Service (SRS) of Public Health England (PHE). This is the national reference laboratory for salmonellas from humans in England and Wales. All isolates were typed phenotypically and were further tested genotypically. A retrospective analysis of all the available clinical and epidemiological data was also carried out on every S. enterica subsp. salamae present in the SRS database between 2004 and 2012. Serotyping. Isolates were cultured and serotyped by SRS staff
using specific antisera. Biochemical analysis and serotyping were in accordance with the White–Kauffmann–Le Minor scheme (Grimont & Weill, 2007). Real-time PCR. Salmonella isolates were screened using an in-house TaqMan real-time PCR assay for identification of the most common subspecies of S. enterica (Hopkins et al., 2009, 2011). MLST. MLST was performed as previously described (Kidgell et al., 2002) by determining the sequences of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA). The data obtained were compared with the Salmonella MLST database at the University College, Cork (http://pubmlst.org) and isolates were assigned
831
S. Nair and others sequence type (ST) numbers as defined by the database. Sequence types were assigned based on the set of allele types derived from each of the seven loci. Flagellar gene typing. Genes encoding flagellin [fliC encodes
the phase 1 flagellar (H1) antigen and fljB encodes the phase 2 flagellar (H2) antigen] are typically conserved at the 59 and 39 ends but variable in the central region in Salmonella and can be used in molecular serology (Mortimer et al., 2004; McQuiston et al., 2004). Partial sequences of the fliC and fljB gene were obtained as previously described by Mortimer et al. (2004) and McQuiston et al. (2004).
RESULTS All three isolates were negative in a simplex 59 nuclease TaqMan assay that targets the hilA gene, indicating that the isolates were not S. enterica subsp. enterica (subsp. I). A duplex 59 nuclease PCR assay for identification of the Arizona group was negative for the LacZ locus but positive results were obtained for the ttrRSBCA locus involved in tetrathionate respiration. This confirmed the presence of Salmonella DNA but indicated that the isolates were not S. enterica subsp. arizonae (subsp. IIIa) or S. enterica diarizonae (subsp. IIIb). A range of biochemical tests were performed together with serum agglutination tests to confirm the presence of less common subspecies of S. enterica. The three isolates were unreactive with a polyvalent Salmonella somatic [O] serum but tested positive with a polyvalent flagellar [H] serum indicative of the presence of Salmonella flagella antigens. Additional agglutination testing identified the phase 1 and phase 2 flagellar antigens (H antigens) to be H15z and H251,5. This was confirmed by amplification and partial sequencing of the fliC and fljB genes (data not shown). Due to the complexity/difficulty in identifying the [O] antigen, further titration against a wider range of groupspecific antisera covering the Group C and Group S serogroups revealed the somatic [O] antigen to be either 6,7 or 41 according to the White–Kauffmann–Le Minor classification. MLST was then used to obtain the possible identity of the Salmonellae involved. MLST clustered the isolates with other subsp. II serotypes within the Salmonella MLST
database, with an identical match to ST1082. The ST1082 isolate in the MLST database is 1709K, the Kauffmann reference strain of S. enterica subsp. salamae 41 : z : 1,5 (formerly serotype Dubrovnik, isolated from a reptile in Yugoslavia in 1964) (F.-X. Weill, Institut Pasteur, personal communication). Although biochemical results were not atypical and confirmed S. enterica subsp. salamae (subsp. II), the techniques were laborious and time consuming, and required long incubation times. Eventually the isolates were serologically classified with the formula 41 : z : 1,5 (somatic antigen [O] : flagellar antigen [H] phase I : phase II) and named as Salmonella subsp. II 41 : z : 1,5 according to current nomenclature (Grimont & Weill, 2007). The possibility of cross-contamination between the isolates from the elderly man and the child was ruled out because the isolates were received and processed by our laboratory at different times. Between January 2004 and December 2012, our laboratory identified 280 isolates of S. enterica subsp. salamae of which 223 were from human infections including three cases of bacteraemia. The majority of these isolations were single occurrences of extremely rare salmonellas, although a few notable exceptions appeared with greater frequency during that period (Table 1). Of the human-derived isolates, 90 % (200/223) were fully sensitive to a wide range of antimicrobials using standard methods of determination (Frost, 1994). Where resistance was present it was usually to a single antimicrobial, with only two records of multidrug resistance (SRS in-house data). The highest incidence of infection with S. enterica subsp. salamae was seen in young children. For isolates from patients of known age, 52 % were from children 4 years of age or less, of which 33 % were from infants younger than 1 year (Fig. 1). Of the 57 non-human isolates, 23 were from animal sources, of which only three were listed as reptiles. Interestingly, of the 16 food isolates, eight were from either herbs or spices. Although six isolates were listed as ‘source unknown’, they all originated from private food laboratories, thus
Table 1. The most prevalent Salmonella enterica subsp. salamae isolated in England and Wales, 2004”2012 Former serovar name Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella
832
Sofia Tranoroa
Hagenbeck Nairobi
New name Subsp. Subsp. Subsp. Subsp. Subsp. Subsp. Subsp.
