Penicillin-Binding Proteins and Associated Protein Mutations Confer Oxacillin/Cefoxitin Tolerance in Borderline Oxacillin-Resistant Staphylococcus aureus

Abstract

Among clinical isolates of Staphylococcus aureus, borderline oxacillin-resistant S. aureus (BORSA), which is mildly resistant to oxacillin (OXA) without harboring the mecA or mecC gene, is considered a risk factor for further resistance against multiple antibiotics. In this study, BORSA isolates and their derivatives were characterized through antibiotic susceptibility testing and mutation analysis of the genes encoding penicillin-binding proteins (PBPs) and their related proteins, including the promoter region. Eight BORSA isolates were confirmed to harbor the blaZ gene, and hyperproduction of blaZ-encoded penicillinase was predicted based on the minimum inhibitory concentrations (MICs). Of these, four derivative strains that were spontaneously selected based on viability on media containing high concentrations of OXA showed higher MICs than the parent isolates. The minimum bactericidal concentrations, MIC ratios, and TDtest results identified many strains with cefoxitin tolerance. Sequencing of pbp1, pbp2, pbp3, pbp4, gdpP, and yjbH, and the promoter of pbp4 revealed mutations in BORSA isolates and derivatives, despite their absence in parent isolates, suggesting that mutations in PBPs confer OXA/cefoxitin tolerance in BORSA strains.

Introduction

The widespread of methicillin-resistant Staphylococcus aureus (MRSA) has become seriously threatened, even in exclusive health care settings. Community-acquired MRSA (CA-MRSA) infections are prevalent among healthy subjects and cause a variety of staphylococcal infections, including asymptomatic infections.^1,2 Screening for MRSA has established that very low-level MRSA is often misdiagnosed as methicillin-susceptible S. aureus (MSSA).³ Although MRSA is defined as harboring mec gene homologs that encode the low-affinity penicillin-binding protein (PBP),⁴ isolates that exhibited low susceptibility to methicillin/oxacillin (OXA) without harboring any mec gene have been found in several studies.^5–8 These S. aureus isolates are broadly defined as borderline oxacillin-resistant S. aureus (BORSA), which has low susceptibility to a large range of β-lactams, especially in penicillinase-resistant penicillin. As a result, these isolates have the potential for further resistance against multiple antibiotics.^8,9 In particular, BORSA is often misidentified as CA-MRSA due to its similar distribution, the condition of the patient, and the fact that the prevalence of BORSA is at most 50% in S. aureus clinical isolates.^8–10 The most common mechanisms underlying antibiotic resistance in BORSA are hyperproduction of blaZ-encoded penicillinase (BHP), production of membrane-bound methicillinase, and amino acid substitutions in the transpeptidase domain in PBPs.^5,10 Thus, although MRSA is the primary focus, BORSA infection should be the next priority, not MSSA. Even though numerous studies regarding MRSA and its distribution are currently available, the characterization of BORSA and its potential resistance against methicillin/OXA are poorly understood.

In this study, the prevalence of BORSA in healthy young adults was determined using the palm stamp method. Subsequently, noteworthy isolates of BORSA were further analyzed for changes in antibiotic susceptibility during spontaneous exposure to an unusually high concentration of OXA. Furthermore, findings on the mutations in genes encoding PBP and related proteins were validated, and a causal relationship between antibiotic resistance, tolerance, and gene mutations was explored.

Materials and Methods

Isolation and typing of borderline OXA-resistant S. aureus

The S. aureus strains that live on palms were isolated with the palm stamp method using mannitol salt agar (MSA; Nissui Pharmaceuticals, Tokyo, Japan). Two hundred healthy young adults between 18 and 41 years of age, who were students in two different career colleges, had their palms stamped on agar, and the MSA was incubated at 37°C for 48 hr. Thereafter, growth colonies surrounded by a yellowish discoloration of the agar were picked and streaked on fresh MSA containing 6 mg/L OXA. Then, detection of the S. aureus-aminoacyltransferase-coding gene femB was used to confirm the identity of the colonies; further analysis with PCR detected mecA and mecC homologs identified in previous studies.^11–14 To distinguish the molecular types of the isolates, spa typing was performed according to the methods of Shopsin et al.¹⁵ and determined using the Ridom SpaServer.¹⁶

