research article microbiological quality of ready-to-eat

Research ArticleMicrobiological Quality of Ready-to-Eat VegetablesCollected in Mexico City Occurrence of Aerobic-MesophilicBacteria Fecal Coliforms and Potentially PathogenicNontuberculous Mycobacteria

Jorge Francisco Cerna-Cortes1 Nancy Leon-Montes1

Ana Laura Cortes-Cueto1 Laura P Salas-Rangel1 Addy Cecilia Helguera-Repetto2

Daniel Lopez-Hernandez3 Sandra Rivera-Gutierrez1

Elizabeth Fernandez-Rendon1 and Jorge Alberto Gonzalez-y-Merchand1

1 Departamento de Microbiologia Escuela Nacional de Ciencias Biologicas-Instituto Politecnico NacionalProlongacion Carpio y Plan de Ayala SN Colonia Casco de Santo Tomas Delegacion Miguel Hidalgo 11340 Mexico DF Mexico

2Departamento de Inmunobioquimica Torre de Investigacion Instituto Nacional de Perinatologıa Isidro Espinosa de los Reyes (INPer)Montes Urales 800 Colonia Lomas de Virreyes 11000 Mexico DF Mexico

3 Departamento de Epidemiologia y Bioestadistica Centro de Investigacion y de Educacion Continua (CENINVEC) Oyameles 30Colonia La Perla 57820 Ciudad Nezahualcoyotl MEX Mexico

Correspondence should be addressed to Jorge Alberto Gonzalez-y-Merchand jgonzal1212yahoocommx

Received 22 August 2014 Accepted 24 September 2014

Academic Editor Petr Kralik

Copyright copy 2015 Jorge Francisco Cerna-Cortes et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The aims of this study were to evaluate the microbiological quality and the occurrence of nontuberculous mycobacteria (NTM)in a variety of salads and sprouts from supermarkets and street vendors in Mexico City Aerobic-mesophilic bacteria (AMB) werepresent in 100 of RTE-salads samples 59 of samples were outside guidelines range (gt517 log

10CFU per g) Although fecal

coliforms (FC) were present in 32 of samples only 8 of them exceeded the permissible limit (100MPNg) Regarding the100 RTE-sprouts all samples were also positive for AMB and total coliforms (TC) and 69 for FC Seven NTM species wererecovered from 7 salad samples they included three M fortuitum two M chelonae one M mucogenicum and one M sp TwelveRTE-sprouts samples harbored NTM which were identified as M porcinum (five) M abscessus (two) M gordonae (two) Mmucogenicum (two) and M avium complex (one) Most RTE-salads and RTE-sprouts had unsatisfactory microbiological qualityand some harbored NTM associated with illness No correlation between the presence of coliforms and NTM was found Overallthese results suggest that RTE-salads and RTE-sprouts might function as vehicles for NTM transmission in humans hence properhandling and treatment before consumption of such products might be recommendable

1 Introduction

According to the definition given by the FAO and the WHO[1] ready-to-eat (RTE) foods include any comestible that isnormally consumed in its raw state Demand for RTE foodhas led to an increase in the amount and selection of differentproducts available for the consumers [2] RTE-salads andRTE-sprouts constitute a suitable and convenient meal for

todayrsquos lifestyles because they need no cooking or furtherpreparation As well as being considered low-calorie foodthey are rich in fiber and provide a great variety of vitaminsminerals and other phytochemicals [3] Their consumptionis encouraged in many countries by government healthagencies to protect people against a range of illnesses such ascancer and cardiovascular diseases [4] Therefore continuedincrease in the consumption of fresh meals has occurred

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 789508 9 pageshttpdxdoiorg1011552015789508

2 BioMed Research International

Gustavo AMadero

Azcapotzalco

Venustiano Carranza

Cuauhteacutemoc

Miguel Hidalgo

IztacalcoBenitoJuaacuterezAacutelvaro

ObregoacutenCuajimalpade

Morelos

MagdalenaContreras

Tlalpan

IztapalapaCoyoacaacuten

Xochimilco

Tlaacutehuac

Milpa Alta

0 10 20 30(km)

Total surface 1525km2

S

N

W E

Figure 1 Boroughs of Mexico City where RTE-salads (e) and RTE-sprouts (◼) were collected

as a result of efforts to promote better nutrition in thepopulation [4] As RTE-salads and RTE-sprouts do not needfurther preparation before consumption they could poten-tially contain pathogens that form part of their microfloraposing a public health problem Fresh vegetables can becomecontaminated by pathogens as Salmonella at any point duringthe food production process During preharvest contact withcontaminated irrigation water soil manure or fecal matterof wild animals may occur These pathogens can both bindto plant leaves andor be internalized via the leaves or theendophytic root system [5 6] During harvest asymptomatichuman carriers might contaminate the products and at thepostharvest level products become contaminated by contactwith polluted water other asymptomatic human carriers orthe production process environment Over the last 30 yearsthere has been at least a 24 of increase in the averageamount of fresh vegetable consumed per person in the USA[7]Moreover the number of gastroenteritis outbreaks causedby foodborne pathogens after consumption of raw vegetablessalads and sprouts has increased worldwide [8ndash11] Eventhough Salmonella is the most common cause of diseaseoutbreaks associated with lettuce and sprouts [12ndash15] thereare other pathogens (Shiga toxin producing E coli O157Norovirus) that have been described as relevant microbial

hazards [16ndash18] For example a large outbreak of hemolytic-uremic syndrome caused by STECO104H4 linked to sproutsoccurred in Germany [19 20]