II II II II II II II
58 : l,z13,z28 : z6 4,12:b:55 : k : z39 30:l,z28 : z6 50 : b : z6 48 : d : z6 42 : r:-
Number of cases 26 20 19 15 12 11 10
Journal of Medical Microbiology 63
S. enterica subsp. II infections in England and Wales
importance as sources of human salmonellosis are not well described and much of the available literature about reptiles and salmonellas focuses on S. enterica subsp. arizonae, S. enterica subsp. diarizonae and S. enterica subsp. houtenae (Warwick et al., 2001; Mermin et al., 2004; Pedersen et al., 2009).
80
No. of reported cases
70 60 50 40 30 20 10 0
Under 1
1–4
5–15 16–45 Age (years)
46–64
65 plus
Fig. 1. Cases of Salmonella enterica subsp. salamae by age in England and Wales, 2004–2012 (N5221).
implicating their origin as from an unknown food. Environmental samples accounted for ten of the remaining isolates. One hundred and seventy-nine of the 223 human cases had no history of foreign travel.
DISCUSSION MLST as a replacement for serotyping in S. enterica has been demonstrated recently (Achtman et al., 2012). In this study, MLST was used to confirm the identity of three strains that were difficult to serotype in a reference laboratory setting. This clearly shows the potential of using MLST in support of routine reference work. In the 1960s, the International Subcommittee on the Taxonomy of the Enterobacteriaceae renamed serovars belonging to S. enterica subsp. salamae (Carpenter, 1968). These serovars are now known as Salmonella subsp. II followed by their antigenic formula (e.g. Salmonella Dubrovnik is now known as Salmonella subsp. II 41 : z : 1,5) (Table 1). Since it was first identified as Salmonella Dubrovnik in 1964 (Kelterborn, 1967) there had been no reports of this serovar in England and Wales until 2004. To date there have only been six isolated cases (one with foreign travel to Italy) of this extremely rare serovar reported in our laboratory although there were four cases reported in Italy between 1994 and 1996 based on a SALM-NET study (Scuderi et al., 2000). Serovars of S. enterica subsp. salamae are typically reported as being associated with cold-blooded hosts, and are cited as rarely causing human infections, but a review of the existing literature does not provide much information on this subspecies, and only one publication specifically names Salmonella Dubrovnik (Wuthe, 1969). Whilst it is noted that turtles, snakes, iguanas and lizards may all carry S. enterica subsp. salamae, the carrier rates and relative http://jmm.sgmjournals.org
While the large majority of human salmonellosis in England and Wales is due to infection with S. enterica subsp. enterica (subsp. I), S. enterica subsp. salamae can still cause gastroenteritis and occasionally systemic disease in warm-blooded hosts. It may also be responsible for severe infection in the young, very old or immunocompromised patients who then require hospitalization (Angulo & Swerdlow, 1995; CDC, 1999). Most of the infections with this subspecies that we saw in our laboratory were accompanied by gastroenteritis, with systemic infection apparent in only a very few cases. Although the outcome of bacteraemia cases may occasionally be fatal, there did not appear to be a problem with antibiotic resistance that would have implications for treatment. The fact that the highest incidence of S. enterica subsp. salamae infection was seen in young children was not unexpected. As noted by Bertrand et al. (2008) the prevalence of infants younger than one year of age may be partly due to the bias in investigating childhood salmonellosis cases more thoroughly. However, our data show that when S. enterica subsp. salamae was compared with other non-typhoidal serovars the incidence was still higher than would be expected for simple age-bias (C. Lane, PHE, personal communication). Most literature states that isolates belonging to subspecies other than S. enterica subsp. enterica (subsp. I) are almost exclusively obtained from exotic sources such as reptiles. It has been suggested that whenever Salmonella belonging to subspecies other than enterica are found in humans, an exotic pet source should be suspected. There are no specific data on reptile ownership in our retrospective dataset, as this information has not been routinely gathered in England and Wales.
