# Phenotypic and Genotypic Characterization of AtypicalListeria monocytogenes and Listeria innocua Isolated from Swine Slaughterhouses and Meat Markets

Authors: Luisa Zanolli Moreno; Renata Paixão; Debora Dirani Sena de Gobbi; Daniele Cristine Raimundo; Thais Sebastiana Porfida Ferreira; Andrea Micke Moreno; Ernesto Hofer; Cristhiane Moura Falavina dos Reis; Glavur Rogério Matté; Maria Helena Matté
Journal: BioMed Research International
 (2014)

---

## Abstract

In the last decade, atypicalListeria monocytogenes and L. innocua strains have been detected in food and the environment. Because of mutations in the major virulence genes, these strains have different virulence intensities in eukaryotic cells. In this study, we performed phenotypic and genotypic characterization of atypical L. monocytogenes and L. innocua isolates obtained from swine slaughterhouses and meat markets. Forty strains were studied, including isolates of L. monocytogenes and L. innocua with low-hemolytic activity. The isolates were characterized using conventional phenotypic Listeria identification tests and by the detection and analysis of L. monocytogenes-specific genes. Analysis of 16S rRNA was used for the molecular identification of the Listeria species. The L. monocytogenes isolates were positive for all of the virulence genes studied. The atypical L. innocua strains were positive for hly, plcA, and inlC. Mutations in the InlC, InlB, InlA, PI-PLC, PC-PLC, and PrfA proteins were detected in the atypical isolates. Further in vitro and transcriptomic studies are being developed to confirm the role of these mutations in Listeria virulence.


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## Body

## 1. Introduction

Listeria monocytogenes andL. innocua are closely related species of the Gram-positive genusListeria. They are widely distributed in the environment and frequently isolated from food.L. monocytogenes is the causative agent of listeriosis, a foodborne disease with a high fatality rate (20–30%) that mostly affects the elderly, neonates, and immunocompromised individuals [1, 2].L. monocytogenes cannot be distinguished from otherListeria species using conventional isolation methods. Standard biochemical methods and selective and differential media are used for the identification ofL. monocytogenes[3, 4]; however, someL. ivanovii,L. innocua, andL. seeligeri strains generate similar results toL. monocytogenesin these tests [5–7]. Therefore, it is necessary to confirm the virulence characteristics ofL. monocytogenes to distinguish theListeria species.The best-characterizedL. monocytogenes virulence factors are listeriolysin O (LLO), phosphatidylinositol phospholipase C (PI-PLC), and the internalins A and B (InlA and InlB). LLO and PI-PLC are encoded by thehly andplcA genes, respectively, which belong to the virulence gene clusterListeria pathogenicity island 1 (LIPI-1), which contains the major virulence genes ofL. monocytogenes [8]. Few atypicalL. innocua strains have been reported to containL. monocytogenes-specific genes and exhibit phenotypic characteristics similar toL. monocytogenes such as weak hemolysis [6, 7, 9]. Furthermore, certain low-hemolyticL. monocytogenes strains retain their virulence despite the presence of mutations in major virulence genes [10–12]. The existence of these atypical strains indicates that traditional phenotypic and genotypic characterization methods must be used with care and that further studies are required to improve the identification ofListeria isolates.This study used phenotypic and genotypic methods to characterize atypicalL. monocytogenes andL. innocua isolates obtained from swine slaughterhouses and meat markets in Sao Paulo State, Brazil.


