CASE REPORT

Human Wound Infection with 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 “ 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 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 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 (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). The diffi- ily and contains six named species, Mannheimia haemolytica, M. culties of confident species identification by MALDI-TOF MS re- glucosida, M. ruminalis, M. varigena, M. granulomatis (4), and M. ported in the current case are similar to those reported for another caviae (10). These fastidious Gram-negative organisms were pre- genus of the family Pasteurellaceae, Avibacterium (26). The failure viously categorized within the genus Pasteurella but were reclassi- of the MicroSeq system to identify the isolate was essentially due to fied in 1999 (11). Mannheimia haemolytica and M. glucosida are the absence of M. glucosida from the database. Diagnostic labora- significant pathogens in livestock and have been associated with tories need to be aware that animal bite-based infections have a ovine mastitis (8, 12) and pneumonia in ruminants (13). high likelihood of yielding members of the family Pasteurellaceae Mannheimia haemolytica has been isolated from oral and respira- (especially Pasteurella multocida) and need to be prepared to use tory mucosae of healthy cattle and sheep. It has been regarded as conventional (non-kit-based) sequencing of key genes such as 16S the major organism involved in the shipping fever complex of rRNA and rpoB and extensive databases such as GenBank or a feedlot cattle and pneumonia, septicemia, and mastitis in sheep more focused, specialized database such as the EzTaxon database (11). Mannheimia glucosida has mostly been associated with (27). sheep, particularly as a cause of mastitis (4), but it has been iso- Antimicrobial susceptibility data for Mannheimia spp. are lim- lated from cases of pneumonia in other ruminants (13). ited, and the available studies have focused on M. haemolytica due Leukotoxin is a known virulence factor for M. haemolytica to its significance as a veterinary pathogen. Tetracyclines are (14). It is a member of the RTX (repeats-in-toxin) family of cyto- widely used in veterinary medicine (1) and are often used as a toxins (15) produced by a number of Gram-negative bacterial first-line treatment for cases of severe ovine mastitis (28). How- species, including Escherichia coli, Actinobacillus pleuropneumo- ever, tetracycline resistance has been described for M. haemolytica, niae, and Aggregatibacter actinomycetemcomitans (16). The leuko- M. glucosida, and M. varigena isolates from cases of pneumonia in toxin operon consists of four genes, designated lktC, lktA, lktB, cattle (29). This resistance is mediated by tetracycline resistance and lktD (17), and aids in bacterial survival by interacting with the (tet) genes found on plasmids and chromosomes in these isolates alveolar macrophages, neutrophils, and lymphocytes (5, 14). and has been demonstrated to be able to transfer horizontally Mannheimia glucosida is phenotypically and genetically a het- between members of different Mannheimia species (29, 30). In erogeneous species (11), which makes identification of the species vitro susceptibility surveys have been reported using different difficult. Often, amplification and sequencing of the 16S rRNA or methods, including broth microdilution, disk diffusion, and cali-

October 2015 Volume 53 Number 10 Journal of Clinical Microbiology jcm.asm.org 3375 Case Report brated dichotomous sensitivity (CDS) testing. Various rates of 14. Davies RL, Whittam TS, Selander RK. 2001. Sequence diversity and resistance to ampicillin, penicillin, and trimethoprim-sulfame- molecular evolution of the leukotoxin (lktA) gene in bovine and ovine thoxazole have been described (29, 31, 32). strains of Mannheimia (Pasteurella) haemolytica. J Bacteriol 183:1394– 1404. http://dx.doi.org/10.1128/JB.183.4.1394-1404.2001. This patient did not receive appropriate empirical antibiotics 15. Lo RY, Strathdee CA, Shewen PE. 1987. Nucleotide sequence of the for a polymicrobial animal bite infection including Pasteurella or leukotoxin genes of Pasteurella haemolytica A1. Infect Immun 55:1987– Mannheimia species. However, the clinical outcome was favor- 1996. able, highlighting the importance of appropriate surgical manage- 16. Aulik NA, Hellenbrand KM, Czuprynski CJ. 2012. Mannheimia haemo- lytica and its leukotoxin cause macrophage extracellular trap formation by ment in these cases. bovine macrophages. Infect Immun 80:1923–1933. http://dx.doi.org/10 Downloaded from Nucleotide sequence accession numbers. Sequences have .1128/IAI.06120-11. been deposited in GenBank under accession no. KT222021 to 17. Davies RL, Arkinsaw S, Selander RK. 1997. Evolutionary genetics of KT222023. Pasteurella haemolytica isolates recovered from cattle and sheep. Infect Immun 65:3585–3593. ACKNOWLEDGMENT 18. Rivera M, Hunter GC, Brooker J. 1994. Aortic graft infection due to Pasteurella haemolytica and group C ␤-hemolytic streptococcus. Clin In- We thank Patrick Blackall, Queensland Alliance for Agriculture and Food fect Dis 19(5):941–943. http://dx.doi.org/10.1093/clinids/19.5.941. Innovation, The University of Queensland, for his scientific support. 19. Yaneza AL, Jivan H, Kumari P, Togoo MS. 1991. Pasteurella haemolytica endocarditis. J Infect 23:65–67. http://dx.doi.org/10.1016/0163-4453(91)

