bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacteriosis caused by Mycobacterium genavense in birds kept in a zoo: 11-year survey
Authors: Portaels F , Realini L , Bauwens L , Hirschel B , Meyers W M , de Meurichy W
Published: January 2020

We report on a disease in 27 birds (1 bird belonging to the order Coraciiformes, 3 to Piciformes, 4 to Galliformes, 7 to Psittaciformes, and 12 to Passeriformes) caused by fastidious mycobacteria. All birds were caged at the Antwerp Zoo and died suddenly between 1983 and 1994. Seventeen birds had no previous signs of disease, and 10 birds showed emaciation. Gross necropsy findings were generally nonspecific, but all the birds were smear positive for acid-fast bacilli (AFB). Histopathologic evaluation performed on 14 birds revealed predominantly intracellular AFB. Extracellular AFB were more abundant in advanced lesions, especially in necrotic areas. In the intestine the mucosal area was generally heavily infiltrated, suggesting an intestinal origin of the infection. There was extensive invasion of the lungs in most birds. In 11 birds sparse growth was obtained after at least 6 months of incubation on Löwenstein-Jensen medium or on Ogawa medium supplemented with mycobactin. Subculture was unsuccessful in all instances. The 16S rRNA gene sequence of the cultured organisms or tissues from seven birds revealed the characteristic signature sequence for Mycobacterium genavense. Direct bird-to-bird transmission in the zoo was unlikely, and the pathogenicity of M. genavense in birds seems to be limited. The source of M. genavense in nature and the epidemiology of the disease in birds remain obscure. As suspected for human cases of M. genavense infection, an oral route of infection has been suggested, and contaminated local water distribution systems may have been the source of the infection. Our study confirms that infections caused by M. genavense should be suspected in birds (especially in Passeriformes and Psittaciformes orders) that die suddenly without previous symptoms and that have AFB in tissues that are difficult to grow on conventional media.
Multiprimer PCR system for differential identification of mycobacteria in clinical samples
Authors: Del Portillo P , Thomas M C , Martínez E , Marañón C , Valladares B , Patarroyo M E , Carlos López M
Published: January 2020

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.
Distribution of capsular types and antibiotic susceptibility of invasive Streptococcus pneumoniae isolated from aborigines in central Australia
Authors: Gratten M , Torzillo P , Morey F , Dixon J , Erlich J , Hagger J , Henrichsen J
Published: January 2020

Streptococcus pneumoniae strains isolated from 203 episodes of invasive disease in central Australian Aborigines were studied. Capsular types from children aged 0 to 4 years (n = 89) belonged most commonly to types 14, 6B, 9V, 4, 18C, and 19F, which together accounted for 67% of the pediatric strains. In adults (n = 98), types 1, 7F, 3, 4, 12F, and 8 contributed 68% of the isolates. Of 114 pneumococci from patients 5 years and older, 102 (89.5%) were types represented in the 23-valent pneumococcal polysaccharide vaccine. The MICs of five antibiotics were determined for 201 strains by using the E-Test (AB Biodisk). No chloramphenicol or ceftriaxone resistance was found, but 46 strains (22.9%) showed diminished susceptibility to one or more of the drugs penicillin, erythromycin, and trimethoprim-sulfamethoxazole. Penicillin resistance occurred in 15.4% of all isolates tested but only within the intermediate range (0.1 to 1.0 microgram/ml). Resistance to trimethoprimsulfamethoxazole affected 13.9% of the pneumococci tested. All type 23F and most type 19F organisms were resistant to one or more antibiotics. Resistance was significantly more common in pediatric isolates than in those from adults (chi 2(1) = 14.1; P < 0.001).
Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward
Authors: Leenders A , van Belkum A , Janssen S , de Marie S , Kluytmans J , Wielenga J , Löwenberg B , Verbrugh H
Published: January 2020

