Atypical detection of group B Streptococcus ST - 17 clone from a diabetic patient with necrotising fasciitis: A case report

Abstract

Streptococcus agalactiae or group B Streptococcus (GBS) is among the leading causes of neonatal infections and illnesses in pregnant women, posing a significant health challenge in both high-income and low- to middle-income countries. Predisposing factors in the elderly, such as diabetes mellitus, immunosuppression, obesity, alcoholism, and nicotine addiction, have been cited. Here, we report a 66-year-old diabetic female patient admitted to the hospital with suspected necrotising fasciitis of the left breast, which developed pus-draining lesions. Empiric treatment with amoxicillin-clavulanic acid was initiated. Culture of a swab sample revealed the presence of beta-haemolytic GBS, which was susceptible to ampicillin/penicillin, erythromycin, clindamycin, and vancomycin. The treatment was de-escalated to ampicillin. The outcome was favourable; the patient was discharged after 23 days of admission. Whole genome sequencing revealed the strain to be a hypervirulent clone ST-17, which carried multiple virulence factors and antimicrobial-associated resistance genes. This is the first-ever whole-genome report of hypervirulent GBS ST-17 in our tertiary hospital. This highlights the need for continuous surveillance, as this strain is known for high rates of morbidity and mortality, especially in neonates and the immunocompromised.

Introduction

Streptococcus agalactiae (Group B Streptococcus; GBS), a facultative anaerobe, catalase negative and Gram-positive coccus [1], [2]. It utilises various virulence factors, such as adhesins, pili, pore-forming toxins (i.e., CAMP factor), and a polysaccharide capsule that is rich in sialic acid to elicit its disease profile [3]. Some strains are categorised as hypervirulent, such as GBS sequence type (ST)-17, which produces the hypervirulent GBS adhesin (hvgA), a specific surface-anchored protein and a critical virulence trait of GBS in neonates [4].

Penicillin remains the treatment of choice for GBS infections, with clindamycin, erythromycin, and vancomycin being alternatives [1], [2]. There is no approved vaccine to prevent GBS infection [5].

Genitourinary carriage rate was found to be 36% and 37% in Denmark and South Africa, respectively [6], [7]. It is a significant cause of morbidity and mortality in neonates, pregnant women and immunocompromised individuals [8], causing meningitis, pneumonia, bacteraemia and skin and soft tissue infections [9]. This is a case report of an atypical necrotising fasciitis due to GBS in a diabetic adult patient.

Materials and methods

Strain isolation, identification, and antimicrobial susceptibility testing

A pus swab was collected intraoperatively from a South African female patient with necrotising fasciitis of the left breast and sent to a tertiary laboratory in Pretoria, South Africa, for routine testing. The sample was cultured on blood agar plates, incubated overnight at 37°C, and examined for growth, colony morphology, and preliminary tests, including Gram stain, catalase test, and rapid latex agglutination test Streptex™ (ThermoFisher Scientific, USA) conducted. Streptococcus agalactiae ATCC 12403 was used as a positive control strain during bacterial isolation. The VITEK®-2 (bioMérieux, Étoiles, France) automated system was used for bacterial confirmation and antimicrobial susceptibility profiling. Results were classified as Susceptible (S), Intermediate (I), or Resistant (R), according to the [10]).

Whole-genome sequencing (WGS) and gene function analysis

Library preparation and nanopore sequencing

The genomic DNA of the isolate was extracted using the Zymo-Quick-DNA Fungal/Bacteria Miniprep kit (Zymo-Research, USA) and quantified using a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). DNA library preparation was performed using the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96, UK) following the Nanopore-Only Microbial Isolate Sequencing Solution (NO-MISS) protocol (Oxford Nanopore Technologies, UK). Sequencing was performed on a MinION MK1C sequencer (Oxford Nanopore Technologies, UK).

