Food commensalmicrobes as a potentiallyimportant avenue

Food commensal microbes as a potentially important avenue

in transmitting antibiotic resistance genes

Hua H.Wang1,2,Michele Manuzon1,Mark Lehman1,Kai Wan1,Hongliang Luo1,

Thomas E.Wittum3,Ahmed Yousef1,2&Lauren O.Bakaletz4

1Department of Food Science and Technology,The Ohio State University,Columbus,OH,USA;2Department of Microbiology,The Ohio State University, Columbus,OH,USA;3Department of Veterinary Preventive Medicine,The Ohio State University,Columbus,OH,USA;and4Department of Pediatrics and Children’s Hospital,The Ohio State University,Columbus,OH,USA

Correspondence:Hua.H.Wang,The Ohio State University,110Parker Food Science& Technology Building,2015Fyffe Road, Columbus,OH43210,USA.Tel.:1614292 0579;fax:16142920218;

e-mail:wang.707@bbde35c5a1c7aa00b52acba9

Received01September2005;revised10 October2005;accepted11October2005. First published online5December2005.

doi:10.1111/j.1574-6968.2005.00030.x

Editor:Robert Burne

Keywords

foods;commensals;antibiotic resistance; horizontal gene transfer;Streptococcus thermophilus.Abstract

The rapid emergence of antibiotic-resistant(ART)pathogens is a major threat to public health.While the surfacing of ART food-borne pathogens is alarming,the magnitude of the antibiotic resistance(AR)gene pool in food-borne commensal microbes is yet to be revealed.Incidence of ART commensals in selected retail food products was examined in this study.The presence of102–107CFU of ART bacteria per gram of foods in many samples,particularly in ready-to-eat,‘healthy’food items,indicates that the ART bacteria are abundant in the food chain.AR-encoding genes were detected in ART isolates,and Streptococcus thermophilus was found to be a major host for AR genes in cheese bbde35c5a1c7aa00b52acba9ctococcus lactis and Leuconostoc sp.isolates were also found carrying AR genes.The data indicate that food could be an important avenue for ART bacterial evolution and dissemination. AR-encoding plasmids from several food-borne commensals were transmitted to Streptococcus mutans via natural gene transformation under laboratory conditions, suggesting the possible transfer of AR genes from food commensals to human residential bacteria via horizontal gene transfer.

Introduction

Resistant pathogens to various antibiotics are emerging rapidly.Surfacing of these resistant pathogens,untreatable by antibiotics,constitutes a real threat to public health. To effectively combat this problem,a comprehensive understanding of the major pathways in antibiotic resistance (AR)gene dissemination as well as the key mechanisms in the evolution of antibiotic-resistant(ART)bacteria is essential.

Horizontal gene transfer among pathogens in the hospital environment has been recognized as an important avenue for the rapid spread of AR genes among pathogens. It is also believed that horizontal transmissions of AR genes between commensal and pathogenic microorganisms in ecosystems are much more likely events than direct AR gene dissemination from one pathogen to another(Andremont, 2003).The presence of AR gene reservoirs in commensal microbes in various environmental and host ecosystems (Gilliver et al.,1999;O¨sterblad et al.,2001;Lancaster et al., 2003;Ready et al.,2003;Nandi et al.,2004;Salyers et al., 2004;Smith et al.,2004),the illustration of commensals as facilitators for AR gene dissemination(Luo et al.,2005b),and the correlation of antibiotics usage in animals with increased AR in human microbiota(Levy et al.,1976;Smith et al.,2002)suggest the importance of commensals in mediating the dissemination of AR genes.The isolation of AR genes in food-borne pathogens from retail products exempli?ed the potential contribution of the food chain in transmitting ART pathogens to humans(Charpentier& Courvalin,1999;Zhao et al.,2001;Luo et al.,2005a).The studies on commensal bacteria,however,are limited and primarily focused on the opportunistic pathogen enterococ-ci(Cocconcelli et al.,2003;Johnston&Jaykus,2004).A standard laboratory enrichment procedure(http:// bbde35c5a1c7aa00b52acba9/cvm/Documents/AppendicesA-6.pdf)is often used to detect the presence of the ART bacteria,masking the real magnitude of the AR problem associated with the food chain.

