Clinical significance of Nosocomiicoccus ampullae isolated from blood cultures

ABSTRACT Nosocomiicoccus species are infrequently isolated from clinical specimens and their clinical significance has not been evaluated. We retrospectively reviewed five cases in which Nosocomiicoccus ampullae was identified in a single blood culture set during routine culture. All patients were admitted for altered mental status and had several comorbidities. Patients improved without antibiotic therapy directed to N. ampullae or their condition declined due to underlying conditions, and N. ampullae isolation was not considered clinically significant in any case. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and/or 16S rRNA gene Sanger sequencing allowed genus identification, but whole genome sequencing was required for accurate species identification. All strains were susceptible to penicillin, oxacillin, cefoxitin, tetracycline, doxycycline, daptomycin, minocycline, clindamycin, nitrofurantoin, vancomycin, and linezolid; they showed high-level resistance to trimethoprim-sulfamethoxazole and erythromycin, and inducible resistance to clindamycin, applying Clinical and Laboratory Standards Institute Staphylococcus spp. guidelines with an extended incubation. They frequently carry genes associated with macrolide resistance. In conclusion, N. ampullae appears to have low pathogenicity and most probably represents a contaminant when isolated from a single blood culture set. IMPORTANCE Nosocomiicoccus species are recently described as members of the Staphylococcaceae family. With their inclusion in commercial matrix-assisted laser desorption/ionization-time of flight mass spectrometry databases, Nosocomiicoccus species can now be identified when Gram-positive cocci in clusters are detected in positive blood cultures. However, their clinical significance is not known, making it difficult for the clinical microbiology laboratory to decide the extent of work-up. Based on our study, Nosocomiicoccus species demonstrate low pathogenicity and opportunistic potential. If isolated from a single blood culture set, limited work-up should be performed to an extent similar to other possible blood culture contaminants.

