Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

An Epidemiological Study of Salmonella enteritidis by Pulsed-Field Gel Electrophoresis (PFGE): Several PFGE Patterns Observed in Isolates from a Food Poisoning Outbreak

An epidemiological analysis of Salmonella enteritidis from a food poisoning was done using pulsed-field gel electrophoresis (PFGE) of BlnI- or XbaI-digested fragments of chromosomal DNA of isolates. S. enteritidis isolates obtained from 19 patients had identical PFGE patterns. Therefore, a strain giving the same pattern was considered to be the causative agent of this outbreak. In addition, four isolates that had different BlnI-digested PFGE patterns were obtained from three patients, suggesting that the observed variations in PFGE patterns might occur as the result of some point mutations of chromosomal DNA during growth or from the existence of several S. enteritidis strains from various sources. Subsequent PFGE analysis of continuously subcultured strains supported the former possibility. These observations indicate that PFGE analysis on multiple numbers of colonies from each patient are necessary for the epidemiologic investigation of S. enteritidis.     

Genome Stability of Bacillus thuringiensis subsp. israelensis Isolates

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred. Received: 8 July 1999 / Accepted: 9 August 1999     

Survey of Phenotypic and Genetic Features of Streptococcus pyogenes Strains Isolated in Northwest Italy

Streptococcus pyogenes (group A Streptococcus [GAS]) is an important pathogen whose virulence is related to the production of exotoxins and the presence of particular surface components. One hundred eighty-two GAS strains were collected in northwestern Italy between 1994 and 2002 and analyzed for phenotypic characteristics (opacity factor, proteolyic activity, and antimicrobial susceptibility) and by polymerase chain reaction for the presence of genes responsible for the production of exotoxins implicated in pathogenesis speA and speF and of prtF₁ (encoding fibronectin-binding protein F1). All strains were speF positive and 19.2% were speA positive and prtF₁ negative, whereas the prtF₁ gene was identified in 39.5% of the other strains. Of these, approximately half revealed the same pulse-field gel electrophoresis (PFGE) pattern but differed in both speA gene and macrolide resistance.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States

Aims: In this study, mechanisms of antimicrobial resistance and genetic relatedness among resistant enterococci from dogs and cats in the United States were determined. Methods and Results: Enterococci resistant to chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, streptomycin, lincomycin, quinupristin/dalfopristin and tetracycline were screened for the presence of 15 antimicrobial resistance genes. Five tetracycline resistance genes [tet(M), tet(O), tet(L), tet(S) and tet(U)] were detected with tet(M) accounting for approx. 60% (130/216) of tetracycline resistance; erm(B) was also widely distributed among 96% (43/45) of the erythromycin‐resistant enterococci. Five aminoglycoside resistance genes were also detected among the kanamycin‐resistant isolates with the majority of isolates (25/36; 69%) containing aph(3′)‐IIIa. The bifunctional aminoglycoside resistance gene, aac(6′)‐Ie‐aph(2″)‐Ia, was detected in gentamicin‐resistant isolates and ant(6)‐Ia in streptomycin‐resistant isolates. The most common gene combination among enterococci from dogs (n = 11) was erm(B), aac(6′)‐Ie‐aph(2″)‐Ia, aph(3′)‐IIIa, tet(M), while tet(O), tet(L) were most common among cats (n = 18). Using pulsed‐field gel electrophoresis (PFGE), isolates clustered according to enterococcal species, source and antimicrobial gene content and indistinguishable patterns were observed for some isolates from dogs and cats. Conclusion: Enterococci from dogs and cats may be a source of antimicrobial resistance genes. Significance and Impact of the Study: Dogs and cats may act as reservoirs of antimicrobial resistance genes that can be transferred from pets to people. Although host‐specific ecovars of enterococcal species have been described, identical PFGE patterns suggest that enterococcal strains may be exchanged between these two animal species.     

New solvent-producing Clostridium sp. strains, hydrolyzing a wide range of polysaccharides, are closely related to Clostridium butyricum

Thirteen new Clostridium strains, previously isolated from soil and found to produce high amounts of solvents from glucose, hydrolyzed a great variety of α- and β-glycans, including raw starch, xylan, pectin, inulin and cellulose. The sequences of the PCR-amplified DNA fragments containing the variable 3′ part of one of the 16S rRNA genes were 99.5% identical. The macrorestriction pattern of two endonucleolytic digests of chromosomal DNA in the pulsed-field gel electrophoresis (PFGE) confirmed their high homogeneity on the DNA level. The complete 16S rRNA gene sequence of three selected strains was 99.8% identical to the 16S rRNA gene sequence from Clostridium butyricum and separates them from C. acetobutylicum. To the closely related four species of solventogenic clostridia a new group of strains has to be added, which has a great potential for the direct fermentation of biomass. Journal of Industrial Microbiology & Biotechnology (2001) 27, 329–335. Received 12 September 2000/ Accepted in revised form 25 July 2001     

Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci

To examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5′ regions of polymerase chain reaction-amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5′emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5′emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation ‘maps’ onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.     

