bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pandemic Spread of an O3:K6 Clone of
Vibrio parahaemolyticus
and Emergence of Related Strains Evidenced by Arbitrarily Primed PCR and
toxRS
Sequence Analyses
Authors: Matsumoto Chiho , Okuda Jun , Ishibashi Masanori , Iwanaga Masaaki , Garg Pallavi , Rammamurthy Thandavarayan , Wong Hin-Chung , Depaola Angelo , Kim Yung Bu , Albert M. John , Nishibuchi Mitsuaki 
Published: December 2019

ABSTRACT

Vibrio parahaemolyticus
O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the
tdh
gene but not the
trh
gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150–3155, 1997). Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the
tdh
and
trh
typing, and AP-PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the
toxRS
sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus
Vibrio
. The
toxRS
sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed. This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method. The
tdh
-positive and
trh
-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their
toxRS
sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of
V. parahaemolyticus
and reports a novel
toxRS
-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.
Quantification of Fungal DNA by Using Fluorescence Resonance Energy Transfer and the Light Cycler System
Authors: Loeffler Juergen , Henke Norbert , Hebart Holger , Schmidt Diethard , Hagmeyer Lars , Schumacher Ulrike , Einsele Hermann 
Published: December 2019

ABSTRACT

The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens,
Candida albicans
and
Aspergillus fumigatus
. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of
C. albicans
and
A. fumigatus
could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 10
1
and 10
4
Aspergillus
conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR–enzyme-linked immunosorbent assay analysis.
Pig and Goat Blood as Substitutes for Sheep Blood in Blood-Supplemented Agar Media
Authors: Anand Chandar , Gordon Rhonda , Shaw Helene , Fonseca Kevin , Olsen Merle 
Published: December 2019

ABSTRACT

In many developing countries sheep and horse blood, the recommended blood supplements in bacteriological media, are not readily available, whereas pig and goat blood are. Therefore, this study examined the use of pig and goat blood as potential substitutes for sheep blood in blood-supplemented bacteriologic media commonly used in clinical microbiology laboratories. In general, the growth characteristics and colony morphologies of a wide range of aerobic and anaerobic bacteria and
Candida albicans
were similar on media containing pig, goat, and sheep blood, although differences were found.
Enterococcus
sp. uniformly produced alpha-hemolysis when incubated in CO
2
, but in anaerobic conditions the hemolysis varied. In contrast, beta-hemolytic streptococci produced identical hemolytic reactions on all three media. Synergistic hemolysis was not observed on pig blood agar in the CAMP test nor on goat blood agar in the reverse CAMP test. The preparation of chocolate agar (heated) with pig blood required heating to a higher temperature than with sheep or goat blood to yield suitable growth of
Haemophilus
species. In general, we conclude that pig and goat blood are suitable alternatives to sheep blood for use in bacteriological media in settings where sheep and horse blood are not readily available.
High Levels of Epstein-Barr Virus DNA in Blood of Solid-Organ Transplant Recipients and Their Value in Predicting Posttransplant Lymphoproliferative Disorders
Authors: Baldanti Fausto , Grossi Paolo , Furione Milena , Simoncini Lavinia , Sarasini Antonella , Comoli Patrizia , Maccario Rita , Fiocchi Roberto , Gerna Giuseppe 
Published: December 2019