Nontuberculous mycobacteria (NTM) are opportunis-tic pathogens found in the environment that cause life-threatening infections in humans other mammals and birds[21 22] The incidence of NTM disease is increasing world-wide [23] in both immunocompetent and immunocompro-mised subjects [22 24 25] As there is no defining evidencefor person-to-person transmission for most of the NTM[24 25] it is therefore important to establish the sourcesand routes of NTM transmission since infections can occurthrough inhalation ingestion gastric reflux or skin trauma[22 26] NTM have been isolated from various kinds offood and many studies support the hypothesis that foodespecially raw or partially cooked products plays a role asa source of NTM for humans primarily in countries withsimilar processing food routes and climates [27ndash29]The aimsof this study were to evaluate the microbiological quality ofRTE-salads and RTE-sprouts collected in Mexico City and toanalyze the occurrence of NTM in these samples in order todeterminewhether theseRTE foodsmay represent a potentialrisk for NTM infection for the consumers

BioMed Research International 3

2 Material and Methods

21 Area of Study and RTE Food Collection The selected areaof study was Mexico City a large urban area that although ithas almost 9 million registered inhabitants during workinghours reaches a population of nearly 25 million Mexico Cityis divided into 16 ldquodelegacionesrdquo or boroughs (Figure 1) FromJanuary to July 2013 a total of 100 RTE-salads samples of rawvegetables (salads from SPM contained mainly lettuce andspinach accompanied by carrot and purple cabbage whilesalads from SVS also contained onion tomatoes cucumberMexican turnip mushroom radish coriander cactus andfruits as strawberry apple and mango) were collected fromdifferent boroughs (Figure 1) 50 samples from differentsupermarkets (SPM) and 50 from street-vendor stalls (SVS)Also 100 RTE-sprouts samples (alfalfa soybean broccolicarrot radish onion amaranth clover arugula lentil wheatmelon turnip or combinations of alfalfa and broccoli alfalfaand clover alfalfa and onion alfalfa and soybean and alfalfaand radish) were purchased (Figure 1) from August 2013 toFebruary 2014 50 from different SPM and 50 from SVSSalads and sprouts collected from SPM were purchased inbags provided by a bigger food processing company Thosevegetables collected from SVS were prepared directly byvendors at the selling spot At the moment of purchase theRTE-salads and RTE-sprouts were packaged in sterile plasticcontainers and were transported to the laboratory for theiranalysis within 2 h after collection

22 Microbiological Analysis According to the FDA [30]50 grams from each sample was placed in ethylene oxidegas-sterilized polypropylene bags (Whirl-Pak Nasco USA)and 450mL of lactose broth was added in order to achievea final dilution 1 10 (10minus1) Samples were homogenizedfor 2min in a stomacher (tissue disrupter) and seriallydiluted (10minus1 to 10minus5) then these dilutions were usedfor quantification (CFUmL) and estimation (MPNg) ofmicroorganisms Each sample was tested for the presenceof aerobic-mesophilic bacteria (AMB) total coliforms (TC)and fecal coliforms (FC) following the methods approved bythe FDArsquos Bacteriological AnalyticalManual [30] All the dataobtained in this work was analyzed according to the official093 guideline [31] which establishes that food samples shouldonly contain up to 517 log

10CFU per g (150000 CFUg) of

AMB and up to 100 MPNg of FC

23 Isolation and Identification of Mycobacteria For bothRTE foods (salads and sprouts) 45mL of supernatant fromdilution 10minus1 was placed in sterile conical centrifugationtubes (Falcon type) of 50mL and then was centrifuged(4000timesg at 25∘C for 20min) Supernatants were discardedand pellets were resuspended in 20mL of 50 ppm chlorinesolution or 20mL of 01 cetylpyridinium chloride for saladsand sprouts respectively The suspensions were incubatedat room temperature for 30min and then neutralized with20mL of phosphate buffer (pH 70) Samples were cen-trifuged as above and pellets were resuspended in 5mLof Dubos medium (Difco Becton Dickinson Sparks MD)

with albumin-dextrose-catalase (ADC Becton DickinsonMexico) 100 120583L of this suspension was inoculated ontoMiddlebrook 7H10 agar (Difco Becton Dickinson) supple-mented with ADC cycloheximide (500120583gmL) and thePANTA cocktail (Becton Dickinson) (40UmL polymyxin B4 120583gmL amphotericin B 16 120583gmL nalidixic acid 4 120583gmLtrimethoprim and 4 120583gmL azlocillin) Plates were incubatedat 35∘C and were examined daily for the first eight days andthereafter once a week for two months Once the bacterialgrowth had been observed on the Middlebrook 7H10 agarthe identification of acid-fast bacilli was carried out byZiehl-Neelsen stain Acid-fast bacilli were subcultured onMiddlebrook 7H10 agar labeled by sampling location andwith a consecutive number

Strains belonging to the genusMycobacterium and to theM tuberculosis complex were identified by two PCR assayspreviously described [32] Briefly 3 120583L aliquots of bacteriallysates were subjected to amplification using a standard Taqpolymerase (Life Technologies Rockville MD) in a totalvolume of 50 120583L of PCRmixture RAC1 and RAC8 andMTB-F andMTB-R primers [32] were used for identification of theMycobacterium genus and of the strains belonging to the Mtuberculosis complex respectively The amplicon producedby the primer combination of RAC1 and RAC8 contains thelast 99 codons of the murA gene the promoter region ofthe rrnA operon and 360 nucleotides from the 51015840 end of the16S rRNA gene As shown by Perez-Martinez et al [33] theamplicon size varies depending on the mycobacteria speciesfrom 934 to 1300 bp MTB-FMTB-R primers amplified aDNA fragment coding for the last five codons ofmurA genethe promoter region of the rrnA operon and the 51015840 end ofthe 16S rRNA a 488 bp fragment characteristic only for Mtuberculosis complex members should be amplified There-fore by exclusion mycobacteria strains that did not belongto the M tuberculosis complex were considered to be NTMTheseNTM species were identified by threemethods (i) PCRrestriction enzyme pattern analysis (PRA) of the 65 kDa heatshock protein gene (hsp65) as described by Telenti et al [34](ii) sequencing of the hypervariable region 2 (V2) of the 16SrRNA gene [35] and (iii) sequencing of the rpoB gene [36]Mycobacterial PRA was performed by PCR amplification ofa 439 bp fragment of the hsp65 gene by using primers Tb11and Tb12 [34] PCR products were digested in two separatereactions with two restriction enzymes BstEII (New EnglandBiolabs) and HaeIII (Invitrogen) Digested products werethen analyzed using the Agilent 2100 bioanalyzer DNA 1000LabChips (Agilent) were used according to manufacturerrsquosprotocol PRA results were interpreted with the algorithmdescribed by Telenti et al [34] which is available on the PRAdatabase [37]