## 2. Material and Methods

### 2.1. Bacterial Strains and Culture Conditions

FortyListeria sp. isolates were studied. Of these, 25 were isolated from pork, slaughterhouses, and markets (15 isolates ofL. monocytogenes and 10 ofL. innocua), 11 isolates ofL. monocytogenes were obtained from human infections, and four were control strains (L. monocytogenes ATCC 19115 and ATCC 19111 andL. innocua ATCC 33090 and CLIP 12612) (Table 1). The environmental and pork isolates were isolated as described by Moreno et al. [13]; the clinical strains andListeria controls were obtained from the Public Health Laboratory (School of Public Health, University of Sao Paulo) and Laboratory of Swine Health (School of Veterinary Medicine and Animal Science, University of Sao Paulo) collections. The environmental and pork isolates were obtained from different swab samples taken from the slaughterhouses environment and carcasses from Sao Paulo State; the clinical isolates were obtained from the blood, placenta, and cerebrospinal fluid samples of different patients from different Brazilian states (Tables 1 and 2).Table 1
Sources and phenotypic and genotypic characteristics of theListeria monocytogenes isolates used in this study.
Isolate
Species
Serotype
Origin
Site
Year
ALOA
Hemolysis
inlA
inlB
inlC
inlJ
*
plcA
plcB
prfA
hly
Lm1
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm2
L. monocytogenes
1/2b
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm3
L. monocytogenes
4b
Market 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm21
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm22
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm23
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm25
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm26
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm27
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm28
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm29
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm30
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm31
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm34
L. monocytogenes
1/2a
Human
Blood
1989
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm35
L. monocytogenes
4b
Human
Blood
2004
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm36
L. monocytogenes
4b
Human
Blood
1977
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm37
L. monocytogenes
4b
Human
CSF
1982
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm38
L. monocytogenes
1/2b
Human
CSF
1983
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm39
L. monocytogenes
1/2a
Human
Placenta
1978
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm39a
L. monocytogenes
1/2a
Human
Placenta
1978
Halo
Positive
+
+
+
+
+
+
+
+
Lm40
L. monocytogenes
1/2a
Human
Blood
1985
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm41
L. monocytogenes
4b
Human
CSF
1997
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm42
L. monocytogenes
4b
Human
CSF
1997
Halo
Positive
+
+
+
+
+
+
+
+
Lm43
L. monocytogenes
1/2a
Human
CSF
1983
Halo
Positive
+
+
+
+
+
+
+
+
Lm4
L. monocytogenes
1/2a
Market 2
Floor
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm33
L. monocytogenes
1/2a
Market 2
Floor
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm10
L. monocytogenes
4b
ATCC 19115
—
—
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm15
L. monocytogenes
1/2a
ATCC 19111
—
—
Halo
Strong positive
+
+
+
+
+
+
+
+
Slaught 1: slaughterhouse 1. CSF: cerebrospinal fluid.*All isolates were positive for fragments of inlJ but presented variable results for hole gene amplification (see Table 4).Table 2
Sources and phenotypic and genotypic characteristics of theListeria innocua isolates used in this study.
Isolate
Species
Serotype
Origin
Site
Year
ALOA
Hemolysis
inlA
x
inlB
inlC
inlJ
plcA
plcB
prfA
hly
x
Lin5
L. innocua
6a
Market 1
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin6
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin7
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin8
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin9
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin16
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin17
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin18
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin19
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin20
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin11
L. innocua
6a
ATCC 33090
—
—
Negative
Negative
−
−
−
−
−
−
−
−
Lin46
L. innocua
6a
CLIP 12612
—
—
Negative
Negative
−
−
−
−
−
−
−
−
Slaught 1: slaughterhouse 1; Slaught 2: slaughterhouse 2.*Subtle halo. ** Very weak positive hemolysis. xAtypical isolates were positive for fragments of inlAB operon and hly but presented variable results for inlA and hly complete amplification (see Table 4).The isolates were maintained in a stock medium containing glycerol at −80°C. The isolates were reactivated in brain-heart infusion (BHI) medium (Difco, Sparks, MD, USA) and plated on tryptone soy agar supplemented with yeast (TSAYE) (Oxoid, Lenexa, USA) to isolate pure colonies before use.


### 2.2. Conventional Listeria Identification Tests

The isolates were serotyped using polyclonal antisera produced against Listeria somatic and flagellar antigens in rabbits, according to the method described by Seeliger and Höhne [14]. The isolates were also characterized by catalase, motility, and biochemical tests including acid production from D-xylose, D-mannitol, L-rhamnose, and α-methyl-D-mannoside. Cultivation in selective agarListeria according to Ottaviani and Agosti (ALOA) (Biolife, Milan, Italy) was used to identifyL. monocytogenes isolates, and β-hemolysis was assessed by sting inoculation on 5% sheep blood agar.


### 2.3. Detection ofL. monocytogenes-Specific Genes

Genomic DNA extraction was performed as described by Ausubel et al. [15]. All isolates were screened for theinlA,inlB,inlC,inlJ,hly,prfA,plcA, andplcB genes. The primers described by Johnson et al. [6], Liu et al. [16], and Jung et al. [17] were used for detection ofprfA,inlCandinlJ,andinlA, respectively. Specific primers were designed for the complete amplification of the virulence genes (Table 3). The PCRs were performed using anEppendorf Mastercycler gradient thermal cycler. Each reaction (25 μL) contained 5 μL of genomic DNA, MilliQ water, 10X PCR buffer, 1.5 mM MgCl2, 200 μM of dNTPs (Fermentas, Burlington, Canada), 200 μM of each primer, and 1.25 U of Taq DNA polymerase (Promega). The PCR programs were as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at primer-specific temperature for 1–1.5 min, elongation at 72°C for 1 min per 1 Kb, and final extension at 72°C for 10 min. The amplified products were separated by electrophoresis on 1.5% agarose gels and stained with ethidium bromide (1 μg/mL). The molecular weights of the products were determined using the 1 Kb Plus DNA Ladder (Fermentas, Burlington, Canada).Table 3
Primers designed in this study for the amplification of theL. monocytogenes virulence genes.
Primer
Sequence 5′-3′
Target
Product (bp)
inlA ext Fw
CGGCTCCGTAGACAGATTAG
inlA
2884
inlA ext Rv
GTGATAGTCTCCGCTTGTAC
inlA In
1
-Fw
GTGAGAAGAAAACGA
1200
inlA Detec-Rv
TGGTGTAAGATCGCT
inlA Detec-Fw
AAGTGATATAACTCC
—
inlB ext Fw
GCTAGATGTGGTTTTCGGACT
inlB
2146
inlB ext Rv
TAAGCAGCGCAAAGGTGATTCCTAC
inlB In-Fw
GTGAAAGAAAAGCAC
1227
inlB Seq
3
-Rv
ATTCCCGCGAATATA
inlB Seq
2
-Fw
TGATGGAACGGTAAT
900
inlB End
3
-Rv
TNATTTCTGTGCCCT
plcB ext Fw
CCATACGACGTTAATTCTTGCAATG
plcB
1039
plcB ext Rv
TATCCACCCGCTAACGAGTG
plcA ext Fw
GAGGTTGCTCGGAGATATAC
plcA
1100
plcA ext Rv
CTGCTGTCCCTTTATCGTCG
plcA Detec-Fw
AACCATTATTATGCG
396
plcA Detec-Rv
TGCAGCATACTGACG
hly ext Fw
CGATAAAGGGACAGCAGGACT
hly
1796
hly ext Rv
AGCCTGTTTCTACATTCTTCACAA
hly Detec-Fw
TAACAACGCAGTAAA
566
hly Detec-Rv
CGTAAGTCTCCGAGG
hly End-Fw
CCTCCTGCATATATC
725
hly End-Rv
TTATTCGATTGGATT
inlC In
1
-Fw
ATGCTAGTNTTAATTGTA
inlC
852
inlC End
2
-Rv
CTAATTCTTGATAGGTTGTG
prfA Detec-Fw
CTGCTAACAGCTGAGCTATG
prfA
404
prfA Detec-Rv
GCTACCGCATACGTTATC
prfA End Rv
ATGAACGCTCAAGCA
—
In: primers corresponding to the beginning of the gene; End: primers corresponding to the end of the gene; Detec: internal primers designed for gene detection; ext: external primers; Seq: internal primers designed for sequencing.


