bluuebunny/crossref_metadata_2025 huggingface dataset:
A Gene Encoding a Holin-Like Protein Involved in Spore Morphogenesis and Spore Germination inBacillus subtilis
Authors: Real Gonçalo , Pinto Sérgio M. , Schyns Ghislain , Costa Teresa , Henriques Adriano O. , Moran Charles P.
Published: September 2005

We report here studies of expression and functional analysis of aBacillus subtilisgene,ywcE, which codes for a product with features of a holin. Primer extension analysis ofywcEtranscription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a σA-type promoter bearing the TG dinucleotide motif of “extended” −10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from theywcEpromoter by purified σA-containing RNA polymerase (EσA), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of theywcEpromoter by EσAduring growth was subjected to repression. Studies with alacZfusion revealed that the transition-state regulator AbrB repressed the transcription ofywcEduring growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise toywcEtranscription. We foundywcEto be required for proper spore morphogenesis. Spores of theywcEmutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid.ywcEwas also found to be important for spore germination.
Modeling the Effects of
prl
Mutations on the
Escherichia coli
SecY Complex
Authors: Smith Margaret A. , Clemons William M. , DeMars Cathrine J. , Flower Ann M.
Published: September 2005

The apparatus responsible for translocation of proteins across bacterial membranes is the conserved SecY complex, consisting of SecY, SecE, and SecG. Prior genetic analysis provided insight into the mechanisms of protein export, as well as the interactions between the component proteins. In particular, the
prl
suppressor alleles of
secE
and
secY
, which allow export of secretory proteins with defective signal sequences, have proven particularly useful. Here, we report the isolation of novel mutations in
secE
and
secY
, as well as the phenotypic effects of combinations of
prl
mutations. These new alleles, as well as previously characterized
prl
mutations, were analyzed in light of the recently published crystal structure of the archaeal SecY complex. Our results support and expand a model of Prl suppressor activity that proposes that all of the
prlA
and
prlG
alleles either destabilize the closed state of the channel or stabilize the open form. These mutants thus allow channel opening to occur without the triggering event of signal sequence binding that is required in a wild-type complex.
The pCoo Plasmid of Enterotoxigenic
Escherichia coli
Is a Mosaic Cointegrate
Authors: Froehlich Barbara , Parkhill Julian , Sanders Mandy , Quail Michael A. , Scott June R.
Published: September 2005

CS1 is the prototype of a class of pili of enterotoxigenic
Escherichia coli
(ETEC) associated with diarrheal disease in humans. The genes encoding this pilus are carried on a large plasmid, pCoo. We report the sequence of the complete 98,396-bp plasmid. Like many other virulence plasmids, pCoo is a mosaic consisting of regions derived from multiple sources. Complete and fragmented insertion sequences (IS) make up 24% of the total DNA and are scattered throughout the plasmid. The pCoo DNA between these IS elements has a wide range of G+C content (35 to 57%), suggesting that these regions have different ancestries. We find that the pCoo plasmid is a cointegrate of two functional replicons, related to R64 and R100, which are joined at a 1,953-bp direct repeat of IS
100
. Recombination between these repeats in the cointegrate generates the two smaller replicons which coexist with the cointegrate in the culture. Both of the smaller replicons have plasmid stability genes as well as genes that may be important in pathogenesis. Examination by PCR of 17 other unrelated CS1 ETEC strains with a variety of serotypes demonstrated that all contained at least parts of both replicons of pCoo and that strains of the O6 genotype appear to contain a cointegrate very similar to pCoo. The results suggest that this family of CS1-encoding plasmids is evolving rapidly.
Activation of thephzOperon ofPseudomonas fluorescens2-79 Requires the LuxR Homolog PhzR,N-(3-OH-Hexanoyl)-l-Homoserine Lactone Produced by the LuxI Homolog PhzI, and acis-ActingphzBox
Authors: Khan Sharik R. , Mavrodi Dmitri V. , Jog Geetanjali J. , Suga Hiroaki , Thomashow Linda S. , Farrand Stephen K.
Published: September 2005