Identification of the mycobacterial species was also car-ried out by automatized sequence of the hypervariable region2 (V2) of the 16S rRNA gene and of the rpoB gene Theamplification of the 16S rRNA gene was performed using theRAC1 and RAC8 primers [32] For the amplification of therpoB gene the Myco-F and Myco-R primers were used toobtain a product of 723 bp [36] Both products of PCR weresequenced using the RAC8 [32] and Myco-F [36] primersrespectively and the big dye terminator ready reaction kit


Table 1 Populations and frequencies of aerobic-mesophilic bacteria (AMB) total coliforms (TC) and fecal coliforms (FC) on RTE-saladsand RTE-sprouts samples

Microorganisms group Minimum Median Maximum Frequency () Number of samples out of the 093guidelinelowast ()

RTE-salads from SPMa

AMB 3 49 66 50 (100) 21 (42)TC lt3 56 gt1100 46 (92) NAFC lt3 lt3 210 7 (14) 2 (4)

RTE-salads from SVSa

AMB 3 61 67 50 (100) 38 (76)TC 3 1100 gt1100 50 (100) NAFC lt3 15 gt1100 25 (50) 6 (12)

RTE-sprouts from SPMa

AMB 61 74 8 50 (100) NATC 61 460 gt1100 50 (100) NAFC lt3 33 gt1100 29 (58) NA

RTE-sprouts from SVSa


a119899 = 50 Minimum median and maximum values are in log

10CFU per g for aerobic-mesophilic bacteria and in most probable number (MPN) per g for total

coliforms and fecal coliforms SPM supermarkets SVS street-vendor stalls and NA not applicable (there is no official guideline for this food)lowastGuideline that establishes that food samples should contain up to 517 log

10CFU per g (150000CFUg) of AMB and up to 100MPNg of FC

(Perkin-Elmer Inc Wellesley MA) The sequences wereanalyzed by ABI PRISM 310 genetic analyzer system (Perkin-Elmer) Nucleotide sequences were compared to knownsequences in the GenBank database by using the Blastnalgorithm Species identifications were based on the 100similarity cut-off for the 16S rRNA gene and ge97 for therpoB gene

The identification of some of the isolated NTM was notpossible using the methods described above therefore full-length 16S rRNA gene was amplified using the 13B8FPLuniversal primers [38] Purified PCR products were directlysequenced in both forward and reverse directions usingthe same primers as for PCR Nucleotide sequences werecompared to known sequences in the GenBank database byusing the Blastn algorithm

24 Statistical Analyses Categorical variables (AMB TC andFC)were compared using the (chi)2 test for the 2 categories oforigin of samples (SPM and SVS)We calculated119875 value fromFisher test for corresponding data The values of median ofCFU of AMB and MPN of TC and FC were compared usingthe Kruskal-Wallis test Two-tailed probability values werecalculated The point-biserial correlation coefficient (119903pb)value was calculated to quantify the relationship betweenthe nominal (presence of NTM) and quantitative variables(concentration of AMB TC and FC) Similarly the phicorrelation coefficient (119903phi) value was calculated to quantifythe relationship between all nominal variables (presenceof NTM and AMB TC and FC) A 119875 value lt 005 was

considered significant All statistical analysis was runwith theStatistical program SPSS for Windows version 21

3 Results

The RTE-salad and RTE-sprout samples had unsatisfactorymicrobiological quality (results outside acceptable microbi-ological limits see reference [39]) A total of 59 of RTE-salads (21 from SPM and 38 from SVS 119875 = 0001) didnot comply with the 093 guideline (see Section 2) (Table 1) Incontrast FCwas detected in 32 of samples of these only 8exceeded the permissible limit of the official guideline Themedian concentration of AMB in RTE-salad samples fromSPM was significantly lower than the one observed in thosefrom SVS (119875 lt 0001) Similarly we found equivalent resultswhen the median concentrations of MPNg of TC and FCwere compared (119875 lt 0001)

AMB and TC were also present in 100 of RTE-sproutssamples analyzed (Table 1) FC were present in 69 ofsamples The median concentration of FC was significantlylower in RTE-sprouts from SPM (33MPNg) than the onefound in SVS (235MPNg) (119875 = 0007) However themedian concentrations of AMB in samples from SPM andSVSwere similar (119875 = 0762) Likewise we observed the sameresult when we compared themedian concentration values ofTC between samples from SPM and from SVS (119875 = 0169)

One hundred RTE-salads were tested for the pres-ence of mycobacteria Seven RTE-salads harbored NTMthree were M fortuitum two were M chelonae one was


