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PubMed2006

Epidemiological typing of MRSA isolates from blood cultures taken in Irish hospitals participating in the European Antimicrobial Resistance Surveillance System (1999-2003).

Between 1999 and 2003, meticillin-resistant Staphylococcus aureus (MRSA) isolates recovered from blood cultures in Irish hospitals that participate in the European Antimicrobial Resistance Surveillance System were investigated by epidemiological typing using antibiogram-resistogram (AR) typing, biotyping, and DNA macrorestriction digestion using SmaI followed by pulsed-field gel electrophoresis (PFGE). PFGE patterns were assigned five-digit pulsed-field type (PFT) numbers, and PFTs of apparently related patterns were abbreviated to two-digit PFT groups (PFGs). AR and PFGE typing results were combined to produce AR-PFG types. Representative isolates of each AR-PFG type recovered in 2002 were typed by multilocus sequence typing and staphylococcal cassette chromosome (SCC) mec analysis. Isolates from 1999 and 2000 were also typed by phage typing. The extent to which epidemiological types of MRSA from blood cultures could be extrapolated to the total MRSA population was investigated by comparing results obtained with isolates from the total MRSA population versus those obtained with blood cultures during three study periods. Over the 5 years from 1999 to 2003, 1,580 blood culture isolates from 1,495 patients were analysed. Typeability and discriminatory indices were as follows: AR typing, 1 and 0.97; phage typing, 0.29 and 0.89; PFGE, 0.99 and 0.95; AR-PFG typing, 1 and 0.95. The most frequently occurring AR-PFG types were 06-01, 07-02, 13-00, and 14-00 and were exhibited by 57, 7, 14, and 12% of isolates, respectively. During the study period, the distribution of AR-PFG type changed markedly, with the prevalence of one type (AR-PFG 06-01) increasing by 880%, from 22% (39/181) in 1999 to 80% (343/430) in 2003. Investigation of whether epidemiological types among blood culture isolates of MRSA were representative of the total MRSA population showed that there was no significant difference in most instances. MLST and SCCmec typing showed that AR-PFG types 06-01, 07-02, 13-00, and 14-00 were ST22-MRSA-IV, ST36-MRSA-II, ST8-MRSA-IID, and ST8-MRSA-IIE, respectively.

Animals↗
PubMed1999

Detection of mutations by automated fluorescence/RNA-based dideoxy fingerprinting (ARddF)

Dideoxy fingerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing to detect the presence of single base changes in a defined fragment of nucleic acid. ddF is no more technically demanding than SSCP, yet it is more sensitive in detecting point mutations. We describe here the adaptation of conventional ddF to an automated sequencing system using fluorescent Cy5 labeled primers. We show that automated RNA-based ddF (ARddF) has several advantages over conventional radioisotope-based ddF, including: (1) analysis of larger nucleic acid fragments (up to 10(3) bp), due to the ability to continuously analyse and compile sequencing information; (2) greater reliability for distinguishing mutant sequences from wild type sequences (particularly when the mutation leads to gain or loss of a dideoxy termination segment); (3) the use of fluorescent labeled primers, making ARddF less hazardous than methods requiring radionucleotides. The use of ARddF in conjunction with new methods for isolating RNA from a [corrected] small number of cells facilitates mutational analysis of small tissue biopsies and other limited samples, and will allow more widespread application of mutational screening in the setting of clinical diagnostic laboratories.

Animals↗
PubMed2005

Pneumonia outbreak associated with group a Streptococcus species at a military training facility.

BACKGROUND: Although group A streptococci (GAS) infections are a major cause of morbidity and mortality, outbreaks of associated pneumonia are rare. We report an outbreak of GAS pneumonia that occurred at a US military training camp. METHODS: Standard epidemiologic and laboratory procedures were used to characterize the outbreak and causative organism(s). A case-control study and determination of the prevalence of GAS infection among camp personnel were also performed. RESULTS: A total of 162 of 4500 Marine Corps personnel were hospitalized for respiratory symptoms during the period of 1 November and 20 December 2002, and 127 (78%) had radiographically confirmed pneumonia. The attack rate was 1.6 cases per 100 person-months. Thirty-four (27%) of 127 patients with pneumonitis had definite or probable GAS pneumonia; an additional 22 (17.3%) were coinfected with GAS and another pathogen. Pathogens, in addition to GAS, included Chlamydia pneumoniae (27 patients), Mycoplasma pneumoniae (19), adenovirus (5), and Streptococcus pneumoniae (2). A survey revealed that the pharyngeal carriage rate of GAS among camp personnel was 16%. Molecular characterization of the GAS isolates found emm type 3, multilocus sequence type 15. The epidemic ended after administration of additional prophylaxis with a single dose of intramuscular benzathine penicillin (1.2 million U) or azithromycin (1 g orally). Because the number of days from the last penicillin injection was correlated with a positive throat culture result and the occurrence of pneumonia, the dosing interval of benzathine penicillin was shortened from every 28-35 days to every 21 days. CONCLUSIONS: This is the largest outbreak of GAS pneumonia reported in >30 years. This outbreak emphasizes the potential for GAS to cause epidemics of severe infection and demonstrates the need for surveillance and consideration of appropriate antibiotic prophylaxis among particularly high-risk populations.

