SpeI Digestion Research Articles

Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum.

Edwardsiella spp. is a gram-negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty-nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed-field gel electrophoresis [PFGE], and virulence-related gene detection). SpeI digestion revealed 10 pulsotypes and I-CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

Molecular markers for differentiation of Fenneropenaeus merguiensis among Penaeus species

The banana shrimp Fenneropenaeus merguiensis is a favourite among consumers. Therefore, overfishing has occurred. Conservation of this shrimp in the wild is essential. Conservation technologies, such as broodstock selection and germ cell transplantation, require molecular markers to discriminate F. merguiensis from other shrimps. This study developed biomarkers to distinguish F. merguiensis from other cultivated shrimps: Penaeus vannamei, Penaeus monodon and Macrobrachium rosenbergii. VASA 160 was used to differentiate F. merguiensis spermatogonia from the other three shrimps. Digestion of the amplified COI (cytochrome oxidase I) fragment with XbaI could discriminate the DNA of F. merguiensis and P. monodon from P. vannamei or M. rosenbergii. In comparison, SpeI digestion of the COI fragment differentiated F. merguiensis from P. monodon. COI266 identified the DNA of F. merguiensis from that of P. vannamei, P. monodon, and the PCR of Fm-15 showed a distinct pattern in F. merguiensis. Besides, the sensitivity of the biomarkers was determined by mixing the DNA of F. merguiensis with that of P. vannamei. VASA160, COI-RFLP and COI266 had a high sensitivity of 1:1000, while Fm 15 was at 1:100. Consequently, 30 processed shrimp foods were identified using these developed markers, and these markers will help monitor these four shrimp species in the future.

Unifying bacteria from decaying wood with various ubiquitous Gibbsiella species as G. acetica sp. nov. based on nucleotide sequence similarities and their acetic acid secretion

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37°C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov.

Characterization of Erwinia amylovora Strains from Bulgaria by Pulsed-Field Gel Electrophoresis

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.

Clonal expansion and genetic diversity of nalidixic acid-resistant Salmonella enterica serotype Paratyphi A

The aim of this study was to understand the clonal expansion and genetic diversity of nalidixic acid -resistant (NAR) Salmonella enterica serotype Paratyphi A (SPA) from Yuxi city, China. Antimicrobial susceptibility testing was performed with 3980 SPA isolates from Yuxi city of China between 1999 and 2007. The incidence of resistance to nalidixic acid (NA) of 1999 and 2000 were 25.0 and 82.2%, respectively. More than 95.0% of the isolates obtained from 2001 to 2007 were resistant to NA. Amplification by PCR and sequencing of the genes within the quinolone resistance-determining region with subsets of 15 NAR strains revealed that the mechanisms of NA resistance resulted from single point mutations in the gyrA gene leading to the Ser83Phe substitution. Subtyping of 120 NAR isolates from seven counties and one Nalidixic acid-susceptible (NAS) isolate were studied using pulsed-field gel electrophoresis (PFGE) analysis, followed by digestion of chromosomal DNA with restriction endonucleases SpeI and XbaI. PFGE patterns were analyzed by cluster analysis. SpeI and XbaI digestion of 121 isolates generated five and four different PFGE patterns with predominance of the SpeI01 and SpeI02 (or the XbaI01) epidemic patterns, respectively. The incidence of resistance to NA of the isolates increased during the study period. NAS isolates predominated in 1999 but was replaced by NAR isolates after 2000. PFGE patterns SpeI01 and SpeI02 (or XbaI01), the main clones of the epidemics, are highly prevalent in Yuxi. Key words: Paratyphoid fever, Salmonella enterica serotype Paratyphi, antimicrobial susceptibility, resistance to nalidixic acid, PFGE, clonal expansion.

