1 Phenotypic and genomic characteristics of oxacillin- and penicillin–clavulanate-susceptible

2 mecA-positive Staphylococcus aureus

3 Vladimir Gostev1,2, Ksenia Ivanova1, Julia Sopova3,4, Olga Kalinogorskaya1, Ofeliia Sulian1, Polina
4 Chulkova1, Maria Velizhanina3,5, Elena Nesterova6, Natalia Trophimova6, Elvira Martens1,2,
5 Sergey Sidorenko1,2
1
6 Pediatric Research and Clinical Center for Infectious Diseases, Saint Petersburg, Russian
7 Federation
2
8 North-Western State Medical University Named After I. I. Mechnikov, Saint Petersburg,
9 Russian Federation
3
10 St. Petersburg State University, Saint Petersburg, Russian Federation
4
11 Vavilov Institute of General Genetics, Saint Petersburg, Russian Federation
5
12 All-Russia Research Institute for Agricultural Microbiology, Saint Petersburg, Russian
13 Federation
6
14 City Skin and Venereal Diseases Dispensary, Saint Petersburg, Russian Federation

15 Abstract

16 Staphylococcus aureus harboring mecA but susceptible to oxacillin (OS-MRSA) is

17 associated with false susceptibility to beta-lactams, even when using cefoxitin-based tests.
18 Here, we characterized 60 isolates of mecA-positive oxacillin- and penicillin–clavulanate-
19 susceptible (MIC < 2 mg/L) S. aureus. OS-MRSA was more prevalent among CA-MRSA (205/49,
20 24%) than among HA-MRSA (575/11, 2%). OS-MRSA isolates belonged to twelve sequence
21 types (ST) with a predominance of ST22-t223-SCCmec-IVc and ST59-t1950-SCCmec-V lineages.
22 OS-MRSA was characterized by mecA promoter mutations at −33 (C→T) or −7 (G→T/A) along
23 with PBP2a substitutions (S225R or E246G). The basal level expression of mecA under oxacillin
24 induction was low compared with control ST8-HA-MRSA isolates. The population analysis
25 profile showed that most OS-MRSA isolates were characterized by oxacillin heteroresistance.
26 Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest
27 positive predictive values (PPV > 0.95) were observed for broth microdilution, VITEK® 2
28 automatic system, and chromogenic media. The OS-MRSA phenotype may be the reason for
29 the mis-prescription of beta-lactams for the treatment of staphylococcal infections.

30 Key Words: Staphylococcus aureus, oxacillin-susceptible mecA-positive Staphylococcus aureus

31 (OS-MRSA), penicillin–clavulanate, oxacillin, cefoxitin, antibiotic susceptibility, promoter
32 mutations

33 Introduction

34 Staphylococcus aureus harboring mecA but susceptible to oxacillin (oxacillin-susceptible

35 methicillin-resistant S. aureus, OS-MRSA) is recognized as a difficult-to-detect pathogen (1). The
36 main concern is reporting false susceptibility to beta-lactams, even when using cefoxitin-based
37 tests, which could result in inappropriate antibiotic prescribing (2). Since the initial description
38 in the early 2000s (3, 4), OS-MRSA isolates have been reported in geographically distinct
39 regions: the USA (5, 6), Brazil (7), the UK (8, 9), Europe (10, 11), Africa (12), Iran (13), India (14),
40 and China (15-17). Recently, OS-MRSA was isolated from pets (18), other animal species (17),
41 and food (19, 20). The population of OS-MRSA is genetically diverse and is represented by
42 different sequence types (ST)—ST1, ST2, ST8, ST59, ST121, ST89, ST22, ST45, and ST30. These
43 features were more strongly associated with methicillin-susceptible S. aureus (MSSA) than with
44 MRSA. It has been suggested that OS-MRSA may represent a stage of phenotypic evolution of
45 MRSA from «pre-MRSA» (21). Mutations in the promoter region of mecA play a crucial role in
46 susceptibility to oxacillin (22, 23). Mutations in blaR1-blaI of the bla operon (24, 25), acquisition
47 of stop codons, and frameshift mutations in mecA have also been described in mecA-positive
48 oxacillin-susceptible isolates (5). Control of mecA expression is dependent on many auxiliary
49 factors that determine methicillin resistance in the core genome of S. aureus (26). Unexpected
50 susceptibility to penicillin–clavulanate combination was observed in mecC- and mecA-positive
51 isolates in the studies of Ba et al. (27) and Harrison et al. (22), respectively. This unusual
52 phenotype results from simultaneous mutations in mecA and its promoter. Treatment with
53 penicillin–clavulanate in a staphylococcal wax moth larvae infection model has shown
54 promising results (22). Several studies in the 1980s discussed the possibility of using this
55 combination to treat MRSA infection (28). However, in vitro, beta-lactams can induce high
56 levels of resistance in OS-MRSA (23, 29). Here, we describe diverse OS-MRSA isolates, collected
57 in Russia during different time periods, that demonstrate susceptibility to penicillin–clavulanate
58 and are characterized by different mutational patterns.

