bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of
Yersinia pestis
Authors: Chapman David A. G. , Zavialov Anton V. , Chernovskaya Tatiana V. , Karlyshev Andrey V. , Zav’yalova Galina A. , Vasiliev Anatoly M. , Dudich Igor V. , Abramov Vyacheslav M. , Zav’yalov Vladimir P. , MacIntyre Sheila
Published: December 2019

The periplasmic molecular chaperone Caf1M of
Yersinia pestis
is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 β-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 β-strands had been deleted, was constructed. Expression of the mutated
caf1M
in
Escherichia coli
resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.
Control of Nitrogen Catabolite Repression Is Not Affected by the tRNA
Gln-CUU
Mutation, Which Results in Constitutive Pseudohyphal Growth of
Saccharomyces cerevisiae
Authors: Beeser Alexander E. , Cooper Terrance G.
Published: December 2019

Saccharomyces cerevisiae
responds to nitrogen availability in several ways. (i) The cell is able to distinguish good nitrogen sources from poor ones through a process designated nitrogen catabolite repression (NCR). Good and poor nitrogen sources do not demonstrably affect the cell cycle other than to influence the cell’s doubling time. (ii) Nitrogen starvation promotes the initiation of sporulation and pseudohyphal growth. (iii) Nitrogen starvation strongly affects the cell cycle; nitrogen-starved cells arrest in G
1
. A specific allele of the
SUP70/CDC65
tRNA
Gln
gene (
sup70-65
) has been reported to be defective in nitrogen signaling associated with pseudohyphal formation, sporulation, and NCR. Our data confirm that pseudohyphal growth occurs gratuitously in
sup70-65
mutants cultured in nitrogen-rich medium at 30°C. However, we find neither any defect in NCR in the
sup70-65
mutant nor any alteration in the control of
YVH1
expression, which has been previously shown to be specifically induced by nitrogen starvation.
Cloning and Characterization of Two Bistructural S-Layer-RTX Proteins from
Campylobacter rectus
Authors: Braun Martin , Kuhnert Peter , Nicolet Jacques , Burnens André P. , Frey Joachim
Published: December 2019

Campylobacter rectus
is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes,
csxA
and
csxB
(for
C. rectus
S-layer and RTX protein) were cloned from
C. rectus
and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of
C. fetus
and Crs of
C. rectus
. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca
2+
. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All
C. rectus
isolates studied contained copies of either the
csxA
or
csxB
gene or both;
csx
genes were absent from all other
Campylobacter
and
Helicobacter
species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.
Mitomycin Resistance in
Streptomyces lavendulae
Includes a Novel Drug-Binding-Protein-Dependent Export System
Authors: Sheldon Paul J. , Mao Yingqing , He Min , Sherman David H.
Published: December 2019

Sequence analysis of
Streptomyces lavendulae
NRRL 2564 chromosomal DNA adjacent to the mitomycin resistance locus
mrd
(encoding a previously described mitomycin-binding protein [P. Sheldon, D. A. Johnson, P. R. August, H.-W. Liu, and D. H. Sherman, J. Bacteriol. 179:1796–1804, 1997]) revealed a putative mitomycin C (MC) transport gene (
mct
) encoding a hydrophobic polypeptide that has significant amino acid sequence similarity with several actinomycete antibiotic export proteins. Disruption of
mct
by insertional inactivation resulted in an
S. lavendulae
mutant strain that was considerably more sensitive to MC. Expression of
mct
in
Escherichia coli
conferred a fivefold increase in cellular resistance to MC, led to the synthesis of a membrane-associated protein, and correlated with reduced intracellular accumulation of the drug. Coexpression of
mct
and
mrd
in
E. coli
resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. Taken together, these data provide evidence that MRD and Mct function as components of a novel drug export system specific to the mitomycins.
Cyclic AMP Can Decrease Expression of Genes Subject to Catabolite Repression in
Saccharomyces cerevisiae
Authors: Zaragoza Oscar , Lindley Chris , Gancedo Juana M.
Published: December 2019

External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a
pde2
mutant of
Saccharomyces cerevisiae
, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1

FBP1

) from
FBP1
, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2

FBP1

. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UAS

FBP1

-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of
FBP1
transcription through cAMP is redundant with other regulatory mechanisms.
Bordetella pertussis waaAEncodes a Monofunctional 2-Keto-3-Deoxy-d-manno-Octulosonic Acid Transferase That Can Complement anEscherichia coli waaAMutation
Authors: Isobe Tomoko , White Kimberley A. , Allen Andrew G. , Peacock Michael , Raetz Christian R. H. , Maskell Duncan J.
Published: December 2019

