bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Relatedness and Superantigen Expression in Group A Streptococcus Serotype M1 Isolates from Patients with Severe and Nonsevere Invasive Diseases
Authors: Chatellier Sonia , Ihendyane Nahla , Kansal Rita G. , Khambaty Farukh , Basma Hesham , Norrby-Teglund Anna , Low Donald E. , McGeer Allison , Kotb Malak
Published: July 2002

The relatedness of group A streptococcal (GAS) strains isolated from 35 Canadian patients with invasive disease of different severity was investigated by a variety of molecular methods. All patients were infected with M1T1 strains and, based on clinical criteria, were classified as severe (n= 21) and nonsevere (n= 14) invasive GAS infection cases. All the M1 strains studied had theemm1.0 allele and the same streptococcal pyrogenic exotoxin (Spe) genotype,speA+speB+speC speF+speG+speH smeZ+ssa. All isolates had the samespeAallotype,speA2. The randomly amplified polymorphic DNA banding pattern with two different primers was identical for all strains, and pulsed field gel electrophoresis analysis showed that 33 and 30 isolates had identical banding patterns after DNA digestion withSfiI orSmaI, respectively; the nonidentical isolates differed from the main pattern by only one band. A relatively high degree of polymorphism in specific regions of thesicgene was observed among isolates; however, this polymorphism was not associated with disease severity. Likewise, although the phenotypic expression of SpeA, SpeB, and SpeF proteins varied among the M1T1 isolates, there was no correlation between the amount of Spe expressed and disease severity. Importantly, mitogenic and cytokine responses induced by partially purified bacterial culture supernatants containing a mixture of expressed superantigens were very similar for isolates from severe and nonsevere cases (P> 0.1). Together, the data indicate that highly related invasive M1T1 isolates, some indistinguishable, can cause disease of varying severity in different individuals. These findings underscore the contribution of host factors to the outcome of invasive GAS infections.
Analysis of Pilus Adhesins from
Haemophilus influenzae
Biotype IV Strains
Authors: Clemans Daniel L. , Marrs Carl F. , Bauer Richard J. , Patel Mayurika , Gilsdorf Janet R.
Published: July 2002

A subset of nontypeable
Haemophilus influenzae
(NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory
H. influenzae
isolates. For this analysis, the presence of
hifA
and
hifE
and their gene products in NTHI biotype IV strains was assessed, the binding of
H. influenzae
biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although
hifA
, which encodes the major structural subunit of hemagglutinating pili, and
hifE
, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of
hifA
and
hifE
) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV
H. influenzae
isolates examined; strain 4162 containing an insertional mutation in
hifA
also showed no surface pili, whereas strain 1595 containing an insertional mutation in
hifB
showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o
−
and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o
−
cells did not correlate with the presence of
hifE
. While both nonpiliated variants and
hifA
and
hifB
(encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o
−
and ME180 cells,
hifA
and
hifB
mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV
H. influenzae
isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.
A 4.1-Kilodalton Polypeptide in the Cultural Supernatant of
Mycoplasma fermentans
Is One of the Substances Responsible for Induction of Interleukin-6 Production by Human Gingival Fibroblasts
Authors: Hasebe Akira , Shibata Ken-Ichiro , Watanabe Tsuguo
Published: July 2002

The cultural supernatant of
Mycoplasma fermentans
induced interleukin-6 production by human gingival fibroblasts. The active entities were divided into hydrophilic and hydrophobic substances. In this study, we purified a 4.1-kilodalton polypeptide from the hydrophilic substances. It reacted with polyclonal antibodies to
M. fermentans
and activated human macrophages.
Identification, Characterization, and Variation in Expression of Two Serologically Distinct O-Antigen Epitopes in Lipopolysaccharides of
Campylobacter fetus
Serotype A Strains
Authors: Brooks Brian W. , Robertson Ruth H. , Lutze-Wallace Cheryl L. , Pfahler Wolfram
Published: July 2002