ABSTRACTEpstein-Barr virus (EBV) DNA was quantitated in peripheral blood mononuclear cells (PBMC) from 25 healthy subjects, 105 asymptomatic solid-organ transplant (SOT) recipients, and 15 SOT recipients with symptomatic EBV infections by using a newly developed quantitative-PCR technique. Patients with symptomatic EBV infections had significantly higher (P< 0.001) median EBV DNA levels than asymptomatic SOT recipients and immunocompetent individuals. In SOT recipients, the positive predictive value of EBV DNA levels of >1,000 genome equivalents (GE)/0.5 μg of total PBMC DNA was 64.7% for symptomatic EBV infection, while the negative predictive value was 96.1%. In 19 of 32 (59.3%) asymptomatic SOT recipients, EBV DNA levels were consistently below 1,000 GE for as long as 18 months, while 10 of 32 (31.2%) patients had 1,000 to 5,000 EBV GE at least once during follow-up. In a minority of patients (3 of 32; 9.3%), ≥5,000 GE could be detected at least once during follow-up. Reduction of immunosuppressive treatment decreased EBV DNA levels by ≥1 log10unit in patients with symptomatic EBV infections. Quantification of EBV DNA is valuable for the diagnosis and monitoring of symptomatic EBV infections in SOT recipients.
Multisite Reproducibility of Etest for Susceptibility Testing of
Mycobacterium abscessus
,
Mycobacterium chelonae
, and
Mycobacterium fortuitum
Authors: Woods Gail L. , Bergmann John S. , Witebsky Frank G. , Fahle Gary A. , Boulet Betty , Plaunt Marianne , Brown Barbara A. , Wallace Richard J. , Wanger Audrey 
Published: December 2019

ABSTRACT

A multicenter study was conducted to assess the inter- and intralaboratory reproducibility of the Etest for susceptibility testing of the rapidly growing mycobacteria. The accuracy also was evaluated by comparing Etest results to those obtained by broth microdilution. Ten isolates (four of the
Mycobacterium fortuitum
group, three of
Mycobacterium abscessus
, and three of
Mycobacterium chelonae
) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and trimethoprim-sulfamethoxazole in each of four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) using common lots of media and Etest strips. Interlaboratory agreement among MICs (i.e., mode ± 1 twofold dilution) varied for the different drug-isolate combinations and overall was best for trimethoprim-sulfamethoxazole (75% for one isolate and 100% for all others), followed by doxycycline and ciprofloxacin. Interlaboratory agreement based on interpretive category also varied and overall was best for doxycycline (100% for all isolates), followed by trimethoprim-sulfamethoxazole and ciprofloxacin. Interlaboratory reproducibility among MICs was most variable for imipenem, and agreement by interpretive category was lowest for imipenem and amikacin. Modal Etest MICs agreed with those by broth microdilution only for doxycycline and the sulfonamides. For all other drugs, the modal MICs by the two methods differed by more than ± 1 twofold dilution for one or more isolates. In all cases, the Etest MIC was higher and would have caused reports of false resistance. In summary, the Etest in this evaluation did not perform as well as broth microdilution for susceptibility testing of the rapidly growing mycobacteria. It was problematic for most species and drugs, primarily because of a trailing endpoint and/or high MICs compared to broth. Its use will necessitate further investigation, including determination of the optimal medium and incubation conditions and clarification of endpoint interpretation.
Problems Related to Determination of MICs of Oximino-Type Expanded-Spectrum Cephems for
Proteus vulgaris
Authors: Ohno Akira , Ishii Yoshikazu , Ma Ling , Yamaguchi Keizo 
Published: December 2019

ABSTRACT

During in vitro susceptibility testing of clinical isolates of
Proteus vulgaris
, we noted that the MICs of several expanded-spectrum cephems were much higher in the broth microdilution method than in the agar dilution method (termed the MIC gap phenomenon). Here we investigated the mechanism of the MIC gap phenomenon. Cephems with the MIC gap phenomenon were of the oximino type, such as cefotaxime, cefteram, and cefpodoxime, which serve as good substrates for inducible class A β-lactamase (CumA) enzymes produced by
P. vulgaris
; this finding suggests a relationship between the MIC gap phenomenon and CumA. Since peptidoglycan recycling shares a system common to that inducing CumA, we analyzed the mechanism of the MIC gap phenomenon using
P. vulgaris
B317 and isogenic mutants with mutations in the peptidoglycan recycling and β-lactamase induction systems. The MIC gap phenomenon was observed in the parent strain B317 but not in B317G (
cumG
-defective mutant; defective peptidoglycan recycling) and B317R (
cumR
-defective mutant; defective CumA transcriptional regulator). No β-lactamase activity was detected in B317G and B317R. β-Lactamase activity and the MIC gap phenomenon were restored in B317G/pMD301 (strain transcomplemented by a cloned
cumG
gene) and B317R/pMD501 (strain transcomplemented by a cloned
cumR
gene). MICs determined by the agar dilution method increased when lower agar concentrations were used. Our results indicated that the mechanism of the MIC gap phenomenon is related to peptidoglycan recycling and CumA induction systems. However, it remains unclear how β-lactamase induction of
P. vulgaris
is suppressed on agar plates.
Comparison of Effects of Medium Composition and Atmospheric Conditions on Detection of
Bilophila wadsworthia
β-Lactamase by Cefinase and Cefinase Plus Methods
Authors: Summanen P. H. 
Published: December 2019