Thephzoperon ofPseudomonas fluorescens2-79, which produces phenazine-1-carboxylate, is preceded by two genes,phzRandphzI, that are homologs of quorum-sensing gene pairs of theluxR-luxIfamily. DeletingphzRandphzIfrom strain 2-79 led to loss of production of the antibiotics, as well as a suite of six acyl-homoserine lactones (acyl-HSLs) that includes four 3-hydroxy- derivatives and two alkanoyl-HSLs. Strain 2-79 accumulatesN-(3-hydroxy-hexanoyl)-l-HSL to levels 20 and 30 times those ofN-(hexanoyl)-l-HSL andN-(3-hydroxy-octanoyl)-HSL, the next most abundant species produced by this isolate. Expression of a clone ofphzIinEscherichia coliandP. fluorescens1855 resulted in the synthesis of all six acyl-HSLs. Maximal activation ofphzAandphzRfused tolacZanduidAreporters, respectively, required PhzR and the acyl-HSL signals. PhzR-mediated expression of thephzA::lacZfusion responded with highest sensitivity and greatest magnitude to pureN-(3-hydroxy-hexanoyl)-l-HSL. When exposed to organic extracts of culture supernatants containing the six acyl-HSLs at their normal levels, the reporter responded strongly toN-(3-hydroxy-hexanoyl)-l-HSL but did not respond to any of the other five acyl-HSLs. The transcriptional start sites for the divergently orientedphzAandphzRgenes were mapped by primer extension analysis. An 18-bp almost perfect inverted repeat, thephzbox, is located between thephzIandphzRpromoters. Disrupting this repeat abolished PhzR-dependent activation ofphzAandphzR. We conclude that PhzI of strain 2-79 synthesizes 3-OH acyl-HSLs and thatP. fluorescens2-79 usesN-(3-hydroxy-hexanoyl)-HSL as its quorum-sensing signal. We also conclude that PhzR, with its quormone, activates expression ofphzAandphzRand that this activation requires an intactphzbox sequence located in the divergent promoter region.
Deletion of the Gene
rpoZ
, Encoding the ω Subunit of RNA Polymerase, in
Mycobacterium smegmatis
Results in Fragmentation of the β′ Subunit in the Enzyme Assembly
Authors: Mathew Renjith , Ramakanth Madhugiri , Chatterji Dipankar
Published: September 2005

A deletion mutation in the gene
rpoZ
of
Mycobacterium smegmatis
causes reduced growth rate and a change in colony morphology. During purification of RNA polymerase from the mutant strain, the β′ subunit undergoes fragmentation but the fragments remain associated with the enzyme and maintain it in an active state until the whole destabilized assembly breaks down in the final step of purification. Complementation of the mutant strain with an integrated copy of the wild-type
rpoZ
brings back the wild-type colony morphology and improves the growth rate and activity of the enzyme, and the integrity of the β′ subunit remains unaffected.
The MutS C Terminus Is Essential for Mismatch Repair Activity In Vivo
Authors: Calmann Melissa A. , Nowosielska Anetta , Marinus M. G.
Published: September 2005

An
Escherichia coli
K-12 strain was constructed with a chromosomal deletion (
mutS
Δ
800
) in the
mutS
gene that produced the removal of the C-terminal 53 amino acids which are not present in the MutS crystal structure. This strain has a MutS null phenotype for mutation avoidance, antirecombination, and sensitivity to cytotoxic agents in a
dam
mutant background.
Prevalence of Surface Swarming Behavior in
Salmonella
Authors: Kim Wook , Surette Michael G.
Published: September 2005

Swarming behavior among 167
Salmonella
sp. isolates, representing all eight groups, was assessed. Only eight strains failed to swarm under standard conditions. Four of the defective strains swarmed on alternate carbon sources, and four harbored general defects in motility or lipopolysaccharide. Thus, swarming may represent an evolutionarily conserved behavior in
Salmonella
spp.
Requirements for Heterologous Production of a Complex Metalloenzyme: the Membrane-Bound [NiFe] Hydrogenase
Authors: Lenz Oliver , Gleiche Andrea , Strack Angelika , Friedrich Bärbel
Published: September 2005

By taking advantage of the tightly clustered genes for the membrane-bound [NiFe] hydrogenase of
Ralstonia eutropha
H16, broad-host-range recombinant plasmids were constructed carrying the entire membrane-bound hydrogenase (MBH) operon encompassing 21 genes. We demonstrate that the complex MBH biosynthetic apparatus is actively produced in hydrogenase-free hosts yielding fully assembled and functional MBH protein.
Iron Starvation Leads to Oxidative Stress in
Anabaena
sp. Strain PCC 7120
Authors: Latifi Amel , Jeanjean Robert , Lemeille Sylvain , Havaux Michel , Zhang Cheng-Cai
Published: September 2005

We establish here that iron deficiency causes oxidative stress in the cyanobacterium
Anabaena
sp. strain PCC 7120. Iron starvation leads to a significant increase in reactive oxygen species, whose effect can be abolished by treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl). Oxidative stress induced by iron starvation could be a common feature of photosynthetic bacteria.
Yersinia pestis
Is Viable with Endotoxin Composed of Only Lipid A
Authors: Tan Li , Darby Creg
Published: September 2005

Lipopolysaccharide (LPS) is the major outer membrane component of gram-negative bacteria. The minimal LPS structure for viability of
Escherichia coli
and
Salmonella enterica
serovar Typhimurium is lipid A glycosylated with 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Here we show that another member of the
Enterobacteriaceae
,
Yersinia pestis
, can survive without Kdo in its LPS.
Characterization of Toxigenic
Corynebacterium ulcerans
Strains Isolated from Humans and Domestic Cats in the United Kingdom
Authors: De Zoysa Aruni , Hawkey Peter M. , Engler Kathy , George Robert , Mann Gina , Reilly William , Taylor David , Efstratiou Androulla
Published: September 2005