Monoclonal antibodies (MAbs) to the lipopolysaccharide (LPS) O-antigens of
Campylobacter fetus
serotype A and B strains were produced. Eight MAbs specific for serotype A LPS were characterized on immunoblots of
C. fetus
serotype A LPS. Two immunoblot patterns were observed and were used to divide the eight MAbs into two groups. MAbs M1177 and M1194 were selected as representative of the two groups and were used in an enzyme-linked immunosorbent assay (ELISA) to examine the LPS O-antigen epitopes of 37 serotype A
C. fetus
subsp.
fetus
and C.
fetus
subsp.
venerealis
strains. Thirty-three strains (89%) reacted with both M1177 and M1194, 2 strains reacted only with M1177, and 2 strains reacted only with M1194. To further characterize the O-antigen epitopes, purified serotype A LPS was treated using various temperature and pH conditions and the effect of the treatments on the reactivity of the LPS with MAbs M1177 and M1194 was evaluated by ELISA. While no difference among several treatments was observed, heating serotype A LPS under alkaline conditions decreased the reaction with M1177 to background levels and increased the reaction with M1194. MAbs M1177 and M1194 were also used with ELISA to investigate in vivo and in vitro expression of the two O-antigen epitopes. There was substantial variation in expression of the two epitopes among weekly isolates of two
C. fetus
serotype A strains recovered from experimentally infected heifers. There was minimal variation in expression of the two epitopes in successive subcultures of three
C. fetus
serotype A strains.
Secretion of Listeriolysin by
Brucella suis
Inhibits Its Intramacrophagic Replication
Authors: Köhler Stephan , Layssac Marion , Naroeni Aroem , Gentschev Ivaylo , Rittig Michael , Liautard Jean-Pierre
Published: July 2002

The introduction into
Brucella suis
1330 of a plasmid allowing the heterologous expression of a hybrid cytolysin containing listeriolysin from
Listeria monocytogenes
, and its export via the
Escherichia coli
hemolysin secretion pathway, resulted in secretion of active listeriolysin monitored by erythrocyte lysis. In contrast to observations with the nonhemolytic control strain, the phagosomes of infected human monocytes containing the hemolytic
B. suis
were partially disrupted, and this strain failed to multiply in human macrophage-like cells. These results added strong evidence supporting the proposal that the phagosome of the macrophage was the predominant niche of brucellae in their mammalian hosts.
Francisella tularensisInduces Cytopathogenicity and Apoptosis in Murine Macrophages via a Mechanism That Requires Intracellular Bacterial Multiplication
Authors: Lai Xin-He , Golovliov Igor , Sjöstedt Anders
Published: July 2002

The murine macrophage-like cell line J774.A1 ingests and allows intracellular growth ofFrancisella tularensis. We demonstrate that, after 24 h of infection, a pronounced cytopathogenicity resulted and the J774 cells were undergoing apoptosis. Despite this host cell apoptosis, no decrease in bacterial numbers was observed. When internalization of bacteria was prevented or intracellularly locatedF. tularensisbacteria were eradicated within 12 h, the progression of host cell cytopathogenicity and apoptosis was prevented.
Nickel-Responsive Induction of Urease Expression in
Helicobacter pylori
Is Mediated at the Transcriptional Level
Authors: van Vliet Arnoud H. M. , Kuipers Ernst J. , Waidner Barbara , Davies Beverly J. , de Vries Nicolette , Penn Charles W. , Vandenbroucke-Grauls Christina M. J. E. , Kist Manfred , Bereswill Stefan , Kusters Johannes G.
Published: July 2002

The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen
Helicobacter pylori
, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of
H. pylori
, expression of urease genes is thought to be constitutive. Here it is demonstrated that
H. pylori
regulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 μM NiCl
2
resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptional
ureA
::
lacZ
fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of
H. pylori
urease, a phenomenon likely to be of importance during the colonization and persistence of
H. pylori
in the gastric mucosa.
Intimin, Tir, and Shiga Toxin 1 Do Not Influence Enteropathogenic Responses to Shiga Toxin-Producing
Escherichia coli
in Bovine Ligated Intestinal Loops
Authors: Stevens Mark P. , Marchès Olivier , Campbell June , Huter Veronika , Frankel Gad , Phillips Alan D. , Oswald Eric , Wallis Timothy S.
Published: July 2002

