bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of histopathologic and bacteriologic changes with cytokine expression in an experimental murine model of bacteremic Staphylococcus aureus infection
Authors: Yao L , Berman J W , Factor S M , Lowy F D
Published: January 2020

Staphylococcus aureus infections are often life threatening. Relatively little is known about the host response to these infections, in particular, the role played by cytokines. We established a mouse model of bacteremic S. aureus infection to correlate bacteriologic findings and pathologic changes with cytokine gene expression. Bacterial density in blood and tissue was highest at 1 h and minimal by 48 h. Despite the rapid clearance of bacteria, pathologic abnormalities and inflammatory cytokines were detected after clearance of the bacteria. The number of infiltrating inflammatory cells, as well as the size of inflammatory foci, increased with time. Interstitial accumulation of inflammatory cells and tissue damage, such as microabscesses, edema, and necrosis progressed following clearance of bacteria from the tissues. Levels of tumor necrosis factor and interleukin-1 protein in serum were detectable at 1 h and peaked at 4 h. Interleukin-6 protein expression showed different kinetics, with low levels detected at 1 h and increasing levels at 72 h postinfection. Tumor necrosis factor and the interleukins were expressed in inflammatory and noninflammatory cells in lung, liver, and heart tissues. Leukocytes in the infected tissues were highly reactive with antibodies to the three cytokines, suggesting that activated leukocytes are a major source of inflammatory cytokines after staphylococcal infection. Expression of interleukin-1 and interleukin-6 in tissue-specific cells and endothelial cells was also detected in infected tissues, indicating that cells other than leukocytes contribute to the elevated cytokine levels in this model. Once initiated, expression of inflammatory cytokines contributes to the pathogenesis of S. aureus disease.
Listeriolysin and IrpA are major protein targets of the human humoral response against Listeria monocytogenes
Authors: Grenningloh R , Darji A , Wehland J , Chakraborty T , Weiss S
Published: January 2020

We have examined the human humoral immune response directed against proteins of Listeria monocytogenes in both healthy individuals and listeriosis patients. Two major targets for an antibody response were found in individuals that did not suffer from listeriosis: listeriolysin (Hly) and the recently described internalin-related protein (IrpA). In contrast, the humoral response in listeriosis patients appears to be more heterogeneous and included Hly, IrpA, InlB, and ActA as major targets.
Identification of a protein in several Borrelia species which is related to OspC of the Lyme disease spirochetes
Authors: Marconi R T , Samuels D S , Schwan T G , Garon C F
Published: January 2020

Using oligonucleotide probes which have previously been shown to be specific for the ospC gene found in the Lyme disease spirochete species Borrelia burgdorferi, B. garinii, and group VS461, we detected an ospC homolog in other Borrelia species including B. coriaceae, B. hermsii, B. anserina, B. turicatae, and B. parkeri. In contrast to the Lyme disease spirochetes, which carry the ospC gene on a 26-kb circular plasmid, we mapped the gene in other Borrelia species to linear plasmids which varied in size among the isolates tested. Some isolates carry multiple copies of the gene residing on linear plasmids of different sizes. The analyses conducted here also demonstrate that these Borrelia species contain a linear chromosome. Northern (RNA) blot analyses demonstrated that the gene is transcriptionally expressed in all species examined. High levels of transcriptional expression were observed in some B. hermsii isolates. Transcriptional start site analyses revealed that the length of the untranslated leader sequence was identical to that observed in the Lyme disease spirochete species. By Western blotting (immunoblotting) with antiserum (polyclonal) raised against the OspC protein of B. burgdorferi, we detected an immunoreactive protein of the same molecular weight as the OspC found in Lyme disease spirochete species. The results presented here demonstrate the presence of a protein that is genetically and antigenically related to OspC which is expressed in all species of the genus Borrelia tested.
Improved purification and biologic activities of staphylococcal toxic shock syndrome toxin 1
Authors: Kum W W , Laupland K B , See R H , Chow A W
Published: January 2020

An improved method for producing highly purified toxic shock syndrome toxin 1 (TSST-1) by preparative isoelectric focusing in a Bio-Rad Rotofor cell and then chromatofocusing is described. Purification to homogeneity was confirmed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 50 micrograms of protein was loaded), by immunoblotting with polyclonal rabbit antiserum raised against the crude culture supernatant used for purification, and by autoradiography after iodination and SDS-PAGE. Biologic activity was demonstrated by mitogenicity and cytokine induction (tumor necrosis factor alpha [TNF-alpha], interleukin 1-beta [IL-1 beta], and IL-6) of human peripheral blood mononuclear cells (PBMCs) and by lethality in New Zealand White rabbits following subcutaneous infusion. In contrast to commercial TSST-1 preparations, our TSST-1 preparation required the presence of both monocytes and T cells for the induction of TNF-alpha and IL-1 beta from human PBMCs. A 46-kDa contaminating protein in the commercial TSST-1 preparation, identified as staphylococcal lipase, was likely responsible for the induction of TNF-alpha and IL-1 beta from human monocytes in the absence of T cells, a biologic activity falsely attributed to purified TSST-1. Our improved purification procedure for TSST-1 provides a high yield and is both more rapid and less labor intensive than previously reported methods. Furthermore, our studies clearly demonstrate the need for stringent methods of purity assessment of TSST-1 preparations before ascribing to them their potent biologic activities.
Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia
Authors: Rudolph K M , Parkinson A J , Black C M , Mayer L W
Published: January 2020