Antibiotic susceptibility and tolerance testing

The minimum inhibitory concentrations (MICs) were determined according to the Clinical and Laboratory Standards Institute guidelines by using a microdilution method and Etest (bioMerieux, Marcy l'Etoile, France). The MICs of ampicillin (AMP), ampicillin/sulbactam (SAM), amoxicillin (AMX), and amoxicillin/clavulanic acid (AMC) were determined using Etest according to the manufacturer's instructions. Furthermore, MICs and disk diffusion of OXA and cefoxitin (FOX) were also measured using a microdilution method and BD BBL Sensi-Disc (OX-1 and FOX-30, respectively; BD, Franklin Lakes, NJ, USA). The remaining S. aureus suspension was spotted onto trypticase soy agar (TSA; BD) and incubated at 37°C for 24 hr to determine the minimum bactericidal concentrations (MBCs) of OXA and FOX. The MBC was defined as the lowest concentration of the antibiotic at which bacterial growth could not be observed on the agar plate. For MIC and MBC determination, the initial number of bacterial cells was adjusted to 5.0 × 10⁴ CFU/mL, and the concentrations of OXA and FOX ranged from 0.0625 to 128 mg/L.

For detection of bacterial tolerance, a TDtest was conducted according to the methods of Gefen et al.¹⁷ with minor modifications reported by Kotková et al.¹⁸ In brief, 100 μL of bacterial suspension (10⁹ CFU/mL) was spread on brain heart infusion agar (BHI; BD), and a disk soaked with 10 μL of 10 mg/L FOX was placed on the center of the BHI plate. After overnight incubation, the disk was replaced with a fresh disk soaked with 20 μL of 40% glucose. The culture plate was incubated for 24 and 48 hr at 35°C. Furthermore, time–kill experiments until the 3 hr mark were performed according to the method of Gefen et al.¹⁷ with minor modifications. In brief, overnight cultures were diluted 1:100 in fresh BHI medium containing 10 mg/L of vancomycin (VAN) or 5 mg/L of FOX and incubated at 35°C for 3 hr with shaking. The bacterial survival fraction was determined by enumeration of growth cells. The antibiotic-susceptible strains of S. aureus ATCC 29213 and the methicillin-resistant strain of S. aureus ATCC 33591 were used for comparative analysis.

Identification of factors affecting β-lactam resistance and PBP-related gene sequencing

To identify the genes affecting antibiotic resistance, the draft genome of isolate B46 was determined by paired-end sequencing using an Illumina HiSeq 2500 with the TrueSeq Nano DNA LT Library Prep kit (Illumina, Inc., San Diego, CA, USA). The Edena software was used to assemble the genome, and the prediction of gene function was conducted using NCBI BLASTp in the NCBI NR and COG databases. From this analysis, the factors affecting β-lactam resistance were identified.

DNA sequencing of the genes coding for PBPs, pbp1, pbp2, pbp3, and pbp4, was performed according to the methods of Fuller et al.¹⁹ Furthermore, the promoter region in pbp4 and the genes encoding the phosphodiesterase c-di-AMP regulator (gdpP) and disulfide stress effector (yjbH) were partially sequenced according to the methods of previous studies.^5,20,21 To exclude substitutions in MSSA, these sequences were compared with those of MSSA reference strains NCTC 8325, MSSA476, and ATCC 25923.