Adolescent↗
PubMed2006

Epidemiological and molecular characteristics of a highly lethal pneumococcal meningitis epidemic in Burkina Faso.

BACKGROUND: Public health and clinical strategies for meningitis epidemics in sub-Saharan Africa usually assume that Neisseria meningitidis infection causes most disease. METHODS: During 24 months from 2002 to 2005, we collected clinical and laboratory information for suspected acute bacterial meningitis cases from 3 districts in Burkina Faso. Streptococcus pneumoniae was identified by culture, polymerase chain reaction, or antigen detection in cerebrospinal fluid. Pneumococcal genotyping was performed on strains using multilocus variable-number tandem repeat typing and multilocus sequence typing. RESULTS: Samples of cerebrospinal fluid were collected from 1686 persons; 249 (15%) had S. pneumoniae identified (annual incidence, 14 cases per 100,000 persons). Of these patients, 115 (46%) died, making S. pneumoniae the most commonly identified organism and responsible for two-thirds of deaths due to bacterial meningitis. During the meningitis epidemic season, an average of 38 cases of S. pneumoniae infection were identified each month, compared with an average of 8.7 cases during other months. Of 48 pneumococci that were tested, 21 (44%) were identified as serotype 1, and the remaining 27 (56%) were identified as 15 different serogroups and/or serotypes. Both serotype 1 and other serogroups and/or serotypes were seasonal. The genotypes of serotype 1 isolates were closely related but diversified over the study period and were similar to, but not identical to, the predominant genotypes found previously in Ghana. CONCLUSIONS: Intervention strategies during the epidemic season in Burkina Faso (and perhaps elsewhere) must now account for pneumococcal meningitis occurring in an epidemic pattern similar to meningococcal meningitis. Although a serotype 1 clone was commonly isolated, over half of the cases were caused by other serogroups and/or serotypes, and genetic diversification increased over a relatively short period.

Adolescent↗
PubMed2000

Molecular epidemiology of methicillin-resistant Staphylococcus aureus strains: state of affairs and tomorrow' s possibilities.

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have posed a clinical threat for nearly 40 years. During these years, an array of additional technologies suited for identification of MRSA below the species level has become available. The technologies, whether they assess phenotype or genotype, provide data that can be used for elucidation of the routes of dissemination of individual MRSA types. This review summarizes the current state of affairs with respect to the quality of the various laboratory techniques and includes descriptions of novel strategies such as binary typing and multilocus sequence typing (MLST). Drawbacks of procedures will be compared, and the value of molecular typing in the elucidation of complex biological phenomena, such as epidemicity, carriage, and reduced vancomycin susceptibility, will be indicated. Means for integrated assessment of bacterial biology, epidemiology, and population structure will be discussed.

Bacterial Typing Techniques↗
PubMed2005

Molecular characterization of methicillin-resistant Staphylococcus aureus bloodstream isolates from Croatia.

OBJECTIVES: The objectives of this study were (i) to investigate the genetic background of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from Croatia and (ii) to monitor the prevalence of Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) among these isolates. METHODS: Eighty-two hospital-acquired MRSA bloodstream isolates, collected in 2001 and 2002 in Croatia, were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). The presence of genes encoding PVL and TSST-1 was investigated by real-time PCR. RESULTS: All strains were multiresistant and were distributed among 16 different similarity groups as determined by PFGE. Two of the groups, groups H and K, harboured the majority of the MRSA strains with 52 and 12%, respectively. The predominant SCCmec type found among the isolates was type I (89%). Eleven per cent of the strains harboured a modified SCCmec type III, which contained, in contrast to the regular type III, an additional dcs region. One strain harboured a novel SCCmec type, containing the ccrC gene in combination with the mecI gene, the dcs region, the locus between pI258 and Tn554 (locus E) and the locus between Tn554 and orfX (locus F). MLST showed the presence of ST111-MRSA-I and ST247-MRSA-I among Croatian MRSA isolates. All isolates were negative for both PVL and TSST-1. CONCLUSIONS: These results indicate the emergence of ST111-MRSA-I and ST247-MRSA-I in Croatia among MRSA bloodstream isolates. The virulence factors PVL and TSST-1 were not present among these isolates.

