Vancomycin MIC and agr dysfunction in invasive MRSA infections in southern Brazil

Abstract

In methicillin-resistant Staphylococcus aureus (MRSA) treatment, the vancomycin minimum inhibitory concentration (MIC) increase, vancomycin heteroresistance (hVISA) presence, and accessory gene regulator (agr) dysfunction are predictors of vancomycin therapy failure. This study evaluated the association between vancomycin MIC (≥ 1.0 μg/mL) and agr dysfunction in invasive MRSA isolates. Vancomycin MIC, hVISA phenotype, agr group, and function were determined in 171 MRSA isolates obtained between 2014 and 2019 from hospitals in Porto Alegre, Brazil. All MRSA were susceptible to vancomycin; 16.4% of these had MIC ≥ 1.0 μg/mL. Seventeen MRSA isolates expressed the hVISA phenotype; 35.3% of them had MIC of 1.5 μg/mL. agr groups I (40.9%) and II (47.1%) were the most found groups for MRSA and hVISA isolates, respectively. The proportion of MRSA with vancomycin MIC ≥ 1.0 μg/mL in agr group II was significantly higher than in agr groups I and III (p = 0.002). agr dysfunction was observed in 4.7% (8/171) of MRSA, especially those with vancomycin MIC ≥ 1.0 μg/mL (p < 0.001). In addition, six isolates (35.3%; 6/17) with hVISA phenotype presented agr dysfunction, which was significantly higher than that in non-hVISA phenotype (p < 0.001). In conclusion, agr dysfunction in MRSA is associated with vancomycin MIC ≥ 1.0 μg/mL and hVISA phenotype, which suggests that agr dysfunction might confer potential advantages on MRSA to survive in invasive infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in the epidemiology of infectious diseases, which causes community- and healthcare-associated MRSA infections with significant morbidity and mortality rates [1]. Vancomycin has been the antimicrobial of choice for MRSA treatment, but in recent years, the emergence of vancomycin resistance has become a challenging public health concern [2]. The Clinical and Laboratory Standards Institute (CLSI) classifies S. aureus vancomycin susceptibility, according to minimum inhibitory concentration (MIC) values, in vancomycin-susceptible S. aureus (VSSA) (MIC ≤ 2 μg/mL), vancomycin-intermediate S. aureus (VISA) (MIC 4–8 μg/mL), and vancomycin-resistant S. aureus (VRSA) (MIC ≥ 16 μg/mL) [3]. In addition, the heterogeneous VISA phenotype (hVISA) represents a subpopulation of cells within a colony with intermediate resistance to vancomycin, while most other cell subpopulations of the colony remain susceptible [4]. The quorum-sensing accessory gene regulator (agr) system regulates the expression of several virulence factors and the heterogeneous resistance of MRSA and contributes to the ability of S. aureus to cause a wide range of infections [5]. The agr locus is present in all staphylococci, with high genetic variability in AgrB, AgrC, and autoinducing thiolactone peptide (AIP), resulting in four agr groups (I–IV) among S. aureus [6]. agr system dysfunctionality results in failure to express the effector molecule RNAIII, which plays a critical role in the regulation of multiple virulence genes, affecting the production of virulence factors associated with invasive diseases [7]. The agr dysfunction has been associated with the adaptation to vancomycin selection pressure and the hVISA/VISA phenotype [8, 9]. Additionally, the increase in vancomycin MIC (i.e., ≥ 1.5 μg/mL) has been associated with higher risk of treatment failure and mortality rates [10]. Considering that vancomycin MIC increase, hVISA phenotype, and agr dysfunction are factors associated with therapeutic failure in MRSA infections, this study aimed to determine the association between vancomycin MIC (≥ 1.0 μg/mL) and agr dysfunction in invasive MRSA infections.