Chromosomal Dynamism in Progeny of Outbreak-Related Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx₂) in the outbreak strain or with the loss of this gene. The two stx₂ alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx₂ genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

Improvement of strain discrimination by combination of RAPD with PFGE for the analysis of the swine isolates of Salmonella enterica serovar Choleraesuis

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) can cause salmonellosis in pigs and humans. Currently, the most common method used for the subtyping of this Salmonella serovar is pulsed field gel electrophoresis (PFGE) using XbaI as a DNA digestion enzyme. In this study, we compared and combined PFGE with the randomly amplified polymorphic DNA method, for the typing of 95 S. Choloraesuis strains isolated from diseased pigs. Using PFGE with XbaI, with AvrII, and with SpeI digested DNA, 29, 74, and 40 patterns, respectively, were obtained. Also, 53, 15, and 35 strains, respectively, belong to the major patterns X1, A1, and S1. When these three digestion patterns were combined, 83 PFGE pattern combinations were obtained. On the other hand, using RAPD with selected primer alone generated 76 patterns, and 11 strains which fell within a single X1A1S1 PFGE combination pattern were discriminated into 10 patterns. Thus, for S. Choloraesuis, PFGE with AvrII allowed higher discrimination than PFGE with XbaI, and some of the PFGE groupings obtained by combining the XbaI, AvrII and SpeI digestion patterns were further subdivided by the RAPD method.     

Cryptic Appearance of a New Clone of Vibrio cholerae Serogroup O1 Biotype El Tor in Calcutta,India

In this study, pulsed-field gel electrophoresis (PFGE) was applied to determine if the Vibrio cholerae O1 strains which reappeared after being temporarily displaced in Calcutta by the O139 serogroup were different from those isolated before the advent of the O139 serogroup. NotI digestion generated a total of 11 different patterns among the 24 strains of V. cholerae randomly selected to represent different time frames. Among the V. cholerae O1 strains isolated after July 1993, 4 PFGE banding patterns designated as H through K were observed with pattern H dominating. Pattern H was distinctly different from all other patterns encountered in this study including patterns A, B and C of V. cholerae O1 El Tor, which dominated before November 1992, and pattern F, which was the dominant V. cholerae O139 pattern. Further, pattern H was also different from the NotI banding patterns of the representative strains of the 4 toxigenic clonal groups of V. cholerae O1 El Tor currently prevailing in different parts of the world. NotI fragments of the new clone of V. cholerae O1 did not hybridize with an O139 specific DNA probe, indicating that there was no O139 genetic material in the new clone of V. cholerae O1. Hybridization data with an O1-specific DNA probe again differentiated between the clones of V. cholerae O1 existing before the genesis of the O139 serogroup and the O1 strains currently prevalent.     

Effects of mixtures of citrus viroids as transmissible small nuclear RNA on tree dwarfing and commercial scion performance on Carrizo citrange rootstock

The term ‘transmissible small nuclear ribonucleic acids' (TsnRNAs) describes well characterised viroid RNA species that do not induce any disease syndromes in specific citrus hosts but rather act as regulatory genetic elements modifying tree performance. Twelve-year-old navel orange and 10-year-old Clementine mandarin trees on Carrizo citrange (Citrus sinensis×Poncirus trifoliata) rootstock treated with a mixture of three TsnRNAs (−Ia, syn. Citrus bent leaf viroid, +IIa, syn. Hop stunt viroid and +IIIb, syn. Citrus dwarfing viroid) were reduced in size by 33% and 43%, respectively. Clementine trees treated with a mixture of TsnRNA−Ia+IIa or −Ia+IIIb also had reduced canopy volume (CV) (∼38 and 31%, respectively), whereas trees treated with TsnRNA−IIa+IIIb showed little effect. The effects of the double TsnRNA treatments −Ia+IIa and −Ia+IIIb on Clementine canopy size and commercial performance were comparable and in some cases superior to that of the triple TsnRNA mixture. The TsnRNA−Ia+IIa treatment had the most attractive commercial traits with increased production of Clementine fruit per CV (23.6%), more fruit with high commercial value (31.7%), and more fruit optimally distributed in the canopy (68% of fruit between 0.5 and 2.5 m). None of the TsnRNA treatments affected the growth of Carrizo rootstock seedlings after 8 years in the field. Navel orange and Clementine scions treated with the same triple TsnRNA mixture expressed different trunk and fruit production patterns although effects on CV were similar.     

Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains showed disappearance of a 70- or 80-kb fragment in their patterns associated with loss of Shiga toxin genes during maintenance or subcultivation. Hybridization experiments with a DNA probe complementary to Shiga toxin sequences revealed that the Shiga toxin genes in the parental strain were located on fragments the same size as the lost fragments from the toxin-negative derivatives. The evidence indicates that PFGE pattern of EHEC O157:H7 may change due to loss of Shiga toxin genes, which is likely to be associated with curing of Shiga toxin gene carrying phages in vitro. Received: 4 May 1998 / Accepted: 19 August 1998     

Comparison of pulsed-field gel electrophoresis patterns of antimicrobial-resistant Escherichia coli and enterococci isolates from the feces of livestock and livestock farmers in Japan

Seven hundred thirty-nine animal strains and 662 livestock-farmer strains, consisting of Escherichia coli and enterococci, were examined for their pulsed-field gel electrophoresis (PFGE) and antimicrobial-resistance patterns. Two hundred fifty-eight and 203 PFGE patterns were found among 739 animal strains isolated from animals comprising broilers, pigs and cattle, and 662 human strains isolated from livestock farmers, respectively, from 27 farms in Japan. These results demonstrated that the PFGE patterns found among E. coli and enterococci strains from animals and livestock-farmers were heterogeneous and considerably diverse. The strains having both the identical PFGE pattern and the same drug-resistance pattern were defined as a single clone in this study. Seven types of E. coli and enterococci clones were shared among animals within the same farms and between the different farms housing the same animal species. The 25 strains (3.4%) of 739 E. coli and enterococci animal strains belonged to these seven types of clones. Only three types of E. coli clones were shared among animals between the different farms housing different animal species, but no identical E. faecalis or E. faecium clones were found between different animal species farms. The 15 strains (2.0%) of 739 E. coli and enterococci animal strains belonged to these three types of clones. Additionally, the 11 strains (1.5%) of 739 E. coli and enterococci strains isolated from animals were identical clones to strains isolated from livestock farmers of the same farm. These results suggest that the transmission of animal clones to livestock farmers or vice versa is less common.     

Identification of putative ancestors of the multidrug-resistant <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar typhimurium DT104 clone harboring the<Emphasis Type="Italic"> Salmonella</Emphasis> genomic island 1

The origin of multidrug-resistant Salmonella enterica serovar typhimurium (S. typhimurium) harboring the Salmonella Genomic Island 1 (SGI1), which was detected for the first time in the mid-1980s is unknown. In this study, we performed microarray genomotyping of four multidrug-resistant SGI1 positive strains and found that unlike the S. typhimurium LT2 strain, the multidrug-resistant strains lacked genes STM0517-0529 allowing the utilization of allantoin as a sole nitrogen source. We extended this observation by PCR screening of additional 120 S. typhimurium field strains and found that this locus was absent in all SGI1 positive and also in 24% of SGI1 negative strains, which were proposed to be the original recipients of SGI1. To prove this hypothesis, we compared the STM0517-0529 negative strains (with or without the SGI1) by PFGE and PCR prophage typing and found that 8 out of 11 of the SGI1 negative strains and 17 out of 22 SGI1 positive strains were of identical PFGE pattern and PCR prophage pattern, while this specific pattern was never observed among STM0517-0529 positive strains. We therefore propose that a lineage of the S. typhimurium DT104 sensitive strain first lost the ability to metabolize allantoin and then acquired SGI1.     

Natural Variation in Susceptibility of Listeria Strains to Class IIa Bacteriocins

Thirty-one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely, enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern in which all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class IIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives. Received: 22 October 1999 / Accepted: 15 December 1999     

Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.     

Molecular Typing by Pulsed-Field Gel Electrophoresis of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> from Root Voles

The root voles intestinal strains of Bacillus thuringiensis, were characterised by pulsed-field gel electrophoresis (PFGE). For 14 isolates, three pulsotypes were found, with the use of SmaI or NotI as restriction enzymes. Strains in each pulsotypes presented identical DNA patterns, indicating that the population structure of B. thuringiensis from root voles is clonal. The similarities in banding patterns were estimated at 56% and 33% for SmaI and NotI digests, respectively. The strains under study differed significantly in the size of their entire genome, which varied between 2.4 and 4.2 Mb. No significant differences were detected among the isolates subjected to biochemical properties determined by API tests. Present study showed that genomic diversity is a common feature of B. thuringiensis originating from one ecological niche. PFGE appears to be a useful technique for use in studies on the spread of B. thuringiensis in the environment. Received: 14 May 2002 / Accepted: 21 June 2002     

Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

Pulsed-field gel electrophoretic analysis and some characteristics of Staphylococcus aureus isolated from retail foods and human hands

This study investigates whether there is a predominant Staphylococcus aureus strain in retail foods and healthy human hands, and examines the relationship between pulsed-field gel electrophoresis (PFGE) banding patterns and the S. aureus characteristics of staphylococcal enterotoxin (SE) type, coagulase type, and β-lactamase activity. Ninety-four strains of S. aureus isolated from retail foods and healthy human hands were analyzed by PFGE. Several strains isolated from the same shop or a chain store showed identical patterns, indicating that the origins of these strains were identical. After excluding these strains showing identical patterns, 54 strains were used for the PFGE analysis. No spread of a particular clone in the environment surrounding the food was apparent. The PFGE analysis of these 54 strains was classified in 6 lineages (L1-L6). There was no relationship between the PFGE banding pattern and coagulase type or SE type. Eleven (84.6%) of the 13 isolates in PFGE banding pattern L5 did not produce β-lactamase, suggesting that the production of β-lactamase influenced a specific PFGE banding pattern.