### 2.4. DNA Sequencing

The amplified fragments were purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare) according to the manufacturer’s protocol and sequenced directly at Genomic (Genomic Engenharia Molecular, Sao Paulo, Brazil). DNA sequencing was performed on an Applied Biosystems 3130xl DNA analyzer using the BigDye Terminator v3.1 cycle sequencing kit.


### 2.5. Detection of Mutations inL. monocytogenes Virulence Genes

Sequence analysis was performed using the BIOEDIT Sequence Alignment Editor 7.0.9 [18]. The obtained sequences of the virulence genes were compared to previously publishedL. monocytogenes sequence accessions from GenBank (NCBI, Bethesda, USA). The sequencing products were edited and compared with the sequences available in the GenBank database by manual alignment and using the ClustalW application. The nucleotide sequences obtained were translated into their corresponding amino acid sequences by the Nucleotide Translate application. Subsequently, the amino acid sequences were analyzed to identify changes in the compositions of their respective proteins, which might modify or eliminate protein functions.


### 2.6. Identification of Protein Domains and Prediction of Secondary Structures

The domains of InlC, InlB, InlA, PI-PLC, PC-PLC, and Hly from reference strainL. monocytogenes EGD-e were determined using the PROSITE database [19] of the ExPASy server (SIB, Swiss Institute of Bioinformatics). The locations of these domains were compared to the mutations identified in the studied isolates.


### 2.7. Species-Level Identification by 16S rRNA Amplification and Phylogenetic Analysis

Species identity was confirmed using 16S rRNA analysis. The primers and amplification protocol described by Thompson et al. [20] were used to amplify complete 16S rRNA genes. The fragments were sequenced and phylogenetic analysis was performed using the Mega 5.10 software [21]. The dendrogram was constructed using the maximum-likelihood method with the Tamura-3-parameter model.


### 2.8. Nucleotide Sequence Accession Numbers

All DNA sequences from this study were deposited in GenBank under the accession numbers KC618415-KC618420, KC666995-KC667019, KC808518-KC808549, and KC808567-KC808583.