To examine the AR risks associated with the food chain, this study aimed at revealing the distribution spectrum and magnitude of ART commensal bacteria and AR gene pool in retail foods,by targeting total food microbiota instead of a particular group of microorganisms or pathogens.There-fore,food samples were analyzed without any laboratory enrichment procedures.Microbial resistance to tetracycline

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(Tet)and erythromycin (Em),which are still used in animal production and human therapy (Chopra &Roberts,2001;Roberts,2004),was investigated.The presence of several AR markers including erm B,erm C,tet S/M and tet A,encoding ribosomal modi?cation and Tet ef?ux mechanisms,in selected food isolates was examined and main AR gene hosts were identi?ed.

Materials and methods

Food sample preparation and enumeration of total and ART populations

Food samples (Fig.1)were purchased from local grocery stores and analyzed within the products’sell-by dates.Fresh raw milk was obtained from the dairy pilot plant at OSU and analyzed the same day as shipped.Five grams of each sample were aseptically removed from the product packaging and placed in disposable plastic bags containing 10mL of sterile 0.1%peptone water.Bagged samples were hand massaged for 10min.Homogenized samples or rinsing liquids were serially diluted and plated on nonselective plate count agar (PCA,Becton Dickinson and Company,Sparks,MD)for nonselective total microbial counting,and on PCA plates containing 16m g mL à1of Tet or 8m g mL à1of Em (Fisher

Biotech,Fair Lawn,NJ).Plates were incubated at conditions as indicated (Fig.1)for up to 48h for assessing Tet-and Em-resistant population.The levels of antibiotics used in selective agar plates were based on that used to screen for ART enterococci (bbde35c5a1c7aa00b52acba9/cvm/Documents/AppendicesA-6.pdf).Serially diluted samples were also plated on Difco Lactobacilli MRS Agar (MRS,Becton Dickinson and Company)and Pseudomonas isolation agar (PIA,EMD Chemicals Inc.,Gibbstown,NJ)plates with proper antibiotics to recover ART lactic acid bacteria and Pseudomonas species,respectively.The cell numbers re-ported (CFU g à1of food,Figs 1a and b)were the mean values from duplicates.

Antibiotic resistance gene detection and host isolates identification

Conventional PCR was conducted to detect AR genes in the ART isolates.Bacterial cells from single colonies were resuspended in 300m L sterile dH 2O containing 100m g of 1:1mixture of 0.5m m diameter and 0.1m m diameter glass beads (Biospec Products Inc.,Bartlesville,OK).The sample mixtures were homogenized using the Mini-Bead-Beater-8(Biospec Products Inc.)for 2min at maximum speed.The resulting cell extracts were placed in a boiling water bath

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Fig.1.Prevalence of antibiotic-resistant mi-crobes in retail foods.(a)Representative cheese samples.(b)Other representative foods.TPC,total plate count;Tet,Tet r population screened on agar plates containing 16m g mL à1tetracy-cline;Em,Em r population screened on agar plates containing 8m g mL à1(a–d)and 50m g mL à1(e)erythromycin.Data were means of duplicated results,and the standard deviations were less than 10%of the mean values.a Mi-croorganisms were recovered by plating on MRS agar plates and incubated anaerobically at 301C.b Microorganisms were recovered by plat-ing on plate count agar (PCA)agar plates and incubated aerobically at 201C.c Microorganisms were recovered by plating on PCA agar plates and incubated aerobically at 371C.d Microor-ganisms were recovered by plating on PCA agar plates and incubated aerobically at 201C.e Mi-croorganisms were recovered from spinach samples by plating on PCA agar plate and incubated anaerobically at 301C.

