Cross-Genera Amplification of prs/hlyA by Multiplex PCR Resulted in Misidentification of Enterococcus faecium as Listeria monocytogenes

20 Importance: Molecular confirmation of Listeria monocytogenes typically employs a multiplex 21 PCR method that targets both genus-specific prs and species-specific hlyA genes. This study 22 assessed the specificity of this assay within Nigeria, where local microbial diversity may influence 23 performance outcomes. 24

Out of eight phenotypically presumptive L. monocytogenes food isolates tested, six 25 produced the expected prs and hlyA amplicons, with five classified as serogroup 1/2b. However, 26 all six isolates tested negative for the crucial virulence regulator prfA, necessitating further 27 investigation. 28

Definitive 16S rRNA gene sequencing revealed that only two of the six PCR-positive 29 isolates were identified as L. monocytogenes, while the remaining four were identified as 30 Enterococcus faecium . This results in a false-positive rate of 66.7% (4/6) for the assay in this 31 particular context. Phylogenetic analysis corroborated the taxonomic distinction, exhibiting a 32 robust clustering of the four E. faecium isolates with reference strains. In contrast, the two 33 confirmed L. monocytogenes isolates formed a separate sub-clade, indicating regional divergence 34 and further underscoring the assay's inability to differentiate between L. monocytogenes and 35 Enterococcus species. 36

