Kit-Wah Leung

Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles

ABSTRACT Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.

Invasive Streptococcus iniae Infections Outside North America

ABSTRACT Streptococcus iniae, a fish pathogen causing infections in aquaculture farms worldwide, has only been reported to cause human infections in North America. In this article, we report the first two cases of invasive S. iniae infections in two Chinese patients outside North America. While the first patient presented with bacteremic cellulitis, which is the most common presentation in previous cases, the second patient represents the first recognized case of S. iniae osteomyelitis. Both S. iniae strains isolated from the two patients were either misidentified or unidentified by three commercial systems and were only identified by 16S rRNA gene sequencing. Since no currently available commercial system for bacterial identification includes S. iniae in its database, 16S rRNA gene sequencing is the most practical and reliable method to identify the bacterium at the moment. In contrast to the distinct genetic profile described previously in clinical isolates from Canada, the present two isolates and a clinical isolate from a Canadian patient were found to be genetically unrelated, as demonstrated by pulsed-field gel electrophoresis. Morphologically, colonies of both isolates were also larger, more beta-hemolytic and mucoid, which differ from the usual morphotype described for S. iniae. Owing to their habit of cooking and eating fresh fish, the Asian population is strongly associated with S. iniae infections. As a result of the difficulty in making microbiological diagnosis in patients with cellulitis and the problem of identification in most clinical microbiology laboratories, the prevalence of S. iniae infections, especially in the Asian population, may have been under-estimated.

Laribacter hongkongensis gen. nov., sp. nov., a Novel Gram-Negative Bacterium Isolated from a Cirrhotic Patient with Bacteremia and Empyema

ABSTRACT A bacterium was isolated from the blood and empyema of a cirrhotic patient. The cells were facultatively anaerobic, nonsporulating, gram-negative, seagull shaped or spiral rods. The bacterium grows on sheep blood agar as nonhemolytic, gray colonies 1 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth also occurs on MacConkey agar and at 25 and 42°C but not at 4, 44, and 50°C. The bacterium can grow in 1 or 2% but not 3, 4, or 5% NaCl. No enhancement of growth is observed with 5% CO2. The organism is aflagellated and nonmotile at both 25 and 37°C. It is oxidase, catalase, urease, and arginine dihydrolase positive, and it reduces nitrate. It does not ferment, oxidize, or assimilate any sugar tested. 16S rRNA gene sequencing showed that there are 91 base differences (6.2%), 112 base differences (7.7%), and 116 base differences (8.2%) between the bacterium andMicrovirgulaaerodenitrificans,Vogesellaindigofera, andChromobacterium species, respectively. The G+C content (mean and standard deviation) is 68.0% ± 2.43%, and the genomic size is about 3 Mb. Based on phylogenetic affiliation, the bacterium belongs to the Neisseriaceae family of the β-subclass ofProteobacteria. For these reasons, a new genus and species, Laribacterhongkongensis gen. nov., sp. nov., is proposed, for which HKU1 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging pathogen.

Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis

Aims: To identify a strain of Gram negative facultative anaerobic curved bacillus, concomitantly isolated with Escherichia coli and Streptococcus milleri, from the blood culture of a 69 year old woman with acute gangrenous appendicitis. The literature on arcobacter bacteraemia and arcobacter infections associated with appendicitis was reviewed. Methods: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacterium was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment. Literature review was performed by MEDLINE search (1966–2000). Results: The bacterium grew on blood agar, chocolate agar, and MacConkey agar to sizes of 1 mm in diameter after 24 hours of incubation at 37°C in 5% CO2. It grew at 15°C, 25°C, and 37°C; it also grew in a microaerophilic environment, and was cytochrome oxidase positive and motile, typically a member of the genus arcobacter. Furthermore, phenotypic testing showed that the biochemical profile of the isolate did not fit into the pattern of any of the known arcobacter species. 16S rRNA gene sequencing showed one to two base differences between the isolate and A butzleri, but 35 to 39 base differences between the isolate and A cryaerophilus, indicating that the isolate was a strain of A butzleri. Only three cases of arcobacter bacteraemia with detailed clinical characteristics were found in the English literature. The sources of the arcobacter species in the three cases were largely unknown, although the gastrointestinal tract is probably the portal of entry of the A butzleri isolated from the present case because the two concomitant isolates (E coli and S milleri) in the blood culture were common flora of the gastrointestinal tract. In addition, A butzleri has previously been isolated from the abdominal contents or peritoneal fluid of three patients with acute appendicitis. Conclusions: 16S rRNA gene sequencing was useful in the identification of the strain of A butzleri isolated from the blood culture of a patient with acute gangrenous appendicitis. Arcobacter bacteraemia is rare. Further studies using selective medium for the delineation of the association between A butzleri and acute appendicitis are warranted.

