Emerging anaerobic and partially acid-fast Lawsonella clevelandensis: extended characterization by antimicrobial susceptibility testing and whole genome sequencing
Authors: D. Goldenberger, M. Naegele, D. Steffens, R. Eichenberger, A. Egli, H.M.B. Seth-Smith
Journal: Clinical Microbiology and Infection (2019)

Lawsonella clevelandensis is an emerging pathogenic bacterium taxonomically described in 2016 [[1]Bell M.E. Bernard K.A. Harrington S.M. Patel N.B. Tucker T.A. Metcalfe M.G. et al.Lawsonella clevelandensis gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from human abscesses.Int J Syst Evol Microbiol. 2016; 66: 2929-2935Crossref PubMed Scopus (22) Google Scholar]. The organism is very slow growing, Gram variable, partially acid fast and facultatively anaerobic. Between 2013 and 2018 a total of eight cases, six from North America and two from Europe, have been reported [2Chudy-Onwugaje K. Vandermeer F. Quezada S. Mimicking abdominal tuberculosis: abdominal abscess caused by Lawsonella clevelandensis in inflammatory bowel disease.Clin Gastroenterol Hepatol. 2019; 17 (e92)Google Scholar, 3Favila Menezes M. Sousa M.J. Paixao P. Atouguia J. Negreiros I. Simoes M.J. Lawsonella clevelandensis as the causative agent of a breast abscess.IDCases. 2018; 12: 95-96Google Scholar, 4Kumaria A. Lucas E.K. Crusz S.A. Howarth S.P.S. Cartmill M. Lawsonella clevelandensis causing spinal subdural empyema.Br J Neurosurg. 2018; https://doi.org/10.1080/02688697.2018.1540767Crossref Scopus (4) Google Scholar]. All cases showed abscess formation with a single bacterial infection in different body sites. To date no antimicrobial susceptibility testing (AST) has been performed on this highly fastidious microorganism, and its normal habitat is unclear. In this report, minimum inhibitory concentrations (MICs) of diverse antimicrobial agents against L. clevelandensis were determined for the first time. In addition, whole genome sequencing (WGS) was performed from our isolate, and retrospective sequence analyses indicate that this novel organism could represent a member of the normal human microbiota. L. clevelandensis strain USB603019-18 was isolated from a patient with mastitis nonpuerperalis. Very tiny and greyish colonies were detected after anaerobic incubation for 6 days on Brucella agar (Becton Dickinson), with Gram stain showing Gram-labile adhering pleomorphic rods (Supplementary Fig. S1). The isolate could not be identified by MALDI-TOF MS. Partial 16S ribosomal RNA (rRNA) gene sequencing (704 bp) showed 100% identity to L. clevelandensis reference sequences (accession nos. NR_151867.1, KM401816.1, KC669624.1). A subsequent direct 16S rRNA gene PCR from DNA extracted from the aspirate confirmed the presence of L. clevelandensis in the sample. The isolate did not grow in thioglycollate broth and showed partially acid-fast property. After repeated culture steps, the strain grew well over 4 to 5 days under anaerobic conditions. In contrast to Müller-Hinton or Brucella agar, Columbia sheep's-blood medium facilitated good and reliable growth. Therefore, the AST was evaluated on this medium. AST was executed twice independently using the gradient diffusion technology (MIC Test Strip; Liofilchem) against 18 antimicrobial agents. The plates were incubated anaerobically at 36°C and were read after 3, 5 and 7 days using a binocular microscope and by eye (after 7 days only). All quality controls using Bacteroides fragilis ATCC 25285 were within the MIC ranges of listed antimicrobial agents in the Clinical and Laboratory Standards Institute guidelines. Susceptibility testing demonstrated low to very low MICs for all substances tested (Table 1).Table 1Antimicrobial susceptibility results from Lawsonella clevelandensis strain USB603019-18AntimicrobialMIC, first testing (mg/L)MIC, second testing (mg/L)MIC, higher value (mg/L)Penicillin0.0030.0020.002Amoxicillin/clavulanic acid<0.016<0.016<0.016Piperacillin/tazobactam<0.016<0.016<0.016Ceftriaxone0.023<0.0160.023Cefuroxime<0.016<0.016<0.016Cefepime0.016<0.0160.016Imipenem<0.002<0.002<0.002Erythromycin0.0320.0230.032Clindamycin0.0640.0160.064Tetracycline0.0470.0230.047Tigecycline0.0230.0160.023Ciprofloxacin0.0470.0230.047Moxifloxacin0.0940.0230.094Vancomycin0.1900.1900.190Gentamicin0.3800.1900.380Rifampicin<0.002<0.002<0.002Linezolid0.0940.0470.094Daptomycin0.0640.0640.064MIC, minimum inhibitory concentration. Open table in a new tab MIC, minimum inhibitory concentration. DNA extracted from USB603019-18 was sequenced on the Illumina MiSeq platform (PE300) following library creation with Nextera XT. The detailed methodology used for WGS is provided in the Supplementary Materials. The assembled genome draft of strain USB603019-18 comprises 26 contigs over 1 877 010 bp, with a G+C percentage of 58.44 and 1606 predicted coding sequences. The data set is available in project PRJEB31421 with annotated assembly under accession number LR584267. No genes associated with conferring virulence or antimicrobial resistance were identified by WGS. The phylogeny of the three sequenced isolates of this species showed a diversity of 7714 single nucleotide polymorphisms across the tree, with