DNA-labelling by multiplex single-primer amplification with biotin-dUTP

Author: Annett Reißig, R&D at Alere Technologies GmbH, Jena, Germany. e-Mail: annett.reissig@clondiag.com ; Download: http://alere-technologies.com/fileadmin/Media/Paper/Poster/DGHM-2015_Salmonella-Lagos.pdf

Automatically generated report with information about the sero- and AMR-genotype

Hybridisation and analysis of the microarray-signals

Annett Reißig(1,5), Sascha Braun(1,5), Stella Smith (2), F. Akintimehin (3), T. Fesobi (4), M. Bamidele (2), AO Cocker (3), Stefan Monecke (1,5,6),Ralf Ehricht (1,5)

(1Alere Technologies GmbH, Germany; ²Molecular Biology and Biotechnology division, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria; ³College of Medicine, University of Lagos, Nigeria; 4Public Health division, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria; 5InfectoGnostics Research Campus, Germany; 6Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Germany)

Introduction: Food-borne salmonellosis is one of the most prevalent zoonotic diseases worldwide. It is important that serotype identification amongst culture-confirmed cases is carried out for epidemiological purposes. Particularly in developing countries, accurate serotype detection is cumbersome and sometimes inaccurate while in Germany, new rules for quality control increase costs of classical serotyping. This makes standardised DNA-microarray-based approaches, i.e., serogenotyping, an attractive option. The aim of the study was to test a recently developed microarray technique by Alere Technologies GmbH (Jena, Germany) by serogenotyping local isolates of Salmonella spp. from food samples in Lagos, Nigeria, as well as to phenotypically and genotypically characterise antimicrobial susceptibility patterns of these isolates. A total of 151 samples of meat, including beef, chicken, pork and goat, were purchased from various abattoirs and markets in six administrative units in the Lagos state.

Methods and Results: Out of 151 samples, 40 isolates were initially confirmed identified Salmonella spp. using the API system, while 33 (82.5 %) of these 40 isolates were confirmed to be Salmonella by VITEK 2. These isolates were subsequently analysed by microarray-based serogenotyping as well as tested for antibiotic susceptibility using agar diffusion assays and the VITEK 2 system. Within this panel, nine different Salmonella serovars were found: S. Amoutive (n=8), S. Bargny (n=5), S. Drac (n=3), S. Ealing (n=5), S. Urbana (n=1), S. Hadar (n=1), S. Nyborg (n=3), S. Anatum (n=5) and S. Havana (n=2). Antibiotic susceptibility testing of 17 antibiotics with the VITEK 2 system showed that all the isolates were susceptible to imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, moxifloxacin, tetracycline, tigecycline, fosfomycin, co-trimoxazole and piperacillin/tazobactam, indicating that, e.g., fluoroquinolones might be used for presumptive therapy. One isolate of serovar S. Nyborg was resistant to ampicillin while another isolate belonging to S. Amoutive was resistant to ampicillin, ampicillin/sulbactam, cefuroxime, ceftazidime, cefotaxime and cefuroxime-axetil. Susceptibility testing of nalidixic acid was done by agar diffusion and revealed five isolates that were resistant (S. Amoutive, S. Drac, S. Bargny, S. Ealing and S. Havana).

Conclusions: The study indicates a presence of Salmonella strains in retail meat samples from Lagos that are considered to be rare elsewhere. Although few isolates proved to be resistant, it is recommended to frequently monitor for antimicrobial resistance. The used system proved to be perfectly suited to replace serotyping for surveillance.

Nal – nalidixic acid, Ap – ampicillin, Ap/Sul – ampicillin/sulbactan, Pip/Tazo – piperacillin/tazobactan, Cefu – cefuroxime, Cefu/Axe - cefuroxime-axetil, Cefo – cefotaxime, Cefta – ceftazidime, Imi – imipenem, Mero – meropenem, Gen – gentamicin, Tobra – tobramycin, Cip – ciprofloxacin, Moxi – moxifloxacin, Tet – tetracycline, Tig – tigecycline, Fosf – fosfomycin, Ctx–co-trimoxazole

aLG: Local Government; colour coding indicates sample sites, as in the map above

Species

Serovar

Serogroup

Antigenic Formula

invA/galF/manC

O-Antigen

H1-Antigen

H2-Antigen

Nig-009 Beef Mushin/Mushin S.e. enterica Havana G (O:13) 1,13,23:f,g,[s]:- +/+/+13,23

f,g -

Nig-014 Chicken Ketu/Kosofe S.e. enterica Anatum E1 (O:3,10) 3,{10}{15}{15,34}:e,h:1,6 +/+/+3,10

