bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Detection of
Stenotrophomonas maltophilia
by rRNA-Directed PCR
Authors: Whitby Paul W. , Carter Karen B. , Burns Jane L. , Royall James A. , LiPuma John J. , Stull Terrence L. 
Published: December 2019

ABSTRACT

Stenotrophomonas maltophilia
has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease,
S. maltophilia
can be cultured from respiratory tract secretions. Identification of
S. maltophilia
can be problematic, and analysis of isolates from the
Burkholderia cepacia
Research Laboratory and Repository showed that several isolates presumptively identified as
B. cepacia
by clinical microbiology laboratories were in fact
S. maltophilia
. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify
S. maltophilia
directly from sputum. The SS-PCR was developed and tested against a panel of 112
S. maltophilia
isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all
S. maltophilia
isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify
S. maltophilia
in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify
S. maltophilia
isolates and which can be used for the direct detection of this organism in CF patient sputum.
Quantitative Analysis of Cytomegalovirus (CMV) Viremia Using the pp65 Antigenemia Assay and the COBAS AMPLICOR CMV MONITOR PCR Test after Blood and Marrow Allogeneic Transplantation
Authors: Boivin G. , Bélanger R. , Delage R. , Béliveau C. , Demers C. , Goyette N. , Roy J. 
Published: December 2019

ABSTRACT

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (
P
= 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.
Antigenic Diversity of
Haemophilus somnus
Lipooligosaccharide: Phase-Variable Accessibility of the Phosphorylcholine Epitope
Authors: Howard Michael D. , Cox Andrew D. , Weiser Jeffrey N. , Schurig Gerhardt G. , Inzana Thomas J. 
Published: December 2019

ABSTRACT

The lipooligosaccharide (LOS) of
Haemophilus somnus
undergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in
H. somnus
has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from
H. somnus
strains and phase variants. Clinical isolates of
H. somnus
exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glycose residues in this region. Our results indicate that outer core epitopes of
H. somnus
LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.
Effects of Amplification Facilitators on Diagnostic PCR in the Presence of Blood, Feces, and Meat
Authors: Abu Al-Soud Waleed , Rådström Peter 
Published: December 2019

ABSTRACT

The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing
rTth
or
Taq
DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing
Taq
DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases.
Taq
DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and
rTth
was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of
rTth
containing meat and of
Taq
DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed
Taq
DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.
Genetic Heterogeneity in
Mycobacterium tuberculosis
Isolates Reflected in IS
6110
Restriction Fragment Length Polymorphism Patterns as Low-Intensity Bands
Authors: de Boer Annette S. , Kremer Kristin , Borgdorff Martien W. , de Haas Petra E. W. , Heersma Herre F. , van Soolingen Dick 
Published: December 2019

ABSTRACT

Mycobacterium tuberculosis
isolates with identical IS
6110
restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS
6110
RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple
M. tuberculosis
strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS
6110
-mediated genetic adaptation of
M. tuberculosis
to changes in the environmental conditions during the dormant state or reactivation thereafter.
Strain Identification of
Trichophyton rubrum
by Specific Amplification of Subrepeat Elements in the Ribosomal DNA Nontranscribed Spacer
Authors: Jackson Colin J. , Barton Richard C. , Kelly Steven L. , Evans E. Glyn V. 
Published: December 2019

ABSTRACT

Trichophyton rubrum
is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the
T. rubrum
ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species,
Trichophyton violaceum
,
Trichophyton gourvilii
, and
Trichophyton soudanense
, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing
T. rubrum
will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.
Duodenal Microflora in Very-Low-Birth-Weight Neonates and Relation to Necrotizing Enterocolitis
Authors: Hoy Christine M. , Wood Christopher M. , Hawkey Peter M. , Puntis John W. L. 
Published: December 2019

ABSTRACT

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in the neonatal period. Small-bowel overgrowth with aerobic gram-negative bacteria has previously been implicated in the development of NEC. This prospective study performed quantitative bacteriology on 422 duodenal aspirates collected from 122 very-low-birth-weight (<1,500-g) newborns, at the time of routine changing of nasogastric tubes. Isolates of
Enterobacteriaceae
were typed by repetitive extragenic, palindromic PCR and pulsed-field gel electrophoresis. One or more samples from 50% of these infants yielded gram-negative bacteria, predominantly
Escherichia coli
,
Klebsiella
spp., and
Enterobacter
spp., with counts up to 10
8
CFU/g. The proportion of samples with gram-negative bacteria increased with postnatal age, while the percentage of sterile samples declined. Molecular typing revealed marked temporal clustering of indistinguishable strains. All infants had been fed prior to isolation of gram-negative organisms. Antibiotic use had no obvious effect on colonization with
Enterobacteriaceae
. There were 15 episodes of suspected NEC (stage I) and 8 confirmed cases of NEC (2 stage II and 6 stage III) during the study period. Duodenal aspirates were collected prior to clinical onset in 13 episodes of NEC. Seven of these yielded
Enterobacteriaceae
, of which five strains were also isolated from infants without NEC. Very-low-birth-weight infants have high levels of duodenal colonization with
Enterobacteriaceae
, with evidence of considerable cross-colonization with indistinguishable strains. There was no association between duodenal colonization with particular strains of
Enterobacteriaceae
and development of NEC.
Characterization of Chloramphenicol and Florfenicol Resistance in
Escherichia coli
Associated with Bovine Diarrhea
Authors: White David G. , Hudson Charlene , Maurer John J. , Ayers Sherry , Zhao Shaohua , Lee Margie D. , Bolton Lance , Foley Thomas , Sherwood Julie 
Published: December 2019