To test the ability of the polymerase chain reaction (PCR) to detect Streptococcus pneumoniae in blood, we generated two sets of nested primers. The first defined 559-bp and 649-bp regions of the pneumolysin gene, and the second defined 445-bp and 553-bp regions of the autolysin gene. These nucleotide segments were detected in DNAs from isolates of all 20 pneumococcal serotypes tested, but they were not detected when used to test DNAs from 41 isolates of nonpneumococcal bacteria and fungi. The sensitivity was evaluated by using purified pneumococcal DNA. We were able to detect 10 fg of S. pneumoniae DNA, or 4.3 genome equivalents. Blood samples were obtained from 16 patients with culture-proven pneumococcal bacteremia and were subjected to PCR analysis. Of eight buffy coat fractions tested, six showed reactivity in the PCR with the pneumolysin primers, and five of the eight produced the expected products when tested with the autolysin primers (sensitivities, 75 and 63%, respectively). Of the eight whole-blood specimens tested, only three produced the expected products with either set of primers. Additionally, we tested 14 samples from patients with bacteremia that were culture positive for nonpneumococcal bacterial species, and 13 were negative (specificity, 93%). This combination of sensitivity and specificity may make detection of S. pneumoniae in blood by PCR in comparison with that by blood culture a very promising alternative for a means of definitive diagnosis.
Isolation of a DNA probe for identification of Mycobacterium kansasii, including the genetic subgroup
Authors: Yang M , Ross B C , Dwyer B
Published: January 2020

In order to develop a DNA-based assay to identify all Mycobacterium kansasii clinical isolates, a specific DNA probe was isolated in plasmid p6123. A total of 145 M. kansasii clinical isolates were collected from several countries and were examined with three probes by DNA hybridization. Of the 145 isolates, 115 (79%) were positive with the previously described probe pMK1-9 (Z. H. Huang, B. C. Ross, and B. Dwyer, J. Clin. Microbiol. 29:2125-2129, 1991), 129 (88%) were positive with the commercial Accu-probe assay (Gen-Probe), and 145 (100%) were positive with the p6123 probe. Southern blot analysis of EcoRI-digested M. kansasii chromosomal DNA with p6123 revealed that all Accu-probe-positive M. kansasii strains exhibited a 3-kb fragment, whereas all Accu-probe-negative M. kansasii strains displayed DNA fragments of variable molecular sizes. These results indicate that, unlike the previously described probes for M. kansasii, the fragment cloned into p6123 identified all 145 biochemically typical strains tested and provides an ideal target for future DNA-based speciation assays.
Identification and characterization of multiple species of vancomycin-resistant enterococci, including an evaluation of Vitek software version 7.1
Authors: Willey B M , Kreiswirth B N , Simor A E , Faur Y , Patel M , Williams G , Low D E
Published: January 2020

A total of 374 clinical isolates of Enterococcus spp. were characterized to determine the species distribution and vancomycin resistance. The ability of the Vitek system (bioMerieux Inc, Hazelwood, St. Louis, Mo.) to identify enterococci to the species level and to recognize vancomycin resistance by using computer software version 7.1 was evaluated. Conventional methods were used for identification and agar dilution was used for susceptibility testing, the results of which were as follows (presented as number of vancomycin-resistant isolates/number of members of that species identified): 219/234 E. faecium, 9/112 E. faecalis, 2/3 E. mundtii, 0/1 E. durans, 0/1 E. hirae, 0/1 E. raffinosis, and 0/1 E. avium. Ten enterococci were in the vancomycin-intermediate category (six E. gallinarum, two E. casseliflavus, and one each of E. faecium and E. faecalis). The Vitek GPI card correctly identified 98% of E. faecium isolates, 99% of E. faecalis isolates, and only two isolates of other enterococcal species. The GPS-TA card was 98% sensitive and 95% specific for the detection of vancomycin resistance, generating a total of four very major (1.6%) and five minor errors (1.3%).
Genetic heterogeneity of oncogenic human papillomavirus type 5 (HPV5) and phylogeny of HPV5 variants associated with epidermodysplasia verruciformis
Authors: Deau M C , Favre M , Jablonska S , Rueda L A , Orth G
Published: January 2020