In the United Kingdom there has been a marked increase in the number of human infections caused by toxigenic
Corynebacterium ulcerans
. During 2002 and 2003 the organism was also isolated from several domestic cats with bilateral nasal discharge. As
C. ulcerans
has never previously been isolated from cats, the 16S rRNA gene from three cat isolates was sequenced to confirm their species identities. Fifty clinical isolates from the United Kingdom isolated from 1986 to 2003 and seven cat isolates were characterized by ribotyping to determine whether the ribotypes of the cat isolates were genotypically related to those found for human clinical isolates. For comparison, the genotypes of 11 overseas isolates and 13 isolates from H. R. Carne's collection isolated between 1933 and 1979 were also determined. Strains isolated from domestic cats were found to exhibit the predominant ribotypes observed among human clinical isolates, suggesting that
C. ulcerans
isolated from cats could be a potential reservoir for human infection.
Genetic Analysis of Noroviruses in Chiba Prefecture, Japan, between 1999 and 2004
Authors: Okada Mineyuki , Ogawa Tomoko , Kaiho Ikuo , Shinozaki Kuniko
Published: September 2005

Noroviruses (NVs) are common pathogens that consist of genetically divergent viruses that induce gastroenteritis in humans and animals. Between September 1999 and June 2004, 1,898 samples obtained from patients showing sporadic or outbreak gastroenteritis in Chiba Prefecture, Japan, were tested for NVs by reverse transcription-PCR. NVs were detected in 603 samples. Approximately 80% were positive for genogroup GII, 13% were positive for genogroup GI, and the remaining 7% were positive for both genogroups. Phylogenetic analysis showed that the GI and GII genogroups could be further divided into 13 and 16 genotypes (including new genotypes), respectively. The GII-4 genotype, which included five small genetic clusters (subtypes), was the most common in this study and was detected in approximately 40% of positive samples. The P2 regions of 10 strains belonging to each of the five GII-4 subtypes showed 5 to 18% amino acid diversity. The amino acid
substitutions accumulated in the protruding (P) region during the 5-year study period. Our data suggest that highly variable NV strains
are circulating in Chiba Prefecture, with a high rate of genetic change observed during the 5-year study period.
Performances of VITEK 2 Colorimetric Cards for Identification of Gram-Positive and Gram-Negative Bacteria
Authors: Wallet Frédéric , Loïez Caroline , Renaux Emilie , Lemaitre Nadine , Courcol René J.
Published: September 2005

The purpose of this study was to evaluate the new VITEK 2 identification
cards that use colorimetric reading to identify gram-positive and
gram-negative bacteria (GP and GN cards, respectively) in comparison to
fluorimetric cards (ID-GPC and ID-GNB, respectively). A total of 580
clinical isolates and stock collection strains belonging to 116 taxa
were included in the study. Of the 249 gram-positive strains tested
with both the ID-GPC and GP cards, 218 (87.5%) and 235 (94.4%) strains
were correctly identified (to the genus and species level),
respectively. Of the 331 gram-negative strains tested with the ID-GNB
and GN cards, 295 (89.1%) and 321 (97%) strains were correctly
identified, respectively. Another focus of the study was to apply the
percentages of correct identifications obtained in this study to the
list of bacteria isolated in our laboratory (32,739 isolates) in the
year 2004. We obtained 97.9% correct identifications with the
colorimetric cards and 93.9% with fluorescent
cards.
Clinical Significance and Epidemiologic Analyses of
Mycobacterium avium
and
Mycobacterium intracellulare
among Patients without AIDS
Authors: Han Xiang Y. , Tarrand Jeffrey J. , Infante Rosa , Jacobson Kalen L. , Truong Mylene
Published: September 2005

The clinical significance and prevalence of
Mycobacterium avium
and
Mycobacterium intracellulare
were analyzed in a cohort of 7,472 patients who, from 1999 to 2003, sought care at the University of Texas M.D. Anderson Cancer Center, Houston, and had cultures performed for mycobacteria. Patients were stratified for age, sex, and underlying diseases, and bacteria were identified by 16S rRNA gene sequencing.
M. avium
was isolated in 62 (0.83%) of 7,472 patients and
M. intracellulare
in 65 (0.87%). Clinically, only 10 of the 62 (16.2%) patients with
M. avium
had probable to definite evidence of infection, whereas the majority (83.8%) had weak evidence of infection. Sex and age did not affect the isolation or infection of
M. avium
. Hematological tumors predisposed to
M. avium
colonization but not infection. In contrast, 41 of the 65 (63.1%) patients with
M. intracellulare
had probable to definite infection, a level much higher than those with
M. avium
(
P
< 0.001).
M. intracellulare
was more prevalent in women (1.33% of 3,311) than in men (0.50% of 4,161) (
P
< 0.001), and underlying diseases had no effect in women. Men with lung cancer had a higher prevalence (1.37%) than men without (0.34%) (4.0-fold;
P
< 0.001), but it was similar to that in women. A marked age trend for the isolation of
M. intracellulare
among women was noted: 0.27% (1-fold) for ages of <50 years, 0.85% (3.1-fold) for ages 50 to 59 years, 1.50% (5.6-fold) for ages 60 to 69 years, and 3.74% (13.9-fold) for ages ≥70 years (trend,
P
< 0.001). The combined rate for women ≥50 was 1.86% (95% confidence interval [1.30 to 2.42%]) (6.9-fold). Together, these results suggest that, among non-AIDS patients,
M. intracellulare
is more pathogenic and tends to infect women increasingly beyond menopause (age ≥50 years) regardless of underlying disease. The prevalence rate of 1.86% in postmenopausal women suggests the need to further investigate the public health significance of
M. intracellulare
.
Quantitative Detection of Hepatitis C Virus (HCV) RNA in Saliva and Gingival Crevicular Fluid of HCV-Infected Patients
Authors: Suzuki Tetsuro , Omata Kazuhiko , Satoh Tazuko , Miyasaka Takahiro , Arai Chiaki , Maeda Munehiro , Matsuno Tomonori , Miyamura Tatsuo
Published: September 2005