Bordetella pertussislipopolysaccharide (LPS) contains a single 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo) residue, whereas LPS fromEscherichia colicontains at least two. Here we report thatB. pertussis waaAencodes an enzyme capable of transferring only a single Kdo during the biosynthesis of LPS and that this activity is sufficient to complement anE. coli waaAmutation.
Function of Coenzyme F
420
in Aerobic Catabolism of 2,4,6-Trinitrophenol and 2,4-Dinitrophenol by
Nocardioides simplex
FJ2-1A
Authors: Ebert Sybille , Rieger Paul-Gerhard , Knackmuss Hans-Joachim
Published: December 2019

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain
Nocardioides simplex
FJ2-1A. Aerobic bacterial degradation of these π-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F
420
. The latter could be replaced by an authentic preparation of coenzyme F
420
from
Methanobacterium thermoautotrophicum
. One of the protein components functions as a NADPH-dependent F
420
reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F
420
to the aromatic system of the nitrophenols. The N-terminal sequence of the F
420
reductase showed high homology with an F
420
-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme.
Identification and Characterization of a New Porin Gene of
Klebsiella pneumoniae
: Its Role in β-Lactam Antibiotic Resistance
Authors: Doménech-Sánchez Antonio , Hernández-Allés Santiago , Martínez-Martínez Luis , Benedí Vicente J. , Albertí Sebastián
Published: December 2019

Klebsiella pneumoniae
porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many β-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated
ompK37
and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in
Escherichia coli
. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient β-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for
K. pneumoniae
porins OmpK36 and OmpK35. This correlated with the susceptibility to certain β-lactam antibiotics, since a
K. pneumoniae
strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to β-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.
Adherence of
Staphylococcus aureus
Is Enhanced by an Endogenous Secreted Protein with Broad Binding Activity
Authors: Palma Marco , Haggar Axana , Flock Jan-Ingmar
Published: December 2019

A novel mechanism for enhancement of adherence of
Staphylococcus aureus
to host components is described. A secreted protein, Eap (extracellular adherence protein), was purified from the supernatant of
S. aureus
Newman and found to be able to bind to at least seven plasma proteins, e.g., fibronectin, the α-chain of fibrinogen, and prothrombin, and to the surface of
S. aureus
. Eap bound much less to cells of
Staphylococcus epidermidis
,
Streptococcus mutans
, or
Escherichia coli
. The protein can form oligomeric forms and is able to cause agglutination of
S. aureus
. Binding of
S. aureus
to fibroblasts and epithelial cells was significantly enhanced by addition of Eap, presumably due to its affinity both for plasma proteins on the cells and for the bacteria.
Characterization of the Major Control Region of
Vibrio cholerae
Bacteriophage K139: Immunity, Exclusion, and Integration
Authors: Nesper Jutta , Blaß Julia , Fountoulakis Michael , Reidl Joachim
Published: December 2019

The temperate bacteriophage K139 is highly associated with pathogenic O1
Vibrio cholerae
strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (
cox
,
cII
,
cI
, and
int
) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter P
L
encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between the
cI
and
int
genes, two additional genes were identified:
orf2
, which has no significant similarity to any other gene, and the formerly characterized gene
glo
. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations in
glo
cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685–701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene
int
, the
attP
site was identified. Upon analysis of the corresponding
attB
site within the
V. cholerae
chromosome, it became evident that phage K139 is integrated between the flagellin genes
flaA
and
flaC
of O1 El Tor and O139
V. cholerae
lysogenic strains.
The C-Terminal Sequence of the λ Holin Constitutes a Cytoplasmic Regulatory Domain
Authors: Bläsi Udo , Fraisl Peter , Chang Chung-Yu , Zhang Ning , Young Ry
Published: December 2019

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of λ S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in
S
and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of
S
is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant
S
A52G, which causes lysis at or before the time when the first phage particle is assembled in the cell. This mutation scrambles the C-terminal sequence of
S
, resulting in a predicted net charge increase of +4, and retards lysis by about 30 min, thus permitting a viable quantity of progeny to accumulate. Thus, the C-terminal domain is not involved in the formation of the lethal membrane lesion nor in the “dual-start” regulation conserved in lambdoid holins. Instead, the C-terminal sequence defines a cytoplasmic regulatory domain which affects the timing of lysis. Comparison of the C-terminal sequences of within holin families suggests that these domains have little or no structure but act as reservoirs of charged residues that interact with the membrane to effect proper lysis timing.
Histidine Operon Deattenuation in
dnaA
Mutants of
Salmonella typhimurium
Correlates with a Decrease in the Gene Dosage Ratio between tRNA
His
and Histidine Biosynthetic Loci
Authors: Blanc-Potard Anne-Beatrice , Figueroa-Bossi Nara , Bossi Lionello
Published: December 2019

