Prolonged postprocedural outbreak of Mycobacterium massiliense infections associated with ultrasound transmission gel  A. Cheng, W.-H. Sheng, Y.-C. Huang, H.-Y. Sun, Y.-T. Tsai, M.-L. Chen, Y.-C. Liu, Y.-C. Chuang, S.-C. Huang, C.-I. Chang, L.-Y. Chang, W.-C. Huang, P.-R. Hsueh, C.-C. Hung, Y.-C. Chen, S.-C. Chang  Clinical Microbiology and Infection  Volume 22, Issue 4, Pages 382.e1-382.e11 (April 2016) DOI: 10.1016/j.cmi.2015.11.021 Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 1 Algorithm for molecular identification and typing of clinical and environmental isolates of mycobacteria. M. abscessus and M. chelonae are phenotypically indistinguishable and were molecularly characterised by concatenating the partial sequences of the hsp65, rpoB, secA1 genes to the subspecies level and discriminating M. massiliense into 12 genotypes. The clonality of the outbreak strain TPE101 was further confirmed by multilocus sequence typing (MLST) (cya, argH, glpK, gnd, murC, pta, purH) and pulsed-field gel electrophoresis (PFGE) of AseI- and XbaI-digested DNA. Clinical Microbiology and Infection 2016 22, 382.e1-382.e11DOI: (10.1016/j.cmi.2015.11.021) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 2 Epidemic curve of post-procedure infections due to a single strain of M. massiliense TPE101, multilocus sequence type (MLST) 48 among patients at two geographically distant medical centers in Taiwan according to month of occurrence. After switch to single-dose sterile gel in February 2012 and recall and replacement of all contaminated ultrasound transmission gel at NTUH in April 2012, there have been no cases in the 18 months of active surveillance since December 2012. Clinical Microbiology and Infection 2016 22, 382.e1-382.e11DOI: (10.1016/j.cmi.2015.11.021) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 3 Pulsed-field gel electrophoresis (PFGE) patterns of AseI-digested chromosomal DNA of representative Mycobacterium massiliense multilocus sequence type (MLST) 48 clinical and environmental isolates (A, top panel) and dendrograms prepared using the BioNumerics program v.5.1 and corresponding genotypes (Mm1-12) determined by sequencing of the hsp65, rpoB, secA1 genes (B, lower panel). A, high-level relativeness of PFGE patterns (indistinguishable or closely related, pulsotype A, TPE101) among case strains and gel strains (lanes 2-18) in contrast to the more diversity among control strains (lanes 19-28). B, showing PFGE discriminated case strains TPE101 (all belonged to genotype 1; Figure 3B, Mm1 and marked in red box) into 8 sub-clones (pulsotype A1-8) with pulsotype A1 and A2 most prevalent. Three subclones were identified from gels (pulsotype A1, A2 and A5). Molecular typing determined by PFGE with 90% similarity was concordant to genotyping. PFGE patterns of case strains are presented by chronological order of occurrence and those of gel strains alongside time- and/or place-linked case strains. Lanes 2, 3, 13, 14 were case strains (case 3 and 6 from Hospital A; case 27 and 28 from Hospital B) with indistinguishable PFGE pattern which belonged to the main subclone (pulsotype A1). Lanes 4, 5, 10 and 11 were case strains (case 10, 17, 36), differing from pulsotype A1 by one band and clustering into the second prevalent subclone (pulsotype A2). Two isolates from case 36 (lane 10, blood isolates; lane 11, pus isolate collected 5 days later at surgical site) demonstrated indistinguishable PFGE pattern which confirmed the primary source of bloodstream infection. Lane 8 was the blood isolate of an adult patient (case 35) and differed from the pulsotype A1 by one band (pulsotype A3). Lanes 6, 7, 9, 12, 17 represented PFGE patterns of strains isolated from in-use (lanes 6, 9) or sealed unopen (lanes 7, 12, 17) ultrasound transmission gel and are classified into pulsotype A1 (lanes 6, 9, 12, and 17) and pulsotype A5 (lane 7) which exhibited one band difference from pulsotype A2. Lane 9 strain obtained from in-use gel which was time- and place- linked to case 35 (lane 8), but exhibited one band difference. Lane 18 was the only one case strain exhibited more than 3 band differences from pulsotype A1 (pulsotype A8) with similarity 89.6%. This pus isolate obtained from an adult patient (case 13) who developed multiple skin and soft tissue infections following cosmetic surgery at local clinic. Lanes 19-28 demonstrated diversity in PEGE patterns of control strains which are unrelated to outbreak strains with 6 or more band difference and are not genotype 1 (Figure 3B, Mm2-8). Lanes 19 and 27 was the cornea isolate and ear isolates, respectively, of two control patients with non-iatrogenic traumatic injury. Lanes 20-26 demonstrate the diversity of PFGE patterns of respiratory isolates of 7 control patients. Lane 28 is a reference strain, M. massiliense BCRC 16916, unrelated to the outbreak strain by PFGE and belonged to genotyping 12 (Mm12). Lanes 1, 15, 16, 29 represented DNA markers. Clinical Microbiology and Infection 2016 22, 382.e1-382.e11DOI: (10.1016/j.cmi.2015.11.021) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions