A case of penicillin-resistant group B Streptococcus isolated from a patient in the UK

Abstract

Objectives

In England, group B streptococci (GBS; Streptococcus agalactiae) are considered universally susceptible to penicillin. Reports from Africa, Asia, North America and a few European countries have described GBS isolates with penicillin MICs above the epidemiological cut-off (0.125 mg/L). Our aim was to characterize a penicillin-resistant GBS (PRGBS) isolate recovered in 2016 from a patient treated with long-term antimicrobials in the UK.

Methods

Antibiotic susceptibility of a referred isolate from a discharging sinus overlying a chronic prosthetic joint infection was determined using gradient strip testing for seven antibiotics. Illumina short read sequencing was carried out using a HiSeq 2500 platform to determine MLST, capsular type, to detect mutations in the pbp genes, and to compare the isolate with contemporaneous GBS isolates circulating in the UK.

Results

The GBS isolate belonged to capsular type Ia and MLST 144. We observed resistance to penicillin (MIC = 1 mg/L) and tetracycline (32 mg/L) with susceptibility to linezolid (1 mg/L), erythromycin (0.064 mg/L), clindamycin (0.064 mg/L), teicoplanin (0.064 mg/L) and vancomycin (0.25 mg/L). Deduced amino acid sequences revealed substitutions and non-synonymous changes in PBP2x and PBP2b. Genomic analysis of contemporaneous cases (n = 34) from across the UK identified single nucleotide polymorphism (SNP) variation ranged from 153–6596 SNPs.

Conclusions

We confirm the first identification of a PRGBS isolate amongst referrals to the UK’s national reference laboratory. Substitutions in pbp1a, pbp2a, pbp2x and pbp2b were identified that likely developed in the face of long-term beta-lactam antibiotic use.

Introduction

Streptococcus agalactiae (group B Streptococcus, GBS) is widely recognized as a leading cause of neonatal sepsis¹ and when a mother is known to be vaginally colonized with GBS, intra-partum antibiotic prophylaxis (IAP) is given during labour to prevent GBS disease in her infant by vertical transmission. GBS is also increasingly recovered from adults with invasive disease, particularly in older people or those with comorbidities.² It is a well-described cause of osteoarticular infection including prosthetic joint infection.³ Penicillin is the first line antibiotic for both IAP and treatment of GBS infection.

EUCAST have determined the epidemiological cut-off (ECOFF) for penicillin in GBS to be 0.125 mg/L. Isolates with MICs above this threshold are considered to have acquired resistance mechanisms. There is growing international literature reporting the isolation of clinical GBS strains that have MICs above the ECOFF (Table 1). In 2008 in Japan,⁴ researchers reported the first molecular characterization of such isolates, demonstrating point mutations in pbp genes (encoding PBP2x, PBP1a, PBP2a and PBP2b), which have subsequently been shown to encode for proteins with a low-affinity for β-lactam binding. Further reports from Japan,^5,8–10 and from Canada,^6,7,15 Korea,¹¹ USA,^13,14 Germany¹² and Sweden,¹⁶ have also identified penicillin-resistant GBS (PRGBS) with mutations in pbp genes (Table 1). The aim of this work was to characterize a PRGBS isolate recovered from a UK patient with a chronic prosthetic joint infection in 2016, by determining antibiotic susceptibility, identifying potential genetic mechanism(s) of resistance and describing the isolate relatedness to other English GBS isolates.

Table 1.

Microbiological and genomic characteristics of our isolate compared to international published reports of isolates with non-wildtype or resistance to penicillin^a amongst Group B Streptococcus isolates

