bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of field inversion gel electrophoresis with contour-clamped homogeneous electric field electrophoresis as a typing method for Enterococcus faecium
Authors: Green M , Barbadora K , Donabedian S , Zervos M J
Published: January 2020

Direct comparisons between contour-clamped homogeneous electric field (CHEF) electrophoresis and field inversion gel electrophoresis (FIGE) to determine the epidemiology of antibiotic-resistant enterococci have not been previously published. Fifty non-beta-lactamase-producing, ampicillin-resistant Enterococcus faecium isolates and 10 vancomycin-resistant E. faecium strains collected from multiple centers were analyzed in a blinded fashion by CHEF electrophoresis and FIGE after digestion with SmaI. Isolates were considered clonally related if there was a difference of three of fewer bands between electrophoretic patterns. Agreement between CHEF electrophoresis and FIGE was seen for 12 of 13 identified groups of ampicillin-resistant E. faecium and 7 of 7 groups of vancomycin-resistant E. faecium. The lone discordance was accounted for by a fourth band difference between two strains recognized near 350 kb by CHEF electrophoresis but not by FIGE, placing them into different clonal groups. Better band separation was noted in the 50- to 200-kb range for FIGE, while CHEF electrophoresis revealed better resolution over 250 kb more reliably, including detection of some bands not seen on FIGE. Molecular epidemiologic investigations of E. faecium by either technique should provide comparable results.
Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes
Authors: Holodniy M , Mole L , Yen-Lieberman B , Margolis D , Starkey C , Carroll R , Spahlinger T , Todd J , Jackson J B
Published: January 2020

This study compared the levels of human immunodeficiency virus (HIV) virion RNA in plasma from whole blood collected in VACUTAINER CPT (cell preparation tube), VACUTAINER PPT (plasma preparation tube), VACUTAINER SST (serum separation tube), and standard VACUTAINER tubes with sodium heparin, acid citrate dextrose, sodium citrate, and potassium EDTA used as anticoagulants. Quantitative plasma HIV RNA levels were measured by branched-DNA signal amplification. Blood from all tubes was either processed within 1 to 3 h after collection or stored at room temperature or at 4 degrees C for analysis at 6 to 8 and 30 h postdraw. Immediately separated plasma from sodium citrate CPT tubes held at 4 degrees C maintained better stability of HIV RNA equivalents than whole blood held at room temperature or 4 degrees C. The highest number of HIV RNA equivalents was seen with EDTA VACUTAINER tubes. HIV RNA equivalents in all types of plasma were significantly higher than in SST tubes. Although a decline in HIV RNA equivalents was seen in all collection devices after 30 h, a significantly greater decline in plasma HIV RNA equivalents occurred in acid citrate dextrose VACUTAINER tubes than in citrate CPT, PPT, and standard EDTA VACUTAINER tubes. In order to minimize the variability of quantitative HIV RNA test results, our data suggest that samples collected for a particular assay should be processed at the same time postdraw using a particular tube type throughout a given study.
Randomly amplified polymorphic DNA analysis provides rapid differentiation of methicillin-resistant coagulase-negative staphylococcus bacteremia isolates in pediatric hospital
Authors: Bingen E , Barc M C , Brahimi N , Vilmer E , Beaufils F
Published: January 2020

Coagulase-negative staphylococci (CoNS) are now recognized as the most common cause of nosocomial bacteremia in pediatric patients. Randomly amplified polymorphic DNA analysis was used to study the relationships among 12 isolates of CoNS obtained from eight patients with catheter-related bacteremia in two distinct wards of our hospital and 6 epidemiologically unrelated strains. With this method, we were able to discriminate isolates that otherwise were indistinguishable by conventional criteria such as biochemical typing and antibiotic susceptibility patterns. Our results indicate that there were episodes of cross-infections among four patients in one ward but independent infectious episodes among four patients in the other ward. Randomly amplified polymorphic DNA analysis is a rapid method which seems particularly well suited to the epidemiological study of CoNS isolates.
Phenotypic characterization of epidemic versus sporadic strains of methicillin-resistant Staphylococcus aureus
Authors: Van Wamel W J , Fluit A C , Wadström T , van Dijk H , Verhoef J , Vandenbroucke-Grauls C M
Published: January 2020

Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%.
Transport and storage conditions for cultural recovery of Chlamydia pneumoniae
Authors: Maass M , Dalhoff K
Published: January 2020

