Division of Listeria monocytogenes serovar 1/2a strains into two groups by PCR and restriction enzyme analysis

Altogether, 100 strains of Listeria monocytogenes serovar 1/2a isolated from humans, animals, food, and the environment were typed by a combination of PCR and restriction enzyme analysis (REA). A PCR product of 2,916 bp, containing the downstream end of the gene inlA (955 bp), the space between inlA and inlB (85 bp), and 1,876 bp of the gene inlB, was cleaved with the enzyme AluI, and the fragments generated were separated by gel electrophoresis. By this method two different cleavage patterns were obtained. Seventy of the 100 strains shared one restriction profile, and the remaining 30 strains shared the second one. No relation was found between the types differentiated by PCR-REA and the origins of the strains.     

The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5′-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3′-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.     

Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.     

Pyrosequencing as a Method for Grouping of Listeria monocytogenes Strains on the Basis of Single-Nucleotide Polymorphisms in the inlB Gene

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763^T and C. mobile DSM 4849^T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766^T and C. piscicola NCDO 2762^T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA^Ile and tRNA^Ala genes. The M-ISR included one tRNA^Ala gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.     

Identification of the Metallo-β-Lactamase Gene from Clinical Isolates of Bacteroides fragilis

Bacteroides fragilis is one of the organisms known to produce carbapenem-hydrolysing metallo-β-lactamase, which can confer resistance to a wide variety of β-lactams. The purpose of this study was to identify carbapenem-hydrolysing metallo-β-lactamase-producing B. fragilis strains by means of PCR assay, nucleotide sequencing and enzyme inhibition studies. Ten β-lactam-resistant B. fragilis isolates were investigated. Four imipenem-resistant strains among the 10 isolates gave positive reactions in the PCR assay. The nucleotide sequences of the PCR products from two imipenem-resistant strains shared >98% similarity with the metallo-β-lactamase gene from B. fragilis TAL 3636, which was used as a control. The amino acid sequence homology between the two imipenem-resistant strains and B. fragilis TAL 3636 was 99.2%. These strains produced high amounts of Zn²⁺-dependent β-lactamases which were inactivated by EDTA.     

Integrin receptors play a role in the internalin B-dependent entry of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> into host cells

Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host’s E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (ΔinlA) carrying a deletion in the gene coding for inlA. The ΔinlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-β₃- and anti-β₁-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-β₂- and anti-β₄-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the β₃- and β₁-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes ΔinlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that β₃- and β₁-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells.     

Molecular Characterization of Yarrowia lipolytica and Candida zeylanoides Isolated from Poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

Distribution of Class 1 Integrons with IS26-Mediated Deletions in Their 3′-Conserved Segments in Escherichia coli of Human and Animal Origin

Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3′-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer.     

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination

Discrimination of Leishmaniainfantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteineproteaseB (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3’ end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.     

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes,Mutability, and Adaptation to Cold Temperatures

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.     

Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10³ CFU ml⁻¹) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

Select Listeria monocytogenes Subtypes Commonly Found in Foods Carry Distinct Nonsense Mutations in inlA, Leading to Expression of Truncated and Secreted Internalin A, and Are Associated with a Reduced Invasion Phenotype for Human Intestinal Epithelial Cells

The surface protein internalin A (InlA) contributes to the invasion of human intestinal epithelial cells by Listeria monocytogenes. Screening of L. monocytogenes strains isolated from human clinical cases (n = 46), foods (n = 118), and healthy animals (n = 58) in the United States revealed mutations in inlA leading to premature stop codons (PMSCs) in L. monocytogenes ribotypes DUP-1052A and DUP-16635A (PMSC mutation type 1), DUP-1025A and DUP-1031A (PMSC mutation type 2), and DUP-1046B and DUP-1062A (PMSC mutation type 3). While all DUP-1046B, DUP-1062A, DUP-16635A, and DUP-1031A isolates (n = 76) contained inlA PMSCs, ribotypes DUP-1052A and DUP-1025A (n = 72) contained isolates with and without inlA PMSCs. Western immunoblotting showed that all three inlA PMSCs result in the production of truncated and secreted InlA. Searches of the Pathogen Tracker database, which contains subtype and source information for more than 5,000 L. monocytogenes isolates, revealed that the six ribotypes shown to contain isolates with inlA PMSCs were overall more commonly isolated from foods than from human listeriosis cases. L. monocytogenes strains carrying inlA PMSCs also showed significantly (P = 0.0004) reduced invasion of Caco-2 cells compared to isolates with homologous 3′ inlA sequences without PMSCs. Invasion assays with an isogenic PMSC mutant further supported the observation that inlA PMSCs lead to reduced invasion of Caco-2 cells. Our data show that specific L. monocytogenes subtypes which are common among U.S. food isolates but rare among human listeriosis isolates carry inlA mutations that are associated with, and possibly at least partially responsible for, an attenuated invasion phenotype.     