## 2.1. Bacterial Strains and Culture Conditions

FortyListeria sp. isolates were studied. Of these, 25 were isolated from pork, slaughterhouses, and markets (15 isolates ofL. monocytogenes and 10 ofL. innocua), 11 isolates ofL. monocytogenes were obtained from human infections, and four were control strains (L. monocytogenes ATCC 19115 and ATCC 19111 andL. innocua ATCC 33090 and CLIP 12612) (Table 1). The environmental and pork isolates were isolated as described by Moreno et al. [13]; the clinical strains andListeria controls were obtained from the Public Health Laboratory (School of Public Health, University of Sao Paulo) and Laboratory of Swine Health (School of Veterinary Medicine and Animal Science, University of Sao Paulo) collections. The environmental and pork isolates were obtained from different swab samples taken from the slaughterhouses environment and carcasses from Sao Paulo State; the clinical isolates were obtained from the blood, placenta, and cerebrospinal fluid samples of different patients from different Brazilian states (Tables 1 and 2).Table 1
Sources and phenotypic and genotypic characteristics of theListeria monocytogenes isolates used in this study.
Isolate
Species
Serotype
Origin
Site
Year
ALOA
Hemolysis
inlA
inlB
inlC
inlJ
*
plcA
plcB
prfA
hly
Lm1
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm2
L. monocytogenes
1/2b
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm3
L. monocytogenes
4b
Market 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm21
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm22
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm23
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm25
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm26
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm27
L. monocytogenes
1/2a
Slaught 1
Floor
2008
Halo
Positive
+
+
+
+
+
+
+
+
Lm28
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm29
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm30
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm31
L. monocytogenes
1/2a
Market 2
Pork
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm34
L. monocytogenes
1/2a
Human
Blood
1989
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm35
L. monocytogenes
4b
Human
Blood
2004
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm36
L. monocytogenes
4b
Human
Blood
1977
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm37
L. monocytogenes
4b
Human
CSF
1982
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm38
L. monocytogenes
1/2b
Human
CSF
1983
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm39
L. monocytogenes
1/2a
Human
Placenta
1978
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm39a
L. monocytogenes
1/2a
Human
Placenta
1978
Halo
Positive
+
+
+
+
+
+
+
+
Lm40
L. monocytogenes
1/2a
Human
Blood
1985
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm41
L. monocytogenes
4b
Human
CSF
1997
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm42
L. monocytogenes
4b
Human
CSF
1997
Halo
Positive
+
+
+
+
+
+
+
+
Lm43
L. monocytogenes
1/2a
Human
CSF
1983
Halo
Positive
+
+
+
+
+
+
+
+
Lm4
L. monocytogenes
1/2a
Market 2
Floor
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm33
L. monocytogenes
1/2a
Market 2
Floor
2008
Halo
Weak positive
+
+
+
+
+
+
+
+
Lm10
L. monocytogenes
4b
ATCC 19115
—
—
Halo
Strong positive
+
+
+
+
+
+
+
+
Lm15
L. monocytogenes
1/2a
ATCC 19111
—
—
Halo
Strong positive
+
+
+
+
+
+
+
+
Slaught 1: slaughterhouse 1. CSF: cerebrospinal fluid.*All isolates were positive for fragments of inlJ but presented variable results for hole gene amplification (see Table 4).Table 2
Sources and phenotypic and genotypic characteristics of theListeria innocua isolates used in this study.
Isolate
Species
Serotype
Origin
Site
Year
ALOA
Hemolysis
inlA
x
inlB
inlC
inlJ
plcA
plcB
prfA
hly
x
Lin5
L. innocua
6a
Market 1
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin6
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin7
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin8
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin9
L. innocua
6a
Slaught 2
Floor
2008
Halo*
Weak positive**
−
−
+
−
+
−
−
+
Lin16
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin17
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin18
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin19
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin20
L. innocua
6a
Slaught 1
Floor
2006
Negative
Negative
−
−
−
−
−
−
−
−
Lin11
L. innocua
6a
ATCC 33090
—
—
Negative
Negative
−
−
−
−
−
−
−
−
Lin46
L. innocua
6a
CLIP 12612
—
—
Negative
Negative
−
−
−
−
−
−
−
−
Slaught 1: slaughterhouse 1; Slaught 2: slaughterhouse 2.*Subtle halo. ** Very weak positive hemolysis. xAtypical isolates were positive for fragments of inlAB operon and hly but presented variable results for inlA and hly complete amplification (see Table 4).The isolates were maintained in a stock medium containing glycerol at −80°C. The isolates were reactivated in brain-heart infusion (BHI) medium (Difco, Sparks, MD, USA) and plated on tryptone soy agar supplemented with yeast (TSAYE) (Oxoid, Lenexa, USA) to isolate pure colonies before use.


## 2.2. Conventional Listeria Identification Tests

The isolates were serotyped using polyclonal antisera produced against Listeria somatic and flagellar antigens in rabbits, according to the method described by Seeliger and Höhne [14]. The isolates were also characterized by catalase, motility, and biochemical tests including acid production from D-xylose, D-mannitol, L-rhamnose, and α-methyl-D-mannoside. Cultivation in selective agarListeria according to Ottaviani and Agosti (ALOA) (Biolife, Milan, Italy) was used to identifyL. monocytogenes isolates, and β-hemolysis was assessed by sting inoculation on 5% sheep blood agar.


## 2.3. Detection ofL. monocytogenes-Specific Genes

Genomic DNA extraction was performed as described by Ausubel et al. [15]. All isolates were screened for theinlA,inlB,inlC,inlJ,hly,prfA,plcA, andplcB genes. The primers described by Johnson et al. [6], Liu et al. [16], and Jung et al. [17] were used for detection ofprfA,inlCandinlJ,andinlA, respectively. Specific primers were designed for the complete amplification of the virulence genes (Table 3). The PCRs were performed using anEppendorf Mastercycler gradient thermal cycler. Each reaction (25 μL) contained 5 μL of genomic DNA, MilliQ water, 10X PCR buffer, 1.5 mM MgCl2, 200 μM of dNTPs (Fermentas, Burlington, Canada), 200 μM of each primer, and 1.25 U of Taq DNA polymerase (Promega). The PCR programs were as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at primer-specific temperature for 1–1.5 min, elongation at 72°C for 1 min per 1 Kb, and final extension at 72°C for 10 min. The amplified products were separated by electrophoresis on 1.5% agarose gels and stained with ethidium bromide (1 μg/mL). The molecular weights of the products were determined using the 1 Kb Plus DNA Ladder (Fermentas, Burlington, Canada).Table 3
Primers designed in this study for the amplification of theL. monocytogenes virulence genes.
Primer
Sequence 5′-3′
Target
Product (bp)
inlA ext Fw
CGGCTCCGTAGACAGATTAG
inlA
2884
inlA ext Rv
GTGATAGTCTCCGCTTGTAC
inlA In
1
-Fw
GTGAGAAGAAAACGA
1200
inlA Detec-Rv
TGGTGTAAGATCGCT
inlA Detec-Fw
AAGTGATATAACTCC
—
inlB ext Fw
GCTAGATGTGGTTTTCGGACT
inlB
2146
inlB ext Rv
TAAGCAGCGCAAAGGTGATTCCTAC
inlB In-Fw
GTGAAAGAAAAGCAC
1227
inlB Seq
3
-Rv
ATTCCCGCGAATATA
inlB Seq
2
-Fw
TGATGGAACGGTAAT
900
inlB End
3
-Rv
TNATTTCTGTGCCCT
plcB ext Fw
CCATACGACGTTAATTCTTGCAATG
plcB
1039
plcB ext Rv
TATCCACCCGCTAACGAGTG
plcA ext Fw
GAGGTTGCTCGGAGATATAC
plcA
1100
plcA ext Rv
CTGCTGTCCCTTTATCGTCG
plcA Detec-Fw
AACCATTATTATGCG
396
plcA Detec-Rv
TGCAGCATACTGACG
hly ext Fw
CGATAAAGGGACAGCAGGACT
hly
1796
hly ext Rv
AGCCTGTTTCTACATTCTTCACAA
hly Detec-Fw
TAACAACGCAGTAAA
566
hly Detec-Rv
CGTAAGTCTCCGAGG
hly End-Fw
CCTCCTGCATATATC
725
hly End-Rv
TTATTCGATTGGATT
inlC In
1
-Fw
ATGCTAGTNTTAATTGTA
inlC
852
inlC End
2
-Rv
CTAATTCTTGATAGGTTGTG
prfA Detec-Fw
CTGCTAACAGCTGAGCTATG
prfA
404
prfA Detec-Rv
GCTACCGCATACGTTATC
prfA End Rv
ATGAACGCTCAAGCA
—
In: primers corresponding to the beginning of the gene; End: primers corresponding to the end of the gene; Detec: internal primers designed for gene detection; ext: external primers; Seq: internal primers designed for sequencing.


