ACCEPTED. Anaerobe Reference Laboratory, Department of Bacterial and Inflammatory Diseases, National

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1 AAC Accepts, published online ahead of print on April 00 Antimicrob. Agents Chemother. doi:./aac Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Antimicrobial susceptibility of Peptostreptococcus anaerobius and the newly described Peptostreptococcus stomatis isolated from various human sources Eija Könönen, 1 * Anne Bryk 1, Päivi Niemi, and Arja Kanervo-Nordström 1 Anaerobe Reference Laboratory, Department of Bacterial and Inflammatory Diseases, National Public Health Institute (KTL), Helsinki, 1 and Kymenlaakso Hospital District, Kotka, Finland Running title: Susceptibility of Peptostreptococcus spp. Key words: gram-positive anaerobic cocci; Peptostreptococcus anaerobius; Peptostreptococcus stomatis; Etest; antimicrobial resistance *Corresponding author. Mailing address: Anaerobe Reference Laboratory, National Public Health Institute (KTL), Mannerheimintie 1, FI-0000 Helsinki, Finland. Phone: +--, Fax: +--, eija.kononen@ktl.fi 0 1

2 ABSTRACT Peptostreptococcus anaerobius sensu lato, currently including two closely related species, P. anaerobius and P. stomatis, is known as more resistant than other gram-positive anaerobic cocci. We re-identified potential Peptostreptococcus isolates and tested their susceptibilities to eight antimicrobials. Notably, P. anaerobius had constantly higher MIC 0 and MIC 0 values than P. stomatis.

3 Gram-positive anaerobic cocci (GPAC) are clinically significant recoveries from mixed infections involving anaerobic bacteria (1, 1). This heterogeneous group has confronted in considerable taxonomic changes, new genera being formed from species previously belonging to the genus Peptostreptococcus (, ). Peptostreptococcus anaerobius remained as a single representative of the genus until Peptostreptococcus stomatis was recently described (). P. anaerobius sensu lato has been frequently cultured from clinical specimens of the mouth, upper respiratory tract, skin and soft tissues, bone and joints, and gastrointestinal and genitourinary tracts (,,, 1, 1, 0,, ). Interestingly, Downes & Wade () suggested that the novel species, P. stomatis, originates from the oral cavity, whereas P. anaerobius sensu stricto has its habitat and is involved in infections under the waistline. GPAC have been considered susceptible to most antimicrobials used against anaerobic infections (1). However, P. anaerobius sensu lato seems to exhibit more resistance than other GPAC (, 1, 1, 1, 1, ). No susceptibility data on P. anaerobius sensu stricto or P. stomatis exist so far. The aim of the present study was to obtain information on their isolation sites and in vitro susceptibilities to eight antimicrobials potentially used to treat anaerobic infections. Sixty-four isolates that had been previously identified as P. anaerobius based on their anaerobic growth, coccoid cell morphology, sensitivity to sodium polyanethol sulfonate (SPS), and major production of isocaproic acid in gas-liquid chromatography (1) were available from various clinical specimens of subjects. Their isolation sites were traced from referrals that were sent together with the isolate to be characterized. The isolates, collected between 1 and 00, were revived from frozen (-0 o C) stocks and subcultured on Brucella blood agar for their purity before further testing. Key phenotypic tests discriminative in separation of P. anaerobius and P. stomatis were the size of the growth inhibition zone around a SPS disk and production of proline arylamidase (). The zone around a SPS disk (Oxoid, Basingstoke, UK) was measured by mm (the minimum size should be 1 mm) on a Brucella agar plate after three days of anaerobic incubation.