Table 2 Characteristics of positive samples for NTM and species identified

Foods Origin Number of positivesamples type Ingredients Number and species of NTM

identified

RTE-salads

SVS 3 mixedLettuce carrot cucumber

Mexican turniptomatoes onion

2M fortuitum1M chelonae

SVS 1 mango Lettuce mangoMexican turnip strawberry 1M fortuitum

SVS 3 nopal Cactus tomatoesonion coriander

1M mucogenicum1M chelonae

1M sp

RTE-sprouts

SPM 3 alfalfa Alfalfa sprouts 2M abscessus1M gordonae

SPM 1 alfalfa and clover Alfalfa sproutsClover sprouts 1M porcinum

SPM 1 alfalfa and onion Alfalfa sproutsonion sprouts 1M porcinum

SVS 4 alfalfa Alfalfa sprouts 1M avium complex3M porcinum

SVS 1 alfalfa and soybean Alfalfa sproutssoybean sprouts 1M mucogenicum

SVS 1 soybean Soybean sprouts 1M mucogenicumSVS 1 broccoli Broccoli sprouts 1M gordonae

SVS street-vendor stalls SPM supermarket

Mmucogenicum and one wasM sp (Table 2) All NTMwereisolated from RTE-salads collected in SVS (119875 lt 005) Nocorrelation between the presence of NTMand the presence ofAMB (119903phi = 0154 119875 = 0234) TC (119903phi = 0056 119875 = 1000)and FC (119903phi = 0064 119875 = 0453) was found Likewise nocorrelation between the presence of NTM and the numberof AMB (119903pb = 0098 119875 = 0330) TC (119903pb = minus0024 119875 =0812) and FC (119903pb = 0162 119875 = 0106) was observedOf the 100 RTE-sprout samples analyzed 12 yielded NTMM porcinum was the most frequently isolated organism (fiveisolates) We have also recovered two strains ofM abscessustwo of M gordonae two of M mucogenicum and one strainbelonging to the M avium complex (Table 2) Comparisonof the number and species of NTM isolated from SPM andSVS showed no significant differences (119875 = 0424) We alsofound no correlation between the presence of NTM and thepresence of AMB TC and FC (119903phi = minus0081 119875 = 0475)in RTE-sprouts Similarly we did not observe correlationbetween the presence of NTM and the median concentrationof TC (119903pb = 0046 119875 = 0653) and FC (119903pb = 0099 119875 =0326) Nevertheless we did observe a significant correlationbetween the presence of NTM and the median concentrationof AMB (119903pb = 0331 119875 = 0001)

4 Discussion

This study shows that AMB were detected in all RTE-saladsregardless of source with limits ranging from 3 to 66 log

10

CFUg AMB counts were found to be higher than thosereported for RTE-salads in Johannesburg South Africa [40]In contrast the number of these microorganisms (AMB) wasfound to be lower than that reported for RTE-salads from

Porto Portugal and Catalonia Spain [41 42] The frequencyof TC on RTE-salads observed here coincides with previousstudies [43ndash45] carried out both in Mexico and in Brazilcountries where proper raw vegetable product handling andsanitation practices need to be promoted and implemented

FC were identified in 32 of RTE-salad samples theirfrequency was substantially lower than that reported forRTE-salads from other developing countries For instancefrequencies of 905 and 89 of FC were reported fromBrazil [45] and Costa Rica [46] respectively In spite ofthat Gomez-Aldapa et al [43] and Castro-Rosas et al [44]reported frequencies of FC of 955 and 99 respectivelycollected from restaurants in our countryWe can assume thatthese higher numbers are the result of the greater numberof people that can possibly be involved in the handling ofthis type of food in these types of places Furthermore thesedifferent numbers compared to the ones found by our workmay be due also to the quality of water used for growingthose vegetables to the different methods of handling themand to the different sampling techniques used Unsatisfactorymicrobiological state of RTE-salads was more frequentlyobserved in salads from SVS than in those from SPM(119875 = 0001) we suggest that these SVS lack adequateappreciation of basic food safety issues that is street vendorskeep the salads at room temperature (up to 27∘C) unlikesupermarkets where the salads are stored at refrigeratedtemperatures (4 to 7∘C) Street vendors often use stands thatare of inefficient construction running water is not easilyaccessible and hand and dish washing are performed in thesame bucket sometimes without soap Wastewater is usuallydiscarded right there in the streets and garbage is likewiseldquoconvenientlyrdquo discarded right next to the stands providing


attraction food and harborage for insects and rodents Inmany cases toilets are not available thus forcing the vendorsto eliminate their body wastes also in areas close by and toreturn to their vending sites without washing their handsSuch conditions and practices are likely to lead to crosscontamination of street food In other cases vendors buy rawmaterials from dubious sources and these materials may becontaminated with foodborne pathogens [47ndash49]

Regarding the microbiological quality of the sproutscollected in our work the frequencies and concentrationsof AMB found coincide with previous studies from differentcountries [3 42 50] It has been reported that vegetableseeds could contain lt2 log

10CFUg of BMA This naturally

occurring population ofmicroorganisms can rapidly increaseduring germination and sprouting because of the favorableconditions for bacterial growth [3] Consequently if seedsbecome contaminated with a pathogen the sprouting processprovides excellent conditions for consequent growth anddistribution

In our study TC were detected in 100 of sproutssamples result which coincides with that reported for beansprouts in Central Mexico where Salmonella and diar-rheagenic Escherichia coli pathotypes were also identified[51] On the other hand FC were detected in the 69 of ourRTE-sprout samples this frequency was found to be lowerthan that reported for RTE-sprouts frommarkets in the townof Pachuca Mexico [51]