CONCLUSION The source of infection in single cases of salmonellosis is rarely identified, especially when the strain is a seldomencountered serovar without any apparent endemic bias preference. Although 44 of the human cases here were known to be associated with foreign countries, especially the African continent (22/44), the majority of cases had no history of foreign travel. Thus it is difficult to identify management strategies without being able to identify potential environmental or food sources. We are not sure whether the case of Salmonella subsp. II 41 : z : 1,5 bacteraemia described here was due to the virulence of the serovar alone or to the underlying health of the elderly patient. As the only link between the two cases is loosely temporal we have to assume that the appearance of both 833
S. Nair and others
cases in a short period of time was coincidental. Although it is generally believed that cold-blooded animals are the major reservoir of S. enterica subsp. salamae, it is also important to consider food-borne infections. If it is assumed that such cases are always sporadic infections from reptiles, either directly or indirectly, they may never be reported to the relevant public health authorities.
Frost, J. A. (1994). Testing for resistance to antibacterial drugs. In
The identification of rare Salmonella serotypes can be hindered due to the difficulty of identifying the somatic [O] antigen or flagella [H] antigens, which might be caused by varying expression levels of the genes involved. MLST was used in this study for rapid and accurate identification of the strains involved. With the current trend for using whole genome sequencing for diagnostic and public health microbiology, MLST will be used in real-time for identifying all Salmonellas within our laboratory in the near future.
Rapid identification of Salmonella enterica subsp. arizonae and S. enterica subsp. diarizonae by real-time polymerase chain reaction. Diagn Microbiol Infect Dis 64, 452–454.
ACKNOWLEDGEMENTS
fever, is approximately 50 000 years old. Infect Genet Evol 2, 39–45.
This work could not have been completed without the help of the staff at the Salmonella Reference Service, Public Health England. We would also like to thank Dr Kathie Grant for critically reading the manuscript. The authors have no conflicts of interest to declare.
REFERENCES Achtman, M., Wain, J., Weill, F. X., Nair, S., Zhou, Z., Sangal, V., Krauland, M. G., Hale, J. L., Harbottle, H. & other authors (2012).
Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog 8, e1002776. Angulo, F. J. & Swerdlow, D. L. (1995). Bacterial enteric infections in persons infected with human immunodeficiency virus. Clin Infect Dis 21 (Suppl 1), S84–S93.
Methods in Practical Laboratory Bacteriology, pp. 73–82. Edited by H. Chart. New York: CRC Press. Grimont, P. A. D. & Weill, F. X. (2007). Antigenic formulas of the
Salmonella serovars. WHO Collaborating Centre for Reference and Research on Salmonella, Institut Pasteur, Paris, France. http://www. pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s-000036-089 Hopkins, K. L., Peters, T. M., Lawson, A. J. & Owen, R. J. (2009).