The search for hepatitis C virus (HCV) in body fluids other than blood is important when assessing possible nonparenteral routes of viral transmission. However, the role of oral fluids in HCV transmission remains controversial. Here we quantitatively determined HCV RNA in saliva and gingival crevicular fluid (GCF) of anti-HCV-positive patients. Most patients (14 of 18; 78%) whose saliva specimens were negative had HCV RNA in their GCF. Most patients (20 of 26; 77%) had higher HCV RNA levels in their GCF than in their saliva. Although there was not a statistically significant correlation between the serum viral load and HCV level in saliva or GCF, patients with low serum HCV loads were less likely to have detectable HCV in their saliva. These findings have important implications for medical personnel and suggest that epidemiological studies designed to understand the significance of the oral route of transmission of HCV are warranted.
Detection of Vi-Negative
Salmonella enterica
Serovar Typhi in the Peripheral Blood of Patients with Typhoid Fever in the Faisalabad Region of Pakistan
Authors: Baker Stephen , Sarwar Yasra , Aziz Hafsa , Haque Asma , Ali Aamir , Dougan Gordon , Wain John , Haque Abdul
Published: September 2005

The synthesis and transportation proteins of the Vi capsular polysaccharide of
Salmonella enterica
serovar Typhi (serovar Typhi) are encoded by the
viaB
operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production:
tviA
and
tviB
. Nine of the sixty strains analyzed (15%) tested negative for both
tviA
and
tviB
; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by
fliC
PCR for serovar Typhi; 4 of these were negative for
tviA
and
tviB
. Three of these samples tested positive for SPI-7. These results demonstrate that
viaB
-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.
Seminational Surveillance of Fungemia in Denmark: Notably High Rates of Fungemia and Numbers of Isolates with Reduced Azole Susceptibility
Authors: Arendrup Maiken Cavling , Fuursted Kurt , Gahrn-Hansen Bente , Jensen Irene Møller , Knudsen Jenny Dahl , Lundgren Bettina , Schønheyder Henrik C. , Tvede Michael
Published: September 2005

The aim of this study was to present the first set of comprehensive data on fungemia in Denmark including the distribution of species and range of susceptibility to major antifungal compounds based on a seminational surveillance study initiated in 2003. The catchment area of the participating hospitals had a population of 2.8 million, or 53% of the Danish population. A total of 303 episodes of fungemia were registered (annual rate, 11 of 100,000 people or 0.49 of 1,000 hospital discharges).
Candida
species accounted for 97.4% of the fungal pathogens.
C. albicans
was the predominant species (63%), but the proportion varied from 57% to 72% among participating departments of clinical microbiology.
C. glabrata
was the second most frequent species (20%; range, 8% to 32%).
C. krusei
was a rare isolate (3%) and occurred only at two of the participating hospitals. Retrospective data retrieved from the Danish laboratory systems documented a continuous increase of candidemia cases since the early 1990s. For the 272 susceptibility-tested isolates, MICs of amphotericin B and caspofungin were within the limits expected for the species or genus. However, decreased azole susceptibility, defined as a fluconazole MIC of >8 μg/ml and/or itraconazole MIC of >0.125 μg/ml, was detected for 11
Candida
isolates that were neither
C. glabrata
nor
C. krusei
. Including intrinsically resistant fungi, we detected decreased susceptibility to fluconazole and/or itraconazole in 87 (32%) current Danish bloodstream fungal isolates. We showed a continuous increase of fungemia in Denmark and an annual rate in 2003 to 2004 higher than in most other countries. The proportion of bloodstream fungal isolates with reduced susceptibility to fluconazole and/or itraconazole was also notably high.
Development and Validation of an Immunoassay for Identification of Recent Human Immunodeficiency Virus Type 1 Infections and Its Use on Dried Serum Spots
Authors: Barin Francis , Meyer Laurence , Lancar Rémi , Deveau Christiane , Gharib Myriam , Laporte Anne , Desenclos Jean-Claude , Costagliola Dominique
Published: September 2005

