bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Effects of Medium Composition and Atmospheric Conditions on Detection of
Bilophila wadsworthia
β-Lactamase by Cefinase and Cefinase Plus Methods
Authors: Summanen P. H.
Published: December 2019

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

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

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

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

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

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

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

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

The
Burkholderia cepacia
complex currently comprises five genomic species, i.e.,
B. cepacia
genomovar I,
B. multivorans
(formerly known as
B. cepacia
genomovar II),
B. cepacia
genomovar III,
B. cepacia
genomovar IV, and
B. vietnamiensis
(also known as
B. cepacia
genomovar V). In the absence of straightforward diagnostic tests for the identification of
B. cepacia
genomovars I, III, and IV, the last two genomic species were not formally classified as novel
Burkholderia
species (genomovar I contains the type strain and therefore retains the name
B. cepacia
). In the present study, we describe differential biochemical tests and a
recA
gene-based PCR assay for the routine identification of strains currently known as
B. cepacia
genomovar IV and propose formal classification of this organism as
Burkholderia stabilis
sp. nov.
B. stabilis
can indeed be differentiated from all other
B. cepacia
complex strains by the absence of beta-galactosidase activity, from strains of
B. cepacia
genomovars I and III and
B. vietnamiensis
by the inability to oxidize sucrose, and from
B. multivorans
by the lack of growth at 42°C. In addition, analysis with the
recA
gene-derived primers BCRG41 (5′-ACCGGCGAGCAGGCGCTT-3′) and BCRG42 (5′-ACGCCATCGGGCATGGCA-3′) specifically allows the detection of
B. stabilis
strains in a conventional PCR assay. Examination of a set of 21
B. stabilis
strains by means of random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis typing suggested that the genome of this organism is highly conserved, which is in sharp contrast to the generally accepted genomic diversity, variability, and plasticity among
B. cepacia
strains.
Clonal and Spontaneous Origins of Fluconazole Resistance in
Candida albicans
Authors: Xu Jianping , Ramos Adela R. , Vilgalys Rytas , Mitchell Thomas G.
Published: December 2019

The genotypes and susceptibilities to fluconazole of 78 strains of the human pathogenic yeast
Candida albicans
were compared. The strains comprised two sets of samples from Durham, N.C.: one from patients infected with the human immunodeficiency virus (HIV) and the other from healthy volunteers. For each strain, the MIC of fluconazole was determined by the standard National Committee for Clinical Laboratory Standards protocol. Genotypes were determined by PCR fingerprinting with five separate primers. The analysis revealed little evidence for genotypic clustering according to HIV status or body site. However, a small group of fluconazole-resistant strains isolated from patients infected with HIV formed a distinct cluster. In addition, two fluconazole-resistant strains were isolated from individuals who never took fluconazole, one from a patient infected with HIV and the other from a healthy person. The results suggest both clonal and spontaneous origins of fluconazole resistance in
C. albicans
.
Two Liquid Medium Systems, Mycobacteria Growth Indicator Tube and MB Redox Tube, for
Mycobacterium tuberculosis
Isolation from Sputum Specimens
Authors: Heifets L. , Linder T. , Sanchez T. , Spencer D. , Brennan J.
Published: December 2019

Two manual liquid medium systems, the Mycobacteria Growth Indicator Tube (MGIT) and MB Redox tube systems, were evaluated in comparison to the radiometric BACTEC-460 semiautomated system for recovery of
Mycobacterium tuberculosis
from sputum specimens. The highest level of recovery, from a total of 77 culture-positive specimens, occurred with the BACTEC-460 system (92.2%), followed by the MB Redox tube (80.5%) and the MGIT (63.6%) systems. The shortest time to detection was observed also among the cultures in BACTEC-460: a mean of 12 days to a growth index (GI) of 10 and 15 days to a GI of 500. The mean times for the other systems were 16 days for the MB Redox tube system and 17.4 days for the MGIT system. The proportion of cultures grown after more than 3 weeks of incubation was only 2.8 or 8.4% in BACTEC-460 (for a GI of 10 or 500) but 17.7% in MB Redox and 22.5% in MGIT. Despite these differences in comparison to the BACTEC-460 system and some differences between the MGIT and MB Redox tube systems, either of the two manual liquid medium systems presents a reasonable alternative to the BACTEC-460 system, especially for laboratories with a limited workload, and a valuable element in the laboratory protocol, in conjunction with solid media, for obtaining rapid detection of growth from about 80% of culture-positive specimens and for better overall recovery of
M. tuberculosis
.
Growth of
Cowdria ruminantium
, the Causative Agent of Heartwater, in a Tick Cell Line
Authors: Bell-Sakyi Lesley , Paxton Edith A. , Munderloh Ulrike G. , Sumption Keith J.
Published: December 2019