These findings highlight a significant lack of specificity, as the prs/hlyA primers 37 exhibited cross-reactivity with non-target E. faecium . The anomalous negative result for prfA 38 served as a critical diagnostic indicator. Consequently, the positive outcomes from this widely 39 utilized confirmatory assay should be regarded as presumptive and necessitate additional 40 verification. 41 42 1.0 INTRODUCTION 43 Prompt and precise identification of Listeria monocytogenes, a significant foodborne pathogen that 44 leads to invasive listeriosis, is crucial for clinical diagnostics and food safety monitoring. Although 45 traditional culture-based methods remain important, molecular techniques offer essential speed and 46 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint sensitivity for verification. Among these, the multiplex polymerase chain reaction (PCR) assay 47 created by Doumith and colleagues—which simultaneously targets the genus-specific prs gene and 48 the L. monocytogenes-specific hlyA gene (which encodes listeriolysin O) —has become a 49 commonly used standard for distinguishing L. monocytogenes from other Listeria species in both 50 clinical settings and food samples [1, 2]. 51 The dependability of a diagnostic assay largely depends on its specificity within the microbial 52 environment in which it is utilized. Primer sequences that have been validated against certain strain 53 collections might not consider the genetic variability present in various geographical or 54 environmental settings, which could result in cross-reactivity and false-positive findings. [3]. This 55 is especially important in areas where the local microbial phylogeny is not well defined and where 56 closely related Gram-positive bacteria (such as Enterococcus spp. and Brochothrix spp.) might 57 have conserved genomic regions in common. In an earlier surveillance study conducted in 58 northeastern Nigeria, we isolated eight strains of L. monocytogenes that were phenotypically 59 presumed from various food samples using standard biochemical methods and automated systems 60 (VITEK 2)[4]. This created an opportunity to genetically analyze these isolates while 61 simultaneously assessing the effectiveness of the Doumith multiplex PCR in this less-explored 62 context. The main aim of this study was not to conduct surveillance, but rather to perform a critical 63 diagnostic evaluation: to determine the agreement between this established PCR method and 64 definitive 16S rRNA gene sequencing for isolates from a region with unique microbial ecology. 65 The study uncovered a significant and surprising lack of agreement, resulting in the identification 66 of considerable cross-genera amplification. This report outlines the assay's specificity failure, 67 where Enterococcus faecium was wrongly identified as L. monocytogenes, and discusses the 68 immediate ramifications for diagnostic practices that depend on this widely used molecular 69 confirmation test. 70 71 2.0 MATERIALS AND METHODS 72 73 2.1. Study Design and Bacterial Isolates 74 This diagnostic evaluation study employed eight isolates of Listeria monocytogenes that were 75 presumptively identified based on their phenotypic characteristics (labeled BO1 –BO8), which 76 were sourced from a previous food surveillance investigation conducted in Maiduguri, Nigeria. [5]. 77 The main goal was to assess the specificity of a standard multiplex PCR assay in comparison to 78 definitive 16S rRNA gene sequencing. Isolates were taken from -80°C storage in Brain Heart 79 Infusion broth containing 20% glycerol, then subcultured on Blood Agar to ensure purity and 80 incubated at 37°C for 24 hours before analysis. A summary of the sample processing workflow 81 from the initial surveillance study can be found in Supplementary Table S1. 82 83 2.2. DNA Extraction 84 Genomic DNA was obtained from fresh, pure colonies utilizing the Quick- DNA™ 85 Fungal/Bacterial Microprep Kit (Zymo Research, USA), following the manufacturer's instructions 86 for bacterial cells. The concentration and purity of the DNA were assessed using a NanoDrop™ 87 One spectrophotometer (Thermo Fisher Scientific). The extracted DNA was then stored at -20°C. 88 89 2.3. Multiplex PCR Assay under Evaluation 90 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint The effectiveness of the commonly utilized Doumith prs/hlyA multiplex PCR test [3] was 91 evaluated. All oligonucleotide primers utilized in this research are detailed in supplementary Table 92 S2. 93 2.3.1. Confirmation of Genus and Species (prs and hlyA) 94 The multiplex reaction was conducted in a 25 µL volume that included: 12.5 µL of 2× OneTaq 95 Quick-Load Master Mix (New England Biolabs), 0.5 µM of each primer, 5 µL of template DNA, 96 along with nuclease-free water. The thermocycling conditions were: an initial denaturation step at 97 95°C for 5 minutes; followed by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 98 45 seconds; and a final extension at 72°C for 5 minutes. 99 100 2.3.2. Detection of Serogroup and Virulence Genes 101 A distinct multiplex PCR was conducted for further characterization of isolates and to examine the 102 prfA virulence regulator, while serogrouping was accomplished using primers for serogroups 1/2a, 103 1/2b, and 4b [3, 6]. The composition of the reaction was the same as described in Section 2.3.1. 104 The cycling conditions included: 94°C for 5 minutes; followed by 35 cycles of 94°C for 40 105 seconds, 53°C for 75 seconds, and 72°C for 75 seconds; concluding with 72°C for 7 minutes. 106 107 2.4 Confirmatory 16S rRNA Gene Sequencing and Phylogenetic Analysis 108 2.4.1. Amplification and Sequencing 109 The near-complete 16S rRNA gene was amplified using the universal primers 27F and 1492R [7] 110 In a 50 µL reaction, the setup included 25 µL of 2× Master Mix, 0.3 µM of each primer, and 5 µL 111 of DNA. The cycling conditions were set at 95°C for 5 minutes; followed by 35 cycles of 95°C for 112 30 seconds, 55°C for 45 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 113 minutes. Amplicons were purified using the QIAquick PCR Purification Kit from Qiagen, and 114 sequencing was performed bidirectionally using Sanger chemistry on an ABI 3500xl Genetic 115 Analyzer from INQABA Biotechnical Industries in Nigeria. 116 For sequence analysis and phylogenetics, forward and reverse reads were assembled and edited 117 using BioEdit version 7.2 [8]. Consensus sequences were matched against the NCBI GenBank 118 database through BLASTn. For the purpose of phylogenetic analysis, sequences were aligned with 119