## 2.4. DNA Sequencing

The amplified fragments were purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare) according to the manufacturer’s protocol and sequenced directly at Genomic (Genomic Engenharia Molecular, Sao Paulo, Brazil). DNA sequencing was performed on an Applied Biosystems 3130xl DNA analyzer using the BigDye Terminator v3.1 cycle sequencing kit.


## 2.5. Detection of Mutations inL. monocytogenes Virulence Genes

Sequence analysis was performed using the BIOEDIT Sequence Alignment Editor 7.0.9 [18]. The obtained sequences of the virulence genes were compared to previously publishedL. monocytogenes sequence accessions from GenBank (NCBI, Bethesda, USA). The sequencing products were edited and compared with the sequences available in the GenBank database by manual alignment and using the ClustalW application. The nucleotide sequences obtained were translated into their corresponding amino acid sequences by the Nucleotide Translate application. Subsequently, the amino acid sequences were analyzed to identify changes in the compositions of their respective proteins, which might modify or eliminate protein functions.


## 2.6. Identification of Protein Domains and Prediction of Secondary Structures

The domains of InlC, InlB, InlA, PI-PLC, PC-PLC, and Hly from reference strainL. monocytogenes EGD-e were determined using the PROSITE database [19] of the ExPASy server (SIB, Swiss Institute of Bioinformatics). The locations of these domains were compared to the mutations identified in the studied isolates.


## 2.7. Species-Level Identification by 16S rRNA Amplification and Phylogenetic Analysis

Species identity was confirmed using 16S rRNA analysis. The primers and amplification protocol described by Thompson et al. [20] were used to amplify complete 16S rRNA genes. The fragments were sequenced and phylogenetic analysis was performed using the Mega 5.10 software [21]. The dendrogram was constructed using the maximum-likelihood method with the Tamura-3-parameter model.


## 2.8. Nucleotide Sequence Accession Numbers

All DNA sequences from this study were deposited in GenBank under the accession numbers KC618415-KC618420, KC666995-KC667019, KC808518-KC808549, and KC808567-KC808583.


## 3. Results

### 3.1. Conventional Listeria Identification Tests

The phenotypic characterization ofListeria sp. isolates is shown in Tables 1 and 2. Five atypicalL. innocua isolates (Lin5–9) and six low-hemolyticL. monocytogenes (Lm4,Lm33, andLm28–31) isolates were observed. The atypicalL. innocua isolates exhibited phenotypic characteristics similar toL. monocytogenes with weak hemolysis and subtle halo in ALOA cultivation. These isolates could be distinguished only by serotyping, which revealed that the atypical isolates wereL. innocua serotype 6a.