Shiga toxin-producing
Escherchia coli
(STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the
stx1
,
eae
, and
tir
genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.
Characterization of the Mouse Beta Defensin 1,
Defb1
, Mutant Mouse Model
Authors: Morrison Gillian , Kilanowski Fiona , Davidson Donald , Dorin Julia
Published: July 2002

Beta defensins are small cationic antimicrobial peptides present in the respiratory system which have been proposed to be dysfunctional in the environment of the cystic fibrosis lung.
Defb1
, a murine homologue to the human beta defensins, has also been found to be expressed in the respiratory system and, in order to examine the function of beta defensins in vivo, gene targeting was used to generate
Defb1
-deficient (
Defb1
tm1Hgu
/

Defb1
tm1Hgu

[
Defb1
−/−
]) mice. The Defb1 synthetic peptide was shown to have a salt-sensitive antimicrobial activity that was stronger against
Staphylococcus aureus
than against
Escherichia coli
or
Pseudomonas aeruginosa. Defb1
−/−
mice were found, however, to be effective in the clearance of the cystic fibrosis relevant pathogen
S. aureus
from the airways after nebulization. Although no overt deleterious phenotype was evident in the
Defb1
−/−
mice, the number of mutant mice found to harbor bacteria of the
Staphylococcus
species in the bladder was significantly higher (
P
= 0.008) than that of controls, suggesting a role for these peptides in resistance to urinary tract infection.
Improved Tuberculosis DNA Vaccines by Formulation in Cationic Lipids
Authors: D'Souza S. , Rosseels V. , Denis O. , Tanghe A. , De Smet N. , Jurion F. , Palfliet K. , Castiglioni N. , Vanonckelen A. , Wheeler C. , Huygen K.
Published: July 2002

Mice were vaccinated with plasmid DNA (pDNA) encoding antigen 85A (Ag85A), Ag85B, or PstS-3 fromMycobacterium tuberculosiseither in saline or formulated for intramuscular injections in VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine) (Vaxfectin; Vical, Inc., San Diego, Calif.) or for intranasal instillations in GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine). These two novel cationic and neutral colipid formulations were previously reported to be effective adjuvants for pDNA-induced antibody responses. The levels of Ag85-specific total immunoglobulin G (IgG) and IgG isotypes were all increased 3- to 10-fold by formulation of pDNA in Vaxfectin. The level of production of splenic T-cell-derived Th1-type cytokines (interleukin-2 and gamma interferon) in response to purified Ag85 and to synthetic peptides spanning the entire Ag85A protein was also significantly higher in animals vaccinated with pDNA formulated in Vaxfectin. Cytolytic T-lymphocyte responses generated by pDNA encoding phosphate-binding protein PstS-3 in Vaxfectin were better sustained over time than were those generated by PstS-3 DNA in saline. Intranasal immunization with Ag85A DNA in saline was completely ineffective, whereas administration in GAP-DLRIE:DOPE induced a positive Th1-type cytokine response; however, the extent of the latter response was clearly lower than that obtained following intramuscular immunization with the same DNA dose. Combined intramuscular and intranasal administrations in cationic lipids resulted in stronger immune responses in the spleen and, more importantly, in the lungs as well. Finally, formulation in Vaxfectin increased the protective efficacy of the Ag85B DNA vaccine, as measured by reduced relative light unit counts and CFU counts in the spleen and lungs from mice challenged with bioluminescentM. tuberculosisH37Rv. These results may be of importance for future clinical use of DNA vaccines in humans.
A New Two-Component Regulatory System Involved in Adhesion, Autolysis, and Extracellular Proteolytic Activity of
Staphylococcus aureus
Authors: Fournier Bénédicte , Hooper David C.
Published: July 2002