strains utilizing ClustalW within MEGA11 [9]. The evolutionary lineage was deduced 120 using the Neighbor-Joining approach [10] with 1,000 bootstrap iterations [11]. Evolutionary 121 distances were calculated using the Maximum Composite Likelihood technique [12]. All uncertain 122 positions were discarded (pairwise deletion option). 123 124 2.5. Gel Electrophoresis 125 PCR products (8 µL) were combined with 2 µL of 6× loading dye and subjected to separation on a 126 1.5% agarose gel in 0.5× TBE buffer, which was stained with Midori Green Advanced DNA Stain. 127 The electrophoresis was carried out at 115 V for 25 minutes. A 100 bp DNA ladder was included 128 to aid in size estimation. The gels were examined using a Bio-Rad Gel Doc™ XR+ system. 129 130 2.6. Data and Performance Analysis 131 The false-positive rate for the prs/hlyA assay was determined using the formula: 132 (Number of isolates that tested PCR-positive but were identified as non- L. monocytogenes through 133 sequencing) / (Total number of PCR-positive isolates) × 100%. 134 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint Sequencing-based identification was deemed conclusive. The 16S rRNA gene sequences obtained 135 in this study have been submitted to GenBank with the accession numbers PV809878.1 to 136 PV809883.1. 137 3.0 RESULTS 138 3.1. The prs/hlyA Multiplex PCR Assay's performance 139 Based on the simultaneous amplification of the anticipated 370 bp and 456 bp fragments, 140 respectively, the multiplex PCR assay targeting the prs and hlyA genes identified six of the eight 141 presumed isolates (BO1 –BO6) as Listeria monocytogenes (Fig 1A). Since isolates BO7 and BO8 142 tested negative for both targets, they were not included in the analysis. A significant abnormality 143 was noted, though: in the supplemental multiplex reaction, none of the six PCR -positive isolates 144 generated an amplicon for the prfA virulence regulator gene (1060 bp). 145 146 3.2. 16S rRNA Gene Definitive Identification Sequencing Exposes Important Disagreement 147 All six PCR -positive isolates underwent 16S rRNA gene sequencing in order to address the 148 taxonomic confusion caused by the unusual prfA-negative phenotype. BLASTn comparison with 149 the NCBI GenBank database yielded a definitive yet discordant outcome (Table 1). 150 151 152 153 154 155 156 157 158 159 160 161 162 163 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint 164 Table 1: Concordance between prs/hlyA multiplex PCR assay and definitive 16S rRNA gene 165 sequencing 166 Isolate Code Original Food prs/hlyA Serogroup prfA PCR 16S rRNA Final Assay Source [5] PCR (PCR) BLASTn Identity Taxonomic Concordance (% Query Cover, Assignment % Identity) BO1 Raw Meat Positive 1/2b Negative Enterococcus Enterococcus False faecium faecium Positive (100%, 99.8%) BO2 Sour Milk Positive 1/2 Negative Listeria Listeria True (Sala) monocytogenes monocytogenes Positive (100%, 99.9%) BO3 Pineapple Positive 1/2b Negative Enterococcus Enterococcus False faecium faecium Positive (100%, 99.7%) BO4 Salad Positive Non-typable Negative Enterococcus Enterococcus False faecium faecium Positive (100%, 99.9%) BO5 Raw Meat Positive 1/2b Negative Listeria Listeria True monocytogenes monocytogenes Positive (100%, 99.9%) BO6 Raw Meat Positive 1/2b Negative Enterococcus Enterococcus False faecium faecium Positive (100%, 99.8%) 167 168 Only two of the six PCR -positive isolates (BO2 and BO5) were identified as L. monocytogenes by 169 sequencing. With 99.7–99.9% similarity, the remaining four isolates (BO1, BO3, BO4, and BO6) 170 were determined to be Enterococcus faecium . In this evaluation, the prs/hlyA PCR assay has a 171 false-positive rate of 66.7% (4/6). 172 173 3.3. Additional Analysis of PCR-Positive Isolates 174 Five of the six PCR -positive isolates (BO1, BO2, BO3, BO5, and BO6) belonged to serogroup 175 1/2b (471 bp amplicon), according to multiplex PCR for major serogroups linked to human 176 listeriosis (Fig 1B). Using the primers, isolate BO4 was not typable. All six isolates consistently 177 lacked the prfA amplicon. 178 179 3.4. Phylogenetic analysis uncovers intricate taxonomic relationships among isolates 180 Phylogenetic analysis based on partial 16S rRNA gene sequences generated a tree with surprising 181 topological characteristics (refer to Fig. 2). Three of the four isolates initially identified as 182 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint Enterococcus faecium—BO1, BO4, and BO6—formed a robust clade (bootstrap ≥ 90%) alongside 183

strains of E. faecium from China (OR795718.1) and Thailand (LC027228.1), validating 184 their classification. However, the fourth presumed E. faecium isolate, BO3, did not fall within this 185 Enterococcus clade. Instead, it was phylogenetically situated between the two Listeria 186 monocytogenes isolates (BO2 and BO5) within the wider Listeria genus clade. The two L. 187 monocytogenes isolates, BO2 and BO5, reliably grouped within a Listeria clade that included 188