The tick-borne rickettsia
Cowdria ruminantium
has been propagated continuously for over 500 days in the
Ixodes scapularis
tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.
Epidemiological Analysis of Non-M-Typeable Group A Streptococcus Isolates from a Thai Population in Northern Thailand
Authors: Pruksakorn Sumalee , Sittisombut Nopporn , Phornphutkul Charlie , Pruksachatkunakorn Chulabhorn , Good Michael F. , Brandt Evelyn
Published: December 2019

Infection with group A streptococci (GAS) can lead to the development of severe postinfectious sequelae such as rheumatic fever (RF). In Thailand, RF and rheumatic heart disease (RHD) remain important health problems. More than 80% of GAS circulating in this population are non-M antigen typeable by conventional M serotyping methods. In this study, we determine the M protein sequence types of GAS isolates found in northern Thailand. Theemmgenes from 53 GAS isolates, collected between 1985 and 1995 from individuals with pharyngitis, impetigo, acute RF (ARF), RHD, or meningitis as well as from individuals without infections, were amplified by PCR and sequenced. Thirteen new sequence types that did not show homology to previously published sequences were characterized. Six of these sequence types could be isolated from both skin and throat sites of impetigo and pharyngitis/ARF patients, respectively. In many cases we could not specifically differentiate skin strains or throat strains that could be associated with ARF or acute glomerulonephritis. Antigenic variations in theemmgene of the isolates investigated, compared to published M protein sequences, were predominantly due to point mutations, small deletions, and insertions in the hypervariable region. One group of isolates with homology to M44 exhibited corrected frameshift mutations. A new M type isolated from an RHD patient exhibited nucleotide sequence corresponding to the N terminus of M58 and the C terminus of M25, suggesting that recombination between the two types may have occurred. This study provided epidemiological data relating to GAS endemic to northern Thailand which could be useful for identification of vaccine candidates in a specific region of endemicity.
Helminthic Transmission and Isolation ofEhrlichia risticii, the Causative Agent of Potomac Horse Fever, by Using Trematode Stages from Freshwater Stream Snails
Authors: Pusterla Nicola , Madigan John E. , Chae Joon-Seok , DeRock Elfriede , Johnson Eileen , Pusterla Jeannine Berger
Published: December 2019

We report successful helminthic transmission ofEhrlichia risticii, the causative agent of Potomac horse fever, using trematode stages collected fromJuga yrekaensissnails. The ehrlichial agent was isolated from the blood of experimentally infected horses by culture in murine monocytic cells and identified asE. risticiiultrastructurally and by characterization of three different genes.
Resolution of the Pathways of Poliovirus Type 1 Transmission during an Outbreak
Authors: Shulman Lester M. , Handsher Rachel , Yang Chen-Fu , Yang Su-Ju , Manor Joseph , Vonsover Ami , Grossman Zehava , Pallansch Mark , Mendelson Ella , Kew Olen M.
Published: December 2019

An outbreak of poliomyelitis with 20 cases occurred in Israel, Gaza, and the West Bank from October 1987 to October 1988. The wild type 1 poliovirus associated with the outbreak was most closely related to viruses found in the Nile Delta. The epidemiologic links among patients involved in the outbreak and patients with community-acquired infections during the outbreak were inferred from the evolutionary relationships among isolates of the outbreak virus. Complete VP1 sequences (906 nucleotides) were determined for 12 clinical and 4 sewage isolates. A total of 58 nucleotide differences were found among the 16 isolates; 74% of all substitutions were synonymous third-position transitions. An evolutionary tree, representing both the pathways of VP1 sequence evolution and the inferred chains of virus transmission during the outbreak, was constructed under the assumption that each substitution had occurred only once. The combined epidemiologic and molecular data suggest that a single founder strain was introduced into Israel from the vicinity of Gaza in the fall of 1987. Poliovirus circulation was apparently localized to southern communities during the winter and spread north by the following summer into the Hadera subdistrict of Israel, where it radiated via multiple chains of transmission into other communities in northern Israel and the West Bank. The close sequence matches (>99%) between clinical and sewage isolates from the same communities confirm the utility of environmental sampling as a tool for monitoring wild poliovirus circulation.
Molecular Analysis of
CAP59
Gene Sequences from Five Serotypes of
Cryptococcus neoformans
Authors: Nakamura Yuka , Kano Rui , Watanabe Shinichi , Hasegawa Atsuhiko
Published: December 2019

