microorganisms

Article Comparison of Common Enrichment Broths Used in Diagnostic Laboratories for Shiga Toxin—Producing Escherichia coli

Michael Bording-Jorgensen 1, Hannah Tyrrell 1, Colin Lloyd 1 and Linda Chui 1,2,*

1 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB T6G 2R3, Canada; [email protected] (M.B.-J.); [email protected] (H.T.); [email protected] (C.L.) 2 Alberta Precision Laboratories-Public Health Laboratory (ProvLab), Edmonton, AB T6G 2J2, Canada * Correspondence: [email protected]; Tel.: +(780)407-8951

Abstract: Acute gastroenteritis caused by Shiga toxin-producing Escherichia coli (STEC) affects more than 4 million individuals in Canada. Diagnostic laboratories are shifting towards culture- independent diagnostic testing; however, recovery of STEC remains an important aspect of surveil- lance programs. The objective of this study was to compare common broth media used for the enrichment of STEC. Clinical isolates including O157:H7 as well as non-O157 serotypes were cultured in tryptic soy (TSB), MacConkey (Mac), and Gram-negative (GN) broths and growth was compared using culture on sheep’s blood agar and real-time PCR (qPCR). In addition, a selection of the same isolates was spiked into negative stool and enriched in the same three broths, which were then evaluated using culture on CHROMagarTM STEC agar and qPCR. TSB was found to provide the

 optimal enrichment for growth of isolates with and without stool. The results from this study suggest  that diagnostic laboratories may benefit from enriching STEC samples in TSB as a first line enrichment

Citation: Bording-Jorgensen, M.; instead of GN or Mac. Tyrrell, H.; Lloyd, C.; Chui, L. Comparison of Common Enrichment Keywords: STEC; enrichment; real-time PCR; broth; culture Broths Used in Diagnostic Laboratories for Shiga Toxin—Producing Escherichia coli. Microorganisms 2021, 9, 503. 1. Introduction https://doi.org/10.3390/ Acute gastroenteritis (AGE), which is defined as vomiting and/or diarrhea for less microorganisms9030503 than 7 days, affects more than 4 million individuals in Canada [1]. Shiga toxin-producing Escherichia coli (STEC) are one of the pathogens responsible for major outbreaks. Although Academic Editor: Hirokazu Kimura their primary reservoir is ruminants, there have been recent outbreaks involving con- taminated food items such as flour, clover sprouts, and cheese [2]. STEC infections are Received: 28 January 2021 associated with hemorrhagic colitis with the possibility of developing hemolytic uremic Accepted: 25 February 2021 Published: 27 February 2021 syndrome [3]. This potentially deadly consequence is due to the production of Shiga toxins (Stx) 1 and/or 2, with Stx 2 having a higher association [4]. The exotoxin genes are located

Publisher’s Note: MDPI stays neutral on a lambda prophage and the toxins are released into the lumen during colonization and with regard to jurisdictional claims in replication resulted in causing damages to the intestinal barrier [5]. STEC can also contain published maps and institutional affil- a variety of virulence factors such as eae and hly, which are localized within the locus of iations. enterocyte effacement pathogenicity island [6]. To date, there have been over 200 serotypes of E. coli identified to contain the Shiga toxin and cause diarrheal disease in humans [7]. E. coli O157:H7 was the first serotype identified in 1982, causing AGE-related morbidity involved in several outbreaks in the United States of America. Recently, other serotypes known as the “Big 6” (O26, O45, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. O103, O111, O121, and O145) have been the cause of outbreak in North America [8]. This article is an open access article Recent genomic comparisons of O145 have shown significant metabolic diversity within a distributed under the terms and particular serotype, highlighting the possibility of a difference in growth requirements for conditions of the Creative Commons enrichment both within and between serotypes, which is required for the identification of Attribution (CC BY) license (https:// serotypes during outbreaks [9]. creativecommons.org/licenses/by/ STEC infection is notifiable in Alberta, indicating its importance for monitoring and 4.0/). control. This is achieved through surveillance programs, which rely on the ability to culture

