NATIONAL SALMONELLA, SHIGELLA & LISTERIA REFERENCE LABORATORY OF IRELAND (HUMAN HEALTH) ANNUAL REPORT FOR 2010 NATIONAL SALMONELLA, SHIGELLA & LISTERIA REFERENCE LABORATORY 1 N N SS R R L L Introduction The National Salmonella Reference Laboratory was established in 2000 with support from the Department of Health and Children to provide reference services related to human health. It is a public service laboratory with 3 full time scientific staff. It operates from the Department of Bacteriology at the Clinical Science Institute, NUI Galway. The NSRL website is at http://www.nuigalway.ie/salmonella_lab/. The reference laboratory uses a number of phenotypic methods (serotyping, antibioticresistance testing and phage typing) and molecular methods (pulse-field gel electrophoresis (PFGE) and multi-locus variable number tandem repeat analysis (MLVA) to precisely characterise isolates of Salmonella, Shigella and Listeria spp. This process can be considered as genetic fingerprinting of food-borne bacteria. The goal of this genetic fingerprinting is to assist relevant agencies in protecting public health by identifying and interrupting chains of transmission of food-borne bacteria. A particular phenomenon that has become very prominent in Ireland in recent years is the increase in the number of isolates that represent a monophasic variant of S. Typhimurium. Cells of S. Typhimurium, as with most other serovars of Salmonella, have genes for 2 different types of flagellar antigens (called phase 1 flagella and phase 2 flagella). In S. Typhimurium the phase 1 flagellar antigen is “i" and the phase 2 flagellar antigen is “2”. There is a process of genetic switching (variation between 2 “phases”) in Salmonella so that a culture of S. Typhimurium comprises a mixture of individual cells some of which are expressing “i" and some of which are expressing “2”. There are variants of S. Typhimurium that only have the capacity to express one type of flagellar antigen (monophasic variants) so that all cells in the culture express a single common flagellar antigen. Such monophasic variants have long been recognised but have been relatively uncommon. This situation has changed quite 2 dramatically in recent years with increasing numbers of monophasic S. Typhimurium in Ireland and throughout Europe. of significant research. The biological basis for this change is the subject The growth of this phenomenon in Ireland is referred to in this report. The Laboratory is committed to providing a high quality and timely service and has achieved accreditation to the ISO15189 standard from the Irish National Accreditation Board (INAB). The continued success of the laboratory is entirely dependent on the support of the staff in the laboratories that submit isolates for typing. My colleagues and I appreciate that the preparation, packing and dispatch of isolates is a significant burden and would like to thank you for your support over the years. I would also like to acknowledge the support of all those agencies with whom we work closely to ensure that the service we provide works as information for action. In particular I would like to thank the Food Safety Authority of Ireland, the Health Protection Surveillance Centre and colleagues in Public Health Departments and Environmental Health Departments throughout the country and to acknowledge the work of colleagues in the National Reference Laboratory Salmonella (Food, Feed and Animal Health)1. If you have any comments or questions arising from the report please feel free to contact me at the email address given below. Martin Cormican Director National Salmonella Reference Laboratory martin.cormican@hse.ie An extract from the 2010 Annual Report of National Reference Laboratory Salmonella (Food, Feed and Animal Health) is available at http://www.agriculture.gov.ie/media/migration/animalhealthwelfare/labservice/nrl/N RL%20Spring%202011%20Newsletter.pdf 3 Salmonella In 2010, 1070 isolates were submitted to the National Salmonella Reference Laboratory. When non-Salmonella, QC contaminants and duplicate isolates were removed a total of 923 Salmonella isolates were typed. This represents a 26% increase in the number of isolates received in 2009. There were 364 human clinical isolates, including 334 faecal isolates, 22 from blood (including 6 S.Typhi, 2 S.Paratyphi A and 1 S.Paratyphi B), 3 other invasive isolates, and 5 urine isolates. S.Typhimurium (n = 113) and its monophasic variant 4,5,12:i:- (n = 19) and S.Enteritidis (n = 