Expression of the histidine operon of
Salmonella typhimurium
is increased in
dnaA
(Ts) mutants at 37°C. This effect requires an intact
his
attenuator and can be suppressed by increasing the gene copy number of the
hisR
locus, which encodes the tRNA
His
. We present data which suggest that the
his
deattenuation defect in
dnaA
(Ts) mutants results from the loss of a gene dosage gradient between the
hisR
locus, close to
oriC
, and the
his
operon, far from
oriC
. Some of the conclusions drawn here may apply to other operons as well.
The C-Terminal Domain of DnaQ Contains the Polymerase Binding Site
Authors: Taft-Benz Sharon A. , Schaaper Roel M.
Published: December 2019

The
Escherichia coli dnaQ
gene encodes the 3′→5′ exonucleolytic proofreading (ɛ) subunit of DNA polymerase III (Pol III). Genetic analysis of
dnaQ
mutants has suggested that ɛ might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (α) subunit. We have created truncated forms of
dnaQ
resulting in ɛ subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain ɛ subunits with the α and θ subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the α subunit while retaining normal binding to the θ subunit. In contrast, the NΔ186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the α subunit but was defective in binding to the θ subunit. A strain carrying the
dnaQ991
allele exhibited a strong, recessive mutator phenotype, as expected from a defective α binding mutant. The data are consistent with the existence of two functional domains in ɛ, with the C-terminal domain responsible for polymerase binding.
Phosphorylation of HPr and Crh by HprK, Early Steps in the Catabolite Repression Signalling Pathway for the
Bacillus subtilis
Levanase Operon
Authors: Martin-Verstraete Isabelle , Deutscher Josef , Galinier Anne
Published: December 2019

Carbon catabolite repression (CCR) of
Bacillus subtilis
catabolic genes is mediated by CcpA and in part by P-Ser–HPr. For certain operons, Crh, an HPr-like protein, is also implicated in CCR. In this study we demonstrated that in
ptsH1 crh1
and
hprK
mutants, expression of the
lev
operon was completely relieved from CCR and that both P-Ser–HPr and P-Ser–Crh stimulated the binding of CcpA to the
cre
sequence of the
lev
operon.
Isolation and Characterization of the
Saccharomyces cerevisiae XPT1
Gene Encoding Xanthine Phosphoribosyl Transferase
Authors: Guetsova M. L. , Crother T. R. , Taylor M. W. , Daignan-Fornier B.
Published: December 2019

A new
Saccharomyces cerevisiae
gene,
XPT1
, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of
XPT1
affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that
XPT1
encodes XPRT in yeast.
Field Evaluation of a Combination of Monospecific Enzyme-Linked Immunosorbent Assays for Type-Specific Diagnosis of Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 Infections in HIV-Seropositive Persons in Abidjan, Ivory Coast
Authors: Nkengasong John N. , Maurice Chantal , Koblavi Stéphania , Kalou Mireille , Bile Celestin , Yavo Daniel , Boateng Emmanuel , Wiktor Stefan Z. , Greenberg Alan E.
Published: December 2019