59 Material and Methods

60 Ethics approval and consent to publication

61 Not required. Only bacterial isolates were subjected for the study. MRSA isolates were
62 collected in participating centers together with record forms. Personal data of patients were
63 not included in record forms.

64 Bacterial isolates

65 Hospital-associated (HA)-MRSA (n=575), isolated from hospitalized patients with nosocomial

66 staphylococcal infections, and community-associated (CA)-MRSA (n=205) were included in the
67 study; CA-MRSA was isolated from healthy adults and children during routine screening for
68 staphylococcal carriage between 2011 and 2019 in 11 cities and 30 medical centers of the
69 Russian Federation. In addition, 21 OS-MRSA isolates from a previous study were included (30).
70 Five HA-MRSA isolates belonging to ST8-t008-SCCmec IVc were used as control strains for the
71 broth microdilution (BMD) tests, population analysis profile (PAP), and mecA expression assays.

72 OS-MRSA detection

73 All isolates were tested for the presence of mecA using PCR. Susceptibility to cefoxitin and
74 oxacillin was evaluated using several approaches: the disc-diffusion method (DDM) with
75 cefoxitin (30 μg) and oxacillin (1 μg) disks (Bio-Rad); the gradient diffusion method (GDM;
 76 ETEST®, bioMérieux); the VITEK® 2 compact system with AST-GP67 cards (bioMérieux); and a
77 chromogenic medium (CM, Brilliance MRSA 2 Agar®; Oxoid, UK). In addition, Mueller–Hinton
78 agar (Bio-Rad) supplemented with 2% of NaCl was used for the DDM and GDM tests. The
79 minimal inhibitory concentrations (MICs) of beta-lactams and other antibiotics (Molekula, UK)
80 were determined using the BMD method, according to ISO 20776-1-2010, and interpreted
81 according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)
82 recommendations; for the oxacillin DDM, a breakpoint of R ≤ 10 mm was used. OS-MRSA was
83 defined as mecA-positive with susceptibility to oxacillin using the BMD method.

84 Population analysis profile with oxacillin

85 PAP was performed according to the microdilution modification described in a previous study
86 (31). Four dilutions of the initial suspension (108 CFU/mL) for each strain were prepared. Three
87 10-µL droplets of each dilution were plated onto oxacillin-containing Brain-Heart Infusion (BHI)
88 agar plates (0.06–32 mg/L). The inoculated plates were incubated for 48 h at 37 °C. Plated
89 droplets containing 5–50 CFUs were selected for counting, and the average number of colonies
90 per oxacillin concentration was determined. Plots showing the number of CFUs per oxacillin
91 concentration were prepared by calculating the area under the curve (AUC).

