Project APVV 0014-274

Project of Slovak Research and Development Agency APVV 0014-274

Small mammals as a potential source of zoonotic bacteria and resistance to antibiotics (duration July 2015- June 2019)

The aim of the project was to obtain new and significantly broaden existing knowledge about the three most important components of natural-focal diseases i.e. 1. small mammals (especially rodents) as reservoir animals; 2. parasitic arthropods (ticks, fleas) as hematophagous parasites and at the same time vectors of agents of diseases; 3. the major bacterial pathogens (Anaplasma, Borrelia, Bartonella, Leptospira, Rickettsia), for which small mammals serve as important reservoirs.

Important part of the project was diagnostics of potential pathogens and pathogens of intestinal and skin microflora of wild rodents and detection of antibiotic resistance in so-called indicator bacteria (Escherichia coli and staphylococci). The evaluation of the role of small mammals and ticks as reservoirs of bacterial antibiotic resistance.

Investigators

Principal investigator:

Responsible investigator:

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Results in 2019

Iron uptake genes  in Escherichia coli from wild small mammals. (Kme V., Handrová L.)

Antibiotic susceptible strains of  faecal Escherichi coli from small mammals (Apodemus agrarius and A. flavicollis)  were investigated for seven ferric iron uptake genes: iha (iron-regulated gene homologue adhesion, Johnson, Stell 2000),  sitA (iron/manganese binding protein, Rodriguez-Siek et al. 2005) , irp2 (iron regulatory protein, Schubert et al. 1998) , fyuA (ferric yersiniabactin uptake, Johnson, Stell 2000), iroN  (Salmochelin receptor Russo et al. 1999),  ireA (iron-responsive element and putative siderophore receptor gene, Russo et al. 2001), iutA  (receptor for aerobactin,  Johnson, Stell 2000) and chuA (hemin receptor, Clermont et al. 2013) and one ferrous gene  feoB-main transmembrane transporter (Lau et al. 2013). 

Screening of Escherichia coli isolates for virulence genes were carried out by PCR:   colicin V – cvaC,  capsular polysialic acid virulence factor - kps (Johnson, Stell 200),  increased serum survival – iss (Foley et al. 2000), temperature sensitive haemagglutinin – tsh (Dozois et al. 2000); P fimbrial adhesion – papC ( Le Bouguénec et al. 1992) and phylogeny typing (Clermont at al. 2013). 

Results showed the presence pathogen (phylogroups D/E or B2)  and commensal (phylogroup B1 and A) faecal Escherichia coli from small mammals. Pathogen E.coli  2441  D/E  group  consist of  chuA, sit A, iha, iroN and ferrous feoB gene and E.coli  30E2 from B2 pathogen group had chuA, sitA, iha, iroN, irp2 and  fyuA. Surprisingly  E.coli 241 from B1 commensals consist of  virulence factors  papC (P fimbrial  adhesion) and iss (increased serum survival)  and  both  group of iron genes  fyuA and feoB. Not  typable  phylogroup of commensals E.coli 30B2 consist of ColV- colicin V (cvaC) virulence factor with  ferric iron genes : sitA, iha, iron, irp2, ireA and fyuA.We have shown circulation of iron uptake genes  in the gut of  small mammals population. The results suggest that wild small mammals are potential bioindicators  of  zoonotic bacterial pathogens (Kmet et al. 2018).

Further work of PCR analysis on more isolates is required, but this study offers the first insight into the virulence of  antibiotic susceptible Escherichia coli from Apodemus species.

In: Symposium Animal Physiology, 2019

https://sites.google.com/view/animal-physiology-conference/home

An update on the Ixodes ricinus microbiome (Kme V., aplová Z.)

 Ixodes ricinus is vector in the transmission of many important infectious diseases in human and animals. There is still minimal information available on the bacterial agents associated with ticks found in Slovakia.

We performed a survey of the bacterial communities associated with Ixodes ricinus collected from forest area near a great city aglomeration. Bacterial 16S rRNA hypervariable region amplicon libraries prepared from the Ixodes ricinus females were sequenced on Illumina MiSeq platform. We detected a total of 107 bacterial genera and order Clostridiales and class Bacilli (with more than 100 reads). Dominant taxa included the tick endosymbionts e.g. spotted fever group (Rickettsia 21998 reads) and Coxiella (10715 reads). Second dominant species was mycoplasma Spiroplasma ixodetis (9554 reads).

Dominant enviromental soil bacteria were Pseudomonas (6741 reads), Nocardiodes (4753 reads), Brevundimonas (2797 reads), Devosia (2797 reads) Aureimonas (1398 reads), Actinomycetospora (964 reads), Terrimonas (962 reads), Pedobacter (1139 reads), Methylobacterium (956 reads), Rhodococcus (945 reads), Williamsia (832 reads), Rhizobium (787 reads), Mesorhizobium (631 reads), Rhizobium (602 reads) and Spirosoma (486 reads) .

Firmicutes were only 280 reads with 151 reads of Clostridia and 128 reads of Bacilli (include Staphylococcus and Bacillus ).

The cultivation and Maldi Tof analysis revealed seven species of coagulase negative staphylococci (CoNS) i.e., Staphylococcus epidermidis, S. capitis, S.haemolyticus, S.hominis, S. pasteuri, S.chromogenes, S.warneri and Micrococcus luteus. Antibiotic resistant CoNS with msrA and blaZ genes were detected. The results of the next generation sequencing revealed a new look at the complexity of the Ixodes ricinus microbiome.

In:  KME V., aplová Z.: An update on the Ixodes ricinus microbiome. J Microbiol Biotech Food Sci ,  8 (6), 1340-1342 ,2019,   doi: 10.15414/jmbfs.2019.8.6.1340-1342

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Results in 2018

Small mammals as sentinels of antimicrobial-resistant staphylococci. (Kme V., uvalová A., Stanko M.)

A total of 39 coagulase-negative staphylococci and seven Staphylococcus aureus strains were isolated from small mammal feces, i.e., the striped field mouse (Apodemus agrarius) and the yellow-necked mouse (A. flavicollis) in two sampling areas, deciduous forest and karst plains.

All strains were susceptible to tetracycline, linezolid, vancomycin, and teicoplanin. Three MRSA strains with the mecA gene were detected. The beta-lactamase gene blaZ was detected in ampicillin-resistant staphylococci and in the high-level resistant strains (oxacillin over 2 mg/L) mecA gene. The mecC gene was not detected by PCR. Erythromycin-resistant staphylococci harbored the ermC gene and/or the efflux gene msrA. There were no detectable dfr genes in trimethoprim-resistant staphylococci and the rifampicin-resistant strains were without mutation in the rpoB gene. In summary, wild small mammals may serve as sentinels of mecA-positive S. aureus with erythromycin resistance genes ermC and efflux msrA. Small mammals appear to be useful indicators of antibiotic resistance.

In:  KME V., uvalová A., Stanko M.: Small mammals as sentinels of antimicrobial-resistant staphylococci. Folia Microbiologica, 63, 665–668, 2018, https://doi.org/10.1007/s12223-018-0594-3

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The information on this site is provided for educational and informational purposes only.
We don`t recommend or endorse any specific products that may be mentioned on the site. Reliance on any information provided by this site is solely at your own risk.

Dr. Vladimir Kmet
webpage: http://home.saske.sk/~kmetv/

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