Chlamydia pneumoniae is characterized by rapidly decreasing viability outside the host cell, and efficient preservation of its infectivity is a prerequisite for subsequent cell culture recovery. Extracellular survival of three C. pneumoniae stock strains and three wild-type strains subjected to simulated conditions of transport was therefore examined in order to establish recommendations for transport and storage of clinical specimens. The presence of fetal calf serum in transport media as well as refrigeration distinctly improved chlamydial retrieval during prolonged transport. Loss of infectivity was kept to a minimum in Eagle's minimal essential medium or sucrose-phosphate-glutamine medium. Storage at 22 degrees C permitted a stock strain recovery of 81% after 12 h. When refrigeration to 4 degrees C was provided, recovery rates of 74% could be achieved after 48 h. Though the strains were from different geographic regions, requirements for good survival were comparable and should therefore apply worldwide. The results indicate that the laboratory strains are not extremely labile. However, comparative examination of the wild-type strains showed less stability: primary isolates were not satisfactorily retrievable beyond 4 h at 22 degrees C or beyond 24 h at 4 degrees C. Further extension of storage times resulted in rapidly decreasing recovery, indicating a requirement to freeze samples at -75 degrees C to preserve viability. Adherence to the shorter storage periods suggested by the data obtained with primary isolates is recommended to ensure successful transport until more extensive testing with clinical materials is available.
Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate
Authors: Tully J G , Rose D L , Baseman J B , Dallo S F , Lazzell A L , Davis C P
Published: January 2020

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.
Concentration of sputum by cytocentrifugation for preparation of smears for detection of acid-fast bacilli does not increase sensitivity of the fluorochrome stain
Authors: Woods G L , Pentony E , Boxley M J , Gatson A M
Published: January 2020

To evaluate the usefulness of cytocentrifugation for detection of acid-fast bacilli (AFB) in sputum specimens, we compared this method to a traditional concentration method for the preparation of smears. A total of 844 sputum specimens (from 579 patients) of adequate volume that were submitted for detection of mycobacteria were evaluated. A portion of each specimen was used for cytocentrifugation; the remainder was processed by our decontamination-concentration protocol (2% sodium hydroxide-N-acetyl-L-cysteine; centrifugation at 3,600 x g for 15 min) for preparation of smears and culture. All smears were stained with auramine O. Ninety-four cultures from 46 patients gave positive results, and AFB were seen in one or both smears from 53 specimens; 3 of the latter specimens (positive by both smear methods) were culture negative. Of the 50 AFB smear-positive and culture-positive specimens, 46 were smear positive by traditional concentration, and 47 were positive by the cytocentrifugation smear (P, not significant). Cultures of all specimens that were smear positive by only one method grew nontuberculous mycobacteria. The routine use of cytocentrifugation for concentrating sputum specimens increases the cost of smear preparation but does not increase detection of AFB by auramine O staining; however, it would be useful in handling emergent requests for AFB smears.
Rapid antigen detection assay for identification of Chlamydia trachomatis infection
Authors: Van der Pol B , Williams J A , Jones R B
Published: January 2020

A rapid antigen detection test was compared with direct fluorescent-antibody staining and with tissue culture isolation for the detection of Chlamydia trachomatis infections in 507 women. The sensitivities observed were 75, 76, and 84%, respectively, with specificities of > 99%.
Brain abscess due to Bacillus macerans following a penetrating periorbital injury
Authors: Bert F , Ouahes O , Lambert-Zechovsky N
Published: January 2020

We report a case of a brain abscess due to Bacillus macerans and Clostridium sp. following a penetrating periorbital injury by a wooden branch. Intracranial penetration by and retention of a foreign body were not suspected initially, and neurological symptoms developed only 2.5 months later. Previously reported cases of brain abscesses due to Bacillus species are reviewed.
Turnover of nonencapsulated Haemophilus influenzae in the nasopharynges of otitis-prone children
Authors: Samuelson A , Freijd A , Jonasson J , Lindberg A A
Published: January 2020

Restriction enzyme analysis of total genomic DNA was applied to study the epidemiology of nontypeable Haemophilus influenzae (NTHI) isolated from the nasopharynges of children with recurrent acute otitis media (AOM). The turnover of strains, as judged from genetic fingerprinting of a total of 213 H. influenzae isolates collected prospectively during a 2-year study period from 38 children under 3 years of age, was examined in relation to episodes of AOM as well as to courses of antibiotic treatment. The children were selected if they had had at least one episode of AOM before 1 year of age and if more than two nasopharyngeal isolates of H. influenzae were recovered. The 213 H. influenzae isolates (90% NTHI) recovered corresponded to 128 different DNA fingerprints. Fifty-eight percent of the fingerprints were observed only once, whereas 42% appeared on two or more occasions in isolates from the same individual or in close relatives, i.e., brothers and sisters. Sixty-seven percent of these strains had a minimum colonization period of 2 months or less. Intermittent nasopharyngeal colonization periods longer than 5 months could be demonstrated for 13% of the strains. The present data suggest that intermittent colonization is due to endogenous reinfections. Genetically identical NTHI strains from unrelated individuals were never identified. As expected from the observation of a relatively high proportion of persistent colonizations, no correlation was found between episodes of AOM and the acquisition of new strains of H. influenzae, nor was any direct relation between antimicrobial therapy and the elimination of nasopharyngeal colonization with a particular strain of H. influenzae observed.
Genetic and biochemical characterization of Citrobacter rodentium sp. nov
Authors: Schauer D B , Zabel B A , Pedraza I F , O'Hara C M , Steigerwalt A G , Brenner D J
Published: January 2020