During a 2-month period, five patients suffering from invasive infections caused by Aspergillus flavus or Aspergillus fumigatus were identified in the Hematology Department of the University Hospital Dijkzigt (Rotterdam, The Netherlands). To study the epidemiological aspects of invasive aspergillosis, strains from these patients and from the hospital environment, isolated during extensive microbiological screening, were subjected to genotyping. A novel DNA extraction technique, involving freezing, grinding, and direct lysis in guanidium isothiocyanate-containing buffers of mycelial material, was applied. DNA isolation was followed by typing by random amplification of polymorphic DNA (RAPD) analysis. This showed that strains isolated from all patients infected with the same fungal species were genotypically distinct, thus providing evidence against the possibility of an ongoing, single-source nosocomial outbreak. Strains could also be differentiated from strains of geographically diverse origins. However, an A. flavus strain from one of the patients was also frequently encountered in the hospital environment. As all environmental strains were collected after this patient had been diagnosed with invasive disease, the epidemiological value of this observation could not be ascertained. Intensive investigations showed no single source of A. flavus or other aspergilli. RAPD genotyping proved that the outbreak of invasive aspergillosis in the hematology ward consisted of a series of unrelated events and was not due to a common source within the hospital. RAPD fingerprinting of aspergilli may greatly facilitate future investigations of the epidemiology of invasive disease caused by these pathogens.
Hospital outbreak of Klebsiella pneumoniae resistant to broad-spectrum cephalosporins and beta-lactam-beta-lactamase inhibitor combinations by hyperproduction of SHV-5 beta-lactamase
Authors: French G L , Shannon K P , Simmons N
Published: January 2020

An aminoglycoside- and ceftazidime-resistant strain of Klebsiella pneumoniae K2 producing the extended-spectrum beta-lactamase SHV-5 infected or colonized 14 pediatric patients at Guy's Hospital. The patients were mostly neonates recovering from cardiac surgery for congenital defects. The organism was also isolated from a nurse and from the father of one of the children. Four patients had septicemia, and two septicemic neonates with postoperative renal failure died. Aminoglycoside and cephalosporin resistance transferred to Escherichia coli in vitro on a 160-kb plasmid, and a similar resistant E. coli strain was isolated from the stools of one of the affected children. The epidemic organism colonized the bowel and skin and was probably transmitted via staff hands. Five wards were involved because of extensive patient movements. The outbreak was controlled by patient isolation and attention to handwashing. All of the isolates of the outbreak strain were identical by phage typing, ribotyping, plasmid profiling, and biochemical and serological testing, but they varied in their production of SHV-5. Some isolates produced normal amounts of SHV-5 and were susceptible to beta-lactam-beta-lactamase inhibitor combinations. Others, including the single isolate of multiresistant E. coli, produced up to five times as much enzyme as "normal" isolates. This hyperproduction resulted in increased resistance to several penicillins and cephalosporins and to the beta-lactam-beta-lactamase inhibitor combinations amoxicillin-clavulanic acid, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-clavulanic acid. The hyperproduction of SHV-5 by K. pneumoniae and E. coli seen in this outbreak suggests that beta-lactam-beta-lactamase inhibitor combinations may be unreliable for the treatment of organisms producing extended-spectrum beta-lactamases.
Rapid characterization of periodontal bacterial isolates by using fluorogenic substrate tests
Authors: Maiden M F , Tanner A , Macuch P J
Published: January 2020

Eighty-nine species of subgingival bacteria, represented by 121 reference strains and 892 patient isolates, including gram-negative, gram-positive, aerobic, facultatively anaerobic, microaerophilic, and anaerobic species, were characterized with a panel of fluorogenic, 4-methylumbelliferyl-linked substrate tests. Identifications of all patient isolates were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins relative to reference strains. Characteristic profiles of positive fluorogenic reactions differentiated most of the species, including five Porphyromonas species, six pigmenting and five nonpigmenting Prevotella species, Bacteroides forsythus, three Capnocytophaga species, six Actinomyces species, four Propionibacterium species, and eight Streptococcus species. Two mannoside isomers differentiated Actinomyces israelii and Actinomyces gerencseriae. In addition to Porphyromonas gingivalis, B. forsythus, and Capnocytophaga species, Fusobacterium alocis, Actinomyces odontolyticus, Actinomyces meyeri, and Bifidobacterium dentium were all positive for so-called trypsin-like activity. Fusobacterium nucleatum, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Campylobacter species were nonreactive with the carbohydrate-based substrates tested. Fluorogenic substrate tests provided a sensitive and simple method for biochemical characterization that could presumptively identify to species level most subgingival isolates within 4 h. The method was ideal for rapidly obtaining presumptive identifications of isolates prior to confirming identifications by definitive methods, such as SDS-PAGE.
Detection of Mycobacterium tuberculosis in cerebrospinal fluid following immunomagnetic enrichment
Authors: Mazurek G H , Reddy V , Murphy D , Ansari T
Published: January 2020

