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Eurosurveillance, Volume 8, Issue 2, 01 February 2003
Editorial
Investigation of human infections with Salmonella enterica serovar Java in Scotland and possible association with imported poultry

Citation style for this article: Brown DJ, Mather H, Browning LM, Coia JE. Investigation of human infections with Salmonella enterica serovar Java in Scotland and possible association with imported poultry. Euro Surveill. 2003;8(2):pii=399. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=399
DJ Brown 1, H Mather 1, LM Browning 2, JE Coia 1.

1. Scottish Salmonella Reference Laboratory (SSRL), Glasgow, Scotland
2. Scottish Centre for Infection & Environmental Health (SCIEH), Glasgow, Scotland


PFGE analysis of S. Java strains (29 from humans, 30 from poultry meat) showed two major clusters. All isolates from poultry imported from the Netherlands belonged to Cluster A, which also comprised 10 human isolates. Thirty-one of the 37 isolates in this cluster had an identical JavX1 pattern, similar to the X8 profile of a particular S. Java clone predominant in poultry production in several European countries. Cluster B comprised 19 human isolates and two poultry isolates of unknown origin. These results combined with epidemiological data and information on the origins of poultry meat strongly suggested that imported poultry meat is an important source of Java infections in humans in Scotland.

Introduction


Poultry and poultry products, including meat and eggs, have long been recognised as an important source of food-borne infections caused by Salmonella enterica (1,2). The global increase in human infections with serovar Enteritidis observed in the late 1980's and early 1990's (3) was almost entirely attributable to the presence of this organism within the poultry production industry worldwide. However, the implementation of national monitoring programmes, together with control measures including vaccination, has resulted in recent years in a reduction in cases of human salmonellosis associated with the consumption of poultry and egg products in the UK (4).
The Scottish Salmonella Reference Laboratory (SSRL) receives all strains of S. enterica isolated from cases of human infection from hospital laboratories. Regional veterinary laboratories, public analysts and water authorities submit isolates of animal, food and environmental origin. All isolates are fully serotyped, phage typed (where applicable), and tested for resistance to 15 antimi-
crobial agents. A selection of isolates is further typed by plasmid profile analysis and pulsed-field gel electrophoresis. Results are reported to the Scottish Centre for Infection & Environmental Health (SCIEH).
In the early summer 2002, a number of isolates of S. enterica serotype Java (S. Java) were examined as part of an exercise to build up a database of plasmid profile and pulsed-field gel electrophoresis (PFGE) data for the top ten serotypes isolated from humans in Scotland at that time (see table 1). Although numbers of isolations of this serotype were relatively small, a small increase was apparent (figure 1). Considerable diversity was observed in plasmid profile, antibiogram, and PFGE pattern, but some isolates from sporadic human cases were found to possess a PFGE pattern indistinguishable from that in some strains of Java isolated from poultry meat. This observation prompted further investigation of other human and poultry isolates of S. Java held in the culture collection at SSRL, and monitoring of all new isolations of this serovar.

Table 1
The top ten ranking serotypes of S. enterica isolated from human infections in Scotland in 2001-02

Rank

Serotype

2001 Total (%)

Serotype

2002 Total

(to week 50)

1

S. Enteritidis

992 (63.1)

S. Enteritidis

616 (54.7)

2

S. Typhimurium

255 (16.2)

S. Typhimurium

217 (19.3)

3

S. Virchow

41 (2.6)

S. Virchow

37 (3.3)

4

S. Hadar

22 (1.4)

S. Hadar

23 (2.0)

5

S. Braenderup

15 (1.0)

S. Agona

22 (2.0)

6

S. Montevideo

15 (1.0)

S. Stanley

17 (1.5)

7

S. Agona

14 (0.9)

S. Java

14 (1.2)

8

S. Java

14 (0.9)

S. Montevideo

11 (1.0)

9

S. Stanley

14 (0.9)

S. Infantis

10 (0.9)

10

S. Infantis

12 (0.8)

Blockley, Derby, Newport, Saint-paul

8 (0.7)

 

Others (62 serotypes)

177 (11.3)

Others

128 (11.4)

 

Total

1571

Total

1127

 