Variants of oncogenic human papillomavirus type 5 (HPV5), specifically associated with epidermodysplasia verruciformis, were recognized on the basis of the genetic heterogeneity of the E6 open reading frame (ORF). To further evaluate the genetic heterogeneity of HPV5, we sequenced the long control region (LCR), the E7 ORF, and the terminal parts of the E2 ORF of five previously characterized HPV5 variants and compared the data with the published HPV5a1 and HPV5b sequences. Alignment of the variants showed 140 (7.6%) variable nucleotides of 1,854 sequenced. Nucleotide substitution rates varied from 3.6% in the E7 ORF to 11% in the E6 ORF. By sequencing the variable region encompassing the LCR 3' part and the E6 ORF of isolates from six additional epidermodysplasia verruciformis patients, we identified three new variants and three already known variants, indicating the stability of HPV5 variants. This stability was further demonstrated by the identity of isolates obtained years later from benign and malignant lesions of three patients. Phylogenetic analysis of the 10 HPV5 variants distributed them into three groups, tentatively defining subtypes a, b, and c. The phylogenetic grouping shows no geographical dependence, a fact that may be related to the host restriction that characterizes HPV5 infections. No differences in the enhancer potential of the LCR or in the transactivating properties of the E2 protein assayed in vitro were observed among HPV5 variants. Whether HPV5 variants possess distinct biological properties in vivo remains to be determined.
Opacification of Middlebrook agar as an aid in identification of Nocardia farcinica
Authors: Flores M , Desmond E
Published: January 2020

One hundred twenty seven human isolates of Nocardia asteroides complex were identified to the species level by drug susceptibility testing, acetamide utilization, thermotolerance studies, studies of arylsulfatase activity, and studies of acid production from rhamnose. N. asteroides complex organisms which were not identifiable as N. farcinica or N. nova were designated N. asteroides sensu stricto. All of the N. farcinica isolates and none of the other isolates produced a milky-white opacity surrounding colonies on Middlebrook 7H10 agar. This finding may facilitate the identification of N. farcinica.
Evaluation of Etest for testing antimicrobial susceptibilities of Neisseria gonorrhoeae isolates with different growth media
Authors: Yeung K H , Ng L K , Dillon J A
Published: January 2020

The MICs for 101 isolates of Neisseria gonorrhoeae obtained by Etest (AB-Biodisk, Solna, Sweden) and the agar dilution method on GC medium base supplemented with 1% Kellog's defined supplement (GCMB) were compared. The overall percent agreement (within 1 log2 dilution) between methods was greater than 97.9. The Pearson's correlation coefficients for penicillin, tetracycline, erythromycin, and ceftriaxone for the two methods were 0.98, 0.97, 0.93, and 0.93 (P = 0.001), respectively, for comparisons on GCMB. The overall percent agreement was lower when hemoglobin-supplemented GCMB was used. Etest is an attractive alternative to the agar dilution method for gonococcal antimicrobial susceptibility testing and should be further analyzed in multicenter studies.
Karyotyping of Cryptococcus neoformans as an epidemiological tool
Authors: Perfect J R , Ketabchi N , Cox G M , Ingram C W , Beiser C L
Published: January 2020

Karyotyping of Cryptococcus neoformans var. neoformans can be used as an epidemiological tool for C. neoformans infections. In this study of over 40 isolates from both clinical and environmental sources, 90% had a unique chromosome banding by pulsed-field electrophoresis. There was no conserved pattern associated with body site of infection, geographical location of the isolate, or human immunodeficiency virus status. Karyotypes of individual isolates remained stable during both in vitro passage and in vivo infections. Karyotype was used to exclude the possibility of nosocomial spread of C. neoformans in one clinical situation and supported relapse in two other cases. Because of its variable sizes between isolates, karyotyping of C. neoformans is a convenient method for molecular identification of different strains.
Comparison of three buffers used in the formulation of buffered charcoal yeast extract medium
Authors: Edelstein P H , Edelstein M A
Published: January 2020

Growth of Legionella spp. on buffered charcoal yeast extract medium supplemented with alpha-ketoglutarate and formulated with 3-(n-morpholino)propanesulfonic acid (MOPS), 3-(n-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), or n-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer was similar. With three exceptions, growth was no different in buffered yeast extract broth supplemented with alpha-ketoglutarate and formulated with MOPS or ACES buffer.
Molecular epidemiology and subgroup analysis of bovine group A rotaviruses associated with diarrhea in South African calves
Authors: da Costa Mendes V M , de Beer M , Peenze I , Steele A D
Published: January 2020

Rotavirus-positive specimens were recovered from 143 Afrikander calves on two farms in the northeastern Cape of South Africa during late 1988 and 1989. The rotavirus strains were analyzed by polyacrylamide gel electrophoresis of the RNA genome, and four rotavirus RNA electrophoretypes, each with a long profile, were identified. A distinct RNA profile was identified on the farms during 1988, but by early 1989, two patterns existed, one unique to each farm. Over the next 8 months a new electrophoretic pattern emerged on one farm, whereas the pattern on the other farm remained unchanged. The rotavirus subgroup I antigen was detected in all specimens examined with subgroup-specific monoclonal antibodies. Non-group A rotaviruses were not identified by RNA genome analysis of 82 specimens from calves with diarrhea negative for group A rotaviruses by an enzyme-linked immunosorbent assay.
Analysis of acquired human cytomegalovirus infections by polymerase chain reaction
Authors: Bale J F , O'Neil M E , Fowler S S , Murph J R
Published: January 2020

