bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specific PCR Identification and Differentiation of the Thermophilic Campylobacters,
Campylobacter jejuni
,
C. coli
,
C. lari
, and
C. upsaliensis
Authors: Fermér Christian , Engvall Eva Olsson 
Published: December 2019

ABSTRACT

A sensitive PCR assay that detects the thermophilic campylobacters
C. jejuni
,
C. coli
,
C. lari
, and
C. upsaliensis
is reported. Furthermore, by digestion of the PCR products with two restriction enzymes, species differentiation was demonstrated. Thus, the present method has the potential to be used for both detection and identification of thermophilic
Campylobacter
species.
Capillary Electrophoresis–Single-Strand Conformation Polymorphism Analysis for Rapid Identification of
Pseudomonas aeruginosa
and Other Gram-Negative Nonfermenting Bacilli Recovered from Patients with Cystic Fibrosis
Authors: Ghozzi Rafiaa , Morand Philippe , Ferroni Agnes , Beretti Jean-Luc , Bingen Edouard , Segonds Christine , Husson Marie-Odile , Izard Daniel , Berche Patrick , Gaillard Jean-Louis 
Published: December 2019

ABSTRACT

We used capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of
Pseudomonas aeruginosa
and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera
Pseudomonas
,
Brevundimonas
,
Burkholderia
,
Comamonas
,
Ralstonia
,
Stenotrophomonas
, and
Alcaligenes
were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (
Escherichia coli
16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002–3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguish
P. aeruginosa
from
Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes
and
Alcaligenes xylosoxidans
from
Alcaligenes denitrificans
. Two hundred seven CF clinical isolates (153 of
P. aeruginosa
, 26 of
Stenotrophomonas maltophilia
, 15 of
Burkholderia
spp., and 13 of
A. xylosoxidans
) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.
Molecular Evidence of Coinfection of Ticks with
Borrelia burgdorferi
Sensu Lato and the Human Granulocytic Ehrlichiosis Agent in Switzerland
Authors: Leutenegger Christian M. , Pusterla Nicola , Mislin Caroline N. , Weber Rainer , Lutz Hans 
Published: December 2019

ABSTRACT

Adult
Ixodes ricinus
ticks were collected in Switzerland and tested for the presence of coinfection with
Borrelia burgdorferi
sensu lato and the human granulocytic ehrlichiosis (HGE) agent by real-time PCR. Of 100 ticks, 49% were positive for
B. burgdorferi
and 2% were positive for the HGE agent. The two HGE agent-positive ticks were also found to be positive for
B. burgdorferi
.
Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates
Authors: Monday Steven R. , Bohach Gregory A. 
Published: December 2019

ABSTRACTStaphylococcus aureusmay contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates ofS. aureusare further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individualS. aureusisolate possesses. We show here, usingS. aureusisolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.
Restriction Fragment Length Polymorphisms of Virulence Plasmids in
Rhodococcus equi
Authors: Takai Shinji , Shoda Masato , Sasaki Yukako , Tsubaki Shiro , Fortier Guillaume , Pronost Stephane , Rahal Karim , Becu Teotimo , Begg Angela , Browning Glenn , Nicholson Vivian M. , Prescott John F. 
Published: December 2019

ABSTRACT

Virulent
Rhodococcus equi
, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases
Bam
HI,
Eco
RI,
Eco
T22I, and
Hin
dIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (
Eco
RI and
Bam
HI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent
R. equi
in the world.
Intravenous Immunoglobulin Therapy: Confounding Effects on Serological Screening for Toxoplasmosis during Pregnancy
Authors: Pelloux Hervé , Fricker-Hidalgo Hélène , Brochier Gilbert , Goullier-Fleuret Andrée , Ambroise-Thomas Pierre 
Published: December 2019