Bacterial Toxins↗
PubMed2006

Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group.

The phylogenetically closely related species Streptococcus salivarius and Streptococcus vestibularis are oral bacteria that are considered commensals, although they can also be found in human infections. The relationship between these two species and the relationship between strains isolated from carriers and strains responsible for invasive infections were investigated by multilocus sequence typing and additional sequence analysis. The clustering of several S. vestibularis alleles and the extent of genomic divergence at certain loci support the conclusion that S. salivarius and S. vestibularis are separate species. The level of sequence diversity in S. salivarius alleles is generally high, whereas that in S. vestibularis alleles is low at certain loci, indicating that the latter species might have evolved recently. Cluster analysis indicated that there has been genetic exchange between S. salivarius and S. vestibularis at three of the nine loci investigated. Horizontal gene transfer between streptococci belonging to the S. salivarius group and other oral streptococci was also detected at several loci. A high level of recombination in S. salivarius was revealed by allele index association and split decomposition sequence analyses. Commensal and infection-associated S. salivarius strains could not be distinguished by cluster analysis, suggesting that the pathogen isolates are opportunistic. Taken together, our results indicate that there is a high level of gene exchange that contributes to the evolution of two streptococcal species from the human oral cavity.

Base Sequence↗
PubMed2002

Introduction of new clones of penicillin-nonsusceptible Streptococcus pneumoniae in Hong Kong.

Analysis of penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) isolates in Hong Kong by use of a combination of antibiogram typing, serotyping, multilocus sequence typing, and pulsed-field gel electrophoresis indicated that the dissemination of PNSP was the result of the spread of international clones: variants of the Spain(23F)-1 or Spain(6B)-2 clones were the predominant PNSP isolates from 1994 to 1997 and remained so, but Taiwan(19F)-14 and Taiwan serotype 6B clones were disseminated in Hong Kong in 1999 and 2000. Concomitant changes in antibiotic susceptibility profiles, with the rate of susceptibility to chloramphenicol rising from 10% in the period from 1994 to 1997 to 31% (P < 0.001) in 1999 and 2000, were noted to accompany the shift of clones.

Anti-Bacterial Agents↗
PubMed2003

Evolution of a vancomycin-intermediate Staphylococcus aureus strain in vivo: multiple changes in the antibiotic resistance phenotypes of a single lineage of methicillin-resistant S. aureus under the impact of antibiotics administered for chemotherapy.

A number of methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent S. aureus bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 micro g/ml for the initial isolate to an MIC of 8 micro g/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates-apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern, spaA type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.

Anti-Bacterial Agents↗
PubMed2004

Widespread skin and soft-tissue infections due to two methicillin-resistant Staphylococcus aureus strains harboring the genes for Panton-Valentine leucocidin.

Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging as a major public health problem. CA-MRSA has been associated previously with skin and soft-tissue infection (SSTI) and with carriage of staphylococcal cassette chromosome mec (SCCmec) type IV and the Panton-Valentine leucocidin (PVL) virulence factor. To assess the clonal distribution of PVL-carrying strains and the association with SSTI in the San Francisco Bay area, we surveyed six collections of S. aureus isolates-671 isolates in all-collected between 1997 and 2002 originating from inpatient and outpatient clinical specimens and from a community-based sampling. Isolates were genotyped by pulsed-field gel electrophoresis, multilocus restriction fragment typing, and multilocus sequence typing and assayed for the PVL virulence factor. The S. aureus populations showed a high proportion of PVL-carrying strains, with frequencies ranging up to 70% in MRSA isolated from jail inmate patients and 69% in MRSA from patients receiving surgical treatment at an outpatient clinic specializing in treating SSTIs. PVL-carrying isolates were identified in nine clonal groups, but 88.5% of the PVL-carrying MRSA isolates belonged to only two clonal groups. These two clonal groups carried the SCCmec type IV resistance determinant and were more likely than other clonal groups to be recovered from SSTI sites than from other sites (P < 0.0001). There is evidence of clonal replacement over the period from 1999 to 2002, with one of these two clonal groups being supplanted by the other.

Bacterial Toxins↗
PubMed2005

High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy.