### 3.2. Detection and Analysis ofL. monocytogenes Virulence Genes

The detection and complete amplification of theinlB,inlC,plcA,plcB,hly, andprfAgenes were performed using previously published primers and primers designed in this study. TheinlA andinlJ genes were only partially amplified using the primersinlA In-Fw/inlA Detec-Rv,designed in this study,andinlJ-Fw/inlJ-Rv, which were described by Liu et al. [16]. AllL. monocytogenes isolates including the six low-hemolytic isolates (Lm4,Lm33,andLm28–31) contained the studied genes. The five atypicalL. innocua isolates (Lin5–9) containedinlC andplcA and fragments of thehlygene (Table 4).Table 4
Distribution of the results of the virulence gene amplification fromListeria species.
Primer
Species
Positive
Negative
N (%)
N (%)
inlC_Liu
1
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlC In–End
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
prfA Johnson–End
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
prfA_Johnson
2
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
prfA Detec
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
plcA ext
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
plcA Detec
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
plcB ext
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlB In–
Seq
3
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlB Seq
2
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlA In–Detec
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlAB_Jung
3
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
hly ext
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
hly End
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
hly_Border
4
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
hly Detec
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
inlJ_Liu
1
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlJ ext
L. monocytogenes
23  (82.1)
5  (17.9)
L. innocua
0
12  (100.0)
1Primers described by Liu et al. [16]. 2Primers described by Johnson et al. [6]. 3Primers described by Jung et al. [17]. 4Primers described by Border et al. [22].Nucleotide substitutions were detected ininlC,inlB,inlA,plcA,plcB, andprfA, only in the six low-hemolyticL. monocytogenes isolates (Lm4,Lm33, andLm28–31). Seven substitutions were detected in theinlC gene; however, only the transition of adenine to cytosine and the inversion of thiamine to adenine at codon 10 led to the mutation Ile10His, and the transition of thiamine to cytosine at codon 12 resulted in the mutation Met12Thr. Ten substitutions were detected inplcA, leading to the mutations Ile17Val and Phe119Tyr in the PI-PLC. In theplcB sequence, only two transitions of thiamine to cytosine were identified at codon 13, which resulted in the mutation Ile13Thr. Seven substitutions were detected ininlB; however, only the transitions of adenine to guanine at codons 117 and 132 resulted in the mutations Ala117Thr and Val132Ile (Figures 1 and 2).Figure 1
Nucleotide substitutions detected in theinlC,plcA,plcB, andinlB genes. TheLm28–31,Lm4, andLm33 isolates were aligned withL. monocytogenes EGDe and the previously described mutant strains. Asterisks indicate the start and stop codons, dots represent identical nucleotides, and numbers indicate the positions of the substitutions.Figure 2
Amino acids substitutions in the InlC, PI-PLC, PC-PLC, InlB, and PrfA proteins. TheLm28–31,Lm4, andLm33 isolates were aligned withL. monocytogenes EGDe and the previously described mutant strains. Asterisks indicate the start and stop codons, dots represent identical amino acids, and numbers indicate the positions of the substitutions.A deletion of five nucleotides was also detected in theprfA sequence, leading to the deletion of codons 236 and 237 in theLm4,Lm33, andLm28–31 isolates. Eight substitutions were detected in theinlA fragments of the low-hemolyticL. monocytogenes isolates, resulting in the mutations Thr51Ala and Ile157Leu (Figure 3). TheLm4,Lm33, andLm28–31 isolates also contained 15 substitutions in thehly sequence, whereas theLin5andLin6–9isolates only contained 14 and 13 of these substitutions, respectively. However, all these atypical isolates contained only the mutations Val438Ile and Lys523Ser (Figure 3).Nucleotide substitutions detected ininlA andhly (a) and mutations identified in InlA and Hly (b). TheLm28–31,Lm4,Lm33, andLin5–9 isolates were aligned withL. monocytogenes EGDe and previously described mutant strains. Asterisks indicate the start and stop codons, dots represent identical amino acids, and numbers indicate the positions of the substitutions. Gaps represent the regions that were not amplified.
(a)
(b)


### 3.3. Identification of Protein Domains

Of the identified mutations, only Ala117Thr and Val132Ile in InlB and Ile157Leu in InlA were located in the leucine-rich repeat (LRR) domains that are characteristic of these proteins. The Phe119Tyr mutation in PI-PLC was also located in the PI-PLC X-box domain. The thiol-activated cytolysin signature motifs in Hly and the zinc-dependent phospholipase C domain in PC-PLC presented distinct locations of the mutations identified in the respective proteins.


### 3.4. Species Confirmation by 16S rRNA Phylogenetic Analysis

From the amplification and analysis of the 16S rRNA genes, a dendrogram was constructed, which allowed the distinction ofL. monocytogenes andL. innocua species. The dendrogram contained three major groups; the first group consisted ofL. grayi andL. murrayi, the second group containedL. rocourtiae, and the third group consisted of clusters ofL. monocytogenes andL. marthii,L. innocua,L. welshimeri,L. seeligeri, andL. ivanovii (Figure 4). The isolatesLin5
–
9 andLin11 were grouped with the standard strains ofL. innocua, whereas the isolatesLm28–31,Lm4, andLm33 were grouped together with the standard strains ofL. monocytogenes.Figure 4
Dendrogram showing the evolutionary relationships among theListeria isolates based on the 16S rRNA nucleotide sequences. The dendrogram was constructed using the maximum-likelihood method (Tamura-3-parameter model) with the MEGA 5.10 software. The bootstrap values ​​presented at corresponding branches were evaluated using 500 replicates.


## 3.1. Conventional Listeria Identification Tests

The phenotypic characterization ofListeria sp. isolates is shown in Tables 1 and 2. Five atypicalL. innocua isolates (Lin5–9) and six low-hemolyticL. monocytogenes (Lm4,Lm33, andLm28–31) isolates were observed. The atypicalL. innocua isolates exhibited phenotypic characteristics similar toL. monocytogenes with weak hemolysis and subtle halo in ALOA cultivation. These isolates could be distinguished only by serotyping, which revealed that the atypical isolates wereL. innocua serotype 6a.