We used the polymerase chain reaction and primers corresponding to three regions of the human cytomegalovirus (HCMV) genome to study HCMVs isolated from 16 children attending a single day-care center and the father of two children in the same center. When we analyzed isolates with primers for the pp65 and major immediate-early genes, we observed nearly uniform amplification yielding products of predicted sizes. By contrast, primers for the a sequence demonstrated variability among HCMV strains, supporting the use of these primers as an epidemiologic tool. Analysis of a-sequence products from two isolates demonstrated 50 to 70% nucleotide homology with the a sequence of HCMV Towne strain DNA. We observed 95% nucleotide homology for the two a-sequence products derived from the father-child pair. Analysis of day-care center isolates indicated that two children excreted two distinct HCMV strains during the study interval.
A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro
Authors: Gebran S J , Newton C A , Yamamoto Y , Klein T W , Friedman H
Published: January 2020

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.
Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase
Authors: Loewy Z G , Mecca J , Diaco R
Published: January 2020

Uracil DNA glycosylases are DNA repair enzymes present in virtually every organism. These enzymes function by excising from DNA uracil residues resulting from either misincorporation of dUMP residues by a DNA polymerase or deamination of cytosine. Recently, the enzyme has been exploited in PCRs as a means for controlling carryover contamination from previously amplified DNA. When the enzyme is used in amplifications of Borrelia burgdorferi target sequences, we have observed an enhancement in signal detected by a microwell plate DNA hybridization assay. This increase in signal is dependent upon the length of the target, is titratable with enzyme concentration, and has been observed with amplifications performed with both symmetric and asymmetric PCR profiles. The enhancement is shown to occur at the level of the target genomic DNA.
Rapid detection of methicillin-resistant Staphylococcus aureus strains not identified by slide agglutination tests
Authors: Kuusela P , Hildén P , Savolainen K , Vuento M , Lyytikäinen O , Vuopio-Varkila J
Published: January 2020

Seventy-nine methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated during 1980 to 1990, were classified as MRSA Aggl- (14 strains) and MRSA Aggl+ (65 strains) strains on the basis of test results in slide agglutination assays designed to detect fibrinogen-binding protein (clumping factor) and protein A on the staphylococcal surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that lysostaphin digests of MRSA Aggl- strains contained a high-molecular-weight protein which was not detected in digests of MRSA Aggl+ strains. Immunization of rabbits with an MRSA Aggl- strain produced an antiserum which agglutinated all MRSA Aggl- strains and also 64 of 65 MRSA Aggl+ strains. Only 1 of 68 coagulase-negative staphylococci showed agglutination in this assay. The anti-MRSA Aggl- antiserum reacted mainly with a 230-kDa staphylococcal surface protein but also with a 175-kDa protein, probably formed by proteolysis of the former and a few slightly smaller proteins. These could not be immunologically detected in lysostaphin digests of MRSA Aggl+ strains. Purified antibodies reacting with the 230-kDa protein agglutinated all MRSA Aggl- strains, indicating that the protein is located on the surfaces of staphylococci. The results suggest a tentative role for the 230-kDa protein or its fragments as a novel target to develop more efficient rapid identification methods for S. aureus, including MRSA.
Evaluation of API NH, a new 2-hour system for identification of Neisseria and Haemophilus species and Moraxella catarrhalis in a routine clinical laboratory
Authors: Barbé G , Babolat M , Boeufgras J M , Monget D , Freney J
Published: January 2020

API NH is a new 2-h system (bioMérieux, La Balme-les-Grottes, France) for the identification of most Neisseria and Haemophilus spp. of clinical significance and of Moraxella catarrhalis and for the detection of penicillinase production. Furthermore, this system allows the biotyping of Haemophilus influenzae and Haemophilus parainfluenzae. Three hundred eighteen strains belonging to these species, previously identified by conventional methods, were tested. Among the 305 strains belonging to species included in the data base, 225 (73.8%) were identified without additional tests, 79 (25.9%) were correctly identified after extra tests, and 1 strain (0.3%) was misidentified. For 131 (90.3%) of the 145 H. influenzae and H. parainfluenzae strains, results of biotyping were in agreement with results of standard methods. API NH is an accurate and reliable method for the routine identification of these bacteria in a clinical laboratory, for biotyping of Haemophilus spp., and for the detection of penicillinase-producing strains. The system is ready to use and time saving; inoculation of the system and reading of results are easy.
Osteomyelitis and intervertebral discitis caused by Blastoschizomyces capitatus in a patient with acute leukemia
Authors: D'Antonio D , Piccolomini R , Fioritoni G , Iacone A , Betti S , Fazii P , Mazzoni A
Published: January 2020

We describe the first known case of vertebral osteomyelitis and discitis caused by Blastoschizomyces capitatus in a leukemic patient and the results of therapy. We also reconfirm the microbiological characteristics which differentiate this species from other yeastlike pathogens.
Characterization of Escherichia coli isolated from the tonsils of cattle
Authors: Frank G H , Briggs R E , Schneider R A
Published: January 2020

