bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Metal Ligands in the
Clostridium histolyticum
ColH Collagenase
Authors: Jung Chang-Min , Matsushita Osamu , Katayama Seiichi , Minami Junzaburo , Sakurai Jun , Okabe Akinobu
Published: December 2019

A
Clostridium histolyticum
116-kDa collagenase has an H
415
EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His
415
or His
419
abolished catalytic activity and zinc binding, while that affecting Glu
416
did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu
447
decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu
446
or Glu
451
had smaller effects on activity and zinc binding. These mutations caused a decrease in
k
cat
but no significant change in

K
m

. These results are consistent with the hypothesis that Glu
447
is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu
446
and Glu
451
suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.
Regulation of Expression of the Fructan Hydrolase Gene of
Streptococcus mutans
GS-5 by Induction and Carbon Catabolite Repression
Authors: Burne Robert A. , Wen Zezhang Thomas , Chen Yi-Ywan M. , Penders Jana E. C.
Published: December 2019

The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the
fruA
gene of
Streptococcus mutans
GS-5. The purpose of this study was to characterize the DNA immediately flanking
fruA
, to explore the regulation of expression of
fruA
by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3′ to
fruA
was an open reading frame (ORF) with similarity to β-fructosidases which was cotranscribed with
fruA
. A transcriptional initiation site, located an appropriate distance from an extended −10-like promoter, was mapped at 165 bp 5′ to the
fruA
structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5′ untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative
cis
elements in gram-positive bacteria, were located close to the promoter. The levels of production of
fruA
mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of
fruA
promoter regions, lacking sequences 5′ or 3′ to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the
fru
operon requires the CRE that is located 3′ to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in
fruA
induction.
trans
-Acting Factors Affecting Carbon Catabolite Repression of the
hut
Operon in
Bacillus subtilis
Authors: Zalieckas Jill M. , Wray Lewis V. , Fisher Susan H.
Published: December 2019

In
Bacillus subtilis
, CcpA-dependent carbon catabolite repression (CCR) mediated at several
cis
-acting carbon repression elements (
cre
) requires the seryl-phosphorylated form of both the HPr (
ptsH
) and Crh (
crh
) proteins. During growth in minimal medium, the
ptsH1
mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (
hut
) enzymes in cells grown in minimal medium showed that neither the
ptsH1
nor the
crh
mutation individually had any affect on
hut
CCR but that
hut
CCR was abolished in a
ptsH1 crh
double mutant. In contrast, the
ptsH1
mutation completely relieved
hut
CCR in cells grown in Luria-Bertani medium. The
ptsH1 crh
double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in
B. subtilis
mutants which synthesize low levels of active glutamine synthetase (
glnA
). In addition, these
glnA
mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of
glnA
mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.
Ferric Enterobactin Binding and Utilization by
Neisseria gonorrhoeae
Authors: Carson Susan D. Biegel , Klebba Philip E. , Newton Salete M. C. , Sparling P. Frederick
Published: December 2019

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both
Neisseria gonorrhoeae
and
Neisseria meningitidis
, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor.
N. gonorrhoeae
FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 μM, but growth stimulation was abolished when an omega (Ω) cassette was inserted within
fetA
or when
tonB
was insertionally interrupted. FA1090 FetA specifically bound
59
Fe-enterobactin, with a

K
d

of approximately 5 μM. Monoclonal antibodies raised against the
Escherichia coli
enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of
fetA
, designated
fetB
, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Ω insertion within
fetB
abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.
Analyses of a Polyhydroxyalkanoic Acid Granule-Associated 16-Kilodalton Protein and Its Putative Regulator in the
pha
Locus of
Paracoccus denitrificans
Authors: Maehara Akira , Ueda Shunsaku , Nakano Hideo , Yamane Tsuneo
Published: December 2019