## 3.2. Detection and Analysis ofL. monocytogenes Virulence Genes

The detection and complete amplification of theinlB,inlC,plcA,plcB,hly, andprfAgenes were performed using previously published primers and primers designed in this study. TheinlA andinlJ genes were only partially amplified using the primersinlA In-Fw/inlA Detec-Rv,designed in this study,andinlJ-Fw/inlJ-Rv, which were described by Liu et al. [16]. AllL. monocytogenes isolates including the six low-hemolytic isolates (Lm4,Lm33,andLm28–31) contained the studied genes. The five atypicalL. innocua isolates (Lin5–9) containedinlC andplcA and fragments of thehlygene (Table 4).Table 4
Distribution of the results of the virulence gene amplification fromListeria species.
Primer
Species
Positive
Negative
N (%)
N (%)
inlC_Liu
1
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlC In–End
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
prfA Johnson–End
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
prfA_Johnson
2
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
prfA Detec
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
plcA ext
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
plcA Detec
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
plcB ext
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlB In–
Seq
3
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlB Seq
2
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlA In–Detec
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlAB_Jung
3
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
hly ext
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
hly End
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
hly_Border
4
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
hly Detec
L. monocytogenes
28  (100.0)
0
L. innocua
5  (41.7)
7  (58.3)
inlJ_Liu
1
L. monocytogenes
28  (100.0)
0
L. innocua
0
12  (100.0)
inlJ ext
L. monocytogenes
23  (82.1)
5  (17.9)
L. innocua
0
12  (100.0)
1Primers described by Liu et al. [16]. 2Primers described by Johnson et al. [6]. 3Primers described by Jung et al. [17]. 4Primers described by Border et al. [22].Nucleotide substitutions were detected ininlC,inlB,inlA,plcA,plcB, andprfA, only in the six low-hemolyticL. monocytogenes isolates (Lm4,Lm33, andLm28–31). Seven substitutions were detected in theinlC gene; however, only the transition of adenine to cytosine and the inversion of thiamine to adenine at codon 10 led to the mutation Ile10His, and the transition of thiamine to cytosine at codon 12 resulted in the mutation Met12Thr. Ten substitutions were detected inplcA, leading to the mutations Ile17Val and Phe119Tyr in the PI-PLC. In theplcB sequence, only two transitions of thiamine to cytosine were identified at codon 13, which resulted in the mutation Ile13Thr. Seven substitutions were detected ininlB; however, only the transitions of adenine to guanine at codons 117 and 132 resulted in the mutations Ala117Thr and Val132Ile (Figures 1 and 2).Figure 1
Nucleotide substitutions detected in theinlC,plcA,plcB, andinlB genes. TheLm28–31,Lm4, andLm33 isolates were aligned withL. monocytogenes EGDe and the previously described mutant strains. Asterisks indicate the start and stop codons, dots represent identical nucleotides, and numbers indicate the positions of the substitutions.Figure 2
Amino acids substitutions in the InlC, PI-PLC, PC-PLC, InlB, and PrfA proteins. TheLm28–31,Lm4, andLm33 isolates were aligned withL. monocytogenes EGDe and the previously described mutant strains. Asterisks indicate the start and stop codons, dots represent identical amino acids, and numbers indicate the positions of the substitutions.A deletion of five nucleotides was also detected in theprfA sequence, leading to the deletion of codons 236 and 237 in theLm4,Lm33, andLm28–31 isolates. Eight substitutions were detected in theinlA fragments of the low-hemolyticL. monocytogenes isolates, resulting in the mutations Thr51Ala and Ile157Leu (Figure 3). TheLm4,Lm33, andLm28–31 isolates also contained 15 substitutions in thehly sequence, whereas theLin5andLin6–9isolates only contained 14 and 13 of these substitutions, respectively. However, all these atypical isolates contained only the mutations Val438Ile and Lys523Ser (Figure 3).Nucleotide substitutions detected ininlA andhly (a) and mutations identified in InlA and Hly (b). TheLm28–31,Lm4,Lm33, andLin5–9 isolates were aligned withL. monocytogenes EGDe and previously described mutant strains. Asterisks indicate the start and stop codons, dots represent identical amino acids, and numbers indicate the positions of the substitutions. Gaps represent the regions that were not amplified.
(a)
(b)


## 3.3. Identification of Protein Domains

Of the identified mutations, only Ala117Thr and Val132Ile in InlB and Ile157Leu in InlA were located in the leucine-rich repeat (LRR) domains that are characteristic of these proteins. The Phe119Tyr mutation in PI-PLC was also located in the PI-PLC X-box domain. The thiol-activated cytolysin signature motifs in Hly and the zinc-dependent phospholipase C domain in PC-PLC presented distinct locations of the mutations identified in the respective proteins.


## 3.4. Species Confirmation by 16S rRNA Phylogenetic Analysis

From the amplification and analysis of the 16S rRNA genes, a dendrogram was constructed, which allowed the distinction ofL. monocytogenes andL. innocua species. The dendrogram contained three major groups; the first group consisted ofL. grayi andL. murrayi, the second group containedL. rocourtiae, and the third group consisted of clusters ofL. monocytogenes andL. marthii,L. innocua,L. welshimeri,L. seeligeri, andL. ivanovii (Figure 4). The isolatesLin5
–
9 andLin11 were grouped with the standard strains ofL. innocua, whereas the isolatesLm28–31,Lm4, andLm33 were grouped together with the standard strains ofL. monocytogenes.Figure 4
Dendrogram showing the evolutionary relationships among theListeria isolates based on the 16S rRNA nucleotide sequences. The dendrogram was constructed using the maximum-likelihood method (Tamura-3-parameter model) with the MEGA 5.10 software. The bootstrap values ​​presented at corresponding branches were evaluated using 500 replicates.


