Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.     

Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis

Forty-three Listeria ivanovii isolates were collected in the UK between 1991 and 1997 from: 35 animal infections; two human infections; five foods; and one environmental source. A further two type strains of L. ivanovii (subsp. ivanovii and subsp. londoniensis) were obtained from a culture collection. These bacteria were characterised by conventional phenotypic methods and by pulsed-field gel electrophoresis (PFGE) using ApaI and SmaI. Forty-two of the isolates from the UK were identified as L. ivanovii subsp. ivanovii and the remaining culture as L. ivanovii subsp. londoniensis. Six and four PFGE profiles were obtained using ApaI and SmaI digestion respectively; six composite profiles were obtained combining the results for both enzymes. The PFGE profile of the UK L. ivanovii subsp. londoniensis (isolated from processed shrimps) was similar to the type strain of this subspecies and differed from all of the L. ivanovii subsp. ivanovii tested. The majority of isolates (38 out of 45) belonged to one profile showing that the UK population of this bacterium is much less genetically diverse than similar studies have shown for Listeria monocytogenes.     

Characterisation of persistent and sporadic Listeria monocytogenes strains by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP)

This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants. In each plant persistent and sporadic strains were selected on the basis of PFGE typing results. A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study. PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information. The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively. The combination of PFGE and AFLP typing results yielded a total of 48 genotypes. Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only. Our results showed that L. monocytogenes strains causing persistent contamination differ from sporadic strains. In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups. These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.     

Genotypic analysis of strains of mutans streptococci by pulsed-field gel electrophoresis

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

Physical and genetic map of the Listeria monocytogenes EGD serotype 1/2a chromosome

Listeria monocytogenes is a facultative intracellular pathogen responsible for both invasive and non-invasive food-borne illness in animals and humans. In this study, macrorestriction analysis following pulsed-field gel electrophoresis was used to show that Listeria monocytogenes serovar 1/2a strain EGD has a single chromosome containing eight NotI fragments of 1100, 850, 365, 320, 275, 40, 30 and 20 kb in size and 11 AscI fragments of 860, 470, 410, 360, 320, 250, 110, 80, 50, 30 and 20 kb. The total genome therefore comprises 3000 +/- 50 kb. The creation of a physical and genetic map of the Listeria genome was achieved by generating NotI linking clones and their use in subsequent hybridisation analysis. Using isogenic mutants harbouring additional artificial NotI restriction sites, we were able to precisely map the positions of all currently known virulence genes on the chromosome.     

脉冲场凝胶电泳对铜绿假单胞菌的分子检测与相关性分析

目的应用PFGE对医院环境物体表面分离到的铜绿假单胞菌进行相关性检测与分析,探讨PFGE在监测和控制医院感染方面的意义。方法选择SpeⅠ酶对铜绿假单胞菌染色体DNA进行酶切,选用适宜的实验条件采用脉冲场凝胶电泳技术分析电泳酶切指纹图谱,了解其流行状况。结果14株铜绿假单胞菌基因组DNA经SpeⅠ限制性内切酶酶切后电泳,在30~800 kb之间产生18~24条大小不同的DNA切割片段区带。可分为9个带型。结论PFGE具有分辨力高,重复性好的特点,是铜绿假单胞菌分子流行病学分析的理想方法。     

Characterization of isolates of Listeria monocytogenes from sludge using pulsed‐field gel electrophoresis and virulence assays

Aims: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. Methods and Results: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque‐forming assay and by mouse virulence assay. Conclusions: Our findings suggest that L. monocytogenes strains found in non‐sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. Significance and Impact of the study: This is the first study dealing with the characterization of L. monocytogenes isolates from non‐sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.     

Genome conservation in Helicobacter mustelae as determined by pulsed-field gel electrophoresis

Abstract Genomic DNA from 15 strains of Helicobacter mustelae was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Pac I and S fi I. H. mustelae genome DNA appeared very similar in all strains examined, whether isolated from ferrets or mink or from animals bred in either the USA or in the UK. The H. mustelae genome size was estimated to be 1.7 Mb, similar in size to that of H. pylori . A minor difference in PacI PFGE pattern and genome size was observed between rifampicin-resistant and rifampicin-susceptible derivatives of H. mustelae F251. Another minor difference in genome pattern based on PFGE with S fi I was observed between an H. mustelae strain used to experimentally infect four ferrets which resulted in loss of an S fi I site in strains obtained from the newly infected ferrets. Thus, although minor differences in PFGE pattern were noted, H. mustelae lacks the genomic diversity observed in H. pylori .     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.     

Differentiation between human and ovine isolates of Bordetellaparapertussis using pulsed-field gel electrophoresis

Abstract The genetic relatedness of 18 human and 29 ovine isolates of Bordetella parapertussis was examined by macrorestriction digestion of DNA with the rarely cutting enzyme Xba I and resolution by pulsed-field gel electrophoresis. There was clear separation of human and ovine isolates and variation within host types. The human isolates were separated into three types as were the 24 Scottish ovine isolates. Species-specific bands were observed with the human isolates at 114, 134, 166, 213, 346 and 372 kb. No species-specific bands were found in the B. parapertussis ovine isolates. Isolates of B. parapertussis recovered from sheep in New Zealand gave a further two DNA banding patterns which were clearly different from the Scottish ovine and the human isolates. These results indicate that human and ovine isolates of B. parapertussis are genetically distinct and that variation exists within isolates from the same host species. Pulsed-field gel electrbphoresis therefore appears to be a powerful discriminatory tool for the classification of B. parapertussis .     