An unusual bacterial pathogen of laboratory mice has been previously classified as an atypical biotype of Citrobacter freundii. Designated C. freundii biotype 4280, this bacterium is the etiologic agent of transmissible murine clonic hyperplasia. An eaeA gene has been shown to be present in this organism and to be necessary for virulence in laboratory mice. However, other biotypes of C. freundii lack DNA homology with the eaeA gene. Because of the recent reclassification in which five named species and three unnamed species, all previously considered C. freundii, were described, we determined the taxonomic status of C. freundii biotype 4280. With a battery of biochemical tests and DNA relatedness studies, three isolates of C. freundii biotype 4280 were shown to be members of an unnamed Citrobacter species, designated species 9. In total, six isolates of Citrobacter species 9, but none of the type strains of the other eight named species or of the two remaining unnamed species of Citrobacter, were shown to possess DNA homology with both the eaeA and the eaeB genes. Species 9 was named Citrobacter rodentium sp. nov.
Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat
Authors: Clarridge J E , Raich T J , Pirwani D , Simon B , Tsai L , Rodriguez-Barradas M C , Regnery R , Zollo A , Jones D C , Rambo C
Published: January 2020

We wished to develop a cost-effective, rapid strategy to detect and identify Bartonella species in the clinical laboratory and to determine the prevalence of Bartonella infection in the Houston veteran population. Bartonella colonies were identified by colony morphology, Gram stain, RapID ANA, repetitive extragenic palindromic-PCR (REP-PCR) and whole-cell fatty acid (CFA) analysis, and these methods were compared for their usefulness. A new test order for "Rochalimaea culture" (the genus Bartonella was previously known as the genus Rochalimaea) was instituted, and in addition, all blood specimens submitted for fungal culture (obtained in an isolator tube) were processed for Bartonella culture. Over a 16-month period we isolated Bartonella henselae from only 0.4% (2 of 533) of total cultures but from 1% (2 of 204) of human immunodeficiency virus-positive patients. After sufficient growth, identification of the Bartonella isolates to the species level could be obtained in 2 days. The REP-PCR allowed discrimination of all known species, whereas CFA analysis distinguished all except B. henselae and Bartonella quintana. The RapID ANA results failed to differentiate between B. henselae and B. quintana, and results for other species differed by only one or two tests. Blood obtained from a kitten which had been introduced into the household of one patient 2 months before the onset of fever yielded a Bartonella strain which was shown to be different from the strain from the patient and distinct from other Bartonella species by a combination of REP-PCR, CFA, and growth characteristics. Subsequent analysis of the citrate synthase gene sequence showed only an 86% similarity with any of the other known Bartonella species, suggesting that this isolate represents a distinct, previously uncharacterized species of Bartonella.
Microbiology of secondary bacterial infection in scabies lesions
Authors: Brook I
Published: January 2020

Aerobic and anaerobic bacteria were grown from specimens obtained from 30 children with secondarily infected scabies lesions. Aerobic or facultative bacteria only were present in 14 (47%) patients, anaerobic bacteria only were present in 6 (20%) patients, and a mixed anaerobic-aerobic flora was present in 10 (33%) patients. Fifty isolates were recovered (1.7 per specimen); 27 were aerobic or facultative bacteria and 23 were strict anaerobes. The predominant aerobic and facultative bacteria were Staphylococcus aureus (nine isolates), group A streptococci (five isolates), and Pseudomonas aeruginosa (three isolates). The predominant anaerobes were Peptostreptococcus sp. (nine isolates) and pigmented Prevotella and Porphyromonas spp. (four isolates). Single bacterial isolates were recovered from nine (30%) patients; five of these were S. aureus. Sixteen organisms isolated from 12 (40%) patients produced the enzyme beta-lactamase. Organisms that resided in the mucous membranes close to or in contact with the lesions predominated in those infections. Enteric gram-negative rods were recovered in leg and trunk lesions. Group A streptococci and S. aureus predominated in finger and hand lesions. Bacteroides fragilis group and Clostridium sp. were isolated from leg lesions, and pigmented Prevotella sp. and Porphyromonas and Fusobacterium spp. were recovered from finger lesions. The polymicrobial etiology of secondarily infected scabies lesions in children and the association of bacterial flora with the anatomical sites of the lesions are demonstrated.
Molecular epidemiology of a fast-food restaurant-associated outbreak of Escherichia coli O157:H7 in Washington State
Authors: Grimm L M , Goldoft M , Kobayashi J , Lewis J H , Alfi D , Perdichizzi A M , Tarr P I , Ongerth J E , Moseley S L , Samadpour M
Published: January 2020