Serologic distinction between human immunodeficiency virus type 1 (HIV-1) and HIV-2 infection is made difficult because of the cross-reactivity and high cost of existing differentiation assays. An evaluation of a strategy based on a combination of monospecific enzyme-linked immunosorbent assays (ELISAs) (CME), was carried out in Abidjan, Ivory Coast, where both HIV-1 and HIV-2 are present, to determine its accuracy and cost-effectiveness. A total of 1,608 (428 HIV-1-positive, 361 HIV-2-positive, 371 dually HIV-1 and HIV-2 [HIV-D] reactive, and 448 HIV-negative) sera that had been serotyped by a line immunoassay (Peptilav) were tested retrospectively by an HIV-1-monospecific (Wellcozyme HIV Recombinant ELISA) and an HIV-2-monospecific (ICE*-HIV-2) assay. The CME strategy gave concordant results for all of the 428 sera scored as HIV-1 by Peptilav. Of the 361 sera scored as HIV-2 by Peptilav, 316 (87.5%) were scored as HIV-2 by CME; the remaining 45 sera were positive by both monospecific ELISAs (mean optical density ratios, 1.36 for Wellcozyme and 11.30 for ICE*-HIV-2) and were classified as HIV-D by CME. Of the 371 sera classified as HIV-D by Peptilav, 344 (92.7%), 21, and 6 were scored as HIV-D, HIV-1, and HIV-2, respectively, by CME. Additional testing of the discrepant samples by two HIV differentiation assays (RIBA and INNO-LIA) gave results that agreed with those by CME for most of the sera. In addition, 267 other sera were tested prospectively by both CME and Peptilav. In the prospective evaluation, CME results agreed with those by Peptilav for all 106 HIV-1 sera and 40 of the 41 HIV-2 sera. However, of the 120 sera scored as HIV-D by Peptilav, 69 (57.5%), 47 (39.2%), and 4 (3.3%) were scored as HIV-D, HIV-1 only, and HIV-2 only, respectively, by CME. All 47 samples scored as HIV-1 by CME and two of four HIV-2 sera gave concordant results by RIBA, whereas 29 of 47 sera scored as HIV-1 by CME and all four HIV-2 sera gave concordant results by INNO-LIA. The reagent cost for the CME strategy was 59% lower than the cost of the Peptilav strategy. These results suggest that a combination of highly sensitive and specific commercially available monospecific ELISAs is a reliable and cost-effective strategy for type-specific serodiagnosis of HIV-1 and HIV-2 infections in HIV-seropositive persons and therefore represents a recommended strategy in areas where both HIV-1 and HIV-2 are endemic.
Interpretation of Trailing Endpoints in Antifungal Susceptibility Testing by the National Committee for Clinical Laboratory Standards Method
Authors: Revankar Sanjay G. , Kirkpatrick William R. , McAtee Robert K. , Fothergill Annette W. , Redding Spencer W. , Rinaldi Michael G. , Patterson Thomas F.
Published: December 2019

Trailing endpoints remain a problem in antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards (NCCLS) method. For isolates for which trailing endpoints are found, MICs of ≤1 μg/ml at 24 h and of >64 μg/ml at 48 h are usually observed. In a study of human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis, we identified three patients with multiple serial isolates for which trailing endpoints were observed with fluconazole. At 24 h, MICs were generally ≤1 μg/ml by both broth macro- and microdilution methods. However, at 48 h, MICs were >64 μg/ml, while the organism remained susceptible by agar dilution testing with fluconazole. Most episodes of oropharyngeal candidiasis with trailing-endpoint isolates responded to doses of fluconazole as low as 100 mg/day. Two patients had both susceptible and trailing-endpoint isolates by NCCLS broth macro- and microdilution testing; these isolates were found to be the same strain by pulsed-field gel electrophoresis using restriction fragment length polymorphisms. Another patient had two different strains, one for which trailing endpoints were observed and one which was susceptible at 48 h. Trailing endpoints may be seen with selected isolates of a strain or may be a characteristic finding for most or all isolates of a strain. In addition, with isolates for which trailing endpoints are observed, reading the endpoint for the NCCLS method at 24 h may be more appropriate.
Detection of Hepatitis G Virus RNA in Persons with and without Known Risk Factors for Blood-Borne Viral Infections in Sweden and Honduras
Authors: Lara Claudia , Halasz Robert , Sönnerborg Anders , Sällberg Matti
Published: December 2019

We analyzed 224 and 163 serum samples from individuals in Sweden and Honduras, respectively, for the presence of the hepatitis G virus (HGV or GB virus-C) RNA. HGV infection in both Sweden and Honduras was related to common risk factors for blood-borne infections, despite a surprisingly high frequency in groups without known risk factors.
Long-Term Predictive Value of a Single Cytomegalovirus (CMV) DNA PCR Assay for CMV Disease in Human Immunodeficiency Virus-Infected Patients
Authors: Walmsley Sharon , Mazzulli Tony , Krajden Mel
Published: December 2019

Universal prophylaxis with oral ganciclovir is not cost-effective for the prevention of cytomegalovirus (CMV) disease in human immunodeficiency virus infection. For a preemptive strategy to be considered, patients at highest risk for CMV disease need to be easily and accurately identified. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of a single CMV DNA PCR assay for the subsequent development of CMV disease were 0.75, 0.89, 0.75, and 0.89, respectively.
Survey of Human Group C Rotaviruses in Japan during the Winter of 1992 to 1993
Authors: Kuzuya Mitsutaka , Fujii Ritsushi , Hamano Masako , Yamada Masao , Shinozaki Kuniko , Sasagawa Akira , Hasegawa Sumiyo , Kawamoto Hiroyoshi , Matsumoto Kazuo , Kawamoto Ayumi , Itagaki Asao , Funatsumaru Sadayuki , Urasawa Syozo
Published: December 2019

Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.
Clinical Evaluation of a New PCR Assay for Detection of
Coxiella burnetii
in Human Serum Samples
Authors: Zhang G. Q. , Nguyen Sa V. , To H. , Ogawa M. , Hotta A. , Yamaguchi T. , Kim H. J. , Fukushi H. , Hirai K.
Published: December 2019

A nested PCR method was developed for the detection of
Coxiella burnetii
in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the
com1
gene encoding a 27-kDa outer membrane protein of
C. burnetii
. The primers amplified the predicted fragments of 21 various strains of
C. burnetii
but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes
Ssp
I and
Sal
I. The utility of the nested PCR was evaluated by testing human serum samples. The
com1
gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the
com1
gene appeared to be a sensitive, specific, and useful method for the detection of
C. burnetii
in serum samples.
Comparison of Performance and Cost-Effectiveness of Direct Fluorescent-Antibody, Ligase Chain Reaction, and PCR Assays for Verification of Chlamydial Enzyme Immunoassay Results for Populations with a Low to Moderate Prevalence of
Chlamydia trachomatis
Infection
Authors: Dean Deborah , Ferrero Dennis , McCarthy Michael
Published: December 2019

Many laboratories use a commercial enzyme immunoassay (EIA) with verification testing to diagnose
Chlamydia trachomatis
infections in an effort to contain costs. This study was designed to compare the performance and cost-effectiveness of direct fluorescent-antibody assay (DFA), commercial PCR, and ligase chain reaction (LCR) for the verification of EIA results. Cervical specimens were screened by EIA. DFA, PCR, and LCR were compared as verification tests for EIA-reactive specimens and negative greyzone (NGZ) specimens at 50% below the cutoff value. These samples were also tested by in-house PCR, which was used in the analysis of verification results. A total of 477 (7%) of 6,571 samples were reactive or within the NGZ. EIA results with verification by DFA testing (EIA/DFA results) agreed with 93% of the true results compared with 97% for EIA/PCR results for one set of 242 samples; there was 97% agreement with true results for EIA/DFA results versus 95% for EIA/LCR results for another set of 235 samples. Ten samples were false positive by LCR. Time and costs were equivalent for EIA with the DFA, PCR, or LCR as the verification test but were two- to threefold greater for PCR or LCR alone than for EIA with verification. Since it is important to balance cost containment with public health objectives, DFA, PCR, and LCR as EIA verification tests for cervical samples offer acceptable sensitivities and specificities at reasonable cost for low- to moderate-risk populations and therefore can be extended to a broader spectrum of at-risk populations.
Restriction Fragment Length Polymorphism Analysis of Some Flagellin Genes of
Salmonella enterica
Authors: Dauga Catherine , Zabrovskaia Anna , Grimont Patrick A. D.
Published: December 2019

Salmonellae often have the ability to express two different flagellar antigen specificities (phase 1 and phase 2). At the cell level, only one flagellar phase is expressed at a time. Two genes,
fliC
, encoding phase-1 flagellin, and
fljB
, encoding phase-2 flagellin, are alternatively expressed. Flagellin genes from 264 serovars of
Salmonella enterica
were amplified by two phase-specific PCR systems. Amplification products were subjected to restriction fragment length polymorphism (RFLP) analysis by using endonucleases
Hha
I and
Hph
I. RFLP with
Hha
I and
Hph
I yielded 64 and 42 different restriction profiles, respectively, among 329 flagellin genes coding for 26 antigens. The phase-1 gene showed 46 patterns with
Hha
I and 30 patterns with
Hph
I. The phase-2 gene showed 23 patterns with
Hha
I and 17 patterns with
Hph
I. When the data from both enzymes were combined, 116 patterns were obtained: 74 for
fliC
, 47 for
fljB
, and 5 shared by both genes. Of these combined patterns, 80% were specifically associated with one flagellar antigen and 20% were associated with more than one antigen. Each flagellar antigen was divided into 2 to 18 different combined patterns. In the sample of strains used, determination of the phase-1 and phase-2 flagellin gene RFLP, added to the knowledge of the O antigen, allowed identification of all diphasic serovars. Overall, the diversity uncovered by flagellin gene RFLP did not precisely match that evidenced by flagellar agglutination.
Massilia timonae
gen. nov., sp. nov., Isolated from Blood of an Immunocompromised Patient with Cerebellar Lesions
Authors: La Scola Bernard , Birtles Richard J. , Mallet Marie-Noëlle , Raoult Didier
Published: December 2019