IMPORTANCE Nosocomiicoccus species are recently described as members of the Staphylococcaceae family.With their inclusion in commercial matrix-assisted laser desorption/ionization-time of flight mass spectrometry databases, Nosocomiicoccus species can now be identified when Gram-positive cocci in clusters are detected in positive blood cultures.However, their clinical significance is not known, making it difficult for the clinical microbiology laboratory to decide the extent of work-up.Based on our study, Nosocomiicoccus species demonstrate low pathogenicity and opportunis tic potential.If isolated from a single blood culture set, limited work-up should be performed to an extent similar to other possible blood culture contaminants.KEYWORDS Nosocomiicoccus, bloodstream infections, blood culture contaminant N osocomiicoccus species are Gram-positive, aerobic cocci, members of the Staphylo coccaceae family (1).There are two species in this genus, Nosocomiicoccus ampullae (2) and Nosocomiicoccus massiliensis (3).A third species, "Candidatus Nosocomiicoccus stercorigallinarum", identified as part of the chicken gut microbiome, has been proposed (4).
Reports on the isolation of these organisms are scarce and their clinical significance is uncertain (Table 1).N. ampullae was first described in 2008 after isolation from the surface of physiological saline bottles used repeatedly for topical washing of wounds (2).Later, it was isolated from a joint aspirate during routine diagnostic analysis (5), from a bloodstream infection in an adult patient (6), from the urine of an adult female with urinary symptoms (7), and from a Nigerian fermented soybean condiment (8).However, based on whole genome sequencing in a later study, it was proposed that the urinary strain was actually N. massiliensis (5).N. massiliensis was first isolated from the fecal microbiota of an individual living with HIV and experiencing acquired immunodeficiency syndrome in 2012 (3) and no additional reports have been published.In this study, we sought to determine the clinical significance of N. ampullae isolated from blood cultures.In addition, we evaluated their antimicrobial susceptibility profiles using several antimicrobial susceptibility testing (AST) methods.
Cases in which Nosocomiicoccus spp.were isolated from blood cultures using the BACTEC system (BD Diagnostics, Inc., Sparks, MD, USA) and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Billerica, MA, USA) and/or 16S rRNA gene Sanger sequencing during standard clinical work-up at the Johns Hopkins Hospital Medical Microbiology Laboratory were found through database search.Medical records were reviewed for clinical presentation, microbiology results, antibiotic therapy, and clinical outcome.
Between April 2014 and May 2022, Nosocomiicoccus spp.were identified in blood cultures from five unique patients without central venous catheters.Nosocomiicoccus spp.were not identified from any other specimen sources.The clinical features of the patients in which Nosocomiicoccus spp.were isolated are summarized in Table 2.A sixth case of isolation of Nosocomiicoccus spp.from blood cultures was identified toward the end of the preparation of this manuscript.The patient characteristics are presented in Table 2, but no further description of this patient/isolate is provided.
Patients were 49-88 years of age (mean 69.3) and 80% were male.All patients were admitted for altered mental status and had various comorbid conditions.There was no overlap in time or hospital location between the patients when/where the blood cultures were drawn.Three blood isolates of Nosocomiicoccus spp.were from a single set out of two or three sets of blood cultures collected within 48 hours, and two from the only set collected.Nosocomiicoccus sp. was the only organism recovered in four cases and these cultures took between 43 and 59 hours to flag positive.Even though Nosocomii coccus sp. has been previously described as aerobic or microaerophilic but unable to grow anaerobically (2), one of the isolates was recovered in the anaerobic bottle.One patient with Nosocomiicoccus sp. in one of three blood culture sets had Staphylococcus simulans in all three sets.Two patients were not diagnosed with infectious processes; one patient had signs of a skin infection without a definitive pathogen and one patient was diagnosed with severe acute respiratory syndrome coronavirus 2 infection and urinary tract infection caused by Proteus mirabilis and Aerococcus urinae.
All isolates were identified as Gram-positive cocci in clusters.Colonies showed a light-yellow pigment and no hemolysis; they were catalase and oxidase positive, consistent with previous reports of N. ampullae (2,3).During standard of care work-up, MALDI-TOF MS was performed on four isolates; two were identified to the genus level (score <2.0) and two were unable to be identified.By 16S rRNA gene Sanger sequencing, one isolate was identified as N. ampullae, one as Nosocomiicoccus sp., two were unable to be identified, and one was not sequenced.Subsequently, all isolates underwent whole genome sequencing by Illumina MiSeq short-read sequencing (Illumina, San Diego, CA, USA) as previously described ( 9), and assembled using SPAdes (10).Nucmer (11) was then used to perform all pairwise alignments between each of the GenBank assemblies (nine sequences) and our isolate assemblies.Using the percent identities calculated by nucmer, the overall percent match between each pair was calculated.From the distance matrix of these nine sequences, a phylogenetic tree was generated, and all five isolates were determined to belong to N. ampullae.
AST was not performed or requested during the standard of care procedures but was performed retrospectively for this study.Since Nosocomiicoccus spp.were recently assigned to the Staphylococcaceae family (1) and we demonstrated that our isolates grew better under ambient air than under air supplemented with 5% CO 2 (Fig. S1), AST was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines and interpretation for testing Staphylococcus spp.other than S. aureus, S. lugdunensis, S. epidermidis, S. pseudointermedius, and S. schleiferi (12).These interpretative criteria were chosen due to the phylogenetic relatedness between Nosocomiicoccus sp. and members of the Staphylococcaceae family, as it has been used for Micrococcus spp.( 13); however, interpretative criteria have not been established for Nosocomiicoccus spp.AST using the BD Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD, USA) was first attempted; however, the isolates failed to grow overnight in the system.Therefore, we performed AST by reference broth microdilution (BMD) in cation-adjusted Mueller-Hinton broth, and gradient diffusion (Etest; bioMerieux, Marcy-l'Étoile, France) and by disk diffusion (DD; BBL disks, BD Diagnostic Systems, Sparks, MD, USA) in Mueller-Hinton agar with reading performed following 48 hours of incubation at 35°C in ambient air, instead of after the 16-24 hours of incubation recommended by CLSI M100 (Table S1) (12,14,15).Based on results from the reference BMD and applying Staphy lococcus spp.interpretations, N. ampullae isolates were 100% susceptible to penicillin, oxacillin, tetracycline, doxycycline, daptomycin, minocycline, clindamycin, vancomycin, and linezolid.Most strains (80%) were resistant to trimethoprim-sulfamethoxazole.By DD, all strains were susceptible to cefoxitin and nitrofurantoin, and four were susceptible to moxifloxacin.Results from BMD and DD were not 100% concordant for erythromycin.By BMD, three strains tested susceptible, one demonstrated an intermediate MIC, and one was resistant to erythromycin; by DD, there was one susceptible strain, one was intermediate, and three were resistant.Further studies are needed to determine which method is more suitable to determine erythromycin resistance within these strains.Four strains showed inducible clindamycin resistance by DD (D-test positive) (Table S1) (12).Clindamycin inducible resistance by BMD was not tested.Interestingly, antimicrobial resistance screening using ABRicate ( 16) on the isolate assemblies revealed four strains that showed resistance to erythromycin and were found to carry the ermY gene (Table S1).This gene encodes for erythromycin ribosome methylase proteins responsible for macrolide resistance (17) and it was also found in the published sequencing data from the N. ampullae strain isolated from joint fluid (5).
None of the patients in this study received directed antimicrobial therapy for Nosocomiicoccus spp.One patient improved with supportive care without receiving antimicrobial treatment, two patients improved while on antibiotics that, although potentially active against N. ampullae, were directed toward other infections, and two patients developed worsening mental status due to underlying conditions with one discharged to hospice care and one expiring on day 8 of admission.Although it is difficult to completely rule out the pathogenic role of the isolates recovered from these patients, no providers noted Nosocomiicoccus spp. to be clinically significant and all patients had other conditions that could explain their symptoms.
In conclusion, N. ampullae appears to have low pathogenicity and opportunistic potential and most probably represents a contaminant when isolated from a single blood culture set.MALDI-TOF MS and sequencing of the first ~500 base pairs of the 16S rRNA gene may not allow discrimination between different species within the Nosocomii coccus genus.If species-level identification is desired, the oxidase test could differentiate between N. massiliensis (negative) and N. ampullae (positive).Additionally, N. ampullae demonstrates high resistance rates to trimethoprim-sulfamethoxazole, erythromycin and inducible resistance to clindamycin, and they can possess antimicrobial resistance genes associated with macrolide resistance.

TABLE 2
Clinical features of cases in which Nosocomiicoccus ampullae was isolated from blood a Patient identified after WGS and antimicrobial susceptibility testing had been performed on the other isolates.Not included in isolate characterization.Observation Microbiology Spectrum November/December 2023 Volume 11 Issue 6 10.1128/spectrum.02179-233