During our studies on tonsillar colonization by Pasteurella haemolytica, we consistently found Escherichia coli to be one of the most prominent and prevalent bacterial species in the tonsils of healthy cattle. Since tonsillar isolates have not been characterized, we grouped 124 isolates from 87 healthy cattle from eight sources by hemolytic zone size and by carbon source utilization and probed them for selected virulence genes. They formed 3 groups by hemolytic zone size and 18 groups (of 2 to 31 isolates) by their metabolic patterns. Most groups included isolates from more than one source. Two isolates contained the Shiga-like toxin gene, and nine others contained the F41 accessory gene.
Development and application of an enzyme immunoassay for coronavirus OC43 antibody in acute respiratory illness
Authors: Gill E P , Dominguez E A , Greenberg S B , Atmar R L , Hogue B G , Baxter B D , Couch R B
Published: January 2020

Study of coronavirus OC43 infections has been limited because of the lack of sensitive cell culture systems and serologic assays. To improve this circumstance, we developed an indirect enzyme immunoassay (EIA) to detect serum antibody to OC43. Antigen (100 ng) prepared by polyethylene glycol precipitation provided optimal results without a postcoat procedure. Evaluation of intraplate variation indicated that a > or = 2.5-fold increase in serum titer was significant. Sixteen of 18 (89%) paired serum samples with previously identified, reproducible increases in the level of hemagglutination inhibition (HAI) antibody to OC43 also showed significant increases as detected by EIA. Specificity for the EIA was established with paired sera obtained from persons given influenza immunizations or experiencing a respiratory infection. No rise in antibody titers occurred among 33 persons with documented coronavirus 229E infection. EIA was then performed on each of 419 paired serum samples from ambulatory chronic obstructive pulmonary disease patients and healthy older adults, from asthmatic adults presenting for emergency room treatment, and from persons hospitalized with acute respiratory symptoms. Twenty-three antibody rises to OC43 were detected; only nine of these were detected by the HAI test, and the HAI test did not detect any increases in antibody titers that were not detected by EIA. Nineteen of 25 coronavirus OC43 infections for which a month of infection could be assigned occurred between November and February. Overall, 4.4% of acute respiratory illnesses in the studied populations were associated with a coronavirus OC43 infection.
Detection of resistance due to inducible beta-lactamase in Enterobacter aerogenes and Enterobacter cloacae
Authors: Huber T W , Thomas J S
Published: January 2020

Thirty-six of 36 strains of Enterobacter cloacae and E. aerogenes with inducible beta-lactamase developed resistance when cefoxitin (inducer) was added to cefuroxime disks. Constitutive beta-lactamase producers (n = 23) were all resistant to cefuroxime. Cefuroxime resistance correlated with the amount of induced or constitutive beta-lactamase. Cefuroxime was a better indicator of induced resistance than cefamandole, cefazolin, cephalothin, ceftriaxone, cefotaxime, ticarcillin with or without clavulanic acid, or cefotetan. Induction by addition of cefoxitin to disks occasionally reduced zone sizes but not enough to change interpretations for ceftazidime, ceftizoxime, aztreonam, cefoperazone with or without sulbactam, and piperacillin with or without tazobactam. Most enterobacters were resistant to cefmetazole. The cefoxitin inducer-cefuroxime indicator method can be used in routine clinical laboratories to detect latent resistance due to chromosomally mediated inducible beta-lactamase in enterobacters.
Quantitative antibiogram typing using inhibition zone diameters compared with ribotyping for epidemiological typing of methicillin-resistant Staphylococcus aureus
Authors: Blanc D S , Lugeon C , Wenger A , Siegrist H H , Francioli P
Published: January 2020

Antibiogram typing of methicillin-resistant Staphylococcus aureus with selected antibiotics was evaluated as a primary epidemiological typing tool and compared with ribotyping. Antibiograms were derived with the Kirby-Bauer disk diffusion method by using erythromycin, clindamycin, cotrimoxazole, gentamicin, and ciprofloxacin. For typing, antibiogram data were analyzed by similarity analysis of disk zone diameters (quantitative antibiogram typing). One hundred seventy-two isolates were typed. Reproducibility reached 98% for the quantitative antibiogram and 100% for ribotyping. With three selected restriction enzymes (EcoRV, HindIII, and KpnI), 40 epidemiologically unrelated isolates could be classified into 21 ribotypes, whereas quantitative antibiogram typing classified these isolates into 19 groups. To evaluate the discriminatory power of the methods, we calculated an index of discrimination from data obtained with these 40 isolates. This index takes into consideration both the number of types defined by the typing method and their relative frequencies. With both ribotyping and quantitative antibiogram typing, high discrimination indices (0.972 and 0.954, respectively) were obtained. When epidemiological links between patients (ward, period of hospitalization, and contacts between staff and patients) were compared with the results of ribotyping or the quantitative antibiogram typing method, it appeared that both methods were able to discriminate epidemiological clusters, with only a few discrepancies. In conclusion, quantitative antibiogram typing, although not necessarily based on genomic markers, is a simple method which enables a reliable workup of methicillin-resistant S. aureus epidemic when sophisticated molecular typing methods are not available.
Hepatitis C virus (HCV) core serotypes in chronic HCV infection
Authors: Mondelli M U , Cerino A , Bono F , Cividini A , Maccabruni A , Aricò M , Malfitano A , Barbarini G , Piazza V , Minoli L
Published: January 2020