ABSTRACT
The serological diagnosis of toxoplasmic infection during pregnancy is intended to prevent congenital infection of the fetus. However, in the context of recurrent pregnancy loss intravenous immunoglobulin therapy can create a biological trap for the interpretation of serological results, with potentially serious consequences for the outcome of the pregnancy.
Emergence and Spread in French Hospitals of Methicillin-Resistant
Staphylococcus aureus
with Increasing Susceptibility to Gentamicin and Other Antibiotics
Authors: Lelièvre Hervé , Lina Gerard , Jones Mark E. , Olive Claude , Forey Françoise , Roussel-Delvallez Micheline , Nicolas-Chanoine Marie-Hélène , Bébéar Cécile M. , Jarlier Vincent , Andremont Antoine , Vandenesch François , Etienne Jerome 
Published: December 2019

ABSTRACT

Oxacillin (methicillin) resistance in methicillin-resistant
Staphylococcus aureus
(MRSA) is associated with an increased incidence of resistance to other antibiotics, which has increased since it was first reported in 1969. In 1992 a new phenotype of MRSA arose in France; this was characterized by a heterogeneous expression of resistance to oxacillin and susceptibility to various antibiotics, including gentamicin but also tetracycline, minocycline, lincomycin, pristinamycin, co-trimoxazole, rifampin, and fusidic acid. In French hospitals a longitudinal nationwide surveillance of antibiotic resistance in
S. aureus
has allowed for the detection of changes in antibiotic susceptibility profiles. Seven French clinical laboratories (six from the mainland and one from the West Indies) reported the results of susceptibility testing of 57,347
S. aureus
strains isolated in their institutes between 1992 and 1998. Over a 7-year period the incidence of isolation of gentamicin-susceptible MRSA (GS-MRSA) strains has steadily increased to represent, in 1998, 46.8 to 94.4% of the MRSA strains, irrespective of the overall incidence of MRSA. Two predominant types recognized by pulsed-field gel electrophoresis (PFGE) accounted for the majority of the GS-MRSA in different mainland hospitals, both differing from the predominant type observed in the French West Indies. Some GS-MRSA and gentamicin-resistant MRSA (GR-MRSA) strains had closely related PFGE profiles, and hybridization studies confirmed the lack in GS-MRSA of the
aac6′-aph2
" gene, which confers resistance to all aminoglycosides, with conservation of the
ant4
′ gene, which confers resistance to kanamycin, tobramycin, and amikacin. Thus, it is likely that certain GS-MRSA strains could have emerged from GR-MRSA strains by excision or deletion of the
aac6′-aph2
" gene.
Development of a PCR Assay for Rapid Detection of Enterococci
Authors: Ke Danbing , Picard François J. , Martineau Francis , Ménard Christian , Roy Paul H. , Ouellette Marc , Bergeron Michel G. 
Published: December 2019

ABSTRACT

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the
tuf
gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the
tuf
gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (
Enterococcus avium
,
E. faecalis
,
E. faecium
, and
E. gallinarum
). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (
E. solitarius
DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2
Abiotrophia
species and several
Listeria
species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61
E. faecium
, 77
E. faecalis
, 9
E. gallinarum
, and 12
E. casseliflavus
isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.
Near Absence of Vancomycin-Resistant Enterococci but High Carriage Rates of Quinolone-Resistant Ampicillin-Resistant Enterococci among Hospitalized Patients and Nonhospitalized Individuals in Sweden
Authors: Torell Erik , Cars Otto , Olsson-Liljequist Barbro , Hoffman Britt-Marie , Lindbäck Johan , Burman Lars G. ,   The Enterococcal Study Group
Published: December 2019