The objective was to develop and to validate an immunossay to identify recent human immunodeficiency virus type 1 (HIV-1) infections that can be used on dried serum spots (DSS). A single, indirect enzyme-linked immunosorbent assay was developed to quantify antibodies toward four HIV-1 antigens: consensus peptides of the immunodominant epitope of gp41 (IDE), consensus V3 peptides, recombinant integrase, and recombinant p24. The parameters of the logistic regression used to classify the samples were estimated on a training sample (210 serum samples) using resampling techniques to get stable estimates and then applied to a validation sample (761 serum samples). The IDE and V3 peptides were the best able to discriminate between the antibodies present in serum from recently (≤6 months) infected individuals and those with long-lasting infection. Combined quantification of antibody binding to these two synthetic antigens allowed us to identify recent infections with an area under the receiver operating characteristic curve of 0.949 and a sensitivity of 88.3%, with a specificity of 97.6% in patients with long-term infection (but not AIDS) and 86.0% in patients suffering from AIDS with a threshold of 0.50 in the validation sample. This simple immunoassay can be used to identify recently HIV-1-infected patients. Its performance is compatible with its use in population-based studies including DSS.
Porphyromonas somerae
sp. nov., a Pathogen Isolated from Humans and Distinct from
Porphyromonas levii
Authors: Summanen Paula H. , Durmaz Bengül , Väisänen Marja-Liisa , Liu Chengxu , Molitoris Denise , Eerola Erkki , Helander Ilkka M. , Finegold Sydney M.
Published: September 2005

Porphyromonas levii
is an anaerobic, pigmented gram-negative bacillus originally isolated from bovine rumen. We describe 58 human clinical strains of
P. levii
-like organisms, isolated from various human clinical specimens that are phenotypically similar to the type strain of
P. levii
, a rumen isolate (ATCC 29147). Our biochemical, comparative 16S rRNA sequence analyses, and DNΑ-DNA relatedness studies indicate that the human
P. levii
-like organisms are similar to each other but genetically different from the
P. levii
type strain isolated from bovine rumen. We therefore propose the name
Porphyromonas somerae
to encompass the human
P. levii
-like organisms.
P. somerae
was predominantly isolated from patients with chronic skin and soft tissue or bone infections, especially in the lower extremities.
Method for Rapid Identification and Differentiation of the Species of the
Mycobacterium chelonae
Complex Based on 16S-23S rRNA Gene Internal Transcribed Spacer PCR-Restriction Analysis
Authors: Khan Izhar U. H. , Selvaraju Suresh B. , Yadav Jagjit S.
Published: September 2005

Members of the
Mycobacterium chelonae
complex (MCC), including
M. immunogenum
,
M. chelonae
, and
M. abscessus
, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region.
Mycobacterium
genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of
M. immunogenum
varied from those of
M. chelonae
and
M. abscessus
. Sequencing of the ∼250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate
M. immunogenum
from
M. chelonae
and
M. abscessus
. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.
Molecular Typing ofMycobacterium tuberculosisby Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis, a More Accurate Method for Identifying Epidemiological Links between Patients with Tuberculosis
Authors: van Deutekom Henk , Supply Philip , de Haas Petra E. W. , Willery Eve , Hoijng Susan P. , Locht Camille , Coutinho Roel A. , van Soolingen Dick
Published: September 2005

IS6110fingerprinting ofMycobacterium tuberculosisis the standard identification method in studies on transmission of tuberculosis. However, intensive epidemiological investigation may fail to confirm transmission links between patients clustered by IS6110-restriction fragment length polymorphism (RFLP) typing. We applied typing based on variable numbers of tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) to isolates from 125 patients in 42 IS6110clusters, for which thorough epidemiological data were available, to investigate the potential of this method in distinguishing epidemiologically linked from nonlinked patients. Of seven IS6110clusters without epidemiological links, five were split by MIRU-VNTR typing, while nearly all IS6110clusters with proven or likely links displayed conserved MIRU-VNTR types. These results provide molecular evidence that not all clusters determined on the basis of multibanded IS6110RFLP patterns necessarily reflect transmission of tuberculosis. They support the use of MIRU-VNTR typing as a more reliable and faster method for transmission analysis.
Development of a Multiplex PCR and SHV Melting-Curve Mutation Detection System for Detection of Some SHV and CTX-M β-Lactamases of
Escherichia coli
,
Klebsiella pneumoniae
, and
Enterobacter cloacae
in Taiwan
Authors: Chia Ju-Hsin , Chu Chishih , Su Lin-Hui , Chiu Cheng-Hsun , Kuo An-Jing , Sun Chien-Feng , Wu Tsu-Lan
Published: September 2005

Infection by extended-spectrum β-lactamase (ESBL)-producing
Enterobacteriaceae
has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify
bla
SHV
,
bla
CTX-M-3
-like, and
bla
CTX-M-14
-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent
bla
SHV
genes (
bla
SHV-1
,
bla
SHV-2
,
bla
SHV-2a
,
bla
SHV-5
,
bla
SHV-11
, and
bla
SHV-12
) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the
bla
SHV
and
bla
CTX-M
genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40
Enterobacter cloacae
, 68
Escherichia coli
, and 91
Klebsiella pneumoniae
, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to
Enterobacter cloacae
, the majority of which produced SHV-type ESBLs,
E. coli
and
K. pneumoniae
were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the
bla
CTX-M-3
-like (CTX-M-15) and
bla
CTX-M-14
-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among
E. coli
,
K. pneumoniae
, and
Enterobacter cloacae
in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.
Human Parainfluenza Virus 4 Outbreak and the Role of Diagnostic Tests
Authors: Lau Susanna K. P. , To Wing-kin , Tse Philomena W. T. , Chan Alex K. H. , Woo Patrick C. Y. , Tsoi Hoi-wah , Leung Annie F. Y. , Li Kenneth S. M. , Chan Paul K. S. , Lim Wilina W. L. , Yung Raymond W. H. , Chan Kwok-hung , Yuen Kwok-yung
Published: September 2005