70) predominated (Table 2). There was marked seasonal variation with the highest number of isolates occurring in months May to November. This coincides with the warmer months of the year and with the peak season for foreign travel (Fig.1) and may be related in part to one or both of these factors. In some cases more than one isolate was received from a patient. For example we may have received an invasive isolate (e.g. from a blood culture) and an isolate from faeces from the same patient. Where invasive and faecal isolates come from the same patient, only the invasive isolate is recorded to avoid duplication. The average turnaround time for testing was 6.3 days (range 2-32 days). An equal number of Salmonella isolates from humans were received in the NSRL in 2010 and 2009 (Table 1). Table 1: Number of Salmonella isolates received in NSRL Year Human Non-human 2010 364 559 2009 364 368 2008 447 815 2007 457 653 2006 430 308 2005 357 494 2004 420 650 2003 486 634 4 2002 394 540 2001 508 574 2000 636 214 Table 2: Top sixteen serotypes of Human Isolates (including Typhi and Paratyphi) Serotype Frequency % Typhimurium 113 31 1 4,5,12:i:- 19 5.2 Enteritidis 70 19.2 Infantis 17 4.7 Newport 8 2.2 Typhi 8 2.2 Braenderup 7 1.9 Dublin 7 1.9 Saintpaul 7 1.9 Montevideo 7 1.9 Java 7 1.9 2 3 0.8 Stanley 5 1.4 Concord 5 1.4 Paratyphi A 4 1.1 Virchow 4 1.2 Others 73 19.8 Total 364 100 1 4,5,12:b:- This antigenic formula is that of S. Typhimurium except that the phase 2 antigen is not expressed. These isolates are generally referred to as monophasic S. Typhimurium. 2 Isolates with this antigenic formula are most likely monophasic variants of S. Java, i.e. phase 2 antigen is not expressed. 5 Seasonal Variation in Salmonella isolates 1 Figure 1 60 no. of isolates 50 Total Salmonella 40 Enteritidis 30 20 Typhimurium/ 4,5,12:i:- 10 Foreign Travel Dec Nov Oct Sep Aug Jul Jun May Apr Mar Feb Jan 0 Month of receipt in NSRL 1. The line “Foreign Travel” describes the number of cases of salmonellosis for which an association with recent foreign travel was reported to NSRL. Reporting of recent foreign travel is likely to be incomplete. It is important to note that there is always an interval between the time of onset of symptoms and date of isolate receipt in the NSRL. This includes time taken for patient to seek medical attention, taking and transporting the sample to the primary laboratory, isolation of Salmonella, and referral to NSRL. Salmonellosis non-typhoidal S.Typhimurium and its monophasic variant. S.Typhimurium 4,5,12;i:2 and its monophasic variant (4,5,12;i:-) together accounted for 36% of all cases of Salmonella. Phage type DT8 was the most common (21%) phage type among human isolates in 2010. This was driven by a single outbreak associated with consumption of duck eggs. Phage types DT104 accounted for 20%, DT104b 11% and DT193 accounted for 14% of the total number of S.Typhimurium/4,5,12:i:- isolates. Apart from DT8 the predominance of phage type DT104 and related phage types are similar to recent years. 6 S.Enteritidis S.Enteritidis accounted for 19% of all cases of Salmonella. The predominant phage types were PT14b (24%), PT1 (20%), PT4 (13%), PT21 (9%) and PT8 (6%). The increase in PT14b was primarily caused by an outbreak (n = 9) of nalidixic acid resistant PT14b. Salmonella Typhi and Paratyphi Eight isolates of S.Typhi, 4 isolates of S.Paratyphi A and 1 isolate of S.Paratyphi B were received. A history of recent travel was recorded for all of the S.Typhi isolates; 7 to the Indian subcontinent and 1 to Guinea (West Africa). Two of the S.Paratyphi A isolates were associated with reported travel to the Indian subcontinent, while 1 isolate had foreign travel to Asia with country not stated. A history of travel to South America (country not stated) was recorded for the S.Paratyphi B isolate. Antimicrobial resistance Fifty-two percent of isolates (n = 189) were susceptible to all antimicrobial agents tested. Thirty percent of isolates (n = 109) were multi-drug resistant (three or more different classes of antibiotics). Of the 30% of multi-drug resistant isolates, 24% (n=26) had the profile of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline (ACSSuT) and were mainly S.Typhimurium DT104 or closely related phage types. This group of S. Typhimurium DT104 has accounted for a high proportion of multidrug-resistant Salmonella isolates for some years now. Ten extended spectrum beta-lactamase (ESBL) producing isolates were detected. All were from patients with a history of foreign travel including 5 S.Concord isolates from children adopted from Ethiopia. The association of ESBL producing S. Concord with children adopted from Ethiopia has been noted in a number of countries and a report of an international collaborative study on this phenomenon in which NSRL participated was published in 2009. Other ESBL producing isolates included those from patients with travel history to Thailand (S.Typhimurium and 4,5,12:i:-), Poland (S.Enteritidis PT4), India (S.Worthington), and Morocco (S.Typhimurium). One AmpC producing isolate was received, a S.Newport, from a patient with no reported history of foreign travel. 