The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of
Paracoccus denitrificans
was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (
phaC
Pd
) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774–779, 1996). Gene walking around
phaC
Pd
revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the
phaP
Pd
gene, encoding GA16 protein, and the other was the
phaR
Pd
gene, encoding a protein that is putatively involved in the regulation of the expression of
phaP
Pd
. Overproduction of PhaP
Pd
was observed in
Escherichia coli
carrying
phaP
Pd
, but the overproduction was not observed in the presence of
phaR
Pd
. Coexpression of
phaP
Pd
and PHA biosynthesis genes in
E. coli
caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. The
phaR
Pd
gene had significant similarities to
stdC
, a possible transcriptional factor of
Comamonas testosteroni
, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria.
Carboxy-Terminal Processing of the Large Subunit of [Fe] Hydrogenase from
Desulfovibrio desulfuricans
ATCC 7757
Authors: Hatchikian E. Claude , Magro Valérie , Forget Nicole , Nicolet Yvain , Fontecilla-Camps Juan C.
Published: December 2019

hydA
and
hydB
, the genes encoding the large (46-kDa) and small (13.5-kDa) subunits of the periplasmic [Fe] hydrogenase from
Desulfovibrio desulfuricans
ATCC 7757, have been cloned and sequenced. The deduced amino acid sequence of the genes product showed complete identity to the sequence of the well-characterized [Fe] hydrogenase from the closely related species
Desulfovibrio vulgaris
Hildenborough (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515–520, 1985). The data show that in addition to the well-known signal peptide preceding the NH
2
terminus of the mature small subunit, the large subunit undergoes a carboxy-terminal processing involving the cleavage of a peptide of 24 residues, in agreement with the recently reported data on the three-dimensional structure of the enzyme (Y. Nicolet, C. Piras, P. Legrand, E. C. Hatchikian, and J. C. Fontecilla-Camps, Structure 7:13–23, 1999). We suggest that this C-terminal processing is involved in the export of the protein to the periplasm.
tRNA Synthetase Mutants of
Escherichia coli
K-12 Are Resistant to the Gyrase Inhibitor Novobiocin
Authors: Milija Jovanovic , Lilic Mirjana , Janjusevic Radmila , Jovanovic Goran , Savic Dragutin J.
Published: December 2019

In previous studies we demonstrated that mutations in the genes
cysB
,
cysE
, and
cls
(
nov
) affect resistance of
Escherichia coli
to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307–311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766–1771, 1995). In this work we expand this list with mutations in
rpoN
(the gene for RNA polymerase subunit ς
54
) and the tRNA synthetase genes
alaS
,
argS
,
ileS
, and
leuS
. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the
cls
gene diminishes resistance to novobiocin but not to mecillinam.
Abiotrophia elegans
sp. nov., a Possible Pathogen in Patients with Culture-Negative Endocarditis
Authors: Roggenkamp Andreas , Abele-Horn Marianne , Trebesius Karl-Heinz , Tretter Ursula , Autenrieth Ingo B. , Heesemann Jürgen
Published: December 2019

We isolated a hitherto undescribed microorganism from a patient with endocarditis. The microscopic appearance, a negative catalase reaction, and growth as satellite colonies next to
Staphylococcus epidermidis
suggested that this microorganism is a member of the genus
Abiotrophia
, formerly known as nutritionally variant streptococci. However, the clinical isolate described herein differed markedly from the known
Abiotrophia
spp.,
A. adiacens
and
A. defectiva
, in terms of its (i) biochemical properties, (ii) restricted growth temperature range, (iii) whole-cell lysate polypeptide profile, and (iv) unique nutritional requirements. In contrast to the type strains of
A. adiacens
and
A. defectiva
, which used
l
-cysteine and pyridoxal hydrochloride as growth factors, the growth of the clinical isolate was only supported by
l
-cysteine hydrochloride and not by pyridoxal hydrochloride when the organism was tested in Todd-Hewitt or casein-soy peptone broth. Comparative 16S rRNA gene sequence analysis revealed that the microorganism was a member of the genus
Abiotrophia
and was most closely related to
A. adiacens
(96.9% homology). Phenotypic and phylogenetic data are consistent with the assumption of a new species within the genus
Abiotrophia
, for which we propose the name
Abiotrophia elegans
sp. nov. The unique nutritional requirements of this strain are of importance for diagnostic laboratories. The media of blood culture systems supplemented only with pyridoxal hydrochloride as a growth factor may fail to promote the growth of
A. elegans
sp. nov., and thus, these systems might not detect this microorganism as a possible cause of endocarditis.
Etiology of Acute Gastroenteritis in Hospitalized Children in Melbourne, Australia, from April 1980 to March 1993
Authors: Barnes Graeme L. , Uren Eric , Stevens Kerrie B. , Bishop Ruth F.
Published: December 2019