The nucleotide sequences of
CAP59
genes from five serotypes of
Cryptococcus neoformans
were analyzed for their phylogenetic relationships. Approximately 600-bp genomic DNA fragments of the
CAP59
gene were amplified from each isolate by PCR and sequenced. The
CAP59
nucleotide sequences of
C. neoformans
showed more than 90% similarity among the five serotypes. By phylogenetic analysis, their sequences were divided into three clusters: serotypes A and AD, serotypes B and C, and serotype D. In addition, the results of reduced amino acid sequences were similar to the nucleotide sequence data. These data revealed that serotype AD was genetically close to serotype A rather than serotype D, although it had been considered to be a mixed type of serotype A and D by serological analysis. Furthermore, the nucleotide sequences of the serotype B and C isolates of
C. neoformans
were very similar to each other. These results indicated that serotype B and C isolates belonging to
C. neoformans
var.
gattii
were genetically homogeneous and closely related. The molecular analysis of the
CAP59
gene will provide useful information for the differentiation of serotypes of
C. neoformans
and for an understanding of their phylogenetic relationships.
Newly Recognized Herpesvirus Causing Malignant Catarrhal Fever in White-Tailed Deer (
Odocoileus virginianus
)
Authors: Li Hong , Dyer Neil , Keller Janice , Crawford Timothy B.
Published: December 2019

Malignant catarrhal fever (MCF) was diagnosed by clinical signs and lesions in five out of six white-tailed deer (
Odocoileus virginianus
) in a North American zoo. The clinical signs and histopathological lesions in these deer were typical of MCF. Antibody to an epitope conserved among the MCF viruses was detected in the sera collected from the deer. PCR failed to amplify viral sequences from DNA extracted from peripheral blood leukocytes (PBL) and/or spleens of the deer with primers specific for ovine herpesvirus 2 (OHV-2) or specific for alcelaphine herpesvirus 1 (AHV-1). By using degenerate primers targeting a conserved region of a herpesviral DNA polymerase gene, a DNA fragment was amplified from the PBL or spleens of all six deer and sequenced. Alignment of the sequences demonstrated that the virus in the deer belongs to the
Gammaherpesvirinae
subfamily, exhibiting 82% identity to OHV-2, 71% to AHV-1, and 60% to a newly identified bovine lymphotropic herpesvirus. This virus, which causes classical MCF in white-tailed deer, is a newly recognized agent belonging to the MCF group of gammaherpesviruses. It is the third reported pathogenic MCF virus, genetically distinct but closely related to OHV-2 and AHV-1. The reservoir for the virus has not been identified.
Genomic Analysis of
Blastocystis hominis
Strains Isolated from Two Long-Term Health Care Facilities
Authors: Yoshikawa Hisao , Abe Niichiro , Iwasawa Mizue , Kitano Syoko , Nagano Isao , Wu Zhiliang , Takahashi Yuzo
Published: December 2019

The genotype
Blastocystis hominis
is highly polymorphic. Therefore, a genetic marker would be a powerful tool for the identification or classification of
B. hominis
subtypes and could be used as a means to resolve the transmission route or origin of the parasite. To this end, 32
B. hominis
isolates were collected from patients and/or staff members of two long-term health care facilities (facilities A and B), and these organisms were subjected to genotype analysis based on diagnostic PCR primers and restriction fragment length polymorphism (RFLP) of small subunit rRNA gene (rDNA). Based on PCR amplification using diagnostic primers which were developed from randomly amplified polymorphic DNA analysis of known strains of
B. hominis
, the 32 isolates of
B. hominis
were classified into three different subtypes. Thirty isolates, including twenty-four that were isolated from patients and a staff member, from facility A and all isolates isolated from six patients from facility B showed the same genotype. Two of six patients of facility B had been transferred from facility A, and these two patients also had the same-genotype
B. hominis
that corresponded to 24 isolates from facility A. This genotype strain may have been transmitted by these two patients from facility A to facility B, suggesting human-to-human transmission. In contrast, 2 of 26 isolates from facility A showed distinct genotypes, suggesting that the colonization by these two isolates is attributable to another infectious route. These different subtypes were subjected to RFLP analysis, and the RFLP profiles were correlated with the results obtained by diagnostic PCR primers. This study presents the first molecular evidence of possible human-to-human
B. hominis
infection between and/or among two small communities.
New
Ehrlichia
Species Closely Related to
Ehrlichia chaffeensis
Isolated from
Ixodes ovatus
Ticks in Japan
Authors: Shibata Shin-ichiro , Kawahara Makoto , Rikihisa Yasuko , Fujita Hiromi , Watanabe Yuriko , Suto Chiharu , Ito Tadahiko
Published: December 2019