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Food commensals in AR transmission

10–15min and5m L of the supernatant were used as PCR templates.The PCR primers tet A-FP50-GCTACAT CCTGCTTGCCTTC-30and tet A-RP50-CATAGATC GCC GTGAAGAGG-30were used to amplify the220bp tet A fragment(Ng et al.,2001),tet S-FP50-CATAGACAAGC CGTTGACC-30and tet S-RP50-ATGTTTTTGGAACGCCA-GAG-30for the667bp tet S/M fragment(Ng et al.,2001), erm B-FP50-GGAACAGGTAAAGGGC-30and erm B-RP50-GGTTTAGGATGAAAGC-30for the389bp erm B fragment (this study),and erm C FP50-GCTAATATTGTTTAAATC GTCAAT-30and erm C RP50-TCAAAACATAATATAGA-TAAA-30for the640bp erm C fragment(Chung et al., 1999).PCR was conducted using reagents and conditions as described previously(Luo et al.,2004).PCR products with expected sizes were puri?ed using the QIAquick s kit (Qiagen,Valencia,CA)following manufacturer’s instruc-tion.DNA sequences of the16S rRNA gene,erm C,tet A gene fragments and50%of the erm B and tet S/M gene fragments were determined using a DNA analyzer(ABI PRISM s3700, Applied Biosystems,Foster City,CA)at the Plant Genome Sequence Facility,The Ohio State University.The DNA sequences were compared with published Tet or Em resis-tance gene sequences deposited in the NCBI database.ART isolates containing the resistance genes were identi?ed by PCR ampli?cation of the16S rRNA gene fragment and sequence analysis following procedures as described pre-viously(Connor et al.,2005).

Minimum inhibition concentration(MIC)profiles of ART isolates

The MIC pro?les of selected ART isolates were determined using a commercial kit(Sensititre s18–24h MIC and Breakpoint Susceptibility Plates;TREK Diagnostic Systems, Cleveland,OH)following the manufacturer’s instructions, with modi?cations.MRS or brain heart infusion(BHI) broth instead of the standard Mueller–Hinton broth was used to culture fastidious organisms.The MIC panels were incubated at either30or371C for24–48h.Additional96-well microtiter plates with wells containing up to256m g mLà1of Tet and Em were used to determine the MIC of Em r or Tet r isolates which exhibited positive growth in wells containing16m g mLà1of Tet or8m g mLà1of Em on the Sensititre s plates.The MICs were reported as the minimum concentration of the antibiotic that inhibited visible growth, as indicated by increased turbidity or by deposition of cells at the bottom of the wells.Control strains used in the study include Staphylococcus aureus ATCC29213(American Type Culture Collection(ATCC),Manassas,VA),Pseudomonas aeruginosa ATCC27853(ATCC),Lactococcus lactis ML3 (Kuhl et al.,1979),and Streptococcus thermophilus LMD-9 (bbde35c5a1c7aa00b52acba9/entrez/query.fcgi?db=gen om eprj&cmd=Retrieve&dopt=Overview&list_uids=13773).Plasmid isolation and natural gene transformation

Lactococcus sp.CZ-T4(Tet r)and CZ-T8(Tet r)were isolated from commercial cheddar cheese,while strain RMK-T14 (Tet r)was obtained from raw milk(this study).The multi-drug-resistant bbde35c5a1c7aa00b52acba9ctis K214was isolated from soft cheese made from raw milk(Perreten et al.,1997).The strains were grown in MRS broth or M17broth with0.5%glucose, supplemented with5m g mLà1Tet,and the mixture were incubated at301C for24h.Plasmids were isolated from these strains following the method of Anderson&McKay (1984)and were used in the natural transformation experi-ments,following procedures as described by Li et al.(2001). For the selection of Tet r transformants,BHI plates were supplemented with5m g mLà1Tet.Plates were incubated in a 5%CO2incubator at371C for48h.Transformation ef?-ciency was calculated based on the ratio of Tet r transfor-mants to the total number of viable cells.