Acute infectious diarrhea is common in children. Control requires knowledge of causes. Few comprehensive long-term studies of etiology have been undertaken in developed countries. This report is of a 13-year survey of 4,637 children from 0 to 14 years of age, admitted to a large children’s hospital for treatment of gastroenteritis, in which viruses, bacteria, and parasites were sought. A recognized enteric pathogen was identified in 56.6% of children. Group A rotaviruses occurred in 39.6% of children overall and in 55% of children 12 to 23 months of age. They were a frequent cause (18.7%) of acute gastroenteritis in children under 6 months and in those aged 5 to 13 years (16%). Rotaviruses were almost entirely responsible for winter admission peaks. Enteric adenovirus types 40 and 41 (6% overall) were more frequent in children under 12 months (9.4%).
Salmonella
spp. (5.8%) and
Campylobacter jejuni
(3.4%) were more common in children over 5 years (13.1% and 6.7%, respectively). The 43.5% of cases (60% in children under 6 months) where no enteric pathogen was identified are cause for concern. The involvement of small viruses (including caliciviruses and astroviruses) may be clarified when molecular biology techniques are utilized to address this gap in our knowledge. This comprehensive 13-year study of the cause of acute infectious diarrhea in children in developed countries reinforces the importance of rotavirus and highlights a large group for whom the etiology remains unknown, an issue of particular concern with babies under 6 months of age. New techniques have the potential to identify old and new pathogens causing disease in these vulnerable infants.
Multicenter Evaluation of the
Clostridium difficile
TOX A/B TEST
Authors: Lyerly D. M. , Neville L. M. , Evans D. T. , Fill J. , Allen S. , Greene W. , Sautter R. , Hnatuck P. , Torpey D. J. , Schwalbe R.
Published: December 2019

Clostridium difficile
, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the “gold standard” for
C. difficile
testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA−/toxB+) isolates were identified. One specimen was from a patient with a clinical history consistent with
C. difficile
infection. Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA−/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins. Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for
C. difficile
disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs.
Confirmation of the Distinct Genotype Groups within the Form Species
Candida parapsilosis
Authors: Roy Basabi , Meyer Sally A.
Published: December 2019

The DNA relatedness and restriction fragment length polymorphism patterns of whole-cell DNA and the general phenotypic properties of 14 isolates of the form species
Candida parapsilosis
representing three diverse genetic groups were compared. The data confirm the unrelatedness of the three groups at the species level.
Association of Borderline Oxacillin-Susceptible Strains ofStaphylococcus aureuswith Surgical Wound Infections
Authors: Kernodle Douglas S. , Classen David C. , Stratton Charles W. , Kaiser Allen B.
Published: December 2019

Staphylococcus aureusisolates which produce type A staphylococcal β-lactamase have been associated with wound infections complicating the use of cefazolin prophylaxis in surgery. To further evaluate this finding, 215 wound isolates from 14 cities in the United States were characterized by antimicrobial susceptibility and β-lactamase type and correlated with the preoperative prophylactic regimen. Borderline-susceptibleS. aureusisolates of phage group 5 (BSSA-5), which produce large amounts of type A β-lactamase and exhibit borderline susceptibility to oxacillin, comprised a greater percentage of the 120 wound isolates associated with cefazolin prophylaxis than they did of the 95 isolates associated with other prophylactic regimens (25% versus 12.6%, respectively;P< 0.05). In contrast, methicillin-resistantS. aureusisolates were distributed evenly between the two groups (8.3% versus 11.6%, respectively). In vitro assays demonstrated that cefazolin was hydrolyzed faster by BSSA-5 strains than by other β-lactamase-producing, methicillin-susceptible strains (1.54 versus 0.50 μg/min/108CFU, respectively;P< 0.0001). These data demonstrate that BSSA-5 strains are a distinct subpopulation of methicillin-susceptibleS. aureuswhich frequently cause deep surgical wound infections. Cefazolin use in prophylaxis is a risk factor for BSSA-5 infection.
Allele-Specific PCR Method Based on
pncA
and
oxyR
Sequences for Distinguishing
Mycobacterium bovis
from
Mycobacterium tuberculosis
: Intraspecific
M. bovis pncA
Sequence Polymorphism
Authors: de los Monteros Luz Elena Espinosa , Galán Juan Carlos , Gutiérrez Montserrat , Samper Sofía , García Marín Juan F. , Martín Carlos , Domínguez Lucas , de Rafael Luis , Baquero Fernando , Gómez-Mampaso Enrique , Blázquez Jesús
Published: December 2019