Recently, two distinct hepatitis C virus (HCV) serologic types have been identified on the basis of amino acid variations in the core region. The two serologic types can readily discriminate between genotypes I-II-V (serotype 1) and III-IV (serotype 2), according to the Okamoto classification. We compared HCV core serotyping with genotyping with sera from 363 anti-HCV-positive patients (309 HCV RNA positive by PCR) using a synthetic core peptide-based enzyme immunoassay and PCR amplification of core region sequences with type-specific primers, respectively. Serologic responses to HCV serotypes were successfully identified in 164 (45%) patients, of whom 153 were viremic. Eighty-nine patients had evidence of exposure to serotype 1: 8 of these were infected with genotype I, 50 were infected with genotype II, 2 were infected with genotype III, 7 were infected with genotype V, 13 had infections with mixed genotypes, 3 were infected with an indeterminate genotype, and 6 were nonviremic. Seventy-four patients had been exposed to serotype 2: 64 were infected with genotype III, 3 were infected with mixed genotypes, 2 were infected with an indeterminate genotype, and 5 were nonviremic. The serum of one patient, infected with genotype III, showed reactivity to both serotypes. Comparative evaluation of HCV core region serotyping and genotyping with sera from 294 viremic patients infected with a known HCV genotype showed a remarkable concordance between HCV core region genotyping and serotyping, with only 2 apparently discordant serum samples (both from patients with genotype III infection) of 148 (1.4%) successfully serotyped samples. Serotype 1 infection was more frequently observed in patients with overt chronic liver disease and accounted for all successfully serotyped samples from intravenous drug abusers. In contrast, serotype 2 was more prevalent in subjects with biochemically silent HCV infection (alanine aminotransferase, < 45 U/liter), in agreement with previous findings at the molecular level. HCV core serologic typing is a simple, inexpensive, and highly reproducible assay that can be applied to more than 50% of viremic HCV antibody carriers prior to the use of more sophisticated molecular typing techniques. Moreover, it may be helpful in tracking transmissions routes, particularly for incorrectly stored samples in which the RNA has degraded or for subjects who have cleared the virus and therefore have only antibodies remaining to testify to a remote infection. The lack of recognition of the core sequence from residues 67 to 81, which contains a minor B-cell epitope used to detect type-specific immunoreactivity, may explain the negative serologic findings for half of the patients.
Monitoring spread of Malassezia infections in a neonatal intensive care unit by PCR-mediated genetic typing
Authors: van Belkum A , Boekhout T , Bosboom R
Published: January 2020

Malassezia furfur and Malassezia pachydermatis were isolated from newborn children and incubators in a neonatal intensive care unit. To assess whether persistence or frequent import of the organisms was the cause of the elevated incidence, genetic typing of the strains was performed by PCR-mediated DNA fingerprinting. By using PCR primers aimed at repeat consensus motifs, six different genotypes could be detected in a collection of six M. furfur reference strains. In the case of 10 M. pachydermatis reference strains, nine different genotypes were detected by three different PCR assays. None of these assays could document genetic differences among the clinical isolates of either M. furfur or M. pachydermatis. On the basis of these results it is concluded that within the neonatal intensive care unit the longitudinal persistence of both an M. furfur and an M. pachydermatis strain has occurred and that Malassezia species can persist on incubator surfaces for prolonged periods of time. It can be concluded that PCR fingerprinting is a Malassezia typing procedure that is to be preferred over the analysis of chromosomal polymorphisms by pulsed-field gel electrophoresis in this genus.
Shigella sonnei strains isolated from U.S. summer students in Guadalajara, Mexico, from 1986 to 1992
Authors: Scerpella E G , Mathewson J J , DuPont H L , Marani S K , Ericsson C D
Published: January 2020

Plasmid DNA analysis and antibiotic susceptibilities were used to study strains of Shigella sonnei isolated from U.S. travelers to Guadalajara, Mexico, over a period of seven years (1986 to 1992). One hundred sixty-one isolates were analyzed. By the use of cluster analysis, eight different plasmid profiles were identified during this interval. At any point in time, three to seven different plasmid profiles were present in this population. The introduction of strains that carried a new plasmid with a molecular mass of 5.1 MDa was coincidental with an increase in isolation of S. sonnei in 1988. This new plasmid was present in 87.5% of the isolates that were resistant to chloramphenicol. Shigellosis in Guadalajara follows a pattern of hyperendemic transmission with transient peaks of high-frequency isolation of S. sonnei. This pattern results from the concurrent presence of a heterogeneous group of strains as opposed to the widespread transmission of one or a few clones.
Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR
Authors: Bricker B J , Halling S M
Published: January 2020

