RlmQ: a newly discovered rRNA modification enzyme bridging RNA modification and virulence traits in Staphylococcus aureus.

rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the rRNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in Escherichia coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralog of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.

The 3'UTR-derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose-6-phosphate uptake in Staphylococcus aureus.

Staphylococcus aureus RsaG is a 3'-untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5'- to 3'-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5'-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.

A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

How Staphylococcus aureus and Pseudomonas aeruginosa Hijack the Host Immune Response in the Context of Cystic Fibrosis.

Cystic fibrosis (CF) is a serious genetic disease that leads to premature death, mainly due to impaired lung function. CF lungs are characterized by ongoing inflammation, impaired immune response, and chronic bacterial colonization. Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are the two most predominant bacterial agents of these chronic infections. Both can colonize the lungs for years by developing host adaptation strategies. In this review, we examined the mechanisms by which SA and PA adapt to the host immune response. They are able to bypass the physical integrity of airway epithelia, evade recognition, and then modulate host immune cell proliferation. They also modulate the immune response by regulating cytokine production and by counteracting the activity of neutrophils and other immune cells. Inhibition of the immune response benefits not only the species that implements them but also other species present, and we therefore discuss how these mechanisms can promote the establishment of coinfections in CF lungs.

Mixed Populations and Co-Infection: Pseudomonas aeruginosa and Staphylococcus aureus.

The human pathogens Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from chronic wounds or cystic fibrosis patient airways. Clinical studies analysing the impact of co-infection on patient clinical outcomes lead to contradictory results. However, laboratory approaches suggest that the two pathogens co-colonize the same infection niches and form a mixed-species biofilm, therefore favouring their resistance to antibiotics and immune response. In parallel, many recent studies have focused on the different interactions between the two bacterial species. It has long been recognized that P. aeruginosa usually outcompetes S. aureus, and the molecular mechanisms involved in this state of bacterial competition are now well understood. However, several recent studies show that interactions between P. aeruginosa and S. aureus can be diverse and evolve over time. Thus, many CF isolates of P. aeruginosa and S. aureus can coexist and develop cooperative behaviours. In this chapter, we will provide an overview of the current knowledge on the mixed populations of P. aeruginosa and S. aureus, from their mechanisms of establishment to their impacts on bacterial physiology and clinical outcomes.

Staphylococcus aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression.

Staphylococcus aureus RNAIII is one of the main intracellular effectors of the quorum-sensing system. It is a multifunctional RNA that encodes a small peptide, and its noncoding parts act as antisense RNAs to regulate the translation and/or the stability of mRNAs encoding transcriptional regulators, major virulence factors, and cell wall metabolism enzymes. In this review, we explain how regulatory proteins and RNAIII are embedded in complex regulatory circuits to express virulence factors in a dynamic and timely manner in response to stress and environmental and metabolic changes.

RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation.

The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5' end while its 3' part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3'UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.

Necrotizing Soft Tissue Infection Staphylococcus aureus but not S. pyogenes Isolates Display High Rates of Internalization and Cytotoxicity Toward Human Myoblasts.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) caused by group A Streptococcus (GAS) and occasionally by Staphylococcus aureus (SA) frequently involve the deep fascia and often lead to muscle necrosis. METHODS: To assess the pathogenicity of GAS and S. aureus for muscles in comparison to keratinocytes, adhesion and invasion of NSTI-GAS and NSTI-SA isolates were assessed in these cells. Bloodstream infections (BSI-SA) and noninvasive coagulase-negative staphylococci (CNS) isolates were used as controls. RESULTS: NSTI-SA and BSI-SA exhibited stronger internalization into human keratinocytes and myoblasts than NSTI-GAS or CNS. S. aureus internalization reached over 30% in human myoblasts due to a higher percentage of infected myoblasts (>11%) as compared to keratinocytes (<3%). Higher cytotoxicity for myoblasts of NSTI-SA as compared to BSI-SA was attributed to higher levels of psmα and RNAIII transcripts in NSTI-SA. However, the 2 groups were not discriminated at the genomic level. The cellular basis of high internalization rate in myoblasts was attributed to higher expression of α5ß1 integrin in myoblasts. Major contribution of FnbpAB-integrin α5ß1 pathway to internalization was confirmed by isogenic mutants. CONCLUSIONS: Our findings suggest a factor in NSTI-SA severity is the strong invasiveness of S. aureus in muscle cells, a property not shared by NSTI-GAS isolates.

The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms.

The virulon of Staphyloccocus aureus is controlled by intricate connections between transcriptional and post-transcriptional regulators including proteins and small non-coding RNAs (sRNAs). Many of the sRNAs regulate gene expression through base-pairings with mRNAs. However, characterization of the direct sRNA targets in Gram-positive bacteria remained a difficult challenge. Here, we have applied and adapted the MS2-affinity purification approach coupled to RNA sequencing (MAPS) to determine the targetome of RsaA sRNA of S. aureus, known to repress the synthesis of the transcriptional regulator MgrA. Several mRNAs were enriched with RsaA expanding its regulatory network. Besides mgrA, several of these mRNAs encode a family of SsaA-like enzymes involved in peptidoglycan metabolism and the secreted anti-inflammatory FLIPr protein. Using a combination of in vivo and in vitro approaches, these mRNAs were validated as direct RsaA targets. Quantitative differential proteomics of wild-type and mutant strains corroborated the MAPS results. Additionally, it revealed that RsaA indirectly activated the synthesis of surface proteins supporting previous data that RsaA stimulated biofilm formation and favoured chronic infections. All together, this study shows that MAPS could also be easily applied in Gram-positive bacteria for identification of sRNA targetome.