Microorganisms 2021, 9, 503. https://doi.org/10.3390/microorganisms9030503 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 503 2 of 10

the organism for further characterization. Although culture-independent diagnostic testing (CIDT) has become more prominent in recent years, culture is essential for surveillance and cluster detection. Isolation of O157 STEC in the diagnostic laboratory can be achieved using sorbitol-MacConkey agar or chromogenic agar (O157 and non-O157), which are both selective media, but they might not support growth of all serotypes [10]. The Centers for Disease Control and Prevention published guidelines for the diagnosis of STEC in which they recommend either MacConkey or Gram-negative broth for enrichment [11]. Cefixime and tellurite are common ingredients used in the selective agar recommended for the isolation of STEC due to the particularly difficult nature regarding the isolation of non- O157 serotypes [10,12,13]. In contrast, the ingredients of the recommended broth for the enrichment of STEC are less selective and can be used for the growth of multiple pathogens. The objective of this study was to evaluate the growth of clinical isolates with and without stool using various broth media (tryptic soy broth, MacConkey, and Gram-negative) by culture and real-time PCR (qPCR).

2. Materials and Methods 2.1. Bacterial Isolate Selection and Enrichment STEC isolates are routinely submitted to the Alberta Precision Laboratories-ProvLab for further characterization. A total of 52 isolates consisting of O157 and non-O157 serotypes as shown in Supplementary Table S1 were included in this study. Archived fingerprinting patterns generated by pulsed-field gel electrophoresis in our database were analysed using BioNumerics software V6.1 (Austin, TX, USA) to ensure that they all have indistinguishable pulsotypes. These isolates were retrieved from skim milk stored at −80 ◦C and cultured on sheep blood agar plates (BAP) (Oxoid, Fisher Scientific, Ottawa, ON, Canada) overnight at 37 ◦C. A single colony was picked, suspended in 250 µL of 1× PBS, and 50 µL was added to 5 mL of tryptic soy broth (TSB, Bacto/BD, Fisher Scientific, Ottawa, ON, Canada), MacConkey (MAC, Dalynn Biologicals, Calgary, AB, Canada) and Gram- negative (GN, Dalynn Biologicals) broths, and incubated overnight at 37 ◦C. A 10-fold serial dilution of each broth were then plated in triplicates onto BAP and colonies were counted the following day.

2.2. Spiking of Negative Stool

Negative stools (n = 3) were screened for the presence of stx1 and stx2 genes using qPCR as well as plated on CHROMagarTM STEC (Dalynn Biologicals, Calgary, AB, Canada) plates to ensure there were no other within the stool that would grow mauve- colored colonies [14], which might be indicative of STEC colonies. These negative stools were then pooled together for the spiking experiments. A subset of STEC isolates (n = 25), which are indicated in Supplementary Table S1, from the broth experiments described above were grown in TSB overnight. Then, 1 mL of a 0.5 O.D. was centrifuged (13,000× g for 10 min) and washed with 1× PBS twice. Dilutions were made to obtain a cell suspension of 1 × 105 CFU/mL, and 100 µL was combined with 150 µL of negative stool and added to three separate 5 mL broth tubes (TSB, Mac, GN). The stool-spiked broth cultures were incubated overnight at 37 ◦C. Ten-fold serial dilutions of each broth were then plated in triplicates on CHROMagarTM STEC (Dalynn Biologicals) and colonies were counted the following day. Production of the Shiga toxin was determined using Shiga Toxin Quik ChekTM (Tech- Lab, Blacksburg, VA, USA) as per manufacturer’s protocol. In brief, 100 µL of broth was added to a tube containing 650 µL diluent and conjugate, and 500 µL was added to the cassette and left at room temperature for 15 min. Next, 300 µL of wash buffer was added followed by substrate and left to develop at room temperature for 10 min. The results were visually read as either positive or negative for Shiga toxin 1 and 2. The stx status of each isolate was already known prior to the experiments; therefore, the Quik ChekTM was used to ensure the growth was due to STEC in the stool experiments (Table S1). Microorganisms 2021, 9, 503 3 of 10