Streptococcus sinensis sp. nov., a Novel Species Isolated from a Patient with Infective Endocarditis

ABSTRACT A bacterium was isolated from the blood culture of a patient with infective endocarditis. The cells were facultative anaerobic, nonsporulating, gram-positive cocci arranged in chains. The bacterium grows on sheep blood agar as alpha-hemolytic, gray colonies of 0.5 to 1 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth also occurs in 10 or 40% bile and on bile esculin agar but not in 6% NaCl. No enhancement of growth is observed in 5% CO2. It is nongroupable with Lancefield groups A, B, C, D, F, or G antisera and is resistant to optochin and bacitracin. The organism is aflagellated and is nonmotile at both 25 and 37°C. It is Voges-Proskauer test positive. It produces leucine arylamidase and β-glucosidase but not catalase, urease, lysine decarboxylase, or ornithine decarboxylase. It hydrolyzes esculin and arginine. It utilizes glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, hemicellulase, mannose, maltose, and starch. 16S rRNA gene sequencing showed that there were 3.6, 3.7, 4.3, 4.7, and 5.9% differences between the 16S rRNA gene sequence of the bacterium and those of Streptococcus gordonii, Streptococcus intermedius, Streptococcus constellatus, Streptococcus sanguis, and Streptococcus anginosus, respectively. The G+C content of it (mean ± standard deviation) was 53.0% ± 2.9%. Based on phylogenetic affiliation, it belongs to the mitis or anginosus group of Streptococcus. For these reasons a new species, Streptococcus sinensis sp. nov., is proposed, for which HKU4 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging cause of infective endocarditis.

Identification of Arcobacter cryaerophilus isolated from a traffic accident victim with bacteremia by 16S ribosomal RNA gene sequencing

Traditional ways of identifying slow growing bacteria is slow and often difficult. In this study, a small, Gram-negative, facultative anaerobic, slow growing bacillus was isolated from the blood culture of a 7-year old traffic accident victim. The bacterium was non-hemolytic, catalase and oxidase positive. An attempt to use the Vitek system (GNI+) and the API system (20NE) to identify the strain was unsuccessful as the growth controls showed negative results. 16S ribosomal RNA gene sequencing showed that there was 1 base difference between the isolate and Arcobacter cryaerophilus (GenBank Accession no. U25805), 1 base difference between the isolate and A. cryaerophilus (GenBank Accession no. U34387), 10 base differences between the isolate and A. cryaerophilus (GenBank Accession no. L14624), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34386), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34387), and 38 base differences between the isolate and A. butzleri (GenBank Accession no. L14626), indicating that the isolate most closely resembled a strain of A. cryaerophilus. Identification of the isolate in our case by conventional methods was difficult, as the absence of a curved morphology has made it confused with other Gram-negative non-fermentative bacteria, and the slow growth rate has made it unidentifiable by both the Vitek and API systems. Although the exact source of infection and route of transmission in our case remains elusive, we speculate that the bacteria were transmitted through the respiratory tract while the boy was suffocated in the mud. The present report represents an example of showing the usefulness of 16S rRNA gene sequencing for identification of slow growing bacteria.

Relatively Alcohol-Resistant Mycobacteria Are Emerging Pathogens in Patients Receiving Acupuncture Treatment

ABSTRACT Acupuncture has been gaining popularity as a form of alternative medicine. In the past, only blood-borne viruses and anecdotal reports of bacterial infections have been associated with acupuncture. We report on four patients with mycobacterial infections complicating acupuncture who were encountered in a 2-year period. All had clinical and/or radiological lesions at acupuncture point- and meridian-specific locations. There was no other history of trauma or other clinical foci of infections, and the chest radiographs were normal. Histological studies of biopsy specimens of all four patients showed changes compatible with chronic inflammation, with granulomatous inflammation present in three patients and acid-fast bacilli present in two. Conventional biochemical tests and whole-cell fatty acid analysis for identification were inconclusive for all four nonpigmented mycobacteria recovered from tissue biopsies. 16S rRNA gene sequencing showed that the strains from two patients were Mycobacterium chelonae and that those from the other two were Mycobacterium nonchromogenicum. Alcohol resistance assay using the quantitative suspension test revealed that all four strains showed prolonged survival in 75% alcohol compared to other skin flora. Mycobacterial infections transmitted by acupuncture are an emerging problem. A high index of suspicion is essential to recognize this clinical syndrome, and strict implementation of proper infection control guidelines for acupuncture is mandatory.