Study (author year)	Country (study period)	Patient/sample	PR-GBS isolates reported (or n/N tested, %)	Serotype	Penicillin MIC	Mutations identified (those identified in PRGBS only are in bold)
						PBP2x	PBP2a	PBP2b	PBP1a
Our findings	UK (2016)	Pus from prosthetic joint infection	1 isolate	Ia	1.0 (n = 1)	I342V V475I P160S Y331H P140L N540D	A27T N741A V744A V541I	V80A	K63E
Kimura 20084	Japan (1995–2005)	Sputum samples from elderly patients	14 isolates	Ib (n = 1) III (n = 8) VI (n = 4) VIII (n = 1)	0.25 (n = 8) 0.5 (n = 5) 1.0 (n = 1)	Q557E V405A	ND	ND	ND
Nagano 20085	Japan (2003–04)	Invasive infections (adults)	8 isolates	Ia (n = 1) Ib (n = 1) VI (n = 5) N-typ (n = 1)	0.25 (n = 1) 0.5 (n = 6) 1.0 (n = 2)	S353F, A374V, F395L, V405A, A400V, R433H, H438Y, A514V, Q557E, G648A, T77I	E63K, T175I, L285F, Y236C	V80A, Y262N, G539E, T567I, G613R	L45P, N163K, Y470F, G527V, N723S
Gaudreau 20106	Canada (2004–07)	Invasive infection (adults)	1 isolate	NR	0.25 (n = 1)	I377V, G627V, N575D	S453N, N682D	V625I, P278L	T526A
Longtin 20117	Canada (2008)	Invasive infection (adults)	1 isolate	II (n = 1)	0.5 (n = 1)	G371D	E636G, S644F, S676F	ND	T546P
Nagano 20128	Japan (2007)	Invasive infections (adults)	10 isolates	VI (n = 10)	0.25 (n = 10)	V405A, F395L, R433H, H438Y, G648A I377V, V510I	NT	T567I	ND
Seki 20159	Japan (2012–13)	Invasive infections	45/306 (14.7%)	NR	0.25 (n = 19) 0.5 (n = 20) 1.0 (n = 6)	V405A, Q557E	NT	NT	NT
Morozumi 201610	Japan (2010–13)	Invasive infections (adults)	9/443 (2.0%)	Ia (n = 2) Ib (n = 1) III (n = 6)	0.125 (n = 5) 0.25 (n = 3) 0.5 (n = 1)	K372E, I377V, G398A, V405A, Q412L, G429D, H438Y, D478A, E513Q, Q557E	NT	NT	NT
Yi 201911	South Korea (NR)	Invasive infections (adults)	2 isolates	NR	0.5 (n = 2)	G398A, V405A, Q557E	NT	NT	NT
Van der Linden 202012	Germany (NR)	Invasive infections (adults)	2 isolates	Ia (n = 2)	0.5 (n = 1) 1.0 (n = 1)	I377V, F395L, V405A, H438Y, V510I, Q557E	ND	V80A	A521V, del719–722, N723S, V726A, T526I
Metcalf 201713	USA (2015)	Invasive infections	6 isolates	II (n = 1) V (n = 1) III (n = 4)	0.25 (n = 6)	1377V G406D Q557E	NT	NR	NR
McGee 202114	USA (2015–17)	Invasive infections	6 isolates	Ia (n = 1) II (n = 1) III (n = 2) V (n = 1)	0.25 (n = 6)	1377V G406D G398A G627V V5101 I510V Q557E L534S G627V	NT	NT	NT

PR-GBS, penicillin-resistant group B Streptococcus; ND, not detected; NR, not reported; NT, not tested; N-typ, non-typable.

^aDefined as penicillin MIC above the EUCAST epidemiological cut-off (ECOFF) of 0.125 mg/L.

Materials and methods

Bacterial culture and antibiotic susceptibility testing

Bacterial swabs collected from a chronic discharging sinus overlying a prosthetic joint infection were cultured on Columbia Blood Agar overnight at 37°C in air supplemented with 5% CO₂. Local genus and species confirmation was carried out using a streptococcal grouping latex kit (Prolex, Prolab Diagnostics, UK) and MALDI-ToF mass spectroscopy (Bruker, Bremen, Germany). Antibiotic susceptibilities were determined by disc diffusion and interpreted in accordance with EUCAST guidelines (http://www.eucast.org).

At the UKHSA (formerly PHE) Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, MICs for penicillin, erythromycin, clindamycin, tetracycline, teicoplanin, vancomycin and linezolid were determined by gradient strip testing (Liofilchem S.r.l., Roseto degli Abruzzi, Italy and bioMérieux, France) using ISO-Sensitest agar supplemented with 5% whole horse blood (Oxoid Ltd, Basingstoke, UK) and interpreted using EUCAST guidelines (http://www.eucast.org; v. 6.0).