ABSTRACT

The influence of growth medium and incubation conditions on the detection of
Bilophila wadsworthia
β-lactamase was tested with Cefinase and Cefinase Plus disks. The tests involved aerobic and anaerobic incubation with conventional disk and quantitative tube assays. The production of β-lactamase was correlated with penicillin G, ampicillin, and ampicillin-sulbactam MICs and inhibition zones on penicillin (2-U) disks. The strains were grown on (i) brucella agar (brucella), (ii) brucella agar supplemented with 1% pyruvate (brucella-pyruvate), and (iii) brucella agar supplemented with 1% taurine (brucella-taurine). With the aerobic disk assay, 100, 100, and 7% of strains were positive after 30 min from growth on brucella-pyruvate, brucella, and brucella-taurine plates, respectively; of strains grown on brucella-taurine, 54% remained negative by the Cefinase assay, and 23% remained negative by the Cefinase Plus assay at 2 h. In quantitative assays, the strains became positive after 30 min from brucella-pyruvate plates and after 1 h from brucella plates. The intensities of the reactions were strongest with brucella-pyruvate plates under anaerobic test conditions. Anaerobic incubation enhanced β-lactamase detection of growth on brucella-taurine: at 3 h, 85% of strains were positive in comparison to 38% with aerobic incubation. All β-lactamase-negative strains were susceptible to penicillin G and ampicillin; all β-lactamase-positive strains were resistant to ampicillin and, with the exception of two strains, penicillin G. In conclusion, β-lactamase production correlated with susceptibility to penicillin G and ampicillin. Brucella agar supplemented with 1% pyruvate was the most reliable medium for testing
B. wadsworthia
β-lactamase, and anaerobic incubation expedited positive results. Brucella agar supplemented with taurine was unsuitable for
B. wadsworthia
β-lactamase testing. Cefinase and Cefinase Plus results were in agreement, but Cefinase Plus yielded faster reactions.
Detection of TT Virus DNA and GB Virus Type C/Hepatitis G Virus RNA in Serum and Breast Milk: Determination of Mother-to-Child Transmission
Authors: Schröter Matthias , Polywka Susanne , Zöllner Bernhard , Schäfer Peter , Laufs Rainer , Feucht Heinz-Hubert 
Published: December 2019

ABSTRACT

To investigate the vertical transmission of the newly described TT virus (TTV), serum and breast milk samples from 46 women as well as sera from their 47 newborns were examined for the presence of TTV DNA by PCR. TTV DNA was detected in 47.8% (
n
= 22) of the women. All but one child born to these women were also viremic for TTV from the first sample onward. TTV DNA was found in 73.9% (
n
= 17) of the breast milk samples derived from TTV viremic mothers. The one TTV-negative child born to a viremic mother remained negative during follow-up, although it was breast-fed. Our data show that TTV is highly effectively transmitted from mothers to their children during pregnancy. Although the majority of breast milk samples from viremic mothers are positive by TTV PCR, there is no need to discourage women from breast-feeding, because most children are TTV viremic even before breast-feeding begins.
Specific and Rapid Detection by Fluorescent In Situ Hybridization of Bacteria in Clinical Samples Obtained from Cystic Fibrosis Patients
Authors: Hogardt Michael , Trebesius Karlheinz , Geiger Anna M. , Hornef Mathias , Rosenecker Josef , Heesemann Jürgen 
Published: December 2019