An allele-specific amplification method based on two genetic polymorphisms to differentiate
Mycobacterium tuberculosis
from
Mycobacterium bovis
was tested. Based on the differences found at position 169 in the
pncA
genes from
M. tuberculosis
and
M. bovis
, a PCR system which was able to differentiate most of the 237
M. tuberculosis
complex isolates tested in one of the two species was developed. All 121
M. tuberculosis
strains showed the expected base (cytosine) at position 169. Most of the
M. bovis
isolates had a guanine at the cited position. Nevertheless, 18 of the 116
M. bovis
isolates, all of them goat isolates, showed the
pncA
polymorphism specific to
M. tuberculosis
. These results suggest that goat
M. bovis
may be the nicotinamidase-missing link at the origin of the
M. tuberculosis
species. Based on the polymorphism found at position 285 in the
oxyR
gene, the same system was used to differentiate
M. tuberculosis
from
M. bovis
. In this case, DNAs from all 121
M. tuberculosis
isolates had the expected base (guanine) at this position. In addition, all 116
M. bovis
isolates, including those from goats, showed the identical polymorphism (adenine). The
oxyR
allele-specific amplification method can differentiate
M. bovis
from
M. tuberculosis
, is rapid (results can be obtained in less than 3 h), and is easy to perform.
Structure and Function of Plasmid pColD157 of Enterohemorrhagic
Escherichia coli
O157 and Its Distribution among Strains from Patients with Diarrhea and Hemolytic-Uremic Syndrome
Authors: Hofinger Clemens , Karch Helge , Schmidt Herbert
Published: December 2019

In this study, pColD157, a 6.7-kb colicinogenic plasmid of enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 strain CL40cu, was characterized by restriction mapping and determination of its complete nucleotide sequence. The sequence consists of 6,675 bp and shows a high degree of similarity to the nucleotide sequence of colicinogenic plasmids pColD-CA23 and pColK. Seven potential genes were located on pColD157, three of which were closely related (>97.9%) to the colicin D structural gene and the corresponding immunity and lysis genes of plasmid pColD-CA23, and these were therefore designated
cda
,
cdi
, and
cdl
, respectively, using the reference extension -CL40 for differentiation. The adjacent 3′ region is related to the origin of replication of pColD-CA23. In contrast, the remaining part of the plasmid harbors a cluster of genes, closely related to the mobilization genes of pColK, which is followed by a 0.3-kb stretch homologous to the pColK resolution function. These determinants were designated
mbdA
,
mbdB
,
mbdC
, and
mbdD
and
cdr
, respectively. Southern blot analysis was performed with a probe specific for the
cda
gene of pColD157 and two groups of EHEC O157:H7 isolates from patients with diarrhea or hemolytic-uremic syndrome resident in Germany. Whereas 16 of 46
E. coli
O157 strains isolated between 1987 and 1991 harbored plasmid pColD157, only 1 of 50 strains isolated during 1996 carried this plasmid. In addition, all strains harboring plasmid pColD157 were shown to have colicinogenic activity.
Development of an Immunomagnetic Method for Selective Isolation of
Actinobacillus pleuropneumoniae
Serotype 1 from Tonsils
Authors: Gagné Annie , Lacouture Sonia , Broes André , D’Allaire Sylvie , Gottschalk Marcelo
Published: December 2019

An immunomagnetic separation technique (IMS) for the selective isolation of
Actinobacillus pleuropneumoniae
serotype 1 was developed. Superparamagnetic polystyrene beads (immunomagnetic beads [IMBs]) were coated with purified rabbit immunoglobulin G specific for
A. pleuropneumoniae
serotype 1. The antibody concentration, the number of IMBs, the incubation time, and the temperature of incubation influenced the recovery of the target bacteria. The sensitivity of the IMS technique was 1,000-fold higher than that of direct culture. When tonsils from animals from infected herds were tested, significantly more positive tonsils were detected by the IMS technique (68%) than by the standard procedures (22%). The method represents an innovative and highly sensitive approach for the isolation of
A. pleuropneumoniae
from carrier animals.
Isolation of
Serpulina pilosicoli
from Rectal Biopsy Specimens Showing Evidence of Intestinal Spirochetosis
Authors: Trivett-Moore Nicolle L. , Gilbert Gwendolyn L. , Law Carmella L. H. , Trott Darren J. , Hampson David J.
Published: December 2019