Owing to the difficulties in isolating the virus and the lack of routine surveillance, the clinical significance of human parainfluenza virus 4 (HPIV-4) is less well defined than that of the other human parainfluenza viruses. We describe the first outbreak of HPIV-4 infection in a developmental disabilities unit, involving 38 institutionalized children and three staff members, during a 3-week period in autumn 2004. Most subjects had upper respiratory tract infections (URTI), while lower respiratory tract infections (LRTI) occurred in three children (7%), one complicated by respiratory failure requiring ventilation support. All patients recovered. Nasopharyngeal aspirates tested for HPIV-4 were positive by reverse transcriptase PCR (RT-PCR) in all 41 cases (100%), by direct immunofluorescence in 29 of 39 tested cases (74%), and by cell cultures in 6 of 37 cases (16%), and serum was positive for antibodies against HPIV-4 in all 35 cases (100%) with serum samples available. In addition, RT-PCR detected HPIV-4 in four children (three LRTI and one URTI) out of 115 patients with community-acquired respiratory tract infection. Molecular analysis of the 1,198-bp phosphoprotein sequences showed that HPIV-4 isolates among the cases were genetically similar, whereas the community controls were more genetically distant, supporting nosocomial transmission of a single HPIV-4 genotype during the outbreak. Moreover, the HPIV-4 causing the outbreak is more closely related to HPIV-4A than HPIV-4B. HPIV-4 may be an important cause of more severe respiratory illness in children. The present RT-PCR assay is a sensitive, specific, and rapid method for the diagnosing HPIV-4 infection. To better define the epidemiology and clinical spectrum of disease of HPIV-4 infections, HPIV-4 should be included in the routine panels of respiratory virus detection on respiratory specimens.
“Bacteroides goldsteiniisp. nov.” Isolated from Clinical Specimens of Human Intestinal Origin
Authors: Song Yuli , Liu Chengxu , Lee Julia , Bolaňos Mauricio , Vaisanen Marja-Liisa , Finegold Sydney M.
Published: September 2005

Phenotypic and phylogenetic studies were performed on an unknown gram-negative, strictly anaerobic, rod-shaped bacterium isolated from human clinical specimens. This organism was indole negative, resistant to 20% bile, produced acetic and a lesser amount of succinic acids as the major end products of glucose metabolism, and possessed a G+C content of approximately 43 mol%. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was a member of theCytophaga-Flavobacter-Bacteroidesphylum of gram-negative bacteria and formed a close association (with an average sequence similarity of 93.6%) with the second subcluster of thePorphyromonascluster in theBacteroidessubgroup. Phylogenetically and phenotypically it resembledBacteroides merdae; however, a 16S rRNA gene sequence divergence of approximately 5.5% between the unknown bacterium andB. merdae, as well as distinguishable biochemical characteristics, demonstrate that the unknown bacterium is genotypically and phenotypically distinct and represents a previously unknown subline within thePorphyromonasphylogenetic cluster. Furthermore, a DNA-DNA reassociation value of 17.8% between isolates WAL 12034T(the type strain of this novel taxon) and ATCC 43184T(B. merdaetype strain) also documented the separateness of the unknown species andB. merdae. Based on the phenotypic and phylogenetic findings, a new species, “Bacteroides goldsteiniisp. nov,” is proposed. The G+C content of the DNA is 43 mol% forBacteroides. The type strain of “B. goldsteinii” is WAL 12034T(= CCUG 48944T= ATCC BAA-1180T).
Discrimination of
Streptococcus pneumoniae
from Viridans Group Streptococci by Genomic Subtractive Hybridization
Authors: Suzuki Nao , Seki Mitsuko , Nakano Yoshio , Kiyoura Yusuke , Maeno Masao , Yamashita Yoshihisa
Published: September 2005

Two oligonucleotide primer sets for the discrimination of
Streptococcus pneumoniae
from “pneumococcus-like” oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between
Streptococcus pneumoniae
strain WU2 and the most closely related oral streptococcus,
Streptococcus mitis
strain 903. We identified 19 clones that contained
S. pneumoniae-
specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of
S. mitis
and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of
S. pneumoniae
were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four
S. pneumoniae
clinical isolates and from 20 atypical organisms classified as
S. mitis
or
S. oralis
, which harbored genes encoding the pneumococcal virulence factors autolysin (
lytA
) or pneumolysin (
ply
), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of
S. pneumoniae
infections.
Comparison of Visual 24-Hour and Spectrophotometric 48-Hour MICs to CLSI Reference Microdilution MICs of Fluconazole, Itraconazole, Posaconazole, and Voriconazole for
Candida
spp.: a Collaborative Study
Authors: Espinel-Ingroff A. , Barchiesi F. , Cuenca-Estrella M. , Fothergill A. , Pfaller M. A. , Rinaldi M. , Rodriguez-Tudela J. L. , Verweij P. E.
Published: September 2005

