Simona Gazzola, Daniela Bassi, Pier Sandro ...

Prevalence of antimicrobial resistant bacteria in the food chain Simona Gazzola, Daniela Bassi, Pier Sandro Cocconcelli Istituto di Microbiologia- Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy - via Milano 24, 26100 Cremona, Italy

Results

Introduction  The Antimicrobial resistance (AMR) is a serious threat to public health worldwide. The surveillance programmes indicate that there is a general world-wide increase of antimicrobial resistance.  During last decades European legislation changed, the use of antibiotic to promote animal growth were banned in animal feed. Nevertheless antibiotics are still extensively used in animals and humans as therapeutic  Less information is available on the spread of these bacteria and of the AMR genes in the food chains and their fate during food fermentation. Moreover, few data are available on the consumer exposure to AMR through foods.

Distribution of antimicrobial resistance

Distribution of AR genes trough different cheeses

Comparative genome analysis

 Plating count technique showed an high number of Gram positive and Gram negative microrganisms with presumptive resistance to tetracycline, erythromycin and ampicillin.

 The most represented genetic determinants involved in antimicrobial resistance are ermB, ermTR, tetM and tetL.

 UC7256 and UC8668 shown higher identity to strains from commensal clade.  UC7267 exhibited regions with high identity to the clade A2 strains E1636 and E1039.

1,00E+05

100,0% 90,0% 80,0%

1,00E+04

1,00E+03

Aim of the work  Evaluate if the food chain could be considered theofmost important vector for Aim the work spreading of resistance between man and animals.

70,0%

blaZ

60,0%

ermB

cheese1

50,0%

cheese2

40,0%

cheese3

ermC ermTR tetM

30,0%

cheese4

20,0%

1,00E+02

 Isolation of Gram positive and Gram negative microrganisms resistant to ampicillin, tetracycline and erithromycin, in curd milk and 4 different kind of cheeses by plating on different selective media added with ampicillin, tetracicline and erythromycin.

0,0%

ermB curd milk

cheese 1

blaZ

cheese 2

cheese 3

cheese 4

Genomic analysis  Starins were identified and the most of them were belong to Enterococcus, Staphylococcus, Lactobacillus, Proteus, Obesobacterium, Klebsiella, Escherichia, Shigella genus.

Enterococcus faecium genome features Strain

 Microbial population in cheese produced with curd (cheese 1, 2 and 3) and pasteurized (cheese 4) milk showed 30% ampicillin resistance, 31% of erythromycin resistance and 39% tetracycline resistance.

 Typing of 263 isolates by RAPD and REP-PCR techniques.  Identification of strains by partial sequence of 16S rRNA and analysis with RDP Release 11.

31%

Isolation Lenght (bp) Contigs Coding sequences

UC7256

cheese

UC30571

cheese

UC8668

cheese

UC7267

cheese

UC7265

Human

UC7266

Hospitalized patient

2,823,689

130

2885

2,774,879

161

2826

2,911,322

106

2918

2,714,726

582

2674

2,842,544

963

2762

2,875,296

1228

2711

Un-rooted Neighbor joining tree based on the core genome after GBLOCKS application using 100 bootstrap replications shows the distribution of strains among commensal associated clade B, hospital associated clade A2 and clade A1. Tree was depicted using the strains selected for this study (boxes) and 26 other E. faecium genomes available in NCBI website. Designations: ef8668=UC8668, ef7256=UC7256, ef8733=UC8733, ef7265=UC7265, ef7267=UC7267, ef7251=UC7251, ef10237=UC10237, ef7266=UC7266.

30% ampR

 Evaluation of the presence of some genes involved in antimicrobial resistance by PCR technique.

tetR eriR

 Genome sequencing by Illumina 36 bp pair ends Quality reads filtery and assembly with Velvet (reseq) Annotation with RAST (rast.nmpdr.org/) and IGS Annotation Engine (ae.igs.umaryland.edu/cgi/manatee_intro.cgi)  Genome analysis: PBP5 analysis CGView Comparison Tool PHAST CRISPRFinder Plasmid finder 1.3

From the outer to inner circle: the CDS, t and r RNA coding stretches (rings 1 and 2); the identities with the compared strains starting from the best matching strain (rings 3-23: TX0082, E1636, E1039, E1162, E1071, U0317, 1 231 501, 1 230 933, E1679, 1 231 410, 1 231 502, Com15, 1 231 408, TX1330, C68, E980, 1 141 733, Com12, TC6, DO, D344SRF); the GC content and the GC skew (rings 24 and 25 respectively). CDS, coding sequences; tRNA, transfer RNA; rRNA, ribosomal RNA.

ermC

1,00E+01

Experimental Design

E. faecium UC7267

ermTR

10,0%

 Evaluate the consumer exposure to AMR through the food chain of fermented products.

tetL

tetL tetM

39%

ampicillin resistant strains

An asterisk (*) indicates a recombinant strain (1_231_408)

Antimicrobial resistance of food isolates  All food isolates are resistant to gentamicin and sensitive to tetracycline, erythromycin.  The food isolates UC7256 and UC8668 are also resistant to streptomycin.

Mobile genetic elements

Ampicillin resistance – analysis of PBP5

erythromycin resistant strains

tetracycline resistant strains

100,00%

 Since hospital associated isolates (MIC > 4 µg/ml ) usually harbour pbp5R and commensal associated isolates (MIC ≤ 4 µg/ml ) carry pbp5S, the entire PBP5 sequences were analyzed: The majority of food isolates have a «hybrid-like» PBP5

 PHAST showed the presence of intact prophage region in UC8668 (763403-824667 kb), in UC7256 (1555019-1599531 kb) and in UC30571 (197419-222795; 1277993-1296790; 1529249-1579994) No intact prophages were found in the putative chromosome of UC30571 and UC7267.

Amino acid at position: Amp MIC Strain (μg/ml) UC7267 4 µg/ml

525 D D E E E D D D

586 V V V V V V L V

629 E E E E E E V V

667 P P P P P P S S

 Analysis by Plasmid finder underlined the presence of one plasmid in UC30571 and in UC7267, two plasmid in UC8668 and UC7256,  UC7265 contains IS116/IS110/IS902 family transposases and transposase IS3/IS911 (HMPREF0351_10878), while UC7267 harbours only transposase IS3/IS911. None of the elements described above were found in the other food isolated strains.  Confirmed CRISPRs have been identified by CRISPRFinder in UC7265 and UC7267, which do not contain prophage sequences.