Clonal expansion and genetic diversity of nalidixic acid-resistant Salmonella enterica serotype Paratyphi A

The aim of this study was to understand the clonal expansion and genetic diversity of nalidixic acid -resistant (NAR) Salmonella enterica serotype Paratyphi A (SPA) from Yuxi city, China. Antimicrobial susceptibility testing was performed with 3980 SPA isolates from Yuxi city of China between 1999 and 2007. The incidence of resistance to nalidixic acid (NA) of 1999 and 2000 were 25.0 and 82.2%, respectively. More than 95.0% of the isolates obtained from 2001 to 2007 were resistant to NA. Amplification by PCR and sequencing of the genes within the quinolone resistance-determining region with subsets of 15 NAR strains revealed that the mechanisms of NA resistance resulted from single point mutations in the gyrA gene leading to the Ser83Phe substitution. Subtyping of 120 NAR isolates from seven counties and one Nalidixic acid-susceptible (NAS) isolate were studied using pulsed-field gel electrophoresis (PFGE) analysis, followed by digestion of chromosomal DNA with restriction endonucleases SpeI and XbaI. PFGE patterns were analyzed by cluster analysis. SpeI and XbaI digestion of 121 isolates generated five and four different PFGE patterns with predominance of the SpeI01 and SpeI02 (or the XbaI01) epidemic patterns, respectively. The incidence of resistance to NA of the isolates increased during the study period. NAS isolates predominated in 1999 but was replaced by NAR isolates after 2000. PFGE patterns SpeI01 and SpeI02 (or XbaI01), the main clones of the epidemics, are highly prevalent in Yuxi. Key words: Paratyphoid fever, Salmonella enterica serotype Paratyphi, antimicrobial susceptibility, resistance to nalidixic acid, PFGE, clonal expansion.

Nalidixic Acid Resistance and Clonal Expansion of <i>Salmonella enterica </i>Serotype Paratyphi a Isolates in Yuxi City, China

The aim of our study was to understand the nalidixic acid resistance and clonal expansion of Salmonella enterica serotype Paratyphi A (SPA) recovered from patients with paratyphoid fever A in Yuxi City from 1999 to 2008. A typing method is needed to determine the epidemic clones of the isolates. Antimicrobial susceptibility testing was performed with 4 060 SPA isolates. 186 isolates were typed by pulsed-field gel electrophoresis (PFGE) with SpeI, and the MICs for nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin, norfloxacin, and gatifloxacin were determined. The incidence of resistance to nalidixic acid of the isolates increased from 12.5% in 1999, 82.2% in 2000, 93.0% in 2001, to100% in 2008. Nalidixic acid-susceptible (NAS) isolates predominated in 1999 but was replaced by nalidixic acid-resistant (NAR) isolates after 2000. The frequencies of intermediary and susceptibility (Kirby-Bauer disc diffusion technique) to ciprofloxacin were 17.0% and 83.0% before 2005, 65.3% and 34.7% in 2008, respectively. A group of 166 NAR and 20 NAS isolates of SPA were randomly selected. SpeI digestion of 186 isolates gave nine different PFGE patterns with predominance of the SpeI01 and SpeI02 epidemic patterns. All NAR isolates studied had a reduced susceptibility to fluoroquinolone, the MICs were higher than that for 20 NAS isolates.We validate the use of the nalidixic acid screening test in the detection of decreased fluoroquinolone susceptibility in SPA. PFGE patterns SpeI01 and SpeI02, the main clones of the epidemics, are highly prevalent and NAR isolates in Yuxi. Recommendation for the emergency treatments of outbreaks and epidemics caused by a fully fluoroquinolone-resistant strain can be made. PFGE with SpeI is a useful technique to differentiate SPA.

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.

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) – PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth.

Emergence and persistence of integron structures harbouring VIM genes in the Children's Memorial Health Institute, Warsaw, Poland, 1998-2006

The aim was to perform a genetically detailed study of the emergence of metallo-beta-lactamase (MBL) genes in Pseudomonas spp. in the Children's Memorial Health Institute over a 9 year period. Carbapenem-resistant Pseudomonas spp. isolates were collected from 1998 to 2006 and screened for MBL production. MBL-positive isolates were further investigated by a combination of genetic techniques including PCR, genomic location experiments using pulsed-field gel electrophoresis (PFGE) of I-Ceu1, S1 and SpeI digests, and sequencing. Of the 20 MBL-containing Pseudomonas isolates collected from 1998 to 2006, 16 Pseudomonas aeruginosa isolates contained an identical class 1 integron structure. Two P. aeruginosa isolates contained the bla(VIM-2) gene, and two Pseudomonas putida isolates harboured the bla(VIM-4) gene cassette in different integron structures. PFGE analysis indicated that all bla(VIM-4)-containing P. aeruginosa isolates were closely related, whereas the P. putida isolates were not. All MBL genes in this study were chromosomally encoded, and all isolates harboured only one class 1 integron. The bla(VIM-2) isolates were clonal, and the genetic structure supporting the bla(VIM-2) gene was found in an identical chromosomal position. MBL gene emergence in this hospital has paralleled a 6-fold increase in carbapenem usage. We have found an increase in MBL gene diversity, MBL host organisms and MBL genetic support structures in the hospital over the 9 year study period. There is also compelling evidence of the persistence of individual strains in the hospital throughout the study period. This suggests that once MBL genes have emerged in a hospital environment, they are difficult to remove.