The detection of Mycobacterium tuberculosis by culture of cerebrospinal fluid (CSF) is unacceptably slow. Low numbers of organisms and the presence of reaction inhibitors may prevent detection of M. tuberculosis by PCR. We used immunomagnetic enrichment to accelerate and enhance the detection of mycobacteria in CSF after demonstrating the utility of the method with pure suspensions. Growth was detected earlier in Bactec cultures of magnetically recovered mycobacteria than in untreated CSF (7 versus 15 days). We detected M. tuberculosis DNA by PCR in the immunomagnetically enriched sample but not in untreated CSF. PCR fingerprintings of the immunomagnetically recovered M. tuberculosis and of the isolate subsequently recovered by culture were identical.
Rapid colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA in gastric biopsy specimens
Authors: Lage A P , Fauconnier A , Burette A , Glupczynski Y , Bollen A , Godfroid E
Published: January 2020

A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.
Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas
Authors: Higgins J A , Radulovic S , Schriefer M E , Azad A F
Published: January 2020

A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.
Comparison of five plating media for isolation of Salmonella species from human stools
Authors: Ruiz J , Nunez M L , Diaz J , Lorente I , Perez J , Gomez J
Published: January 2020

A comparative study was carried out to evaluate the performances of different culture media for the recovery of Salmonella spp. from 1,000 routine samples of human stools. By direct plating we tested Salmonella-Shigella agar (SS), Hektoen enteric agar (HE), bismuth sulfite agar (BS), novobiocin-brilliant green-glycerol-lactose agar (NBGL) and SM-ID medium (SM), and after selenite enrichment, we tested all of the media except HE. C8-esterase and oxidase tests were used for the screening of Salmonella spp. on SS and HE. The total number of Salmonella isolates from direct culture was 74, with respective sensitivities and positive predictive values (PPVs) of 78.4 and 61%, 64.9 and 18.7%, 36.5 and 34.2%, 55.4 and 20.7%, and 39.2 and 43.9% for NBGL, SS, HE, BS, and SMID, respectively. After enrichment, the total number of Salmonella isolates was 88. The respective sensitivities and PPVs obtained were 90.9 and 62.5%, 92 and 17%, 90.9 and 32% and 93.2 and 71.3% for NBGL, SS, BS, and SM, respectively. According to our results, NBGL in direct plating was the medium with the highest sensitivity with respect to the sensitivities of the other media, with significant statistical differences (P < 0.05). Likewise, the PPV for NBGL was also the highest (61%). After enrichment in selenite broth, the sensitivities of the four media tested were similar, with the best PPV obtained with SM (71.3%); this was followed by NBGL (62.5%). When C8-esterase was used on SS or HE, the PPVs improved from less than 40% to about 100%.
Factors influencing the vitek gram-positive susceptibility system's detection of vanB-encoded vancomycin resistance among enterococci
Authors: Jett B , Free L , Sahm D F
Published: January 2020

Studies were conducted to identify factors contributing to the inability of the Vitek Gram-Positive Susceptibility system (GPS; bioMerieux, Vitek, Inc., Hazelwood, Mo.) to reliably detect vanB-mediated vancomycin resistance among enterococci. To some extent the accuracy of the GPS depended on a particular strain's level of resistance, as all isolates for which vancomycin MICs were > or = 128 mu g/ml were readily detected but detection of resistance expressed by several strains for which MICs were < or = 64 mu g/ml was sporadic. Factors besides the level of resistance were studied in two vanB strains. For one strain (Enterococcus faecium U8304), the ability of GPS to detect resistance was accurate and consistent, while for the other (Enterococcus faecalis V583), GPS results were inconsistent and unreliable. Using these isolates, we established that growth medium had the most notable effect on the detection of resistance. In the absence of vancomycin, Vitek GPS broth supported growth comparable to that obtained with brain heart infusion broth for both E. faecium U8304 and E. faecalis V583. However, in the presence of vancomycin the growth patterns changed dramatically so that neither VanB strain grew well in Vitek broth, and growth of V583 was barely detectable after 8 h of incubation. In contrast, good growth of both strains was observed in brain heart infusion broth supplemented with vancomycin. Additionally, the same medium effect was observed with other inducibly resistant VanB strains. In conclusion, although Vitek broth can support good enterococcal growth, this medium does not sufficiently support expression of vancomycin resistance by certain strains to allow them to be detected by the Vitek automated system. Furthermore, this observation establishes that the type of growth medium used can substantially influence the expression of vancomycin resistance and indicates that medium-based strategies should be explored for the enhancement of resistance detection among commercial systems.
Two novel genital human papillomavirus (HPV) types, HPV68 and HPV70, related to the potentially oncogenic HPV39
Authors: Longuet M , Beaudenon S , Orth G
Published: January 2020