Genome assembly, annotation, and data analysis

FASTQ files were uploaded to the Oxford Nanopore cloud-based EPI2ME software and analysed using the bacterial genome workflow. De novo assembly of bacterial genomes was done using Flye v2.9.5-b1801. Consensus sequences were polished using Medaka v2.0.0. The annotation of the resulting contigs was carried out with Prokka (v1.14.5). In-silico antimicrobial resistance (AMR) genes were identified using ResFinder v3.10. Multilocus sequence type (MLST) was identified using MLST v2.23. Genome mapping was constructed using Proksee genome analysis (https://proksee.ca/), which predicted resistance genes using a comprehensive antibiotic resistance database (CARD) and resistance gene identifier (RGI) software v6.0.3. Different virulence genes were predicted using Coding DNA Sequence (CDS). The mobile genetic elements were predicted using mobileOG-db v1.1.3, and finally, the map was filtered to show the G+C contents and GC skew of the isolate. The GC content value was determined using Bakta Web v1.11.3.

Results

Case presentation

A 66-year-old diabetic female on metformin for the treatment of type 2 diabetes mellitus presented to the hospital with a one-week history of pain and a sore on the left breast. On examination, she was pyrexial (i.e., 38-degree Celsius) and had a draining mass lesion in the left breast with the overlying necrotic skin. Debridement was performed in theatre, and a diagnosis of necrotising fasciitis was made. A pus swab sent for microbiology for evaluation isolated a pure growth of GBS that was susceptible to ampicillin/penicillin, erythromycin, clindamycin, and vancomycin, but resistant to tetracycline. Empiric treatment with amoxicillin-clavulanic acid was de-escalated to ampicillin. The outcome was favourable, and the patient was discharged after 23 days of admission. The clinical presentation in our patient correlates with what the literature describes. A rare clinical manifestation that is not well defined in a patient who is immunocompromised by diabetes and old age (Kardos et.al.2019).

Bacterial isolation and identification

Direct microscopy (Gram stain) showed Gram-positive cocci in chains, and 3 + neutrophils were observed. The pus swab was inoculated on routine agar plates. Within a day, β-hemolytic colonies appeared on the blood agar plate, and they agglutinated with the group B reagent of Streptex. VITEK®-2 automated system confirmed the identification of this strain as GBS.

Sequence analysis

The GBS isolate was fully sequenced. The resulting genome size was 2.13 Mb, encoding 2271 genes with a GC content of 47.48%. Analysis on PubMLST revealed a 100% match to S. agalactiae sequence type (ST)-17, with seven housekeeping genes/alleles encoding adhP (2), atr (1), glcK (1), glnA (2), pheS (1), sdhA (1), and tkt (1). Genome mapping constructed using Proksee software (Fig. 1) revealed that the genome consisted of tRNAs (88), rRNAs (27), tmRNA (1), Small Open Reading Frame (2227), CRISPR element (1), and repeat region (1); and 180 different mobile genetic elements were associated with the GBS isolate, including Integration/Excision (n = 58), Replication/Recombination/Repair (n = 47), Phage (n = 46), Stability/Transfer/Defense (n = 15), and Transfer (T) (n = 20). The map was filtered to show different virulence genes, resistance genes, G+C content, and GC skew of the GBS isolate and the GC content value was 35.8% (Fig. 1).

Fig. 1.

Circular genome map of Streptococcus agalactiae ST-17 from a necrotising fasciitis patient, with a genome size of 2.13 Mb, showing virulence genes (CDS) in blue and resistance genes (CARD) in red.

Antibiotic susceptibility results, resistance and virulence genes detected

The isolate was found to be susceptible to penicillin, ampicillin, erythromycin, clindamycin, moxifloxacin, linezolid, tigecycline, and vancomycin, but resistant to tetracycline. The genome sequence of the isolate harboured four antibiotic resistance-associated genes, including mreA, tet(M), mprF, and vanG cluster. Distinct virulence factors were detected in the genome sequence of the isolate, as shown in Supplementary material 1.