Identification by 16s ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient

Aims—To ascertain the clinical relevance of a strain of Enterobacteriaceae isolated from the stool of a bone marrow transplant recipient with diarrhoea. The isolate could not be identified to the genus level by conventional phenotypic methods and required 16S ribosomal RNA (rRNA) gene sequencing for full identification. Methods—The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GNI+) and API (20E) systems. Genotypically, the 16S bacterial rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by multiple sequence alignment. Results—Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The isolate was identified as Salmonella arizonae (73%) and Escherichia coli (76%) by the Vitek (GNI+) and API (20E) systems, respectively. 16S rRNA sequencing showed that there was only one base difference between the isolate and E coli K-12, but 48 and 47 base differences between the isolate and S typhimurium (NCTC 8391) and S typhi (St111), respectively, showing that it was an E coli strain. The patient did not require any specific treatment and the diarrhoea subsided spontaneously. Conclusions—16S rRNA gene sequencing was useful in ascertaining the clinical relevance of the strain of Enterobacteriaceae isolated from the stool of the bone marrow transplant recipient with diarrhoea.

Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species with ambiguous biochemical profile from a renal transplant recipient

Traditional ways of identification of bacteria by phenotypic characteristics cannot be used for non-cultivable organisms and organisms with unusual biochemical profiles. In this study, an Enterobacteriaceae was isolated in pure growth from the mid-stream urine of a 67-year old renal transplant recipient with urinary tract infection. Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The Vitek system (GNI+) showed that it was 18% Leclercia adecarboxylata and 55% Klebsiella ozaenae; whereas the API system (20E) showed that it was 99.8% Rahnella aquatilis. 16S ribosomal RNA gene sequencing showed that there was 7 base differences between the isolate and Enterobacter cloacae, 18 base differences between the isolate and Enterobacter asburiae, 17 base differences between the isolate and Enterobacter cancerogenus, 35 base differences between the isolate and K. ozaenae, 27 base differences between the isolate and L. adecarboxylata, and 72 base differences between the isolate and R. aquatilis, indicating that the isolate most closely resembled a strain of E. cloacae. Identification of the organism in this study is important, as the choice of antibiotics would be radically different. In this case, cephalosporins should be avoided regardless of in-vitro susceptibility as cephalosporins are well-known to select for AmpC derepressed mutants in Enterobacter, and previous administration of third-generation cephalosporins is more likely to be associated with multidrug resistant Enterobacter isolates than is administration of antibiotics that do not include a third-generation cephalosporin.

Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing

Aims—To ascertain the clinical importance of a strain of slide coagulase positive but tube coagulase negative staphylococcus species isolated from the blood culture of a 43 year old patient with refractory anaemia with excessive blasts in transformation who had neutropenic fever. Methods—The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GPI) and API (Staph) systems. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacteria was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment. Results—Conventional biochemical tests did not reveal a pattern resembling a known staphylococcus species. The Vitek system (GPI) showed that it was 94% S simulans and 3% S haemolyticus, whereas the API system (Staph) showed that it was 86.8% S aureus and 5.1% S warneri. 16S rRNA gene sequencing showed that there was a 0 base difference between the isolate and S aureus, 28 base difference between the isolate and S lugdunensis, 39 base difference between the isolate and S schleiferi, 21 base difference between the isolate and S haemolyticus, 41 base difference between the isolate and S simulans, and 23 base difference between the isolate and S warneri, indicating that the isolate was a strain of S aureus. Vancomycin was subsequently prescribed and blood cultures taken four days after the start of treatment were negative. Conclusions—16S rRNA gene sequencing was useful in ascertaining the clinical importance of the strain of slide coagulase positive but tube coagulase negative staphylococcus species isolated from blood culture and allowing appropriate management.