The genomes of two novel human papillomavirus (HPV) types, HPV68 and HPV70, were cloned from a low-grade cervical intraepithelial neoplasia and a vulvar papilloma, respectively, and partially sequenced. Both types are related to HPV39, a potentially oncogenic virus. HPV68 and HPV70 were also detected in genital intraepithelial neoplasia from three patients and one patient, respectively. Comparison with sequence data in the literature indicates that the subgenomic ME180-HPV DNA fragment, cloned from a carcinoma cell line, corresponds to an HPV68 subtype and that several HPV DNA fragments amplified by PCR from genital neoplasia represent worldwide distributed variants of HPV68 and HPV70.
Comparison of five selective media for identifying fecal carriage of vancomycin-resistant enterococci
Authors: Landman D , Quale J M , Oydna E , Willey B , Ditore V , Zaman M , Patel K , Saurina G , Huang W
Published: January 2020

There is no uniformly accepted method for detecting colonization with vancomycin-resistant enterococci (VRE). The sensitivities of five culture methods were determined for patients known to harbor VRE. Of 189 inpatients, 101 were found to harbor VRE by at least one method. Three methods detected fewer than half of the cultures. Campylobacter agar identified 70% of patients. Enterococcosel broth (containing vancomycin and aztreonam) identified 88% and may be preferred over other media for routine surveillance.
Neisseria elongata subsp. elongata, a case of human endocarditis complicated by pseudoaneurysm
Authors: Nawaz T , Hardy D J , Bonnez W
Published: January 2020

We report an unusual case of endocarditis caused by Neisseria elongata subsp. elongata. The illness was complicated by a ruptured mycotic aneurysm of the right brachial artery, with compression of the brachial plexus. A cure was achieved after aneurysm resection and treatment with intravenous ceftriaxone and gentamicin.
Specimen contamination in mycobacteriology laboratory detected by pseudo-outbreak of multidrug-resistant tuberculosis: analysis by routine epidemiology and confirmation by molecular technique
Authors: Wurtz R , Demarais P , Trainor W , McAuley J , Kocka F , Mosher L , Dietrich S
Published: January 2020

A cluster of multidrug-resistant tuberculosis sputum isolates led to the detection of specimen contamination in a hospital mycobacteriology laboratory. Thirteen specimens were smear negative but culture positive from one specimen only; 12 appeared to be contaminated. Each of these specimens was processed in the same batch as one or more smear- and culture-positive isolates. Molecular analysis confirmed the traditional epidemiologic, laboratory, and clinical methods of evaluating presumed mycobacterial contamination.
Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis
Authors: Lin A W , Usera M A , Barrett T J , Goldsby R A
Published: January 2020

A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Salmonella enteritidis isolates. A total of 65 arbitrary primers were screened with S. enteritidis isolates of different phage types. This allowed selection of a panel of primers capable of detecting DNA polymorphisms among S. enteritidis isolates. This panel was used to examine a panel of 29 isolates of S. enteritidis which had been previously characterized by other subtyping methods, including phage typing (PT) (n = 7), ribotyping (RT) (n = 13), and pulsed-field gel electrophoresis (PFGE). Applied collectively, these three methods resolved the collection into 20 different subtypes. However, by the RAPD fingerprinting method alone, 14 RAPD subtypes were revealed. Eight isolates of S. enteritidis phage type 8 that failed to be discriminated by other typing methods (PT, RT, and PFGE) were resolved into three different subtypes by RAPD analysis. In contrast, isolates that were derived from the same sources were not differentiated by any of the subtyping methods employed, including PT, RT, PFGE, and RAPD analysis. This RAPD approach to S. enteritidis subtyping provided more discriminatory power than did any of several other subtyping methods applied individually. Once the challenging step of primer identification was accomplished, determinations of the appropriate concentrations of arbitrary primer, DNA template, and MG2+ ion were also necessary for optimal discriminatory power. The bacterial DNA used in this RAPD protocol was obtained by boiling the bacterial sample. This simple procedure yielded DNA that produced fingerprint patterns as consistent as those obtained from phenol-chloroform-extracted DNA. Clearly, when appropriately constituted primer sets are identified and employed, RAPD analysis provides a simple, rapid, and powerful subtyping method for S. enteritidis.
DNA fingerprinting and serotyping of Campylobacter jejuni isolates from epidemic outbreaks
Authors: Lind L , Sjögren E , Melby K , Kaijser B
Published: January 2020

The aim of the present investigation was to compare DNA fingerprinting and serotyping (heat-stabile and heat-labile antigens) of isolates from epidemic outbreaks as well as of solitary isolates. Campylobacter jejuni isolates from two epidemic outbreaks in Sweden, one milkborne (35 isolates) and one waterborne (17 isolates), and one waterborne outbreak in Norway (11 isolates), as well as 30 solitary isolates from Swedish patients with gastroenteritis, were analyzed. A total of 93 isolates were analyzed. In the waterborne outbreak in Norway, only one serotype with one DNA pattern was found. In the milkborne outbreak in Sweden, two serotypes (HS2:HL4 and HSNT:HL4) with two different DNA patterns were found. The isolates from the waterborne outbreak in Sweden were different serotypes. For two isolates of the same serotype, different DNA patterns were seen. This was also recorded for isolates from solitary cases. It was concluded that serotyping is a useful tool in most epidemiological situations but sometimes lacks sufficient discriminatory power. DNA fingerprinting can add valuable epidemiological information to that supplied by serotyping and can in some situations provide sufficient epidemiological information when used alone.
Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci
Authors: Beall B , Facklam R , Thompson T
Published: January 2020

Rapid sequence analysis of specific PCR products was used to accurately deduce emm types corresponding to the majority of the known group A streptococcal (GAS) M serotypes. The study involved 95 M type reference GAS strains and a survey of 74 recent clinical isolates. A high percentage of agreement between M type serology and the previously published 5' sequences of the emm genes of M type reference strains was noted. The 5' sequences for six established M protein genes--the emm-32, emm-34, emm-38, emm-40, emm-42, and emm-71 genes--were determined to supplement the existing emm sequence database. Rapid sequence analysis differentiated serologically M-nontypeable strains and was used to establish the probable.
Serum rotavirus neutralizing-antibody titers compared by plaque reduction and enzyme-linked immunosorbent assay-based neutralization assays
Authors: Ward R L , Kapikian A Z , Goldberg K M , Knowlton D R , Watson M W , Rappaport R
Published: January 2020

Comparisons in rotavirus neutralizing-antibody responses were made with sera collected from vaccinated infants. The methods were a plaque reduction assay and a new enzyme-linked immunosorbent assay-based neutralization assay. Agreement of 94% was found in detecting at least fourfold seroresponses, and correlation coefficients between titers obtained by the two methods showed excellent agreement, indicating that either could be used reliably.
Ribotyping: a tool for taxonomy and identification of the Nocardia asteroides complex species
Authors: Laurent F , Carlotti A , Boiron P , Villard J , Freney J
Published: January 2020

Ribotyping has been evaluated as a taxonomic tool for studying bacteria belonging to the Nocardia asteroides complex. The probe used was obtained by PCR from DNA extracted from the type strain Nocardia asteroides ATCC 19247, a sequence that codes partly for 16S rRNA. Interpretation of hybridization after EcoRI restriction of total DNA of 21 strains of the N. asteroides complex allowed for the identification of only four different ribotypes: two related to N. asteroides sensu stricto, one related to N. farcinica and one related to N. nova. These results confirm the classification established by Tsukamura in 1982 (M. Tsukamura, Microbiol. Immunol. 26:1101-1119, 1982) as well as the heterogeneity of N. asteroides sensu stricto species. Ribotyping may also provide a major identification technique for the species belonging to the N. asteroides complex.
Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci
Authors: Barbier N , Saulnier P , Chachaty E , Dumontier S , Andremont A
Published: January 2020

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.
Isolation and characterization of a unique group of slowly growing mycobacteria: description of Mycobacterium lentiflavum sp. nov
Authors: Springer B , Wu W K , Bodmer T , Haase G , Pfyffer G E , Kroppenstedt R M , Schröder K H , Emler S , Kilburn J O , Kirschner P , Telenti A , Coyle M B , Böttger E C
Published: January 2020

A distinct group of slowly growing mycobacteria was identified on the basis of growth characteristics, biochemical and lipid profiles, and nucleic acid analyses. The isolates showed growth at 22 to 37 degrees C, yellow pigmentation, and negative tests for Tween 80 hydrolysis, nicotinic acid, nitrate reductase, and urease; tests for arylsulfatase, pyrazinamidase, and heat-stable catalase were variable. Analysis of cellular fatty acids by gas-liquid chromatography and mycolic acids by thin-layer chromatography and high-performance liquid chromatography indicated a distinctive pattern which was unlike those of other species. Determination of the 16S rRNA gene sequence showed a unique sequence closely related to Mycobacterium simiae and M. genavense. On the basis of DNA homology studies, we suggest that these organisms are representatives of a novel species, for which the name M. lentiflavum sp. nov. is proposed.
Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis
Authors: Chaves F , Yang Z , el Hajj H , Alonso M , Burman W J , Eisenach K D , Dronda F , Bates J H , Cave M D
Published: January 2020

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.
Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis
Authors: Mahenthiralingam E , Campbell M E , Foster J , Lam J S , Speert D P
Published: January 2020

Pseudomonas aeruginosa isolates recovered from chronically colonized patients with cystic fibrosis (CF) are phenotypically different from those collected from other patients or from the environment. To assess whether alterations in motility, mucoidy, and serum susceptibility represented an adaptation to chronic infection or replacement by a new strain, sequential P. aeruginosa isolates of known phenotype collected from 20 CF patients were typed by random amplified polymorphic DNA (RAPD) analysis. A total of 35 RAPD strain types were found among 385 isolates from 20 patients, and only two patients had P. aeruginosa strains of the same RAPD fingerprint. Eight strain pairs representative of the first eight RAPD types were also analyzed by SpeI macrorestriction followed by pulsed-field gel electrophoresis (PFGE); the strain types found by both fingerprinting techniques correlated exactly. In 11 of 20 patients, the RAPD types of serial P. aeruginosa isolates remained stable despite alterations in isolate motility, colonial morphology, and lipopolysaccharide phenotype. However, in isolates collected from one CF patient, a single band change in RAPD fingerprint and CeuI PFGE profile correlated with the appearance of an RpoN mutant phenotype, suggesting that the altered phenotype may have been due to a stable genomic rearrangement. Secretion of mucoid exopolysaccharide, loss of expression of RpoN-dependent surface factors, and acquisition of a serum-susceptible phenotype in P. aeruginosa appear to evolve during chronic colonization in CF patients from specific adaptation to infection rather than from acquisition of new bacterial strains.
Genomic analysis of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by isoenzyme analysis and pulsed-field gel electrophoresis
Authors: Feizabadi M M , Robertson I D , Cousins D V , Hampson D J
Published: January 2020

Initially, multilocus enzyme electrophoresis was used to examine genetic relationships among 63 isolates of Mycobacterium bovis and 13 other members of the M. tuberculosis complex. The isolates were divided into five electrophoretic types, with a mean genetic diversity of 0.1. The strains were genetically homogenous, indicating that members of the complex were closely related. This supported the suggestion that they should be considered as subspecies of a single species. Pulsed-field gel electrophoresis (PFGE) was then used to differentiate these isolates, as well as 59 additional isolates of M. bovis from different parts of the world. PFGE differentiated these strains into 63 patterns (53 patterns for M. bovis). Isolates of M. bovis from Western Australia (n = 46) were more homogenous than isolates from other regions. Eight strains were identified in that state, and one predominantly bovine strain was isolated from two human beings and a feral pig. Although M. bovis isolates from different parts of the world had distinct DNA patterns, some were very similar. PFGE is a highly discriminatory technique for epidemiological studies of bovine tuberculosis. For example, it allowed differentiation between isolates of M. bovis cultured from animals in separate outbreaks of tuberculosis, it suggested the transmission of infection between certain properties, and it demonstrated the existence of multiple infections with different strains at certain farms.
Preservation of Neisseria gonorrhoeae at -20 degrees C
Authors: Harbec P S , Turcotte P
Published: January 2020