ABSTRACT

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected
Pseudomonas aeruginosa
,
Burkholderia cepacia
,
Stenotrophomonas maltophilia
,
Haemophilus influenzae
, and
Staphylococcus aureus
within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 10
5
CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with
H. influenzae
,
S. aureus
, and
P. aeruginosa
exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.
Capsule Expression by Bovine Isolates ofStaphylococcus aureusfrom Argentina: Genetic and Epidemiologic Analyses
Authors: Sordelli D. O. , Buzzola F. R. , Gomez M. I. , Steele-Moore L. , Berg D. , Gentilini E. , Catalano M. , Reitz A. J. , Tollersrud T. , Denamiel G. , Jeric P. , Lee J. C. 
Published: December 2019

ABSTRACTStaphylococcus aureusis an important cause of bovine mastitis worldwide, and effective preventive or therapeutic modalities are lacking. Although most humanS. aureusisolates produce capsular polysaccharides (CPs), few reports have described the prevalence of capsules on bovine isolates. This information is important for the rational design of a vaccine for the prevention of staphylococcal mastitis. We serotyped 195S. aureusstrains isolated between 1989 and 1997 from the milk of mastitic cows in Argentina. Only 14 (7.1%) of the strains were serotype 5, and all were recovered between 1989 and 1992. Thirteen serotype 8 strains were identified, and 12 of these were isolated between 1991 and 1994. The remaining 168 isolates were nonreactive (NR) with CP serotype 5 (CP5)- or CP8-specific antibodies. Hybridization studies performed with genomic DNA from eight NR strains revealed that only three of them carried the capsule genes. Pulsed-field gel electrophoresis (PFGE) performed with 127 of the 195S. aureusisolates revealed that most (86%) strains belonged to one of four major PFGE groups. Although 8 of 14 CP5 isolates showed a common PFGE pattern (arbitrarily defined as A1), 31 other A1 isolates from the same time period (1989 to 1992) were not CP5 positive. In contrast, only nine PFGE type B3 isolates were recovered between 1990 and 1994, and eight of these were positive for CP8 (P< 0.0003). The results of this study underscore the variability in capsule expression byS. aureusstrains isolated from different geographical regions and cast doubt on the roles of CP5 and CP8 in the pathogenesis and immunoprophylaxis of bovine mastitis in Argentina.
Comparison of Blood Collected in Acid-Citrate-Dextrose and EDTA for Use in Human Immunodeficiency Virus Peripheral Blood Mononuclear Cell Cultures
Authors: Fiscus Susan A. , Chakraborty Hrishikesh , Shepard Robin , Goodman Melissa 
Published: December 2019

ABSTRACT
Paired blood samples collected in acid-citrate-dextrose and EDTA were compared for human immunodeficiency virus (HIV) infectivity on the day of collection or after 1 day of storage at room temperature. No significant differences between the anticoagulants were observed. Culture positivity was significantly associated with HIV RNA viral loads for both anticoagulants.
PCR-Based Assay for Discrimination between Invasive and Contaminating
Staphylococcus epidermidis
Strains
Authors: Frebourg Noëlle Barbier , Lefebvre Sophie , Baert Stéphanie , Lemeland Jean-François 
Published: December 2019