Hopkins, K. L., Lawson, A. J., Connell, S., Peters, T. M. & de Pinna, E. (2011). A novel real-time polymerase chain reaction for identification
of Salmonella enterica subspecies enterica. Diagn Microbiol Infect Dis 70, 278–280. Kelterborn, E. (1967). Salmonella Species: First Isolations, Names
and Occurrences, 1st edn. Springer, London. Kidgell, C., Reichard, U., Wain, J., Linz, B., Torpdahl, M., Dougan, G. & Achtman, M. (2002). Salmonella typhi, the causative agent of typhoid McQuiston, J. R., Parrenas, R., Ortiz-Rivera, M., Gheesling, L., Brenner, F. & Fields, P. I. (2004). Sequencing and comparative
analysis of flagellin genes fliC, fljB, and flpA from Salmonella. J Clin Microbiol 42, 1923–1932. Mermin, J., Hutwagner, L., Vugia, D., Shallow, S., Daily, P., Bender, J., Koehler, J., Marcus, R., Angulo, F. J. & Emerging Infections Program FoodNet Working Group (2004). Reptiles, amphibians, and human
Salmonella infection: a population-based, case-control study. Clin Infect Dis 38 (Suppl 3), S253–S261. Mortimer, C. K. B., Peters, T. M., Gharbia, S. E., Logan, J. M. J. & Arnold, C. (2004). Towards the development of a DNA-sequence based
approach to serotyping of Salmonella enterica. BMC Microbiol 4, 31. Pedersen, K., Lassen-Nielsen, A. M., Nordentoft, S. & Hammer, A. S. (2009). Serovars of Salmonella from captive reptiles. Zoonoses Public
Health 56, 238–242.
Bertrand, S., Rimhanen-Finne, R., Weill, F. X., Rabsch, W., Thornton, L., Perevoscikovs, J., van Pelt, W., Heck, M. & Editorial team (2008).
Peters, T., Hopkins, K. L., Lane, C., Nair, S., Wain, J. & de Pinna, E. (2010). Emergence and characterization of Salmonella enterica serovar
Salmonella infections associated with reptiles: the current situation in Europe. Euro Surveill 13.
Scuderi, G., Fantasia, M. & Niglio, T. (2000). Results of the three-year
Carpenter, K. P. (1968). Report and minutes of the meeting of the
Typhimurium phage type DT191a. J Clin Microbiol 48, 3375–3377. surveillance by the Italian SALM-NET System: human isolates of Salmonella serotypes. Eur J Epidemiol 16, 377–383.
International Enterobacteriaceae subcommittee, Moscow 1966. Int J Syst Bacteriol 18, 191–196.
Warwick, C., Lambiris, A. J., Westwood, D. & Steedman, C. (2001).
Centers for Disease Control and Prevention (CDC) (1999). Reptile-
Reptile-related salmonellosis. J R Soc Med 94, 124–126.
associated salmonellosis-selected states, 1996–1998. MMWR Morb Mortal Wkly Rep 48, 1009–1013.
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Wuthe, H. H. (1969). [Occurrence of Salmonella dubrovnik and Salmonella epicrates in humans]. Arch Hyg Bakteriol 153, 176–178.
Journal of Medical Microbiology 63
DOI 10.1099/jmm.0.072702-0
Salmonella enterica subspecies II infections in England and Wales – the use of multilocus sequence typing to assist serovar identification Satheesh Nair,1 John Wain,2 Steve Connell,1 Elizabeth de Pinna1 and Tansy Peters1 Correspondence
1
Satheesh Nair
2
[email protected]
Received 31 December 2013 Accepted 30 March 2014
Salmonella Reference Service, Public Health England, Colindale, London NW9 5EQ, UK Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK
Identifying rare Salmonella serotypes by conventional serotyping can be a problem for diagnostic or reference microbiology laboratories. We report two cases of the seldom encountered serovar Salmonella Dubrovnik, which is known as Salmonella subspecies II 41 : z : 1,5, in an elderly man and a young child. Multilocus sequence typing, a technique that is being used more frequently in our laboratory, was used to assist serovar identification as serotyping proved to be inconclusive. To our knowledge, these cases were not linked geographically and there were no known associations between them although they occurred within a very short time of each other.