The First Metallo-β-Lactamase Identified in Norway Is Associated with a TniC-Like Transposon in a Pseudomonas aeruginosa Isolate of Sequence Type 233 Imported from Ghana

Metallo-β-lactamases (MBLs) are an emerging problem among various clinically important gram-negative bacilli, such as Pseudomonas aeruginosa, Acinetobacter spp., and Enterobacteriaceae (8). Scandinavian countries are renowned for their low level of antibiotic resistance (1), and previous reports on the emergence of new resistance mechanisms have been associated with strain import, such as with the first Swedish MBL, derived from Greece (3). As part of an ongoing national study of MBLs in clinical isolates of P. aeruginosa, the National Reference Centre received a carbapenem-resistant isolate (K34-7) from the Ulleval University Hospital in the autumn of 2006. The isolate was recovered from tracheal secretions upon admission of a patient who transferred to the hospital after prolonged hospitalization in Ghana. The isolate is therefore likely to have been imported to Norway from Ghana. Susceptibility testing of the isolate using Etests (AB Biodisk, Solna, Sweden) showed that the isolate was susceptible only to colistin, intermediate to aztreonam, and resistant to other β-lactams (imipenem-meropenem MIC, >32 μg/ml), aminoglycosides, and fluoroquinolones according to EUCAST clinical breakpoints. The isolate had a positive MBL Etest ratio, and MBL production was confirmed by spectrophotometric analysis of imipenem hydrolysis by crude cell extracts and subsequent inhibition by EDTA (11). The sequence of the blaVIM-2 gene was confirmed by PCR using consensus primers for blaVIM, and sequence analysis of the genetic context using oligonucleotides for the 5′ conserved sequence (5′CS), the 3′CS, blaVIM, and tniC showed that the blaVIM-2 gene was located in an unusual class 1 integron flanked by the tni module similar to Tn402 (7) and not the normal 3′CS end (fused qacEΔ1-sul1). PCR linking blaVIM-2 to tniA, orf6, and tniB and sequencing confirmed that the whole tni module was present. The gene cassette array of aacA7-blaVIM-2-dhfrB5-aacC-A5 is identical to other TniC-like transposons found in isolates from the United States (6), Russia (GenBank accession no. {type:entrez-nucleotide,attrs:{text:DQ522233,term_id:102231610,term_text:DQ522233}}DQ522233), and Taiwan (12) and almost identical to a TniC-like transposon found in an Indian isolate (10). Multilocus sequence typing showed that K34-7 belonged to ST233, which is not part of any clonal complexes; however, Russian isolates with the same transposon belong to ST235, which is part of a clonal complex harboring MBL isolates from several countries in Europe (2, 4, 9). Further, pulsed-field gel electrophoresis (PFGE) analysis (SpeI digestion) and serotyping of K34-7 and isolates possessing TniC-like transposons from Russia (GenBank accession no. {type:entrez-nucleotide,attrs:{text:DQ522233,term_id:102231610,term_text:DQ522233}}DQ522233) and Taiwan (12) showed that the isolates had different PFGE profiles and were of different serotypes (Russian isolate, O11; Taiwan isolate, O2; and K34-7, O6). Thus, the appearance of this TniC-like transposon in unrelated P. aeruginosa isolates suggests that the transposon is itself transferable and also responsible for the dissemination of blaVIM-2. The chromosomal location of the TniC-like transposon was confirmed by hybridization of a radiolabeled blaVIM-2 probe to a chromosomal band larger than 1 megabase after I-Ceu-1 digestion of K34-7 genomic DNA and PFGE (5; data not shown). In conclusion, this study highlights the importance of TniC-like transposons in the global dissemination of blaVIM-2 and also the contribution of human population dynamics in spreading MBL genes. (Part of this study was presented at the 17th European Congress of Clinical Microbiology and Infectious Diseases, Munich, Germany.)