2.3. DNA Extraction and qPCR of Broth Enrichment Enriched TSB, Mac, and GN broths without (n = 52) and with (n = 25) stool were extracted using rapid lysis buffer (100 mM NaCl, 10 mM Tris-HCL pH 8.3, 1 mM EDTA pH 9.0, 1% Triton X-100). A 250 µL volume of enriched culture was centrifuged (13,000× g for 10 min) and the pellet was resuspended in 100 µL of rapid lysis buffer and heated to 95 ◦C for 15 min using a heating block. The samples were then centrifuged (13,000× g for 10 min), and the supernatant was stored at 4 ◦C until further testing via qPCR. The primers and probes (Integrated DNA Technology, Coralville, IA, USA) for detect- ing stx genes are shown in Table1. The total reaction contained 12.5 µL of 1× PrimeTime® Gene Expression Master Mix (Integrated DNA Technology), 0.33 µM of each primer, 0.22 µM probe, 5 µL DNA template and molecular biology grade water in a total of 25 µL reaction volume. A negative template control and O157 positive control DNA was included in each run. qPCR amplification conditions consisted of 95 ◦C for 1 min followed by 40 cy- cles of 95 ◦C for 5 s and 58 ◦C for 45 s performed on the 7500 FAST real-time PCR system (Applied Biosystems, Foster City, CA, USA). Using a crossing threshold of 0.1, all Ct values below 30 were considered positive.

Table 1. Primer and probe sequences used in this study [15].

Reference Gene, Primer/Probe Sequence 50-30

stx1-F TTT GTY ACT GTS ACA GCW GAA GCY TTA CG

stx1-R CCC CAG TTC ARW GTR AGR TCM ACR TC

stx1-P CTG GAT GAT CTC AGT GGG CGT TCT TAT GTA A

stx2-F TTT GTY ACT GTS ACA GCW GAA GCY TTA CG

stx2-R CCC CAG TTC ARW GTR AGR TCM ACR TC

stx2 -P TCG TCA GGC ACT GTC TGA AAC TGC TCC In the sequences: Y is (C, T), S is (C, G), W is (A, T), R is (A, G), M is (A, C).

2.4. Statistics Statistical analysis was performed using Prism8 for Mac (Graph Pad, San Diego, CA, USA). One-way ANOVA with Holm–Sidak’s multiple comparison test were used for statistical comparisons of media growth within each isolate. Bar graphs represent the mean ±SEM and all comparisons with p ≤ 0.05 were considered significant.

3. Results 3.1. Comparing Enrichment of STEC in Different Broths with and without the Presence of Stool STEC isolates (n = 52) were grown in TSB, GN, and Mac broth to determine if broth composition would influence growth. Overall, growth in TSB was significantly (p ≤ 0.5) higher than GN or Mac broth (Figure1A). GN broth also showed significantly ( p ≤ 0.5) higher growth compared to Mac broth (Figure1A). A subset of isolates ( n = 25) was then grown in the same broth with the presence of STEC negative stool. TSB showed significantly higher (p ≤ 0.5) growth as compared to GN and Mac in the presence of stool (Figure1B). There was no significant growth difference between GN and Mac broth (Figure1B). Growth was overall lower for each of the broths containing stool as compared to no stool; however, there was no statistical difference (Figure1A,B). Microorganisms 2021, 9, x FOR PEER REVIEW 4 of 10 Microorganisms 2021, 9, 503 4 of 10