the genome of USB603019-18 having a distance of 4161 single nucleotide polymorphisms from that of the type strain (Supplementary Fig. S2). A search for matches to the type L. clevelandensis 16S rRNA gene reference sequence accession number NR_151867.1 in the GenBank database of uncultured 16S rRNA gene sequences identified two identical sequence accession numbers, EF703946 and EF703981 (100%, 693/693, nucleotides each), from a microbiome analysis of the human small intestine in 2007; GQ025374 and JF205374 (100%, 703/703 nucleotides each) from two human skin microbiome studies; and LT685863 (100%, 736/736 nucleotides) representing a human head sample from Cleveland, OH, USA (unpublished data). Our patient represents the ninth case described worldwide and the third from Europe [2Chudy-Onwugaje K. Vandermeer F. Quezada S. Mimicking abdominal tuberculosis: abdominal abscess caused by Lawsonella clevelandensis in inflammatory bowel disease.Clin Gastroenterol Hepatol. 2019; 17 (e92)Google Scholar, 3Favila Menezes M. Sousa M.J. Paixao P. Atouguia J. Negreiros I. Simoes M.J. Lawsonella clevelandensis as the causative agent of a breast abscess.IDCases. 2018; 12: 95-96Google Scholar, 4Kumaria A. Lucas E.K. Crusz S.A. Howarth S.P.S. Cartmill M. Lawsonella clevelandensis causing spinal subdural empyema.Br J Neurosurg. 2018; https://doi.org/10.1080/02688697.2018.1540767Crossref Scopus (4) Google Scholar]. Among the five case publications, the agent could not be cultivated in two [2Chudy-Onwugaje K. Vandermeer F. Quezada S. Mimicking abdominal tuberculosis: abdominal abscess caused by Lawsonella clevelandensis in inflammatory bowel disease.Clin Gastroenterol Hepatol. 2019; 17 (e92)Google Scholar, 4Kumaria A. Lucas E.K. Crusz S.A. Howarth S.P.S. Cartmill M. Lawsonella clevelandensis causing spinal subdural empyema.Br J Neurosurg. 2018; https://doi.org/10.1080/02688697.2018.1540767Crossref Scopus (4) Google Scholar], indicating that this organism is difficult to propagate in a clinical laboratory. As a result of this organism's fastidiousness and slow growth, no AST has previously been performed on this novel bacterium. In the published case reports, therefore, antimicrobial therapy was provided empirically. Amoxicillin/clavulanate was used successfully in five cases; ciprofloxacin, levofloxacin, metronidazole, vancomycin, clindamycin, sulfamethoxazole-trimethoprim, cefuroxime, ertapenem and tigecycline were provided in variable combinations. Here we report antimicrobial susceptibility results from our L. clevelandensis strain showing low to very low MICs to all antimicrobial agents tested, a reproducible finding despite longstanding incubation. These phenotypic findings are corroborated by our WGS data, as no known resistance markers were found in the assembled genome, although it remains a possibility that unidentified determinants are present, given the unusual species. Recommendations for therapy of L. clevelandensis are missing, but because of the very low growth rate of L. clevelandensis, the duration of antimicrobial therapy should probably be adapted. L. clevelandensis 16S rRNA gene sequences can be found in several human microbiome studies of the small intestine, skin microbiome and human head. Two recently published studies found that L. clevelandensis may be part of the human skin microbiota [5Escapa I.F. Chen T. Huang Y. Gajare P. Dewhirst F.E. Lemon K.P. New insights into human nostril microbiome from the Expanded Human Oral Microbiome Database (eHOMD): a resource for the microbiome of the human aerodigestive tract.mSystems. 2018; 3 (e00187-18)Crossref PubMed Google Scholar, 6Francuzik W. Franke K. Schumann R.R. Heine G. Worm M. Propionibacterium acnes abundance correlates inversely with Staphylococcus aureus: data from atopic dermatitis skin microbiome.Acta Derm Venereol. 2018; 98: 490-495Crossref PubMed Scopus (38) Google Scholar]. Escapa et al. [[5]Escapa I.F. Chen T. Huang Y. Gajare P. Dewhirst F.E. Lemon K.P. New insights into human nostril microbiome from the Expanded Human Oral Microbiome Database (eHOMD): a resource for the microbiome of the human aerodigestive tract.mSystems. 2018; 3 (e00187-18)Crossref PubMed Google Scholar] demonstrated that the bacterium is highly abundant in oily skin sites, such as the alar crease and the glabella. On the basis of this information, L. clevelandensis may represent an organism of the human skin and mucosal microbiota. Damage or irritation of this organs (e.g. during surgery) could presumably lead to soft tissue infection or abscess formation. All authors report no conflicts of interest relevant to this article. We thank C. Kiessling, M. Schneider, E. Schultheiss, R.-M. Vesco, S. Heimberg and C. Straub (University Hospital Basel) for excellent technical assistance in sequencing and MIC determination. The critical review provided by V. Hinić (University Hospital Basel) is acknowledged, with great thanks. The following are the Supplementary data to this article: Download .pdf (.6 MB) Help with pdf files Multimedia component 1 Download .pdf (.14 MB) Help with pdf files Multimedia component 2 Download .docx (.01 MB) Help with docx files Multimedia component 3