Results

Prevalence of ART bacteria in food samples Antibiotic-resistant microbes were detected in majority of the retail foods examined,from raw food materials such as meat and shrimp to ready-to-eat items such as cheeses and salad.No detectable ART microbes were found in processed cheese(heat treated during manufacture)and yogurt sam-ples,with representative data illustrated in Fig.1.Twenty out of the23cheese samples analyzed contained Tet r and/or Em r microbes ranging from102to107CFU gà1of food, which are equivalent to103–108CFU ART microbes per slice of cheese(about20g).In general,the number of Tet r microbes was greater in cheeses than that of Em r bacteria. It is worth noting that the study was conducted using limited incubation conditions,and the antibiotic concentra-tions used to screen for resistant organisms might not be optimal for all bacteria.

Detection of AR genes and ART isolates identification

To con?rm that most of the ART organisms detected by growth on the selective agar plates were resistant bacteria because of the possession of AR determinants,conventional PCR was conducted to detect the presence of selected AR genes in these organisms and the results were summarized in Table1.Among the Tet r isolates recovered from cheese, about10%contained the tet S/M gene.Seven out of11tet S/ M1cheese isolates identi?ed were Staphylococcus thermo-philus;two tet S/M1isolates were found to be Lactococcus lactis.Two additional cheese isolates CZ-T4and CZ-T8had 97%16S rRNA gene sequence identity to unidenti?ed

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228H.H.Wang et al.

Lactococcus sp.,and particularly had 93–94%identity to Lactococcus garvieae and bbde35c5a1c7aa00b52acba9ctis ,similar with that of the raw milk ART isolate RMK-T14,suggesting this might be a common organism from milk.Therefore,it is possible that the Lactococcus sp.tet S/M 1isolates from cheese originated from milk (pasteurized but not sterile)or dairy processing environment during cheese fermentation.Another tet S/M 1isolate from raw milk was identi?ed as Leuconostoc sp.In addition,the tet A gene was found in two cheese isolates CZ-T3,CZ-T7and several isolates from raw pork meat.These isolates were all identi?ed as Pseudomonas sp.

Among the Em r isolates from cheese,more than 50%contained the erm B gene,and the carrier organisms identi-?ed so far include Staphylococcus sp.(?ve out of 28)and S .thermophilus (23out of 28).Both tet S/M and erm C genes were found in the isolate CX-I EM from packaged sliced chicken lunchmeat,suggesting a multidrug resistance phe-notype of the strain.CX-I EM was identi?ed as Pseudomonas sp.ART bacteria were isolated sporadically in lunchmeat (data not shown),which is probably because of occasional contamination during the processing of the meat.

Minimum inhibition concentration analysis Minimum inhibition concentration tests of selected cheese isolates showed that Lactococcus sp.CZ-T4and CZ-T8(tet S/M 1)were resistant to at least 128m g mL à1Tet,and S.thermophilus E4(erm B 1)was resistant to Em (4256m g mL à1),clarithromycin (48m g mL à1),and clindamycin (44m g mL à1).S.thermophilus BOR-COCZ-T19(tet S/M 1)was resistant to Tet (4128m g mL à1).Staphylococcus sp.C202was resistant to both Em (4256m g mL à1)and Tet (432m g mL à1),suggesting the possible possession of both resistance determinants in this isolate.The control strains Lactococcus lactis ML3,Staphylococcus thermophilus LMD-9,two other commercial S.thermophilus starters and Staphylo-coccus aureus ATCC 29213were sensitive (o 2m g mL à1)to the above antibiotics.

