Mongolian National University of Medical Sciences Institute of

Mongolian National University of Medical Sciences Institute of Veterinary Medicene Result of Listeria monocytogenes differentials аnd to determine antibiotic resistant Leader: B. Ariunsanaa Ph. D B. Narangerel Ph. D Presenter: E. Enkhtaivan

Background In the European Union listeriosis follows an upward trend that began in 2008, causing 2, 161 confirmed cases and 210 reported deaths in 2014, 16% more than in 2013. For our country, the population official data diagnosed with Listeria is not yet available but the livestock such as goat, sheep of 21 provinces is diagnosed. Population might be risk contaminated with food products from animal and listeriosis epidemic among population of locality having an epicenter. Therefore, our research aim is to detect Listeria monocytogenes those risks by the Mongolian of listeriosis.

About pathogen Listeria monocytogenes is pathogenic, foodborne bacterium that causes listeriosis. Listeria monocytogenes is Gram positive, motility bacillus. In the environment it is common in soil and water.

• The genus Listeria has containing about 10 species. • Both L. ivanovii and L. monocytogenes are pathogenic in animal, but only L. monocytogenesis consistently associated with human illness. • The 13 serotypes of L. monocytogenes can cause disease, but more than 90% of human isolates belong to only three serotypes: 1/2 a, 1/2 b, and 4 b.

• Listeriosis affects mainly pregnant women, newborns, the elderly, and adults with impaired immune systems. • In pregnant women, the infection can result in miscarriage, premature delivery, serious infection of the newborn, or even stillbirth.

What are the symptoms? • The symptoms of listeriosis include fever, muscle aches, and sometimes nausea or diarrhea. • If infection spreads to the nervous system, symptoms occur such as headache, stiff neck, confusion, loss of balance, or convulsions can occur.

Transmitted Infections • Infection risk of food products from animal and plant products especially from the fruits and vegetables.

Our aim • To detect Listeria monocytogenes • Determination of susceptibility to antibiotics • Define the serotype and virulence gene

Study of objective • Selection sample risk group of people and detecting Listeria monocytogenes by bacteriological test. • Check Listeria monocytogenes determination of antibiotic susceptibility. • Detection of antibiotic resistance, virulence genes and serotype the pathogen.

MATERIALS AND METHODS incubation 35ºC 48 h Sample selection. Total 62 incubation 35ºC, 48 h The PCR products were further analyzed by electrophoresis in 1. 2 -1. 5% agarose gel for 40 min in Tris-acetate buffer PCR Lis. iap gene 1500 bp L. m iap gene 400 bp Gram’s method DNA extraction Boil, Phenolchloroform

MATERIALS AND METHODS Listeria spp. iap gene L. mono iap gene 37ºC, 24 h hly gene 234 bp Prf A ген 467 bp Serotype 4 b, 1/2 a, 1/2 b aad 6 - streptomycin, П Г У Cat- chloramphenicol msr(A)-macrolid 37ºC, 24 h Bla. TEM- амpicillin tet(K)- tetracycline Sul 1. 2 - sulphanilamid vancomycin, амоxicillin, амpicillin, nalidixic acid, streptomycin, tetracycline, cefotaxim, chloramphenicol, erythromycin, gentamycin, nitrofurantoin penicillin G

MATERIALS AND METHODS Nucleotide sequences of primer sets used in this study. Target gene Primer Sequence Product Size (bp) Listeria. spp iap GCTACAGCTGGGATTGCGGT gene TTATACGCGACCGAAGCCAA CAAACTGCTAACACAGCTACT L. мono iap gene GCACTTGAATTGCTGTTATTG 1500 L. mono hly gene 234 CGGAGGTTCCGCAAAAGATG CCTCCAGAGTGATCGATGTT L. mono prf. A gene ACCAATGGGATCCACAAGA CAGCTGAGCTATGTGCGAT 400 467