Those high frequencies and concentrations of AMB TCand FC found in our RTE-sprouts may mean that they areso heavily contaminated that traditional sanitation practiceswould not be enough to reduce bacterial contamination Ithas been suggested that an alternative which can be usedonly by big food companies for reducing the bacterial loadin sprouts is the use of ionizing radiation since it hasbeen reported that a dose of 15 and 2 kGy can significantlyreduce E coli O157H7 and Salmonella to nondetectablelimits in bean and radish sprouts [52] Therefore our resultsconclude together with some others [43 44 51] reported forour country the highlighted need for implementing stricterhygienic control standards and measures for vegetables andsprouts grown in Mexico

In this study we combined conventional and molecularmethods and detected and identified NTM in 7 RTE-saladsand 12 RTE-sprouts All NTM identified in this study havealso been found in Mexican water samples [53] and in watersamples from other countries [21 54]Therefore it is possiblethat water may be the original source of NTM transmissionto vegetables (used in the salads) or sprouts while growingor during harvesting washing slicing soaking packagingand preparation NTM are opportunistic pathogens found inwater and soil as normal flora Therefore another possibilityof the presence of NTM in RTE-salads or RTE-sprouts comesfrom the soil and the water used for the growing and theirrigation respectively of those vegetables

A significant correlation between the presence of NTMand the median concentration of AMB (119875 = 0001) wasobserved This may be due to the fact that NTM as wellas AMB are environmental organisms that have features in

common such as growing in moderate to warm tempera-tures (20 and 45∘C) and in aerobiosis conditions

Some of theNTM identified in our study included speciesthat have been frequently associated with human illness inother countries that isM aviumM fortuitumM abscessusM chelonae and M mucogenium [55 56] In the case of Mavium it has been suggested that the most common portalsof entry of this microorganism are the gastrointestinal andthe respiratory tracts [57 58] In AIDS patients M avium isacquired predominantly via the gastrointestinal tract whereit is able to invade the intestinal mucosa to infect andmultiply within submucosal macrophages and to cause bac-teremia leading to the dissemination of the microorganismto the liver spleen and bone marrow [58] Regarding Mmucogenium it has been associated with infection of thegastrointestinal tract in patients with a diagnosis of Crohnrsquosdisease [59]

In Mexico City the prevalence of NTM infections ispoorly known and only a few studies have been publishedAmong these studies Lopez-Alvarez et al [60] reported in2010 that 15 of mycobacterial strains isolated from 67 HIVpatients belonged to NTM (10 strains were identified as Mavium and 1 strain was identified as M intracellulare) Inanother recent study Cortes-Torres et al in 2013 [61] reportedthat 37 of 96 patients in a Mexico City Hospital sufferingfrom various immunodeficiencies presented several strainsof NTM including M avium M simiae M gordonae andM kansasii Although no combined conclusion could bereached between these findings and ours further studies ofDNA fingerprinting of NTM should be carried out in orderto confirm that NTM isolated from RTE-salads and RTE-sprouts are the same as those isolated from patients

5 Conclusions

Most RTE-salads and RTE-sprouts analyzed in this study hadunsatisfactory microbiological quality and some harboredNTM associated with illness Measures to diminish or elimi-nate NTM strains from these food items might be advisablesuch as a proper handling andwashing before consumption ofthese products RTE-salads and sprouts could be consideredas potential sources for NTM infections in humans

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thankDr T Horn-CopelandMDfor the helpful English language review of the paper Thestudy was supported by CONACyT Mexico Grant CB 2010-156347-M (Jorge Alberto Gonzalez-y-Merchand) IPN SIP20130531 and 20140732 (Jorge Francisco Cerna-Cortes) andIPN SIP 20140970 (Jorge Alberto Gonzalez-y-Merchand)Sandra Rivera-Gutierrez Jorge Francisco Cerna-Cortes andJorge Alberto Gonzalez-y-Merchand are fellows of COFAA


and EDI IPN and Elizabeth Fernandez-Rendon is fellow ofCOFAA and EDD IPN

References

[1] Food and Agriculture Organization and World HealthOrganization Risk Assessment of Listeria monocytogenes inReady-to-Eat Foods Microbiological Risk Assessment Series4 Interpretive Summary 2004 httpappswhointirisbit-stream106654287419241562617pdfua=1

[2] N A Almualla L C Laleye A A Abushelaibi R A Al-Qassemi A A Wasesa and J Baboucarr ldquoAspects of themicrobiological quality and safety of ready-to-eat foods insharjah supermarkets in the United Arab Emiratesrdquo Journal ofFood Protection vol 73 no 7 pp 1328ndash1331 2010

[3] SD Saroj R Shashidhar VDhokane SHajare A Sharma andJ R Bandekar ldquoMicrobiological evaluation of sprouts marketedin Mumbai India and its suburbsrdquo Journal of Food Protectionvol 69 no 10 pp 2515ndash2518 2006

[4] B Mercanoglu Taban and A K Halkman ldquoDo leafy greenvegetables and their ready-to-eat [RTE] salads carry a risk offoodborne pathogensrdquo Anaerobe vol 17 no 6 pp 286ndash2872011

[5] G Gu J Hu J M Cevallos-Cevallos S M Richardson J ABartz and A H C van Bruggen ldquoInternal colonization ofSalmonella enterica serovar Typhimurium in tomato plantsrdquoPLoS ONE vol 6 no 11 Article ID e27340 2011

[6] S Kutter A Hartmann andM Schmid ldquoColonization of barley(Hordeum vulgare) with Salmonella enterica and Listeria spprdquoFEMS Microbiology Ecology vol 56 no 2 pp 262ndash271 2006

[7] S Pollack ldquoConsumer demand for fruit and vegetables the USexamplerdquo 2001 httpwwwersusdagovpublicationswrs011wrs011hpdfVCppelfd1dI

[8] L H Gould K A Walsh A R Vieira et al ldquoSurveillancefor foodborne disease outbreaksmdashUnited States 1998ndash2008rdquoMMWR Surveillance Summaries vol 62 no 1 pp 1ndash34 2013