Noncoding transcription controls downstream promoters to regulate T-cell receptor α recombination

The T early alpha (TEA) promoter in the murine Tcra locus generates noncoding transcripts that extend across the 65 kb Jalpha array. Here, we have analyzed the significance of TEA transcription for Tcra locus regulation through the targeted introduction of a transcription terminator downstream of the TEA promoter. We demonstrate that noncoding transcription driven by this single promoter can instruct both positively and negatively the activity of downstream Jalpha promoters, and can similarly instruct alterations in Jalpha chromatin structure and Jalpha recombination. TEA transcription activates promoters associated with relatively proximal Jalpha segments and stimulates histone acetylation, histone methylation and chromatin accessibility in this region. In contrast, at more distal locations, TEA transcription inhibits promoter activity through transcriptional interference and suppresses chromatin accessibility. In combination, these effects target initial Valpha-to-Jalpha recombination to TEA-proximal Jalpha segments and promote the ordered usage of the Jalpha array. The ability of TEA transcription to coordinate the activity of multiple downstream promoters maximizes the biological potential of the Jalpha array and diversifies the Tcra repertoire.

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

Enhanced Shutoff of Phototransduction in Transgenic Mice Expressing Palmitoylation-deficient Rhodopsin*♦

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.

Antimicrobial Susceptibilities and Epidemiological Analysis of Salmonella Enteritidis Isolates in Korea by Phage Typing and Pulsed-Field Gel Electrophoresis

A total of 81 isolates of Salmonella Enteritidis were analyzed by antibiotic susceptibility, phage typing, and pulsed-field gel electrophoresis (PFGE). Thirty-two isolates came from broiler carcasses and pig feces, and 49 isolates were from humans in Seoul and suburbs of Seoul, Korea. Antibiotic resistance was most prevalent among human isolates. Of human isolates, 89.8% were resistant to more than two antibiotics, while 64.7% of poultry isolates and 13.3% of pig isolates showed multiple resistance to more than two antibiotics. The most common phage type (PT) was PT1, followed by PT30 or 33, PT21 and PT20a. The isolates showed six PFGE patterns with XbaI or SpeI digestion, and five PFGE patterns with NotI digestion. But a single pattern, PFGE X1, S1, or N1, was predominant and the rest of the PFGE patterns differed by only one or two bands. Results indicated the spread of a genetically related clone of Salmonella Enteritidis in foods and humans in Korea and that phage typing as well as PFGE may offer an improved level of discrimination for the epidemiological investigation of Salmonella Enteritidis.

Pulsed field gel electrophoresis for animal Salmonella enterica serovar Typhimurium isolates in Taiwan

Salmonella enterica subspecies enterica serovar Typhimurium is a common pathogen for humans and animals. In order to trace the clonal relationship and to find the circulating strains between human and animal isolates, chromosomal DNAs from 87 serovar Typhimurium strains isolated from animals (pigs were the majority) were subjected to XbaI and SpeI digestion and pulsed field gel electrophoresis (PFGE). For the 87 animal isolates, 38 PFGE pattern combinations were obtained. As the subtyping results from animal isolates were compared with those from the 45 human isolates, it was found that 14 of the animal isolates and 13 of the human isolates shared a common PFGE pattern combination, i.e., pattern XgSf (or called X5S4). When these human and animal isolates were subjected to antibiotic susceptibility test using 11 antibiotics, it was found that strains of pattern XgSf (X5S4) belong to a common antibiogram pattern which is tetracycline, gentamicin, ampicillin, streptomycin and chloramphenicol resistant. Since most of the animal and human strains in pattern XgSf were originally isolated from various areas over different years, strains of this PFGE pattern may be the most epidemic strains which circulating between human and animal sources.

Molecular Detection and Differentiation of Erwinia pyrifoliae and Host Range Analysis of the Asian Pear Pathogen.