Detection of the blaZ-encoding gene

To detect type A β-lactamase (blaZ) in isolates, blaZ-targeted PCR was performed using the following primers: primer 1, 5-TCATCACCCATAATTGCACGG-3; primer 2, 5-TGCCGTATCCAAACATGTGC-3′. The optimized PCR mixture contained 10 × PCR amplification buffer, 25 mmol/L MgCl₂, 2 mmol/L dNTP, 25 mmol/L per primer, 1.25 U Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA), and 1 μL of template DNA in a 50 μL PCR mixture. Thermal cycling was performed with a Takara PCR Thermal Cycler Dice (Takara Bio, Shiga, Japan) and consisted of initial denaturation at 95°C for 30 sec, denaturation at 95°C for 15 sec, primer annealing at 55°C for 15 sec, and extension at 72°C for 30 sec. After the initial denaturation, the reaction was performed for 30 cycles. A 180-bp amplification product was confirmed on 1.5% agarose gels stained with ethidium bromide.

Spontaneous exposure to high concentrations of antibiotics

To ascertain the viability of the isolates in high concentrations of antibiotics, a 10 μL loop of BORSA colony was picked up and linearly streaked on TSA containing a 6–30 mg/L OXA gradient concentration. This passage was repeated every 3 days; putative derivative strains that were tolerant and viable on agar with a final concentration of 30 mg/L OXA were successfully isolated from the four-parent BORSA isolates. These strains were used for comparative analysis.

Nucleic acid accession numbers

The draft genome sequence of strain B46 has been deposited in DDBJ/EMBL/GenBank under the accession numbers BLXM01000001–BLXM01000018. The version mentioned in this study is the first noncorrected version.

Results

Antibiotic susceptibility characteristics of BORSA strains

Of the 200 healthy young adults, 8 S. aureus strains that were thought to be BORSA and non-MRSA were isolated and screened based on viability on media containing 6 mg/L of OXA. Thereafter, the isolates were passaged on media containing a gradient of increasing concentrations of OXA, up to 30 mg/L. Consequently, four viable strains derived from strain no. 026, SAS, SAH, and SGS were isolated; in contrast, the remaining four isolates were not viable on media containing a high concentration of OXA. Table 1 summarizes the spa typing and presence of mecA, mecC, and blaZ genes in the strains used in this study. A prime with the parent isolate name in Table 1 indicates the derivative strain, which was streaked on media containing high concentrations of OXA, originating from the same isolate. The spa types from the Ridom SpaServer varied among the isolates, excluding SA1 and SAH that were isolated from identical classes and colleges, and 026 was revealed to be a nontypable strain based on the spa typing. Using PCR methods, all the isolates and derivatives were confirmed to harbor the blaZ gene and lack the mecA gene and its related homologs.

Table 1.

spa Typing and Presence of Gene Coding for mecA, mecC, and blaZ in Borderline Oxacillin-Resistant Staphylococcus aureus Isolates and Their Derivatives

Isolate and derivative^a	spa typing^b	Gene positive for
Isolate and derivative^a	spa typing^b	mecA	mecC	blaZ
Isolate
B46	t1767	−	−	+
B51	t002	−	−	+
A11	t251	−	−	+
SA1	t3345	−	−	+
026	NT	−	−	+
SAS	t189	−	−	+
SAH	t3345	−	−	+
SGS	t16497	−	−	+
Derivative
026′	NT	−	−	+
SAS′	t189	−	−	+
SAH′	t3345	−	−	+
SGS′	t16497	−	−	+

Same name with a prime indicates derivative clone of homonymous parent isolate.

spa types from the Ridom SpaServer are shown.

NT, nontypeable.

The MICs and disk diffusion diameters of the 12 parent isolates and derivative strains are summarized in Table 2. The MICs of AMP and AMX were much higher than those of SAM and AMC, respectively, indicating that all isolates and derivatives had putative hyperproduction of BHP. MICs of OXA and FOX ranged from 0.5 to 4.0 mg/L. Four derivative strains (026′, SAS′, SAH′, and SGS′) were tolerant and viable on agar plates containing 30 mg/L of OXA; nevertheless, MICs of OXA and FOX were one to four times higher for derivative strains than those of the parent isolates. Although the MICs of BORSA were slightly increased for OXA/FOX, the diameters of OXA and FOX disk diffusion tests showed that all the isolates and derivatives were susceptible to both antibiotics.

Table 2.