ABSTRACT

The discrimination between
Staphylococcus epidermidis
strains that contaminate and infect blood cultures is a daily challenge for clinical laboratories. The results of PCR detection of putative virulence genes were compared for contaminating strains, sepsis-related strains, catheter strains, and saprophytic strains. Multiplex PCR was used to explore the
atlE
gene, which is involved in initial adherence, the intercellular adhesion gene cluster (
ica
), which mediates the formation of the biofilm, and the
agrA
,
sarA
, and
mecA
genes, which might contribute to the pathogenicity of
S. epidermidis
. Whereas the
atlE
,
agrA
, and
sarA
genes were almost ubiquitously amplified, the
ica
and
mecA
genes were detected significantly more in infecting strains than in contaminating strains (
P
≤ 0.02) and thus appeared to be related to the potential virulence of
S. epidermidis
.
Failure To Detect
Chlamydia pneumoniae
in Brain Sections of Alzheimer's Disease Patients
Authors: Gieffers Jens , Reusche Erich , Solbach Werner , Maass Matthias 
Published: December 2019

ABSTRACT

A recent North American study detected
Chlamydia pneumoniae
in 17 of 19 brains of Alzheimer's patients and supposed a
C. pneumoniae
infection to be a risk factor for Alzheimer's disease (AD). In this study, we analyzed paraffin-embedded tissue samples of 20 AD patients by nested PCR and immunocytochemistry with a panel of antichlamydial antibodies and could detect neither
C. pneumoniae
-specific DNA nor chlamydial antigens. From our data, the presence of
C. pneumoniae
in the brains of Alzheimer's patients is not a common phenomenon; an association remains questionable.
PCR Using 3′-Mismatched Primers To Detect A2142C Mutation in 23S rRNA Conferring Resistance to Clarithromycin in
Helicobacter pylori
Clinical Isolates
Authors: Alarcón Teresa , Domingo Diego , Prieto Nuria , López-Brea Manuel 
Published: December 2019

ABSTRACT

Twenty-five clarithromycin-resistant
Helicobacter pylori
strains (selected by agar dilution) were studied to detect A2142G and A2143G mutations in the 23S rRNA gene by a PCR-restriction fragment length polymorphism method and an A2142C mutation by PCR using a 3′-mismatched specific primer. A 700-bp amplified fragment was obtained by the mismatched PCR only in strains without an A2142G or A2143G mutation, indicating that those strains had the A2142C mutation.
Identification and Population Structure of
Burkholderia stabilis
sp. nov. (formerly
Burkholderia cepacia
Genomovar IV)
Authors: Vandamme P. , Mahenthiralingam E. , Holmes B. , Coenye T. , Hoste B. , De Vos P. , Henry D. , Speert D. P. 
Published: December 2019

ABSTRACT

The
Burkholderia cepacia
complex currently comprises five genomic species, i.e.,
B. cepacia
genomovar I,
B. multivorans
(formerly known as
B. cepacia
genomovar II),
B. cepacia
genomovar III,
B. cepacia
genomovar IV, and
B. vietnamiensis
(also known as
B. cepacia
genomovar V). In the absence of straightforward diagnostic tests for the identification of
B. cepacia
genomovars I, III, and IV, the last two genomic species were not formally classified as novel
Burkholderia
species (genomovar I contains the type strain and therefore retains the name
B. cepacia
). In the present study, we describe differential biochemical tests and a
recA
gene-based PCR assay for the routine identification of strains currently known as
B. cepacia
genomovar IV and propose formal classification of this organism as
Burkholderia stabilis
sp. nov.
B. stabilis
can indeed be differentiated from all other
B. cepacia
complex strains by the absence of beta-galactosidase activity, from strains of
B. cepacia
genomovars I and III and
B. vietnamiensis
by the inability to oxidize sucrose, and from
B. multivorans
by the lack of growth at 42°C. In addition, analysis with the
recA
gene-derived primers BCRG41 (5′-ACCGGCGAGCAGGCGCTT-3′) and BCRG42 (5′-ACGCCATCGGGCATGGCA-3′) specifically allows the detection of
B. stabilis
strains in a conventional PCR assay. Examination of a set of 21
B. stabilis
strains by means of random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis typing suggested that the genome of this organism is highly conserved, which is in sharp contrast to the generally accepted genomic diversity, variability, and plasticity among
B. cepacia
strains.