strains from Germany, Poland, China, and India. They formed a unique sub-clade with 189 moderate bootstrap support (54–57%), suggesting potential regional lineage differentiation. 190 191 3.5. Overview of Assay Performance Metrics 192 Using sequencing as the reference standard, the performance metrics of the prs/hlyA multiplex 193 PCR assay in this study were: 194 • Sensitivity: 100% (2 out of 2 true L. monocytogenes isolates detected) 195 • Specificity (for E. faecium): 0% (0 out of 4 non-target E. faecium isolates correctly excluded) 196 • False-Positive Rate: 66.7% (4 out of 6 PCR-positive results were incorrect) 197 • Positive Predictive Value (PPV): 33.3% (only 2 out of 6 PCR -positive results were true L. 198 monocytogenes) 199 The 16S rRNA gene sequences obtained in this study have been submitted to GenBank with 200 accession numbers PV809878.1 (BO1) to PV809883.1 (BO6). 201 202 4.0 DISCUSSION 203 204 This research highlights a significant specificity issue in a commonly utilized confirmatory PCR 205 assay, illustrating that the Doumith prs/hlyA multiplex PCR may yield false-positive results for 206 Listeria monocytogenes due to cross-reactivity with Enterococcus faecium. With a false-positive 207 rate of 66.7% (4 out of 6 isolates) observed in this assessment, our results have important 208 ramifications for diagnostic and food safety labs that depend on this assay as a conclusive test. The 209 most likely reason for this misidentification is the binding of primers in a non-specific manner. 210 Although the prs gene is used as a target specific to the Listeria genus, it encodes a highly 211 conserved housekeeping enzyme (phosphoribosylpyrophosphate synthetase) that is found 212 throughout bacteria, including Enterococcus [3]. The primer sequences probably display enough 213 similarity with the relevant regions in the E. faecium genomes present in our sample set, resulting 214 in successful amplification. This highlights a crucial concept in molecular diagnostics: tests that 215 have been validated with certain strain collections from a specific geographical or ecological 216 context may lose their specificity when used in another area with different microbial populations 217 [3]. Additionally, recorded proof of horizontal gene transfer among Firmicutes in common 218 environments, like food, might lead to shared genetic segments that cause cross-reactivity [14, 15]. 219 The persistent lack of a prfA amplicon in all six PCR-positive isolates raised the initial and 220 significant diagnostic concern. The prfA gene functions as the primary transcriptional activator of 221 the L. monocytogenes pathogenicity island and is regarded as a vital, conserved element of virulent 222 strains [12]. While there are attenuated or avirulent strains that lack a functional prfA gene, finding 223 a complete absence of an amplicon in a hlyA-positive isolate is quite uncommon and should raise 224 concerns about a potential misidentification, which was confirmed through sequencing in this 225 instance. For the two isolates that were definitively confirmed as L. monocytogenes (BO2, BO5), 226 the negative prfA PCR result may suggest mismatches between the primer and template due to 227 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint locally varying prfA alleles, emphasizing a secondary limitation of the assay's published primer set 228 that is dependent on the strain [16]. 229 The phylogenetic analysis based on the 16S rRNA gene offered crucial, albeit intricate, taxonomic 230 insights that were integral to our diagnostic assessment. This analysis effectively resolved the 231 majority of the isolates, confirming E. faecium isolates (BO1, BO4, BO6) and L. monocytogenes 232 isolates (BO2, BO5) to their anticipated genera, but it also uncovered a notable phylogenetic 233 inconsistency. The classification of the presumed E. faecium isolate BO3 within the Listeria clade 234 contradicts its original phenotypic classification and highlights a key limitation of depending on 235 single-marker phylogenetics for definitive identification in certain cases. 236 This discrepancy emphasizes the well-recognized limitations of the 16S rRNA gene, which, 237 despite being regarded as a gold standard for broad taxonomic categorization and differentiation 238 between genera like Listeria and Enterococcus, can produce ambiguous or misleading outcomes 239 when sequences display unusual evolutionary paths. For BO3, possible explanations could be 1) an 240 initial misidentification, 2) the acquisition of a Listeria-like 16S rRNA gene through horizontal 241 gene transfer, or 3) the occurrence of a chimeric PCR artifact. Regardless of the reason, this 242 observation prompted the assay's failure to accurately identify this isolate, as the molecular target 243 diverged from the anticipated Enterococcus sequence. 244 The grouping of the two confirmed L. monocytogenes isolates (BO2, BO5) into a distinct sub-245 clade, separate from global reference strains, implies potential local genetic divergence that aligns 246 with the formation of region-specific ecotypes. However, the primary significance of this 247 phylogenetic analysis was its function as a high-resolution comparator. It not only confirmed the 248 assay’s effectiveness for isolates with clear 16S rRNA phylogeny but, more crucially, uncovered 249 its diagnostic failure by indicating a major taxonomic inconsistency that simpler comparative 250