ABSTRACT

Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary
Escherichia coli
isolates incriminated in bovine diarrhea. The
flo
gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified in
Photobacterium piscicida
and
Salmonella enterica
serovar Typhimurium DT104. The
flo
gene product is closely related to the CmlA protein identified in
Pseudomonas aeruginosa
. The
cmlA
gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight
E. coli
isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both
flo
and
cmlA
genes. Forty-two of the 44 isolates for which florfenicol MICs were ≥16 μg/ml were positive via PCR for the
flo
gene. All
E. coli
isolates for which florfenicol MICs were ≤8 μg/ml were negative for the
flo
gene (
n
= 4). Twelve
E. coli
isolates were positive for
cmlA
, and chloramphenicol MICs for all 12 were ≥32 μg/ml. Additionally, eight isolates were positive for both
flo
and
cmlA
, and both florfenicol and chloramphenicol MICs for these isolates were ≥64 μg/ml. DNA sequence analysis of the
E. coli flo
gene demonstrated 98% identity to the published GenBank sequences of both serovar Typhimurium

flo
St

and
P. piscicida pp-flo
. The
flo
gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistant
E. coli
isolates. However, not all of the florfenicol-resistant
E. coli
isolates tested contained the large
flo
-positive plasmids. This suggests that several of the
E. coli
isolates may possess a chromosomal
flo
gene. The
E. coli flo
gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine
E. coli
isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.
Evaluation of Random Amplified Polymorphic DNA Typing of
Pseudomonas aeruginosa
Authors: Campbell Maureen , Mahenthiralingam Eshwar , Speert David P. 
Published: December 2019

ABSTRACT

A method for distinguishing among
Pseudomonas aeruginosa
strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power. A total of 200 isolates, blinded in triplicate, were evaluated by RAPD. All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.
Antimicrobial Resistance ofSalmonellaIsolates from Swine
Authors: Gebreyes Wondwossen A. , Davies Peter R. , Morrow W. E. Morgan , Funk Julie A. , Altier Craig 
Published: December 2019

ABSTRACTWe examined the antimicrobial resistance of 1,257 isolates of 30 serovars ofSalmonella entericasubsp.entericaisolated from swine. Serovars Typhimurium and Typhimurium var. Copenhagen were widespread and were frequently multidrug resistant, with distinct resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline and to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, respectively.
High-Pathogenicity Island of
Yersinia
spp. in
Escherichia coli
Strains Isolated from Diarrhea Patients in China
Authors: Xu Jian-guo , Cheng Bokun , Wen Xuanqin , Cui Shuyu , Ye Changyun 
Published: December 2019

ABSTRACT

The high-pathogenicity island (HPI) of
Yersinia
has been observed in 93% of 60 enteroadhesive
Escherichia coli
strains and 80% of
E. coli
strains isolated from blood samples. In the present study we investigated 671 fecal samples from patients with diarrhea in Shandong Province, China, and isolated HPI-harboring
E. coli
from 6.26% of the samples. The isolation rates for patients with diarrhea in three age groups, 10 to 20, 30 to 40, and 50 to 60 years, were 6.70, 12.35, and 10.81%, respectively. Therefore, HPI-harboring
E. coli
is the third most frequently isolated enteric pathogen from patients with diarrhea. Vomiting and abdominal pain were recorded for 33.33 and 66.67% of the patients, respectively. Stools with blood were observed for 9.52% of the patients. Twenty-four of 42 (57%) patients experienced a temperature over 37.4°C. These observations indicate that HPI-harboring
E. coli
is one of the major causes of diarrheal disease in China and that the clinical symptoms caused by HPI-harboring
E. coli
differ from those caused by enteroadhesive
E. coli.
Simplified Hepatitis C Virus Genotyping by Heteroduplex Mobility Analysis
Authors: White Peter A. , Zhai Xinyan , Carter Ian , Zhao Yue , Rawlinson William D. 
Published: December 2019