7 Two isolates of Salmonella resistant to ciprofloxacin were detected. These were both S.Kentucky, one with travel history to East Africa, and the other with no report of foreign travel. Resistance to ciprofloxacin is rare among Salmonella but a ciprofloxacin-resistant clone of S. Kentucky has arisen and spread from North Africa in the last decade. In addition 61 isolates resistant to nalidixic acid and with reduced susceptibility to ciprofloxacin were detected; an increase from 50 in 2009. Travel related infection A history of recent foreign travel was recorded in 40% (147) of human cases of infection (Table 3), Ireland was noted as the country of infection in 38.5% (140) of cases while 21% (77) had no country of infection recorded. Spain was the most commonly recorded travel destination (recorded in 24 cases) with S. Enteritidis (n = 15) accounting for the majority of these cases. More than half (61%) of S.Enteritidis isolates were associated with foreign travel compared to 19% for S.Typhimurium and its monophasic variant combined. Although NSRL does not have access to data on the number of Irish people who travel to each country it is likely that the number of cases associated with each country is at least in part accounted for by the popularity of the country as a holiday destination. Table 3: Foreign travel history for Salmonella isolates Continent Country Number Europe (n = 54) Spain 24 Turkey 8 UK 5 Poland 4 Malta 3 Italy 2 Bulgaria 1 France 1 Germany 1 Lithuania 1 Greece 1 8 Croatia 1 Croatia/Bosnia 1 The Netherlands 1 Africa (n = 36) Ethiopia 6 Nigeria 6 Tunisia 6 Egypt 4 Morocco 3 S.Africa 1 Algeria 1 Chad 1 Ghana 1 Guinea 1 Sudan 1 Zambia 1 Tanzania 1 Tanzania/Zanzibar 1 Mauritius 1 Mauritius/Africa 1 Australasia (n = 51) Thailand 16 India 9 Indonesia 4 Pakistan 3 Bangladesh 3 Phillipines 2 UAE 2 Vietnam 2 Cambodia 2 Asia * 2 Nepal 1 9 Bahrain 1 Australia 1 Australia/Malaysia 1 Malaysia/Thailand 1 Maldives/Dubai 1 Americas (n = 6) Carribean/USA 2 USA 1 Mexico 1 South America * 1 Dominican Republic 1 * Country not stated Clusters Twenty-six clusters of cases involving 106 isolates were identified in 2010. S.Typhimurium was involved in 13 clusters while S.Enteritidis was implicated in 3 clusters. Eight of the clusters were family outbreaks, i.e. all patients affected were from one family, while four outbreaks were associated with foreign travel (all patients had travelled to same country). A point source outbreak involving a S.Infantis strain was linked to a self-catered confirmation party and turkey was the suspected vehicle. Fourteen of thirty people were sick after the event while 9 S.Infantis isolates were received in the NSRL. Detailed subtyping detected a S.Montevideo outbreak (n = 5) occurring in the East of the country. PFGE proved useful in distinguishing sporadic cases from the outbreak strain. The outbreak of S.Typhimurium phage type DT8 that began in August 2009 continued in 2010. A total of 28 DT8 isolates from human cases were typed in the NSRL in 2010. This 10 phage type is associated with ducks and several of the cases were linked epidemiologically with ducks or consumption of duck eggs. The UK has also seen a similar outbreak which is also linked to ducks. The NSRL has liased with the Central Veterinary Research Laboratory, the Food Safety Authority of Ireland, the Health Protection Surveillance Centre and other agencies in the investigation and control of this outbreak and a detailed account is in preparation in association with other partners involved in managing this issue. The NSRL liaises with the European Centre for Disease Control (ECDC) in the investigation of outbreaks that may have an international dimension. The UK declared an outbreak of S.Bareilly involving 224 isolates since August 2010 linked to the consumption of beansprouts. The NSRL typed a S.Bareilly