92 Whole genome sequencing

93 A Nextera Flex Kit (Illumina, San Diego, CA) was used for DNA library preparation, followed by
94 sample indexing and amplification, according to the manufacturer’s protocol. The DNA libraries
95 were sequenced using MiSeq (Illumina, San Diego, CA). Genomic data have been deposited in
96 the NCBI Sequence Read Archive (SRA), under BioProject PRJNA872007.

97 mecA expression assay

98 mecA expression was analyzed using RT-PCR in BHI broth (Bio-Rad) and induction was achieved
99 by addition of 0.016 mg/L of oxacillin. Samples were collected at the late-exponential phase of
100 growth and incubated with RNAprotect reagent (Qiagen, Germany). Prior to RNA extraction,
101 bacterial cells were mechanically disrupted in a cool environment (4 °C) for 30 min using glass
102 beads (Sigma-Aldrich, USA). Total RNA was extracted and purified using the PureLink™ RNA
103 Mini Kit (ThermoFisher Scientific, USA). DNase treatment and reverse transcription were
104 performed using a cDNA Synthesis Kit (K1681; ThermoFisher Scientific, USA). The probes mecA
105 (target) and nuc, which codes the thermonuclease protein (reference internal control), labeled
106 with FAM and ROX dyes, respectively, were used in a single multiplex PCR (probes and primer
107 sequences are presented in Supplementary Table S1). Eight OS-MRSA isolates and one control
108 (HA-MRSA-ST8) were included in the experiment. Measurements were performed in triplicate.
109 Expression fold-changes were calculated using the 2-∆∆Ct method (32).

110 Bioinformatics and statistical analysis

111 The reads were filtered and trimmed using Trimmomatic (33). De novo contigs were assembled
112 using SPAdes (34). Phylogenetic analysis, molecular typing, and single nucleotide
113 polymorphisms (SNP) calling were performed with Snippy
114 (https://github.com/tseemann/snippy), mlst (https://github.com/tseemann/mlst), and
115 ABRicate (https://github.com/tseemann/abricate) and bowtie2 (35), respectively. Statistical
116 analyses were performed in R using the Mann–Whitney U test and Spearman’s correlation
117 analysis with the corrplot package (https://cran.r-project.org/web/packages/corrplot).

118 Results and Discussion

119 Among the 575 HA-MRSA and 205 CA-MRSA isolates, 11 (2%) and 49 (24%) OS-MRSA
120 were detected, respectively. Of the 60 OS-MRSA isolates (Table 1), 21 (35%) belonged to ST22
121 (Gaza Strip clone), and most of them were drawn from healthy carriers. Ten isolates (17%)
122 belonged to the ST59 clone; nine were from healthy carriers and one from a hospital-acquired
123 skin and soft tissue infection (SSTI). The remaining 29 isolates belonged to ten different STs; 20
124 were from healthy carriers and nine from patients with SSTI, osteomyelitis, and bacteremia.
125 Both dominant OS-MRSA lineages are globally found mainly among healthy carriers: ST22, in
126 the Middle East (30, 36), and ST59, in Taiwan and continental China (15, 37). Phylogenetic
127 analysis based on the alignment of 117 726 core-SNP revealed that the clustering of the isolates
128 included in the current study fully corresponded to their sequence types (Supplementary Figure
129 S1), which confirms the polyclonal structure of the population.

130 The complete characterization of the genotypes and phenotypes of the isolates is
131 presented in Supplementary Table S2. OS-MRSA demonstrated a low level of resistance to non-
132 beta-lactam antibiotics, and, on average, each isolate was resistant to two groups of antibiotics.
133 Resistance to macrolides was mediated by ermB and ermC; to tetracyclines, by tetK and tetM;
134 to phenicols, by cat and fexA; to fusidic acid, by fusC; to aminoglycosides, by aac(6')-aph(2''),
135 ant(6)-Ia, and aph(3')-III; and mutations in gyrA (S84L) and parC (S80F) mediated resistance to
136 fluoroquinolones. All isolates were susceptible to vancomycin, daptomycin, linezolid,
137 tigecycline, and ceftaroline. The presented results are consistent with previously published data
138 on the predominant affiliation of OS-MRSA with CA-MRSA and the relatively low frequency of
139 their resistance to non-beta-lactam antibiotics (38). Presence of virulence genes has been
140 associated with specific STs; lukFS (PVL) was identified in ST2704 isolates, tsst in ST22, and seb
141 in ST59.

