Restriction Enzyme Pattern Analysis Research Articles

Emerging Infectious Disease Implications of Invasive Mammalian Species: The Greater White-Toothed Shrew (Crocidura russula) Is Associated With a Novel Serovar of Pathogenic Leptospira in Ireland.

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.

Occurrence of potentially pathogenic nontuberculous mycobacteria in Mexican household potable water: a pilot study

BackgroundNontuberculous mycobacteria (NTM) are environmental opportunistic pathogens found in natural and human-engineered waters, including drinking water distribution systems and household plumbing. This pilot study examined the frequency of occurrence of NTM in household potable water samples in Mexico City. Potable water samples were collected from the “main house faucet” and kitchen faucet. The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC), fecal coliforms (FC) and NTM species were determined. Mycobacteria species were identified by PCR restriction enzyme pattern analysis (PRA) of the 65-kDa heat shock protein gene (hsp65) and sequencing of the hypervariable region 2 (V2) of the 16S rRNA gene and of the rpoB gene.ResultsAMB (<100 CFU/ml) were present in 118 out of 120 samples; only two samples were outside guidelines ranges (>100 CFU/ml). TC and FC were detected in four and one samples, respectively. NTM species were recovered from 16% samples (19/120) and included M. mucogenicum (nine), M. porcinum (three), M. avium (three), M. gordonae (one), M. cosmeticum (one), M. fortuitum (one), and Mycobacterium sp (one). All household water samples that contained NTM complied with the standards required to grade the water as “good quality” potable water.ConclusionHousehold potable water may be a potential source of NTM infection in Mexico City.

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil.

BackgroundMycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli.ResultsOf the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds.ConclusionThe microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products.

First Isolation of Mycobacterium kyorinense from Clinical Specimens in Brazil

In this article, the first isolation of Mycobacterium kyorinense specimens in Brazil is described. M. kyorinense is a recently identified species, with a few strains reported only in Japan. The Brazilian isolates were initially identified as Mycobacterium celatum by PCR restriction enzyme pattern analysis (PRA) with hsp65. However, biochemical tests indicated the same profile of M. kyorinense and distinguished them from M. celatum and Mycobacterium branderi. The sequencing of the hsp65, rpoB, and 16S rRNA genes allowed the accurate identification of isolates as M. kyorinense.

Mycobacterium noviomagense sp. nov.; clinical relevance evaluated in 17 patients

Eighteen isolates of a nonchromogenic, slowly growing, non-tuberculous species of the genus Mycobacterium were cultured from respiratory specimens obtained over the last eight years from 17 patients in the Netherlands. These isolates were grouped because they revealed a unique 16S rRNA gene sequence and were related to Mycobacterium xenopi. None of the 17 patients met the American Thoracic Society diagnostic criteria for non-tuberculous mycobacterial disease, which distinguishes the novel isolates from the related species, M. xenopi. A polyphasic taxonomic approach, including identification by biochemical and phenotypical analysis, hsp65 gene sequencing and PCR restriction enzyme pattern analysis, and sequence analyses of the rpoB gene and 16S-23S internal transcribed spacer supported the separate species status of the novel isolates. The name Mycobacterium noviomagense sp. nov. is proposed for the novel strains. The type strain is NLA000500338(T) (=DSM 45145(T)=CIP 109766(T)). A more distinctive taxonomy of NTM is a prerequisite for the assessment of their clinical relevance.

Genotype-based differentiation of theCryptococcus neoformansserotypes by combined PCR-RFLP analysis of the capsule-associated genesCAP10andCAP59

This report describes an indirect identification method for Cryptococcus neoformans serotypes developed using combined restriction enzyme pattern analysis of two PCR-amplified portions of the capsule-associated genes CAP10 and CAP59. The method relies on the recognition of the sequence conformation of nine serotype-related polymorphic sites by the analysis of four restriction profiles. A 610 nucleotides long trait of the CAP10 gene was digested with the enzymes Sty I or Sal I and a 597 nucleotides long trait of the CAP59 gene was digested with the enzymes Sal I or EcoRV+PstI. The resulting profiles, reported as a string of four numbers, defined for each strain an intrinsically coherent allelic profile closely correlated to the serotype. We analyzed by this method 172 C. neoformans strains obtained from different sources. All the serotype A strains examined and all the strains of the B-C serotypes group were recognized by specific allelic profiles, but serotypes B and C could not be distinguished from each other. Of the serotype D strains, 84% were characterized by a unique allelic pattern, while the remaining 16% were genotypically indistinguishable from the AD serotype organisms among which differences in the ploidy number and evidence of recombination could be recognized.