4 Enzyme activity patterns were determined by the Rapid ID A test kit (API system; biomérieux, Marcy l Étoile, France) according to the manufacturer s instructions. For isolates with discrepant phenotypic characteristics, partial sequencing of the 1S rrna gene was performed (1) and compared to sequences in the GenBank (). Antimicrobial susceptibilities to amoxicillin, amoxicillin/clavulanic acid, cefoxitin, ertapenem, azithromycin, clindamycin, metronidazole, and moxifloxacin were tested by the Etest (AB Biodisk, Solna, Sweden). Bacteria were harvested from a h anaerobic culture on prereduced Brucella and FAA (fastidious anaerobe agar) agars both supplemented with % defibrinated sheep blood, hemin, and vitamin K 1. Bacterial suspensions made in 0.% NaCl and adjusted to a turbidity of 1- of a McFarland standard were applied to fresh Brucella and FAA plates. The plates with Etest strips were incubated in an anaerobic chamber at C for h (for some slow-growing strains for h) before reading of the MICs. Susceptibility breakpoints were determined as follows: amoxicillin (0. µg/ml), amoxicillin/clavulanic acid ( µg/ml), cefoxitin (1 µg/ml), ertapenem ( µg/ml), clindamycin ( µg/ml), metronidazole ( µg/ml), and moxifloxacin ( µg/ml) (). No breakpoint for azithromycin has been approved for anaerobes. β-lactamase production was tested using the nitrocefin disk (AB Biodisk) test. Of the isolates, the identity of 0 isolates was confirmed as P. anaerobius based on the size of SPS inhibition zone between 1 <1 mm (here: 1 1 mm) and the Rapid ID A code , including positive α-glucosidase and proline arylamidase reactions (). Thirty-two isolates were re-identified as P. stomatis based on the size of SPS inhibition zone 1 mm (here: 1 mm) and the Rapid ID A code , including positive α-glucosidase and negative proline arylamidase reactions (). The identification of two isolates remained uncertain by biochemical testing; one blood isolate was proline arylamidase-negative but with SPS inhibition zone of 1 mm and another isolate from an abscess of the scrotum showed typical key characteristics of P. anaerobius but was negative for α-glucosidase production. The sequencing

5 identified the former isolate as P. stomatis and the latter isolate as most closely related to Peptoniphilus. P. anaerobius isolates originated from miscellaneous clinical specimens representing various anatomical sites, especially from ulcer and skin specimens of the lower extremities (%) and pus specimens of the genitourinary tract (%), but only occasionally from specimens of the head and gastrointestinal tract, whereas % of the P. stomatis isolates originated from oropharyngeal specimens (six oral and six pharyngeal isolates) and % of the isolates from gastrointestinal specimens (1 appendicidal and three fecal isolates). One P. anaerobius isolate and two P. stomatis isolates were from blood. The susceptibility data presented in Table come from 1 Peptostreptococcus isolates, since two P. stomatis isolates failed repeatedly to grow with the Etest. P. stomatis was susceptible to all drugs except for one isolate with a reduced susceptibility (MIC µg/ml) to clindamycin, whereas four P. anaerobius isolates demonstrated intermediate/resistant MICs to one or more drugs: one isolate to amoxicillin, amoxicillin/clavulanic acid, cefoxitin, and azithromycin (,,, and µg/ml, respectively), one isolate to amoxicillin, amoxicillin/clavulanic acid, and cefoxitin (1,, and µg/ml, respectively), one isolate to amoxicillin and amoxicillin/clavulanic acid ( and µg/ml, respectively), and one isolate to moxifloxacin (1 µg/ml). Although no breakpoint for azithromycin has been approved for anaerobes, the MIC µg/ml indicates resistance. Among isolates collected during 10 s, no resistance was seen, whereas 1/ isolates (%) from 10 s and /1 (1%) isolates from 000 s showed resistance. P. anaerobius had constantly higher MIC 0 and MIC 0 values than P. stomatis (Table). No beta-lactamase production was detected. Instead of grouping microorganisms together, the accurate characterization of bacterial species can reveal differences in their presumed habitats or infection patterns. Downes & Wade () suggested that P. stomatis originates from the oral cavity, whereas P. anaerobius sensu stricto is involved in infections under the waistline. Their suggestion was consistent with a previous report of