A total of 171 MRSA from invasive infection cases, comprising pneumonia (47.4%; 81/171), bacteremia (38%; 65/171), and osteomyelitis (14.6%; 25/171), were isolated between January 2014 and January 2019 at four hospitals in Porto Alegre, Brazil (Institutional Ethics Committee: 2.770.338). S. aureus was identified in these samples by conventional methods, such as colony morphology on sheep blood agar, Gram stain, catalase activity, production of coagulase, and growth on mannitol salt agar. Methicillin resistance was confirmed by cefoxitin disk diffusion and PCR detection of the mecA gene [3, 11]. S. aureus ATCC^®25923 and S. aureus ATCC^®43300 were used as mecA-negative and mecA-positive controls, respectively [3]. Vancomycin MIC was evaluated according to CLSI guidelines [3] using the microdilution method on Mueller-Hinton broth. All MRSA were screened for hVISA in brain heart infusion agar (BHIA) containing 6 μg/mL vancomycin (BHIA-6 V) according to the CLSI protocol [3]. The control strains Enterococcus faecalis ATCC^®29212 (vancomycin-susceptible) and Enterococcus faecalis ATCC^®51299 (vancomycin-resistant) were included in each analysis [3]. All screenings were performed in triplicate. hVISA was confirmed using population analysis profile/area under the curve (PAP/AUC), using VSSA (ATCC^®29213), hVISA Mu3 (ATCC^®700698), and VISA Mu50 (ATCC^®700699) as control strains, as previously described [12]. The agr groups (I–IV) were identified using multiplex-PCR [13]. Positive (agr I: S. aureus COL; agr II: S. aureus N315; agr III: S. aureus ATCC^®25923; and agr IV: S. aureus A920210) and negative (PCR mixture components without DNA template) controls were included in the analysis. Agr functionality, measured by delta-hemolysin activity, was evaluated by cross-streaking MRSA perpendicularly to RN4220 strains (hyperproducers of beta-hemolysin but not of alpha- and delta-hemolysin) [14]. An enhanced area of hemolysis at the intersection of MRSA and RN4220 streaks indicated delta-hemolysin activity, resulting from the synergetic effects of delta- and beta-hemolysins and, consequently, a functional agr system. In contrast, agr dysfunction was defined as the complete absence of delta-hemolysin activity. Statistical analyses were conducted using Chi-square or Fisher’s exact test. All p < 0.05 were considered to be statistically significant.

The MIC results (Table 1) showed that all MRSA were susceptible to vancomycin with MIC of 0.25–1.50 μg/mL, 16.4% (28/171) of these isolates with MIC ≥ 1.0 μg/mL. Takesue et al. [15] revealed that the efficacy of vancomycin in patients with bacteremia by MRSA was significantly lower in the strains that presented MIC of 2 μg/mL in comparison with strains MIC of 1 μg/mL (30.0% vs. 78.8%, p < 0.001), whereas a meta-analysis study reported an increase in vancomycin treatment failure and mortality rates for vancomycin-susceptible MRSA, particularly those with MIC ≥ 1.5 μg/mL, irrespective of the source of infection or method of MIC determination [10]. The PAP/AUC analysis confirmed 9.9% (17/171) of the MRSA expressed the hVISA phenotype, six of which had vancomycin MIC of 1.5 μg/mL. No VISA phenotype was observed. Chen et al. [16] found that the hVISA phenotype increased in MRSA when vancomycin MIC increased from 1 to 2 μg/mL, agreeing with other studies that suggest a direct relation between hVISA incidence and vancomycin MIC increase [17, 18]. For the agr grouping, group I (40.9%) was the most prevalent, followed by groups II (36.3%) and III (22.8%). No sample was identified for agr group IV. Other studies also found a higher incidence of agr group I among MRSA [19–21]. Most of the samples with the hVISA phenotype belonged to agr group II (47.1%; 8/17), followed by agr groups I (29.4%; 5/17) and III (23.5%; 4/17). Moreover, the percentage of MRSA with vancomycin MIC ≥ 1.0 μg/mL in agr group II (64.3%; 18/28) was significantly higher than in agr groups I and III (35.7%; 10/28) (p = 0.002). Park et al. [19] concluded that bloodstream MRSA with vancomycin MIC of 2 μg/mL were more common in agr group II than in other agr groups. Other studies have also shown agr group II to be more predominant among S. aureus, with reduced susceptibility to vancomycin by an intrinsic survival advantage during exposure to glycopeptides [22]. Consequently, agr group II is associated with reduced vancomycin susceptibility, higher treatment failure rate, and higher mortality in patients with MRSA bacteremia in critical condition who were treated with vancomycin [20, 23]. Most MRSA with the hVISA phenotype in agr group II were associated with reduced vancomycin susceptibility. Based on the delta-hemolysin activity test (Table 2), 4.7% of the MRSA (8/171) presented with agr dysfunction. These MRSA were isolated from bacteremia (75%; 6/8), pneumonia (12.5%; 1/8), and osteomyelitis (12.5%; 1/8) at four different hospitals in the years of 2014 (25%; 2/8), 2015 (12.5%; 1/8), 2016 (25%; 2/8), 2017 (25%; 2/8), and 2018 (12.5%; 1/8). The number of agr dysfunctional isolates were four, two, and two in agr group III (10.2%; 4/39), II (3.2%; 2/62), and I (2.8%; 2/70), respectively. In previous reports, the frequency of agr dysfunctional ranged from 3.79 to 13.0% of MRSA, and these isolates were associated with poor healthcare, multidrug resistance, deleterious outcomes, and increased mortality [21, 24]. The prevalence of agr dysfunction in healthcare settings is associated with attenuated vancomycin activity in hVISA and VISA strains, which potentially confers an advantage on S. aureus for survival in hospital settings [25, 26]. Additionally, a fitness advantage in agr dysfunctional isolates was observed, justifying the efficient transmission and persistence at infection sites and, consequently, the chronic course and deleterious outcomes of the disease [21]. The presence of agr dysfunction in MRSA with vancomycin MIC ≥ 1.0 μg/mL (21.4%; 6/28) was significantly higher than in MRSA with MIC < 1.0 μg/mL (1.4%; 2/143) (p < 0.001). Also, six hVISA (35.3%; 6/17) were agr dysfunctional, which were significantly higher than isolates without the hVISA phenotype (1.3%; 2/154) (p < 0.001). Harigaya et al. [27] reported five times more agr dysfunction in hVISA strains than in VSSA strains. The advantages of a dysfunctional agr system in hVISA and VISA strains are related to the development of vancomycin resistance and biofilm production, which consequently enhance the survival of these strains [28]. Furthermore, the resistance of hVISA is not related to a specific genetic mechanism but to several mutations in multiple genes, mainly in the genes vraSR, graSR, walKR, and tcaRAB [29–31]. The development of the hVISA phenotype in VSSA gradually occurs, which results in mutations that accumulate and affects the reduction of susceptibility to vancomycin [32].