Mutations in the kinase domain (KD) of BCR-ABL are the leading cause of acquired imatinib resistance. In some cases, identical mutations were detected at relapse and in pretherapeutic specimens, consistent with selection of resistant clones in the presence of drug. However, the incidence of KD mutations in imatinibnaive patients, irrespective of response to therapy, is unknown. We studied mutation frequency in 66 patients with chronic myelogenous leukemia (CML), using cDNA sequencing and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) assays for 8 common mutations. Thirteen patients were positive by ASO-PCR only, 1 by ASO-PCR and sequencing, and 1 by sequencing only (overall frequency, 22.7%). T315I was most frequent (12% of patients). Eleven of the 14 patients with positive ASO-PCR had follow-up samples available for sequencing. Wild-type sequence was detected in 6 of 11, 2 different mutations in 1 of 11, and identical mutations in 4 of 11 patients, 2 of whom had achieved major cytogenetic response. In multivariate analysis mutation detection was associated with clonal cytogenetic evolution, exposure to 6-Thioguanine, and a low platelet count, but not with response to imatinib, event-free survival, and overall survival. KD mutants present at low levels do not invariably lead to relapse, and additional factors are required to induce a fully drug-resistant phenotype.

Adult↗
PubMed2004

[Molecular methods as a diagnostic tool for fungal infections and their prospect].

The current status of molecular biological methods to control mycoses was described. New tools for detecting of specific fungal DNA by reaction at a constant temperature with Loop-mediated isothermal amplification of DNA (LAMP, Eiken Chemical Co., Ltd.), and for strain typing by multilocus sequence typing (MLST) will greatly aid in managing complex fungal infection.

Humans↗
PubMed1999

Divergence of virulence factors of Helicobacter pylori among clinical isolates does not correlate with disease specificity.

The aim of this study was to examine the relation between disease specificity and the virulence factors of Helicobacter pylori isolated from patients with gastric cancer (GC), duodenal ulcer (DU), and gastritis (GS). Altogether 18 isolates obtained from patients with GC, 28 isolates from DU patients, and 13 isolates from GS patients were analyzed. All isolates were tested for the presence of the cagA gene, and genotyping of the vacA gene was done by the polymerase chain reaction. Production of VacA protein and expression of vacuolating cytotoxic activity in the H. pylori culture supernatant were examined. The serum antibody titers against purified VacA and CagA proteins were determined by enzyme-linked immunosorbent assay (ELISA). Interleukin-8 (IL-8) production by AGS cells in response to H. pylori isolates was measured by an hIL-8 ELISA kit. Genetic analysis of vacA revealed that most of the clinical isolates were classified into the S1a type by signal sequence typing. There were no differences in cagA detection rates, vacuolating cytotoxin activity, or mean antibody titers against VacA and CagA protein among the three groups. The mean IL-8 concentrations in the supernatants of AGS cells were similar in the three groups. In this study, there was no difference in virulence factors of H. pylori among isolates from GC, DU, and GS.

Antigens, Bacterial↗
PubMed2003

Short report: Echinococcus granulosus from Xinjiang, PR China: cDNAS encoding the EG95 vaccine antigen are expressed in different life cycle stages and are conserved in the oncosphere.

The EG95-based vaccine protects sheep from infection with the dog tapeworm Echinococcus granulosus. The EG95 encoding gene is a member of a multigene family, several members of which are expressed in the oncosphere, believed to be the target of immunity induced by the vaccine. E. granulosus exhibits extensive intraspecific (strain) variation, and variability of the eg95 gene in different isolates of E. granulosus may directly impact the effectiveness of the EG95-based vaccine. We analyzed the eg95 gene from E. granulosus collected in Xinjiang, in northwest China, where hydatid disease is hyperendemic. The gene is expressed in oncospheres, protoscoleces, and immature and mature adult worms, and the eg95 gene family was shown to comprise two basic sequence types. Very limited sequence variation was evident in the EG95 protein from oncospheres. This high degree of sequence conservation predicts that the vaccine will continue to be effective in China and elsewhere.

Amino Acid Sequence↗
PubMed2004

[Pharyngeal carriage of Neisseria meningitidis in a school of Niamey, Niger].

This study of pharyngeal carriage of Neisseria meningitidis in a school in Niamey, Nigeria was carried out to confirm the feasibility of evaluating the impact of conjugate vaccine on the meningococcal carriage. All 90 pupils attending the school were examined during the dry season in February 1998. All children had been vaccinated using polysaccharide A/C in 1996. Samples were collected from the soft palate and immediately seeded on selective medium. After incubation at 37 degrees C for 24 hours, suspicious colonies were re-seeded on Miller-Hinton medium. Identification of N. meningitidis was based on standard biochemical criteria, agglutination grouping and DNA fingerprinting. Seven carriers of N. meningitidis X:NT:P1.5 were found. One of these carriers also presented a strain of N. meningitis A:4:P1.9. The high prevalence of serogroup X strains coincided with an outbreak of meningitis involving the same sub-type and sequence-type in Niamey.

Adolescent↗
PubMed1990

Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site.

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.

Amino Acid Sequence↗