Discussion

Necrotising fasciitis is a severe infection of the soft tissues, mostly caused by beta-haemolytic group A Streptococcus bacterium and, in some cases, other bacteria [11]. It is classified into four categories based on the microorganisms involved and they are as follows: Type I (polymicrobial which accounts for 70–90%), Type II (monomicrobial mainly due to Group A streptococcus and Staphylococcus aureus), Type III (monomicrobial with Gram negative bacteria especially marine pathogens) and Type IV (fungal infections with Candida and zygomycetes being most common) [12]. In this case report, our patient had Type II necrotising fasciitis with a rare anatomical involvement (breast) and causative bacteria (GBS) [12].

WGS analysis revealed that this GBS isolate possessed several virulence genes (Fig. 1). MLST further identified the isolate as sequence type (ST)-17, a hypervirulent clone previously associated with neonatal infections specifically linked to meningitis, due to the production of hvgA gene [4], [13]. In Hungary, Kardos et.al found that the colonization prevalence of this hypervirulent strain to be as high as 21.9% in non-pregnant women and men [14].

The identification of ST-17 in this adult case highlights the broader clinical spectrum of this clone and underscores its capacity to cause severe disease beyond the neonatal period. In this isolate, hvgA gene was not detected. This suggests the use of other virulence factors (supplementary Table) to cause infection in adult patients. Tazi et al., [4], [13].

The antimicrobial susceptibility results showed the GBS isolate was sensitive to most antimicrobial agents, including ampicillin and penicillin, the preferred treatments for GBS infections and intrapartum prophylaxis [14]. No phenotypic resistance to erythromycin and clindamycin agents, used as alternative options for individuals allergic to penicillin, was observed [2]. However, despite being susceptible to most antibiotics, some resistance genes were detected (possibly still suppressed), suggesting possibility of this clone becoming difficult to manage in the future. Data from the ResFinder software and CARD database identified resistance genes, including mreA, tet(M), mprF, and the vanG cluster [15], [16]. The discrepancy between phenotypic and molecular findings may be related to the expression levels of these genes, their silencing or inactivation, or the bacteria's genetic background [14]. Furthermore, the presence of genotypic elements known to facilitate resistance should underscore the need to emphasise the judicious use of antibiotics so that strains do not induce/activate these underlying mechanisms of resistance.

In this case study, necrotising fasciitis caused by GBS was identified in a 66-year-old female patient with type 2 diabetes mellitus. Similar GBS-related cases have been reported, including four diabetic women in Singapore [17]. The presence of GBS in necrotising fasciitis patients in the present report and other studies suggests that the microbiological differential diagnosis of monomicrobial necrotising fasciitis should be expanded to include GBS [17].

Conclusion

This case report identified a hypervirulent GBS (ST-17) clone in an adult diabetic patient with necrotising fasciitis of the breast. The GBS isolate carried multiple virulence factors and resistance genes, highlighting its pathogenic potential. Notably, this is the first whole-genome report of hypervirulent GBS ST-17 amongst GBS isolates in South Africa. Monitoring GBS virulence and resistance genes remains valuable for guiding rational antimicrobial use.

Author contributions

All authors contributed to the revision and final approval of the manuscript. MDM and XOB provided clinical data while JYB, AMM and KMM conducted molecular analysis. JYB and MDM provided the first draft. MN, SIM and AMM supervised the study.

CRediT authorship contribution statement

Maphoshane Nchabeleng: Writing – review & editing, Supervision, Conceptualization. Musyoki Andrew M: Writing – review & editing, Resources, Investigation, Formal analysis, Conceptualization. Masemola Mmathathe: Writing – original draft, Formal analysis, Data curation. Mulaudzi Selinah: Writing – review & editing, Investigation, Formal analysis, Conceptualization. Bekebu Xanti: Writing – review & editing, Formal analysis, Data curation. John Y. Bolukaoto: Writing – review & editing, Writing – original draft, Methodology, Investigation. Malemela Kholofelo: Writing – review & editing, Investigation, Formal analysis, Data curation.

Ethical considerations

Ethical approval to conduct this study was obtained from the Research and Ethics Committee of Sefako Makgatho Health Sciences University, South Africa under an ongoing GBS study with reference number SMUREC/M/212/2025:PG.

Funding

The authors have not declared a specific grant for this research from any funding agency in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

Supplementary material