Several PCR assays which identify the genus Brucella but do not discriminate among species have been reported. We describe a PCR assay that comprises five oligonucleotide primers which can identify selected biovars of four species of Brucella. Individual biovars within a species are not differentiated. The assay can identify three biovars (1, 2, and 4) of B. abortus, all three biovars of B. melitensis, biovar 1 of B. suis, and all B. ovis biovars. These biovars include all of the Brucella species typically isolated from cattle in the United States, a goal of the present research. The assay exploits the polymorphism arising from species-specific localization of the genetic element IS711 in the Brucella chromosome. Identity is determined by the size(s) of the product(s) amplified from primers hybridizing at various distances from the element. The performance of the assay with U.S. field isolates was highly effective. When 107 field isolates were screened by the described method, there was 100% agreement with the identifications made by conventional methods. Six closely related bacteria (Agrobacterium radiobacter, Agrobacterium rhizogenes, Ochrobactrum anthropi, Rhizobium leguminosarum, Rhizobium meliloti, and Rhodospirillum rubrum) and two control bacteria (Bordetella bronchiseptica and Escherichia coli) tested negative by the assay.
Characterization of a hospital outbreak of imipenem-resistant Acinetobacter baumannii by phenotypic and genotypic typing methods
Authors: Tankovic J , Legrand P , De Gatines G , Chemineau V , Brun-Buisson C , Duval J
Published: January 2020

During a 13-month period, 31 patients hospitalized primarily in two intensive care units (ICUs) were either colonized or infected by imipenem-resistant Acinetobacter baumannii. Typing of the isolates by three methods (antibiotyping, biotyping, and pulsed-field gel electrophoresis) revealed that two distinct strains were involved in the first 9 cases of the outbreak and that one of these strains, which had acquired a higher level of imipenem resistance as well as resistance to all aminoglycosides, accounted for 21 of 22 cases in the second part of the outbreak. ICU environmental contamination was recognized as an important reservoir of this epidemic strain. The outbreak ceased only after the ICUs were closed for complete cleaning and disinfection.
Novel, ultrasensitive, Q-beta replicase-amplified hybridization assay for detection of Chlamydia trachomatis
Authors: Shah J S , Liu J , Smith J , Popoff S , Radcliffe G , O'Brien W J , Serpe G , Olive D M , King W
Published: January 2020

A sensitive, nonisotopic hybridization assay termed "dual capture" is described. The assay rapidly and specifically detects very low levels of target nucleic acids and organisms. The assay is based on the principles of sandwich hybridization, reversible target capture, and Q-Beta replicase amplification. The assay can be completed in less than 4 h, and in the described model format, it detects Chlamydia trachomatis rRNA or rDNA. Up to 96 samples can be analyzed simultaneously. The assay employs two types of probes: a test-specific capture probe, which mediates the cycling of the target probe complex on and off derivatized magnetic beads, and a replicatable RNA detector molecule containing a sequence complementary to and adjacent to the capture probe site on the target. Following reversible target capture, detection of the signal is accomplished by replication of the detector molecule by Q-Beta replicase in the presence of propidium iodide. A specific assay signal can be detected from as few as 1,000 molecules above the background. In a limited study of 94 urogenital samples the assay detected five of the six culture-positive samples and did not detect the C. trachomatis target in 85 of the 88 culture-negative samples.
Comparison of Chlamydia pneumoniae isolates by western blot (immunoblot) analysis and DNA sequencing of the omp 2 gene
Authors: Wagels G , Rasmussen S , Timms P
Published: January 2020

The 60-kDa cysteine-rich outer membrane protein gene (omp 2) from nine Chlamydia pneumoniae isolates (TW-183, CM-1, CWL-050, CWL-011, IOL-207, FIL, Kajaani-6, Helsinki-12, and Parjaanonen) was amplified by PCR and sequenced from positions 1 to 580. In contrast to the sequence differences previously observed in this gene in other chlamydial species, all nine C. pneumoniae isolates were 100% identical. However, when sera from C. pneumoniae microimmunofluorescence-positive patients (with clinical signs of persistent cough or asthma) were immunoblotted against five C. pneumoniae isolates, distinct antigenic differences were observed. TW-183 was characterized by major bands at 35 and 43 kDa. In contrast, the other four isolates tested produced similar, though not identical, immunoblot profiles, characterized by strong bands at 18, 25, 29, 40, 46, and 53 kDa. These data support the fact that significant differences do exist between C. pneumoniae isolates, but unlike the case with other chlamydial species, these differences do not reside in either of the commonly studied outer membrane protein genes, omp 1 or omp 2.
Simple test of synergy between ampicillin and vancomycin for resistant strains of Enterococcus faecium
Authors: Green M , Barbadora K , Wadowsky R M
Published: January 2020

The combination of ampicillin and vancomycin kills some but not all strains of ampicillin- and vancomycin-resistant Enterococcus faecium. We compared a simple test for synergy utilizing a commercially available microdilution susceptibility system with time-kill studies and determined acceptable breakpoints for this test for 20 strains of ampicillin- and vancomycin-resistant E. faecium. The combination of ampicillin and vancomycin was tested for synergy by time-kill, broth macrodilution, and broth microdilution procedures. Repeat testing of isolates by macro- and microdilution synergy methods yielded MICs that were within one twofold dilution of each other for both intra- and intertest comparisons. Synergy was always detected by time-kill studies when the MIC of ampicillin in the combination synergy screen was < or = 8 micrograms/ml in the presence of vancomycin. No synergy was detected when the MIC was > 16 micrograms/ml in the combination microdilution synergy screen. The determination of the synergy by the broth microdilution procedure appears to be simple, convenient, and accurate.
Clostridium bifermentans bacteremia with metastatic osteomyelitis
Authors: Scanlan D R , Smith M A , Isenberg H D , Engrassia S , Hilton E
Published: January 2020