isolate with an indistinguishable pulsed field profile. This patient had travelled to London in September and had eaten beansprouts. An outbreak of S.Enteritidis PT14b with resistance to nalidixic acid in which the first cases were seen here in November 2009 continued until March 2010 (11 cases in 2009 and 9 cases in 2010). A similar increase in isolation of this strain in the UK was considered to be associated with consumption of Spanish eggs. (http://www.hpa.org.uk/NewsCentre/NationalPressReleases/2010PressReleases/100914coo perationcurtailedsalmonellaoutbreak/). Irish isolates have been provided to colleagues in the UK to assist in further research on this strain. Evidence of Links between S. Typhimurium in Humans, Food and Animals. Multi locus variable number tandem repeat analysis (MLVA) is a relatively new technology that allows for very fine discrimination between isolates that appear very closely related by other methods including PFGE. This precise discrimination has proved a useful technique in showing persuasive evidence of links between human S.Typhimurium clusters and individual cases and recent food and animals sources. Table 4 illustrates molecular and other laboratory evidence of links between human infection and food /food animal types. In some cases the link is complete from animal, to animal related food product and to human cases. Interpretation is not always straightforward as illustrated in footnotes 5 and 6. 11 The importance of using standardised and readily comparable methods for “fingerprinting” human, food and animal isolates of Salmonella not just within Ireland but across Europe and the world is illustrated by this body of work over the past year. Table 4 MLVA profiles of human, food and animal S. Typhimurium isolates Isolate no. Source Phage type Resistance MLVA profile 28 Isolates Human DT8 none 2-10-NA-12-2121,2 119 Isolates Duck3 DT8 none 2-10-NA-12-2122,4 MS100758 Human Untypable none 2-15-17-11-212 MS100800 Human Untypable none 2-15-17-11-212 (N = 3) Bovine faeces Untypable none 2-15-17-11-212 MS1008045 Dairy product Untypable none 2-15-17-11-212 Group 1 Group 2 Group 3 MS100796 Human(Typhimurium) DT120 ASSuT 3-11-10-NA-211 MS100926 Human (4,5,12:i:-) ASSuT 3-11-10-NA-211 (N = 4) Bovine faeces (4,5,12:i:-) DT193 ASSuT 3-11-10-NA-211 MS100185 Human DT104 ACSSuT 3-13-15-14-311 MS100504 Bovine faeces DT104 Not tested 3-13-15-14-311 MS100054 Human (4,5,12:i:-) DT193 ASSuT 3-13-9-NA-211 MS100080 Human (4,5,12:i:-) DT193 ASSuT 3-13-9-NA-211 MS1006866 Human (4,5,12:i:-) DT193 ACSSuTCazCtx 3-13-9-NA-211 MS1006427 Human (4,5,12:i:-) U323 ASSuT 3-13-9-NA-211 MS100911 Human (Typhimurium) DT120 none 3-13-9-NA-211 (N = 2) Bovine faeces(4,5,12:i:-) DT193 ASSuT 3-13-9-NA-211 DT193 Group 4 Group 5 12 (N = 2) Chicken (4,5,12:i:-) DT193 ASSuT 3-13-9-NA-211 (N = 6)8 Swine (4,5,12:i:-) Untypable ASSuT 3-13-9-NA-211 MS100018 Human (4,5,12:i:-) U311 ASSuT 3-14-10-NA-211 (N = 18)9,10 Swine U311 ASSuT 3-14-10-NA-211 MS101008 Human DT104b ACSSuTTm 3-14-6-11-311 MS101024 Human DT104b ACSSuTTm 3-14-6-11-311 MS101047 Human DT104b ACSSuTTm 3-14-6-11-311 MS101048 Human DT104b ACSSuTTm 3-14-6-11-311 MS100342 Swine faeces DT104b ACSSuT MS100513 Human DT104b ACSSuTTm 3-14-6-12-311 MS100167 Bovine DT104b ACSSuT 3-14-6-13-311 (N = 5) 12 Swine DT104b ASu 3-14-6-12-311 MS100891 Human DT104 SSu 3-15-17-11-311 MS100908 Human DT104 SSu 3-15-17-11-311 MS100914 Bovine faeces DT104 SSu 3-15-17-11-311 MS100220 Human DT104b ACSSuT 3-15-6-13-311 (N = 5) 13 Swine DT104b14 ACSSuT 3-15-6-12-311 (N = 2) 13 Swine DT1214 ACSSuT 3-15-6-12-311 MS100534 Human DT104b ACSSuT 3-16-5-12-311 MS100645 Bovine faeces DT104b ACSSuT 3-16-5-12-311 Group 6 Group 7 3-14-6-11-311 Group 8 11 Group 9 Group 10 Group 11 13 1. A small number of isolates exhibited antimicrobial resistance as follows, 2 resistant to streptomycin, 1 resistant to ampicillin and 1 resistant to Ampicillin, sulphonamide and trimethoprim 2. A small number of isolates showed a degree of variation in the MLVA pattern for example, 2-9-NA-12212 3. In addition there was a single isolate each of S. Typhimurium DT8 from feline and bovine sources 4. A small number of isolates exhibited antimicrobial resistance as follows, 2 resistant to streptomycin, 1 resistant to streptomycin, sulphonamide, tetracycline and trimethoprim 5. This isolate was from a previously opened product and post-opening contamination was suspected. 