Intracoronary delivery of recombinant TIMP-3 after myocardial infarction: effects on myocardial remodeling and function.

Ischemia-reperfusion (IR) and myocardial infarction (MI) cause adverse left ventricular (LV) remodeling and heart failure and are facilitated by an imbalance in matrix metalloproteinase (MMP) activation and the endogenous tissue inhibitors of metalloproteinase (TIMPs). We have identified that myocardial injections of recombinant TIMP-3 (rTIMP-3; human full length) can interrupt post-MI remodeling. However, whether and to what degree intracoronary delivery of rTIMP-3 post-IR is feasible and effective remained to be established. Pigs (25 kg) underwent coronary catheterization and balloon occlusion of the left anterior descending coronary artery (LAD) for 90 min whereby at the final 4 min, rTIMP-3 (30 mg, n = 9) or saline was infused in the distal LAD. LV echocardiography was performed at 3-28 days post-IR, and LV ejection fraction (EF) and LV end-diastolic volume were measured. LV EF fell and LV end-diastolic volume increased from baseline (pre-IR) values (66 ± 1% and 40 ± 1 ml, respectively, means ± standard deviation) in both groups; however, the extent of LV dilation was reduced in the rTIMP-3 group by 40% at 28 days post-IR (P < 0.05) and the fall in LV EF was attenuated. Despite equivalent plasma troponin levels (14 ± 3 ng/ml), computed MI size at 28 days was reduced by over 45% in the rTIMP-3 group (P < 0.05), indicating that rTIMP-3 treatment abrogated MI expansion post-IR. Plasma NH2-terminal pro-brain natriuretic peptide levels, an index of heart failure progression, were reduced by 25% in the rTIMP-3 group compared with MI saline values (P < 0.05). Although the imbalance between MMPs and TIMPs has been recognized as a contributory factor for post-MI remodeling, therapeutic strategies targeting this imbalance have not been forthcoming. This study is the first to demonstrate that a relevant delivery approach (intracoronary) using rTIMP can alter the course of post-MI remodeling.NEW & NOTEWORTHY Myocardial ischemia and reperfusion injury remain significant causes of morbidity and mortality whereby alterations in the balance between matrix metalloproteinase and tissue inhibitor of metalloproteinase have been identified as contributory biological mechanisms. This novel translational study advances the concept of targeted delivery of recombinant proteins to modify adverse myocardial remodeling in ischemia-reperfusion injury.

Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir.

Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.

Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence.

Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in hlgCB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on hlgC promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of hlgCB mRNA strongly impaired hlgC translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. IMPORTANCE S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in pvl-negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of hlgCB mRNA induce the differential expression of hlgCB, drastically impacting hlgC mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.

Targeted proteomics links virulence factor expression with clinical severity in staphylococcal pneumonia.

Introduction: The bacterial pathogen Staphylococcus aureus harbors numerous virulence factors that impact infection severity. Beyond virulence gene presence or absence, the expression level of virulence proteins is known to vary across S. aureus lineages and isolates. However, the impact of expression level on severity is poorly understood due to the lack of high-throughput quantification methods of virulence proteins. Methods: We present a targeted proteomic approach able to monitor 42 staphylococcal proteins in a single experiment. Using this approach, we compared the quantitative virulomes of 136 S. aureus isolates from a nationwide cohort of French patients with severe community-acquired staphylococcal pneumonia, all requiring intensive care. We used multivariable regression models adjusted for patient baseline health (Charlson comorbidity score) to identify the virulence factors whose in vitro expression level predicted pneumonia severity markers, namely leukopenia and hemoptysis, as well as patient survival. Results: We found that leukopenia was predicted by higher expression of HlgB, Nuc, and Tsst-1 and lower expression of BlaI and HlgC, while hemoptysis was predicted by higher expression of BlaZ and HlgB and lower expression of HlgC. Strikingly, mortality was independently predicted in a dose-dependent fashion by a single phage-encoded virulence factor, the Panton-Valentine leucocidin (PVL), both in logistic (OR 1.28; 95%CI[1.02;1.60]) and survival (HR 1.15; 95%CI[1.02;1.30]) regression models. Discussion: These findings demonstrate that the in vitro expression level of virulence factors can be correlated with infection severity using targeted proteomics, a method that may be adapted to other bacterial pathogens.

All Staphylococcus aureus bacteraemia-inducing strains can cause infective endocarditis: Results of GWAS and experimental animal studies.

OBJECTIVES: We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB). METHODS: A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves. RESULTS: The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU. CONCLUSION: All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream.

Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity.