A * B * * 109 109 * * 108 108 107 107 106 106 105 105 104 104 3

3 Growth (CFU/mL) 10

Growth (CFU/mL) Growth 10 102 102 1

1 E. coli 10

E. coli coli E. 10 100 100 TSB GN MAC TSB GN MAC Broth without Stool Broth with Stool

Figure 1. Effect of the tryptic soy broth (TSB), Gram-negative (GN), and MacConkey (MAC) broths Figure 1. Effect of the tryptic soy broth (TSB), Gram-negative (GN), and MacConkey (MAC) broths for E. coli (STEC) enrichment in the absence ((A), n = 52 isolates) or presence ((B), n = 25 isolates) for E. coli (STEC) enrichment in the absence ((A), n = 52 isolates) or presence ((B), n = 25 isolates) of of stool. Bars represent the CFU/mL calculated from serial dilutions on blood agar plates (A) or stool. Bars represent the CFU/mL calculated from serial dilutions on blood agar plates (A) or CHROMagarTM (B). Error bars represent the standard error of the mean. * Indicates growth is CHROMagarTM (B). Error bars represent the standard error of the mean. * Indicates growth is sig- ≤ nificant;significant; ANOVA ANOVA p ≤ 0.05.p 0.05. 3.2. Comparison of Isolates within Each Serotype Group Independent of Stool 3.2. Comparison of Isolates within Each Serotype Group Independent of Stool We compared the growth of all the isolates (n = 52) to determine if there was a difference betweenWe compared or within the each growth of the of serotype all the isolates groups (n when = 52) enrichedto determine in each if there of the was broth a differ- used. enceSTEC between were grown or within on BAP each plates of the and serotype enumerated groups for when comparison enriched between in each broths. of the TSB broth was used.found STEC to significantly were grown (71%, on BAPp ≤ 0.5)plates improve and enumerated growth compared for comparison to GN broth between for 38 broths. isolates TSB(Figure was2 found, Table 2to). significantly TSB was shown (71%, to improvep ≤ 0.5) improve growth significantly growth compared ( p ≤ 0.5) to asGN compared broth for to 38Mac isolates broth (Figure for 81% 2, (Tablen = 42) 2). of TSB the isolateswas shown (Figure to improve2, Table2 growth). There significantly was significant (p ≤ growth 0.5) as comparedimprovement to Mac in broth GN broth for 81% (p ≤ (n0.5) = 42) when of the compared isolates (Figure to Mac 2, broth Table for 2). 28 There (54%) was isolates sig- nificant(Figure 2growth, Table2 improvement). Surprisingly, in GN GN showed broth significant(p ≤ 0.5) when ( p ≤ compared0.5) growth to as Mac compared broth for to TSB28 (54%)in isolates isolates #29 (Figure (O145) 2, and Table #34 (O118)2). Surprisingly, (Figure2, TableGN showed2). Growth significant was found (p ≤ to0.5) be growth decreased as comparedin Mac broth to TSB for allin isolates isolates, #29 although (O145) there and was#34 (O118) no significant (Figure difference 2, Table 2). in brothGrowth type was for foundsix (12%) to be isolates decreased (#10 in (O103), Mac broth #12 (O103), for all isolates, #18 (O111), although #22 (O121), there #38was (O71), no significant and #51 (O5))dif- ference(Figure in2). broth type for six (12%) isolates (#10 (O103), #12 (O103), #18 (O111), #22 (O121), #38 (O71), and #51 (O5)) (Figure 2). Table 2. Percentage of isolates with significantly different growth in each broth (n = 52).

Broth TSB GN Mac TSB NA 38 (71%) 42 (81%) GN 2 (3%) NA 28 (54%) Mac 0 0 NA No Significance 6 (12%) Tryptic Soy Broth (TSB), Gram-negative Broth (GN), MacConkey Broth (Mac). MicroorganismsMicroorganisms 2021, 92021, x FOR, 9, 503PEER REVIEW 5 of 105 of 10

* * * * * * * * 10 * * * 10 * * * 10 * * TSB 10 * * * * * * 10 9 9 * * * 10 * * * 10 10 TSB 9 * * ** 8 GN 8 10 TSB 10 10 8 7 7 GN 10 10 MAC 10 7 GN 6 6 10 10 10 MAC 6 5 5 10 10 10 5 MAC 4 4 10 10 10 4 103 3 10 O45 Growth (CFU/mL) Growth O45 10 3 2 2 10 10 Growth (CFU/mL) O26 10 2

1 1 O103 Growth (CFU/mL) 10 10 10 1

E. coli E. coli 0 0 10 10 10 0 123 E. coli 10

456789 E. coli 10 11 12 13 14 15 Bacterial Isolate Bacterial Isolate Bacterial Isolate

* * 10 * * * * * * * 10 * * * * 10 * * * * * 10 * * * * 10 * * 9 TSB 10 9 10 * * * 9 * * * * TSB 10 TSB 8 10 8 10 GN 8 10 7 10 GN 7 GN 10 MAC 7 10 6 10 6 10 6 MAC 10 5 10 5 MAC 10 5 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10

2 (CFU/mlLO121 Growth 10 2 O111 Growth (CFU/mL) Growth O111 10 1 (CFU/mL) O145 Growth 10 1 10 1 10 0 10 100 10 100 E. coli E. coli 16 17 18 19 20 21 22 23 24 25 26 coli E. 27 28 29 30 31 Bacterial Isolate Bacterial Isolate Bacterial Isolate

* * * * * 10 9 * * * * 10 * * * 10 TSB 10 * ** 9 * * * TSB 8 10 * 10 10 9 TSB 8 7 GN 10 10 10 8 7 GN 6 10 GN 10 MAC 7 6 10 10 10 5 6 MAC 5 MAC 10 10 10 4 5 4 10 10 10 3 3 10 104 10 2 3