A transposition mutant of
Staphylococcus aureus
was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5′ terminus of the gene
arlS
. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that
arlS
forms an operon with upstream gene
arlR
. The predicted product of
arlR
is a member of the OmpR-PhoB family of response regulators. The
arlS
mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the
arlS
mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the
arlS
mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus
arlR-arlS
strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence of
S. aureus
.
The Trimethylamine Methyltransferase Gene and Multiple Dimethylamine Methyltransferase Genes of
Methanosarcina barkeri
Contain In-Frame and Read-Through Amber Codons
Authors: Paul Ligi , Ferguson Donald J. , Krzycki Joseph A.
Published: July 2002

Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of
mttB
, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene,
mtbB1
. However, two other nearly identical copies of
mtbB1
, designated
mtbB2
and
mtbB3
, were also found in the genome. A 6.8-kb transcript was detected with probes to
mttB
and
mtbB1
, as well as to
mtbC
and
mttC
, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to
mttP
, encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order
mtbC
,
mttB
,
mttC
,
mttP
, and
mtbB1
, form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of
mtbC
. The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from
Methanosarcina barkeri
contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of
mtbB1
,
mtbB2
, or
mtbB3
if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The
mttB
gene from
Methanosarcina thermophila
contained a UAG codon at the same position as the
M. barkeri mttB
gene. The UAG codon is also present in
mttB
transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.
Bacterial Diversity in Human Subgingival Plaque
Authors: Paster Bruce J. , Boches Susan K. , Galvin Jamie L. , Ericson Rebecca E. , Lau Carol N. , Levanos Valerie A. , Sahasrabudhe Ashish , Dewhirst Floyd E.
Published: July 2002

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into
Escherichia coli
. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as
Porphyromonas gingivalis
,
Bacteroides forsythus
, and
Treponema denticola
were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.
PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in
Bacillus megaterium
Authors: McCool Gabriel J. , Cannon Maura C.
Published: July 2002

Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for
phaE
in addition to the
phaC
gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from
Bacillus megaterium
required PhaC (PhaC
Bm
) and PhaR (PhaR
Bm
) for activity in vivo and in vitro. PhaC
Bm
showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaR
Bm
had no obvious homology to PhaE. Previously we showed that PhaC
Bm
, and here we show that PhaR
Bm
, is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaC
Bm
to produce an active PHA synthase.
Mutations in the 16S rRNA Genes ofHelicobacter pyloriMediate Resistance to Tetracycline
Authors: Trieber Catharine A. , Taylor Diane E.
Published: July 2002

Low-cost and rescue treatments forHelicobacter pyloriinfections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged inH.pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensusH.pylori16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance onH.pylori26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of severalH.pyloristrains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance inH.pylori.
Respiratory Viral Infections among Pediatric Inpatients and Outpatients in Taiwan from 1997 to 1999
Authors: Tsai Huey-Pin , Kuo Pin-Hwa , Liu Ching-Chuan , Wang Jen-Ren
Published: July 2002