The recently described pathogen Erwinia pyrifoliae, isolated from Nashi pear fruit trees in Korea, resembles the fire blight pathogen Erwinia amylovora in some of its properties. The two pathogens were classified into different species by DNA hybridization kinetics and microbiological criteria. From the nucleotide sequences of the 16S rRNA and the internal transcribed spacer (ITS) region as well as extracellular polysaccharide (EPS)-encoding genes, polymerase chain reaction (PCR) primers were designed that specifically detect E. pyrifoliae but not the fire blight pathogen Erwinia amylovora, and these primers were also applied to identify E. pyrifoliae in necrotic plant material. The genomes of several strains were digested with the restriction enzyme SpeI, and the DNA fragments were analyzed by pulsed-field gel electrophoresis (PFGE). Three groups of patterns could be distinguished for the isolated E. pyrifoliae strains, all different from various E. amylovora strains, which produce a relatively homogeneous PFGE pattern after SpeI digests. Typical fire blight host plants were assayed in a growth chamber or an experimental field for their susceptibility to E. pyrifoliae. A strong preference was found for pear varieties, whereas apple, cotoneaster, hawthorn, or raspberry rarely produced necrotic symptoms. E. pyrifoliae was readily detected in samples from pear orchards in South Korea during 1995 to 1998; however, the Asian pear pathogen was not recovered in necrotic plant tissue from 1999 and 2000.

Molecular analysis of Salmonella enteritidis isolates from the Caribbean by pulsed-field gel electrophoresis.

Using pulsed-field gel electrophoresis (PFGE), between 1987 and 1996 we analyzed Salmonella enteritidis isolates from gastroenteritis cases in four Caribbean countries: Barbados, Saint Kitts and Nevis, Saint Lucia, and Trinidad and Tobago. We also determined the resistance of the isolates to 12 antimicrobial agents. Of the 129 isolates of S. enteritidis available for testing, DNA digested by XbaI revealed 13 distinctive PFGE patterns. The most prevalent XbaI PFGE patterns were group 1 (88 of 129 isolates, 68.2%) and group 2 (26 of 129, 20.2%). The patterns found among S. enteritidis isolates correlated with the geographical origin of the isolates. Of the 28 isolates from Barbados, 20 of them (71.4%) belonged to XbaI PFGE group 2, and of the 93 isolates from Trinidad and Tobago, 78 of them (83.9%) belonged to group 1. SpeI digestion of S. enteritidis genome was not as discriminatory as XbaI. Overall, of the 129 isolates, 67 of them (51.9%) exhibited resistance to one or more of the 12 antimicrobial agents that we tested. The prevalence of resistance was 53.8% for the S. enteritidis isolates tested from Trinidad and Tobago, 50.0% for those from Barbados, 28.6% for those from Saint Lucia, and 100.0% for one isolate from the island of Saint Kitts. Resistance was highest to triple sulfur (59 of 129 isolates, 45.7%), followed by furadantoin (10 of 129, 7.8%), ampicillin (7 of 129, 5.4%), and carbamycin (5 of 129, 3.9%).

Gonorrhea among men who have sex with men: outbreak caused by a single genotype of erythromycin-resistant Neisseria gonorrhoeae with a single-base pair deletion in the mtrR promoter region.

During 1995-1997, an outbreak of 66 cases of gonorrhea caused by an erythromycin-resistant (Ery(r); MIC >/=1.0 microgram/mL) prototrophic (proto) auxotype IB-1 serovar of Neisseria gonorrhoeae occurred in King County, Washington; 65 cases involved men who have sex with men (MSM), which accounted for approximately 37% of infections among MSM during this period. Isolates from 19 of these 65 cases of infection were analyzed by DNA sequencing of the polymerase chain reaction-amplified promoter region of the mtrR gene and by pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA after NheI and SpeI digestion. Eighteen of the 19 isolates had a 1-bp A/T deletion in a 13-bp inverted repeat of the mtrR promoter region and shared a single PFGE type. Among MSM who provided data about sexual behavior, 37 (64%) of 58 MSM infected by the proto/IB-1 Ery(r) strain reported having >2 sex partners during the past 60 days, compared with 32 (30%) of 106 MSM infected by other strains (P<.001). This clonal outbreak of gonorrhea illustrates the ongoing need for behavioral preventive interventions among MSM.