2 O71 Growth (CFU/mL) 10 10 O118 Growth (CFU/mlL Growth O118 10 1 2 101 10 10 0 0 101 10 10 0 E. coli E. E. coli E. coli 32 33 34 35 36 37 38 10 39 40 41

Bacterial Isolate Bacterial Isolate (CFU/mL Growth STEC 6 Non-Top Bacterial Isolate

* * * * * * * * * ** * 10 * * 10 * * * * 9 * * * 10 109 * TSB 108 TSB 8 7 10 GN 10 GN 107 6 10 MAC 106 MAC 105 105 104 104 3 103 10 2 2 O157 Growth (CFU/mL 10 10 1 1 10 10 0 0 10

10 coli E. 46 47 48 49 50 42 43 44 45 51 52

Non-Top 6 STECGrowth(CFU/mL) 6 Non-Top Bacterial Isolate Bacterial Isolate

FigureFigure 2. Effects 2. Effects of different of different enrichments enrichments broths broths tryptic tryptic soy br soyoth broth(TSB), (TSB), GN, and GN, MacConkey and MacConkey (MAC) (MAC) on the on growth the growth of of STECSTEC isolates isolates (n = 52 (n =isolates). 52 isolates). Bars Bars represent represent the theCFU/mL CFU/mL calculated calculated from from serial serial dilutions dilutions plated plated in in triplicate triplicate on on BAP. Error Error bars represent the standard error of the mean. * Growth is significant; ANOVA p ≤ 0.05. bars represent the standard error of the mean. * Growth is significant; ANOVA p ≤ 0.05.

Table 2. Percentage of isolates with significantly different growth in each broth (n = 52). 3.3. Comparing Isolate Growth in the Presence of Stool SelectedBroth isolates (n = 25) from theTSB previous experimentGN were grown inMac the same broth type in the presenceTSB of stool to determine NA if their growth 38 (71%) would be impacted 42 (81%) in the presence of competingGN bacteria. The stool was 2 (3%) first confirmed NA to be negative for 28 STEC(54%) as well as no growthMac on CHROMagar TM STEC from0 other bacteria.0 Blood agar platesNA were not used for enumerationNo Significance as they support growth of commensal 6 (12%)E. coli and other enteric bacteria foundTryptic in stools. Soy Broth CHROMagar (TSB), Gram-negativTM STEC wase Broth selected (GN), MacConkey for the culture Broth media (Mac). for performing plate counts. Growth in TSB was found to be significantly (p ≤ 0.5) higher compared to 3.3. Comparingboth GN Isolate (23 isolates, Growth 92%) in the and Presence Mac brothsof Stool (25 isolates, 100%) (Figure3, Table3). GN Selectedbroth improved isolates growth(n = 25) significantlyfrom the previous (p ≤ 0.5) experiment when compared were grown to Mac in the broth same for broth isolates #3 type (O45),in the presence #5 (O26), of # 7stool (O26), to #22determine (O121), if #29 their (O145), growth #34 would (O118), be #50 impacted (O157), in and the #51 pres- (O5), as ence ofillustrated competing in Figurebacteria.3. The stool was first confirmed to be negative for STEC as well as no growth on CHROMagarTM STEC from other bacteria. Blood agar plates were not used for enumeration as they support growth of commensal E. coli and other enteric bac- teria found in stools. CHROMagarTM STEC was selected for the culture media for perform- ing plate counts. Growth in TSB was found to be significantly (p ≤ 0.5) higher compared to both GN (23 isolates, 92%) and Mac broths (25 isolates, 100%) (Figure 3, Table 3). GN broth improved growth significantly (p ≤ 0.5) when compared to Mac broth for isolates #3 Microorganisms 2021, 9, x FOR PEER REVIEW 6 of 10

Microorganisms 2021, 9, 503 (O45), #5 (O26), # 7 (O26), #22 (O121), #29 (O145), #34 (O118), #50 (O157), and #51 (O5),6 of as 10 illustrated in Figure 3.