Minimum Inhibitory Concentration, Zone Diameter of Disk Diffusion, and Minimum Bactericidal Concentration in Borderline Oxacillin-Resistant Staphylococcus aureus Isolates and Their Derivatives

Isolate and derivative^a	Minimum inhibitory concentration, MIC (mg/L)^b						Zone diameter (mm)^c		Minimum bactericidal concentration, MBC (mg/L)^b
Isolate and derivative^a	OXA	FOX	AMP	SAM	AMX	AMC	OXA	FOX	OXA	FOX
Isolate
B46	2	2	12	0.5	24	0.75	15	24	4	32
B51	2	2	4	1	24	0.75	14	22	8	32
A11	2	2	4	0.5	12	0.5	21	28	4	32
SA1	2	2	8	1	32	0.5	14	22	4	32
026	2	2	8	3	24	1.5	15	22	8	64
SAS	1	1	1	0.13	2	05	20	27	8	64
SAH	2	2	4	2	8	1	14	22	16	64
SGS	0.5	0.5	4	1.5	16	1.5	19	28	4	32
Derivative
026′	2	2	16	2	24	1.5	13	22	8	64
SAS′	2	2	1	0.5	1.5	0.75	15	22	8	64
SAH′	4	4	12	4	12	2	14	22	16	64
SGS′	2	2	6	1.5	16	1.5	16	25	4	32

Same name with a prime indicates derivative clone of homonymous parent isolate.

Oxacillin (OXA), cefoxitin (FOX), ampicillin (AMP), ampicillin/sulbactam (SAM), amoxicillin (AMX), amoxicillin/clavulanic acid (AMC).

Zone diameter was determined by disk diffusion method using OXA and FOX disk.

The MBCs of OXA ranged from 4 to 16 mg/L; in contrast, the MBCs of FOX were 32 or 64 mg/L, indicating that, compared with OXA, FOX is needed at a much higher concentration for it to act as a bactericide against BORSA derivative strains rather than the parent strains (Table 2). Furthermore, in selected strains, the TDtest and time–killing experiment until the 3 hr mark showed the presence of exiguous persister cells under the bactericidal conditions, indicating that almost all isolates have tolerance for FOX (Supplementary Fig. S1).

Mutations in PBP-related genes and gene products

Table 3 shows amino acid and nucleotide substitutions in the atypical BORSA strains. Almost all mutations were unique in this study, and the isolates without any point mutations were strain 026 and SGS. In PBP2, a large insertion was found in strain B46, and its origin was mainly PBP2 of Staphylococcus hominis and Staphylococcus haemolyticus (Supplementary Fig. S2). Of these mutations, L256V in PBP2, P233L in PBP3, T104I in GdpP, and S267L in YjbH were identified in ceftobiprole-resistant and high-level β-lactam-resistant MRSA.^22,23 In a survey of BORSA, D105N in GdpP, found in strain B51, was identified in MRSA isolates.^5,21 An italicized mutation means that it is absent in parent isolates and is only found in derivative strains. In brief, in strain SGS′, mutations in R300C and Q387H in PBP4 and T144P in YjbH were not found in the parent strain. Although MBCs of strain SGS′ were identical to those of the parent strain SGS, the MICs of OXA and FOX for SGS′ were 2.0 mg/L compared with 0.5 mg/L for SGS, indicating that spontaneous exposure to a high concentration of OXA induces enhancement of resistance after these point mutations.

Table 3.