We studied the molecular epidemiology of the recent fast-food restaurant chain-associated Escherichia coli O157:H7 outbreak in Washington State. Genomic DNAs prepared from strains isolated from 433 patients were probed with radiolabelled Shiga-like toxin (SLT) I and SLT II genes and bacteriophage lambda DNA and were subsequently analyzed for their restriction fragment length polymorphism (RFLP) patterns. The SLT RFLP and lambda RFLP profiles of an E. coli O157:H7 strain isolated from the incriminated beef and prototype patient were compared with those of the patient isolates for determination of the concordance between patterns. Of the 377 patients with primary and secondary cases of infection epidemiologically linked to the outbreak, isolates from 367 (97.3%) of the patients displayed SLT RFLP and lambda RFLP profiles identical to those of the outbreak strains. Isolates from 10 of the 377 (2.6%) patients possessed SLT RFLP and lambda RFLP profiles different from those of the outbreak strains, and the patients from whom those isolates were obtained were subsequently characterized as having non-outbreak-related infections. The E. coli O157:H7 strains isolated from 31 of 44 (70.4%) patients who were epidemiologically excluded from the outbreak were linked to the outbreak by RFLP typing. Our results indicate that SLT RFLP and lambda RFLP analyses are stable and sensitive methods, and when they are used in conjunction with an epidemiological investigation they could result in an earlier recognition of outbreaks and their sources, hence prompting measures to prevent the continued transmission of E. coli O157:H7.
Comparative evaluation of microagglutination test and serum agglutination test as supplementary diagnostic methods for brucellosis
Authors: Baum M , Zamir O , Bergman-Rios R , Katz E , Beider Z , Cohen A , Banai M
Published: January 2020

The diagnosis of brucellosis in cattle and small ruminants requires the use of more than one serological test. The complement fixation test (CFT), the rose bengal test (RBT), and the serum agglutination test (SAT) are among the most useful tests for routine diagnosis. The microagglutination test (MAT) was developed as a simpler and more efficient test than the SAT. The relative efficacy of this test compared with that of the SAT was evaluated by using brucella-free sheep and goats prior to and after vaccination treatment. The specificities of the MAT and the SAT were 100%. Of the ewes and goats with a vaccination history, one ewe, expectedly a negative responder, had reactions in the MAT, the complement fixation test, and the rose bengal test but not in the SAT, suggesting a lower sensitivity of the SAT in this case. The calculated sensitivities of the MAT and the SAT were 93.9%. The agreement between MAT and SAT results from nonresponders was examined by using sera from unvaccinated lambs and kids (95.2% agreement), unvaccinated ewes and goats (84.4%), and ewes and goats with a vaccination history (43.9%). For the latter group higher levels of agglutination units were observed by the MAT than by the SAT in 51.5% of the samples. In testing sera from positive reactors after vaccination neither method was superior (MAT values were greater than SAT values for 23.5% of the samples, and MAT values were less than SAT values for 21.9% of the samples). Comparison of the methods on the individual sample level revealed a significant correlation between the MAT and the SAT (r = 0.96 +/- 0.005; P < 0.001). Since the MAT is simpler to perform than the SAT and can potentially be automated, the inclusion of the MAT as a supplementary test in brucellosis control programs is recommended.
Genetic diversity in clinical isolates of the dimorphic fungus Blastomyces dermatitidis detected by a PCR-based random amplified polymorphic DNA assay
Authors: Yates-Siilata K E , Sander D M , Keath E J
Published: January 2020

Blastomyces dermatitidis is a dimorphic fungus causing localized or systemic infection in areas where the organism is endemic in the central and southeastern United States. In this study, 19 independent isolates of B. dermatitidis from Little Rock, Ark., were grouped into three classes based on restriction fragment length polymorphism patterns in mitochondrial DNA with a heterologous probe from Histoplasma capsulatum. One large class of 15 isolates and two smaller classes (classes 2 and 3), each consisting of two isolates, were observed in BglII digests. Strain-specific arrays of PCR-amplified DNA products were obtained with arbitrarily selected primers (18 to 29 nucleotides long; G+C contents, 33 to 56%). In the large class 1 group, 13 isolates could be differentiated by the random amplified polymorphic DNA (RAPD) method with various primers. The two remaining class 1 isolates were obtained from the same patients and produced identical RAPD arrays. Dissimilar RAPD patterns were obtained from the smaller class 2 group but not from the class 3 isolates. Significant genetic diversity in clinical isolates of B. dermatitidis was observed; this may underscore a similar environmental diversification. Further application of the typing techniques may provide significant insight into the epidemiology of blastomycosis and aid in the assessment of specific virulence phenotypes.
Sorbitol-negative phenotype among enterohemorrhagic Escherichia coli strains of different serotypes and from different sources
Authors: Ojeda A , Prado V , Martinez J , Arellano C , Borczyk A , Johnson W , Lior H , Levine M M
Published: January 2020