INTRODUCTION Classification and identification of Salmonella are complex, and are mainly based on serological detection of antigens. The complexity is often due to the presence of Salmonella strains that do not fully express their antigens and hence confuse the identification process (Grimont & Weill, 2007; Peters et al., 2010; Achtman et al., 2012). Therefore reference laboratories frequently receive isolates that are difficult to type and require a combination of microbiological and molecular methods for identification. Our laboratory screens Salmonella isolates using TaqMan realtime PCR for identification of the most common subspecies of Salmonella enterica (subsp. I and III) (Hopkins et al., 2009, 2011), as well as multilocus sequence typing (MLST) for problematic strains. Between July and August 2010, three unusual isolates were referred to the Public Health England Salmonella Reference Service (formerly the Health Protection Agency Salmonella Reference Unit) for confirmation and typing. Two isolates came from the same elderly male patient (83 years of age) admitted to hospital with fever. The first isolate was from a blood culture and had a presumptive identification from the sending laboratory of Salmonella sp., while the second isolate from a faecal culture had a presumptive identification of Salmonella arizonae. The third isolate was from faeces of a 3-year-old female hospitalized with severe diarrhoea. There was no known link between the cases, and they were hospitalized in geographically separated regions. Despite treatment with appropriate antibiotics the elderly male deteriorated and died following admission. Abbreviations: MLST, multilocus sequence typing; ST, sequence type.
072702 G 2014 Crown Copyright
Printed in Great Britain
In order to establish whether the isolates from these two cases belonged to the less common S. enterica subsp. salamae (subsp. II), S. enterica subsp. houtenae (subsp. IV) or Salmonella bongori (previously subsp. V), a range of biochemical tests were performed together with serum agglutination tests, MLST (Kidgell et al., 2002) and fljB and fliC genotyping (Mortimer et al., 2004; McQuiston et al., 2004).
METHODS Bacterial isolates and identification. The two isolates from the
elderly male were submitted on 9 and 22 July 2010, and the isolate from the child was submitted on 11 August 2010, to the Salmonella Reference Service (SRS) of Public Health England (PHE). This is the national reference laboratory for salmonellas from humans in England and Wales. All isolates were typed phenotypically and were further tested genotypically. A retrospective analysis of all the available clinical and epidemiological data was also carried out on every S. enterica subsp. salamae present in the SRS database between 2004 and 2012. Serotyping. Isolates were cultured and serotyped by SRS staff
using specific antisera. Biochemical analysis and serotyping were in accordance with the White–Kauffmann–Le Minor scheme (Grimont & Weill, 2007). Real-time PCR. Salmonella isolates were screened using an in-house TaqMan real-time PCR assay for identification of the most common subspecies of S. enterica (Hopkins et al., 2009, 2011). MLST. MLST was performed as previously described (Kidgell et al., 2002) by determining the sequences of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA). The data obtained were compared with the Salmonella MLST database at the University College, Cork (http://pubmlst.org) and isolates were assigned
831
S. Nair and others sequence type (ST) numbers as defined by the database. Sequence types were assigned based on the set of allele types derived from each of the seven loci. Flagellar gene typing. Genes encoding flagellin [fliC encodes
the phase 1 flagellar (H1) antigen and fljB encodes the phase 2 flagellar (H2) antigen] are typically conserved at the 59 and 39 ends but variable in the central region in Salmonella and can be used in molecular serology (Mortimer et al., 2004; McQuiston et al., 2004). Partial sequences of the fliC and fljB gene were obtained as previously described by Mortimer et al. (2004) and McQuiston et al. (2004).
RESULTS All three isolates were negative in a simplex 59 nuclease TaqMan assay that targets the hilA gene, indicating that the isolates were not S. enterica subsp. enterica (subsp. I). A duplex 59 nuclease PCR assay for identification of the Arizona group was negative for the LacZ locus but positive results were obtained for the ttrRSBCA locus involved in tetrathionate respiration. This confirmed the presence of Salmonella DNA but indicated that the isolates were not S. enterica subsp. arizonae (subsp. IIIa) or S. enterica diarizonae (subsp. IIIb). A range of biochemical tests were performed together with serum agglutination tests to confirm the presence of less common subspecies of S. enterica. The three isolates were unreactive with a polyvalent Salmonella somatic [O] serum but tested positive with a polyvalent flagellar [H] serum indicative of the presence of Salmonella flagella antigens. Additional agglutination testing identified the phase 1 and phase 2 flagellar antigens (H antigens) to be H15z and H251,5. This was confirmed by amplification and partial sequencing of the fliC and fljB genes (data not shown). Due to the complexity/difficulty in identifying the [O] antigen, further titration against a wider range of groupspecific antisera covering the Group C and Group S serogroups revealed the somatic [O] antigen to be either 6,7 or 41 according to the White–Kauffmann–Le Minor classification. MLST was then used to obtain the possible identity of the Salmonellae involved. MLST clustered the isolates with other subsp. II serotypes within the Salmonella MLST
database, with an identical match to ST1082. The ST1082 isolate in the MLST database is 1709K, the Kauffmann reference strain of S. enterica subsp. salamae 41 : z : 1,5 (formerly serotype Dubrovnik, isolated from a reptile in Yugoslavia in 1964) (F.-X. Weill, Institut Pasteur, personal communication). Although biochemical results were not atypical and confirmed S. enterica subsp. salamae (subsp. II), the techniques were laborious and time consuming, and required long incubation times. Eventually the isolates were serologically classified with the formula 41 : z : 1,5 (somatic antigen [O] : flagellar antigen [H] phase I : phase II) and named as Salmonella subsp. II 41 : z : 1,5 according to current nomenclature (Grimont & Weill, 2007). The possibility of cross-contamination between the isolates from the elderly man and the child was ruled out because the isolates were received and processed by our laboratory at different times. Between January 2004 and December 2012, our laboratory identified 280 isolates of S. enterica subsp. salamae of which 223 were from human infections including three cases of bacteraemia. The majority of these isolations were single occurrences of extremely rare salmonellas, although a few notable exceptions appeared with greater frequency during that period (Table 1). Of the human-derived isolates, 90 % (200/223) were fully sensitive to a wide range of antimicrobials using standard methods of determination (Frost, 1994). Where resistance was present it was usually to a single antimicrobial, with only two records of multidrug resistance (SRS in-house data). The highest incidence of infection with S. enterica subsp. salamae was seen in young children. For isolates from patients of known age, 52 % were from children 4 years of age or less, of which 33 % were from infants younger than 1 year (Fig. 1). Of the 57 non-human isolates, 23 were from animal sources, of which only three were listed as reptiles. Interestingly, of the 16 food isolates, eight were from either herbs or spices. Although six isolates were listed as ‘source unknown’, they all originated from private food laboratories, thus
Table 1. The most prevalent Salmonella enterica subsp. salamae isolated in England and Wales, 2004”2012 Former serovar name Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella
832
Sofia Tranoroa
Hagenbeck Nairobi
New name Subsp. Subsp. Subsp. Subsp. Subsp. Subsp. Subsp.
II II II II II II II
58 : l,z13,z28 : z6 4,12:b:55 : k : z39 30:l,z28 : z6 50 : b : z6 48 : d : z6 42 : r:-
Number of cases 26 20 19 15 12 11 10
Journal of Medical Microbiology 63
S. enterica subsp. II infections in England and Wales
importance as sources of human salmonellosis are not well described and much of the available literature about reptiles and salmonellas focuses on S. enterica subsp. arizonae, S. enterica subsp. diarizonae and S. enterica subsp. houtenae (Warwick et al., 2001; Mermin et al., 2004; Pedersen et al., 2009).
80
No. of reported cases
70 60 50 40 30 20 10 0
Under 1
1–4
5–15 16–45 Age (years)
46–64
65 plus
Fig. 1. Cases of Salmonella enterica subsp. salamae by age in England and Wales, 2004–2012 (N5221).
implicating their origin as from an unknown food. Environmental samples accounted for ten of the remaining isolates. One hundred and seventy-nine of the 223 human cases had no history of foreign travel.