Seven
Ehrlichia
strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult
Ixodes ovatus
collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain,
Ehrlichia chaffeensis
(human monocytic ehrlichiosis agent), and
E. muris
, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of
E. chaffeensis
, the Anan strain, and
E. canis
, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or
E. muris
strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of
E. canis
genogroup are naturally present in
Ixodes
ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by
E. canis
genogroup.
Heterogeneous Antimicrobial Resistance Patterns in Polyclonal Populations of Coagulase-Negative Staphylococci Isolated from Catheters
Authors: García de Viedma Darío , Martín Rabadán Pablo , Díaz Marisol , Cercenado Emilia , Bouza Emilio
Published: December 2019

Most cases of nosocomial bacteremia are catheter related, and coagulase-negative staphylococci (CoNS) are the microorganisms most frequently associated with these infections. Subtle morphological differences are frequently found among CoNS colonies cultured from infected catheters. The aim of this study was to analyze the significance of the morphological heterogeneity observed in these CoNS populations. With this purpose in mind, the clonal composition of the CoNS populations obtained from a selection of nine catheters was analyzed by two different molecular techniques, arbitrarily primed-PCR and DNA macrorestriction analysis by pulsed-field gel electrophoresis. Twenty CoNS morphotypes were included for analysis, and four single colonies representative of each morphotype were selected. Morphological differences between colonies were found to correlate in all cases with differences at the molecular level. Unique fingerprints were also obtained for some isolates which were indistinguishable from other representatives of the same morphotypes. Differences in the molecular patterns among the isolates were associated in most of the cases with differences in the antimicrobial susceptibility patterns. The frequent isolation of polyclonal CoNS populations from catheters, with heterogeneous antimicrobial susceptibility patterns, has relevant epidemiologic and therapeutic implications in the context of catheter-related infections.
Rapid Enzyme Immunoassay for Determination of Toxigenicity among Clinical Isolates of Corynebacteria
Authors: Engler Kathryn H. , Efstratiou Androulla
Published: December 2019

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (
tox
) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the
tox
gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.
Identification of
Aspergillus
Species Using Internal Transcribed Spacer Regions 1 and 2
Authors: Henry Travis , Iwen Peter C. , Hinrichs Steven H.
Published: December 2019

Aspergillus
species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species,
Aspergillus flavus
,
A. fumigatus
, and
A. terreus
, account for most cases of invasive aspergillosis (IA), with
A. nidulans
,
A. niger
, and
A. ustus
being rare causes of IA. The ability to distinguish between the various clinically relevant
Aspergillus
species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify
Aspergillus
at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1–5.8S–ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from
Aspergillus
species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of
Aspergillus
at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with
Aspergillus
ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA.
Comparison of Enhanced
Mycobacterium tuberculosis
Amplified Direct Test with COBAS AMPLICOR
Mycobacterium tuberculosis
Assay for Direct Detection of
Mycobacterium tuberculosis
Complex in Respiratory and Extrapulmonary Specimens
Authors: Scarparo Claudio , Piccoli Paola , Rigon Alessandra , Ruggiero Giuliana , Scagnelli Mariuccia , Piersimoni Claudio
Published: December 2019

The new Roche COBAS AMPLICOR
Mycobacterium tuberculosis
Assay was compared to the Gen-Probe enhanced
Mycobacterium tuberculosis
Amplified Direct Test (AMTDII). A total of 486 specimens (296 respiratory and 190 extrapulmonary) collected from 323 patients were tested in parallel with both assays. Results were compared with those of acid-fast staining and culture, setting the combination of culture and clinical diagnosis as the “gold standard.” After resolution of discrepant results, the sensitivity, specificity, and positive and negative predictive values for AMTDII were 85.7, 100, 100, and 90.4% for respiratory specimens and 82.9, 100, 100, and 95.5% for extrapulmonary specimens, respectively. The corresponding values for AMPLICOR were 94.2, 100, 100, and 96.6% for respiratory specimens and 85, 100, 100, and 96.1% for extrapulmonary specimens, respectively. No significant differences were observed between the results of both assays or, within each one, between respiratory and extrapulmonary specimens. The difference between AMTDII and AMPLICOR sensitivities was related to the presence of inhibitory samples, which the former assay, lacking an internal amplification control (IAC), could not detect. The overall inhibition rate for the AMPLICOR assay was 3.9% (19 specimens). It is concluded that, although both amplification assays proved to be rapid and specific for the detection of
M. tuberculosis
complex in clinical samples, AMPLICOR, by a completely automated amplification and detection procedure, was shown to be particularly feasible for a routine laboratory setting. Finally, AMTDII is potentially an excellent diagnostic technique for both respiratory and extrapulmonary specimens, provided that an IAC is included with the assay.
Genetic Structure of Population of
Bacillus cereus
and
B. thuringiensis
Isolates Associated with Periodontitis and Other Human Infections
Authors: Helgason Erlendur , Caugant Dominique A. , Olsen Ingar , Kolstø Anne-Brit
Published: December 2019

The genetic diversity and relationships among 35
Bacillus cereus
and
Bacillus thuringiensis
isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of
B. cereus
and
B. thuringiensis
are associated with oral infections.
Fluorescent Amplified-Fragment Length Polymorphism Genotyping of
Salmonella enterica
subsp.
enterica
Serovars and Comparison with Pulsed-Field Gel Electrophoresis Typing
Authors: Lindstedt Bjørn-Arne , Heir Even , Vardund Traute , Kapperud Georg
Published: December 2019

We have performed the fluorescently labeled amplified-fragment length polymorphism (FAFLP) method on 97 strains of the food-borne pathogen
Salmonella enterica
subsp.
enterica
comprising seven different serovars using the restriction enzymes
Eco
RI and
Mse
I. From the total FAFLP fingerprinted strains, 81 were compared with pulsed-field gel electrophoresis (PFGE) typing of the same strains. The FAFLP method showed a discriminatory power equal to that of PFGE. We report a fast, robust, and high-resolution adaptation of the AFLP assay for fingerprinting
S. enterica
subsp.
enterica
serovars with capillary electrophoresis that can be scaled to high throughput on automated analysis instruments.
Molecular Phylogenetic Evidence for Noninvasive Zoonotic Transmission ofStaphylococcus intermediusfrom a Canine Pet to a Human
Authors: Tanner Michael A. , Everett Christine L. , Youvan Douglas C.
Published: December 2019

rRNA-based molecular phylogenetic techniques were used to identify the bacterial species present in the ear fluid from a female patient with otitis externa. We report the identification ofStaphylococcus intermediusfrom the patient and a possible route of transmission. Analysis of 16S ribosomal DNA restriction fragment length polymorphisms indicated that the dominant species present wasS. intermedius. A pet dog owned by the patient also was tested and found to harborS. intermedius. In humans, the disease is rare and considered a zoonosis. Previously,S. intermediushas been associated with dog bite wounds, catheter-related injuries, and surgery. This study represents the first reported case of a noninvasive infection withS. intermedius.
Characterization of a Recurrent Clonal Type of
Escherichia coli
O157:H7 Causing Major Outbreaks of Infection in Scotland
Authors: Allison Lesley J. , Carter Philip E. , Thomson-Carter Fiona M.
Published: December 2019

A particular recurrent clonal type of
Escherichia coli
O157 has been isolated from multiple clinical, veterinary, food, and environmental sources throughout Scotland since 1989. Significant genotypic variation was detected among isolates from distinct outbreaks, with the presence or absence of single fragments being sufficient to delineate outbreak groups within the clonal type.
Molecular Characterization of Multidrug-Resistant
Streptococcus pneumoniae
Isolates in Korea
Authors: Song Jae-Hoon , Yang Ji-won , Jin Joung Hwa , Kim Shin Woo , Kim Choon Kwan , Lee Hyuck , Peck Kyong Ran , Kim Sungmin , Lee Nam Yong , Jacobs Michael R. , Appelbaum Peter C. ,   The Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study Group
Published: December 2019

Pulsed-field gel electrophoresis, ribotyping, and fingerprinting analysis of 22 invasive isolates of multidrug-resistant (MDR) pneumococci from Korea showed that 59 to 82% were genetically related. DNA sequencing of the PBP 2B gene showed relatively uniform alterations in nucleotides (5.4 to 7.8%) and amino acids (3.0 to 4.3%), while Asn-276→Lys, Arg-285→Cys and Ser-305→Phe substitutions were unique to Korean MDR strains, suggesting the spread of a few epidemic clones of resistant pneumococci within Korea.
PCR Assay for Species-Specific Identification of
Bacteroides thetaiotaomicron
Authors: Teng Lee-Jene , Hsueh Po-Ren , Tsai Jui-Chang , Chiang Feng-Lin , Chen Ching-Yi , Ho Shen-Wu , Luh Kwen-Tay
Published: December 2019

Bacteroides thetaiotaomicron
is the second most frequently encountered species of the anaerobes isolated from clinical specimens. We developed a PCR-based assay for the rapid identification of
B. thetaiotaomicron
. Specific primers were based on shared amplicons of about 1.2 kb generated from
B. thetaiotaomicron
by randomly amplified polymorphic DNA. This 1.2-kb fragment was sequenced and then used to design a set of PCR amplification primers. This PCR generated an amplification product of 721 bp, which was unique to all 65 isolates of
B. thetaiotaomicron
tested. There was no amplification with isolates of other bacterial species. Restriction enzyme digestion of the amplification product and dot blot hybridization further verified the specificity of the assay. These results suggest that this PCR assay targets a nucleotide sequence that is strongly conserved in
B. thetaiotaomicron
. This simple and rapid PCR assay provides a rapid and accurate method for identification of
B. thetaiotaomicron
and shows promise for the detection of
B. thetaiotaomicron
in clinical samples.
Evaluation of the PASCO Strep Plus Broth Microdilution Antimicrobial Susceptibility Panels for Testing
Streptococcus pneumoniae
and Other Streptococcal Species
Authors: Mohammed M. Jasmine , Tenover Fred C.
Published: December 2019

Antimicrobial resistance continues to increase worldwide among isolates of
Streptococcus pneumoniae
and other species of streptococci. Increasing rates of penicillin resistance, particularly in viridans group streptococci, and resistance to multiple classes of antimicrobial agents, including β-lactams, macrolides, and fluoroquinolones, in pneumococci have increased the importance of having accurate antimicrobial susceptibility testing results for guiding therapy. One commercial method of assessing resistance in streptococci is the PASCO Strep Plus panel. This broth microdilution-based method has recently been expanded to include a variety of newer antimicrobial agents. Therefore, we compared the results of the new PASCO Strep Plus panels for 26 antimicrobial agents against the results generated using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method for 75 pneumococci and 68 other streptococcal isolates. Only 4 (0.2%) very major errors (all with pneumococci and each with a different antimicrobial agent) were observed. There were 5 (0.3%) major errors observed with pneumococci (each with a different antimicrobial agent), but only 1 major error with nonpneumococcal streptococci. All of the very major and major errors resolved on retesting. Of the 65 (3.9%) and 17 (1.6%) minor errors observed with pneumococci and other streptococci, respectively, all were within 1 dilution of the broth microdilution reference MIC result. Thus, the PASCO Strep Plus panel has comparable accuracy to the NCCLS broth microdilution reference method.
Use of Clindamycin Disks To Detect Macrolide Resistance Mediated by
ermB
and
mefE
in
Streptococcus pneumoniae
Isolates from Adults and Children
Authors: Waites Ken , Johnson Crystal , Gray Barry , Edwards Kathryn , Crain Marilyn , Benjamin William
Published: December 2019

