The Recombinant Bacteriophage Endolysin HY-133 Exhibits In Vitro Activity against Different African Clonal Lineages of the Staphylococcus aureus Complex, Including Staphylococcus schweitzeri

Abstract

HY-133 is a recombinant bacteriophage endolysin with bactericidal activity against Staphylococcus aureus. Here, HY-133 showed in vitro activity against major African methicillin-susceptible and methicillin-resistant S. aureus lineages and ceftaroline/ceftobiprole- and borderline oxacillin-resistant isolates. HY-133 was also active against Staphylococcus schweitzeri, a recently described species of the S. aureus complex. The activity of HY-133 on the tested isolates (MIC₅₀, 0.25 μg/ml; MIC₉₀, 0.5 μg/ml; range, 0.125 to 0.5 μg/ml) was independent of the species and strain background or antibiotic resistance.

TEXT

Bacteriophage endolysins are peptidoglycan hydrolases with strong species-specific bactericidal activity (1). Endolysins are considered a potential new class of antimicrobial agents which could be used both for decolonization of methicillin-resistant Staphylococcus aureus (MRSA) and invasive infections, as animal models have revealed (1). Indeed, there is a need for the development of new antimicrobial agents, in particular against S. aureus infections, as resistance rates are rising not only in industrialized countries but also in so-called developing regions, such as sub-Saharan Africa (2).

Similar to its progenitor PRF-119 (3), HY-133 (owned by Hyglos GmbH, Bernried, Germany) is an optimized, recombinantly produced chimeric bacteriophage endolysin which has a pronounced bactericidal activity against S. aureus and an increased proteolytic stability due to a shortened linker peptide (4). These chimeric endolysins consist of an enzymatic active domain (EAD), a cysteine- and histidine-dependent aminopeptidase/hydrolase (CHAP) from the endolysin of phage K, and a cell wall-binding domain (CBD) from lysostaphin (3, 4).

While the progenitor molecule PRF-119 demonstrated activity against all clinical S. aureus isolates collected from multiple centers in a European country (3), the present study aimed at challenging the optimized bacteriophage endolysin HY-133 with isolates belonging to the S. aureus complex from sub-Sahara Africa, including the recently described species Staphylococcus schweitzeri (5, 6). This species shows a peptidoglycan type which is distinct from that of S. aureus sensu stricto (5).

Here, we tested the in vitro activity of HY-133 against a highly diverse collection of African S. aureus complex lineages (n = 61). This collection consists of major African methicillin-susceptible S. aureus (MSSA), as well as methicillin-, ceftaroline/ceftobiprole-, and borderline oxacillin-resistant S. aureus from humans (colonization and infection) and animals (colonization) (Table 1). The most common African MSSA and MRSA included in this study represent lineages which have been found to be predominant in sub-Saharan Africa based on a recent review (7). We also included 25 isolates from different clonal lineages of S. schweitzeri. The multilocus sequence types (ST) of the isolates included in this study are given in Table 1. The activity of HY-133 was determined using the broth microdilution method in accordance with ISO 20776-1 guidance. The testing was performed in triplicate, and the median MIC was taken for analysis. MIC₅₀, MIC₉₀, and MIC ranges were separately calculated for the different groups of isolates (Table 1). S. aureus ATCC 29213 was used as a quality control (QC) strain. While no official QC ranges are available for HY-133, MICs for the QC strain were within the in-house-established limits throughout the testing.

TABLE 1.

In vitro activity of the bacteriophage endolysin HY-133 against a highly diverse collection of African isolates belonging to the Staphylococcus aureus complex^c

Group (no. of isolates)	Colonization/infection (no. of isolates)	MLST sequence types	Country(ies)	MIC (μg/ml)			Reference(s)
				50%	90%	Range
Most common African MSSA isolates (12)	6/6	ST15, ST30, ST121, ST152	Gabon, Nigeria	0.25	0.5	0.25–0.5	9, 10
Most common African MRSA isolates (19)a	7/12	ST5, ST8, ST88, ST241	Côte d'Ivoire, DR Congo, Gabon, Nigeria	0.25	0.5	0.25–0.5	6, 9, 11–13
Staphylococcus schweitzeri (25)	25/0	ST1822, ST1857, ST1872, ST1873, ST1874, ST2022, ST2071, ST2067, ST2295, ST2074, ST2295, ST2296, ST2474, ST2475, ST2476, ST2477, ST2478, ST2946	Côte d'Ivoire, DR Congo, Gabon	0.25	0.25	0.25–0.5	6, 11
Othersb (5)	5/0	ST2265, ST2353	DR Congo, Gabon	NA	NA	NA	6
Ceftobiprole/ceftaroline-resistant MRSA (11)	0/11	ST241, ST15	Nigeria	0.5	0.5	0.25–0.5	9, 14
Total				0.25	0.50	0.125–0.5

^aIncluding ceftobiprole/ceftaroline-resistant MRSA isolates.

^bComprising borderline oxacillin-resistant S. aureus (BORSA) isolates from a remote Pygmy population (n = 4) and a highly divergent S. aureus strain (ST2353; n = 1). The calculation of MIC₅₀ and MIC₉₀ was not feasible due to the small sample size. All isolates showed a MIC of 0.25 μg/ml.

^cDR Congo, Democratic Republic of the Congo. NA, not applicable.

HY-133 was active against all isolates tested. The MIC ranged between 0.125 μg/ml and 0.5 μg/ml, independent of the clonal lineages or the antimicrobial resistances. As our strain collection covers different lineages and resistance phenotypes from sub-Saharan Africa, there is good evidence that HY-133 has a bactericidal activity against African S. aureus similar to that demonstrated for the progenitor PRF-119 against S. aureus isolates from Europe (3).

It is noteworthy that HY-133 was also active against the novel staphylococcal species S. schweitzeri, which belongs to the S. aureus complex. The HY-133 MIC₅₀ and MIC₉₀ for S. schweitzeri were even one doubling dilution lower than those for ceftaroline/ceftobiprole-resistant MRSA. Binding of lysostaphin depends on the length and composition of peptidoglycan interpeptide bridges (8). For this reason, the high activity of HY-133 against S. schweitzeri is worth mentioning, as the peptidoglycan [A3α type, A11.8 type; l-Lys–L-Ala–(Gly)4-5] is distinct from that of S. aureus [A3α type, A11.2 type; l-Lys–(Gly)4-5] (5). This indicates that an l-Ala residue in the interpeptide bridge does not interfere with lysostaphin binding and suggests that the species-specific action of HY-133 also includes other species within the S. aureus complex.

In conclusion, our study demonstrates that the bacteriophage endolysin HY-133 is universally active against S. aureus complex members irrespective of the strains' genetic background and resistance to antibiotics.

Funding Statement

The study was supported in part by the BMBF-DZIF (German Center for Infection Research, TTU 08.807).