A multicenter (six-center) study evaluated the performance (interlaboratory reproducibility, compatibility with reference methods, and categorical agreement) of 24-h visual and 48-h spectrophotometric MICs. MICs of fluconazole, itraconazole, voriconazole, and posaconazole were compared to reference 48-h microdilution broth visual MICs (CLSI [formerly NCCLS] M27-A2 document) for 71 isolates of
Candida
spp. that included 10 fluconazole-resistant strains. Twenty readings (5%) were reported as showing no growth at 24 h, mostly for
Candida dubliniensis
and from a single center. The overall interlaboratory agreement of 24-h visual readings and 48-h spectrophotometric MICs, as well their compatibility with reference values, were excellent with the four triazoles for most of the species (93 to 99%, within 3 dilutions). The categorical agreement between the investigational reading conditions and reference values was good with fluconazole and voriconazole (93 to 97%) but lower with itraconazole (86 to 88%), due primarily to minor errors. There were only 0 to 3% very major errors with these three triazoles; the number of substantial errors (more than three dilutions) was also low (<2%) with posaconazole. These data suggest that the performance of both investigational MIC readings gives results similar to those of reference MICs. Since spectrophotometric MICs are more objective and the 24-h time period would shorten the MIC determination of azoles, the description of either of these two reading conditions in the M27-A2 document should be considered by the CLSI subcommittee in addition to or instead of the longer, less practical, and more subjective 48-h visual MIC reading.
Correlation of Penicillin Binding Protein 2a Detection with Oxacillin Resistance in
Staphylococcus aureus
and Discovery of a Novel Penicillin Binding Protein 2a Mutation
Authors: Bressler Adam M. , Williams Teresa , Culler Elizabeth E. , Zhu Wenming , Lonsway David , Patel Jean B. , Nolte Frederick S.
Published: September 2005

We compared a rapid slide latex agglutination test (LAT; Oxoid, Basingstoke, United Kingdom) that detects penicillin binding protein 2a (PBP2a) with MicroScan conventional panels (Dade Behring, West Sacramento, CA) for detection of oxacillin resistance in
Staphylococcus aureus
. The PBP2a LAT demonstrated 99% agreement with MicroScan oxacillin MIC results for 388 isolates of
S. aureus
. All 249 oxacillin-resistant isolates gave strong positive reactions in the LAT (100% sensitivity). Three of the 139 oxacillin-susceptible isolates were also strongly positive and one was weakly positive in the LAT (97.1% specificity). The three oxacillin-susceptible isolates with strongly positive reactions were further characterized. The
mecA
gene was detected in all three by PCR; one isolate was determined to be resistant to oxacillin by reference broth microdilution testing (MIC, 8 μg/ml), one isolate was inducibly resistant to oxacillin (MIC of 16 μg/ml after overnight induction), and one isolate remained susceptible regardless of the method used for testing. Sequence analysis of a 2.1-kb gene fragment of the
mecA
gene from the susceptible isolate revealed a one-base substitution at nucleotide position 1449 which results in a Met-to-Ile change for amino acid residue 483. This amino acid substitution has not been previously reported and may be associated with a change in the function of PBP2a resulting in oxacillin susceptibility. An additional 487 isolates were tested in parallel with the both the LAT and MicroScan panels using criteria in which only strong (3 to 4+) or repeatedly weak (1 to 2+) LAT reactions were considered positive, and the results showed 99.4% agreement. The PBP2a LAT provided rapid and reliable detection of oxacillin resistance and proved a useful adjunct to the phenotypic method. Both methods provided reliable detection of oxacillin-resistant
S. aureus
and facilitated the discovery of a novel, functionally impaired form of PBP2a.
Salmonella enterica
Serovar Typhi O:1,9,12 Polysaccharide-Protein Conjugate as a Diagnostic Tool for Typhoid Fever
Authors: Zuñiga Jessica , Lillo Luis , Shin Junghee J. , Machavarapu Rajya L. , Quiroga Teresa , Goycoolea Manuela , Matsuhiro Betty , Aron-Hott L. , Godfrey Henry P. , Cabello Felipe C.
Published: September 2005