Lactococcus sp.RMK-T14(tet S/M 1)from raw milk was resistant to Tet (4128m g mL à1),Em (464m g mL à1),clarithromycin (48m g mL à1),and clindamycin (44m

g mL à1).Therefore this isolate likely carried multidrug-resistant determinants or multidrug-resistant mechan-ism(s).The raw milk isolate Streptococcus uberis RMK-T22W exhibited resistance to Tet (4128m g mL à1).

All of the Pseudomonas tet A 1isolates recovered from pork and cheese exhibited resistance to Tet (4128m g mL à1)and vancomycin (432m g mL à1);The Pseudomo-nas sp.CX-I EM (erm C 1tet S/M 1)from packaged sliced chicken lunchmeat was resistant to Tet (464m g mL à1)and its tolerance to Em (4256m g mL à1)was much higher than the control strain P.aeruginosa ATCC 27853(Tet o 16m g mL à1,Em o 32m g mL à1).

Horizontal transfer ofthe AR gene from food isolates to oral residential bacterium

The tet S/M-containing lactococcal isolates CZ-T4and CZ-T8,recovered from cheese,and RMK-T14,isolated from raw milk,contained a plasmid with an approximate size of 20–25kb.To assess the potential risk of the food-borne ART bacteria in disseminating AR genes to human micro-biota,plasmids isolated from the above strains were used for natural transformation of the oral cariogenic pathogen Streptococcus mutans under laboratory conditions.The tet S/M gene was successfully transferred to S.mutans UA159at frequencies ranging from 1.9?10à7to 2.8?10à5,4.7?10à7to 2.3?10à6,and 3.8?10à7to 2.1?10à6trans-formants per recipient cell using CZ-T4,CZ-T8and RMK-T14plasmid extracts,respectively.In addition,the multi-drug-resistant plasmid pK214from the cheese isolate bbde35c5a1c7aa00b52acba9ctis K214was also successfully transformed into S.mutans UA159at frequencies of 1.1?10à6–1.2?10à5transfor-mants per recipient cell.PCR ampli?cation con?rmed the presence of the tet S/M gene in the streptococcal transfor-mants.MIC test showed that the transformants had sig-ni?cantly increased resistance to Tet (4128m g mL à1)compared with the parental strain UA159(o 4m g mL à1).These results illustrated that the tet S/M gene from food isolates can lead to resistance in residential host bacteria or pathogens,if acquired by horizontal gene transfer.

Table 1.Screening of antibiotic resistance genes from dairy samples and identi?cation of host strains Food Antibiotic-resistant trait Resistance gene (no.of carriers/no.of isolates screened)16S rRNA gene identity (no.of organisms/no.of identi?ed)

Cheese

Tet

tet S/M (11/113)Lactococcus sp.(2/11)Streptococcus thermophilus (7/11)Lactococcus lactis (2/11)(including Lactococcus sp.CZ-T8,CZ-T4,S.thermophilus BOR-COCZ-T19)tet A (2/33)Pseudomonas sp.(2/2)(including Pseudomonas sp.CZ-T3,CZ-T7)

Em

erm B (32/56)Staphylococcus sp.(5/28)Streptococcus thermophilus .(23/28)(including S.thermophilus E4,Staphylococcus sp.C202)

Raw milk

Tet

tet S/M (9/108)

Lactococcus sp.(3/9)Streptococcus sp.(2/9)Leuconostoc sp.(1/9)(including Lactococcus sp.RMK-T14,Streptococcus uberis RMK-T22W)

