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PubMed2006

Clonal composition of Staphylococcus aureus isolates at a Brazilian university hospital: identification of international circulating lineages.

In only a few instances has the clonal composition of Staphylococcus aureus collections that include methicillin-susceptible S. aureus (MSSA) been extensively characterized. In order to investigate the clonal composition of MSSA and methicillin-resistant S. aureus (MRSA) and examine whether the infections diagnosed at our hospital were related to internationally distributed S. aureus lineages, we collected 89 clinical S. aureus isolates from patients at a public university hospital in Rio de Janeiro, Brazil, from September 1999 to June 2000. All S. aureus isolates were genotyped by pulsed-field gel electrophoresis and multilocus restriction fragment typing (MLRFT), and a subset (n = 17) was further characterized by multilocus sequence typing (MLST). The 34 MRSA isolates were additionally characterized by SCCmec typing. The MSSA population (n = 55) was grouped into 18 restriction fragment types (RFTs); of these, five RFTs accounted for 67% (37) of the MSSA isolates. MRSA isolates were clustered into only three RFTs (P = 0.02). The majority of MSSA RFTs were related to sequence type 30 (ST30) (12 isolates, 22%), ST1, ST188, and ST432 (6 isolates, 11% each). The predominant MRSA RFT comprised 31 (91%) of 34 isolates; four randomly selected isolates of this RFT were ST239, the previously described widely disseminated Brazilian clone. However, a fifth isolate belonging to this RFT was the ST644, a new single locus variant of ST239. By applying MLRFT and MLST, we found evidence for a clonal structure in MSSA isolates and detected the dissemination of MSSA clonal complexes 1, 5, 8, 30, and 45.

Adolescent↗
PubMed2006

Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing.

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogen Campylobacter jejuni. SNPs were identified in silico using the program 'Minimum SNPs', which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-D SNPs identified in this study were derived from the combined C. jejuni/Campylobacter coli multilocus sequence typing (MLST) database. Seven SNPs were found that provided a D of 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-D SNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69 C. jejuni isolates were subjected to MLST and flagellin A short variable region (flaA SVR) sequencing and combined with a population of 84 C. jejuni and C. coli isolates previously characterized by these methods. Within this collection of 153 isolates, 19 flaA SVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST and flaA SVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided a D of 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of the flaA SVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaA SVR. This investigation has shown that a seven-member C. jejuni SNP typing assay, used in combination with sequencing of the flaA SVR, efficiently discriminates C. jejuni isolates.

Alleles↗
PubMed2026

Whole-Genome Analysis Reveals Antimicrobial Resistance and Population Structure of Environmental and Veterinary Acinetobacter baumannii.

Acinetobacter (A.) baumannii is an important multidrug-resistant pathogen increasingly recognized across animal and environmental settings, and carbapenem-resistant A. baumannii (CRAB) is classified as a critical-priority pathogen by the World Health Organization. This study investigated the antimicrobial resistance (AMR) and genomic characteristics of 122 A. baumannii isolates comprising 72 veterinary and 50 environmental isolates collected in Andhra Pradesh, India. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), resistance and virulence gene profiling, multilocus sequence typing (MLST), core-genome analysis, single nucleotide polymorphism (SNP) phylogeny, and pan-genome analysis were performed. Overall, 58.2% of isolates were multidrug-resistant (MDR), and 41.8% were extensively drug-resistant (XDR). Sequence type (ST) 52 predominated among veterinary isolates, whereas ST2 was more frequent among environmental isolates. The presence of carbapenem-resistant isolates along with the ST2 lineage enhances the similarity to clinical A. baumannii. Several intrinsic resistance genes, including blaOXA-23, armA, aph(3″)-Ib, aph(6)-Id, tet(B), mph(E), and msr(E), were more prevalent in the ST2-associated population. Virulence-associated determinants were widely conserved. Core-genome MLST (cgMLST) and core-genome SNP (cgSNP) analyses identified highly related isolates within both lineages, while pairwise SNP differences were 0-7. Pan-genome analysis identified 4204 gene clusters and distinct accessory gene patterns between ST2 and ST52. These findings indicate that resistance gene distribution was closely associated with lineage structure and support integrated genomic surveillance of A. baumannii across animal and environmental reservoirs.

Acinetobacter baumannii↗
PubMed2002

Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates.

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

Alleles↗
PubMed2003

PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks.

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.

Amebiasis↗
PubMed2005

mef(A), mef(E) and a new mef allele in macrolide-resistant Streptococcus spp. isolates from Norway.