might overlook. 251 The practical ramifications for laboratory operations are considerable. Laboratories that rely solely 252 on the prs/hlyA multiplex PCR as a confirmatory test risk producing inaccurate results, which 253 could lead to unnecessary product recalls, inflated epidemiological statistics, and misallocation of 254 public health resources. Thus, we recommend a revised diagnostic protocol in which positive 255

from this assay are treated as presumptive. It is essential to confirm findings through an 256 orthogonal, sequence-based method. For resource-limited environments, sequencing the 16S rRNA 257 gene of a representative subset of amplicons presents a cost-effective approach for periodic assay 258 validation. Where possible, adding a second highly specific target (such as iap) into a confirmatory 259 PCR is advisable to improve reliability. 260 In conclusion, this case study underscores the importance of context-specific validation for even 261 established molecular assays. It demonstrates how an anomalous result ( prfA-negativity) can act as 262 a crucial indicator of a more profound diagnostic issue. By combining targeted PCR with definitive 263 sequencing, laboratories can greatly enhance the accuracy of L. monocytogenes reporting, thus 264 reinforcing food safety measures and public health diagnostics. 265 266 ACKNOWLEGEMENT 267 We thank the Department of Medical Microbiology University of Maiduguri Teaching Hospital, 268 Department of Veterinary Public Health, Ahmadu Bello University Zaria, and the Department of 269 Microbiology, University of Nigeria Nsukka, for their technical support. 270 271 FUNDING 272 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint This study did not receive any specific funding from public, commercial, or not-for-profit 273 organizations. 274 275 CONFLICT OF INTEREST 276 The authors state that there are no conflicts of interest. 277 278 AUTHOR CONTRIBUTIONS 279 H.B.A carried out the conceptualization, methodology, investigation and wrote the original draft. 280 A.S.K supervised, validated and reviewed the methodology. A.J.M., Y.U., A.S.B. and M.U carried 281 out investigation, validation and reviewed the write up. All authors approved the final manuscript. 282 283 DATA AVAILABILITY 284 The sequences of the 16S rRNA gene produced in this study have been submitted to the NCBI 285 GenBank database with accession numbers PV809878.1 to PV809883.1. 286 287

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Use of a tissue culture assay to detect 334 invasive Listeria monocytogenes among wild-type strains isolated from food and humans. J Appl 335 Bacteriol. 1991;70(2):121–126. 336 337 338 Figure Legends 339 340 Figure 1. Assessment of the prs/hlyA multiplex PCR assay 341 (A) Gel electrophoresis used for identifying the Listeria genus (prs, 370 bp) and the L. 342 monocytogenes species (hlyA, 456 bp). Lane M: 100 bp DNA marker; Lane PC: Positive control 343 (L. monocytogenes ATCC 19115); Lane NC: Negative control (nuclease-free water); Lanes 1-6: 344 Isolates BO1 to BO6. (B) Serogroup analysis for lineages 1/2a (691 bp), 1/2b (471 bp), and 4b 345 (597 bp). Lane identifications are the same as in (A). A 471 bp band (serogroup 1/2b) is detected 346 in lanes 1, 2, 3, 4, and 6. 347 348 Figure 2. Phylogenetic analysis utilizing 16S rRNA gene sequences 349 A Neighbor-Joining tree illustrates the relationship between the study isolates (highlighted in bold) 350 and reference sequences sourced from GenBank. The four Enterococcus faecium isolates (BO1, 351 BO3, BO4, BO6) group within the Enterococcus clade. The two Listeria monocytogenes isolates 352 (BO2, BO5) group within the Listeria clade. Bootstrap values (≥50%) derived from 1000 iterations 353 are presented. The scale bar represents 0.05 nucleotide substitutions per site. 354 355 356 357 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700535doi: bioRxiv preprint