## 4. Discussion

Studies onListeria virulence mechanisms have become important in recent decades because this microorganism is used as a model of intracellular infection.L. monocytogenes virulence factors have been described, and their mechanisms of action and respective genes have been studied using distinct molecular techniques andin vivo andin vitro experiments. In addition to the use ofListeria as a model organism, there is great interest in studying this organism because of the increasing incidence of listeriosis in the United States of America (USA) and Europe [23, 24].Our results using conventionalListeria identification tests are consistent with the subjectivity and ambiguity of phenotypic tests that have been discussed in the last decade [6, 7]. Although these conventional methods are still utilized, biochemical and phenotypic tests yield variable results during the identification ofListeria species and serotypes, and the emergence of atypical isolates has further increased the uncertainty of the application of these tests. From a public health perspective, a drastic measure could be adopted to classify all isolates with doubtful hemolytic status asL. monocytogenes or as isolates with pathogenic risk without major efforts to identify the species and serovars. However, for better epidemiological, microbiological, and evolutionary understanding, it is important to identify and characterize the phenotypes and molecular features of these atypical isolates.This study aimed to detect thehly,plcA,plcB,prfA,inlA,inlB,inlC, andinlJ genes inL. monocytogenes andL. innocua isolates. These genes are characteristic ofL. monocytogenes and are essential for intracellular infection. The presence of these genes in isolates from meat and the environment suggests the pathogenic potential of these isolates and a risk to human health. We detected these virulence genes in allL. monocytogenes isolates including the six low-hemolytic isolates (Lm4,Lm33, andLm28–31); additionally, the five atypicalL. innocua isolates (Lin5–9) contained theinlC,hly, andplcA genes.Our results are consistent with the data of Johnson et al. [6] and Volokhov et al. [7], who identified someL. monocytogenesvirulence genes inL. innocua strains with atypical hemolysis. Therefore, the use of traditional PCR methods based mostly on the detection ofhly andplcA for the distinction ofListeria pathogenic species should be reconsidered because these methods do not enable the distinction of atypical isolates. Accurate identification ofListeria species was possible only by the complete sequencing and phylogenetic analysis of the 16S rRNA gene (Figure 4). We propose that the detection ofprfA,plcB, andinlBmight be a better and reliable alternative to enable the rapid distinction ofL. monocytogenesandL. innocua. We also suggest that analysis of the complete 16S rRNA gene sequences is important for the accurate identification ofListeria species.TheinlCandplcA genes from the atypicalL. innocua isolates did not contain nucleotide substitutions and mutations in their respective proteins. The only mutations identified in these isolates were the Val438Ile and Lys523Ser in Hly. Thehly gene could not be completely amplified, but this might be due to insertions or deletions between the detected fragments. However, the hemolytic phenotypes of these atypical isolates suggest that despite the difficulty in amplifying this locus there were no gross alterations in Hly function. Further studies will be carried out to confirm and quantifyhly expression.Because the atypicalL. innocua isolates presented the low-hemolytic phenotype and halo in ALOA cultivation, we concluded that these isolates produce at least Hly and PI-PLC. Since the only detected mutations were not located in the thiol-activated cytolysin signature motifs in Hly, the low expression of thehly andplcA genes might be due to altered promoter activity. As theprfA gene was also not detected in these isolates, we suggest that a secondary promoter might activate the expression ofhly andplcA and originate the observed phenotype. However, furtherin vitro and proteomic studies are necessary to verify the activity and integrity of these virulence factors.The mutations detected in InlB and PI-PLC in the low-hemolyticL. monocytogenes isolates (Lm4,Lm33, andLm28–31) are consistent with results from previous studies on low-virulentL. monocytogenes field strains [10–12]. The mutations Ala117Thr and Val132Ile in InlB are located in the LRR domains of this protein, which are directly related to the interaction of this internalin with the Met cellular receptor and might compromise the adhesin function of InlB [11, 12]. The Ile17Val and Phe119Tyr mutations in PI-PLC are located in the signal sequence and the X-box domain, respectively, whereas the Thr262Ala mutation causes the introduction of an amino acid with different physicochemical properties, which might inhibit PI-PLC activity [12].The mutations identified in PC-PLC, InlC, InlA, PrfA, and Hly are novel. The Ile13Thr mutation in PC-PLC is not located at the zinc-dependent phospholipase C domain of this protein, and the Ile10His and Met12Thr mutations in InlC are not located in the LRR domains of this internalin. The Thr51Ala and Ile157Leu mutations in InlA are also novel, and although they do not cause the truncation of InlA [11, 12], they are located in the LRR domains; therefore, these mutations might compromise the internalization ofL. monocytogenes in epithelial cells. Further expression studies are required to confirm whether these mutations affect the expression and function of these virulence factors.The low-hemolyticL. monocytogenes isolates contained the same Hly mutations as the atypicalL. innocua; theconsequence of this observation is unclear. The deletion inprfAin the low-hemolyticL. monocytogenes isolates might underlie the reduced hemolytic activity in these strains because PrfA is the activator of the LIPI-1 cluster. However, the impairment ofprfA would result in the reduced expression of all LIPI-1 genes. Therefore, further transcriptomic studies are required to completely characterize these atypical isolates, enhance our knowledge of their evolution and impact on public health, and develop more efficient methods for the identification and distinction ofListeria species.



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*Source: 742032-2014-05-28.xml*