Genomic sequencing

WGS was undertaken at UKHSA, on the PRGBS isolate and compared with WGS data from 34 capsular type Ia contemporaneous sporadic isolates (selected from the same region as the PRGBS isolate to determine any genomic relationship with isolates of the same serotype) recovered in England between 2014 and 2020, the GenBank reference GBS type Ia isolate CP000114.1 (PUBMED: 16172379) and two published PRGBS isolates ERR3464559 and ERR3464558¹² (to compare resistance-conferring mutations). Sequencing was carried out using a HiSeq 2500 platform (Illumina; San Diego, CA, USA). Nucleotides with a Phred score less than Q30 at the ends of the reads were removed with Trimmomatic.¹⁷ SNPs were determined using the PHEnix pipeline (GitHub. phe-bioinformatics/PHEnix. San Francisco: GitHub; available from: https://github.com/phe-bioinformatics/PHEnix). Reads were mapped to reference strain CP000114.1 using bwa (version 0.7.12). Variants were called using Genome Analysis Toolkit (GATK) 2.6.5 and parsed to retain high-quality SNPs (conditions: depth of coverage ≥ 10, AD ratio (ratio between variant base and alternative bases) ≥ 0.9, Mapping Quality ≥ 040, ratio of reads with MQ0 to total number of reads ≤ 0.1). Positions that fulfilled the filtering criteria in >0.9 of the samples were joined to produce a multiple FASTA format file where the sequence for each strain consists of the concatenated variants. The FASTQ files for the isolates described in this study were submitted to the European Nucleotide Archive (ENA) study number ID PRJEB56528. GUBBINS (Genealogies Unbiased By recomBinations In Nucleotide Sequences version 2.0.0) was used to remove SNPs from suspected regions of recombination (high SNP density), and distances were calculated using the reduced FASTA, creating a pairwise distance matrix with SNP-dists (GitHub. Tseemann/snp-dists. Available from: https://github.com/tseemann/snp-dists). MLST profile was determined by the mapping tool MOST.¹⁸ Mutations in pbp1a, pbp2a, pbp2x and pbp2b were identified using GeneFinder, an in-house software where sequenced reads are mapped to reference sequences with bowtie2 and mpileup file generated with SAMtools version 0.1.18.¹⁹

Results

Case details

In January 2016, a 47-year-old woman presented with a chronic infection at the site of distal femoral replacement. Forty years previously, she had undergone total femoral replacement following a diagnosis of osteosarcoma. In the 10 years preceding presentation, she had undergone multiple revisions for peri-prosthetic fracture and prosthetic bone and joint infection. All of her surgery had taken place in the UK. Since 2007, multiple chronic discharging sinuses had developed over the tibia and distal femur, however she remained well in herself. She declined further surgery, which would have involved amputation or hip disarticulation and little chance of eradicating the infection, preferring to stay on long-term antibiotic suppressive therapy. This consisted of doxycycline and trimethoprim for 7 years, then doxycycline and penicillin for 2 years, followed by clindamycin. Swabs of pus from the sinus had previously grown GBS, MSSA and Staphylococcus pseudintermedius. By 2016, these organisms were doxycycline and clindamycin resistant, and she was not able to tolerate trimethoprim. Clindamycin was therefore changed to co-amoxiclav in January 2016, and a swab was taken to guide further suppressive therapy. PRGBS was isolated on this single occasion and has not been identified since. As there were limited alternative oral antibiotic options, a decision was made to continue co-amoxiclav, which she has remained on ever since. At her most recent clinic visit in February 2024, she had ongoing copious sinus discharge and had developed ipsilateral inguinal lymphadenopathy but remained systemically well.

Bacteriology

The GBS isolate was found to have a reduced penicillin disc diffusion zone at the local microbiology laboratory and was sent to the UKHSA AMRHAI Reference Unit for further testing. The isolate was confirmed as Lancefield group B Streptococcus serotype Ia by agglutination with specific antisera and was resistant to penicillin (MIC = 1 mg/L) and tetracycline (32 mg/L) but susceptible to linezolid (1 mg/L), erythromycin (0.064 mg/L), clindamycin (0.064 mg/L), teicoplanin (0.064 mg/L) and vancomycin (0.25 mg/L).

Whole genome sequencing

The PRGBS isolate was identified as capsular type Ia and MLST 144 (belonging to CC23) by WGS analysis. SNP variation ranged from 153–6596 SNPs when the sequence of this isolate was compared with contemporaneous UK GBS sequences, suggesting that this was not closely related to the serotype Ia isolates from the same region. Genomic analysis of the current English PRGBS isolate and two published Japanese PRGBS isolates found differences in pbp1a, pbp2a, pbp2b and pbp2x genes in comparison with the sequence from penicillin-susceptible reference strain CP000114.1 genes (Tables S1 and S2, available as Supplementary data at JAC Online, respectively), revealing amino acid substitutions in pbp1a, pbp2a, pbp2x and pbp2b. We observed in total 55 SNPs, which include 18 non-synonymous changes between the penicillin binding protein genes from the three penicillin-resistant isolates and the reference CP000114.1. In pbp2x, 15 synonymous mutations were observed, as well as four new non-synonymous mutations in the English isolate (P160S, Y331H, P410L, N540D), four mutations unique to either published PRGBS isolates (F360L, V370A, H403Y, Q522E) and two mutations conserved across the English isolate and the published PRGBS isolates (I343V, V374I) (Table S1). Seven synonymous changes were identified in pbp2b compared to the penicillin-susceptible reference strain, and one non-synonymous mutation was identified in all PRGBS sequences (V80A) (Table S2). For pbp1a, nine synonymous mutations were observed, as well as one non-synonymous mutation that was conserved across the English isolate and the published PRGBS isolates (K63E) (Table S3). In pbp2a, eight synonymous mutations were observed, as well as one new non-synonymous mutation in the English isolate (V541I), two non-synonymous mutation in ERR3464558A (V541A, T546I) and four non-synonymous mutations that were conserved across the English isolate and the published PRGBS isolates (A27T, N741A, V744A) (Table S4).

