Detection of Mycobacteria by Culture and DNA-Based Methods in Animal-Derived Food Products Purchased at Spanish Supermarkets

Abstract

Mycobacteria include obligate and opportunistic pathogens that cause significant human and animal disease. The burden of tuberculosis has been largely reduced in developed territories but remains a huge problem worldwide. The significance of nontuberculous mycobacteria is growing considerably, especially in developed regions with higher life expectancy and more therapy-related immunosuppressed individuals. Due to their robustness mycobacteria can contaminate animal products by direct transmission from infected individuals or by environmental contamination during processing. The situation at market level is poorly known. Most studies analyzing commercially available foods are limited to a small or local scale and mainly focused on a particular mycobacterial species. There is a need to investigate if animal products that have passed the established controls to be for sale at main supermarkets could represent a route of contact with any mycobacteria. Thus, our goal was to study the prevalence of mycobacteria in these foods to assess if this could represent a source of human exposure. Five stores from the main supermarket chains in Spain were selected. 138 dairy and 119 meat products were purchased. All were processed using culture and multiplex real-time PCR methods. Additional molecular methods were used to specifically identify any positive result. Mycobacterium avium subsp. hominissuis (2), M. avium subsp. avium (1), and M. fortuitum (1) were isolated from powdered infant formula and ground beef, chicken sausage, and mortadella cold cut, respectively. Mycobacterial DNA (M. avium, M. tuberculosis complex and other nontuberculous mycobacteria) was detected in 15% of dairy products and 2% of meat products. These results show that the prevalence of viable mycobacteria in foods of animal origin obtained at the supermarket was not substantial although a considerable proportion of them contained mycobacterial DNA. Contact with mycobacteria through this route could be ensured over time. Further investigation is necessary to determine the real impact of foodborne mycobacterial exposure on human health and identify critical points in the food production system to enable setting up more stringent control measures.

Tuberculosis outbreak caused byMycobacterium capraein a rabbit farm in Spain

“…DNA extracted from all positive cultures was tested by a tetraplex qPCR (Sevilla et al, ) for simultaneous MTBC confirmation or detection of other mycobacteria. For species identification of MTBC positive samples, a panel of singleplex PCR assays previously described (Sevilla et al, ) to detect the regions of difference (RD) and spoligotyping (Kamerbeek et al, ) were used. Amplification of RD 1, 4, 9 and 12 was carried out using the primers (RD1_F_5′‐CCC TTT CTC GTG TTT ATA CGT TTG A‐3′, RD1_R_5′‐GCC ATA TCG TCC GGA GCT/T3′, RD4_F_5′‐CCA CGA CTA TGA CTA GGA CAG CAA‐3′, RD4_R_5′‐AAG AAC TAT CAA TCG GGC AAG ATC‐3′, RD9_F_5′‐TGC GGG CGG ACA ACT C‐3′, RD9_R_5′‐CAC TGC GGT CGG CAT TG‐3′, RD12_F_5′‐CGT TGG AAC GCG AAA TAC G‐3′, RD12_R_5′‐CCA GGA TAT GGG CGC AAA/T3′) reported earlier by Halse, Escuyer, & Musser, () in independent conventional singleplex PCR assays.…”

Detection of Wood Mice (Apodemus sylvaticus) Carrying Non-Tuberculous Mycobacteria Able to Infect Cattle and Interfere with the Diagnosis of Bovine Tuberculosis

“…DNA extraction from tissue homogenate aliquots (0.25 mL) was performed using a modified protocol of the Speedtools Tissue DNA extraction kit (BioTools, B&M Labs S. A., Madrid, Spain) as described previously [23,24]. DNA was extracted from all MGIT cultures regardless of having positive or negative BACTEC time to detection (TTD) readouts.…”

“…A previously described [23] and modified [24] tetraplex real-time PCR was performed for the screening of DNA extracted from MGIT cultures and homogenized tissue pools. This technique allows for the simultaneous detection of the Mycobacterium genus, all four M. avium subspecies, and MTC.…”

“…The reaction was carried out in a total volume of 25 µL, containing 3 µL of extracted DNA and 22 µL of mastermix. Amplification was carried out in a 7500 Real-Time PCR thermal cycler (Applied Biosystems, Foster City, CA, USA) under previously described conditions [23,24]. The estimation of valid cycle threshold (CT) and baseline was calculated automatically with the SDS software v. 1.5.1 (Applied Biosystems, Foster City, CA, USA), visually confirmed by checking amplification plots, and manually adjusted if needed.…”

“…The strategy outlined for the identification of MTC-positive samples included standard spoligotyping [28] as well as amplification of the regions of difference (RD) 1, 4, 9, and 12 of M. tuberculosis using previously described primers [29] in independent conventional singleplex PCR assays [24]. The RD signature patterns for MTC species identification have been specified earlier [29].…”