Osteomyelitis caused solely by an anaerobic organism is uncommon. We report a case of recurrent Clostridium bifermentans bacteremia resulting in metastatic osteomyelitis involving the sacrum, spine, and ribs. The emergence of resistance of this organism to imipenem and metronidazole is noteworthy because of the usual susceptibility of clostridial species to these antibiotics.
rRNA gene restriction patterns of Streptococcus pyogenes: epidemiological applications and relation to serotypes
Authors: Bruneau S , de Montclos H , Drouet E , Denoyel G A
Published: January 2020

The use of rRNA gene restriction fragment length polymorphism analysis (ribotyping) to subtype Streptococcus pyogenes strains was investigated. Sixty-eight S. pyogenes strains, including 17 reference strains and 51 isolates from blood, acute or chronic pharyngitis, and food-borne outbreaks, were characterized by determination of both their rDNA restriction fragment length polymorphism profiles and their serotypes (T and M). Total DNA was cleaved with five selected restriction enzymes and then probed with a digoxigenin-labeled stretch of 1,063 bp hybridizing with 16S rRNA genes. Fifteen and nine distinct patterns were generated with SacI and XhoI, respectively, and five patterns were generated with each of the three additional restriction enzymes. With the combination SacI-XhoI, a total of 21 distinct ribotypes were obtained among the 68 isolates. This number was not increased by the results obtained with the other restriction enzymes. All strains tested were typeable. All isolates from each food-borne outbreak belonged to the same ribotype, and all isolates (pre- and posttreatment) from each child with chronic pharyngitis also belonged to the same ribotype, suggesting antibiotic treatment failures. A discriminatory index was calculated for the 47 isolates which were epidemiologically unrelated, using the Hunter-Gaston formula. This index reached 0.955 when the combination SacI-XhoI was used, showing the good discriminatory power of this typing method. Therefore, ribotyping proved to be a molecular method of interest to subtype S. pyogenes. Moreover, there was some correlation between ribotyping and serotyping, as several ribotypes were related to a unique distinct M serotype.
PCR identification of four medically important Candida species by using a single primer pair
Authors: Jordan J A
Published: January 2020

A single pair of primers was used in a PCR assay to amplify and identify the DNAs from four medically important Candida species: C. albicans, C. parapsilosis, C. tropicalis, and C. (Torulopsis) glabrata. The report describes the first successful amplification of a chitin synthase-specific fragment from the four Candida species responsible for more than 90% of all cases of neonatal candidemia. The primer pair sequence was based on that from the C. albicans chitin synthase gene, CHS1 (J. Au-Young and P.W. Robbins, Mol. Microbiol. 4:197-207, 1990). Each of the four amplified products is a single band of a different size. The DNA sequence of each PCR product was determined, and four species-specific probes were synthesized. The DNAs from as few as 10 organisms in 100 microliters of plasma could be detected after amplification and Southern blot analysis. In a retrospective study of 27 paired blood samples from 16 patients with culture-proven candidemia, PCR analysis was successful at detecting and correctly identifying to the species level 26 of the 27 Candida isolates. The speed and accuracy of this PCR-based technology make it a very powerful tool for detecting and diagnosing candidemia. Implementation of this assay for analyzing blood samples should result in the more timely treatment of neonatal candidemia, thereby reducing morbidity and mortality.
Rapid identification of bacteria by PCR-single-strand conformation polymorphism
Authors: Widjojoatmodjo M N , Fluit A C , Verhoef J
Published: January 2020

A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.
Rapid identification of Candida albicans by using Albicans ID and fluoroplate agar plates
Authors: Rousselle P , Freydiere A M , Couillerot P J , de Montclos H , Gille Y
Published: January 2020

Two commercially available agar media, Albicans ID and Fluoroplate, that use a chromogenic or a fluorogenic substrate for the detection and identification of Candida albicans were evaluated. From 1,006 clinical samples containing 723 yeast strains, 352 C. albicans strains were detected with either of the two media. The sensitivity of each of the two media was 93.8% and the specificity was 98.6%, with five false-positive reactions for Candida tropicalis and no false-negative reactions.
Serological cross-reaction between Legionella spp. and Capnocytophaga ochracea by using latex agglutination test
Authors: Chen S , Hicks L , Yuen M , Mitchell D , Gilbert G L
Published: January 2020

Cross-reactivity between Legionella spp. and Capnocytophaga ochracea was noted by latex agglutination tests (Serobact Legionella; Disposable Products, Adelaide, Australia). Four of 11 (36%) C. ochracea isolates agglutinated with latex reagents designed to identify Legionella pneumophila serogroups. C. ochracea isolated on buffered charcoal yeast extract media may give false-positive results in this Legionella latex agglutination assay.