[9] C N Berger S V Sodha R K Shaw et al ldquoFresh fruit andvegetables as vehicles for the transmission of humanpathogensrdquoEnvironmental Microbiology vol 12 no 9 pp 2385ndash2397 2010

[10] M F Lynch R V Tauxe and C W Hedberg ldquoThe growingburden of foodborne outbreaks due to contaminated freshproduce Risks and opportunitiesrdquo Epidemiology and Infectionvol 137 no 3 pp 307ndash315 2009

[11] C L Little and I A Gillespie ldquoPrepared salads and publichealthrdquo Journal of AppliedMicrobiology vol 105 no 6 pp 1729ndash1743 2008

[12] T Lienemann T Niskanen S Guedes A Siitonen M Kuusiand R Rimhanen-Finne ldquoIceberg lettuce as suggested sourceof a nationwide outbreak caused by two salmonella serotypesnewport and reading in Finland in 2008rdquo Journal of FoodProtection vol 74 no 6 pp 1035ndash1040 2011

[13] K Nygard J Lassen L Vold et al ldquoOutbreak of SalmonellaThompson infections linked to imported rucola lettucerdquo Food-borne Pathogens and Disease vol 5 no 2 pp 165ndash173 2008

[14] S Werner K Boman I Einemo et al ldquoOutbreak of SalmonellaStanley in Sweden associated with alfalfa sprouts July-August2007rdquo Euro Surveillance vol 12 no 10 Article ID E07101822007

[15] Y T H P van Duynhoven M-A Widdowson C M de Jageret al ldquoSalmonella enterica serotype Enteritidis phage type 4boutbreak associated with bean sproutsrdquo Emerging InfectiousDiseases vol 8 no 4 pp 440ndash443 2002

[16] S Ethelberg M Lisby B Bottiger et al ldquoOutbreaks of gas-troenteritis linked to lettuce Denmark January 2010rdquo EuroSurveillance vol 15 no 6 2010

[17] I Friesema G Sigmundsdottir K van der Zwaluw et al ldquoAninternational outbreak of Shiga toxin-producingEscherichia coliO157 infection due to lettuce September-October 2007rdquo EuroSurveillance vol 13 no 50 2008

[18] A Soderstrom P Osterberg A Lindqvist et al ldquoA largeEscherichia coliO157 outbreak in Sweden associatedwith locallyproduced lettucerdquo Foodborne Pathogens and Disease vol 5 no3 pp 339ndash349 2008

[19] U Buchholz H Bernard D Werber et al ldquoGerman outbreakof Escherichia coli O104H4 associated with sproutsrdquo The NewEngland Journal ofMedicine vol 365 no 19 pp 1763ndash1770 2011

[20] O Bezuidt R Pierneef K Mncube G Lima-Mendez and ON Reva ldquoMainstreams of horizontal gene exchange in enter-obacteria consideration of the outbreak of enterohemorrhagicE coli O104H4 in Germany in 2011rdquo PLoS ONE vol 6 no 10Article ID e25702 2011

[21] J O Falkinham ldquoImpact of human activities on the ecology ofnontuberculous mycobacteriardquo Future Microbiology vol 5 no6 pp 951ndash960 2010

[22] J O Falkinham III ldquoEpidemiology of infection by nontubercu-lous mycobacteriardquo Clinical Microbiology Reviews vol 9 no 2pp 177ndash215 1996

[23] B A Kendall and K LWinthrop ldquoUpdate on the epidemiologyof pulmonary nontuberculous mycobacterial infectionsrdquo Semi-nars in Respiratory and Critical Care Medicine vol 34 no 1 pp87ndash94 2013

[24] C Piersimoni and C Scarparo ldquoExtrapulmonary infectionsassociated with nontuberculous mycobacteria in immunocom-petent personsrdquo Emerging Infectious Diseases vol 15 no 9 pp1351ndash1358 2009

[25] C Piersimoni and C Scarparo ldquoPulmonary infections associ-ated with non-tuberculous mycobacteria in immunocompetentpatientsrdquo The Lancet Infectious Diseases vol 8 no 5 pp 323ndash334 2008

[26] R M Thomson J G Armstrong and D F Looke ldquoGastroe-sophageal reflux disease acid suppression and Mycobacteriumavium complex pulmonary diseaserdquo Chest vol 131 no 4 pp1166ndash1172 2007

[27] C Argueta S Yoder A E Holtzman et al ldquoIsolation andidentification of nontuberculous mycobacteria from foods aspossible exposure sourcesrdquo Journal of Food Protection vol 63no 7 pp 930ndash933 2000

[28] S Yoder C Argueta A Holtzman et al ldquoPCR comparisonof Mycobacterium avium isolates obtained from patients andfoodsrdquo Applied and Environmental Microbiology vol 65 no 6pp 2650ndash2653 1999

[29] C F von Reyn R D Arbeit A N A Tosteson et al ldquoTheinternational epidemiology of disseminated Mycobacteriumavium complex infection inAIDSrdquoAIDS vol 10 no 9 pp 1025ndash1032 1996

[30] U S Food and Drug Administration (FDA) ldquoBacteriologi-cal analytical manual onlinerdquo 2013 httpwwwfdagovFoodFoodScienceResearchLaboratoryMethodsucm2006949htm

[31] ldquoNorma oficial mexicana nom-093-ssa1-1994 bienes yservicios Practicas de higiene y sanidad en la preparacionde alimentos que se ofrecen en establecimientos fijosrdquo 1995httpconapescagobmxworksitesconaresourcesLocalCo-ntent82704NOM093SSA11994pdf