6. History of recent travel to Thailand – it is unlikely that these strains are epidemiologically related – this could reflect convergence of unrelated strains on a common MLVA profile 7. History of recent travel to Spain – it is unlikely that these strains are epidemiologically related – this could reflect convergence of unrelated strains on a common MLVA profile. 8. Group 5 included porcine sponge (n=5) and pig meat (n=1). 9. There were in addition a number of isolates other then U311 with indistinguishable MLVA profile as follows; (a) phage untypable (ASSuT), (b) DT193 (ASSuT) and (c) DT104 (ACSSuT). These isolates may be related to this group. 10. Group 6 included swine (n=7) and pork (n=11). 11. Likely to be closely related to Group 7. 12. Group 8 included swine (n=3), swine food (n=1) and sausages (n=1). 13. Group 10 included swine animals (n=4) and swine meat (n=3). 14. Phage types DT104, DT104b and DT12 are closely related Animal Contact A history of animal contact was recorded for 75 patients with salmonellosis including contact with turtles, lizards, snakes, fish, pet birds, horses, dogs and farm animals (Table 5). Dogs were the most common contact animal (n = 34) while contact with cats was less common (n = 11). As dogs and cats are very common companion animals contact with these animals is not persuasive evidence of a link between the infection and the animal species. However in some cases typing of isolates from patients and individual animals they had contact with, e.g. lizards (S.Oranienburg and S.subsp.IV 48:g,z51) showed them to be indistinguishable. 14 Other strong links included a family outbreak of S. Newport and pet turtles; single cases of S.Muenchen and a serovar 4,5,12:i:- (monophasic Typhimurium) DT191A associated with turtle contact; contact with lizards and cases of S.Itami, S.Muenchen, S.Muenster and S.subsp. IV 45:g,z51. There were also 2 cases of S.Typhimurium DT135 linked to pet canaries. This strain was also linked with pet canaries in previous years. However an indistinguishable strain has also been isolated from cattle. Although public information on the risk (particularly to children) of contact with reptiles has been circulated it appears that this may not be reaching relevant sections of the population or may not have resulted in modification of risk behaviour. It may be appropriate to consider if further steps to limit exposure of children to risk of salmonellosis from contact with reptiles and other exotic pets is appropriate. In total 19 isolates of Salmonella (approx. 5.2 % of all human cases) were associated with contact with exotic animals, predominately reptiles. Among people that had a recorded history of living on farms or working with farm animals (n = 24), S.Typhimurium/4,5,12:i:- predominated (n =15), especially the DT104 phage group (n = 6). Three patients with S.Typhimurium DT8 had contact with ducks. In the absence of more detailed analysis this must be interpreted with caution however it is noteworthy particularly as S. Typhimurium is more closely associated with acquisition in Ireland than the other major serovar S. Enteritidis. Many of the patients that had a history of animal contact also had other risk factors, e.g. recent history of foreign travel or consumption of particular food products such as duck eggs. It is important to note that Salmonella is primarily a foodborne disease and only a certain percentage of these animal contacts resulted in the patients’ infections. 15 Table 5: Animal contact history or strains associated with animal contact NSRL no. Source Sub1. Strain Animal Contact MS100327 Human I Newport Turtle MS100400 Human I Newport Turtle MS100722 Human I Muenchen Turtle MS100035 Human I 4,5,12:i:- DT191A Turtle MS100244 Human I Itami Lizards, Corn snake MS101025 Human I Muenchen Lizard MS100175 Human I Typhimurium DT10 Lizard MS100790 Human IV 45:g,z51 Lizard MS100020 Human I Oranienburg Lizard MS100021 Human I Oranienburg Lizard I Oranienburg - MS100044 1 Lizard faeces I Oranienburg - MS100150 Human I Oranienburg Lizard MS100375 Human I Muenster Bearded dragon (lizard) MS100501 Human IV 48:g,z51 Lizard MS100425 2 Lizard IV 48:g,z51 - MS100901 Human I Typhimurium DT135 Canaries MS101012 Human I Typhimurium DT135 Canary, cats, dogs MS100050 Human II 13,23:g,s,t Bird MS100011 Human I Enteritidis PT1 Zoo MS100102 Human I Enteritidis PT13a Octopus MS101023 Human I Enteritidis PT21 Bird MS100397 Human I Java Fish aquarium MS100647 Human II 1 42:r:- None Exotic animals MS100043 1 Lizard hide Farm or Food animals (including horses) MS100428 Human I Dublin