A fastidious, slowly growing, strictly aerobic, gram-negative bacterium was isolated from a culture of blood from a 25-year-old man with common variable immunodeficiency. The man had been admitted to hospital with febrile progressive cerebellar ataxia. The use of standard phenotypic schemes did not lead to identification, but sequence analysis demonstrated that the 16S rRNA gene of the isolate was most similar to those of the environmental bacteria
Duganella zoogloeoides
(formerly
Zoogloea ramigera
115) and
Telluria mixta
. Further characterization of the bacterium by biochemical analysis, electron microscopy, G+C content estimation, and fatty acid analysis demonstrated significant differences between the bacterium and
D. zoogloeoides
and
Telluria
species; thus, we propose it as a new taxon with the name
Massilia timonae
gen. nov., sp. nov.
Evaluation of the ESP Culture System II for Testing Susceptibilities of
Mycobacterium tuberculosis
Isolates to Four Primary Antituberculous Drugs
Authors: Bergmann John S. , Woods Gail L.
Published: December 2019

The reliability of the ESP Culture System II (herein referred to as ESP II) for testing susceptibilities of
Mycobacterium tuberculosis
isolates to isoniazid, rifampin, ethambutol, and streptomycin was evaluated by comparing results to those of the method of proportion (MOP), which was considered the reference method, for 20 clinical isolates and 30 challenge strains provided by the Centers for Disease Control and Prevention (CDC). Clinical isolates also were tested with the BACTEC TB 460 system; these results agreed with those obtained by the MOP for all isolates and all drugs, except the high concentration of isoniazid, for which agreement was 95%. After resolution of discrepancies, levels of agreement between ESP II and MOP for the clinical isolates were 95 and 100%, respectively, for the low and high concentrations of isoniazid, 100% for rifampin and ethambutol, and 95% for streptomycin. For the 30 challenge isolates, ESP II results for both concentrations of isoniazid agreed with the expected results in all cases, whereas agreement was 93% for both rifampin and streptomycin and 90% for ethambutol. All discrepancies with the CDC isolates were due to failure of ESP II to correctly classify resistant strains. By testing isolates yielding discrepant ethambutol and streptomycin results with a lower concentration of both drugs in the ESP II system, agreement increased to 93% for ethambutol and 100% for streptomycin. For the clinical isolates, the times to an ESP II result of susceptible (means ± standard errors of the means) were 8.47 ± 0.12 days (range, 7 to 10 days) and 8.73 ± 0.29 days (range, 5 to 11 days) when the inoculum was prepared from a McFarland equivalent and from a seed bottle, respectively. The time to an ESP II result of resistant varied by drug and method of inoculum preparation, ranging from 5.50 ± 0.22 days for ethambutol with the inoculum prepared from a McFarland standard to 8.0 days for ethambutol with the inoculum prepared from a seed bottle. These data suggest that the ESP II system is a rapid and reliable method for testing susceptibilities of
M. tuberculosis
isolates to isoniazid and rifampin. Performance, however, may be suboptimal for ethambutol and streptomycin. Testing additional ethambutol-resistant and streptomycin-resistant strains with two concentrations of both drugs is necessary.
Detection of Specific Antibodies to an Antigenic Mannoprotein for Diagnosis of
Penicillium marneffei
Penicilliosis
Authors: Cao Liang , Chen Da-Liang , Lee Cindy , Chan Che-Man , Chan King-Man , Vanittanakom Nongnuch , Tsang Dominic N. C. , Yuen Kwok-Yung
Published: December 2019

The disseminated and progressive fungal disease
Penicillium marneffei
penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of
P. marneffei
. Evaluation of the test with guinea pig sera against
P. marneffei
and other pathogenic fungi indicated that this assay was specific for
P. marneffei
. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.
Seropositivity for and Intestinal Colonization with
Entamoeba histolytica
and
Entamoeba dispar
in Individuals in Northeastern Brazil
Authors: Braga Lucia L. , Mendonca Yacy , Paiva Clece A. , Sales Andrea , Cavalcante Andre L. M. , Mann Barbara J.
Published: December 2019

In a slum community in northeastern Brazil 20% of a sample population was colonized with
Entamoeba histolytica
or
Entamoeba dispar
and 10.6% was colonized with
E. histolytica
alone. No correlation between seropositivity for anti-GalNAc lectin antibody and colonization was found. These results suggest that colonization does not necessarily produce immunity to reinfection.
Rapid Diagnosis of Pulmonary Tuberculosis with the LCx
Mycobacterium tuberculosis
Assay and Comparison with Conventional Diagnostic Techniques
Authors: Rohner Peter , Jahn Esther I. M. , Ninet Beatrice , Ionati Concetta , Weber Rainer , Auckenthaler Raymond , Pfyffer Gaby E.
Published: December 2019