REFERENCES 94085-X. http://jcm.asm.org/ 1. Korczak B, Christensen H, Emler S, Frey J, Kuhnert P. 2004. Phylogeny 20. Takeda S, Arashima Y, Kato K, Ogawa M, Kono K, Watanabe K, Saito of the family Pasteurellaceae based on rpoB sequences. Int J Syst Evol T. 2003. A case of Pasteurella haemolytica sepsis in a patient with mitral Microbiol 54:1393–1399. http://dx.doi.org/10.1099/ijs.0.03043-0. valve disease who developed a splenic abscess. Scand J Infect Dis 35:764– 2. Kuhnert P, Capaul SE, Nicolet J. 1996. Phylogenetic positions of Clos- 765. http://dx.doi.org/10.1080/00365540310016385. tridium chauvoei and Clostridium septicum based on 16S rRNA gene se- 21. Watanabe T, Sato M, Abe T, Oda Y. 1998. Bacterial croup caused by quences. Int J Syst Bacteriol 46:1174–1176. http://dx.doi.org/10.1099 Pasteurella haemolytica. Acta Paediatr Jpn 40:360–361. http://dx.doi.org /00207713-46-4-1174. /10.1111/j.1442-200X.1998.tb01948.x. 3. Omaleki L, Browning GF, Barber SR, Allen JL, Srikumaran S, Markham 22. Gautier A-L, Dubois D, Escande F, Avril J-L, Trieu-Cuot P, Gaillot O. PF. 2014. Sequence diversity, cytotoxicity and antigenic similarities of the 2005. Rapid and accurate identification of human isolates of Pasteurella

