CASE REPORT Human Wound Infection with Mannheimia glucosida following Lamb Bite Jillian S. Y. Lau,a Lida Omaleki,b Conny Turni,b Stuart Richard Barber,c Glenn Francis Browning,c Michelle J. Francis,d a,d a,d,e Maryza Graham, Tony M. Korman Downloaded from Monash Infectious Diseases, Monash Health, Clayton, Victoria, Australiaa; Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Queensland, Australiab; Faculty of Veterinary and Agricultural Science, The University of Melbourne, Parkville, Victoria, Australiac; Department of Microbiology, Monash Health, Clayton, Victoria, Australiad; Department of Medicine, Monash University, Clayton, Victoria, Australiae Mannheimia spp. are veterinary pathogens that can cause mastitis and pneumonia in domestic cattle and sheep. While Mannheimia glucosida can be found as normal flora in oral and respiratory mucosa in sheep, there have been no reported cases of human infection with this organism. http://jcm.asm.org/ CASE REPORT Partial 16S rRNA gene sequencing was performed using the MicroSeq 500 bacterial identification kit (Perkin-Elmer/Applied 64-year-old man sustained a bite on the right thumb from a Biosystems, Foster City, CA), with sequence analysis performed 10-month-old lamb. The lamb’s teeth punctured his thumb- A on 500 nucleotides using MicroSeq 500 (version 2.2). A result for nail while he was holding the lamb’s mouth open during anthel- M. haemolytica was reported, with a specimen score of 43 and a minthic treatment with a malfunctioning dosing device. He ap- 98.4% match (consensus length of 488 bp out of the 489-bp library plied epoxy resin to the nail to prevent the shattered edges from length). Sequence analysis using GenBank BLAST version 2.0 on September 15, 2016 by University of Queensland Library catching. Following this application, he developed pain around demonstrated 93% homology with M. glucosida (accession no. the wound, and 10 days after the injury, he presented to the hos- DQ301921.1) as the top match. The MicroSeq database includes pital. On examination, the nail was discolored, with localized M. haemolytica but not M. glucosida. wound tenderness but no discharge, and there was erythema Given the uncertainty surrounding the identity of the iso- tracking from the right thumbnail up to the axilla. The wound was explored surgically, the nail plate was removed, and pus was late, amplification and sequencing were performed on two washed out and sent to the laboratory for microscopy and culture. housekeeping genes, 16S rRNA (1,464 bp; GenBank accession The patient was treated with intravenous flucloxacillin for 24 h, no. KT222023) and partial rpoB (534 bp; accession no. followed by oral cephalexin for 7 days. On review 1 week later, the KT222022), as well as one virulence gene, lktA (2,862 bp; accession erythema had resolved and the wound had healed well. no. KT222021), using primers and protocols described previously Microscopy revealed large numbers of polymorphonuclear (1–3). The sequences were then compared and aligned with those cells, but no organisms were seen on Gram staining. After 1 day of obtained previously from ovine mastitis isolates (4), using Clust- incubation on horse blood agar, there was heavy growth of a alW in Geneious version 8.0 (Biomatters Ltd., Auckland, New Gram-negative bacillus that was catalase positive, oxidase posi- Zealand). The analysis showed that the 16S rRNA gene from the tive, and indole negative. It was hemolytic on 5% horse blood agar isolate was 99.91% identical to that of M. glucosida isolates F1 and and Mueller-Hinton agar containing 5% sheep blood. After a sec- H2, obtained previously from cases of mastitis in sheep (4). A lower level of identity (98.47%) was found with M. haemolytica ond day of incubation, a lighter growth of Escherichia coli, Entero- T coccus faecalis, coagulase-negative staphylococci, and mixed an- ATCC 33396 . Moreover, the isolate was 100% identical to the aerobes was apparent. two Mannheimia glucosida isolates in their partial rpoB gene se- With the use of the Vitek 2 GN card (bioMérieux, Marcy quences (4) and shared 98.96% identity with the M. haemolytica l’Etoile, France), the Gram-negative bacillus was identified as Sph- type strain ATCC 33396. The lktA gene was 100% identical to that ingomonas paucimobilis (97% match). Notably, the trehalose re- of M. glucosida isolate H2, obtained previously from a case of action was negative, the beta-glucosidase reaction was positive, mastitis in a sheep (3), and contained only one nonsynonymous and the ornithine decarboxylase reaction was negative on the Vi- tek card. Matrix-assisted laser desorption ionization–time of Received 18 May 2015 Returned for modification 10 June 2015 flight mass spectrometry (MALDI-TOF MS)-based identification Accepted 10 July 2015 was performed using the Microflex LT mass spectrometer (Bruker Accepted manuscript posted online 22 July 2015 Daltonik), and the results were analyzed using the MALDI Bio- Citation Lau JSY, Omaleki L, Turni C, Barber SR, Browning GF, Francis MJ, Graham typer software program (version 4.0.0.1). The top score for this M, Korman TM. 2015. Human wound infection with Mannheimia glucosida Gram-negative organism was Mannheimia haemolytica, with a following lamb bite. J Clin Microbiol 53:3374–3376. doi:10.1128/JCM.01249-15. score of 2.177 and the accompanying comment “species of this Editor: B. W. Fenwick genus have very similar patterns, therefore distinguishing their Address correspondence to Jillian S. Y. Lau, [email protected]. species is difficult.” Other Mannheimia species appeared in the top Copyright © 2015, American Society for Microbiology. All Rights Reserved. 