DISCUSSION MLST as a replacement for serotyping in S. enterica has been demonstrated recently (Achtman et al., 2012). In this study, MLST was used to confirm the identity of three strains that were difficult to serotype in a reference laboratory setting. This clearly shows the potential of using MLST in support of routine reference work. In the 1960s, the International Subcommittee on the Taxonomy of the Enterobacteriaceae renamed serovars belonging to S. enterica subsp. salamae (Carpenter, 1968). These serovars are now known as Salmonella subsp. II followed by their antigenic formula (e.g. Salmonella Dubrovnik is now known as Salmonella subsp. II 41 : z : 1,5) (Table 1). Since it was first identified as Salmonella Dubrovnik in 1964 (Kelterborn, 1967) there had been no reports of this serovar in England and Wales until 2004. To date there have only been six isolated cases (one with foreign travel to Italy) of this extremely rare serovar reported in our laboratory although there were four cases reported in Italy between 1994 and 1996 based on a SALM-NET study (Scuderi et al., 2000). Serovars of S. enterica subsp. salamae are typically reported as being associated with cold-blooded hosts, and are cited as rarely causing human infections, but a review of the existing literature does not provide much information on this subspecies, and only one publication specifically names Salmonella Dubrovnik (Wuthe, 1969). Whilst it is noted that turtles, snakes, iguanas and lizards may all carry S. enterica subsp. salamae, the carrier rates and relative http://jmm.sgmjournals.org
While the large majority of human salmonellosis in England and Wales is due to infection with S. enterica subsp. enterica (subsp. I), S. enterica subsp. salamae can still cause gastroenteritis and occasionally systemic disease in warm-blooded hosts. It may also be responsible for severe infection in the young, very old or immunocompromised patients who then require hospitalization (Angulo & Swerdlow, 1995; CDC, 1999). Most of the infections with this subspecies that we saw in our laboratory were accompanied by gastroenteritis, with systemic infection apparent in only a very few cases. Although the outcome of bacteraemia cases may occasionally be fatal, there did not appear to be a problem with antibiotic resistance that would have implications for treatment. The fact that the highest incidence of S. enterica subsp. salamae infection was seen in young children was not unexpected. As noted by Bertrand et al. (2008) the prevalence of infants younger than one year of age may be partly due to the bias in investigating childhood salmonellosis cases more thoroughly. However, our data show that when S. enterica subsp. salamae was compared with other non-typhoidal serovars the incidence was still higher than would be expected for simple age-bias (C. Lane, PHE, personal communication). Most literature states that isolates belonging to subspecies other than S. enterica subsp. enterica (subsp. I) are almost exclusively obtained from exotic sources such as reptiles. It has been suggested that whenever Salmonella belonging to subspecies other than enterica are found in humans, an exotic pet source should be suspected. There are no specific data on reptile ownership in our retrospective dataset, as this information has not been routinely gathered in England and Wales.
CONCLUSION The source of infection in single cases of salmonellosis is rarely identified, especially when the strain is a seldomencountered serovar without any apparent endemic bias preference. Although 44 of the human cases here were known to be associated with foreign countries, especially the African continent (22/44), the majority of cases had no history of foreign travel. Thus it is difficult to identify management strategies without being able to identify potential environmental or food sources. We are not sure whether the case of Salmonella subsp. II 41 : z : 1,5 bacteraemia described here was due to the virulence of the serovar alone or to the underlying health of the elderly patient. As the only link between the two cases is loosely temporal we have to assume that the appearance of both 833
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cases in a short period of time was coincidental. Although it is generally believed that cold-blooded animals are the major reservoir of S. enterica subsp. salamae, it is also important to consider food-borne infections. If it is assumed that such cases are always sporadic infections from reptiles, either directly or indirectly, they may never be reported to the relevant public health authorities.
Frost, J. A. (1994). Testing for resistance to antibacterial drugs. In
The identification of rare Salmonella serotypes can be hindered due to the difficulty of identifying the somatic [O] antigen or flagella [H] antigens, which might be caused by varying expression levels of the genes involved. MLST was used in this study for rapid and accurate identification of the strains involved. With the current trend for using whole genome sequencing for diagnostic and public health microbiology, MLST will be used in real-time for identifying all Salmonellas within our laboratory in the near future.
Rapid identification of Salmonella enterica subsp. arizonae and S. enterica subsp. diarizonae by real-time polymerase chain reaction. Diagn Microbiol Infect Dis 64, 452–454.
ACKNOWLEDGEMENTS
fever, is approximately 50 000 years old. Infect Genet Evol 2, 39–45.
This work could not have been completed without the help of the staff at the Salmonella Reference Service, Public Health England. We would also like to thank Dr Kathie Grant for critically reading the manuscript. The authors have no conflicts of interest to declare.
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