Restriction Enzyme Pattern Analysis of Mycobacteria DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

A new method for rapidly detecting restriction enzyme pattern of mycobacterium deoxyribonucleic acid (DNA) by capillary electrophoresis with laser induced fluorescence detection (CE-LIFD) was developed. Polymerase chain reaction was used to amplify a 439 bp fragment of 65,000 (Mr) heat shock protein gene (hsp65) of mycobacterium. After digesting the amplification products by BstE II and Hae III respectively, the patterns of enzyme cleavaged products were detected by both CE-LIFD and agarose gel electrophoresis (AGE). The experimental parameters of CE were optimized. The restriction enzyme patterns of mycobacterium DNA can be detected under the optimum electrophoresis conditions: a coated capillary column with the length of 50 cm and 100 microm i. d., electrophoresis buffer of 45 mmol/L TBE (trihydroxymethyl aminomethane (Tris)-boric acid-ethylenediaminetetraacetic acid (EDTA)) and 11 kV running voltage. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of the DNA segments were less than 3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means for DNA restriction enzyme pattern analysis.

Investigation of genetic heterogeneity in Mycobacterium tuberculosis isolates from tuberculosis patients using DNA fingerprinting

DNA fingerprinting of Mycobacterium tuberculosis (MTB) based on IS6110 has been shown to be a powerful epidemiologic tool. Restriction enzyme analysis (REA) is a fingerprinting technique, which is used for differentiation and investigation of genetic diversity among mycobacterial species. To investigating the genetic heterogeneity in MTB isolates in Ahvaz, Iran. It was a cross-sectional study conducted in Ahvaz, Iran. One hundred and eighty clinical isolates of MTB were collected from TB reference unit, PHLS, Ahvaz, Iran. The PCR-REA employed uses a simple DNA extraction followed by a PCR step involving a single primer based on the insertion sequence IS6110. Restriction enzyme analysis was performed on the amplification products using HaeIII enzyme. Data was analyzed using SPSS software and chi-square test/Fishers' exact test was applied wherever applicable. The isolates were divided into four clusters based on their REA patterns. Cluster I contained 71.1% of strains with two fragments of 72 and 118. Cluster II with three fragments of 72, 118, and 194; cluster III with three fragments of 118, 194, and 234; and cluster IV with four fragments of 72, 118, 194, and 234 base pairs. As many as 73.8% of the identical fingerprint patterns were seen in male patients. Accounting the men as the major population in the study, there was no significant difference between REA patterns and sex; similarly, with age, patients' occupation and degree of smear positivity. However, we found significant correlation between REA patterns and patients' origin. As many as 61.6% of identical patterns were found in the patients who were lived in the same suburb. By PCR-based REA typing, the isolates studied were grouped into four clusters each containing between two and four fragments. However, in order to ascertain the level of heterogeneity of MTB isolates in their sample, further testing with a more discriminatory method is needed.

Pasteurella multocida from outbreaks of avian cholera in wild and captive birds in Denmark.

An outbreak of avian cholera was observed among wild birds in a few localities in Denmark in 2001. The highest mortalities were among breeding eiders (Somateria mollissima) and gulls (Larus spp.). Pulsed-field gel electrophoresis (PFGE) was conducted using ApaI and SmaI as restriction enzymes and restriction enzyme analysis (REA) using HpaII. The Pasteurella multocida subsp. multocida strain isolated from birds in this outbreak was indistinguishable from a strain that caused outbreaks in 1996 and 2003. Most isolates from domestic poultry had other PFGE patterns but some were indistinguishable from the outbreak strain. Among 68 isolates from wild birds, only one PFGE and one REA pattern were demonstrated, whereas among 23 isolates from domestic poultry, 14 different SmaI, 12 different ApaI, and 10 different HpaII patterns were found. The results suggest that a P. multocida strain has survived during several years among wild birds in Denmark.