Laboratory studies


A total of 59 strains of S. Java were included in the study. Thirty isolates originated from 29 human cases between June 1994 and November 2002. All human isolates from 2001 and 2002 were examined while clones isolated previously were selected based on resistance to antibiotics. The remaining 29 isolates were all isolated from poultry meat or poultry skin. Poultry isolates were submitted to SSRL by commercial poultry companies after recovery during routine in-house sampling. Although information on these isolates is incomplete, it is thought that thirteen of them had been recovered from poultry meat originating in the Netherlands, and sampled on 6 different occasions between July 1997 and September 2002.
Plasmid profiling was carried out as previously described (2). PFGE was carried out using a standardised protocol which has been implemented in a number of European reference laboratories under the European Union funded Salm-gene project (see article pp46-50). This was done to allow ease of comparison with other countries. Briefly, chromosomal DNA plugs were digested with the restriction endonuclease XbaI, and subjected to electrophoresis using a CHEF DR-II® apparatus (BioRad, USA). Running conditions were 1% agarose in 0.5x TBE, 6 V/cm for 22 hours, initial pulse 2 seconds, final pulse 64 seconds. Analysis of PFGE patterns was carried out using Phoretix 1-D Advanced® (v 5.0) software (Nonlinear Dynamics, England). Antimicrobial resistance was determined by growth on solid media containing antibiotics at breakpoint concentrations (ampicillin 50 mg/ml, cefotaxime 1mg/ml, chloramphenicol 20mg/ml, ciprofloxacin (high level) 0.5mg/ml, ciprofloxacin (low level) 0.12 mg/ml, furazolidone 20mg/ml, gentamicin 20mg/ml, kanamycin 20mg/ml, nalidixic acid 40mg/ml, netilmicin 20mg/ml, spectinomycin 100mg/ml, streptomycin 20mg/ml, sulphamethoxazole 100mg/ml, tetracycline 10mg/ml, trimethoprim 2mg/ml).

Results and discussion


When comparing typing data for isolates of S. Java, the initial impression was that we were dealing with a highly diverse organism (see table 2). All but 13 of the isolates examined for plasmid content contained at least one plasmid and up to five different plasmids. It was also noted that isolates recovered from poultry meat sampled at the same time did not all possess the same plasmid profile. These isolates contained between 3 and 5 plasmids in different combinations. While these differences may at first seem to complicate matters, it should be noted that variation in plasmid profile in salmonellae with close epidemiological connections has been reported previously (2,5).
The results from antibiotic resistance screening also revealed wide variability. All isolates from poultry products were multiresistant (resistant to 3 or more antibiotics) while 11 human isolates were fully sensitive, one was resistant to two antibiotics and 18 were multiresistant. Antimicrobial resistance data from the SSRL database on all human isolates of S. Java confirmed that the number of multiresistant strains has been on the increase since the mid-1990s (figure 1).

Table 2
Summary of typing results for isolates of S. enterica Java recovered from humans and poultry products in Scotland

Ref. Number

Origin

PFGE cluster

Plasmid profile (kbp)

Antibiotic resistance1

Comments

942590

Human, 1994

B

Plasmid free

ApCmSpStSuTc

 

970652

Human, 1997

B

110;3.6

ApSuTm

 

973591

Chicken fillet 1997

A

110

ApNaStTcCpL

Imported Holland

982035

Chicken fillet 1998

A

105;2.1

FuKmSpStSuTcTm

Unknown origin

992264

Human, 1999

A

110

ApFuSpStSuTm

 

995029

Human, 1999

B

Plasmid free

ApCmSpStSuTc

 

003337

Human, 2000

A

160;6.5

CmFuSpStSuTcTm

 

004082

Human, 2000

B

Plasmid free

ApCmSpStSuTc

 

004250

Chicken fillet, 2000

A

110

ApSpStSuTm

Unknown origin

010300

Human, 2001

B

Plasmid free

ApCmSpStSuTc

 

011304

Human, 2001

B

4.4;3.7

Sensitive

 

011405

Human, 2001

A

200;110;4.5;2.2

ApFuSpStSuTm

 

011589

Human, 2001

B

Plasmid free

ApCmSpStSuTc

 

011667

Human, 2001

B

Plasmid free

Sensitive

Travel associated

012082

Human, 2001

B

Plasmid free

ApCmSpStSuTc

 

012388

Chicken skin, 2001

A

100;4.4

ApSpStSuTm

Unknown origin

012551

Human, 2001

A

150;6.7

CmSpStSuTcTm

 

012737

Human, 2001

B

Plasmid free

Sensitive

Travel associated

013612

Human, 2001

B

Plasmid free

Sensitive

 

013944

Human, 2001

B

80

Sensitive

Travel associated

014536

Human, 2001

B

90;4.4;3.7

ApCmSpStSuTc

 

014602

Chicken fillet, 2001

A

100

NaSpStTcTmCpL

Unknown origin

014749

Human, 2001

B

100

ApSu

 

020036

Human, 2002

A

100;4.2

ApSpStSuTm

 