Enterohemorrhagic Escherichia coli (EHEC) strains detected with DNA probes (for virulence plasmid and Shiga-like toxins) from subjects with hemolytic-uremic syndrome (n = 19) or diarrhea (n = 41) or asymptomatic carriers (n = 29) were examined for sorbitol fermentability, as were enterotoxigenic (n = 40), enteropathogenic (n = 40), and enteroinvasive (n = 40) E. coli and urinary tract infection (n = 40) strains and normal flora E. coli strains (n = 40). Sorbitol negativity was common only in EHEC, particularly among strains from severe clinical infections. All 19 EHEC strains from patients with hemolytic-uremic syndrome, irrespective of O:H serotype or Shiga-like toxin genotype, were sorbitol negative.
Corynebacterium seminale sp. nov., a new species associated with genital infections in male patients
Authors: Riegel P , Ruimy R , de Briel D , Prévost G , Jehl F , Bimet F , Christen R , Monteil H
Published: January 2020

We studied 12 coryneform isolates having similar biochemical profiles which did not permit their assignment to any recognized taxa. Human semen was the source for seven of these strains, whereas the other strains were isolated from urethra, urine, and blood specimens of adult male patients. These bacteria were found in significant quantities (10(4) to 10(5) CFU/ml) in semen specimens from infertile male patients with the diagnosis of prostatitis. These strains had characteristics of the genus Corynebacterium, such as 60 mol% G + C in the DNA and corynemycolic acids, meso-diaminopimelic acid, arabinose, and galactose in the cell wall. Quantitative DNA-DNA hybridizations (S1 nuclease procedure) and phylogenies based on comparisons of almost-complete small-subunit ribosomal DNA sequences confirmed that these strains constitute a single new species within the genus Corynebacterium. All 12 strains showed similar phenotypic features, i.e., good growth on sheep blood agar in contrast with poor growth on the same medium supplemented with 1% Tween 80, a positive CAMP test in the presence of Staphylococcus aureus, glucose and sucrose fermentation, and the presence of beta-glucuronidase. Some strains reduced nitrate and hydrolyzed urea or esculin. These features allowed us to distinguish these strains from members of any other coryneform taxon, and the proposed name is Corynebacterium seminale with strain IBS B12915 (CIP 104297) as the type strain. The description and delineation of these strains as a new species should be useful for further studies, including evaluations of their prevalence among the normal flora and their clinical implications.
Comparison of Mycoplasma arthritidis strains by enzyme-linked immunosorbent assay, immunoblotting, and DNA restriction analysis
Authors: Washburn L R , Voelker L L , Ehle L J , Hirsch S , Dutenhofer C , Olson K , Beck B
Published: January 2020

Twenty Mycoplasma arthritidis strains or isolates were compared by a combination of enzyme-linked immunosorbent assay by an antiserum adsorption technique, Western immunoblotting, and restriction analysis of chromosomal DNA. Antigenic markers that defined strains related to strains 158p10p9, PG6, and H606 were identified. In addition, restriction analysis allowed all 20 strains to be divided into six groups. Results of restriction analysis corresponded generally with antigenic similarities, although the former did not allow grouping with as fine a precision as the latter. However, intrastrain antigenic variability, which is common among many Mycoplasma species, including M. arthritidis, introduced a complicating factor into our attempts at antigenic analysis. While serologic and antigenic analyses remain useful, we recommend that they be used with caution and in combination with other techniques for identifying and characterizing new isolates and newly acquired strains. Combinations of these techniques have proven to be useful in our laboratory for quality control and for uncovering interesting relationships among strains subjected to animal passage and their less virulent antecedents and among strains originally classified as the same but obtained from different sources and maintained, sometimes for decades, in different laboratories.
Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois
Authors: Picken R N , Cheng Y , Han D , Nelson J A , Reddy A G , Hayden M K , Picken M M , Strle F , Bouseman J K , Trenholme G M
Published: January 2020

We have characterized 33 isolates of Borrelia burgdorferi from northern Illinois (32 isolates) and Wisconsin (1 isolate) representing the largest series of midwestern isolates investigated to date. The techniques used for molecular analysis of strains included (i) genospecies typing with species-specific PCR primers, (ii) plasmid profiling by pulsed-field gel electrophoresis of total genomic DNA, (iii) large-restriction-fragment pattern (LRFP) analysis by pulsed-field gel electrophoresis of MluI-digested genomic DNA (J. Belfaiza, D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons, J. Clin. Microbiol. 31:2873-2877, 1993), (iv) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins, (v) microsequencing of high-performance liquid chromatography-purified peptides derived from proteins showing high levels of expression, (vi) amino acid composition analysis of proteins, and (vii) immunological analysis of proteins with a polyclonal antiserum of human origin. Five reference strains as well as two atypical tick isolates from California (DN127) and New York (25015) were included for comparison. All of the Illinois and Wisconsin isolates were typed as B. burgdorferi sensu stricto with genospecies-specific PCR primers. The isolates were found to be heterogeneous with regard to their plasmid and protein profiles. One isolate from Illinois possessed two large-molecular-size plasmids instead of the usual 49-kb plasmid. Fragment patterns resulting from MluI digestion of genomic DNA from the 33 isolates and strains DN127 and 25015 were separable into six distinct LRFPs, five of which have not previously been described. Strain 25015 and an isolate from Illinois (CT39) shared an unusual LRFP that is not typical of other B. burgdorferi sensu stricto strains, suggesting that they may represent a fifth species of B. burgdorferi sensu lato. Five of the 33 isolates and strains DN127 and 25015 showed high-level expression of proteins with molecular masses of approximately 22 kDa. Investigation of these proteins by microsequencing of individual peptides and total amino acid composition analysis indicated that the 22-kDa proteins expressed by the seven strains were polymorphic OspC proteins. By using a polyclonal serum of human origin, expression of OspC could be detected in all 33 Illinois and Wisconsin isolates.
Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates
Authors: Marconi R T , Liveris D , Schwartz I
Published: January 2020

