Diagnosis of pelvic actinomycosis by 16S ribosomal RNA gene sequencing and its clinical significance

doi:10.1016/S0732-8893(02)00375-9

Diagnostic Microbiology and Infectious Disease

Volume 43, Issue 2, June 2002, Pages 113-118

https://doi.org/10.1016/S0732-8893(02)00375-9 Get rights and content

Abstract

Traditional ways of identification of anaerobic Gram-positive non-sporulating bacilli by isolation of the organism and studying it phenotypically by elucidation of its morphologic and biochemical characteristics and metabolic end products are associated with a need for special equipment and expertise, and strains that are “unidentified” because of ambiguous biochemical profiles. In this study, an anaerobic Gram-positive non-sporulating bacterium was isolated from the intrauterine contraceptive device of a 36-year old woman with pyosalpinx. The Vitek system (ANI) showed that it was 99% Propionibacterium granulosum; whereas the API system (20A) showed that it was 78% Actinomyces meyeri/odontolyticus. The 16S ribosomal RNA gene of the strain was amplified and sequenced. There was 0 base difference between the isolate and A. odontolyticus (GenBank Accession no. AJ234047), indicating the isolate most closely resembled a strain of A. odontolyticus. Identification of the organism in this study was important because the duration of antibiotic therapy would be entirely different. In the present case, identification of the bacterium as A. odontolyticus inferred that the patient suffered from an intermediate form of pelvic actinomycosis. A prolonged course of antibiotics would be more desirable, as the relapse rate of actinomycosis after a short course of antibiotics is high.

Introduction

Identification of anaerobic Gram-positive non-sporulating bacilli in the clinical microbiology laboratory is traditionally performed by isolation of the organism and studying it phenotypically by elucidation of its morphologic and biochemical characteristics and metabolic end products. However, these methods have two major drawbacks. First, for organic acid analysis using gas chromatography, special equipment and expertise are required, but are generally not available in most routine clinical microbiology laboratories. Second, we are occasionally faced with organisms with biochemical characteristics that do not fit into patterns of any known genus and species.

Since the discovery of polymerase chain reaction (PCR) and DNA sequencing, comparison of the gene sequences of bacterial species showed that the 16S ribosomal RNA (rRNA) gene is highly conserved within a species and among species of the same genus, and hence can be used as the new gold standard for speciation of bacteria. Using this new standard, phylogenetic trees, based on base differences between species, are constructed; and bacteria are classified and re-classified into new genera Olsen et al 1992, Olsen and Woese 1993. Furthermore, non-cultivable organisms and organisms with ambiguous biochemical profiles can be classified and identified Relman et al 1990, Relman et al 1992, Tang et al 1998, Patel et al 2000. Recently, we have reported the application of 16S rRNA gene sequencing in the identification of clinical isolates with ambiguous biochemical profiles Woo et al 2000a, Woo et al 2000b, Woo et al 2001a, Woo et al 2001e, and a bacterium that was non-cultivable (Cheuk et al., 2000). In this study, we report the application of such a technique in the identification of a strain of Actinomyces odontolyticus isolated from the intrauterine contraceptive device (IUCD) of a patient with pyosalpinx, and the significance of identifying the organism is discussed.

Section snippets

Patient and microbiologic methods

All clinical data were collected prospectively as described in our previous publication (Luk et al., 1998). Clinical specimens were collected and handled according to standard protocols. The Vitek System (ANI) (bioMerieux Vitek, USA) and API system (20A) (bioMerieux Vitek, Hazelwood, Mo., USA) were used for the identification of the bacterial isolate in this study.