Table 1.

Distribution of agr groups between vancomycin MICs in MRSA isolates

agr group	Vancomycin MIC (μg/mL)					Total
	0.25	0.5	0.75	1.0	1.5
I	31 (48.4%)	17 (37.0%)	16 (48.5%)	5 (22.7%)	1 (16.7%)	70 (40.9%)
II	16 (25.0%)	14 (30.4%)	14 (42.4%)	14 (63.6%)	4 (66.7%)	62 (36.3%)
III	17 (26.6%)	15 (32.6%)	3 (9.1%)	3 (13.6%)	1 (16.7%)	39 (22.8%)
Total	64 (37.4%)	46 (26.9%)	33 (19.3%)	22 (12.9%)	6 (3.5%)	171 (100%)

agr accessory gene regulator, MIC minimum inhibitory concentration, MRSA methicillin-resistant Staphylococcus aureus. The data are presented by number of isolates (%)

Table 2.

Characteristics of MRSA isolates stratified by agr functionality

Characteristics	agr functionality		Total
	Dysfunctional	Functional
phenotype
hVISA	6 (35.3%)	11 (64.7%)	17 (9.9%)
Non-hVISA	2 (1.3%)	152 (98.7%)	154 (90.1%)
agr group
I	2 (2.9%)	68 (97.1%)	70 (40.9%)
II	2 (3.2%)	60 (96.8%)	62 (36.3%)
III	4 (10.3%)	35 (89.7%)	39 (22.8%)
MICvan (μg/mL)
0.25	0	64 (100%)	64 (37.4%)
0.5	1 (2.2%)	45 (97.8%)	46 (26.9%)
0.75	1 (3.0%)	32 (97.0%)	33 (19.3%)
1.0	1 (4.5%)	21 (95.5%)	22 (12.9%)
1.5	5 (83.3%)	1 (16.7%)	6 (3.5%)

agr accessory gene regulator, hVISA heterogeneous vancomycin-intermediate Staphylococcus aureus, MIC_van minimum inhibitory concentration of vancomycin, MRSA methicillin-resistant Staphylococcus aureus. Data are presented by number of isolates (%)

In conclusion, agr system dysfunction in MRSA is associated with vancomycin MIC ≥ 1.0 μg/mL and the hVISA phenotype, which suggests that agr dysfunction may confer potential advantages on MRSA for survival in invasive infections.

Authors’ contributions

Adriana Medianeira Rossato—conception and planning of the study; obtaining, analyzing, and interpreting the data; statistical analysis; elaboration and writing of the manuscript; effective participation in research orientation; critical review of the literature and the manuscript; and approval of the final version of the manuscript. Muriel Primon-Barros—elaboration and writing of the manuscript; critical review of the literature and the manuscript; and approval of the final version of the manuscript. Cícero Armídio Gomes Dias—critical review of the manuscript and approval of the final version of the manuscript. Pedro Alves d’Azevedo—conception and planning of the study; effective participation in research orientation; critical review of the literature and the manuscript; and approval of the final version of the manuscript.

Funding

This study was funded by Research Foundation of the State of Rio Grande do Sul (FAPERGS - 02/2017 PqG).

Data availability

Not applicable.

Compliance with ethical standards

Conflicts of interest/competing interests

All authors declare no conflicts of interest.

Ethics approval

The study was approved by Federal University of Health Sciences of Porto Alegre (UFCSPA) Institutional Ethics under number 2.770.338.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Code availability

Not applicable.