Histologic evidence of intestinal spirochetosis (IS) was found in 22 of 41 (53.7%) rectal biopsy specimens from homosexual men attending a sexually transmitted diseases clinic.
Serpulina pilosicoli
was cultured from 11 of the IS-positive biopsy specimens (50%) and from 2 specimens (10.5%) in which spirochetes were not observed. The association between seeing spirochetes in biopsy specimens and isolating
S. pilosicoli
was statistically significant, clearly indicating that this spirochete is the agent of IS.
Species-Specific Plasmid Sequences for PCR Identification of the Three Species of
Borrelia burgdorferi
Sensu Lato Involved in Lyme Disease
Authors: Misonne Marie-Christine , Hoet Philippe Pierre
Published: December 2019

Species-specific sequences were shown to be carried by plasmids of the three main species of
Borrelia burgdorferi
sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of
B. burgdorferi
sensu stricto,
Borrelia garinii
, and
Borrelia afzelii
, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for
B. burgdorferi
sensu stricto and 1,000 spirochetes for
B. garinii
and
B. afzelii
. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.
Premarket Evaluation of a Commercial Glycoprotein G-Based Enzyme Immunoassay for Herpes Simplex Virus Type-Specific Antibodies
Authors: Ashley Rhoda L. , Wu Linxian , Pickering Jerry W. , Tu Ming-Chieh , Schnorenberg Laura
Published: December 2019

A new commercial glycoprotein G-based enzyme immunoassay (gG-EIA) was compared with Western blotting (WB) for detection of herpes simplex virus type 1 (HSV-1) or HSV-2 type-specific antibodies in 193 serum samples. Sensitivity for HSV-1 was 95%; specificity was 96%. Sensitivity for HSV-2 was 98%; specificity was 97%. Twelve of 13 serum samples with equivocal gG-EIA results were serotyped by WB.
Evaluation of the BIOMIC Video Reader System for Determining Interpretive Categories of Isolates on the Basis of Disk Diffusion Susceptibility Results
Authors: Korgenski E. Kent , Daly Judy A.
Published: December 2019

The BIOMIC System (Giles Scientific, New York, N.Y.) includes software and a video-assisted plate reader that functions with a personal computer to automate, speed read, and interpret standard antibiotic disk diffusion test plates. The video reader helps standardize endpoints, speeds quantitative measurements by 40 to 90%, and reduces fatigue and transcription and interpretation errors (H. Wei-Fang, Am. Clin. Lab. 13:28–29, 1994). Organisms tested were isolated from patient specimens collected at Primary Children’s Medical Center and included rapidly growing gram-positive and gram-negative strains that fulfill the National Committee for Clinical Laboratory Standards guidelines for disk diffusion susceptibility testing. A comparison of the plate reader-determined zones and visually measured zones for 3,339 organism-antimicrobial agent combinations was performed. The results demonstrated 0.1% (4 of 3,339) false-susceptible reads and 0.2% (6 of 3,339) false-resistant reads by the video reader compared with visual reads. Minor discrepancies (4.7% [156 of 3339]), resulting in category interpretation changes of intermediate to resistant or susceptible or changes of resistant or susceptible to intermediate, were also encountered. Of the discrepant results, 80.8% (139 of 172) resulted from a 3-mm or less zone diameter difference between the two different techniques. We conclude that the video-assisted plate reader is a reliable system for determining interpretative categories from zone diameters of standard antibiotic disk diffusion test plates.
Absolute Copy Number and Relative Change in Determinations of Human Immunodeficiency Virus Type 1 RNA in Plasma: Effect of an External Standard on Kit Comparisons
Authors: Brambilla Donald , Leung Sheila , Lew Judy , Todd John , Herman Steven , Cronin Mike , Shapiro David E. , Bremer James , Hanson Celine , Hillyer George V. , McSherry George D. , Sperling Rhoda S. , Coombs Robert W. , Reichelderfer Patricia S.
Published: December 2019