Extraction of bacterial DNA for 16s ribosomal RNA gene sequencing

Bacterial DNA extraction was modified from our previous published protocol (Woo et al., 1997). Briefly, 80 μL of NaOH (0.05 M) was

Patient

A 36-year old Chinese woman was admitted to hospital because of left lower quadrant pain for three weeks. She had an IUCD inserted for more than one year. On admission, her oral temperature was 39.8°C. Examination of the abdomen revealed left lower quadrant tenderness. Total white cell count was 19.1 × 10⁹/L, with neutrophil 16.2 × 10⁹/L, lymphocyte 1.3 × 10⁹/L and monocyte 1.5 × 10⁹/L. Hemoglobin level was 10.6 g/dL and platelet count 269 × 10⁹/L. Pelvic ultrasound confirmed the presence of a

Discussion

We describe the identification of a strain of A. odontolyticus isolated from the IUCD of a patient with pyosalpinx by 16S rRNA gene sequencing. Anaerobic Gram-positive non-sporulating rods have often presented problems with identification. In particular, the morphologic, biochemical and microecological characteristics of Actinomyces species appear to be similar to those of Eubacterium species (Hill et al., 1987). In the present case, commercially available kits failed to identify the IUCD

Acknowledgements

This work was partly supported by the University Research Grant Council and Committee of Research and Conference Grants, The University of Hong Kong.