142 All isolates included in the study demonstrated an oxacillin MIC of ≤ 2.0 mg/L, determined
143 using BMD. The isolates had high MIC values of penicillin (MIC90 8 mg/L) and amoxicillin (MIC90
144 64 mg/L), but meropenem MIC did not exceed 2.0 mg/L. When clavulanate was added to the
145 penicillins, a decrease in MICs was detected (penicillin MIC90, 2.0 mg/L; amoxicillin MIC90, 16
146 mg/L). The combination of penicillin with clavulanate reached the Clinical and Laboratory
147 Standards Institute (CLSI)/EUCAST susceptibility breakpoint (0.125 μg/mL) only in two isolates.
148 However, an epidemiological cutoff value (ECOFF) ≤ 2 μg/mL has been proposed for penicillin–
149 clavulanic acid (22), and most isolates were within the limit of 2 gm/μL. The OS-MRSA
150 phenotypes were significantly different from those of typical MRSA (HA-MRSA), which were
151 characterized by high MIC values for both penicillins and meropenem and lack of synergism
152 between penicillins and clavulanate (Supplementary Table S2).
153 OS-MRSA harbored SCCmec types IV and V, and seven isolates were blaZ-negative (Table
154 1). A reduction in resistance to penicillins–clavulanate was detected only in blaZ-positive
155 isolates. The bla genes (blaZ, blaR1, and blaI) were carried by plasmids, except in the ST22 and
156 ST1 isolates wherein blaZ was located on the chromosome. We noted no differences in the
157 phenotypes between isolates with plasmids and the chromosomal localization of blaZ.

158 In all OS-MRSA, we identified SNPs in the mecA promoter at positions −33 (C→T) or −7
159 (G→T/A). We did not detect any isolates harboring both substitutions simultaneously. In one
160 isolate, a mutation at −38 (A→G) was found in addition to the substitution in position −7
161 (G→T/A). This genotype has been associated with penicillin–clavulanate resistance, and similar
162 results were obtained by Chen et al. (39). Most ST22 isolates (17 of 21) harbored the wildtype
163 mecA gene. The remaining ST22 isolates and those of other STs carried E246G amino acid
164 substitutions (AAS) and different combinations of five additional AAS in PBP2a. We did not
165 detect any insertions, deletions, or stop codons. The identified mutations did not directly affect
166 the function of the mecA gene; however, the basal level expression of mecA in OS-MRSA was
167 extremely low (p < 0.05) compared with that in the control ST8-HA-MRSA isolate (Fig. 1).
168 Induction by oxacillin increased the level of mecA transcripts but fold-change did not reach the
169 control isolate level. The isolates with the mecA promoter mutation at position −33
170 demonstrated lower levels of mecA expression than the control (p < 0.05); however, under
171 induction, the level of fold-change compared with that of isolates with a mutation at −7 was
172 not statistically significant (p = 0.3).

173 In a recent study, Harrison et al. demonstrated that penicillin–clavulanate susceptibility in

174 a significant proportion of MRSA is mediated by a combination of two different mutations in
175 the mecA promoter region (at position −7 or −33) and by either one of two substitutions in
176 PBP2a (E246G or M122I); inhibition of the staphylococcal β-lactamase was proposed as the
177 mechanism of synergism (18). In another recent study, Zhuang et al. showed that isolates with
178 a combination of upstream mutation at position −7 and S225R+E246G substitution in PBP2a
179 had a minimal penicillin–clavulanate MIC of 0.03–1 μg/mL (40). Our results confirm the role of
180 blaZ inhibition as a mechanism of synergism between penicillins and clavulanate; however, the
181 decrease in the MIC of penicillins in isolates with wildtype mecA and mutations in the promoter
182 region of this gene indicates a superior role for the latter in the formation of the OS-MRSA
183 phenotype.