Use of PCR-Restriction Fragment Length Polymorphism of inlA for Rapid Screening of Listeria monocytogenes Strains Deficient in the Ability To Invade Caco-2 Cells

A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA. inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA. Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen L. monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L. monocytogenes strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L. monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.     

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.     

Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris

Aspergillus niger lipases are important biocatalysts for a broad range of industrial applications. To enhance the expression level of a newly cloned lipase gene lip2 of A. niger in Pichia pastoris, we applied codon optimization and synthesized the full length codon-optimized gene by a two-step gene synthesis strategy. This strategy consists of an assembly PCR for several small DNA fragments and enzymatic digestion and ligation steps to ligate these fragments into the full-length gene. First, the full-length lip2 gene was divided into three fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) with the additions of proper restriction sites, and separately amplified by assembly PCR reactions. Second, three PCR amplified fragments were digested and ligated into the full-length lip2 gene. In the two-step gene synthesis, synthesis of smaller DNA fragments resulted in a significant lower level of nonspecific mismatching among oligonucleotides and a very low mutational rate of the PCR products, demonstrating the superiority of the method. When compared with the originally cloned lip2 gene of A. niger, the new codon optimized lip2 gene expressed at a significantly higher level in yeasts after methanol induction for 72 h, and both the enzyme activity and protein content reached maximal levels of 191 U/ml and 154 mg/1, with 11.6- and 5.3-fold increases, respectively.     

Listeria monocytogenes Prevalence and Characteristics in Retail Raw Foods in China

The prevalence and levels of Listeria monocytogenes in retail raw foods covering most provincial capitals in China were studied with testing of 1036 samples of vegetables, edible mushrooms, raw meat, aquatic products and quick-frozen products from September 2012 to January 2014. The total prevalence of Listeria monocytogenes was 20.0% (207/1036), and the most probable number (MPN) values of 65.7% of the positive samples ranged from 0.3 to 110 MPN/g. Geographical differences were observed in this survey, and the results of both qualitative and quantitative methods indicated that the levels in the samples from North China were higher than those in the samples from South China. A total of 248 isolates were analyzed, of which approximately half belonged to molecular serogroup 1/2a-3a (45.2%), followed by 1/2b-3b-7 (30.6%), 1/2c-3c (16.1%), 4b-4d-4e (5.2%) and 4a-4c (2.8%). Most of the isolates carried hly (100%), inlB (98.8%), inlA (99.6%), inlC (98.0%) and inlJ (99.2%), and 44.8% of the isolates were llsX-positive. Seventeen epidemic clones (ECs) were detected, with 7 strains belonging to ECI (2.8%) and 10 belonging to ECIII (4.03%). Resistance to clindamycin (46.8%) was commonly observed, and 59 strains (23.8%) were susceptible to all 14 tested antibiotics, whereas 84 (33.9%) showed an intermediate level of resistance or were resistant to two or more antibiotics, including 7 multi-resistant strains that exhibited resistance to more than 10 antibiotics. The data obtained in the present study provides useful information for assessment of the possible risk posed to Chinese consumers, and this information will have a significant public health impact in China. Furthermore, the presence of virulence markers, epidemic clones, as well as the antibiotic resistance amongst the isolates strongly implies that many of these strains might be capable of causing listeriosis, and more accurate treatment of human listeriosis with effective antibiotics should be considered. This research represents a more full-scale and systematical investigation of the prevalence of L. monocytogenes in retail raw foods in China, and it provides baseline information for Chinese regulatory authorities that will aid in the formulation of a regulatory framework for controlling L. monocytogenes with the aim of improving the microbiological safety of raw foods.     

Reproductive compatibility and genetic variation between two strains of Aphelinus albipodus (Hymenoptera: Aphelinidae), a parasitoid of the soybean aphid, Aphis glycines (Homoptera: Aphididae)

Aphelinus albipodus Hayat and Fatima is a potential biological control agent of the soybean aphid, Aphis glycines Matsumura, which is a newly introduced soybean pest in the United States. We compared the reproductive compatibility and molecular genetic variation between two geographic strains of A. albipodus. One strain was collected from soybean aphids in Japan and the other recovered from Russian wheat aphid, Diuraphis noxia (Mordvilko), in the western U.S., populations of which were established with parasitoids imported from Eurasia. We present results of crossing experiments between the two strains, genetic differences based on RAPD-PCR markers, rDNA ITS1 and ITS2 gene sequences, and presence of Wolbachia in the two strains using PCR amplification of the wsp gene. We found no reduction in the production of females in reciprocal crosses between strains, but a significant reduction in fecundity when F₁ females stemming from one of the reciprocal crosses were backcrossed to males from either source. The two strains differed by 3.4% in the rDNA ITS1 sequence and by presence/absence of one RAPD-PCR marker from a total of 20 RAPD primers screened, but their rDNA ITS2 sequences were identical. We used restriction enzyme analysis to separate the strains by differential digestion of the ITS1 PCR product. Wolbachia was present in 100% of males and females of both strains of A. albipodus.