ABSTRACT
Heteroduplex mobility analysis (HMA) was used to genotype hepatitis C viruses (HCV) with PCR fragments derived from the 5′ untranslated region (5′-UTR) or the NS5b region. HCV 5′-UTR fragments were amplified from 296 serum samples by use of a combined reverse transcription-PCR assay, and the genotypes of isolates were determined by sequencing. HCV genotype distributions in Australia were 39% for genotype 1a, 15% for 1b, 3% for 1a/b, <1% for 2a/c, 5% for 2b, 34% for 3a, <1% for 3b, and 1% for 4, and 1% of patients were infected with more than one genotype. Pairwise HMA of subtypes 1a, 1b, 2a/c, 2b, 3a, 3b, 4a, and 6a demonstrated that five distinct heteroduplex patterns were formed between the eight subtypes. A reference panel that contained a representative of each pattern (1a, 2b, 3a, 4a, and 6a) was used for genotyping. The pattern of heteroduplexes formed when a test isolate was mixed with the five reference isolates was correlated with the genotype, as determined by sequencing. Genotypes determined by HMA correlated exactly with sequencing results within the groups 1, 2, 3a, 3b/4, and 6. HMA was also used to simplify the identification of mixed infection with two HCV genotypes. In further studies, with amplicons from the NS5b region, HMA classified isolates into their respective subtypes, and the heteroduplex mobility ratio correlated closely with nucleotide sequence variation at the isolate, subtype, and genotype levels. HMA provides an adaptable, inexpensive, and rapid method of genotyping HCV that requires fewer resources than DNA sequencing.
Sequence Analysis and Clinical Significance of the
iceA
Gene from
Helicobacter pylori
Strains in Japan
Authors: Ito Yoshiyuki , Azuma Takeshi , Ito Shigeji , Suto Hiroyuki , Miyaji Hideki , Yamazaki Yukinao , Kato Takuji , Kohli Yoshihiro , Keida Yoshihide , Kuriyama Masaru 
Published: December 2019

ABSTRACT

The
Helicobacter pylori iceA
gene was recently identified as a genetic marker for the development of peptic ulcer in a Western population. To assess the significance of
iceA
subtypes of
H. pylori
in relation to peptic ulcer, 140 Japanese clinical isolates (88 from Fukui and 52 from Okinawa) were characterized. Sequence analysis of the
iceA1
gene from 25 representative Japanese strains was also carried out to identify the differences in
iceA
between the ulcer group and the gastritis group. The
iceA1
genotype was not correlated with the presence of peptic ulceration in either area. In addition, sequence analysis led to identification of five deletions and five point mutations (a nonsense mutation or a 1-bp insertion) within the
iceA1
open reading frame corresponding to previously published sequences. These mutations were identified in both clinical groups (ulcer and gastritis groups) in each area. Local DNA sequence analysis revealed that the endpoints of all five deletions coincided with direct repeats. We also found four strains that carried longer
iceA1
open reading frames compared with that for strain 60190. In conclusion, carriage of an
iceA1
strain does not seem to be a risk factor for peptic ulcer in Japanese subjects. The critical mutations in the
iceA1
gene in some isolates from patients with peptic ulcers suggested that IceA does not participate in the pathogenesis of peptic ulcer in Japan. We also found deletion hot spots that were associated with direct repeats in
iceA1
and that favored a small-deletion model of slipped mispairing events during replication. We showed that
iceA1
sequence variations may be useful tools for analysis of the population genetics of
H. pylori
.
Antimicrobial Resistance of
Neisseria gonorrhoeae
and High Prevalence of Ciprofloxacin-Resistant Isolates in Japan, 1993 to 1998
Authors: Tanaka Masatoshi , Nakayama Hiroshi , Haraoka Masashi , Saika Takeshi , Kobayashi Intetsu , Naito Seiji 
Published: December 2019

ABSTRACT

To assess the antimicrobial resistance of
Neisseria gonorrhoeae
isolated from 1993 through 1998 in Japan, susceptibility testing was conducted on 502 isolates. Selected isolates were characterized by auxotype and analysis for mutations within the quinolone resistance-determining region (QRDR) in the
gyrA
and
parC
genes, which confer fluoroquinolone resistance on the organism. Plasmid-mediated penicillin resistance (penicillinase-producing
N. gonorrhoeae
) decreased significantly from 1993–1994 (7.9%) to 1997–1998 (2.0%). Chromosomally mediated penicillin resistance decreased from 1993–1994 (12.6%) to 1995–1996 (1.9%) and then increased in 1997–1998 (10.7%). Chromosomally mediated tetracycline resistance decreased from 1993–1994 (3.3%) to 1997–1998 (2.0%), and no plasmid-mediated high-level tetracycline resistance was found. Isolates with ciprofloxacin resistance (MIC ≥ 1 μg/ml) increased significantly from 1993–1994 (6.6%) to 1997–1998 (24.4%). The proline-requiring isolates were less susceptible to ciprofloxacin than the prototrophic or arginine-requiring isolates. Ciprofloxacin-resistant isolates contained three or four amino acid substitutions within the QRDR in the GyrA and ParC proteins.