Antigenic and restriction enzyme analysis of Campylobacter spp associated with abortion in sheep

Abstract Objective To determine the scope of strain variations among Campylobacter spp associated with abortion in sheep. Design To examine Campylobacter spp isolated from cases of abortion for biochemical, antigenic, and genetic differences. Sample Population 15 isolates of Campylobacter spp isolated from cases of abortion during a single lambing season. Procedure Isolates were examined, using biochemical tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins (OMP), and DNA restriction enzyme analysis (REA). Results Eight strain variants were detected among the 15 isolates. 14 of the isolates were C jejuni, 13 of which were biotype I and 1 biotype II, and the remaining isolate was identified as C fetus subsp fetus. Five sodium dodecyl sulfate-polyacrylamide gel electrophoresis OMP patterns had distinctive profiles for C fetus subsp fetus and C jejuni biogroup-II isolates and 3 variants within the C jejuni bio-group-I isolates. Examination of REA patterns of DNA from the 15 isolates digested with Cfo I indicated clear differences correlating with species and biogroups and 4 REA variants among biotype-l isolates. Conclusions Marked antigenic and genetic heterogeneity of Campylobacter isolates were associated with ovine abortion within a defined geographic area. Clinical Relevance Representatives of differing OMP and REA profile groups should be considered for incorporation in vaccines to optimize protection in this region and possibly other geographic areas.

Outbreak of gentamicin-resistant Acinetobacter baumanii in an intensive care unit: clinical, epidemiological and microbiological features

The clinical, epidemiological and microbiological features of an outbreak of infection and colonisation caused by gentamicin-resistant Acinetobacter baumanii (GRAB) in an 18-bed intensive care unit (ICU) of a 680-bed adult teaching hospital are described. A retrospective review of medical, laboratory and infection control records was followed by prospective surveillance. Typing of isolates was performed by restriction enzyme analysis (REA) of chromosomal DNA. The incidence of GRAB in the ICU increased from 1.26 cases per 1000 occupied bed days (OBDs) for January to June 1993, to 6.62 per 1000 OBDs for July to December 1993 (Chi square = 4.8, P < 0.05), confirming the existence of an outbreak. For the two year period, 1993 and 1994, a total of 45 cases of GRAB infection or colonisation was identified. Males and females were equally represented, with an age range of 16-79 years and a mean age of 51 years. Admitting diagnoses varied, with multiple trauma and head injury predominating (ten cases). For 35 of the 45 cases the initial site of GRAB isolation was sputum or other respiratory tract specimen. Specific treatment for GRAB was initiated in 23 patients, however no deaths were directly attributable to GRAB infection. The period of time between admission to the ICU and first isolation of GRAB ranged from three to 70 days with a median of nine days. Overall, ten (11%) of 91 staff hand samples and one of 37 (3%) environmental samples yielded GRAB. All GRAB isolates produced similar biochemical profiles and antibiotic resistance patterns, except for a group of five which were ciprofloxacin resistant. Thirty patient isolates, all ten staff hand isolates and the environmental isolate produced identical REA patterns. The remaining five patient isolates (all ciprofloxacin resistant) which were available for typing produced a different REA pattern. Our study has documented a moderate-sized outbreak of GRAB in an ICU setting. Typing of isolates using REA was useful in delineating outbreak strains. Carriage of GRAB on staff hands was demonstrated as the most likely source of infection. Despite institution of infection control measures GRAB now appears endemic in the ICU.

Expression of the MAGE gene family in human head‐and‐neck squamous‐cell carcinomas

The MAGE genes encode certain tumor-associated antigens recognized by cytotoxic T lymphocytes. We investigated the expression of the MAGE-1, -2, -3, -4, -41, and -6 genes in 88 head-and-neck squamous-cell carcinomas (83 fresh tumor samples and 5 cell lines), using a reverse-transcription-polymerase-chain-reaction assay, followed by dot-blot hybridization with sequence-specific oligonucleotides and/or restriction enzyme-pattern analysis. The MAGE-1, -2, -3, -4, -41 and -6 genes were expressed at the mRNA level in 27, 34, 36, 22, 16 and 35, respectively, of 83 fresh tumor samples. At least one of these genes was expressed in 59 of the 83 samples. Neither non-tumor inflammatory cells nor normal tissues were positive for these genes. The MAGE-1 gene was expressed relatively frequently in SCC of the oropharynx, hypopharynx and maxillary sinus, but at lower rates in SCC of the larynx and of the tongue and oral cavity. MAGE-1 was frequently expressed in poorly differentiated SCC, somewhat less frequently in moderately differentiated SCC, and only infrequently in well-differentiated SCC. The expression levels of the other MAGE genes also varied with the anatomic site as well as the degree of differentiation. Our results suggest that specific immunotherapy against MAGE gene products may be useful for patients with head-and-neck carcinomas.