The present study examined the association of specific virus infections with acute respiratory tract conditions among hospitalized and outpatient children in a subtropical country. A total of 2,295 virus infections were detected in 6,986 patients between 1997 and 1999, including infections caused by respiratory syncytial virus (RSV) (1.7%), parainfluenza virus (2.0%), influenza B virus (2.6%), adenovirus (4.0%), herpes simplex virus type 1 (4.4%), influenza A virus (5.5%), and enterovirus (12.7%). There were 61 mixed infections, and no consistent seasonal variation was found. One or more viruses were detected among 24.8% of hospitalized patients and 35.0% of outpatients. The frequencies and profiles of detection of various viruses among in- and outpatients were different. The occurrence of enterovirus infections exceeded that of other viral infections detected in 1998 and 1999 due to outbreaks of enterovirus 71 and coxsackievirus A10. RSV was the most prevalent virus detected among hospitalized children, whereas influenza virus was the most frequently isolated virus in the outpatient group. Most respiratory viral infections (39.3%) occurred in children between 1 and 3 years old. RSV (
P
< 0.025) and influenza A virus (
P
< 0.05) infections were dominant in the male inpatient group. In addition, most pneumonia and bronchiolitis (48.4%) was caused by RSV among hospitalized children less than 6 months old. Adenovirus was the most common agent associated with pharyngitis and tonsilitis (45.5%). These data expand our understanding of the etiology of acute respiratory tract viral infections among in- and outpatients in a subtropical country and may contribute to the prevention and control of viral respiratory tract infections.
Epidemiological Validation of Pulsed-Field Gel Electrophoresis Patterns for Methicillin-Resistant
Staphylococcus aureus
Authors: Blanc D. S. , Struelens M. J. , Deplano A. , De Ryck R. , Hauser P. M. , Petignat C. , Francioli P.
Published: July 2002

To determine the stability of pulsed-field gel electrophoresis (PFGE) patterns of methicillin-resistant
Staphylococcus aureus
in the nosocomial setting, we analyzed isolates from long-term carriers (>1 month) and from patients involved in well-defined nosocomial epidemics. The number of fragment differences between the first isolate and subsequent isolates in long-term carriers showed a bimodal distribution, with one group having 0 to 6 fragment differences and the other group having 14 to 24 fragment differences. The PFGE patterns of isolates involved in epidemics also presented a similar bimodal distribution of the number of fragment differences. Typing these isolates with another molecular method (inter-IS
256
PCR) showed that isolates of the first group (i.e., with 1 to 6 fragment differences) were clonally related, whereas the second group (with 14 to 24 fragment differences) could be considered genetically different. Among long-term carriers with clonally related isolates, 74 of 84 (88%) of consecutive isolates showed indistinguishable patterns, whereas 10 of 84 (12%) showed related patterns differing by one to six fragments. Moreover, the frequency of apparition of related patterns is higher when the time between the first and the subsequent isolate is longer. During seven nosocomial epidemics lasting from 1 to 15 months, only 2 of 120 isolates (1.7%) showed a pattern which was different, although related, from the predominant one involved in each of these outbreaks.
Evaluation of Efficiency of Screening Extended-Spectrum β-Lactamase-Producing
Escherichia coli
and
Klebsiella pneumoniae
in Hospitals Where the Bacteria Are Increasingly Prevalent
Authors: Lee K. , Lim J. K. , Yong D. , Yum J. , Chong Y. , Okamoto R. , Inoue M.
Published: July 2002

The disk screening methods for extended-spectrum β-lactamase-producing strains were evaluated. The confirmatory work is reduced significantly in settings such as those in this study, by changing the cefpodoxime breakpoint to ≤20 mm and by not testing cefoxitin-resistant isolates. Cefotaxime and ceftazidime disk screening is reliable, and the laboratory-prepared cefotaxime- and ceftazidime-clavulanic acid disks are stable at −20°C for 12 weeks.
Evaluation of BacT/ALERT System for Detection of
Mycoplasma hominis
in Simulated Blood Cultures
Authors: Waites Ken B. , Canupp Kay C.
Published: July 2002