* * * * * * 9 * *** * * 9 * * * * * * 10 * *** * * 10 * * * TSB TSB 108 108 * * GN GN 107 107 106 MAC 106 MAC 5 105 10 104 104 103 103 2 2

10 Growth Bacterial 10 Bacterial Growth 1 1 with Stool (CFU/mL) with Stool (CFU/mL) Stool with 10 10 100 100 34571315 17 20 22 25 29 30 O45 O26 O103 O111 O121 O145 Bacterial Isolate Bacterial Isolate

* * * * * 1010 109 * * * * * * * 9 * * * * * * 8 * * 10 * * * * * * 10 8 * TSB TSB 10 107 107 GN 6 GN 6 10 10 5 5 MAC 10 MAC 10 4 4 10 10 3 103 10 2 2 Bacterial Growth Bacterial Growth 10 10 1 1 with Stool (CFU/mL) with Stool (CFU/mL) Stool with 10 10 100 100 34 35 36 37 39 41 42 45 48 49 50 51 52 O118 O71 O146 O85 O38 O22 O157 OR Bacterial Isolate Bacterial Isolate

Figure 3. Effects of different enrichments broths tryptic soy broth (TSB), GN, and MacConkey (MAC) on the growth of Figure 3. Effects of different enrichments broths tryptic soy broth (TSB), GN, and MacConkey (MAC) on the growth of STECSTEC isolates isolates ( n = 25 25 isolates) isolates) with stool. The bacterial serotype is indicated. Bars represent thethe CFU/mL calculated calculated from from TM serialserial dilution dilution plates in triplicatetriplicate onon CHROMagarCHROMagarTM plates.plates. ErrorError barsbars representrepresent the the standard standard error error of of the the mean. mean. * Growth* Growth is issignificant; significant; ANOVA ANOVAp ≤p ≤0.05. 0.05.

TableTable 3. PercentagePercentage of of isolates with significantly significantly di differentfferent growth in each broth with stool (n = 25).

BrothBroth TSBTSB GNGN Mac Mac TSB NA 23 (92%) 25 (100%) TSB NA 23 (92%) 25 (100%) GN 0 NA 8 (32%) GNMac 00 NA0 8NA (32%) MacNo Significance 0 0 0 NA No SignificanceTryptic Soy Broth (TSB), Gram-negative Broth (GN), 0 MacConkey Broth (Mac) Tryptic Soy Broth (TSB), Gram-negative Broth (GN), MacConkey Broth (Mac). 3.4. Comparing Ct Values Targeting the stx Gene for Isolates Inoculated into Broth with and without3.4. Comparing Stool Ct Values Targeting the Stx Gene for Isolates Inoculated into Broth with and withoutDNA Stool was extracted from the TSB, GN, and Mac broths from both pure cultures and stool-spikedDNA was enrichments extracted fromand the the relative TSB, GN, abundance and Mac of broths STEC from was bothcompared pure culturesusing qPCR and crossingstool-spiked threshold enrichments (Ct) values. and theSimilar relative to the abundance observations of STEC using was colony compared enumeration, using qPCR Ct valuescrossing were threshold consistently (Ct) values. lower with Similar DNA to extracted the observations from isolates using grown colony in enumeration, TSB (14.7; 95% Ct CIvalues 13.64-15.31) were consistently as compared lower to GN with (16.14; DNA 95% extracted CI 15.38–16.90) from isolates or Mac grown (17.55; in TSB95% (14.7;CI 17.55– 95% 19.32)CI 13.64–15.31) broths (Figure as compared 4) was observed. to GN (16.14;The Ct 95%values CI were 15.38–16.90) higher with or Mac DNA (17.55; from isolates 95% CI grown17.55–19.32) in all broth broths types (Figure in 4the) was presence observed. of stool; The Cthowever, values werethe trend higher remained with DNA the fromsame withisolates TSB grown (17.11; in 95% all brothCI 16.46, types 17.75), in the showing presence lower of stool; Ct values however, as compared the trend to remained GN (17.67; the 95%same CI with 16.85, TSB 18.49) (17.11; and 95% Mac CI (21.18; 16.46, 95% 17.75), CI showing20.03, 22.33) lower (Figure Ct values 4). as compared to GN (17.67; 95% CI 16.85, 18.49) and Mac (21.18; 95% CI 20.03, 22.33) (Figure4). Microorganisms 2021, 9, 503 7 of 10 Microorganisms 2021, 9, x FOR PEER REVIEW 7 of 10