SystemicPasteurella HaemolyticaInfection as a Rare Sequel to Avirulent LivePasteurella HaemolyticaVaccination in Cattle

Eleven cases of systemic Pasteurella haemolytica infection in cattle were identified from routine diagnostic laboratory submissions during the falls of 1988, 1989, and 1991. All cases came with a history of recent vaccination with an avirulent live culture P. haemolytica product. Nine of 11 cases involved cattle vaccinated between 2 and 18 days previously with this product. Ten of 11 cases involved 182-227-kg beef calves that were vaccinated between September and November during routine processing for entry into feedlots. The morbidity and mortality was generally low. The major pathologic findings included meningitis, injection site abscessation and/or cellulitis, and polyarthritis. Systemic infection was indicated in all cases by the isolation of P. haemolytica from 2 or more organs or distinct anatomical sites. In 6 cases, the vaccine injection site was cultured, and in all 6 cases, P. haemolytica was isolated. Three separate P. haemolytica isolates from 2 cases were further studied by restriction enzyme analysis (REA). These isolates were from tissues with suppurative inflammation, including the brain, joint, and injection site. The REA patterns of each of these 3 isolates were identical to the REA pattern of the vaccine masterseed, which strongly suggested that the organisms causing systemic infection were the same as the organism used to produce the vaccine. Because the overall incidence was quite low, other factors, such as stress, probably played a major role in the expression of this syndrome.

Use of DNA analysis of Pasteurella haemolytica biotype T isolates to monitor transmission in bighorn sheep (Ovis canadensis canadensis)

Pneumonia has been identified as a major cause of poor lamb survival in indigenous herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in central Idaho. Pasteurella haemolytica was isolated from five adult Rocky Mountain bighorn ewes captured from a free-ranging herd in central Idaho. The lambs from two of these ewes delivered by cesarean section were free of P. haemolytica until 40 days of age and after repeated contact with their dams. The lambs subsequently developed signs of pneumonia, and P. haemolytica was isolated from nasal, pharyngeal, and transtracheal wash samples from each lamb. All P. haemolytica biotype T isolates from the ewes and lambs, as well as those from a 9-month-old lamb of the same herd from which samples for culture were obtained 2 years earlier, were subjected to HaeIII restriction enzyme analysis (REA) and ribotyping. Two ribotypes and seven REA patterns were visually distinguishable by these procedures. Similarity coefficients (SAB) of 0.09 to 0.95 were calculated for the seven REA patterns. The REA patterns of the isolates from the lambs were identical (SAB = 1.0). The isolates from the lambs also had SAB values of 1.0, which was indicative of identity with one of the seven isolates cultured from the ewes at the time of capture and with the organism isolated from the 9-month-old lamb. These procedures have the discriminatory capabilities necessary to monitor the transmission of specific strains of bacteria within and between animal populations.

Characterization of the Virulence Regions in the Plasmids of Three Live Salmonella Vaccines

Three live Salmonella vaccines, Zoosaloral "Dessau", Bovisaloral "Dessau", and Suisaloral "Dessau" successfully used in veterinary medicine in eastern Germany were analysed for their plasmid DNA content. Plasmids of 60 MDa (S. typhimurium), 55 MDa and 26 MDa (S. dublin) and 34 MDa (S. choleraesuis) were found. The three large plasmids contained a virulence region as shown by restriction enzyme analysis and hybridization studies with virulence-region-specific DNA probes. Restriction enzyme pattern analysis of the whole plasmids revealed differences between vaccine strains and parental strains. We wanted to know if there were alterations within the virulence region of the vaccine strain plasmids compared with parental strains caused by the attenuation procedure. Therefore, the 6 kb-Cla I fragments of several plasmids were cloned and characterized by restriction enzyme fingerprinting. The expression of the Cla I fragment-encoded proteins was analysed in the minicell-producing strain DS 410. The experiments revealed that the restriction pattern of the 6 kb-Cla I fragments of Zoosaloral and Bovisaloral as well as the PAGE pattern of virulence-region-encoded proteins were in complete accordance with one another, between vaccine strains and the parental strain of Zoosaloral, and with a wild-type strain of S. typhimurium.