We used simulated blood cultures inoculated with clinical isolates of
Mycoplasma hominis
to determine whether liquid media of the BacT/ALERT (Organon Teknika, Durham, N.C.) will support growth of this fastidious organism and whether its presence can generate a positive signal with the instrument. Viability of clinical isolates of
M. hominis
was maintained for 7 days in BacT/ALERT media, and organisms were able to multiply when 1% gelatin was added to neutralize the mycoplasmastatic effects of the sodium polyanetholsulfonate anticoagulant. Without the addition of gelatin to BacT/ALERT bottles, the mycoplasmas declined in numbers or became completely nonviable. Mycoplasmal growth was further enhanced in BacT/ALERT PF both supplemented with gelatin, arginine, and DNA in comparison to broth with only gelatin added. No BacT/ALERT bottles containing
M. hominis
in simulated blood cultures were flagged positive by the instrument, despite growth of microorganisms of up to 10
7
CFU/ml after incubation for up to 7 days, suggesting that inadequate CO
2
production or some other mechanism prevents the instrument from recognizing the presence of the organism and its metabolic products. The fastidious cultivation requirements and relatively slow growth of
M. hominis
warrant that dependence on automated systems and techniques designed to detect conventional bacteria will not be reliable for recovery of
M. hominis
and that specialized media and incubation conditions designed for optimum cultivation of mycoplasmas should be employed when this organism is suspected on clinical grounds.
Molecular Typing of
Penicillium marneffei
Isolates from Thailand by
Not
I Macrorestriction and Pulsed-Field Gel Electrophoresis
Authors: Trewatcharegon Sompong , Sirisinha Stitaya , Romsai Amparat , Eampokalap Boonchaoy , Teanpaisan Rawee , Chaiyaroj Sansanee C.
Published: July 2002

Penicillium marneffei
is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64
P. marneffei
isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme
Not
I. Among the 69 isolates fingerprinted by PFGE, 17 were compared by
Hae
III restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with
Hae
II. Moreover, an impressive diversity of
P. marneffei
isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of
P. marneffei
. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the
P. marneffei
isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and
Not
I macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional
P. marneffei
isolates.
Evaluation of a Fluorescence In Situ Hybridization Assay for Differentiation between Tuberculous and Nontuberculous
Mycobacterium
Species in Smears of Lowenstein-Jensen and Mycobacteria Growth Indicator Tube Cultures Using Peptide Nucleic Acid Probes
Authors: Hongmanee Poonpilas , Stender Henrik , Rasmussen Ole F.
Published: July 2002

A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of
Mycobacterium tuberculosis
complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evaluated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as well as Mycobacteria Growth Indicator Tube (MGIT) liquid cultures from 80 AFB-positive sputum samples.
Mycobacterium
species could be identified from a total of 53 LJ cultures and 77 MGIT cultures. The diagnostic specificities of the MTB and NTM probes were 100% for both cultures. The diagnostic sensitivities of the MTB probe for the LJ and MGIT cultures were 98 and 99%, respectively, whereas the sensitivities of the NTM probe were 57 and 100%, respectively. The relatively low sensitivity of the NTM probe was due to a high proportion of
M. fortuitum
, which is not identified by the probe.
Yield of Vancomycin-Resistant Enterococci and Multidrug-Resistant
Enterobacteriaceae
from Stools Submitted for
Clostridium difficile
Testing Compared to Results from a Focused Surveillance Program
Authors: Hacek Donna M. , Bednarz Patrice , Noskin Gary A. , Zembower Teresa , Peterson Lance R.
Published: July 2002

It has been suggested that a method of performing surveillance for vancomycin-resistant enterococci (VRE) is to screen specimens submitted for
Clostridium difficile
testing. We compared this approach to our focused surveillance program of high-risk units during October 1997 to compare the yield of VRE and multidrug-resistant
Enterobacteriaceae
(MDRE) with both methods. Of the stools submitted for
C. difficile
testing, 14% were positive for VRE or MDRE, whereas rectal swabs from routine surveillance yielded 11% VRE- or MDRE-positive results. Although stools submitted for
C. difficile
testing resulted in a higher percentage of positive cultures, 14 VRE- and 2 MDRE-positive patients from our high-risk population were missed because many patients had no stool submitted for
C. difficile
testing. Therefore, while screening stools submitted for
C. difficile
testing cannot replace our focused surveillance program, it appears advantageous to assess these stools at various intervals to detect new patient reservoirs of drug-resistant organisms that may benefit from routine surveillance cultures.
Possible Horizontal Transfer of the
vanB2
Gene among Genetically Diverse Strains of Vancomycin-Resistant
Enterococcus faecium
in a Korean Hospital
Authors: Lee Wee Gyo , Jernigan John A. , Rasheed James K. , Anderson Gregory J. , Tenover Fred C.
Published: July 2002