FigureFigure 4. 4.qPCR(A) Ct represent values of broth stx from without STEC Stool, isolates (B) enriched represent in broth tryptic with soy Stool.broth (TSB), qPCR GN, Ct values and of MacConkeystx from STEC (MAC) isolates with enrichedand with inout tryptic stool. soyDNA broth was (TSB),extracted GN, fr andom the MacConkey STEC enriched (MAC) broth with and andwithout qPCR stool. was done. DNA Each was extractedsymbol represents from the STECa single enriched isolate. broth n = 25 and isolates. qPCR was done. Each symbol represents a single isolate. n = 25 isolates. 4. Discussion 4. DiscussionSTEC is a major cause of global AGE and is responsible for many notable foodborne outbreaks.STEC Recently, is a major there cause has of been global an AGE increased and is prevalence responsible of for serotypes many notable other than food- O157:H7borne outbreaks. implicated Recently, in these there outbreaks has been [16]. an These increased associations prevalence indicate of serotypes an urgency other thanfor increasedO157:H7 surveillance implicated inof thesethese outbreaksserotypes, [particularly16]. These associationsthose deemed indicate the “Big an 6” urgency in North for Americaincreased (O26, surveillance O45, O103, of O111, these serotypes,O121, and particularlyO145) [17]. The those use deemed of CIDT the is becoming “Big 6” in more North widespreadAmerica (O26, due O45, to its O103, fast turnaround O111, O121, time and O145)for reporting, [17]. The as use well of CIDTas the ishigh becoming sensitivity more andwidespread specificity. due However, to its fast culture turnaround still remain times for essential reporting, for surveillance as well as the purposes, high sensitivity epide- miologicaland specificity. investigations, However, and culture early still cluster remains detection. essential Culturing for surveillance from a purposes,stool sample epidemi- can beological particularly investigations, challenging and due early to clusterthe pres detection.ence of the Culturing patient’s from own a microbiome; stool sample there- can be fore,particularly it is crucial challenging that the optimal due to theculture presence media of and the patient’sconditions own be microbiome;applied for the therefore, specific it pathogenis crucial involved. that the optimal To our culture knowledge, media this and conditionsis the first bestudy applied to publish for the data specific comparing pathogen enrichmentinvolved. Tobroths our knowledge,used in diagnostic this is thelaborato first studyries for to the publish culturing data comparingof STEC from enrichment patient stoolbroths samples. used in diagnostic laboratories for the culturing of STEC from patient stool samples. TheThe first first aim aim of this studystudy waswas to to determine determine whether whether known known STEC STEC isolates isolates would would have havedifferent different growth growth dynamics dynamics with respectwith respec to eacht to of each the broth of the tested. broth GN tested. and MacGN and broths Mac are selective media commonly used for enteric bacteria such as E. coli due to the basic nutrients broths are selective media commonly used for enteric bacteria such as E. coli due to the provided [18,19] as compared with TSB, which is a general medium. E. coli is usually basic nutrients provided [18,19] as compared with TSB, which is a general medium. E. coli regarded as a non-fastidious organism that grows well in most conditions; however, as is usually regarded as a non-fastidious organism that grows well in most conditions; how- illustrated in our data, we have found both GN and Mac broths are limited in their ability to ever, as illustrated in our data, we have found both GN and Mac broths are limited in their support the growth of certain STEC, as expected. Mac broth consistently showed reduced ability to support the growth of certain STEC, as expected. Mac broth consistently showed growth as compared to TSB and GN, although there was no indication that this occurred reduced growth as compared to TSB and GN, although there was no indication that this within a particular serotype. Instead, the data suggest that Mac broth may be lacking occurred within a particular serotype. Instead, the data suggest that Mac broth may be a particular nutrient as compared to the others, which these isolates require for growth. lacking a particular nutrient as compared to the others, which these isolates require for One such possibility is that Mac broth contains lactose, whereas TSB and GN broth have growth. One such possibility is that Mac broth contains lactose, whereas TSB and GN glucose as the main carbon source. Another speculation is that these isolates might have brothmutations have glucose within theas the Lac main operon carbon which source limits. Another their ability speculation to utilize is lactose that these as efficiently isolates mightas their have main mutations carbon source,within the which Lac isoperon not unique, which aslimits other their mutations ability to have utilize been lactose found as in efficientlySTEC that as affect their theirmain metabolic carbon source, profile which [20]. Futureis not unique, study of as the other biochemical mutations pathways have been of foundthese in organisms STEC that will affect help their to understand metabolic the pr growthofile [20]. performance Future study of theseof the isolates biochemical in such pathwaysmedia. E. of coli theseare normallyorganisms lactose will fermentinghelp to understand organisms, the which growth allows performance for their distinction of these isolatesfrom in suchspp media. when E. diagnosingcoli are normally diarrheal lactose infections. fermenting However, organisms, there haswhich been allows evidence for theirthat distinction the stx lambdoid from Shigella phage can spp disrupt when diagnosing the metabolic diarrheal pathways, infections. particularly However, in those there with has been evidence that the stx lambdoid phage can disrupt the metabolic pathways, par- stx2a [21]. We did not subtype the stx gene in these isolates; however, some of the STEC ticularly in those with stx2a [21]. We did not subtype the stx gene in these isolates; however, Microorganisms 2021, 9, 503 8 of 10