Use of a common set of human immunodeficiency virus type 1 (HIV-1) RNA standards eliminated differences among absolute HIV-1 RNA copy number estimates made with three commercially available assays. The relative changes in the viral RNA levels determined by the commercial assays were similar and were unaffected by the use of a common set of standards.
Evaluation of a New Method for Rapid Drug Susceptibility Testing of
Mycobacterium avium
Complex Isolates by Using the Mycobacteria Growth Indicator Tube
Authors: Piersimoni Claudio , Nista Domenico , Bornigia Stefano , De Sio Giuseppina
Published: December 2019

The reliability of the Mycobacteria Growth Indicator Tube (MGIT [BBL]) for rapid drug susceptibility testing of
Mycobacterium avium
complex (MAC) isolates was evaluated. MICs of amikacin, clarithromycin, clofazimine, ethambutol, and rifabutin were determined by the MGIT system for 16 MAC strains. The results were compared with those obtained by the BACTEC broth macrodilution method. The turnaround times were 6 to 8 days (median, 7 days) for the MGIT and 5 to 7 days (median, 6 days) for the BACTEC system. Agreements with BACTEC system-determined MICs, within ±1 log
2
dilution, were 100, 100, 88, 63, and 44% for amikacin, clofazimine, rifabutin, clarithromycin, and ethambutol, respectively. Within ±2 log
2
dilutions, agreement with BACTEC system-determined MICs increased to 100% for all the tested drugs. In addition, if MGIT-determined MICs were evaluated according to the thresholds adopted for the interpretation of BACTEC system-determined ones, ethambutol was the only drug for which susceptible strains were frequently misclassified as resistant. It is concluded that the MGIT system is a promising, nonradiometric alternative to the BACTEC method for rapid susceptibility testing of MAC isolates; however, additional studies are required to confirm our results and to determine the optimal criteria for the interpretation of ethambutol MICs.
Molecular Epidemiology of Recent Belgian Isolates of
Neisseria meningitidis
Serogroup B
Authors: Van Looveren M. , Vandamme P. , Hauchecorne M. , Wijdooghe M. , Carion F. , Caugant D. A. , Goossens H.
Published: December 2019

In Belgium an increase in the incidence of meningococcal disease has been noted since the early 1990s. Four hundred twenty clinical strains isolated during the period from 1990 to 1995, along with a set of 30 European reference strains, and 20 Dutch isolates were examined by random-primer and repetitive-motif-based PCR. A subset was investigated by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis. The data were compared with results obtained by serotyping (M. Van Looveren, F. Carion, P. Vandamme, and H. Goossens, Clin. Microbiol. Infect. 4:224–228, 1998). Both phenotypic and molecular epidemiological data suggest that the lineage III of
Neisseria meningitidis
, first encountered in The Netherlands in about 1980, has been introduced in Belgium. The epidemic clone, as defined by oligonucleotide D8635-primed PCR, encompasses mainly phenotypes B:4:P1.4 and B:nontypeable:P1.4, but strains with several other phenotypes were also encountered. Therefore, serotyping alone would underestimate the prevalence of the epidemic clone.
Prevalence of
Candida dubliniensis
Isolates in a Yeast Stock Collection
Authors: Odds Frank C. , Van Nuffel Luc , Dams Géry
Published: December 2019