10 identifications listed, including Mannheimia glucosida, the doi:10.1128/JCM.01249-15 highest score for which was 1.305. 3374 jcm.asm.org Journal of Clinical Microbiology October 2015 Volume 53 Number 10 Case Report substitution compared with M. glucosida isolate PH498 (GenBank rpoB genes alone can be inconclusive for identification of this accession no. AF314518), an isolate originally obtained from a species and needs to be combined with phenotypic tests. Identifi- sheep in the United Kingdom (5). Pairwise nucleotide differences cation of M. haemolytica, however, can be achieved by amplifica- of 6.6 and 16.0% between the leukotoxins of M. glucosida and tion and sequencing of the rpoB gene, as M. haemolytica isolates those of the different alleles of M. haemolytica have previously from sheep mastitis have been found to be 100% identical in their been reported (5). On the basis of the similarity of the 16S rRNA, partial rpoB gene sequences (4). partial rpoB, and lktA genes of known M. glucosida isolates and This is the first case of M. glucosida infection in humans iden- reference strains, the isolate was identified as M. glucosida. tified using modern molecular methods. The older literature does Downloaded from Antimicrobial susceptibility testing was performed by disk dif- report human infections with Pasteurella haemolytica, including fusion on Mueller-Hinton sheep blood agar, and the results were an aortic graft infection (18), infective endocarditis (19, 20), re- interpreted using Clinical and Laboratory Standards Institute spiratory infections (21), and superficial wound infections (22). (CLSI) criteria for Pasteurella spp. (document M45-A2) (6). The The actual identification of the causative agent in this older liter- isolate was resistant to penicillin but susceptible to ceftriaxone, ature is clouded by the taxonomic rearrangements that have oc- tetracycline, and trimethoprim-sulfamethoxazole. The penicillin curred with Pasteurella haemolytica and the lack of molecular tests MIC as determined by Etest (bioMérieux, Marcy l’Etoile, France) at that time. Trehalose-negative strains of the Pasteurella haemo- was 0.75 ␮g/ml (breakpoint by CLSI criteria, 0.5 ␮g/ml), which lytica complex were transferred to the genus Mannheimia in 1999 http://jcm.asm.org/ confirmed the resistance categorization by disk diffusion. Using (11). In detail, P. haemolytica biogroup 1 became M. haemolytica, the criteria for M. haemolytica in Performance Standards for Anti- containing reference strains of serovars 1, 2, 5 to 9, 12 to 14, and 16 microbial Disk and Dilution Susceptibility Tests for Bacteria Isolated of the former P. haemolytica. Biotypes 3A to 3H and 9 and also from Animals (CLSI document VET01-A4) (7), the isolate was also serovar 11 of the former P. haemolytica were reclassified as M. found to be resistant to penicillin (breakpoint, 0.25 ␮g/ml). The glucosida (13). Biotypes and serotypes were not clearly reported in result for beta-lactamase testing using the nitrocefin disk test these previously published cases, making any retrospective con- (Becton Dickinson, Franklin Lakes, NJ) was negative after1hof version to the modern taxonomy impossible. incubation. In the current case, a number of phenotypic (Vitek and MALDI- TOF MS) and genotypic (MicroSeq 500) commercial identifica- on September 15, 2016 by University of Queensland Library tion systems failed to confidently identify the M. glucosida isolate. It is well recognized that commercial identification systems can Mannheimia spp. are known to be an important cause of mas- have databases that focus on common medical pathogens and titis and respiratory tract infections in sheep and other livestock have deficiencies for bacteria encountered more rarely in medical (4, 8). In this report, we present a case of severe M. glucosida soft cases, such as the Pasteurellaceae (23). While MALDI-TOF MS has tissue infection following a lamb bite. We are aware of only one been shown to confidently identify some species within the genus report of human bacterial infection following a sheep bite: Acti- Mannheimia, this prior work was limited to just three species, M. nobacillus lignieresii was isolated from an infected finger wound granulomatis, M. haemolytica, and M. varigena (24). Misidentifi- following a sheep bite (9). cation by MALDI-TOF MS has been associated with insufficient The genus Mannheimia is a member of the Pasteurellaceae fam- numbers of reference strains within the database (25).