To explore the feasibility of preserving Neisseria gonorrhoeae at -20 degrees C, we studied its viability quantitatively and qualitatively for 12 and 18 months, respectively, in the following media: a gelatin-based medium used mainly to prepare dried gelatin discs (S. Yamai, Y. Obara, T. Nikkawa, Y Shimoda, and Y. Miyamoto, Br. J. Vener. Dis. 55:90-93, 1979), a simplified version (LSPQ preservation medium), and Trypticase soy broth with 10% (vol/vol) glycerol, a medium commonly used for preservation at -70 degrees C. The latter was studied for 4 months only. Four reference strains and two clinical isolates of N. gonorrhoeae were used. The storage temperature was rigorously preadjusted and monitored at -20 +/- 1 degree C during the entire project. After 12 months of storage, all strains remained viable in both gelatin-based media, whereas a significant loss of viability was observed in Trypticase soy broth-10% glycerol after only 4 months. After 18 months, five strains were still viable in both gelatin-based media and no significant difference was observed between antimicrobial susceptibility results and those of the original strains preserved at -70 degrees C. On the basis of these results, we believe that LSPQ preservation medium represents a good alternative for the storage of N. gonorrhoeae at -20 degrees C for at least a year. Furthermore, it is easy to prepare and use and can by stored at 4 to 8 degrees C for a year prior to use.
Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains
Authors: van Belkum A , Sluijuter M , de Groot R , Verbrugh H , Hermans P W
Published: January 2020

Typing data obtained by specifically targeting a single, high-stringency PCR at the pneumococcal BOX repeat element for 28 strains of Streptococcus pneumoniae completely corroborated the resolutions attained by five genotypic procedures as described by Hermans et al. (P.W.M. Hermans, M. Sluijter, T. Hoogenboezem, H. Heersma, A. van Belkum, and R. de Groot, J. Clin. Microbiol. 33:1606-1612, 1995). All pairs of strains, except one, derived from both the cerebrospinal fluid and blood of the same individual were shown to be identical. Moreover, other, epidemiologically unrelated isolates were demonstrated to be unique. Considering the combined data from the five typing techniques applied previously as the "gold standard," the single BOX PCR test demonstrated excellent resolving powers while maintaining epidemiological linkage.
Restriction fragment length polymorphism of flagellin genes of Campylobacter jejuni and/or C. coli isolates from Egypt
Authors: Mohran Z S , Guerry P , Lior H , Murphy J R , el-Gendy A M , Mikhail M M , Oyofo B A
Published: January 2020

The conservation of flagellin genes from thermophilic Campylobacter spp. strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay. The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups. The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup. In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed. The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates.
Endocarditis of native aortic and mitral valves due to Corynebacterium accolens: report of a case and application of phenotypic and genotypic techniques for identification
Authors: Claeys G , Vanhouteghem H , Riegel P , Wauters G , Hamerlynck R , Dierick J , de Witte J , Verschraegen G , Vaneechoutte M
Published: January 2020

Endocarditis of native aortic and mitral valves due to an organism identified as Corynebacterium accolens developed in a 73-year-old patient without predisposing factors. The organism was identified as C. accolens by biochemical identification, amplified rRNA gene restriction analysis, and DNA-DNA hybridization. This is the first case of C. accolens endocarditis reported, adding to the increasing number of Corynebacterium-related cases of endocarditis.
Comparison of urinary bladder and ear biopsy samples for determining prevalence of Borrelia burgdorferi in rodents in central Europe
Authors: Petney T N , Hassler D , Brückner M , Maiwald M
Published: January 2020

PCR was used to compare urinary bladder and ear biopsy samples from four European species of wild rodents for the presence of Borrelia burgdorferi sensu lato. From 60 paired comparisons of bladder and ear biopsy samples, the PCR results were concordantly positive or negative in 43 samples (71.7%). Of the 17 which differed, 14 bladder samples were positive and ear samples were negative while the converse occurred for three samples. Thus ear biopsy samples led to a significantly lower estimate of infection than bladder biopsy samples. This suggests that the use of ear biopsy samples in epidemiological studies of B. burgdorferi in Central European rodents is likely to lead to underestimates of the prevalence.
Comparison of BacT/Alert FAN medium with BACTEC NR660 Plus 26A medium
Authors: Snyder J W , Lude S K
Published: January 2020