Cattle MS100503 Human I Dublin Cattle- farmer 16 MS100793 Human I Dublin Farmer MS100358 Human I 4,5,12:i:- DT193 Vet assistant, pets MS100291 Human I 4,5,12:i:- DT193 Ponies, cats MS100348 Human I Typhimurium DT104 Horses, cattle, dogs MS100562 Human I Typhimurium DT104 Cattle, dogs MS100578 Human I Typhimurium DT104 Chickens MS100581 Human I Typhimurium DT104 Lives on farm MS100636 Human I Typhimurium DT104 Horses MS100891 Human I Typhimurium DT104 Meat worker MS100105 Human I Typhimurium DT120 Horse, dog, rabbit MS100911 Human I Typhimurium DT120 Horses, dogs MS100749 Human I Typhimurium DT8 Duck, dog, horse MS100860 Human I Typhimurium DT8 Ducks, hens, goat MS100894 Human I Typhimurium DT8 Ducks, hens, goat MS100523 Human I Typhimurium PT untypable Horses, dogs MS100758 Human I Typhimurium PT untypable Father has contact with Human I Enteritidis PT4 Horses,Cows, dogs, cats MS100489 Human I Enteritidis PT4 Cattle MS100036 Human I Enteritidis PT14b Horses, Dogs MS100543 Human I London Pigs MS100186 Human I Nima Cattle- calving MS100019 Human I Saintpaul Hens, Dogs MS100003 Human I Enteritidis PT1 Dog MS100088 Human I Enteritidis PT14b Hamster MS100756 Human I Enteritidis PT14c Dog MS100410 Human I Typhimurium DT104 Dog, cat MS100454 Human I Typhimurium DT104 Dogs MS100670 Human I Typhimurium DT104 Dogs MS100691 Human I Typhimurium DT104 Dogs MS100653 Human I Typhimurium DT104 Dog, cat horses MS100075 and hens Domestic pets 17 MS100757 Human I Typhimurium DT104 Dog, cat MS100100 Human I Typhimurium DT104b Dog MS100538 Human I Typhimurium DT8 Dogs MS100058 Human I Typhimurium DT8 Dog, cat MS100085 Human I Typhimurium DT8 Cat MS100695 Human I Typhimurium DT8 Cats MS100910 Human I Typhimurium DT8 Dog MS100702 Human I Typhimurium DT2 Dog MS100754 Human I Typhimurium PT untypable Dog MS100406 Human I 4,5,12:i:- PT untypable Dog MS100436 Human I 4,5,12:i:- DT193 Pets MS100443 Human I Kentucky Dogs MS100611 Human I Kentucky Rabbit MS101046 Human I Senftenberg Dogs MS100740 Human I Infantis Dogs MS100332 Human I Umbilo Dogs MS100594 Human I Javiana Dogs MS100741 Human I Panama Dog MS100755 Human I Unnamed (4,12:-:-) Dog MS100806 Human I Saintpaul Dog MS100698 Human I Newport Dog, cat MS100013 Human I Hadar Cat 1. Sub = Subspecies. There are over 2500 Salmonella serotypes of which approximately 60% belong to subspecies I. These account for about 99% of human infections. Subspecies I is present in both warm and cold blooded animals while the other Salmonella subspecies are generally associated with cold-blooded animals. 1 Isolates from pet lizard of patients MS110020 & MS110021. 2 Isolate from pet lizard of patient MS100501 18 Non-Human isolates In 2010, 559 isolates of Salmonella of non-human origin were submitted to the NSRL. This represents an increase of 52% on the number of non-human isolates received in 2009. The majority of isolates were from swine (n = 193) and poultry (n = 221) while there were 65 isolates from bovine sources. S.Typhimurium/4,5,12;i:- (n = 381) and S.Kentucky (n = 63) were the most prevalent serovars (Table 6). The increase in the S.Typhimurium isolates tested was due to the investigation of the duck-associated DT8 outbreak. Table 6 Top fifteen serotypes among non-human isolates Serotype Frequency Typhimurium %3 222 39.7 49 8.8 110 19.7 Kentucky 63 11.3 Dublin 18 3.2 Mbandaka 10 1.8 Virchow 7 1.3 Tennessee 6 1.0 Montevideo 6 1.0 Agona 4 0.7 Derby 4 0.7 Oranienburg 4 0.7 Livingstone 3 0.5 Ohio 3 0.5 50 8.9 1 4,5,12:i:- 2 Not serotyped Others Total 559 100 1 Most likely monophasic variants of S. Typhimurium 2 These are S. Typhimurium or 4,5,12:i:- (monophasic S. Typhimurium) isolates received from CVRL for phage typing only. 3 Approximate figures. 19 Salmonella serotypes and correlation with Human Infection S.Typhimurium S.Typhimurium and its monophasic variant 4,5,12;i:- accounted for 68.2% of all nonhuman isolates and were isolated from a variety of sources predominantly swine (n = 172) but also including poultry (n = 141) and bovine (n = 39) sources. Phage types DT104b (n = 33), DT193 (n = 32), Untypable (n = 22), U311 (n = 25), U302 (n = 21), DT120/DT120 low (n = 12) and DT104 (n = 8), were the most common phage types from swine. Phage type DT104 (n = 18) was the most common phage type among bovine isolates. As illustrated in Table 4 (above) there is evidence from molecular typing of a significant link between isolates from pigs and pig products and also with cattle and human infection. Salmonella Enteritidis. No