To establish the historical prevalence of the novel yeast species
Candida dubliniensis
, a survey of 2,589 yeasts originally identified as
Candida albicans
and maintained in a stock collection dating back to the early 1970s was undertaken. A total of 590 yeasts, including 93 (18.5%) β-glucosidase-negative isolates among 502 isolates that showed abnormal colony colors on a differential chromogenic agar and 497 other isolates, were subjected to DNA fingerprinting with the moderately repetitive sequence Ca3. On this basis, 53 yeasts were reidentified as
C. dubliniensis
(including the
C. dubliniensis
type strain, included as a blind control in the panel of yeasts). The 52 newly found isolates came from 36 different persons, and a further 3
C. dubliniensis
isolates were detected by DNA fingerprinting of previously untested isolates from one of these individuals. The prevalence of
C. dubliniensis
among yeasts in oral and fecal samples was significantly higher than that among yeasts from other anatomical sites and was significantly higher among human immunodeficiency virus (HIV)-infected individuals than among known or presumed HIV-negative individuals. However, a single vaginal isolate and two oral isolates from healthy volunteers confirmed that the species is restricted neither to gastrointestinal sites nor to patients with overt disease. The oldest examples of
C. dubliniensis
were from oral samples of three patients in the United Kingdom in 1973 and 1975. In comparison with age-matched control isolates of
C. albicans
, the
C. dubliniensis
isolates showed slightly higher levels of susceptibility in vitro to amphotericin B and flucytosine and slightly lower levels of susceptibility to three azole antifungal agents.
Fluconazole Susceptibility Testing of
Cryptococcus neoformans
: Comparison of Two Broth Microdilution Methods and Clinical Correlates among Isolates from Ugandan AIDS Patients
Authors: Jessup C. J. , Pfaller M. A. , Messer S. A. , Zhang J. , Tumberland M. , Mbidde E. K. , Ghannoum M. A.
Published: December 2019

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of
Cryptococcus neoformans
isolates to fluconazole. A total of 149 isolates of
C. neoformans
var.
neoformans
from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing
C. neoformans
against fluconazole.
Comparison of In Vitro Activities of the New Triazole SCH56592 and the Echinocandins MK-0991 (L-743,872) and LY303366 against Opportunistic Filamentous and Dimorphic Fungi and Yeasts
Authors: Espinel-Ingroff Ana
Published: December 2019

The in vitro antifungal activities of SCH56592, MK-0991, and LY303366 against 83 isolates of
Acremonium strictum
,
Aspergillus flavus
,
Aspergillus fumigatus
,
Aspergillus terreus
,
Bipolaris
spp.,
Blastomyces dermatitidis
,
Cladophialophora bantiana
,
Fusarium oxysporum
,
Fusarium solani
,
Histoplasma capsulatum
,
Phialophora
spp.,
Pseudallescheria boydii
,
Rhizopus arrhizus
,
Scedosporium prolificans
, and
Sporothrix schenckii
were compared. The in vitro activities of these agents against 104 isolates of yeast pathogens of
Candida
spp.,
Cryptococcus neoformans
, and
Trichosporon beigelii
were also compared. MICs were determined by following a procedure under evaluation by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution testing of the filamentous fungi (visual MICs) and the NCCLS M27-A broth microdilution method for yeasts (both visual and turbidimetric MICs). The in vitro fungicidal activity of SCH56592 was superior (minimum fungicidal concentrations [MFCs], 0.25 to 4 μg/ml for 7 of 18 species tested) to those of MK-0991 and LY303366 (MFCs, 8 to >16 μg/ml for all species tested) for the molds tested, but the echinocandins had a broader spectrum of fungicidal activity (MFCs at which 90% of strains are inhibited [MFC
90
s], 0.5 to 4 μg/ml for 6 of 9 species tested) than SCH56592 (MFC
90
s, 0.25 to 8 μg/ml for 4 of 9 species tested) against most of the yeasts tested. Neither echinocandin had in vitro activity (MICs, >16 μg/ml) against
C. neoformans
and
T. beigelii
, while the SCH56592 MICs ranged from 0.12 to 1.0 μg/ml for these two species. The MICs of the three agents for the other species ranged from <0.03 to 4 μg/ml. These results suggest that these new agents have broad-spectrum activities in vitro; their effectiveness in the treatment of human mycoses is to be determined.
Comparison of Different DNA Fingerprinting Techniques for Molecular Typing of
Bartonella henselae
Isolates
Authors: Sander Anna , Ruess Michael , Bereswill Stefan , Schuppler Markus , Steinbrueckner Bernhard
Published: December 2019

Seventeen isolates of
Bartonella henselae
from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the
B. henselae
isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping
B. henselae
strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of
B. henselae
strains is high. The methods applied were found useful for typing
B. henselae
isolates, providing tools for epidemiological and clinical follow-up studies.
Plaque Formation by and Plaque Cloning of
Chlamydia trachomatis
Biovar Trachoma
Authors: Matsumoto Akira , Izutsu Hiroshi , Miyashita Naoyuki , Ohuchi Masanobu
Published: December 2019