020593

Human, 2002

B

90

Sensitive

 

020898

Chicken joint, 2002

B

90

NaSpStSuTmCpH

Unknown origin

020947

Chicken joint, 2002

B

100

ApFuNaSpStSuTmCpH

Unknown origin

020969

Human, 2002

A

Plasmid free

ApCmSpStSuTc

 

021169

Chicken joint, 2002

A

100

ApNaSpStSuTmCpH

Imported Holland

021175

Human, 2002

A

100

ApCmSpStSuTc

 

021274

Human, 2002

B

Plasmid free

Sensitive

 

021481

Chicken meat, 2002

A

110;5.4;5.1;5.0;2.1

ApSpStSuTm

Imported Holland

021485

Chicken joint, 2002

A

110;6.5

FuKmNaSpStSuTcTmCpL

Imported Holland

021626

Human, 2002

B

100

Sensitive

Travel associated

021637

Chicken joint, 2002

A

110

ApSpStSuTm

Unknown origin

021715

Chicken meat

A

110

SpStTm

Unknown origin

021790

Human, 2002

B

ND

Sensitive

Travel associated

021816

Chicken joint, 2002

A

ND

ApSpSuTm

Unknown origin

021857

Human, 2002

B

ND

Sensitive

 

022432

Human, 2002

ND

Plasmid free

Sensitive

 

022528

Chicken joint, 2002

A

110;6.0

SpStSuTm

Unknown origin

022634

Human, 2002

A

110

ApSpStSuTm

 

2 x Isolates

Chicken joint, 05/09/2002

A

110;5.4;5.1;5.0;2.1

ApSpStSuTm

Imported Holland

9 x isolates

Chicken joint 10/09/2002

A

Various combinations

ApSpStSuTm

5 confirmed imported Holland

4 x isolates

Chicken joint

13/09/2002

A

Various combinations

ApSpStSuTm

1 confirmed imported Holland

023030

Human, 09/2002

A

100

ApFuNaSpStSuTcTmCpH

See 023338

023338

Human, 10/2002

A

100

ApNaSpStSuTmCpH

Repeat isolate from patient


ND - Not Done
Ap ampicillin; Cm chloramphenicol; CpH ciprofloxacin (high level); CpL ciprofloxacin (low level); Fu furazolidone; Km kanamycin; Na nalidixic acid; Sp spectinomycin;
St streptomycin; Su sulphonamide; Tc tetracycline; Tm trimethoprim


Pulsed-field gel electrophoresis has been demonstrated to be a valuable tool in the investigation of outbreaks of salmonellosis, and in the identification of sources of infection. Digestion of chromosomal DNA with XbaI provided a good range of fragment sizes and easily discernible patterns under these running conditions (figure 2). Comparison of fragment patterns by the Dice coefficient and using UPGMA (unweighted pair-group method using arithmetic averages) clustering generated two major clusters (Figure 3). Cluster A comprised 10 of the 29 human isolates examined, the earliest from 1999, and 28 of the 30 poultry isolates. All isolates from poultry imported from Holland belonged to this cluster. PFGE patterns within this cluster were relatively homogeneous, 30 isolates had an identical fragment pattern (designated JavX1) with the remaining variants differing by a small number of bands. Cluster B comprised 19 human isolates and 2 poultry isolates of undetermined origin. This cluster was much more heterogeneous with only 3 patterns being represented by more than a single isolate.