e,h 1,6

Nig-015 Goat Ketu/Kosofe S.e. enterica Anatum E1 (O:3,10) 3,{10}{15}{15,34}:e,h:1,6 +/+/+3,10

e,h 1,6

Nig-017 Beef Ketu/Kosofe S.e. enterica Anatum E1 (O:3,10) 3,{10}{15}{15,34}:e,h:1,6 +/+/+3,10

e,h 1,6

Nig-026 Beef Yaba/Mainland S.e. enterica Ealing O (O:35) 35:g,m,s:- +/+/+35

g,m,s -

Nig-027 Beef Mushin/Mushin S.e. enterica Nyborg E1 (O:3,10) 3,{10}{15}:e,h:1,7 +/+/+3,10

e,h 1,7

Nig-028 Goat Mushin/Mushin S.e. enterica Ealing O (O:35) 35:g,m,s:- +/+/+35

g,m,s -

Nig-032 Beef Mushin/Mushin S.e. enterica Nyborg E1 (O:3,10) 3,{10}{15}:e,h:1,7 +/+/+3,10

e,h 1,7

Nig-045 Chicken Berger/Ikeja S.e. enterica Hadar C2-C3 (O:8) 6,8:z10:e,n,x +/+/+8

z10 e,n,x

Nig-057 Beef Mushin/Mushin S.e. enterica Urbana N (O:30) 30:b:e,n,x +/+/+30

b e,n,x

Nig-063 Chicken Yaba/Mainland S.e. enterica Drac n.d. n.d.:l,v:e,n,x +/+/+47

l,v e,n,x

Nig-077 Beef Surulere/Surulere S.e. enterica Ealing O (O:35) 35:g,m,s:- +/+/+35

g,m,s -

Nig-079 Beef Surulere/Surulere S.e. enterica Bargny C2-C3 (O:8) 8,20:i:1,5 +/+/+8,20

i 1,5

Nig-082 Beef Surulere/Surulere S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-087 Goat Mushin/Mushin S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-102-1 Beef Surulere/Surulere S.e. enterica Bargny C2-C3 (O:8) 8,20:i:1,5 +/+/+8,20

i 1,5

Nig-102-2 Beef Surulere/Surulere S.e. enterica Bargny C2-C3 (O:8) 8,20:i:1,5 +/+/+8,20

i 1,5

Nig-107 Chicken Mushin/Mushin S.e. enterica Drac n.d. n.d.:l,v:e,n,x +/+/+47

l,v e,n,x

Nig-111 Beef Yaba/Mainland S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-120 Chicken Ojota/Kosofe S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-121 Beef Ojota/Kosofe S.e. enterica Anatum E1 (O:3,10) 3,{10}{15}{15,34}:e,h:1,6 +/+/+3,10

e,h 1,6

Nig-122 Pork Ojota/Kosofe S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-124 Chicken Ketu/Kosofe S.e. enterica Havana G (O:13) 1,13,23:f,g,[s]:- +/+/+13,23

f,g -

Nig-125 Chicken Surulere/Surulere S.e. enterica Bargny C2-C3 (O:8) 8,20:i:1,5 +/+/+8,20

i 1,5

Nig-129-1 Chicken Yaba/Mainland S.e. enterica Bargny C2-C3 (O:8) 8,20:i:1,5 +/+/+8,20

i 1,5

Nig-129-2 Chicken Yaba/Mainland S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-134 Beef Mushin/Mushin S.e. enterica Anatum E1 (O:3,10) 3,{10}{15}{15,34}:e,h:1,6 +/+/+3,10

e,h 1,6

Nig-135 Chicken Mushin/Mushin S.e. enterica Nyborg E1 (O:3,10) 3,{10}{15}:e,h:1,7 +/+/+3,10

e,h 1,7

Nig-144 Goat Surulere/Surulere S.e. enterica Drac n.d. n.d.:l,v:e,n,x +/+/+47

l,v e,n,x

Nig-145 Beef Surulere/Surulere S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-147-2 Beef Surulere/Surulere S.e. enterica Amoutive M (O:28) 28:d:1,5 +/+/+28

d 1,5

Nig-149 Beef Mushin/Mushin S.e. enterica Ealing O (O:35) 35:g,m,s:- +/+/+35

g,m,s -

Nig-150 Beef Mushin/Mushin S.e. enterica Ealing O (O:35) 35:g,m,s:- +/+/+35

g,m,s -

StrainSample

sourceSample sites/LG

a

Results of microarray based Serotyping Results of classical Serotyping