The LCx MTB amplification assay is a nucleic acid amplification test intended for the direct detection of
Mycobacterium tuberculosis
complex in respiratory specimens. We evaluated its performance on 2,001 consecutive respiratory specimens; 78 were culture positive for
M. tuberculosis
. Sensitivity, specificity, and positive and negative predictive values of this assay for all specimens compared to culture results were 88.5, 97.7, 60.5, and 99.5%, respectively. When referred to resolved clinical diagnosis of active tuberculosis, these values improved to 90.2, 98.4, 72.8, and 99.5%, respectively.
Urine Specimens from Pregnant and Nonpregnant Women Inhibitory to Amplification of
Chlamydia trachomatis
Nucleic Acid by PCR, Ligase Chain Reaction, and Transcription-Mediated Amplification: Identification of Urinary Substances Associated with Inhibition and Removal of Inhibitory Activity
Authors: Mahony J. , Chong S. , Jang D. , Luinstra K. , Faught M. , Dalby D. , Sellors J. , Chernesky M.
Published: December 2019

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of
Chlamydia trachomatis
nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for
C. trachomatis
detection. Aliquots were spiked with the equivalent of one
C. trachomatis
elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%;
P
= 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and −70°C overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or −70°C and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.
Genospecies Identification and Characterization of Lyme Disease Spirochetes of Genospecies
Borrelia burgdorferi
Sensu Lato Isolated from Rodents in Taiwan
Authors: Shih Chien-Ming , Chang Han-Ming , Chen Show-Li , Chao Li-Lian
Published: December 2019

Lyme disease spirochetes of the genospecies
Borrelia burgdorferi
sensu lato were identified and characterized for the first time in Taiwan. Seven isolates, designated TWKM1 to TWKM7, were purified from the ear tissues of three species of rodents captured from seven localities of Taiwan. The immunological characteristics of these Taiwan isolates were compared with those of other genospecies of Lyme disease spirochetes by analyzing the protein profiles and reactivities with
B. burgdorferi
-specific monoclonal antibodies (MAbs). The genospecies of these Taiwan isolates were also identified by the similarities in their plasmid profiles and differential reactivities with genospecies-specific PCR primers. Although two distinct protein profiles were observed among the seven Taiwan isolates, the MAb reactivities against the outer surface proteins of
B. burgdorferi
of all of these isolates were consistent with those of
B. burgdorferi
sensu lato. The similarities of the plasmid profiles also confirmed the identities of these Taiwan isolates. PCR analysis indicated that all of these Taiwan isolates were genetically related to the genospecies
B. burgdorferi
sensu stricto. These results demonstrate the first identification of Lyme disease spirochetes in Taiwan and also highlight the increasing demand for defining the reservoirs and vector ticks of
B. burgdorferi
. A serosurvey for Lyme disease infection in the human population of Taiwan may also be required.
Sequencing of
Escherichia coli
O111 O-Antigen Gene Cluster and Identification of O111-Specific Genes
Authors: Wang Lei , Curd Heather , Qu Wenjia , Reeves Peter R.
Published: December 2019

Shiga toxin (Stx)-producing
Escherichia coli
strains of serogroup O111 are the most frequently isolated non-O157 strains causing outbreaks of gastroenteritis with hemolytic-uremic syndrome. The O111 O-antigen gene cluster had been cloned and about half of it has been sequenced; we have now sequenced the remainder of the gene cluster, which is 12.5 kb in length and which comprises 11 genes. On the basis of sequence similarity, we have identified all the O-antigen genes expected, including five sugar biosynthetic pathway genes, three transferase genes, the O-unit flippase gene, and the O-antigen polymerase gene. By PCR testing with
E. coli
strains representing all 166 O-antigen forms, some randomly selected gram-negative bacteria, and
Salmonella enterica
serovar Adelaide, we showed that four O-antigen genes are highly specific to O111. This work provides the basis for a sensitive test for the rapid detection of
E. coli
O111. This is important both for decisions related to patient care, because early treatment may reduce the risk of life-threatening complications, and for the detection of sources of contamination.
Extraintestinal Salmonellosis in a General Hospital (1991 to 1996): Relationships between
Salmonella
Genomic Groups and Clinical Presentations
Authors: Rodríguez Mercedes , de Diego Isabel , Mendoza M. Carmen
Published: December 2019