ABSTRACT

Rates of colonization with enterococci with acquired resistance to vancomycin (vancomycin-resistant enterococci [VRE]) and ampicillin (ampicillin-resistant enterococci [ARE]) were determined by using fecal samples from 670 nonhospitalized individuals and 841 patients in 27 major hospitals. Of the hospitalized patients, 181 (21.5%) were carriers of ARE and 9 (1.1%) were carriers of VRE. In univariate analyses, length of hospital stay (odds ratio [OR], 4.6; 95% confidence interval [CI], 2.5 to 8.9) and antimicrobial therapy (OR, 4.7; 95% CI, 3.3 to 6.7) were associated with ARE colonization, as were prior treatment with penicillins (OR, 3.1; 95% CI, 1.8 to 5.5), cephalosporins (OR, 2.9; 95% CI, 1.7 to 5.0), or quinolones (OR, 2.7; 95% CI, 1.5 to 4.7). In logistic regression analysis, antimicrobial therapy for at least 5 days was independently associated with ARE carriage (adjusted OR, 3.8; 95% CI, 2.6 to 5.4). Over 90% of the ARE isolates were fluoroquinolone resistant, whereas 14% of the ampicillin-susceptible
Enterococcus faecium
isolates were fluoroquinolone resistant. ARE carriage rates correlated with the use of fluoroquinolones (
P
= 0.04) but not with the use of ampicillin (
P
= 0.68) or cephalosporins (
P
= 0.40). All nine VRE isolates were
E. faecium vanB
and were found in one hospital. Seven of these isolates were related according to their types as determined by pulsed-field gel electrophoresis. Among the nonhospitalized individuals, the ARE carriage rate was lower (6%;
P
< 0.05), and only one person, who had recently returned from Africa, harbored VRE (
E. faecium vanA
). The absence of VRE colonization in nonhospitalized individuals reflects an epidemiological situation in Sweden radically different from that in countries in continental Europe where glycopeptides have been widely used for nonmedical purposes.
Evaluation of a Bacteriophage-Based Assay (Phage Amplified Biologically Assay) as a Rapid Screen for Resistance to Isoniazid, Ethambutol, Streptomycin, Pyrazinamide, and Ciprofloxacin among Clinical Isolates of
Mycobacterium tuberculosis
Authors: Eltringham I. J. , Wilson S. M. , Drobniewski F. A. 
Published: December 2019

ABSTRACT

Rapid molecular assays for the detection of mutations associated with rifampin resistance in
Mycobacterium tuberculosis
are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of
M. tuberculosis
. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in
M. tuberculosis
isolates, especially in developing countries.
Identification of
Fusarium
Species Involved in Human Infections by 28S rRNA Gene Sequencing
Authors: Hennequin C. , Abachin E. , Symoens F. , Lavarde V. , Reboux G. , Nolard N. , Berche P. 
Published: December 2019

ABSTRACT

Fusarium
spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against
Fusarium
isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33
Fusarium
isolates belonging to six
Fusarium
species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera
Acremonium
and
Cylindrocarpon
. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of
Fusarium
species isolated from patients.
Glycopeptide-Intermediate
Staphylococcus aureus
: Evaluation of a Novel Screening Method and Results of a Survey of Selected U.S. Hospitals
Authors: Hubert Susannah K. , Mohammed Jasmine M. , Fridkin Scott K. , Gaynes Robert P. , McGowan John E. , Tenover Fred C. 
Published: December 2019

ABSTRACT

Isolates of
Staphylococcus aureus
with decreased susceptibilities to glycopeptide antimicrobial agents, such as vancomycin and teicoplanin, have emerged in the United States and elsewhere. Commercially prepared brain heart infusion agar (BHIA) supplemented with 6 μg of vancomycin per ml was shown in a previous study to detect glycopeptide-intermediate
S. aureus
(GISA) with high sensitivity and specificity; however, this medium, when prepared in-house, occasionally showed growth of vancomycin-susceptible control organisms. This limitation could significantly impact laboratories that prepare media in-house, particularly if they wished to conduct large surveillance studies for GISA. Therefore, a pilot study to detect GISA was performed with vancomycin-containing Mueller-Hinton agar (MHA) prepared in-house in place of commercially prepared BHIA. MHA was selected for this study because this medium is widely available and well standardized. The results of the pilot study showed that supplementation of MHA with 5 μg of vancomycin per ml was both a sensitive and a specific method for screening for GISA isolates. This method was used to screen for GISA among 630 clinical isolates of methicillin-resistant
S. aureus
collected during 1997 from 33 U.S. hospitals. Although 14
S. aureus
isolates grew on the screening agar, all were vancomycin susceptible (MICs were ≤1 μg/ml) by broth microdilution testing. Population analyses of five isolates revealed two with a subpopulation for which vancomycin MICs were 8 μg/ml. In summary, the MHA screen plate containing 5 μg of vancomycin per ml prepared in-house provides a sensitive and cost-effective method for large-scale screening for GISA for which vancomycin MICs are 8 μg/ml. However, confirmation of isolates as vancomycin resistant is critical. This study suggests that GISA was not a widespread problem in the United States in 1997.
Rapid Identification of
Burkholderia pseudomallei
in Blood Cultures by a Monoclonal Antibody Assay
Authors: Pongsunk Supinya , Thirawattanasuk Nittaya , Piyasangthong Nuanchan , Ekpo Pattama 
Published: December 2019