[32] L Cobos-Marın J Montes-Vargas S Rivera-Gutierrez ALicea-Navarro J A Gonzalez-Y-Merchand and I Estrada-Garcıa ldquoA novel multiplex-PCR for the rapid identification ofMycobacterium bovis in clinical isolates of both veterinary andhuman originrdquo Epidemiology and Infection vol 130 no 3 pp485ndash490 2003

[33] I Perez-Martinez A Ponce-De-Leon M Bobadilla et alldquoA novel identification scheme for genus Mycobacterium Mtuberculosis complex and sevenmycobacteria species of humanclinical impactrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 27 no 6 pp 451ndash459 2008

[34] A Telenti F Marchesi M Balz F Bally E C Bottger and TBodmer ldquoRapid identification of mycobacteria to the specieslevel by polymerase chain reaction and restriction enzymeanalysisrdquo Journal of Clinical Microbiology vol 31 no 2 pp 175ndash178 1993

[35] P Kirschner B Springer U Vogel et al ldquoGenotypic identifica-tion of mycobacteria by nucleic acid sequence determinationreport of a 2-year experience in a clinical laboratoryrdquo Journal ofClinical Microbiology vol 31 no 11 pp 2882ndash2889 1993

[36] T Adekambi P Colson and M Drancourt ldquorpo B -basedidentification of nonpigmented and late-pigmenting rapidlygrowing mycobacteriardquo Journal of Clinical Microbiology vol 41no 12 pp 5699ndash5708 2003

[37] ldquoIdentification of mycobacteriardquo PRA Database httpappchuvchprasiteindexhtml

[38] D A Relman T M Schmidt R P Macdermott and S FalkowldquoIdentification of the uncultured bacillus of Whipples diseaserdquoNew England Journal of Medicine vol 327 no 5 pp 293ndash3011992

[39] ldquoGuidelines for microbiological examination of ready-to-eat foodsrdquo httpwwwfoodstandardsgovaupublicationsdoc-umentsGuidelines20for20Micro20exampdf

[40] F M Mosupye and A von Holy ldquoMicrobiological quality andsafety of ready-to-eat street-vended foods in JohannesburgSouth Africardquo Journal of Food Protection vol 62 no 11 pp1278ndash1284 1999

[41] J Campos J Mourao N Pestana L Peixe C Novais andP Antunes ldquoMicrobiological quality of ready-to-eat saladsan underestimated vehicle of bacteria and clinically relevantantibiotic resistance genesrdquo International Journal of FoodMicro-biology vol 166 no 3 pp 464ndash470 2013

[42] M Abadias J Usall M Anguera C Solsona and I VinasldquoMicrobiological quality of fresh minimally-processed fruitand vegetables and sprouts from retail establishmentsrdquo Interna-tional Journal of FoodMicrobiology vol 123 no 1-2 pp 121ndash1292008

[43] C A Gomez-Aldapa E Rangel-Vargas and J Castro-RosasldquoFrequency and correlation of some enteric indicator bacteriaand salmonella in ready-to-eat raw vegetable salads from Mex-ican restaurantsrdquo Journal of Food Science vol 78 no 8 ppM1201ndashM1207 2013

[44] J Castro-Rosas J F Cerna-Cortes EMendez-Reyes D Lopez-Hernandez C A Gomez-Aldapa and T Estrada-Garcia ldquoPres-ence of faecal coliforms Escherichia coli and diarrheagenic Ecoli pathotypes in ready-to-eat salads from an area where cropsare irrigated with untreated sewage waterrdquo International Journalof Food Microbiology vol 156 no 2 pp 176ndash180 2012

[45] H Froder C G Martins K L O De Souza M Landgraf B DGM Franco andM TDestro ldquoMinimally processed vegetablesalads microbial quality evaluationrdquo Journal of Food Protectionvol 70 no 5 pp 1277ndash1280 2007

[46] E Rodrıguez-Cavallini C Rodrıguez M del Mar Gamboaand M L Arias ldquoMicrobiological evaluation of ready-to-eatfoods manufactured by small Costa Rican industriesrdquo ArchivosLatinoamericanos de Nutricion vol 60 no 2 pp 179ndash183 2010

[47] S Rane ldquoStreet vended food in developing world hazardanalysesrdquo Indian Journal of Microbiology vol 51 no 1 pp 100ndash106 2011

[48] D H Tambekar V J Jaiswal D V Dhanorkar P B Gulhaneand M N Dudhane ldquoIdentification of microbiological hazardsand safety of ready-to-eat food vended in streets of AmravatiCity Indiardquo Journal of Applied Biosciences vol 7 pp 195ndash2012008

[49] E O Ekanem ldquoThe street food trade in Africa safety and socio-environmental issuesrdquo Food Control vol 9 no 4 pp 211ndash2151998

[50] D Althaus E Hofer S Corti A Julmi and R StephanldquoBacteriological survey of ready-to-eat lettuce fresh-cut fruitand sprouts collected from the swiss marketrdquo Journal of FoodProtection vol 75 no 7 pp 1338ndash1341 2012

[51] J F Cerna-Cortes C A Gomez-Aldapa E Rangel-VargasE Ramırez-Cruz and J Castro-Rosas ldquoPresence of indicatorbacteria Salmonella and diarrheagenic Escherichia coli patho-types on mung bean sprouts from public markets in PachucaMexicordquo Food Control vol 31 no 2 pp 280ndash283 2013

[52] M L Bari M I Al-Haq T Kawasaki et al ldquoIrradiation to killEscherichia coliO157H7 and Salmonella on ready-to-eat radishand mung bean sproutsrdquo Journal of Food Protection vol 67 no10 pp 2263ndash2268 2004

[53] I Perez-Martinez D A Aguilar-Ayala E Fernandez-Rendonet al ldquoOccurrence of potentially pathogenic nontuberculousmycobacteria in Mexican household potable water a pilotstudyrdquo BMC Research Notes vol 6 no 1 article 531 2013