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Discussion and conclusion

Despite the fact that this current study only screened for a limited number of resistance markers,it illustrated the prevalence of ART commensals and AR genes in retail foods. Many ART bacteria-containing ready-to-eat products are consumed without further cooking or processing.Conse-quently,human are routinely inoculated with ART bacteria through daily food intake,including opportunistic patho-gens and commensals such as Pseudomonas sp.,Streptococcus sp.and Staphylococcus sp.The detection of high numbers (i.e.up to108CFU per serving of food)in several products is alarming,suggesting that food can be a potentially impor-tant avenue transmitting ART bacteria.This?nding is in agreement with a previous report showing that consuming sterile foods can signi?cantly decrease the presence of ART bacteria in the gastrointestinal system(Levy,1998).While further research is needed to establish the direct correlation between the ART microbes from foods and the ART population in the host ecosystems,it is evident that a constant supply of ART bacteria,partnered with occasional colonization and horizontal gene transfer,are at least partially responsible for the increased AR pro?les seen in humans.Such an intrinsic AR gene pool could have sig-ni?cant impact on pathogen resistance in susceptible popu-lation,particularly those receiving antibiotic treatment. Oral cavity could be an important area where many initial interactions between food microbes and human microbiota, including horizontal gene transfer events such as conjuga-tion and transformation,might take place during the reten-tion of food residues in the oral cavity.Our data are consistent with results from recent studies showing that the microbiota in children and adults is becoming increasingly resistant to antibiotics,even in the absence of antibiotic treatment(Lancaster et al.,2003;Ready et al.,2003;Ville-dieu et al.,2004).In fact,the tet S/M and erm B genes were found to be abundant in bacteria isolated from foods,which is in agreement with the prevalence of these Tet-and Em-resistance genes in human oral micro?ora(Roberts,1998). Successful transmission of the resistance genes from the food isolates to the oral residential bacterium S.mutans,by natural gene transformation,further con?rmed the func-tionality of the mobile resistance-encoding elements from food isolates,if acquired by horizontal gene transfer. Identi?cation of the key pathways in AR gene transfer is critical,but developing a strategy to combat this problem is even more important.The identi?cation of ART bacteria in cheeses often associated with raw milk,such as Lactococcus sp.,Streptococcus sp.and Staphylococcus sp.,suggests that cheese fermentation is a susceptible process during which ART bacteria could evolve and proliferate.Improving sani-tation and milk heat treatment are thereby essential steps in reducing ART bacteria.While it is a major challenge to track the direct and indirect gene transfer events among microbes in complicated ecosystems(Andremont,2003),identifying key AR gene host organisms in foods,and likely in other ecosystems,not only reveals the ultimate consequence of these events in the food chain and the organisms involved in horizontal gene transfer,but opens the door for further characterization of conditions in these ecosystems that might facilitate horizontal gene transfer and features of the organisms that might grant their?tness in such ecological niches(Luo et al.,2005a).Such understanding would be critical for effective counteractive strategies to interfere with the detrimental gene swapping in both natural and host ecosystems.An industrially important lactic acid bacterium, S.thermophilus,was found to be a dominant host for both Tet and Em genes.ART bbde35c5a1c7aa00b52acba9ctis was also isolated from cheese.Genetic screening and MIC tests of three commercial S.thermophilus starter cultures as well as the control bbde35c5a1c7aa00b52acba9ctis strain showed that they are free of the above AR genes, suggesting the susceptibility of these starter cultures to horizontal gene transfer,at least during certain cheese fermentation processes.The potential health impact of these organisms thus needs to be carefully evaluated.Although it would be a tedious and likely long-term effort to clean up the AR gene pool in the environment,interrupting the transmission of ART bacteria into human by focusing our efforts on the food chain could be an effective strategy to combat the AR challenge in humans. Acknowledgements

The authors thank Drs John Reeve,Steve Schwartz(OSU), Peter Greenberg(UW),Paul Kolenbrander(NIH/NIDCR), Stuart Levy(Tufts)and David White(FDA)for helpful discussions.This project was sponsored by OSU start-up fund for H.H.Wang.M.Lehman is supported by US Air Force Institute of Technology.

Disclaimer:The views expressed in this article are those of the author(s)and do not re?ect the of?cial policy or position of the United States Air Force,Department of Defense,or the bbde35c5a1c7aa00b52acba9ernment.The authors MM,KL, ML,KW and HL contributed equally to this work. References

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