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

How Bacterial Adaptation to Cystic Fibrosis Environment Shapes Interactions Between Pseudomonas aeruginosa and Staphylococcus aureus.

Pseudomonas aeruginosa and Staphylococcus aureus are the two most prevalent bacteria species in the lungs of cystic fibrosis (CF) patients and are associated with poor clinical outcomes. Co-infection by the two species is a frequent situation that promotes their interaction. The ability of P. aeruginosa to outperform S. aureus has been widely described, and this competitive interaction was, for a long time, the only one considered. More recently, several studies have described that the two species are able to coexist. This change in relationship is linked to the evolution of bacterial strains in the lungs. This review attempts to decipher how bacterial adaptation to the CF environment can induce a change in the type of interaction and promote coexisting interaction between P. aeruginosa and S. aureus. The impact of coexistence on the establishment and maintenance of a chronic infection will also be presented, by considering the latest research on the subject.

Staphylococcus aureus Arsenal To Conquer the Lower Respiratory Tract.

Staphylococcus aureus is both a commensal and a pathogenic bacterium for humans. Its ability to induce severe infections is based on a wide range of virulence factors. S. aureus community-acquired pneumonia (SA-CAP) is rare and severe, and the contribution of certain virulence factors in this disease has been recognized over the past 2 decades. First, the factors involved in metabolism adaptation are crucial for S. aureus survival in the lower respiratory tract, and toxins and enzymes are required for it to cross the pulmonary epithelial barrier. S. aureus subsequently faces host defense mechanisms, including the epithelial barrier, but most importantly the immune system. Here, again, S. aureus uses myriad virulence factors to successfully escape from the host's defenses and takes advantage of them. The impact of S. aureus virulence, combined with the collateral damage caused by an overwhelming immune response, leads to severe tissue damage and adverse clinical outcomes. In this review, we summarize step by step all of the S. aureus factors implicated in CAP and described to date, and we provide an outlook for future research.

From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment.

Pseudomonas aeruginosa is one of the main microbial species colonizing the lungs of cystic fibrosis patients and is responsible for the decline in respiratory function. Despite the hostile pulmonary environment, P. aeruginosa is able to establish chronic infections thanks to its strong adaptive capacity. Various longitudinal studies have attempted to compare the strains of early infection with the adapted strains of chronic infection. Thanks to new '-omics' techniques, convergent genetic mutations, as well as transcriptomic and proteomic dysregulations have been identified. As a consequence of this evolution, the adapted strains of P. aeruginosa have particular phenotypes that promote persistent infection.

Avian Cell Line DuckCelt®-T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac®.

The development of a live-attenuated vaccine (LAV) for the prevention of human metapneumovirus (HMPV) infection is often hampered by the lack of highly efficient and scalable cell-based production systems that support eventual global vaccine production. Avian cell lines cultivated in suspension compete with traditional cell platforms used for viral vaccine manufacture. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel), previously described as an efficient production system for several influenza strains, could also be used to produce a new HMPV LAV candidate (Metavac®, SH gene-deleted A1/C-85473 HMPV). To that end, we characterized the operational parameters of MOI, cell density, and trypsin addition to achieve the optimal production of Metavac®, and demonstrated that the DuckCelt®-T17 cell line is permissive and well-adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, and its capacity to induce efficient neutralizing antibodies in a BALB/c mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for the scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

Fractional nonablative 1,540-nm laser resurfacing of atrophic acne scars. A randomized controlled trial with blinded response evaluation.

The efficacy of nonablative fractional laser resurfacing of acne scars has been described in case reports and uncontrolled trials. The present study is the first randomized controlled trial in this field. The aim of this study was to examine the efficacy and adverse effects of 1,540-nm nonablative fractional laser treatment of acne scars. Ten patients with acne scars were included. Two intraindividual areas of similar size and appearance within contralateral anatomical regions were randomized to (1) 3-monthly laser treatments with a StarLux 1,540-nm fractional handpiece, and (2) no treatment. Blinded on-site clinical evaluations were performed before treatment, and at 4 and 12 weeks after the final treatment. End-points were overall change in scar texture (from score 0, even texture, to 10, worst possible scarring), adverse effects, change in skin colour (from score 0, absent, to 10, worst possible), and patient satisfaction (from score 0, no satisfaction, to 10, best imaginable satisfaction). Before treatment, scars were moderately atrophic and uneven in texture on both treated and untreated sides (median score 6.5, interquartile range 4.5-8; P=1). After treatment, laser-treated scars appeared more even and smooth than untreated control areas (4.5, 2-6.5, versus 6.5, 4.5-8, P=0.0156, at 4 weeks; 4.5, 2.5-6.5, versus 6.5, 4.5-8, at 12 weeks; P=0.0313). Patients were satisfied with the treatment (5.5, 1-7, after 12 weeks) and five of the ten patients evaluated their acne scars as moderately or significantly improved. No differences were found in skin redness or pigmentation between before and after treatment. Patients experienced moderate pain, erythema, oedema, bullae, and crusts. No adverse effects were seen in untreated control areas. The nonablative 1,540-nm fractional laser improves acne scars with a minimum of adverse effects.