Borrelia spp. associated with Lyme disease possess an rRNA gene organization consisting of a single 16S rRNA gene followed by a spacer of several kilobases and a tandem repeat of a 23S (rrl)-5S (rrf) rRNA gene cluster. The restriction fragment length polymorphism (RFLP) patterns for these genes have been widely used to classify Lyme disease spirochete isolates. We analyzed the rRNA gene organization and sequences for two Ixodes ovatus isolates from Japan (IKA2 and HO14) and two group 21038 isolates associated with Ixodes dentatus ticks or rabbits from North America (isolates 21038 and 19857). This analysis revealed unique polymorphisms not previously described in other Lyme disease spirochete isolates. The molecular basis of these polymorphisms was determined by Southern blotting and PCR analyses. Only one continuous copy of the rrl-rrf gene cluster was identified in isolates IKA2, 19857, and 21038. The second rrl-rrf gene cluster is entirely absent from the IKA2 genome. In isolates 19857 and 21038, an intervening sequence is present, resulting in a fragment rrlB gene. The insertion site of this intervening sequence element differed in each isolate. While isolates 19857 and 21038 were found to carry a fragmented rrlB gene, they lacked rrfB. To determine if these rRNA polymorphisms were indicative of an underlying phylogenetic divergence, sequence analysis of the 16S rRNA (rrs) genes was conducted. The phylogenies inferred from rrs sequence analysis suggest that the polymorphisms resulted from recent mutational events. In addition, the phylogenetic analyses also support the proposed species status of Borrelia japonica sp. nov. and indicate that isolates of genomic group 21038 belong to a previously undescribed species for which we propose the nomenclature Borrelia andersonii sp. nov.
Evaluation of BACTEC 9240 blood culture system by using high-volume aerobic resin media
Authors: Schwabe L D , Thomson R B , Flint K K , Koontz F P
Published: January 2020

The BACTEC 9240 blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) is one of three automated, continuous-monitoring systems that is widely used in clinical laboratories. The BACTEC 9240 was compared with the BACTEC NR 660 for the detection of organisms and bacteremic episodes; time to detection of positive cultures; number of false-positive and false-negative cultures; and time needed to load, process, and perform quality control functions by using high-volume aerobic media. Blood specimens (5,282) were inoculated in equal volumes (5 to 10 ml per bottle) into BACTEC Plus Aerobic/F (9240 system) and BACTEC Plus NR26 (660 system) bottles. Clinically significant isolates were detected in 6.6% of cultures, representing 348 microorganisms and 216 bacteremic episodes. Two hundred forty-eight microorganisms were detected by both systems, 48 by the 9240 only and 52 by the 660 only (P = not significant). Of the bacteremic episodes, 158 were detected by both systems, 27 by the 9240 only and 31 by the 660 only (P = not significant). Analysis of data by month revealed equivalent recovery rates for both systems, with the exception of a 30-day period at one study site during which the 660 system detected significantly more microorganisms. Following a proprietary hardware design retrofit of the 9240 instrument, detection rates were again equivalent for the remaining three months at this study site. Positive cultures detected by both systems were detected an average of 4.3 h faster by the 9240 system (21 versus 25.3 h). The numbers of false-positive cultures for the 9240 and 660 systems were 40 (1.0%) and 9 ( < 1.0%), respectively. The numbers of false-negative cultures were five for the 9240 system and three for the 660 system. The 9240 system required 23 s less technologist time per bottle to operate during the 5-day protocol. In conclusion, the BACTEC 9240 used with high-volume Aerobic/F medium is equivalent to the BACTEC 660 used with high volume NR26 medium for the detection of microorganisms and bacteremic episodes. In addition, the 9240 system detects positive cultures more rapidly than the 660 system but requires further evaluation to ensure reliability of instrument components.
Phenotypic characteristics of 31 strains of Corynebacterium striatum isolated from clinical samples
Authors: Martínez-Martínez L , Suárez A I , Winstanley J , Ortega M C , Bernard K
Published: January 2020