6 Riggio and Lennon () who failed to detect P. anaerobius in oral specimens by species-specific PCR. In line with these studies was our finding that most P. anaerobius isolates were from miscellaneous infectious specimens of the lower extremities and genitourinary tract. In contrast, the majority of the P. stomatis isolates were from oral and pharyngeal but also from gastrointestinal specimens. An interesting observation was that P. anaerobius had constantly higher MIC 0 and MIC 0 values than P. stomatis. In studies dealing with antimicrobial susceptibilities of anaerobes, GPAC usually appear as an otherwise non-specified group. However, an accurate identification can reveal unexpected differences in susceptibility patterns of related species within a genus (1, ). In the present study, a considerable number of isolates representing both P. anaerobius and P. stomatis was available for testing. In general, the drugs demonstrated excellent activities, especially against P. stomatis. However, 1% of the P. anaerobius isolates were resistant to one or more drugs. The combination of amoxicillin/clavulanic acid has been reported to be less effective against P. anaerobius than other GPAC (, 1). Indeed, % of the present P. anaerobius isolates showed resistance to amoxicillin/clavulanic acid. Since no β-lactamase production was detected, a similar resistance pattern for amoxicillin/clavulanic acid was expected as for amoxicillin alone. A novel parenteral carbapenem antibiotic, ertapenem, is reported to be active against most anaerobic species, including GPAC (1), and this was the case among the present Peptostreptococcus isolates. Moxifloxacin belongs to fluoroquinolones that have shown broad-spectrum activity also against anaerobic organisms, including peptostreptococci, MICs usually being.0 µg/ml (1). However, in the present study, one P. anaerobius isolate from a surgical wound specimen demonstrated repeatedly a resistant MIC value (1 µg/ml) to moxifloxacin. Metronidazole proved to have an excellent activity against both Peptostreptococcus species. Of concern may be that nim genes, which encode nitroimidazole resistance, are frequently present in GPAC, among those P. anaerobius ().

7 In conclusion, both Peptostreptococcus species are involved in infections, including bacteremia, and they may become increasingly resistant to antibiotics. It is important that clinicians and microbiology laboratories are aware of differences between these two species. Recently, an easy-to-use flow-chart for identification of GPAC has been created for clinical microbiology laboratories (). If the species identification is not considered that may result in poor therapy outcomes, especially, when treating infections with the involvement of P. anaerobius sensu stricto. REFERENCES 1. Ackermann, G., R. Schaumann, B. Pless, M. Claros, E. J. C. Goldstein, and A. C. Rodloff Comparative activity of moxifloxacin in vitro against obligately anaerobic bacteria. Eur. J. Clin. Microbiol. Infect. Dis. 1:-.. Aldridge, K. E., C. V. Sanders, A. C. Lewis, and R. L. Marier. 1. Susceptibility of anaerobic bacteria to beta-lactam antibiotics and beta-lactamase production. J. Med. Microbiol. 1:-.. Benson, D. A., I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, and D. L. Wheeler. 00. GenBank. Nucleic Acids Res. :D1-0.. Brazier, J. S., V. Hall, T. E. Morris, M. Gal, and B. I. Duerden. 00. Antibiotic susceptibilities of Gram-positive anaerobic cocci: results of a sentinel study in England and Wales. J. Antimicrob. Chemother. :-.. Brook, I. 1. Recovery of anaerobic bacteria from clinical specimens in 1 years at two military hospitals. J. Clin. Microbiol. :1-.. Brook, I. 1. Peptostreptococcal infection in children. Scand. J. Infect. Dis. :0-.

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11 TABLE. Antimicrobial susceptibilities of the tested strains of P. anaerobius and P. stomatis. P. anaerobius P. stomatis (n = 0) (n = 1) Antimicrobial agent Range of MICs MIC 0 MIC 0 Range of MICs MIC 0 MIC 0 (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) Amoxicillin < < Amoxicillin/clavulanate < < Cefoxitin < Ertapenem Azithromycin < Clindamycin < <0.01 < Metronidazole < < Moxifloxacin