A total of 25 isolates of
vanB
-containing
Enterococcus faecium
were recovered from patients in a single Korean hospital over a 20-month period. There were two distinct
vanB2
patterns among the 11 pulsed-field gel electrophoresis types; 17 contained the prototype
vanB2
and 8 contained a novel
vanB2
with a 177-bp deletion in

vanY
B

. Both
vanB2
genes were transmissible in vitro at a mean frequency of 1.1 × 10
−8
transconjugants/donor. These results suggest the horizontal spread of
vanB2
is occurring among genetically diverse strains of
E. faecium
in Korean hospitals.
Recurrent Self-Limited Fungemia Caused by
Yarrowia lipolytica
in a Patient with Acute Myelogenous Leukemia
Authors: Chang Chulhun Ludgerus , Park Tae Hee , Lee Eun Yup , Lim Young Tak , Son Han Chul
Published: July 2002

Yarrowia lipolytica
is a weakly pathogenic yeast that is rarely isolated from the blood. We observed transient recurrent catheter-related fungemia attributable to this organism in a leukemic patient. The fungemia and accompanying fever subsided spontaneously. The data suggest that it might be possible to withhold specific treatment for
Y. lipolytica
fungemia even in an immunocompromised patient.
Controlled Clinical Comparison of BACTEC Plus Anaerobic/F to Standard Anaerobic/F as the Anaerobic Companion Bottle to Plus Aerobic/F Medium for Culturing Blood from Adults
Authors: Wilson Michael L. , Mirrett Stanley , Meredith Frances T. , Weinstein Melvin P. , Scotto Vincenzo , Reller L. Barth
Published: July 2002

To determine the optimal anaerobic companion bottle to pair with BACTEC Plus Aerobic/F medium for recovery of pathogenic microorganisms from adult patients with bacteremia and fungemia, we compared Plus Anaerobic/F bottles with Standard Anaerobic/F bottles, each of which was filled with 4 to 6 ml of blood. The two bottles were paired with a Plus Aerobic/F bottle filled with 8 to 12 ml of blood. A total of 14,011 blood culture sets were obtained. Of these, 11,583 sets were received with all three bottles filled adequately and 12,257 were received with both anaerobic bottles filled adequately. Of 818 clinically important isolates detected in one or both adequately filled anaerobic bottles, significantly more staphylococci (
P
< 0.001), streptococci (
P
< 0.005),
Escherichia coli
isolates (
P
< 0.02),
Klebsiella pneumoniae
isolates (
P
< 0.005), and all microorganisms combined (
P
< 0.001) were detected in Plus Anaerobic/F bottles. In contrast, significantly more anaerobic gram-negative bacilli were detected in Standard Anaerobic/F bottles (
P
< 0.05). Of 397 unimicrobial episodes of septicemia, 354 were detected with both pairs, 30 were detected with Plus Aerobic/F–Plus Anaerobic/F pairs only, and 13 were detected with Plus Aerobic/F–Standard Anaerobic/F pairs only (
P
< 0.05). Significantly more episodes of bacteremia caused by members of the family
Enterobacteriaceae
(
P
< 0.05) and aerobic and facultative gram-positive bacteria (
P
< 0.025) were detected with Plus Anaerobic/F bottles only. In a paired-bottle analysis, 810 of 950 isolates were recovered from both pairs, 90 were recovered from Plus Aerobic/F–Plus Anaerobic/F pairs only, and 50 were recovered from Plus Aerobic/F–Standard Anaerobic/F pairs only (
P
< 0.001). Paired Plus Aerobic/F–Plus Anaerobic/F bottles yielded significantly more staphylococci (
P
< 0.001), streptococci (
P
< 0.05), and members of the family
Enterobacteriaceae
(
P
<0.001). We conclude that Plus Anaerobic/F bottles detect more microorganisms and episodes of bacteremia and fungemia than Standard Anaerobic/F bottles as companion bottles to Plus Aerobic/F bottles in the BACTEC 9240 blood culture system.
Nested Restriction Site-Specific PCR To Detect and Type Hepatitis C Virus (HCV): a Rapid Method To Distinguish HCV Subtype 1b from Other Genotypes
Authors: Krekulova Laura , Rehak Vratislav , Wakil Adil E. , Harris Eva , Riley Lee W.
Published: July 2002

Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a “fingerprint” pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5′ noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5′ NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.
Practical Strategies for Detecting and Confirming Vancomycin-Intermediate
Staphylococcus aureus
: a Tertiary-Care Hospital Laboratory's Experience
Authors: Marlowe Elizabeth M. , Cohen Martin D. , Hindler Janet F. , Ward Kevin W. , Bruckner David A.
Published: July 2002

The clinical microbiology laboratory plays a critical role in the detection of
Staphylococcus aureus
with decreased susceptibility to vancomycin. Staff education and rapid laboratory response are of utmost importance. We report on our laboratory's experience and provide recommendations for the identification and confirmation of vancomycin-intermediate
S. aureus
.
Fluoroquinolone Resistance Is a Poor Surrogate Marker for Type II Topoisomerase Mutations in Clinical Isolates of
Streptococcus pneumoniae
Authors: Millichap John J. , Pestova Ekaterina , Siddiqui Farida , Noskin Gary A. , Peterson Lance R.
Published: July 2002

The association between fluoroquinolone susceptibility and DNA mutations coding for amino acid substitutions in the quinolone resistance-determining region was assessed with 44 clinical isolates of
Streptococcus pneumoniae
. Twenty-three strains bore at least one amino acid substitution. Only seven strains with mutations were suggested by diminished susceptibility to ciprofloxacin (MIC, ≥2 μg/ml).
Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5.8S Ribosomal DNA Typing in Ocular Infections
Authors: Ferrer Consuelo , Colom Francisca , Frasés Susana , Mulet Emilia , Abad José L. , Alió Jorge L.
Published: July 2002

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.
Identification of the Hexon Region of an Adenovirus Involved in a New Outbreak of Keratoconjunctivitis
Authors: Imai Yasuo , Kameya Shuhei , Ohkoshi Masatoshi , Yamaki Kunihiko , Sakuragi Shozo
Published: July 2002

We tested 15 adenovirus (Ad)-positive patients involved in a case of nosocomial spread of keratoconjunctivitis. A neutralization test, PCR-restriction fragment length polymorphism analysis, and sequencing of the hypervariable regions of the hexons were performed in order to identify the type of Ad involved. The serotype of the Ad was not identical to any published Ad sequence by either method.
Simplified Microneutralization Test for Serotyping Adenovirus Isolates
Authors: Malasig Marietta D. , Goswami Pulak R. , Crawford-Miksza Leta K. , Schnurr David P. , Gray Gregory C.
Published: July 2002

A simplified microneutralization procedure is described that uses an empirically determined virus challenge dose, a single dilution of antiserum, and observation of cytopathic effect to determine the adenovirus serotype. The simplified test has faster turnaround time and was 96% concordant with a confirmatory test using serial dilutions of type-specific sera. This method will find utility in high-volume serotyping work.
Fatal Pulmonary
Nocardia farcinica
Infection
Authors: De La Iglesia Pedro , Viejo Guillermo , Gomez Beatriz , De Miguel Dolores , Del Valle Asuncion , Otero Luis
Published: July 2002

Nocardia farcinica
infections are rare and potentially life threatening. Identification is based on growth at 45°C, opacification of Middlebrook 7H10 agar, and resistance to antibiotics. We describe a case of fatal pulmonary
N. farcinica
infection in a patient with pneumoconiosis that was diagnosed by culture of sputum onto selective media.