ABSTRACT

Burkholderia pseudomallei
is the causative agent of melioidosis. In northeast Thailand, this gram-negative bacterium is a major cause of mortality from septicemia. The definitive diagnosis of this disease is made by bacterial culture. In this study, we produced a monoclonal antibody (MAb) specific to the 30-kDa protein of
B. pseudomallei
by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen. The MAb could directly agglutinate with all 243 clinical isolates of
B. pseudomallei
but not with other gram-negative bacteria, except for one strain of
Burkholderia mallei
. However, the MAb cross-reacted with the gram-positive
Bacillus
sp. and
Streptococcus pyogenes. B. pseudomallei
in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay. The sensitivity and specificity of direct agglutination compared to the “gold standard,” the culture method, were 94.12 and 98.25%, respectively. However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system. The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens. The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively. The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds. Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory. This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method. In addition, the MAb is stable at room temperature for 2 weeks and at 4, −20, and −70°C for at least 1 year. The latex reagent was stable for at least 6 months at 4°C.
Genotypic Survey of Recent β-Lactam-Resistant Pneumococcal Nasopharyngeal Isolates from Asymptomatic Children in Chile
Authors: Gherardi Giovanni , Inostrozo Jaime S. , O'ryan Miguel , Prado Valeria , Prieto Susana , Arellano Carolina , Facklam Richard R. , Beall Bernard 
Published: December 2019

ABSTRACT

To assess pneumococcal strain variability among young asymptomatic carriers in Chile, we used serotyping, antibiotic susceptibility testing, and genotyping to analyze 68 multidrug-resistant pneumococcal isolates recovered from 54 asymptomatic children 6 to 48 months of age. The isolates represented capsular serotypes 19F (43 isolates), 14 (14 isolates), 23F (7 isolates), 6B (3 isolates), and 6A (1 isolate). Genotypic analysis, which included pulsed-field gel electrophoresis (PFGE) of chromosomal digests, penicillin binding protein (PBP) gene fingerprinting, and
dhf
gene fingerprinting, revealed that the isolates represented six different genetic lineages. Clear circumstantial evidence of capsular switching was seen within each of four of the genetically related sets. The majority of the isolates, consisting of the 43 19F isolates and 2 type 6B isolates, appeared to represent a genetically highly related set distinct from previously characterized pneumococcal strains. Each of three other genetically defined lineages was closely related to one of the previously characterized clones Spain
6B
-2, France
9V
-3, or Spain
23F
-1. A fifth lineage was comprised of four type 23F isolates that, by the techniques used for this study, were genetically indistinguishable from three recent type 19F sterile-site isolates from the United States. Finally, a sixth lineage was represented by a single type 23F isolate which had a unique PFGE type and unique PBP and
dhf
gene fingerprints.
Case of Peritonitis Caused by
Ewingella americana
in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis
Authors: Kati Chryssa , Bibashi Evangelia , Kokolina Elisabeth , Sofianou Danai 
Published: December 2019

ABSTRACT

Reports of serious infections caused by
Ewingella americana
have been rare. A case of
E. americana
peritonitis in a patient receiving continuous ambulatory peritoneal dialysis is described. This is the first report of
E. americana
causing such an infection.