Most isolates recovered in our laboratory are identified by phenotypic criteria with an automated microbiology system, which likely reflects the present situation at most teaching hospital laboratories worldwide. It is well known that the microbiology systems from Siemens MicroScan WalkAway 96 plus System, BD Phoenix 100 Automated Microbiology System and BioMerieux Vitek 2 Compact have been extensively applied clinically in different regions. However, some isolates that are uncommon or atypical clinically have difficulty in identifying by the above systems. Genetic methods for the identification and taxonomic classification of microorganisms have thus been established in clinical microbiology laboratories in recent years.
The sequence analysis of 16S rDNA is the most common molecular method for the identification of bacteria , but most studies have mainly focused on method development and anecdotal case reports. On the basis of key reports and previous clinical work, we selected a set of universal reference primers to amplify 16S rDNA and identify previously “difficult-to-identify” bacteria in clinical settings. In addition, the ITS region (especially ITS2) is heavily used for the identification of fungi . The identification of standard strains and common clinical isolates by PCR and sequencing in this study demonstrates that this method is consistent with conventional laboratory methods. This molecular identification method offers numerous desirable features: (1) convenience, as common molecular biology methods and techniques are used in the procedure (including efficient one-step DNA extraction, PCR, electrophoresis, commercial sequencing, and sequence analysis); (2) Availability, as it is based on well established theories and methods (including amplicon preparation by PCR, commercial sequencing, and GeneBank verification); and (3) cost-effectiveness, as the cost of the procedure can be lower than ever for the development of information and technology. If laboratories haven’t the extensive expertise to use other methods by phenotypic criteria, the use of the molecular methods as an alternative to identify “difficult to identify” microbes can be considered.
Various bacteria were identified through sequencing in this study. The overall performance of sequence analysis is excellent, as it enables the identification of “difficult-to-identify” bacterial isolates. Thus, the sequencing of 16S rDNA can serve as a supplement in regular clinical work for bacteria identification, especially for poorly described, biochemically deficient or fastidious organisms. However, the method does have a little potential limitation that it can not identify all of bacteria successfully. This is due to insufficient base heterogeneity in the 16S rDNA of some organisms, resulting in the inadequate discrimination of some species, such as Shigella sonnei and Escherichia fergusonii. In this case, epidemiological, clinical, and phenotypic microbiological data of an isolate can be collected and combined with sequence analysis from a more variable region, such as intergenic 16S-23S rRNA spacer regions.
The conventional identification of fungi is mainly based on a combination of morphological and biochemical features. Morphologies at all levels require careful expert evaluation for correct species assignment because of excessive morphological variability. Our study demonstrates the feasibility of ITS2 sequencing for the identification of clinically important and rare fungi. This method identified 31 isolates as 15 species or genera, including Aspergillus, Cryptococcus, Exophiala, Rhizopus, and Trichosporon. Almost all clinically relevant species could be identified by ITS2 region sequencing, which is highly specific .
We conducted a systematic search in PubMed, GenBank, and Chinese National Knowledge Infrastructure (CNKI) databases to review case reports related to rare clinical isolates we identified. One of these rare isolates, from a female patient with acute pancreatitis, is Agrobacterium tumefaciens, a rod shaped, Gram-negative soil bacterium and the causal agent of crown gall disease. Although the international literature has reported Agrobacterium tumefaciens in clinical catheter-related infection , this clinical infection has not been reported in China. Another rare isolate, from puncture fluid of a lung cancer patient with pleural effusion, Bilophila wadsworthia, is a unique Gram-negative anaerobic rod bacterium that has not been reported in mainland China but in Taiwan . International literature has reported this isolate from a variety of specimens like bile, pus, and blood [16, 17]. Capnocytophaga sputigena is a Gram-negative rod bacterium in the oral flora of healthy humans that likely causes systemic infections in immunocompromised patients with granulocytopenia and oral ulcerations, especially in children [18, 19]. In this study, three isolates were recovered at our hospital from three children (under 10 years old) suffering from lymphoblastic leukemia or thalassemia. Dialister pneumosintes, a small, non-fermentative, Gram-negative anaerobic rod bacterium, is considered a commensal organism of the oral cavity, nasopharynx, intestine, and vaginal flora [20, 21]. This pathogen was responsible for various infections in immunocompromised patients . A 50-year-old woman suffering from cerebral trauma was infected by D. pneumosintes isolated from cerebrospinal fluid in this study. A species of Massilia, Massilia timonae, a nonfermentative aerobic Gram-negative rod bacterium, was isolated from the blood and CSF [23, 24]. We first reported Massilia sp. from clinical specimens in China.
Pontibacter sp. has been mainly reported in the environment, including water, garbage dumps, and soil. There are no clinical reports on this species in the international literature to date. Bacillus niabensis is also a species commonly found in the environment that rarely causes clinical infections and human colonization. Here, the specimens from bathystixis contained B. niabensis in a patient. The genus Janibacter is typically isolated from sludge and the name Janibacter refers to the changing morphology of the microorganisms during growth [25, 26]. We identified Janibacter sp. from an adult with a fever over 38°C, indicating it may be isolated from clinical specimens, even blood, in China. Leifsonia sp. is an aquatic coryneform rod rarely associated with human infections. Some species, like Leifsonia aquatica, have been accidentally found in the clinic in other countries, but no species previously reported in China [27–29]. We report two cases of bloodstream infection in a female patient with osteogenic sarcoma and a 45-day-old infant. Paenibacillus massiliensis is a Gram-positive, facultative anaerobic rod bacterium isolated from blood culture . We are the first to report the species from clinical specimens in China. In this hospital, the sample containing acid-fast bacilli will be sent to Guangzhou Antituberculosis Station to identify the microbes after our laboratory checks it positive by acid-fast staining. This Mycobacterium tuberculosis in Table 1 was used to validate the molecular method of 16S rDNA sequencing.
Phomopsis sp. is a frequent fungal parasite of plants around the world, but only a limited number of genera have been documented to cause human disease [31–33]. We report the first human case due to Phomopsis sp. in China. Rhizopus oryzae can cause invasive fungal infections called mucormycosis in humans . Here we report a serious case of disseminated type mucormycosis caused by Rhizopus oryzae in a patient suffering diabetes with orbital cellulitis, which is a rare clinical case. Trichomonascus ciferrii known as Candida ciferrii has morphological characteristics that differ from more common Candida species and is emerging as a causative agent of opportunistic infections. This is the first case reported in China, from a patient with chronic suppurative otitis media.
In this study, all “difficult-to-identify” isolates were correctly identified and the etiological agent identified. Therefore, sequencing 16S rDNA and ITS 2 region is a good supplemental for clinical conventional methods in the identification of microbes.