A total of 6,010 blood culture sets were obtained from adult patients with suspected bacteremia or fungemia. The overall recovery of organisms was equivalent in two systems used, BacT/Alert FAN medium and BACTEC NR660 Plus 26A medium.
Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA
Authors: Quentin R , Ruimy R , Rosenau A , Musser J M , Christen R
Published: January 2020

Previous genetic analysis of Haemophilus influenzae strains isolated from genital and neonatal infections identified a group of biotype IV that constitutes a cryptic genospecies only distantly related to H. influenzae and H. Haemolyticus. Small-subunit rRNA genes of two representative strains of this genital Haemophilus genospecies (strains 16N and 2406) were sequenced. The analysis indicated that these strains form a monophyletic unit with H. haemolyticus and H. influenzae biogroups Influenzae and Aegyptius and are more closely related to H. haemolyticus than to H. influenzae biogroups Influenzae and Aegyptius. 16S rRNA gene sequences were used to formulate primers for PCR-based identification of cryptic genital Haemophilus organisms. A 242-bp fragment was amplified from strains belonging to the genital Haemophilus genospecies but not from strains of 12 other Haemophilus species, including strains of H. influenzae biotype IV sensu stricto.
Longitudinal study of molecular epidemiology of small round-structured viruses in a pediatric population
Authors: Levett P N , Gu M , Luan B , Fearon M , Stubberfield J , Jamieson F , Petric M
Published: January 2020

Small round-structured viruses (SRSV), recently designated members of the family Caliciviridae, can now be readily subtyped by amplification of a defined portion of their genome by reverse transcription-PCR and then by identification of the amplicons with specific probes by Southern blotting. A longitudinal survey (from 1991 to 1995) was conducted to determine the genotypes of the SRSV present in pediatric stool specimens from patients with sporadic cases of gastroenteritis. It was found that almost all viruses were of the G-2 genotype, and on probing, the subtype P2-B was predominant but the frequencies of the different subtypes varied from year to year. A survey of the genotypes of SRSV from community outbreaks from 1995 showed that the G-2 genotype was also predominant and that the distribution of its subtypes was similar to that seen in sporadic cases of diarrhea in pediatric patients over the same time period. It was concluded that there is a succession of subtypes of SRSV in our pediatric population over time. This distribution of genotypes in sporadic cases of pediatric gastroenteritis may reflect the distribution in community outbreaks occurring at the same time.
Discrepancies between results by E-test and standard microbroth dilution testing of Streptococcus pneumoniae for susceptibility to vancomycin
Authors: Hashemi F B , Schutze G E , Mason E O
Published: January 2020

Vancomycin susceptibility testing of Streptococcus pneumoniae by the E-test consistently resulted in MICs that were at the upper limit of the 1-microgram/ml susceptible category defined by current National Committee for Clinical Laboratory Standards guidelines and were always higher than MICs obtained by microbroth dilution. Three of five E-test results for S. pneumoniae ATCC 49619 were higher than the acceptable limits.
Testing of susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by mycobacterium growth indicator tube method
Authors: Walters S B , Hanna B A
Published: January 2020

We tested isolates of Mycobacterium tuberculosis recovered from 117 patients for their susceptibilities to isoniazid (INH) and rifampin (RIF) by the Centers for Disease Control and Prevention's disk modification of the indirect method of proportions (MOP) test and a three-tube mycobacteria growth indicator tube (MGIT; BBL) antimycobacterial susceptibility test (AST). Sixty-seven of the M. tuberculosis isolates were recovered from Lowenstein-Jensen (BBL) subcultures, and 50 of the isolates were recovered from MGIT cultures of samples from various body sites. For the MGIT AST method, 0.5 ml of test organism suspension was inoculated into an MGIT with 0.1 micrograms of INH per ml, an MGIT with 1.0 micrograms of RIF per ml, and growth control MGIT. The tubes were incubated at 37 degrees C and were examined daily. The MGIT AST results were interpreted as follows: susceptible if the tubes containing INH or RIF did not fluoresce within 2 days of the time that the positive growth control fluoresced and resistant if the tubes containing INH or RIF did fluoresce within 2 days of the time that the positive growth control fluoresced. The mean time fluorescence for the positive growth control was 5.5 days. The two methods were in agreement for 114 of the 117 isolates from patients, while for 3 isolates there were minor discordant results.