During a 34-month period (January 1991 to October 1993), 31 Corynebacterium striatum stains recovered from clinical samples from 24 patients were characterized. Twenty (64%) strains were isolated from wound exudates, 5 (16%) were isolated from bronchial aspirates, 2 (7%) were isolated from urine, 2 (7%) were isolated from endotracheal tubes, 1 (3%) was isolated from a catheter, and 1 (3%) was isolated from empyema. The organisms were identified by conventional culture and phenotypic characterization, the API CORYNE system, and cellular fatty acid composition analyses. The colonies of C. striatum could be confused with those of coagulase-negative staphylococci upon primary isolation from clinical material. A consistent phenotypic pattern was observed: all strains reduced nitrate, hydrolyzed tyrosine, and produced acid from glucose, fructose, and sucrose but not from maltose. API CORYNE profile numbers were 3100105 (28 strains) and 3000105 (3 strains). Susceptibility testing of C. striatum was performed by disk diffusion. All strains were susceptible to both imipenem and vancomycin and resistant to fosfomycin; most strains were susceptible to ampicillin and cephalosporins and resistant to clindamycin, erythromycin, and tetracycline. Performing a Gram stain of fosfomycin-resistant "Staphylococcus-like" colonies was critical in order to identify C. striatum.
Streptococcus pneumoniae types 19A and 19F and Streptococcus suis capsular type 8 share common capsular epitopes
Authors: Gottschalk M , Kolberg J , Charland N , Jacques M
Published: January 2020

Two monoclonal antibodies (MAbs) to Streptococcus pneumoniae types 19A and 19F were tested with the 35 reference strains and 334 field strains of Streptococcus suis by dot blotting. Both MAbs reacted with the capsular type 8 reference strain, and one reacted with 69% and one reacted with 100% of 81 S. suis capsular type 8 field strains tested. Epitopes recognized by both MAbs are capsular in origin.
Detection of Cowdria ruminantium in blood and bone marrow samples from clinically normal, free-ranging Zimbabwean wild ungulates
Authors: Kock N D , van Vliet A H , Charlton K , Jongejan F
Published: January 2020

Cowdria ruminantium causes severe, often fatal disease in domestic ruminants, whereas wildlife species usually are not affected. Blood and bone marrow samples from healthy, free-ranging Zimbabwean ungulates were taken during translocation from areas harboring Amblyomma ticks and tested for the presence of C. ruminantium, using a PCR assay based on the C. ruminantium map1 gene. Positive reactions were obtained in tsessebe (Damaliscus lunatus), waterbuck (Kobus ellipsiprymnus), and impala (Aepyceros melampus). Wildlife species may therefore be a reservoir for C. ruminantium thus contributing to the spread of cowdriosis.
Species-specific Mycobacterium genavense DNA in intestinal tissues of individuals not infected with human immunodeficiency virus
Authors: Dumonceau J M , Fonteyne P A , Realini L , Van Gossum A , Van Vooren J P , Portaels F
Published: January 2020

Mycobacterium genavense species-specific DNA was detected in intestinal tissues from two of nine individuals not infected with human immunodeficiency virus. This newly described microorganism is well documented as a causative agent of disseminated infections in AIDS patients. Our results suggest that it may colonize the guts of individuals not infected with human immunodeficiency virus.
Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche PCR-microwell plate hybridization method (AMPLICOR MYCOBACTERIUM) for direct detection of mycobacteria
Authors: Ichiyama S , Iinuma Y , Tawada Y , Yamori S , Hasegawa Y , Shimokata K , Nakashima N
Published: January 2020

We evaluated the clinical utility of an rRNA amplification-based Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) system and a PCR-based Roche AMPLICOR MYCOBACTERIUM system for direct detection of Mycobacterium tuberculosis, M. avium, and M. intracellulare. Of the 422 sputum samples from 170 patients, 137 (121 of M. tuberculosis, 14 of M. avium-M. intracellulare complex [MAC], 2 of mycobacterium other than M. tuberculosis or MAC) were culture positive with the Septi-Chek AFB system. One sample of a contaminated culture results was excluded for further analyses. The AMTD system detected all of the 121 samples which grew M. tuberculosis (sensitivity, 100%). Of the 284 culture negative samples, 28 were positive by this system (specificity, 90.1%). After resolution of the discrepant samples, based on a positive history for culture of the patient, the specificity of this system increased to 99.3%. On the other hand, the AMPLICOR system gave a positive result for 132 out of the 135 culture positive samples for M. tuberculosis or MAC (sensitivity, 97.8%). Of the 284 culture-negative samples, 37 were positive by this system (specificity, 87.0%). The specificity for this system after resolution of the discrepant samples increased to 98.9%. The agreement between the results from the AMTD system and the AMPLICOR system was 98.7%. Both of the systems are highly sensitive and specific for detecting M. tuberculosis and/or MAC directly from sputum samples within hours, and they should be recommended for routine use in the clinical microbiology laboratory.
Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis
Authors: Rossetti O L , Arese A I , Boschiroli M L , Cravero S L
Published: January 2020