Antibiotic-resistant genes determination used primer m. PCR. Target gene Primer Sequence Product Size (bp) aad 6 AGAAGATGTAATAATATAG CTGTAATCACTGTTCCCGCCT 978 Cat GAACAGGAATTAATAGTGAG GGTAACCATCACATAC 384 erm(A) CTTCGATAGTTTATTAATATTAGT TCTAAAAAGCATGTAAAAGAA 645 Bla. TEM ATGAGTATTCAACATTTCCG ACCAATGCTTAATCAGTGAG 692 Sul 1. 2 GCGCTCAAGGCAGATGGCATT GCGTTTGATACCGGCACCCGT 293 tet(k) CGATAGGAAGAGCAGTATGG TTAGCCCACCAGAAAACC 614

Serotype of primer sets used in this study. Target gene Primer Sequence Product Size (bp) Lmo 0737 AGGGCTTCAAGGACTTACCC ACGATTTCTGCTTGCCATTC 691 Lmo 1118 AGGGGTCTTAAATCCTGGAA CGGCTTGTTCGGCATACTTA 906 ORF 2819 AGCAAAATGCCAAAACTCGT CATCACTAAAGCCTCCCATTG 471 GCTGAAGAGATTGCGAAAGAAG CAAAGAAACCTTGGATTTGCGG 370 AGTGGACAATTGGTGAA CATCCCTTACTTTGGAC 597 prs ORF 2110

Bacteriological test results • Total 62 sample • 13 sample positive

Result in selective media • Black colonies like listeria. PALCAM and OXFORD agar.

Morphological result Gram positive, bacillus.

Molecular biological test results The result of PCR determined by Listeria. spp Listeria. spp iap gene M- Маrker 1 - negative control /water/ 2 -L. iva 3 -EGDE 4 - Lis V 2009 5 - Lis I 6 - isolated cultures

The result of PCR determined by L. monocytogenes L. mono iap gene M- Маrker 1 -Lis V 2004 2 -Lis V 2009 3 -Lis I 4 - isolated cultures 5, 6 - food sample 7 - negative control /L. iva/ 8 - negative control /water/

L. monocytogenes serotypes determined by m. PCR M- Marker 1 - EGDE 2 -negative control /L. iva/ 3 - Lis V 2004 4 - Lis V 2009 5 - Lis I 6 - isolated cultures L. monocytogenes serotype 1/2 b 4 b 597 х. н 471 х. н 370 х. н 1/2 b 1/2 a 471 х. н 370 х. н 691 х. н

Virulence gene determines PCR result M- Маrкеr 1 - EGDE 2 - negative control L. iva 3 - Lis V 2004 4 - Lis V 2009 5 - Lis I 6 - isolated cultures Virulence hly gene

Virulence gene determined by PCR result Virulence prf(A) gene M- Маrker 1 - EGDE 2 - negative control L. iva 3 - Lis V 2004 4 - Lis V 2009 5 - Lis I 6 - isolated cultures

Virulence verified with biological test • We have infected 10 laboratory mice to check virulence. After 24 -48 h all mice dead. So we confirmed that Listeria monocytogenes is toxic.

Results of sensitivity to antibiotics Antibiotic use in study isolated culture mm EUCAST V. 7. 1 mm result Gentamycin 26 21 S tetracyclin 34 27 -31 S Erthyromycin 34 25 S Vancomycin 25 16 S Nalidixic acid R R R Амоxicillin 24 16 S Амpicillin 20 16 S Penicillin G 22 16 S Streptomycin 20 12 -20 S Cefotaxim 12 29 -35 R Nitrofurantoin 23 20 -25 I

Conclusion • This study validated PCR in the analysis and detection of L. monocytogenes in human samples. • Listeriosis can be treated by tetracycline, streptomycin, vancomycin, ampicillin, amoxicillinis but cefotaxime shown to be ineffective. • The 1/2 b serotype of Listeria monocytogenes were detected in people who were infected.

Thank you for attention.