Discussion

This is the first report of a penicillin-resistant group B Streptococcus (PRGBS) isolate amongst referrals to the national reference laboratory in England, UK. This may represent in-host evolution of resistance in the face of exposure to prolonged antibiotic use. Ongoing surveillance of routine laboratory surveillance data in England has recorded no additional confirmed instances of PRGBS.²⁰

PRGBS isolates with MICs above the ECOFF have been reported sporadically around the world since 1994; however, reports from European countries have been less common.¹⁶ Several previous reports describe patients with similar case histories: individuals with chronic prosthetic joint infections who had failed surgical attempts to eradicate infection and had received long-term suppressive antibiotic therapy.^6,7,12 This highlights the risk of point mutation accumulation with long-term antibiotic selection pressure.

Several previous publications have noted amino acid changes in the pbp2x genes V405A and Q557E.^4,5,9–12 These mutations have been considered key in the development of PRGBS due to their proximity to the conserved transpeptidase domain active site.^4,9,11,12 However, the Q557E mutation has been reported in penicillin-susceptible GBS with elevated MICs (0.125 mg/L), and it has been suggested that it is a common first step in the development of resistance through the additive accumulation of subsequent pbp2x mutations.²¹ However, V405A and Q557E mutations were not observed in the isolate described here. Two non-synonymous pbp2x mutations that were conserved across all PRGBS isolates (I343V and V374I) and four new non-synonymous pbp2x mutations were identified (P160S, Y331H, P410L, N540D). To our knowledge, these mutations have not previously been reported in PRGBS isolates. We identified one pbp2b mutation (V80) that has previously been described.^5,12 It is likely that the mechanism of resistance in this isolate is a stepwise accumulation of mutations in PBP1a, PBP2a, PBP2b and PBP2x.^4–11 However, further work is required to determine the significance of mutations identified in genes other than pbp2x in terms of the development of penicillin resistance. Limitations of this work include that growth characteristics and MLST types were not compared.

The clinical significance of this PRGBS isolate is uncertain, and no further PRGBS isolates were recovered from this patient despite continued selection pressure from long-term beta-lactam use. However, the impact of antimicrobial resistance is clear from this case in which development of resistance to tetracyclines in the GBS isolate, in conjunction with resistance to macrolides and lincosamides in the other organisms implicated in her infection left her with few options for effective treatment. Resistance to these antibiotic classes, which are often considered second-line agents in GBS therapy, is increasing,²⁰ and commonly reported in conjunction with PRGBS.^8,9,22 Repeated sampling of sites of chronic infection should be considered for patients taking long-term suppressive antibiotics to monitor any development of antibiotic resistance. In addition, penicillin remains the first line drug of choice for intra-partum antibiotics used in the prevention of maternal and neonatal invasive GBS disease.²³ There may be considerable selection pressure from IAP, particularly in countries with universal screening for GBS such as the USA where 30% of labouring women receive antibiotics in labour.²⁴ While PRGBS have not been identified in surveillance studies of neonatal invasive GBS isolates in the USA and UK, continued surveillance of penicillin-susceptibility in GBS isolates is essential to guide therapeutic and preventative measures.^25–27 The importance of ongoing surveillance of penicillin-susceptibility in GBS isolates is highlighted by the establishment of PRGBS in the WHO Bacterial Priority Pathogens List, 2024.²⁸ GBS is also a common pathogen in animals, and a One Health approach to antimicrobial stewardship is crucial in stemming the tide of the development of antimicrobial resistance.^29,30 Resistance to penicillin in GBS is classed as an exceptional phenotype by EUCAST; non-wildtype isolates (penicillin MIC ≥ 0.25 mg/L) should be referred to the AMRHAI Reference Unit for confirmation and further characterization.

Ethics

Informed patient consent to publish these data was obtained by clinical team.

Funding

This work was supported by internal UKHSA funding and data generated as part of the routine work of the AMRHAI Reference Unit, UKHSA.

Transparency declarations

None to declare.

Supplementary data

Tables S1–S4 are available as Supplementary data at JAC Online.