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The commercially available chemical identification test kits have proven unreliable due to incomplete and obsolete databases [10,11]. 16S rRNA sequencing has been proven useful in differentiating Actinomyces spp. from non-Actinomyces anaerobic Gram-positive bacilli [7,12,13]. The availability of 16S rRNA sequencing has also led to a dramatic change in the classification of the genus Actinomyces [14].
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for bacterial identification. This study compared the Bruker MALDI-TOF BioTyper MS (MBT) and 16S rRNA gene sequencing for the identification of Actinomyces and Actinotignum spp. The MBT identified 68/77 (88.3%) of Actinomyces isolates to the genus-level and 44/77 (57.1%) of Actinomyces isolates to the species-level using the manufacturer's identification criteria. The MBT did not yield reliable identification for only 1/77 (1.3%) and generated no identification for 8/77 (10.4%) of the isolates. No misidentifications were found. Discordance at the species level was observed for eight isolates. Overall, the MBT demonstrated good concordance with the 16S rRNA gene sequencing with the exception of the closely related species A. naeslundii, A. viscosus and A. oris. A variety of Actinomyces spp. were isolated from orocervicofacial/dental specimens, but only a limited number of species were isolated from urine or intra-abdominal specimens. This study confirms the utility of MBT in the identification of Actinomyces spp. and describes the diversity and anatomic niche of species in human clinical specimens from various body sites.
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In addition, accurate identification is also crucial for the determination of the choice and duration of antimicrobial agents for treatment as well as the infection control procedures. For example, identifying a bacterial isolate as an Actinomyces species in a case of pelvic inflammatory disease implies that the patient is suffering from pelvic actinomycosis, which requires prolonged antibiotic treatment (Woo, Fung, Lau, Hon, & Yuen, 2002; Woo, Fung, Lau, Teng, et al., 2003). At the population scale, this is important for defining the epidemiology of infectious diseases, antibiotic resistance patterns of bacteria and hence the choice of empiric antimicrobials, as well as outcome and prognosis of the infections.
The most important task of the clinical microbiology laboratory is to accurately identify the responsible pathogens to species level. Phenotypic methods for bacterial identification are associated with many problems, such as strains with unusual biochemical profiles and slowly growing bacterial species. In the past few decades, gene sequencing has evolved to be an objective method for bacterial identification. Among the various gene targets, the 16S rRNA gene is the most common target used. However, for some groups of bacteria, 16S rRNA gene sequencing is not discriminative enough for discriminating closely related bacterial species. In such cases, other additional genes, such as groEL and rpoB, are used as the gene targets. In addition to identification, gene sequencing has led to the discovery of numerous novel bacterial genera and species. In the last few years, user-friendly databases and software have been developed for analysing 16S rRNA gene sequences, which has enabled the more common use of this technology in clinical microbiology laboratories feasible. Such databases have been validated using complete bacterial genome sequences. Moreover, the availability of abundant complete bacterial genome sequences has also facilitated the design of polymerase chain reaction primers for specific amplification of particular bacterial species for identification or directly from clinical or environmental specimens. Although matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry is an emerging technology for bacterial identification in clinical laboratories, it is not feasible to manually interpret the raw data in case of ambiguous results. Therefore, gene sequencing or a polyphasic approach for bacterial identification is still the standard to resolve difficult cases.
Evaluation of 16SpathDB 2.0, an automated 16S rRNA gene sequence database, using 689 complete bacterial genomes
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In view of these problems, we have developed our own database, which included the most representative 16S rRNA sequence of all medically important bacteria listed in the 9th edition of the Manual of Clinical Microbiology (Murray et al., 2007), for identification of medically important bacteria using 16S rRNA sequencing. In the first version of this database, 16SpathDB (Woo et al., 2002b), we sought to create an automated user-friendly platform that indicated the most likely identity of the 16S rRNA sequence of a medically important bacterium and other medically important bacteria with similar 16S rRNA sequences that may be the alternative identity which the user should be aware of. In our preliminary evaluation of 16SpathDB using 91 non-duplicated bacterial isolates collected in our clinical microbiology laboratory, the true identities of which were determined by a combination of phenotypic tests and 16S rRNA sequencing, it was shown that 16SpathDB is an automated, user-friendly, efficient, and accurate database for 16S rRNA sequence interpretation in clinical microbiology laboratories (Woo et al., 2011).
Interpretation of 16S rRNA sequences is a difficult problem faced by clinical microbiologists and technicians. In this study, we evaluated the updated 16SpathDB 2.0 database, using 689 16S rRNA sequences from 689 complete genomes of medically important bacteria. Among these 689 16S rRNA sequences, none was wrongly identified, with 35.8% reported as a single bacterial species having >98% identity with the query sequence (category 1), 63.9% reported as more than 1 bacterial species having >98% identity with the query sequence (category 2), 0.3% reported to the genus level (category 3), and none reported as no match (category 4). For the 16S rRNA sequences of non-duplicated bacterial species reported as category 1 or 2, the percentage of bacterial species reported as category 1 was significantly higher for anaerobic Gram-positive/Gram-negative bacteria than aerobic/facultative anaerobic Gram-positive/Gram-negative bacteria. 16SpathDB 2.0 is a user-friendly and accurate database for 16S rRNA sequence interpretation in clinical laboratories.
Actinomyces graevenitzii bacteremia in a patient with alcoholic liver cirrhosis