S.Enteritidis isolates were received from non-human sources in the NSRL in 2010. It is of interest that S. Enteritidis is rarely detected from poultry or poultry products in Ireland but that S. Enteritidis is the first or second most common serovar isolated from human infections. In this context it is worth noting that a history of travel outside of Ireland is reported in relation to 61% of cases S. Enteritidis compared with only 19% of S. Typhimurium/4,5,12:i:-. Sources of Isolates Swine A total of 9 serovars accounted for the 193 isolates received from swine with S.Typhimurium/ 4,5,12:i:- (n = 172) accounting for the majority of isolates. Poultry A total of 221 isolates comprising 13 serovars were received from poultry sources. S.Typhimurium/ 4,5,12:i:- (n = 141) and S. Kentucky were the most common serovars (n = 56) and most isolates were susceptible to all antimicrobial agents tested. The vast majority of the S.Typhimurium isolates were from ducks while the S. Kentucky isolates were mainly from chickens. S.Virchow was also a common serovar from poultry sources (n = 7). There 20 were 4 human cases of S.Virchow in 2010 but no specific product was implicated in these infections. Bovine A total of 65 isolates were received from bovine sources. S.Typhimurium/4,5,12:i:accounted for the majority of isolates (n = 39) with DT104 (n = 18) being the most common phage type. There were also 14 S.Dublin isolates. There were 7 human cases of S.Dublin. No specific product was implicated in these infections but 3 were farmers. Antimicrobial Resistance Antimicrobial susceptibility testing was performed on 437 of the 559 non-human isolates (122 were referred for phage typing only). Fifty-six percent of isolates were susceptible to all antimicrobial agents tested. Thirty-four percent were multi-drug resistant (three or more different classes of antibiotics). Ten percent of isolates had the profile of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline (ACSSuT) and most of these typed as S.Typhimurium DT104 or closely related phage types. None of the non-human isolates were resistant to third generation cephalosporins or ciprofloxacin; however 26 isolates exhibited resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. Laboratory Contamination False-positive Salmonella results due to laboratory cross-contamination are a serious problem for laboratories and can be difficult to detect. Cross-contamination in a laboratory can result in inappropriate diagnosis of patient infection or in unfounded concerns regarding the safety of a food product. Detailed subtyping of isolates by the NSRL helps in detection and confirmation of laboratory contamination incidents (Role of Subtyping in Detecting Salmonella Cross Contamination in the Laboratory; BMC Microbiology: 9; 155). The NSRL assisted in recognising 9 such incidents (14 isolates) with Salmonella in 2010 with 3 of the incidents involving the laboratory positive control strain, S.Nottingham, and 1 incident involving a S.Alachua control isolate. Two of the S.Nottingham incidents involved food laboratories while the other involved a human clinical sample. The S.Alachua incident 21 occurred in a food laboratory. There was an incident of S.Enteritidis contamination (4 isolates) involving an external quality assessment sample in another laboratory. Four contamination incidents occurred with human clinical samples where the contaminating isolate arose from another patient’s sample. We would like to reiterate our request that all laboratories involved in testing Salmonella from any source use Salmonella Nottingham NCTC 7382 as their positive control. 22 Listeria monocytogenes Fig. 2 Dendrogram of L. monocytogenes 4b isolates digested with AscI Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-100.0%] 100 95 90 PFGE-AscI 85 80 PFGE-AscI MQ100005 Swine MQ100011 Sausage MQ100022 Sausage- Swine MQ100019 Sliced cooked ham MQ100007 Goats cheese starter MQ100018 CSF MQ100015 Ice cream from machine MQ100016 Blood culture MQ100030 Cooked duck The NSRL received 30 Listeria monocytogenes isolates and 1 Listeria innocua isolate in 2010. These included 4 human clinical isolates; 3 from blood cultures and 1 from CSF. Two of the isolates from humans typed as serotype 4b while 2 typed as serotype 1/2. Pulsed field gel electrophoresis (PFGE) with ApaI and AscI was performed on the L.monocytogenes isolates. Comparison of the serotype 4b patterns showed that MQ100016 matched human and food isolates from this and previous years (2004-10) with both AscI and ApaI. MQ100018 was an unusual profile that was similar (AscI 100% and ApaI 90%) to an isolate from goats cheese. This is not a basis for concluding that goats cheese was the vehicle of infection in this instance. Among the serotype 1/2 isolates the PFGE patterns of MQ100029 matched those of isolates from mushrooms, beef, pork and swine from 2006-09 and were very similar to a human case from 2009 (L07-029). 