isolates with stx1 also showed decreased growth in MAC broth, suggesting this may not be limited to stx2a. The addition of stool further complicated the growth of many of the isolates we used; however, TSB remained as the most supportive enrichment broth. Stool samples are complex matrices due to the patient’s microbiome, which may affect the growth of STEC. The family found within the microbiome is of particular importance as they can be easily cultured on MacConkey agar [22]. This competition between microbiome and STEC may be the reason why the growth in Mac broth was significantly lower with the addition of stool compared to enrichment using TSB and GN broth. The addition of stool introduces competition between STEC and organisms present in the microbiome for the limited nutrients included in the broth, which would explain why some of the isolates had considerably lower growth in Mac broth. Isolates #20 and #30 were interesting because there was more growth in Mac broth as compared to GN, suggesting that these isolates could not compete well with the organisms found in the stool when enriched in GN broth as compared to Mac broth, as shown in Figure3, and such a phenomenon was not observed when this isolate was enriched in the absence of stool (Figure2). As CIDT use has increased over the last few years, we performed qPCR to determine if we would see differences between different enrichment broths using a molecular assay. We found that the Ct values reflected the same trend, as was observed by plate enumeration in the absence of stools. Using rapid lysis buffer on the broths containing stool is considered a “crude” method, and there is a chance for PCR inhibition; however, an increase in the Ct values between the different broths corresponded to the decrease in CFU by colony count- ing. It is most likely that any PCR inhibitors were diluted in the broth during enrichment and therefore did not affect our results. Amplification using DNA from isolates grown in TSB broth showed a mean lower Ct values as compared to both GN and Mac broths. Therefore, if an overnight enrichment is required for the stool samples for the detection of STEC using an enzyme immunoassay, Mac and GN may not be the broths of choice, because they might not provide the optimal growth of STEC. Consequently, depending on the sensitivity of the EIA being used by the diagnostic laboratory, the toxin level might not be sufficient enough to be detected by the assay. Therefore, it is important that the sample be enriched in the appropriate media to ensure an accurate diagnosis.

5. Conclusions This study highlights the incredible diversity found both within and between STEC serotypes in terms of their enrichment requirements. The enrichment broth chosen by the diagnostic laboratory can greatly influence how well they are able to culture STEC for their detection and further analysis. Based on the results of this study, we suggest that diagnostic laboratories currently using GN or Mac broth may benefit from switching to TSB, which was more supportive of STEC growth. In addition, we hope to alert manufacturers that GN and Mac broth are not optimized for the enrichment of STEC and instead recommend the use of TSB.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-260 7/9/3/503/s1, Table S1: Bacterial Isolate Characteristics. Author Contributions: Conceptualization, M.B.-J. and L.C.; data curation, H.T.; formal analysis, M.B.-J.; investigation, M.B.-J., H.T. and C.L.; methodology, M.B.-J. and L.C.; supervision, L.C.; writing—original draft, M.B.-J.; writing—review and editing, H.T., C.L. and L.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received commercial grant funding from TechLab Inc. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Microorganisms 2021, 9, 503 9 of 10

Acknowledgments: The authors would like to thank Christina Ferrato and Joanne Callfas for their help in providing isolate identification. Conflicts of Interest: Linda Chui has received research grants from TechLab.

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