Episodes of extraintestinal salmonellosis treated at a general hospital (1,522 beds) over a 6-year period (1991 to 1996) were characterized by the analysis of phenotypic and genotypic traits of
Salmonella
organisms and clinical data from medical reports. Extraintestinal salmonellosis accounted for 8% of all salmonellosis episodes. Fifty-two medical reports, dealing with 6 cases of typhoid fever, 32 cases of bacteremia, and 14 focal infections, were reviewed. All cases of typhoid fever except 1, 7 cases of bacteremia, and 5 focal infections were not related to any underlying disease or predisposing factors, while 25 cases of bacteremia and 9 focal infections were associated with some of these risk factors. All typhoid isolates and 65.4% of the nontyphoid isolates were susceptible to antimicrobials. Fifty-one nontyphoid strains were analyzed and assigned to 21 genomic groups, which were defined by serotype, combined ribotype, and combined randomly amplified polymorphic DNA type (each genomic group could include organisms differing in some phenotypic traits). The relationships between genomic groups and clinical presentations were traced. Organisms causing 22 episodes (17 episodes of bacteremia, 2 of pneumonia, 1 of peritonitis, 1 of pyelonephritis, and 1 of cystitis) belonged to a prevalent
Salmonella enterica
serotype Enteritidis genomic group, which included organisms assigned to four phage types, five biotypes, and four resistance patterns, causing infections in patients with and without risk factors. Seven other genomic groups, 4 Enteritidis groups (associated with both bacteremia and focal infections), 2 Typhimurium groups (one associated with bacteremia and the other with focal infections) and 1 Brandenburg group (associated with bacteremia) included two or more strains, and the remaining 13 genomic groups consisted of only one strain each.
Identification of New Verocytotoxin Type 2 Variant B-Subunit Genes in Human and Animal
Escherichia coli
Isolates
Authors: Piérard D. , Muyldermans G. , Moriau L. , Stevens D. , Lauwers S.
Published: December 2019

The sequence of a verocytotoxin 2 (VT2) variant gene that was untypeable by the B subunit PCR and restriction fragment length polymorphism analysis (PCR-RFLP) method described by Tyler et al. (S. D. Tyler, W. M. Johnson, H. Lior, G. Wang, and K. R. Rozee, J. Clin. Microbiol. 29:1339–1343, 1991) was determined and compared with published sequences. It was highly homologous to two recently reported VT2 variant sequences. The PCR-RFLP method described by Tyler et al. was extended to include these new sequences. New VT2 variants were identified in 65 of 359 VT-producing
Escherichia coli
(VTEC) with newly designed primers (VT2-cm and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f). The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler’s PCR-RFLP method because no amplification was obtained with the primers used with this method (VT2-c and VT2-d). The last isolate harbored the new variant gene in addition to VT2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were negative for the
eaeA
gene and were significantly less frequently enterohemolytic or positive for the enterohemorrhagic
E. coli
(EHEC) virulence plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to identify less-virulent VTEC isolates and for VT genotyping in epidemiological studies with non-O157 strains.
Efficacy of API 20C and ID 32C Systems for Identification of Common and Rare Clinical Yeast Isolates
Authors: Ramani Rama , Gromadzki Sally , Pincus David H. , Salkin Ira F. , Chaturvedi Vishnu
Published: December 2019

The abilities of the API 20C and ID 32C yeast identification systems to identify 123 common and 120 rare clinical yeast isolates were compared. API 20C facilitated correct identification of 97% common and 88% rare isolates while ID 32C facilitated correct identification of 92% common and 85% rare isolates.
In Vitro Activities of Ampicillin-Sulbactam and Amoxicillin-Clavulanic Acid against
Acinetobacter baumannii
Authors: Pandey Anita , Kapil Arti , Sood Seema , Goel Vikas , Das Bimal , Seth Pradeep
Published: December 2019

In vitro susceptibility patterns of newer β-lactamase-inhibiting antibiotics ampicillin-sulbactam (A/S) and amoxicillin-clavulanic acid (A/C) for 100 consecutive isolates of
Acinetobacter baumannii
obtained from various clinical samples were studied. The A/C MIC for 86% of the strains was more than 16/8 μg/ml, whereas there was an A/S MIC of more than 16/8 μg/ml for only 38% of the strains. This showed that A/S has significantly superior in vitro activity compared to A/C against
A. baumannii
, although, theoretically, both should have similar activities. The therapeutic superiority of A/S over A/C needs to be studied, or else the breakpoints for these agents in in vitro tests need to be redefined.