23 The majority of the food isolates serotyped as 1/2 (n = 19), while 7 isolates were serotype 4b. The NSRL is working with colleagues in food and veterinary microbiology in Ireland and with colleagues in Europe to build a library of typing data that may help to identify sources of human infection. typing. A critical limiting factor is the availability of human isolates for The total number of human clinical isolates in Ireland per year is very small therefore it is critical that all such isolates are available for typing and we appeal for all isolates to be forwarded for typing. 24 Shigella species Fig. 3 Dendrogram of PFGE of S.sonnei digested with XbaI Dice (Opt:0.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-98.3%] 100 98 96 94 92 90 88 86 84 PFGE-XbaI 82 80 78 PFGE-XbaI MH100009 SSuTTm MH100010 SSuTTm MH100011 SSuTTm MH100015 SSuTTm MH100030 Cameroon SSuTTm MH100048 Egypt SSuTTm MH100021 Nigeria SSuTTm MH100025 TmNaCp MH100027 TmNaCp MH100012 SSuTTmNaCp MH100026 India SSuTTmNaCp MH100033 India SSuTTmNaCp MH100017 MH100018 SSuTTm South America MH100037 MH100016 AST ASSuTTm India SSuTTmNa A total of 49 isolates were referred to the NSRL in 2010 for Shigella typing. When nonShigella, QC and duplicate isolates were removed a total of 39 Shigella isolates were typed. These included 17 S.sonnei, 18 S.flexneri, 3 S.boydii and 1 Shigella unidentifiable (did not react with any Shigella antisera). The S.flexneri isolates were further divided into 1 S.flexneri 1a, 3 S.flexneri 1b, 1 S.flexneri 1c, 2 S.flexneri 2a, 3 S.flexneri 3a, 2 S.flexneri 3b, 3 S.flexneri 4c, and 3 S.flexneri 6. Ninety-seven percent of isolates (n = 38) were multi-drug resistant (three or more different classes of antibiotics). A S.boydii isolate, where the patient had a history of recent travel to Egypt, was an ESBL producer while 9 isolates were resistant to ciprofloxacin. The ciprofloxacin resistant isolates included 6 Shigella sonnei, 2 of whom had travelled to India, and 2 S.flexneri 6 isolates, also with history of travel to India. A combination of pulsed field gel electrophoresis and antimicrobial susceptibility testing proved useful in outbreak detection and investigation. There were 2 suspected Shigella sonnei outbreaks in 2010. 25 Nineteen patients had a recorded history of recent foreign travel, including Africa (n = 10), Australasia (n = 7) and the Americas (n = 2). Nine patients had Ireland recorded as their country of infection while there were no details for 11 patients. Table 7: Foreign travel history for Shigella isolates Continent Country Number Africa (n = 10) Egypt 4 Nigeria 2 Cameroon 1 Ethiopia 1 Africa * 2 Australasia (n = 7) India 5 China 1 Pakistan 1 Americas (n = 2) South America Haiti 1 1 * Country not stated 26 NSRL Publications and Presentations 2010 Poster Presentations Comparative Genomic Analysis of Salmonella enterica Serovar Enteritidis Typing Phages SETP3, SETP7 and SETP13. I3S Conference, Saint-Malo, France. June 2010. Papers Nicolay, N., Garvey, P., DeLappe,N., Cormican,M., and P. McKeown. Completeness and timeliness of Salmonella notifications in Ireland in 2008: a cross sectional study. BMC Public Health. 2010. v.10: 568. Garvey, P., McKeown, P., DeLappe, N., and M.Cormican. Number of shigellosis cases in Ireland remain low. Epi-Insight. 2010. v.11: Issue 1. Garvey, P., Skally, M., McKeown, P., DeLappe, N., Doran, G., Ward, J., and M.Cormican. Indigenous salmonellosis low in Ireland. Epi-Insight. 2010. v.11: Issue 3. Boyle, F., Morris, D., O Connor,J., DeLappe, N., Ward, J., and M.Cormican. First Report of Extended-Spectrum –-Lactamase-Producing Salmonella enterica Serovar Kentucky Isolated from Poultry in Ireland. AAC. 2010. v.54: p.551-553. Nicolay, N., Thornton, L., Cotter, S., Garvey, P., Bannon, O., McKeown, P., Cormican, M., Fisher, I., Little, C., Boxall, N., De Pinna, E., Peters, T.M., Cowden, J., Salmon, R., Mason, B., Irvine, N., Rooney, P., and D.O Flanagan. Salmonella enterica serovar Agona European outbreak associated with a food company. Epidemiol. Infect.;. 2010 Oct 18: 1-9. 27