Serologic tests play an important role in diagnosis of typhoid fever. In an effort to develop a more defined reagent for these tests, purified
Salmonella enterica
serovar Typhi (ST) O:1,9,12 polysaccharide was conjugated to human serum albumin (HSA), and the conjugate was purified chromatographically to yield a reagent with 2 moles ST O polysaccharide per mole HSA. In 40 patients with bacteriologically confirmed typhoid fever, significant dot immunobinding titers (≥20,000) were present in 28 (70%) tested with 100 ng of ST O antigen-HSA (ST O-HSA) conjugate, in 38 (95%) tested with 100 ng of ST lipopolysaccharide, and in 16 (40%) tested with purified unconjugated ST O chains. In sera from 22 patients with other nontyphoid fevers, 2 (9.1%) had such reactivities with 100 ng of ST O-HSA, 1 (4.5%) had such reactivity with 100 ng of ST lipopolysaccharide (4.5%), and none reacted with 100 ng of unconjugated ST O chains. None of the 17 healthy-control sera reacted significantly with any of the ST reagents. None of the patient or control sera reacted with unconjugated HSA. The sensitivity of dot immunobinding for typhoid fever was 70% with 100 ng of ST O-HSA, somewhat lower than that with 100 ng of ST lipopolysaccharide (95%) but similar to that of the Widal H agglutination test with a ≥1/160 cutoff (74%). Specificities of these tests were 91%, 95%, and 86%, respectively. These preliminary results suggest that ST O polysaccharide-protein conjugates could provide a nontoxic, easily quality-controlled synthetic reagent for analysis of human immune responses to ST as well as for the development of new diagnostics and vaccines for typhoid fever.
Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays
Authors: Dreier Jens , Störmer Melanie , Kleesiek Knut
Published: September 2005

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated.
Human Coronavirus NL-63 Infections in Children: a 1-Year Study
Authors: Bastien Nathalie , Robinson Joan L. , Tse Alena , Lee Bonita E. , Hart Laura , Li Yan
Published: September 2005

Human coronavirus NL63 (HCoV-NL63), a newly discovered coronavirus, has been associated with acute respiratory tract infections (ARI). Important questions pertaining to the contribution of HCoV-NL63 to ARI and its impact on public health remain. We reviewed HCoV-NL63 in specimens collected from 13 November 2002 to 31 December 2003 from the Stollery Children's Hospital on patients of <17 years of age to assess the role of this virus in ARI in children. Twenty-six of 1,240 specimens (2.1%) from seven outpatients and 19 inpatients aged 7 days to 9.5 years tested positive for HCoV-NL63 by reverse transcription-PCR. The majority of outpatients (86%) had upper respiratory tract infections, while the majority of inpatients (58%) had bronchiolitis. Peak HCoV-NL63 activity occurred in March. These results provide further evidence of the importance of HCoV-NL63 in ARI in children.
Depletion of Resident
Chlamydia pneumoniae
through Leukoreduction by Filtration of Blood for Transfusion
Authors: Ikejima Hideaki , Friedman Herman , Leparc German F. , Yamamoto Yoshimasa
Published: September 2005

Current studies indicate that a significant percentage of healthy blood donors carry
Chlamydia pneumoniae
in their blood. Although the clinical significance of such findings is unknown, eradication of such bacteria from blood components may contribute to transfusion safety. Deletion of
C. pneumoniae
in Red Blood Cell (RBC) units was accomplished through leukoreduction by filtration. The presence of bacteria in RBC units before and after leukoreduction was assessed by real-time PCR using primers specific for
C. pneumoniae
16S rRNA. The eluates of filters used for leukoreduction were also assessed by PCR and immunostaining with fluorescein isothiocyanate-conjugated chlamydial monoclonal antibodies specific for
C. pneumoniae
determination. Nineteen of 30 RBC units tested showed the presence of
C. pneumoniae
DNA. Leukofiltration resulted in a marked reduction of leukocytes as well as
C. pneumoniae
in terms of bacterial number and positive rate for the bacteria. The eluates of filters showed trapped bacteria determined by both PCR and immunostaining assays. Thus, leukoreduction with a filter is an effective method to significantly reduce resident
C. pneumoniae
levels in RBC components but may not be completely sufficient for total eradication of this pathogen.
Phenotypic and Genotypic Characteristics of
Streptococcus porcinus
Isolated from Human Sources
Authors: Duarte Rafael S. , Barros Rosana R. , Facklam Richard R. , Teixeira Lúcia M.
Published: September 2005

The phenotypic and genotypic characteristics of 25
Streptococcus porcinus
isolates recovered from human sources were investigated and compared to the characteristics of 17 reference strains obtained from nonhuman sources. All of the
S. porcinus
isolates were beta-hemolytic (wide zones), susceptible to vancomycin, gave positive results for the leucine aminopeptidase and
l
-pyrrolidonylarylamidase tests, and produced acids from mannitol and sorbitol. Most of them were positive for the CAMP test and resistant to bacitracin. The isolates were susceptible to most of the 14 antimicrobials tested, except for tetracycline, for which 80% of the human isolates and 35.2% of the nonhuman strains were resistant. The
tet
(M) and the
tet
(O) genes were detected in 23 (88.5%) and 8 (30.8%) of the 26 tetracycline-resistant isolates, respectively. Analysis of whole-cell protein profiles obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high similarity among the profiles. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI and by random(ly) amplified polymorphic DNA (RAPD)-PCR using primer 1254. Analysis of SmaI-restricted genomic DNA revealed the substantial genetic diversity among
S. porcinus
isolates from nonhuman sources, which were also serologically more diverse. Most of the human isolates belonged to serogroup NG1 and shared highly related PFGE profiles that were distinct from profiles of isolates from nonhuman sources. These results were in agreement with those obtained by analysis of amplicons after RAPD-PCR, indicating the potential ability of these techniques for typing
S. porcinus
and suggesting the occurrence of a few clonal groups of
S. porcinus
strains adapted to the human host.