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But, it is so difficult and unreliable to identify Actinomyces by conventional methods [3,8]. Furthermore, many recently discovered species of the genus Actinomyces are not included in most commercial identification systems [14]. So 16S rRNA gene sequencing is needed to precisely identify rare or unusual bacteria.
Cerebral actinomycosis pseudotumor: A case report
2011, Revue Neurologique
L’actinomycose cérébrale est rare et de diagnostic difficile.
Nous rapportons le cas d’un patient âgé de 45 ans, VIH négatif, hospitalisé pour des convulsions associées à une hémiparésie gauche et à une fièvre. La tomodensitométrie cérébrale a objectivé des lésions nodulaires occipitales, hyperdenses, prenant le contraste de façon annulaire avec un œdème péri-lésionnel, évocatrices de métastases cérébrales. Le diagnostic d’actinomycose cérébrale a été porté par l’étude anatomopathologique de la pièce opératoire. La recherche d’une porte d’entrée a permis d’objectiver des granulomes dentaires. Le patient a gardé une hémiparésie séquellaire après 12 mois d’antibiothérapie associée aux soins dentaires et à une corticothérapie de courte durée.
L’actinomycose doit être évoquée devant toute lésion cérébrale chez un patient ayant une porte d’entrée dentaire. L’antibiothérapie seule est généralement suffisante et la chirurgie est réalisée souvent dans un but diagnostique.
Cerebral actinomycosis is rare and difficult to diagnose.
We report a case of a 45-year-old men hospitalized for seizures associated with fever and left hemiparesis. The white cell count and C-reactive protein were elevated. HIV serology was negative. Blood cultures remained sterile. The CT scan revealed hyperdense nodular lesions in the occipital area, with annular contrast uptake and peripheral edema causing a mass effect, suggestive of brain metastasis. The pathology examination of a surgical specimen disclosed cerebral actinomycosis. A dental origin of the infection was suspected. Hemiparesis remained after a 12-month antibiotic regimen associated with dental care and short-term corticosteroid therapy.
Actinomycosis should be discussed as a possible diagnosis for all cerebral lesions, particularly in patients with a potential dental infection. Histology is required for positive diagnosis. Antibiotic therapy alone is generally sufficient; surgery is often performed for diagnostic purposes.
Tumoral form of abdominal actinomycosis: A retrospective case series of seven patients
2010, Revue de Medecine Interne
L’actinomycose abdominale est une infection granulomateuse chronique peu fréquente, due à un bacille à Gram positif anaérobie du genre Actinomyces. Ce germe, habituellement saprophyte du tube digestif et des muqueuses génitales, peut néanmoins être responsable d’infections digestives à type de lésions simulant une affection néoplasique. L’objectif de cette étude rétrospective était de rappeler les difficultés diagnostiques que peut poser l’actinomycose abdominale et d’en analyser les aspects bactériologiques, cliniques et thérapeutiques.
De janvier 1995 à décembre 2007, les données rétrospectives concernant les patients atteints d’actinomycose abdominale pris en charge au CHU Sahloul (Sousse, Tunisie) ont été analysées. Le diagnostic de certitude a été retenu sur (1) la culture en anaérobiose d’une espèce d’Actinomyces à partir de produit de suppuration ou (2) la présence de grains ou follicules actinomycosiques sur tissus d’excision chirurgicale. Les caractéristiques cliniques, les moyens diagnostiques, les traitements proposés et l’évolution des patients ont été relevés à partir des dossiers cliniques.
Sept cas d’actinomycose abdominale ont été diagnostiqués au cours de la période d’étude. Tous les patients présentaient lors de l’examen clinique une masse abdominale. Le diagnostic a été posé chez la totalité des patients en postopératoire, dans six cas par l’étude histologique et dans deux cas par isolement bactériologique de l’Actinomyces responsable. Pour les cinq patients pris en charge par une antibiothérapie adaptée et prolongée, l’évolution a été favorable.
Le diagnostic d’actinomycose doit être évoqué devant toute masse abdominale invasive d’apparence néoplasique.
Abdominal actinomycosis is an uncommon chronic infectious disease due to Actinomyces, a Gram-positive bacteria. This saprophytic bacteria of digestive tract and genital mucosa can occasionally become pathogenic mimicking a digestive neoplasia. The aim of this study was to underline diagnostic features of abdominal actinomycosis and to summarize data about clinical, diagnostic and therapeutic approach of this type of infection.
From January 1995 to December 2007, retrospective data concerning patients with abdominal actinomycosis who were followed-up in the University Hospital Sahloul (Sousse, Tunisia) were analysed.
Seven patients with abdominal actinomycosis were identified during the study period. All presented with an abdominal mass. The diagnosis of actinomycosis was obtained after surgical resection in all cases. The histological study permitted the diagnosis in six cases, and the surgical samples grew up Actinomyces in two patients. For the five patients who received prolonged and adapted antibiotic therapy, a favourable outcome was observed.
Actinomycosis must be included in the differential diagnosis of invasive abdominal lesions with “malignant appearance”.

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MycologyDiagnosis of pelvic actinomycosis by 16S ribosomal RNA gene sequencing and its clinical significance

Abstract

Introduction

Section snippets

Patient and microbiologic methods

Extraction of bacterial DNA for 16s ribosomal RNA gene sequencing

Patient

Discussion

Acknowledgements

Eur J Obstet Gynecol Reprod Biol

Eur J Obstet Gynecol Reprod Biol

Diagn Microbiol Infect Dis

Intestinal inflammatory pseudotumor with regional lymph node involvementidentification of a new bacterium as the etiologic agent

J Pathol

Enterobacter bacteremiaclinical features and emergence of antibiotic resistance during therapy

Ann Intern Med

Systemic Actinomyces infection. A potential complication of intrauterine contraceptive devices

JAMA

Bilateral female pelvic actinomycosis

Acta Obstet Gynecol Scand

Eubacterium nodatum mimics Actinomyces in intrauterine device-associated infections and other settings within the female genital tract

Obstet Gynecol

Characteristics and sites of infection of Eubacterium nodatum. Eubacterium timidum, Eubacterium brachy, and other asaccharolytic eubacteria

J Clin Microbiol

Pelvic actinomycosis and usage of intrauterine contraceptive devices

Yale J Biol Med