Point Mutations of Amino Acids and Nucleotides Substitution in Borderline Oxacillin-Resistant Staphylococcus aureus Isolates and Their Derivatives

Isolate and derivative^a	Amino acid or nucleotide substitution
Isolate and derivative^a	PBP1	PBP2	PBP3	PBP4	GdpP	YjbH	Upstream of pbp4 start codon
Isolate
B46	—	F130_G131ins^b	P233L	Y83H	—	—	—
B51	—	—	T117I	—	D105N	—	—
A11	V602A	R249H R717del	K345T	F356V E399Q	—	—	171 C to T 259 C to T
SA1	—	—	P509S	—	—	—	—
026	—	—	—	—	—	—	—
SAS	—	Y255S L256V	—	—	T104I A176V	S267L	—
SAH	—	S175L	F608L	—	—	T144P	—
SGS	—	—	—	—	—	—	—
Derivative
026′	—	—	—	—	—	—	—
SAS′	—	Y255S L256V	—	—	T104I A176V	S267L	—
SAH′	—	S175L	F608L	—	—	T144P	—
SGS′	—	—	—	R300C ^c Q387H ^c	—	T144P ^c	—

Same name with a prime indicates derivative clone of homonymous parent isolate.

p.F130_G131insGAIGKNLTGGFGYKRLF (sequence alignment is shown in Supplementary Fig. S2).

Italicized substitution indicates derivative clone-specific mutation that is absent in parent isolate.

Discussion

In this study, the antibiotic tolerance in BORSA was explored with regard to gene mutation. Bacterial tolerance to antibiotics is thought to lead to failure of chemotherapy and an important issue that could be resolved. Bacterial tolerance and persistence are also caused by multiple factors, including environmental triggers.²⁴ The decrease in bacterial susceptibility to antibiotics demonstrates bacterial resistance, bacterial tolerance, and persistence, implying that bacteria survive even under conditions of exposure to a higher concentration of antibiotics than the MIC without changing the parent MIC.²⁵ In contrast to bacterial tolerance, persistence is a heterogeneous response against antibiotics to form clonal persistent and nonpersistent subpopulations.²⁶ Although this phenomenon is generally not heritable, the heterogeneous response against antimicrobials is repeated as the surviving subpopulation is grown in the same antibiotics.²⁷ When spontaneously exposed to high concentrations of OXA, derivative strain was found to be mutated in the pbp4 and yjbH genes, indicating that spontaneous mutation induces either bacterial tolerance or persistence. Although large sequence that was partially identical to PBP2 of S. hominis and S. haemolyticus was found in strain B46, this sequence was found in only a few strains of S. aureus. The relationship between this insertion and antibiotics susceptibility needs be clarified.

Furthermore, a low growth rate and reduced cellular metabolism are reportedly involved in bacterial tolerance.^27–29 In particular, deletion of the gene encoding the Clp proteolytic system directly leads to a reduction in the stress response, growth rate, and antibiotic susceptibility.^30,31 Changes in growth rate and phase are known to increase antibiotic tolerance, and mutation of tricarboxylic acid (TCA) cycle-related genes in S. aureus causes a slow growth rate and persister cells under antibiotic conditions.^32,33 As one of the candidate mechanisms involves the TCA cycle, incomplete cellular process due to low levels of ATP may cause evasion of bactericidal action.

Mutations in the sequenced genes could not be identified in three of the four derivative strains, even though their MICs were greater than the MICs of the parent isolates. The ATP-binding cassette (ABC) transporter family protein, AbcA, releases energy through ATP hydrolysis; its encoding gene abcA cooperates with pbp4 and has been reported to contribute to resistance to β-lactams.^34,35 Furthermore, genomic analysis of mecA-negative S. aureus with high β-lactam resistance revealed mutations in the cation multidrug efflux transporters, AcrB, PBP4, and GdpP.⁶ These results indicate that mutations in upstream and downstream regulator genes of PBPs may alter susceptibility to β-lactams, and the role of these causative genes should be clarified in future studies. In this study, all the isolates were predicted as BHP based on the MIC ratio of AMP and AMX to SAM and AMC, respectively.⁵ Although protein expression analysis was conducted to attempt clarification of BHP traits, this experiment could not detect defined hyperproduction of β-lactamase. The experimental detection and identification of hyperproduction of β-lactamase in BHP may need be developed for better understanding characteristics of BHP.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Joint Research Program of Juntendo University, Faculty of Health and Sports Science. This study was also supported, in part, by JSPS KAKENHI, Grant Nos. 16K07095 and 20K08846.

Supplementary Material

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