It has recently been reported that a particular clone of S. Java has become predominant in poultry production in
Germany in the latter half of the 1990s (6), and this can be distinguished by its XbaI PFGE profile (X8). Preliminary comparison strongly suggested that this pattern was the same as the JavX1 pattern reported in this study. Moreover, a similar degree of variability in plasmid profile and antibiogram was described in the German study, and the authors had identified isolates from Belgium and Holland as belonging to the same clone.
Seventeen of the thirty isolates recovered from poultry were recovered from meat of unknown origin. The latest available report on the isolation of salmonella from livestock in the United Kingdom records only a single isolation of S. Java from cattle, sheep, pigs or poultry, including the statutory monitoring of breeding flocks and hatcheries, in the period from the beginning of 1997 to the end of 2001 (7). This isolation was made from ducks or geese in 2000. From the same report, no isolations of S. Java were made from animal feedstuffs during 2000 or 2001. It would therefore seem unlikely that the poultry meat infected with S. Java originated in the United Kingdom.
The potential for poultry meat to act as a vehicle for multiresistant strains is a matter of concern for public health. In recent years, the number of cases of human infection caused by multiresistant Java strains with the type A PFGE profile has increased in Scotland (one in 1999, one in 2000, two in 2001 and five in 2002). In particular, the presence of resistance to quinolone antimicrobials, at high as well as low levels of resistance, is important. Fluoroquinolone antibiotics such as ciprofloxacin are the drugs of choice in cases of invasive salmonellosis in humans. Cross-resistance between quinolones and fluoroquinolones is well documented (8). In a recent Danish study, an increased mortality rate was observed in patients in a two-year period following infection with S. Typhimurium when the infecting strain was resistant to quinolones (9). From the Netherlands, it has been reported that levels of infection with S. Java have increased in poultry from 2% of all isolates prior to 1996 to 40% in 2001, and the clone responsible has been demonstrated as the X8/JavX1 PFGE type (10). Resistance to flumequin, a quinolone antibiotic currently licensed for use in livestock in the EU, has increased in S. Java in Holland from 3% between 1996-99 to 20% between 2000-02.
Plasmid profiling and antibiogram typing have previously given valuable information on the source of salmonella infections. However, in this case PFGE proved to be invaluable in building an association between cases of human infection and isolates from poultry meat. The evidence from PFGE analysis, together with the epidemiological information available for human infections, and the origins of poultry meat, strongly implicate imported poultry meat as an important source of S. Java infections in humans in Scotland. Despite the recent reports on the high levels of S. Java infections in poultry flocks in Germany and the Netherlands (6,10), no reports of a similar increase in human cases have appeared and there have been suggestions that the current poultry associated clone may be of reduced virulence in man. In this study, we demonstrate that infection in humans, although relatively rare, does occur. The same multiresistant clone found in poultry in Germany and the Netherlands has been responsible for a significant proportion of human cases of S. Java infection in Scotland, particularly since 2000. All new cases of S. Java in the human population in Scotland will continue to be monitored.
The application of standardised protocols to facilitate the investigation of international outbreaks has previously been advocated (11). This approach was invaluable in allowing the direct comparison of PFGE typing results with those of colleagues in Germany and the Netherlands.

Acknowledgements

This work was supported in part by grants from the European Commission (Salm-gene project QLK2-CT-2001-01940).

Références

1. Baggesen DL, Wegener HC. Phage types of Salmonella enterica ssp. enterica serovar Typhimurium isolated from production animals and humans in Denmark. Acta Vet Scand 1994; 35:349-354.
2. Olsen JE, Sørensen M, Brown DJ, Gaarslev K, Bisgaard M. Plasmid profiles as an epidemiological marker in Salmonella enterica serovar berta infections. Comparison of isolates obtained from humans and poultry. APMIS 1992; 100:221-228.
3. Rodrigue DC, Tauxe RV, Rowe B. International increase in Salmonella enteritidis: A new pandemic? Epidemiol Infect 1990; 105:21-27.
4. Kessel AS, Gillespie IA, O'Brien SJ, Adak GK, Humphrey TJ, Ward LR. General outbreaks of infectious intestinal disease linked with poultry, England and Wales, 1992-1999. Commun Dis Public Health 2001; 4:171-177.
5. Brown DJ, Threlfall EJ, Rowe B. Instability of multiple drug resistance plasmids in Salmonella typhimurium isolated from poultry. Epidemiol Infect 1991; 106:247-257.
6. Miko A, Guerra B, Schroeter A, Dorn C, Helmuth R. Molecular characterization of multiresistant d-tartrate-positive Salmonella enterica serovar Paratyphi B isolates. J Clin Microbiol 2002; 40:3184-3191.
7. Anon. Salmonella in Livestock Production in GB, 2001. 2002; Weybridge, Surrey, UK: Veterinary Laboratory Agency.
8. Piddock LJ. Fluoroquinolone resistance in Salmonella serovars isolated from humans and food animals. FEMS Microbiol Rev 2002; 26:3-16.
9. Helms M, Vastrup P, Gerner-Smidt P, Molbak K. Excess mortality associated with antimicrobial drug-resistant Salmonella typhimurium. Emerg Infect Dis 2002; 8:490-495.
10. van Pelt W, van der Zee H, Wannet WJB, van der Giessen AW, Mevius DJ, Bolder NM, et al. An explosive increase of Salmonella Java in poultry in the Netherlands: Is it a public health threat? Infectieziekten Bulletin 2002; 13:260-265.
11. Lindsay EA, Lawson AJ, Walker RA, Ward LR, Smith HR, Scott FW, et al. Molecular characterization of a multiresistant strain of Salmonella enterica serotype Typhimurium DT204b responsible for an international outbreak of salmonellosis: importance of electronic exchange of gel data for outbreak investigations. Emerg Infect Dis 2002; 8:732-734.



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