184 PAP showed that most OS-MRSA isolates were represented by heterogeneous
185 populations. Isolates with a promoter mutation at position -7 demonstrated higher AUC values
186 than those of mecA-negative isolates, lower AUC values than those of the control mecA-positive
187 HA-MRSA, and contained a subpopulation with an oxacillin MIC of 32.0 mg/L (Supplementary
188 Figure S2-A). Isolates with a promoter mutation at position −33 were represented by two
189 subgroups (Supplementary Figure S2-B). One group did not differ substantially from mecA-
190 negative isolates (MSSA-like), while the other showed a slight increase in AUC and contained a
191 subpopulation with an oxacillin MIC of 8 mg/L. The presence of a subpopulation with high MICs
192 threatens the transformation of OS-MRSA into MRSA, with a high level of resistance to beta-
193 lactams, as has been demonstrated in previous studies (23, 41).
194 Comparative analysis of different antimicrobial susceptibility tests showed that cefoxitin-
195 based tests are highly specific for the detection of mecA-positive phenotypes. In particular, the
196 highest positive predictive values (PPV > 0.95) was obtained using BMD, the VITEK® 2 automatic
197 system, and chromogenic media (Table 2). Adding of 2% sodium chloride to the media
198 significantly increased PPV (up to 0.92) in oxacillin-based tests; sodium chloride has already
199 been used for OS-MRSA detection (42). According to previous studies (1, 43), OS-MRSA can be
200 divided into two subgroups: cefoxitin-resistant/oxacillin-susceptible and cefoxitin-
201 susceptible/oxacillin-susceptible isolates. However, in most previous studies, interpretations of
202 cefoxitin-based tests included DDM or automatic systems, without multiple comparisons. In the
203 present study, comparative analysis showed discordance among the various methods:
204 interpretations of diffusion methods did not match those of dilution methods or other
205 approaches; that is, different tests could be interpreted differently. Similar results have been
206 described in previous studies (15, 44, 45). Using correlation analysis, correlation between
207 different methods for methicillin detection was found not for all tests (Supplementary Figure
208 S3-A). In particular, the FOX BMD data were not correlated with cefoxitin-based DDM, GDM,
209 and GDM with oxacillin tests. Correlation between the level of penicillin–clavulanate MICs and
210 other antibiotic susceptibility tests was either low or absent. We observed a tendency (p=0.044)
211 for promoter mutation positions to influence the distribution of inhibition zones in FOX DDM
212 (Supplementary Figure S3-B). For isolates with a mutation at position −33, the diameters of
213 inhibition zones were in the range of 17–22 mm, whereas, in isolates with a mutation at −7,
214 values were more widely distributed (14–25 mm).

215 Conclusion

216 Mutations in the promoter region and a low level of mecA expression likely mediate beta-
217 lactam susceptibility of OS-MRSA isolates. The OS-MRSA phenotype (false susceptibility to beta-
218 lactams) may account for the mis-prescription of beta-lactam antibiotics for the treatment of
219 staphylococcal infections. Cefoxitin-based tests provide the best predictions for the detection
220 of methicillin-resistant phenotypes among oxacillin-susceptible mecA-positive isolates. OS-
221 MRSA is characterized by susceptibility to penicillin–clavulanic acid; however, administration of
222 this combination for the treatment of OS-MRSA infections is not advisable, given that OS-MRSA
223 frequently consists of subpopulations with high oxacillin-resistant levels.

224 ACKNOWLEDGMENTS

225 This study was funded by the Russian Science Foundation (grant no. 18-75-10114-P).

226 Supplementary material

227 Supplementary Table S1. Sequence of primers and probes for the mecA expression assay.

228 Supplementary Table S2. Phenotypes and genotypes of OS-MRSA.

229 Supplementary Figure S1. Phylogenetic analysis of mecA-positive oxacillin-susceptible S.

230 aureus.