Brucella spp. are the causative agents of brucellosis in many different hosts, including humans. Most of the serological methods of diagnosis are based on the detection of antilipopolysaccharide antibodies, which makes the differentiation of vaccinated animals from infected animals difficult. By using molecular biology techniques, a gene that encodes a 26-kDa protein (BP26) was isolated from a Brucella abortus S19 genome lambda gt11 library. This protein is in the periplasm of B. abortus and in transformed Escherichia coli. It is exported to the periplasm via a preprotein of 29 kDa with a signal sequence of 28 amino acids. The nucleotide and amino acid sequences of this gene and protein did not show any similarity with those of previously sequenced genes. The use of this protein in Western blotting allowed the differentiation between vaccinated bovines from infected bovines and the detection of infected rams: on the other hand, sera from human patients with active brucellosis were positive, while sera from human patients with chronic brucellosis or without clinical signs were nonreactive. BP26 might be of value as an antigen for serological diagnosis of brucellosis in different mammals.
Clinical comparison of difco ESP, Wampole isolator, and Becton Dickinson Septi-Chek aerobic blood culturing systems
Authors: Cockerill F R , Torgerson C A , Reed G S , Vetter E A , Weaver A L , Dale J C , Roberts G D , Henry N K , Ilstrup D M , Rosenblatt J E
Published: January 2020

The ESP 80A aerobic blood culture of the ESP automated blood culture system (Difco Laboratories. Detroit, Mich.) was compared with two manual aerobic blood culture systems, the Isolator (Wampole Laboratories, Cranbury, N.J.) and the Septi-Chek (Becton Dickinson, Cockeysville, Md.) systems, for the detection of bloodstream microorganisms from 5,845 blood samples for culture collected from adult patients with suspected septicemia. The bottles were incubated for 7 days, and the sediment from the Isolator tube was inoculated onto solid medium and this medium was incubated for 72 h. A total of 609 microorganisms were recovered from 546 blood cultures. There was no statistically significant difference in the total recovery of microorganisms for the ESP 80A system when compared with that for the Septi-Chek system (P = 0.083); however, the Isolator system recovered significantly more microorganisms overall than either the ESP 80A (P < 0.001) or the Septi-Chek (P < 0.001) system. When assessing individual probable pathogens, the Isolator system detected statistically significantly more Staphylococcus aureus and Candida spp. than either the ESP 80A or the Septi-Chek system (P < 0.05). Similarly, the Isolator system detected statistically significantly more bloodstream infections (septic episodes) caused by S. aureus and Candida spp. than either the ESP 80A or the Septi-Chek system (P < 0.05). In blood culture sets which produced growth of the same probable pathogens in the ESP 80A and the Isolator systems, there was no statistically significant difference in the median times to detection for all pathogens combined (P = 0.067). However, a similar comparison showed the Isolator and the ESP 80A systems to have statistically significantly shorter median detection times for all pathogens combined (P < 0.001) when they were independently compared with the Septi-Chek system. The ESP 80A system had 29 (0.5%) false-positive signals. The ESP system required less processing time than the Isolator system and eliminates the hands-on time for the detection of positive cultures required by the manual systems.
Comparison of rectal and perirectal swabs for detection of colonization with vancomycin-resistant enterococci
Authors: Weinstein J W , Tallapragada S , Farrel P , Dembry L M
Published: January 2020

Patients whose gastrointestinal tracts are colonized with vancomycin-resistant enterococci (VRE) may serve as a reservoir for nosocomial transmission. We compared the sensitivities and concordance of several methods used to detect VRE colonization. Eighty-two paired rectal and perirectal swabs were obtained from 13 patients over a 9-day period. The sensitivity of both rectal and perirectal swabs was 79%. There was 100% concordance of culture results between simultaneously obtained rectal and perirectal swabs, and the quantities of growth were similar by these two methods of detection. Our data suggest that rectal and perirectal swabs are equally sensitive for the detection of VRE colonization.
PCR monitoring of response to liposomal amphotericin B treatment of systemic candidiasis in neutropenic mice
Authors: van Deventer A J , Goessens W H , van Belkum A , van Etten E W , van Vliet H J , Verbrugh H A
Published: January 2020

When a diagnosis of invasive candidiasis has been made, treatment with toxic fungicidal agents is inevitable. The crucial decision of when to stop such treatment is difficult to make, because cultures are often negative despite ongoing invasive candidiasis and can therefore not be used as a reliable parameter of effective therapy. In the present study, the use of PCR in monitoring the therapeutic efficacy of antifungal treatment with liposomal amphotericin B was evaluated by using neutropenic mice with systemic candidiasis. Blood cultures of infected mice treated with different doses of liposomal amphotericin B were only positive at the early onset of the infection process and became sterile within 3 days; this was true even with mice treated with 1 mg of liposomal amphotericin B per kg of body weight that experienced a relapse of infection 14 days later. A significant correlation between presence of Candida albicans in the kidneys and PCR results obtained with blood was demonstrated. Thus, PCR results obtained with blood samples correlated well with the therapeutic efficacy of antifungal treatment.