[54] R M Thomson R Carter C Tolson C Coulter F Huygensand M Hargreaves ldquoFactors associated with the isolation ofNontuberculous mycobacteria (NTM) from a large municipalwater system in Brisbane Australiardquo BMCMicrobiology vol 13no 1 article 89 2013

[55] A R F da Costa J O Falkinham III M L Lopes et al ldquoOccur-rence of nontuberculous mycobacterial pulmonary infectionin an endemic area of tuberculosisrdquo PLoS Neglected TropicalDiseases vol 7 no 7 Article ID e2340 2013

[56] P M Cassidy K Hedberg A Saulson E McNelly and K LWinthrop ldquoNontuberculous mycobacterial disease prevalenceand risk factors a changing epidemiologyrdquo Clinical InfectiousDiseases vol 49 no 12 pp e124ndashe129 2009

[57] C A Benson ldquoDisease due to the Mycobacterium aviumcomplex in patients with AIDS epidemiology and clinicalsyndromerdquo Clinical Infectious Diseases vol 18 supplement 3pp S218ndashS222 1994

[58] B Damsker and E J Bottone ldquoMycobacterium avium-Mycobacterium intracellulare from the intestinal tracts ofpatients with the acquired immunodeficiency syndrome con-cepts regarding acquisition and pathogenesisrdquo Journal of Infec-tious Diseases vol 151 no 1 pp 179ndash181 1985

[59] C Y Tzen and T Y Wu ldquoDetection of mycobacteria in Crohnrsquosdisease by a broad spectrum polymerase chain reactionrdquo Jour-nal of the FormosanMedical Association vol 105 no 4 pp 290ndash298 2006

[60] R Lopez-Alvarez C Badillo-Lopez J F Cerna-Cortes et alldquoFirst insights into the genetic diversity of Mycobacteriumtuberculosis isolates from HIV-infected Mexican patients and


mutations causing multidrug resistancerdquo BMC Microbiologyvol 10 article 82 2010

[61] N Cortes-Torres J A Gonzalez-y-Merchand C Gonzalez-Bonilla and G Garcıa-Elorriaga ldquoMolecular analysis ofmycobacteria isolated in Mexican patients with differentimmunodeficiencies in a tertiary care hospitalrdquo Archives ofMedical Research vol 44 no 7 pp 562ndash569 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Gustavo AMadero

Azcapotzalco

Venustiano Carranza

Cuauhteacutemoc

Miguel Hidalgo

IztacalcoBenitoJuaacuterezAacutelvaro

ObregoacutenCuajimalpade

Morelos

MagdalenaContreras

Tlalpan

IztapalapaCoyoacaacuten

Xochimilco

Tlaacutehuac

Milpa Alta

0 10 20 30(km)

Total surface 1525km2

S

N

W E

Figure 1 Boroughs of Mexico City where RTE-salads (e) and RTE-sprouts (◼) were collected

as a result of efforts to promote better nutrition in thepopulation [4] As RTE-salads and RTE-sprouts do not needfurther preparation before consumption they could poten-tially contain pathogens that form part of their microfloraposing a public health problem Fresh vegetables can becomecontaminated by pathogens as Salmonella at any point duringthe food production process During preharvest contact withcontaminated irrigation water soil manure or fecal matterof wild animals may occur These pathogens can both bindto plant leaves andor be internalized via the leaves or theendophytic root system [5 6] During harvest asymptomatichuman carriers might contaminate the products and at thepostharvest level products become contaminated by contactwith polluted water other asymptomatic human carriers orthe production process environment Over the last 30 yearsthere has been at least a 24 of increase in the averageamount of fresh vegetable consumed per person in the USA[7]Moreover the number of gastroenteritis outbreaks causedby foodborne pathogens after consumption of raw vegetablessalads and sprouts has increased worldwide [8ndash11] Eventhough Salmonella is the most common cause of diseaseoutbreaks associated with lettuce and sprouts [12ndash15] thereare other pathogens (Shiga toxin producing E coli O157Norovirus) that have been described as relevant microbial

hazards [16ndash18] For example a large outbreak of hemolytic-uremic syndrome caused by STECO104H4 linked to sproutsoccurred in Germany [19 20]

Nontuberculous mycobacteria (NTM) are opportunis-tic pathogens found in the environment that cause life-threatening infections in humans other mammals and birds[21 22] The incidence of NTM disease is increasing world-wide [23] in both immunocompetent and immunocompro-mised subjects [22 24 25] As there is no defining evidencefor person-to-person transmission for most of the NTM[24 25] it is therefore important to establish the sourcesand routes of NTM transmission since infections can occurthrough inhalation ingestion gastric reflux or skin trauma[22 26] NTM have been isolated from various kinds offood and many studies support the hypothesis that foodespecially raw or partially cooked products plays a role asa source of NTM for humans primarily in countries withsimilar processing food routes and climates [27ndash29]The aimsof this study were to evaluate the microbiological quality ofRTE-salads and RTE-sprouts collected in Mexico City and toanalyze the occurrence of NTM in these samples in order todeterminewhether theseRTE foodsmay represent a potentialrisk for NTM infection for the consumers






























3 Results







identified

RTE-salads







1M sp

RTE-sprouts









4 Discussion


10
















5 Conclusions




Acknowledgments




References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























3 Results







identified

RTE-salads







1M sp

RTE-sprouts









4 Discussion


10
















5 Conclusions




Acknowledgments




References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




identified

RTE-salads







1M sp

RTE-sprouts









4 Discussion


10
















5 Conclusions




Acknowledgments




References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













5 Conclusions




Acknowledgments




References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article microbiological quality of ready-to-eat

Documents