We studied 198 macrolide-resistant
S. pneumoniae
isolates obtained from adults and children to evaluate whether 2-μg clindamycin disks can distinguish between isolates manifesting
ermB
- versus
mefE
-mediated resistance to clarithromycin and to determine the relative frequency with which each resistance mechanism occurred in these populations. The
mefE
gene was predominant among 109 isolates from children, occurring in 73.4% versus 50.6% of 89 isolates from adults. Three isolates (1.5%) did not amplify either gene. Among 125
mefE
+
isolates, the MIC of clarithromycin at which 90% of the isolates tested were inhibited, determined by Etest, was 32 μg/ml versus >256 μg/ml in 70
ermB
+
isolates. All
ermB
+
isolates were highly resistant to clindamycin (MICs >256 μg/ml), whereas all
mefE
+
isolates were susceptible to clindamycin using the 2-μg disk. Testing
S. pneumoniae
from the respiratory tract for susceptibility to clindamycin by agar disk diffusion is an easy and inexpensive method to estimate the frequency of resistance mediated by
ermB
in specific patient populations. Macrolide resistance mediated by
ermB
is usually of greater magnitude than that due to
mefE
. Clinical studies are needed to determine the significance of high- versus low-level macrolide resistance in
S. pneumoniae
.
Distribution of Genes Encoding Putative Transmissibility Factors among Epidemic and Nonepidemic Strains of
Burkholderia cepacia
from Cystic Fibrosis Patients in the United Kingdom
Authors: Clode Fiona E. , Kaufmann Mary E. , Malnick Henry , Pitt Tyrone L.
Published: December 2019

In the last 15 years,
Burkholderia cepacia
has emerged as a significant pathogen in cystic fibrosis (CF) patients, mainly due to the severity of infection observed in a subset of patients and the fear of transmission of the organism to noncolonized patients. Although patients who deteriorate rapidly cannot be predicted by microbiological characteristics, three genetic markers have been described for strains that spread between patients. These are the
cblA
gene, encoding giant cable pili; a hybrid of two insertion sequences, IS
1356
and IS
402
; and a 1.4-kb open reading frame known as the
B. cepacia
epidemic strain marker (BCESM). The latter two are of unknown function. An epidemic strain lineage was previously identified among CF patients in the United Kingdom that apparently had spread from North America and that was characterized by a specific random amplified polymorphic DNA (RAPD) pattern. We searched for the described genetic markers using specific PCR assays with 117 patient isolates of
B. cepacia
from 40 United Kingdom hospitals. Isolates were grouped according to genomovar and epidemic strain lineage RAPD pattern with a 10-base primer, P272. A total of 41 isolates from patients in 12 hospitals were classified as the epidemic strain, and 40 of these were distributed in genomovars IIIa (11 isolates), IIIb (1 isolate), and IIIc (28 isolates). All isolates of the epidemic strain were positive for the
cblA
gene and BCESM, but two lacked the insertion sequence hybrid. None of the 76 sporadic isolates contained
cblA
or the insertion sequence hybrid, but 11 of them were positive for BCESM. Nonepidemic isolates were distributed among genomovars I or IV (9), II (49), IIIa (11), IIIb (3), and IIIc (4). There were three clusters of cross-infection (one involving two patients and two involving three patients) with isolates of genomovar II. We conclude that in the United Kingdom, a single clonal lineage has spread between and within some hospitals providing care for CF patients. The presence of the
cblA
gene is the most specific marker for the epidemic strain. We recommend that all isolates of
B. cepacia
from CF patients should be screened by PCR to influence segregation and infection control strategies.
Occurrence and Detection of AmpC Beta-Lactamases among
Escherichia coli
,
Klebsiella pneumoniae
, and
Proteus mirabilis
Isolates at a Veterans Medical Center
Authors: Coudron Philip E. , Moland Ellen S. , Thomson Kenneth S.
Published: December 2019

AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of β-lactam drugs and that may thereby create serious therapeutic problems. Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases in
Escherichia coli
,
Klebsiella pneumoniae
, and
Proteus mirabilis
remains unknown. We tested a total of 1,286 consecutive, nonrepeat isolates of these three species and found that, overall, 45 (3.5%) yielded a cefoxitin zone diameter less than 18 mm (screen positive) and that 16 (1.2%) demonstrated AmpC bands by isoelectric focusing. Based on the species, of 683
E. coli
, 371
K. pneumoniae
, and 232
P. mirabilis
isolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively, demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands. Cefoxitin resistance was transferred for all but 8 (
E. coli
) of the 16 AmpC producers. We also describe a three-dimensional extract test, which was used to detect phenotypically isolates that harbor AmpC beta-lactamase. Of the 45 cefoxitin-resistant isolates, the three-dimensional extract test accurately identified all 16 AmpC producers and 28 of 29 (97%) isolates as non-AmpC producers. Interestingly, most (86%) isolates in the latter group were
K. pneumoniae
isolates. These data confirm that, at our institution,
E. coli
,
K. pneumoniae
, and
P. mirabilis
harbor plasmid-mediated AmpC enzymes.