leukotoxin of isolates of Mannheimia species from mastitis in domestic and related species by sequencing the sodA gene. J Clin Microbiol 43: on September 15, 2016 by University of Queensland Library sheep. Vet Microbiol 174:172–179. http://dx.doi.org/10.1016/j.vetmic 2307–2314. http://dx.doi.org/10.1128/JCM.43.5.2307-2314.2005. .2014.08.009. 23. Blackall PJ, Norskov-Lauritsen N. 2008. Pasteurellaceae: the view from 4. Omaleki L, Barber SR, Allen JL, Browning GF. 2010. Mannheimia the diagnostic laboratory, p 227. In Kuhnert P, Christensen H (ed), Pas- species associated with ovine mastitis. J Clin Microbiol 48:3419–3422. teurellaceae: biology, genomics and molecular aspects. Horizon Scientific http://dx.doi.org/10.1128/JCM.01145-10. Press, Norwich, United Kingdom. 5. Davies RL, Campbell S, Whittam TS. 2002. Mosaic structure and mo- 24. Kuhnert P, Bisgaard M, Korczak BM, Schwendener S, Christensen H, lecular evolution of the leukotoxin operon (lktCABD)inMannheimia Frey J. 2012. Identification of animal Pasteurellaceae by MALDI-TOF (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella treha- mass spectrometry. J Microbiol Methods 89:1–7. http://dx.doi.org/10 losi. J Bacteriol 184:266–277. http://dx.doi.org/10.1128/JB.184.1.266-277 .1016/j.mimet.2012.02.001. .2002. 25. van Veen SQ, Claas ECJ, Kuijper EJ. 2010. High-throughput identifica- 6. Clinical and Laboratory Standards Institute. 2010. Methods for an- tion of bacteria and yeast by matrix-assisted laser desorption ionization– timicrobial dilution and disk susceptibility testing of infrequently iso- time of flight mass spectrometry in conventional medical microbiology lated or fastidious bacteria, 2nd ed. Approved standard M45-A2. CLSI, laboratories. J Clin Microbiol 48:900–907. http://dx.doi.org/10.1128 Wayne, PA. /JCM.02071-09. 7. Clinical and Laboratory Standards Institute. 2013. Performance stan- 26. Alispahic M, Christensen H, Bisgaard M, Hess M, Hess C. 2014. dards for antimicrobial disk and dilution susceptibility tests for bacteria MALDI-TOF mass spectrometry confirms difficulties in separating spe- isolated from animals, 4th ed. Approved standard VET01-A4. CLSI, cies of the Avibacterium genus. Avian Pathol 43:258–263. http://dx.doi Wayne, PA. .org/10.1080/03079457.2014.916038. 8. Barber SR, Allen JL, Mansell P, Browning GF. 2006. Mastitis in the ewe, 27. Chun J, Lee J-H, Jung Y, Kim M, Kim S, Kim BK, Lim Y-W. 2007. p 127–132. In Proceedings of the Australian Sheep Veterinarians 2006 EzTaxon: a web-based tool for the identification of prokaryotes based on Conferences, vol 16. Australian Sheep Veterinarians, Eight Mile Plains, 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57:2259– Australia. 2261. http://dx.doi.org/10.1099/ijs.0.64915-0. 9. Peel MM, Hornidge KA, Luppino M, Stacpoole AM, Weaver RE. 1991. 28. Mavrogianni VS, Menzies PI, Fragkou IA, Fthenakis GC. 2011. Princi- Actinobacillus spp. and related bacteria in infected wounds of humans ples of mastitis treatment in sheep and goats. Vet Clin North Am Food bitten by horses and sheep. J Clin Microbiol 29:2535–2538. Anim Pract 27:115–120. http://dx.doi.org/10.1016/j.cvfa.2010.10.010. 10. Christensen H, Bojesen AM, Bisgaard M. 2011. Mannheimia caviae sp. 29. Kehrenberg C, Salmon SA, Watts JL, Schwarz S. 2001. Tetracycline nov., isolated from epidemic conjunctivitis and otitis media in guinea resistance genes in isolates of Pasteurella multocida, Mannheimia haemo- pigs. Int J Syst Evol Microbiol 61:1699–1704. http://dx.doi.org/10.1099/ijs lytica, Mannheimia glucosida and from bovine and .0.026518-0. swine respiratory disease: intergeneric spread of the tet(H) plasmid 11. Angen O, Mutters R, Caugant DA, Olsen JE, Bisgaard M. 1999. Taxo- pMHT1. J Antimicrob Chemother 48:631–640. http://dx.doi.org/10.1093 nomic relationships of the [Pasteurella] haemolytica complex as evaluated /jac/48.5.631. by DNA-DNA hybridizations and 16S rRNA sequencing with proposal of 30. Kehrenberg C, Catry B, Haesebrouck F, de Kruif A, Schwarz S. 2005. Mannheimia haemolytica gen. nov., comb. nov., Mannheimia granuloma- tet(L)-mediated tetracycline resistance in bovine Mannheimia and Pasteu- tis comb. nov., Mannheimia glucosida sp. nov, sp. rella isolates. J Antimicrob Chemother 56:403–406. http://dx.doi.org/10 nov. and Mannheimia varigena sp. nov. Int J Syst Bacteriol 49(1):67–86. .1093/jac/dki210. http://dx.doi.org/10.1099/00207713-49-1-67. 31. Mohammadi GR, Ghazvini K, Abbas Panah H. 2006. Antimicrobial 12. Omaleki L, Browning GF, Allen JL, Barber SR. 2011. The role of susceptibility testing of Mannheimia haemolytica and Pasteurella multo- Mannheimia species in ovine mastitis. Vet Microbiol 153:67–72. http://dx cida isolated from calves with dairy calf pneumonia. Arch Razi Inst 61(2): .doi.org/10.1016/j.vetmic.2011.03.024. 91–96. 13. Angen O, Ahrens P, Bisgaard M. 2002. Phenotypic and genotypic char- 32. Schwarz S, Kehrenberg C, Salmon SA, Watts JL. 2004. In vitro activities of acterization of Mannheimia (Pasteurella) haemolytica-like strains isolated spectinomycin and comparator agents against Pasteurella multocida and from diseased animals in Denmark. Vet Microbiol 84:103–114. http://dx Mannheimia haemolytica from respiratory tract infections of cattle. J An- .doi.org/10.1016/S0378-1135(01)00439-4. timicrob Chemother 53:379–382. http://dx.doi.org/10.1093/jac/dkh059.

3376 jcm.asm.org Journal of Clinical Microbiology October 2015 Volume 53 Number 10