OBJECTIVES: To type mef genes in a nationwide collection of clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes as well as pharyngeal carrier strains of viridans streptococci in Norway. METHODS: Erythromycin-resistant mef-positive multilocus sequence-typed (MLST) clinical isolates of S. pneumoniae (n = 36) and S. pyogenes (n = 12) from the National Surveillance Program for Antimicrobial Resistance (NORM) as well as viridans streptococci (n = 20) from healthy adults were included. PCR-amplified mef genes were initially discriminated by BamHI digestion. Selected mef genes from representatives of different sequence types (STs) of S. pneumoniae (n = 11) and S. pyogenes (n = 4), and viridans group streptococcal species (n = 8) were typed by sequencing and their strains examined for co-resistances. Hydropathy plots of different mef-encoded proteins were performed. RESULTS: A predominance of mef(A) was detected in S. pneumoniae (23/36) and S. pyogenes (9/12) due to the clonal spread of ST9 and ST39, respectively. mef(E) was the most widely distributed mef determinant occurring in nine different STs of S. pneumoniae and in four different viridans species. A new mef allele was identified in two STs of S. pyogenes. CONCLUSIONS: mef(E) is the most widely distributed mef determinant in Norwegian clinical strains of S. pneumoniae and pharyngeal carrier strains of various viridans streptococci. However, mef(A) is more prevalent in S. pneumoniae and S. pyogenes due to clonal spread. A new mef allele was found in two different STs of S. pyogenes.

Amino Acid Sequence↗
PubMed1990

Perinatal infection of human T-lymphotropic virus type I, the etiologic virus of adult T-cell leukemia/lymphoma. DNA amplification of specific human T-lymphotropic virus type I sequences.

A gene amplification technique, polymerase chain reaction, was used to detect human T-lymphotropic virus type I (HTLV-I), the etiologic agent of adult T-cell leukemia/lymphoma, in mononuclear cells in peripheral blood and breast milk of ten HTLV-I carrier gravida. The DNA in umbilical cord blood mononuclear cells of the neonates born to the HTLV-I carrier gravida were also amplified and examined for the possibility of HTLV-I infection via placenta during pregnancy. The HTLV-I sequences were detected both in the peripheral blood and milk of all ten carrier gravida by Southern blot analysis of amplified DNA. However, HTLV-I proviral DNA could not be detected in the cord blood of the carriers' neonates, indicating that transplacental infection of HTLV-I should be rare and that postpartum infection via breast milk is a likely major perinatal transmission route.

Antibodies, Neoplasm↗
PubMed2003

Polymorphism of a specific region in gene p23 of Citrus tristeza virus allows discrimination between mild and severe isolates.

The pathogenicity determinants of Citrus tristeza virus (CTV) are presently unknown, although transgenic Mexican limes over-expressing CTV p23, an RNA-binding protein involved in regulating the asymmetrical accumulation of viral RNA strands, display typical CTV symptoms. Here we compared the predominant sequence variants of gene p23 from 18 CTV isolates of different geographic origin and pathogenicity characteristics. Phylogenetic analysis of these sequences revealed three groups of isolates: i) mild, inducing only symptoms in lime and/or decline of citrus species grafted on sour orange rootstock, ii) severe, causing additionally stem pitting on sweet orange and/or grapefruit, and iii) an atypical group of isolates inciting variable symptoms. The sequences of the isolates located at the periphery of each group were recombinants. Pairwise comparisons of the predicted amino acid sequences showed that residues at positions 78-80 were characteristic of each group of isolates. Group-specific primers based on these differences allowed RT-PCR detection of each sequence type in dsRNA-rich preparations from infected tissues. While mild isolates contained only the sequence characteristic of this group, most severe isolates contained the sequences characteristic of their group, and additionally, sequences characteristic of the mild and/or the atypical groups, suggesting that the severe phenotype is associated with the presence of the severe and/or the atypical sequence types. This association can be exploited for quick detection of potentially damaging sequence variants and for monitoring cross protection.

Amino Acid Sequence↗
PubMed2005

Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill.

A 16S rDNA-based molecular study was performed to determine the nature of the bacterial constituents of the leachate from a closed municipal solid waste landfill. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified and cloned. Recombinant rDNA clones in the library were randomly selected, and they were sequenced for a single run and then grouped. A total of 76 sequence types representing 138 randomly selected nonchimeric clones were identified. Full-length sequencing and phylogenetic analysis of the sequence types revealed that more than 90% of the screened clones were affiliated with low-G+C gram-positive bacteria (38.4%), Proteobacteria (35.5%), the Cytophaga Flexibacter Bacteroides group (11.6%), and Spirochaetes (5.1%). Minor portions were affiliated with Verrucomicrobia (2.9%), candidate division OP11 (2.2%), and the green nonsulfur bacteria, Cyanobacteria and the Deinococcus Thermus group (each <1.0%). Although some rDNA sequences clustered with genera or taxa that were classically identified within anaerobic treatment systems and expected with known functions, a substantial fraction of the clone sequences showed relatively low levels of similarity with any other reported rDNA sequences and thus were derived from unknown taxa. These results suggest that bacterial communities in landfill environment are far more complex than previously expected and remain largely unexplored.

Bacteria↗
PubMed2004

Identification of a variant "Rome clone" of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit.

We describe the identification of a variant of the "Rome clone" of methicillin-resistant Staphylococcus aureus (MRSA), responsible for an outbreak involving 5 patients in a Cardiac Surgery Intensive Care Unit (CS-ICU) of a tertiary-care University Hospital in Rome. All strains isolated from patients and from nasal swabs obtained from four members of the CS-ICU personnel, belonged to the same identified clone. The characteristics of this clone were: (1) resistance to ampicillin, oxacillin, gentamicin, ciprofloxacin, erythromycin, clindamycin, rifampin, spectinomycin, and tetracycline; (2) vancomycin and teicoplanin MICs respectively of 2 and 4 mg/L; (3) heteroresistant subpopulations in the presence of 4 and 6 mg/L of vancomycin (10(-3) and 10(-5), respectively); (4) clonal type I::J::C determined following an established protocol (mec A::Tn 554 ::PFGE); (5) sequence type ST247 (3-3-1-12-4-4-16), obtained by multilocus sequence typing (MLST); and (6) the staphylococcal cassette chromosome mec (SCC) IA, obtained by multiplex PCR method. This new strain had different characteristics from the epidemic clone circulating in the same hospital from 1997 and designed "Rome clone," which was susceptible to erythromycin, clindamycin, and spectinomycin and belonged to the II::NH::C genetic background. A high genetic similarity between this Rome clone and the previously classified Archaic and Iberian clones was found, because they shared the same allelic profile (ST247), probably originating from the same S. aureus ancestor of the Iberian MRSA strains. Therefore, the strains responsible for the outbreak, with vancomycin MICs 2-4 mg/L, are variant clones, showing the genotype of the "Rome clone," the ST247 in association with SCC mec type IA (ST247-MRSA-IA), and are characterized by a uniform susceptibility to fosfomycin.

Anti-Bacterial Agents↗
PubMed2000

Carried meningococci in the Czech Republic: a diverse recombining population.

Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.

Adolescent↗
PubMed1994

Use of DNA moderately repetitive sequence to type Aspergillus fumigatus isolates from aspergilloma patients.

Polymorphism of forty-seven sequential clinical isolates from 3 patients with an aspergilloma was analyzed. DNA from each isolate was digested with EcoRI and hybridized in Southern blots with a 32P-labeled nonribosomal DNA repetitive sequence. Most isolates from each patient displayed the same hybridization pattern. Southern blot patterns obtained with DNA repetitive sequences can be used to type clinical isolates of Aspergillus fumigatus and have shown that aspergilloma patients are most probably infected by a single strain.

Aspergillosis↗
PubMed1993

Extensive N nucleotide addition in junctional region of T cell receptor V gamma 5 genes rearranged in fetal liver-derived thymocytes in radiation chimera mice.

The V gamma 5 chain of T cell receptor gamma delta is preferentially expressed by murine fetal thymocytes. The fetal V gamma 5 gene is known to be homogeneous and lacks N nucleotide addition. We previously reported that V gamma 5+ cells were detected among donor-derived thymocytes from irradiated C3H/He mice soon after reconstitution with AKR/J fetal liver (FL) cells, but that only a few V gamma 5+ were detected among donor-derived thymocytes from irradiated C3H/He mice reconstituted with adult bone marrow (ABM) cells. The results suggest that preferential use of V gamma 5 is determined at the level of T cell precursor generation. In the present report, we analyzed whether the junctional region of FL-derived V gamma 5 genes in the FL chimeras is of fetal type. Sequencing analysis showed that V gamma 5 genes from FL chimeras contained extensive N nucleotide addition and were diverse both in nucleotide sequences and deduced amino acid sequences. V gamma 5 genes from donor-derived thymocytes of ABM chimeras, which by polymerase chain reaction were revealed to be less frequent than those of FL chimeras, also showed extensive N addition. Furthermore, the terminal deoxynucleotidyl transferase (TdT) gene, which encodes an enzyme that adds N nucleotides into the junctional region, was expressed at high level in the donor-derived thymocytes of FL chimeras and normal 8-week-old AKR/J thymocytes but at low level in day 17 fetal AKR/J thymocytes. Our results suggest that the preferential rearrangement of the V gamma 5 gene is determined at the level of T cell precursor generation but the homogeneous V gamma 5 sequence of fetal type is generated only in the fetal thymic microenvironment where TdT gene expression is low or absent. The nomenclature of murine TcR gamma chains is according to Reilly et al. (Nature 1986. 321: 878). The relationship between the different nomenclature systems is summarized in Takagaki et al. (J. Immunol. 1989. 141: 2112).

Animals↗