231 Supplementary Figure S2. Oxacillin population analysis profile of OS-MRSA.

232 Supplementary Figure S3. Correlation analysis of different antibiotic susceptibility tests.

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376

377

378
379 Table 1. Genotypes and mutations associated with oxacillin susceptibility in mecA-positive S. aureus

mecA promoter PBP2a MIC, mg/L

MLST, ST Spa SCCmec N blaZ
-38 -33 -7 S225R A228T E246G D323N A468V S590P PEN PEN-CL
ST22 t223 IVa, IVc 17 + - - + - - - - - - 1–8 0.5 – 4
ST22 t223 IVa, IVc 3 +/- (1) * - - + - - + - - - 1–8 0.5 – 2
ST22 t223 IVc 1 - - - + - - + + - - 0.5 0.5
ST59 t1950 Vb 10 +/- (2) - + - + - + - - - 0.125 – 2 0.25 – 0.5
ST1 t321 IVa 5 + - - + - - + - + - 2–4 0.5 – 1
ST1 t127 IVa 1 + - - + - - + - - - 4 0.5
ST8 t008 IVc 3 + - - + - - + - - - 8 – 32 1–2
ST8 t008 IVc 1 - + - + - - + - - - 8 8
ST8 t3308 V 1 + - + - + - + - - - 8 0.5
ST5 t688 V 5 +/- (1) - + - + - + - - + 0.5 – 4 0.25 – 0.5
ST2704 t002 IVc 5 + - - + - - + - - - 2–8 0.5 – 1
ST97 t359 IVb 1 + - - + - - + - - - 8 2
ST97 t9129 V 1 + - + - + - + - - - 16 1
ST6 t1950 V 1 + - + - + - + - - - 1 0.25
ST6 t304 IVa 1 + - - + - - + - - - 8 2
ST45 NT Vc 1 + - + - + - + - - - 16 0.25
ST398 t034 Vc 1 - - + - + + + - - - 0.25 0.125
ST1535 t084 V 1 + - + - + - + - - - 1 0.5
ST7 t091 IVa 1 + - - + - - + - - - 16 4
380 Most prevalent spa and SCCmec types are shown.

381 *In brackets, number of blaZ-negative isolates, PEN: penicillin, PEN–CL: penicillin-clavulanic acid.

382

383
384 Table 2. Comparison of different antibiotic susceptibility tests for detection of the methicillin-
385 resistant phenotype in mecA-positive S. aureus

MIC (mg/L) or DDM (mm)

Tests SA (n) RA (n) PPV MIC50 MIC90
Range Geo. mean
OXA BMD 60 0 0 0.25 – 2 0.7 0.5 2
OXA GDM 49 11 0.18 0.5 – 24 1.6 1.5 4
VITEK® 2 OXA 53 7 0.11 ≤0.25 – >4 NA NA NA
OXA DDMB 41 19 0.32 0 – 23 14 NA NA
OXA GDM Salt 13 47 0.78 1 – 256 4.7 4 16
FOX GDM 12 48 0.8 3 – 64 9.7 8 16
FOX GDM Salt 10 50 0.83 4 – 48 9.1 8 16
FOX DDM 7 53 0.9 0 – 25 18 NA NA
B
OXA DDM Salt 5 55 0.92 0 – 19 0 NA NA
FOX DDM Salt 5 55 0.92 8 – 24 18.5 NA NA
FOX BMD 2 58 0.96 2 – 32 11 8 16
CM 2 58 0.96 NA NA NA NA
VITEK® 2 FOX 1 59 0.98 NA NA NA NA
A
386 : according to EUCAST 2022 breakpoints; B: breakpoint R ≤ 10 mm.

387 Geo. mean: geometric mean, NA: not applicable, PPV: positive predictive value.

388
389

390

391 Fig 1. mecA expression assay data in isolates with different promoter mutations, BHI: brain
392 heart infusion medium. Oxacillin (0.016 mg/L) was added for induction of mecA expression.