Rapid determination of multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis

This report presents a new method for identifying multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis (μ-TGGE). Genomic comparison of L. monocytogenes serovar 1/2a strains EGD-e and F6854 allowed selection of novel polymorphic sequences lmo0386 and lmo0428 as optimum regions for μ-TGGE analysis, in addition to the previously identified lmo0297 gene. Sequence analysis of a total of 48 standard strains revealed that the strains could be grouped into 7 (lmo0386), 8 (lmo0428) and 12 (lmo0297) sequence types. The PCR products from 2, 4 and 4 sequence types of the lmo0386, lmo0428 and lmo0297 genes were selected as marker alleles, and μ-TGGE analysis of the mixture revealed adequate band separation on a single gel. Furthermore, the primer sets could be successfully mixed in a single tube for multiplex PCR, yielding a rapid and easy strategy for sequence type identification. For practical application, multiplex PCR was performed with Cy3-labeled primers against a sequence type-unknown sample isolated from meat. The resulting products were mixed with Cy5-labeled products of marker alleles whose sequence types were known, and μ-TGGE analysis was performed. Overlapping Cy3 and Cy5 patterns allowed identification of the sequence types at all 3 loci on a single gel. Moreover, the μ-TGGE analysis step took only 9 min. Thus, this novel method of multiplex PCR followed by μ-TGGE analysis could prove useful as a rapid and discriminative tool for tracing the strain types, contamination routes and sources of L. monocytogenes.     

Characterization of DNA rearrangements of N-myc gene amplification in three neuroblastoma cell lines by pulsed-field gel electrophoresis

We characterized N-myc gene amplification in three human neuroblastoma cell lines (IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rare-cutting enzymes completely digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case. We estimated that the minimal distance between neighboring N-myc genes is at least several hundred kilobases. Our data suggest that amplification units contain several DNA fragments derived from ditterent loci, but that they are homogeneous.     

Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis

The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 α-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest that human and animal isolates of S. dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.     

Sizing of the Leptospira genome by pulsed-field agarose gel electrophoresis

Pulsed-field gel electrophoresis allowed the determination of the size of the genome of Leptospira, a bacterium of the spirochete family. The three restriction enzymes, NotI (5'GC/GGCCGC), NheI (5'G/CTAGC), ApaI (5'-GGGCC/C) generated DNA fragments of suitable size. The results are compatible with a size of 5000 kb for the chromosome of both the pathogenic and the saprophytic species of Leptospira.     

The diversity of Listeria monocytogenes strains from 10 Icelandic sheep farms

AIMS: The purpose of this study was to examine the diversity of Listeria monocytogenes strains from healthy sheep, winter feed and environment of sheep farms in Iceland. METHODS AND RESULTS: A total of 104 L. monocytogenes isolates from animals, winter feed and environment on 10 Icelandic sheep farms were compared by serotyping, ribotyping, and pulsed-field gel electrophoresis with ApaI and AscI. The isolates were divided into 24 genotypes, all identified as serovars 1/2a, 1/2b, or 4b. Nine genotypes were detected on more than one farm. On three of the farms there seemed to be a dominant strain of L. monocytogenes. Isolates from incidents of listeriosis in animals occurring on two of the farms belonged to the genotype most commonly found on the particular farm. Nine of the 24 genotypes found on the sheep farms have been associated with disease in animals and/or humans elsewhere in Iceland. CONCLUSIONS: Certain strains of L. monocytogenes seem to be widely distributed on Icelandic sheep farms. On some farms there appears to be a dominant strain of L. monocytogenes. Incidents of listeriosis in animals may tend to be associated with strains commonly found on the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the diversity of L. monocytogenes present in healthy sheep and their environment.     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

Molecular and experimental virulence of Listeria monocytogenes strains isolated from cases with invasive listeriosis and febrile gastroenteritis

We analyzed 27 Listeria monocytogenes strains of serotypes 1/2b and 4b, from invasive and gastroenteric listeriosis, for molecular and experimental virulence. Molecular virulence was tested by PCR for the presence of 8 major virulence-associated genes and genetic polymorphisms through restriction enzyme analysis; genomic DNA typing using pulsed-field gel electrophoresis was also performed. Experimental virulence was evaluated through intra-peritoneal and intra-gastric mouse virulence assays. Our results showed no significant differences in the virulence-related molecular properties of the strains analyzed. All strains were equally pathogenic following intra-peritoneal inoculation of mice. In mice inoculated intra-gastric with 4 representative strains of the 2 types of listeriosis, there were no significant differences in the bacterial count when comparing invasive and gastroenteric strains, suggesting that the strains were comparable in terms of mean oral infectivity.     

Characterization of the Legionella anisa population structure by pulsed-field gel electrophoresis

We analysed 38 French isolates of Legionella anisa by means of pulsed-field gel electrophoresis (PFGE) with single or double digestion. Double digestion was more discriminatory than single digestion, and can thus be useful for epidemiological studies of L. anisa. Several isolates from different parts of France clustered together on the basis of their PFGE patterns (similarity cutoff of 80%), suggesting that the L. anisa population structure is homogenous or that a few clones of L. anisa strains have spread widely in France.