A new technique for the induction of plaque formation by
Chlamydia trachomatis
biovar trachoma applicable to the titration of infectivity and cloning of biovar trachoma was established. Three novel strains were cloned and confirmed to be free of glycogen inclusions. The lack of glycogen accumulation correlated with the absence of a 7.5-kb plasmid, which is highly conserved in other strains of
C. trachomatis
. Although the growth efficiency of these plasmid-free strains was slightly lower than that of plasmid-positive strains, possession of the plasmid and glycogen accumulation were not essential for the survival of
C. trachomatis
.
Genotyping of 27 Human Papillomavirus Types by Using L1 Consensus PCR Products by a Single-Hybridization, Reverse Line Blot Detection Method
Authors: Gravitt P. E. , Peyton C. L. , Apple R. J. , Wheeler C. M.
Published: December 2019

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5
+
/6
+
) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of β-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132–152,
in
C. S. Herrington and J. O. D. McGee (ed.),
Diagnostic Molecular Pathology: a Practical Approach
, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78,
P
< 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/μl) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.
IS
1245
Restriction Fragment Length Polymorphism Typing of
Mycobacterium avium
Isolates: Proposal for Standardization
Authors: van Soolingen Dick , Bauer Jeanett , Ritacco Viviana , Leão Sylvia Cardoso , Pavlik Ivo , Vincent Veronique , Rastogi Nalin , Gori Andrea , Bodmer Thomas , Garzelli Carlo , Garcia Maria J.
Published: December 2019

Mycobacterium avium
has become a major human pathogen, primarily due to the emergence of the AIDS epidemic. Restriction fragment length polymorphism (RFLP) typing, using insertion sequence IS
1245
as a probe, provides a powerful tool in the molecular epidemiology of
M. avium
-related infections and will facilitate well-founded studies into the sources of
M. avium
infections in animal and environmental reservoirs. The standardization of this technique allows computerization of IS
1245
RFLP patterns for comparison on a local level and the establishment of
M. avium
DNA fingerprint databases for interlaboratory comparison. Moreover, by combining international DNA typing results of
M. avium
complex isolates from a broad spectrum of sources, long-lasting questions on the epidemiology of this major agent of mycobacterial infections will be answered.
Staphylococcal Scalded-Skin Syndrome Complicating Wound Infection in a Preterm Infant with Postoperative Chylothorax
Authors: Peters Bjoern , Hentschel Juliane , Mau Harald , Halle Elke , Witte Wolfgang , Obladen Michael
Published: December 2019

The course of infection in a 3-week-old premature newborn suffering from extensive dermatitis with flaccid blisters is described.
Staphylococcus aureus
was recovered from a local wound infection around a chest tube inserted to drain a postoperative chylothorax. The strain isolated tested positive for the
eta
gene for exfoliative toxin A, the causative agent of staphylococcal scalded-skin syndrome (SSSS). In this case, prematurity and loss of chylus with consecutive lymphopenia may have contributed to development of SSSS.
Genotyping of
Chlamydia trachomatis
in Urine Specimens Will Facilitate Large Epidemiological Studies
Authors: Morré Servaas A. , Moes Robert , Van Valkengoed Irene , Boeke Joan P. , van Eijk Jacques T. M. , Meijer Chris J. L. M. , Van den Brule Adriaan J. C.
Published: December 2019

The serovars of
Chlamydia trachomatis
-positive urine specimens (
n
= 81; as detected by PCR and ligase chain reaction) were successfully analyzed in 94% of cases by
omp1
PCR-based RFLP analysis. The use of urine specimens and this simple and sensitive typing method will greatly facilitate epidemiological studies of
C. trachomatis
serovar distribution in asymptomatic
C. trachomatis
infections in both females and males.
Detection of Lassa Virus Antinucleoprotein Immunoglobulin G (IgG) and IgM Antibodies by a Simple Recombinant Immunoblot Assay for Field Use
Authors: ter Meulen J. , Koulemou K. , Wittekindt T. , Windisch K. , Strigl S. , Conde S. , Schmitz H.
Published: December 2019

The nucleoprotein of Lassa virus, strain Josiah, was expressed in
Escherichia coli
as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 μg of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 μg. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative predictive value, a single IgM immunoblot result will be valuable for excluding acute Lassa fever for cases of FUO in areas where Lassa fever is endemic.