Antibiotic heteroresistance in ESKAPE pathogens, from bench to bedside.

BACKGROUND: Heteroresistance refers to subpopulation-mediated differential antimicrobial susceptibility within a clonal bacterial population. Usually, it designates a resistant subpopulation identified within an isolate considered susceptible by classical antimicrobial susceptibility testing. Heteroresistance lacks a uniform microbiological definition for diagnostic laboratories, and its clinical impact remains unclear for most bacterial species. OBJECTIVES: This narrative review aims to provide a practical overview on the latest developments in the field of heteroresistance for both clinical microbiologists and physicians, with a particular focus on ESKAPE pathogens. SOURCES: A literature search was performed on Pubmed and Google with the key words heteroresistance (heterogeneity OR heterogeneous) AND antibiotic resistance. Among the 836 publications selected based on their abstracts, the most relevant for the detection, epidemiology and clinical impact of heteroresistance in ESKAPE pathogens are discussed here. CONTENT: Heteroresistance is only clearly defined for heterogeneous vancomycin intermediate Staphylococcus aureus. We compiled a larger microbiological definition to be applicable to other bacterial species and antibiotics in the clinical context. We highlighted the key technical points of population analysis profile, which is the reference standard for detecting heteroresistance. Heteroresistance to polymyxins, ß-lactams (carbapenems, cefiderocol), fosfomycin, tigecycline and aminoglycosides is frequently reported in multidrug-resistant gram-negative pathogens. Treatment failure due to heteroresistance has been described in case reports or retrospective studies, so far confirmed by meta-analyses in the case of heterogeneous vancomycin intermediate S. aureus only. Finally, to treat pandrug-resistant bacterial infections, the option of targeting susceptible subpopulations of resistant isolates using tailored antibiotic combinations is also discussed. IMPLICATIONS: Systematic heteroresistance screening by clinical laboratories is not currently recommended. Nevertheless, we should be aware of this phenomenon, and in specific cases, such as treatment failure, heteroresistance should be tested by reference laboratories. Additional studies using standardized methods are needed to improve our understanding of heteroresistance and further assess its clinical impact.

Characterization of Amino Acid Substitution W20S in MgrB Involved in Polymyxin Resistance in Klebsiella pneumoniae.

In the major human pathogen Klebsiella pneumoniae, MgrB inactivation by disruptive insertion sequence (IS) elements and mutations leading to early termination are known to play an important role in polymyxin resistance. In this study, we examined a collection of invasive blaKPC-2-producing K. pneumoniae isolates belonging to the high-risk clone sequence type 258 (ST258) displaying high rates of resistance to many antimicrobials, including polymyxins. We identified a deleterious substitution (W20S) in MgrB and confirmed by genetic complementation analysis that this variant was inactive, leading to increased polymyxin B and colistin MICs. IMPORTANCE Carbapenem-resistant Gram-negative bacteria are designated critical pathogens by the World Health Organization. Polymyxins (i.e., polymyxin B and colistin) are last-resort antibiotics and particularly useful against these multidrug-resistant bacteria. In Klebsiella pneumoniae, the inactivation of MgrB, a negative regulator of PhoPQ, was shown to be the major pathway leading to colistin resistance. While gene disruption by insertion sequence (IS) elements and mutations leading to early termination (stop codons) are frequent, deleterious mutations are not observed frequently and have not been characterized. Here, we identified a deleterious substitution (W20S) in MgrB among a collection of bloodstream infection, blaKPC-2-producing K. pneumoniae sequence type 258 (ST258) isolates, displaying high rates of resistance to polymyxins and associated with a high mortality rate. The dissemination of such a MgrB-W20S mutation leading to polymyxin resistance within the ST258 high-risk clone background is problematic and thus warrants particular attention.

Immediate Effect of Dry Needling on the Viscoelastic Properties of a Trigger Point on the Infraspinatus Muscle Measured with MyotonPRO.

Purpose: This article investigates the immediate effects of a dry needling (DN) puncture on the viscoelastic properties (tone, stiffness, elasticity) of a trigger point (TP) in the infraspinatus muscle in non-traumatic chronic shoulder pain. Method: Forty-eight individuals with non-traumatic chronic shoulder pain were recruited. The presence of a TP in the infraspinatus muscle was confirmed by a standardized palpatory exam. The viscoelastic properties were measured with a MyotonPRO device at baseline (T1), immediately after DN (T2), and 30 minutes later (T3). A DN puncture was applied to the TP to obtain a local twitch response while performing the technique. Results: Analyses of variance showed significant decreases in tone (p < 0.001) and stiffness (p = 0.003) across time after the DN technique. Post hoc tests revealed a significant reduction in tone and stiffness from T1 to T2 (p ≤ 0.004) and no significant changes from T2 to T3 (p ≥ 0.10). At T3, only stiffness remained significantly lower compared to T1 (p = 0.013). Conclusions: This study brings new insights on the immediate mechanical effect of DN on tone and stiffness of TPs. Whether these effects are associated with symptom improvement and long-term effects still needs to be verified.

Objectif : examiner les effets immédiats de la puncture avec aiguille sèche (AS) sur les propriétés viscoélastiques (tonus, raideur, élasticité) d'un point gâchette (PG) du muscle infra-épineux dans des cas de douleur chronique d'origine non traumatique de l'épaule. Méthodologie : les chercheurs ont recruté 48 personnes avec présence de douleur chronique d'origine non traumatique à l'épaule. Ils ont confirmé la présence d'un PG dans le muscle infra-épineux avec un examen palpatoire standardisé. Au moyen de l'appareil MyotonPRO, ils ont mesuré les propriétés viscoélastiques en début d'étude (T1), immédiatement après l'intervention par AS (T2), puis 30 minutes plus tard (T3). Durant l'application de la technique de AS, des manoeuvres étaient effectuées avec l'aiguille dans le but d'obtenir une contraction musculaire réflexe involontaire. Résultats : les analyses de variance ont révélé une diminution importante du tonus (p < 0,001) et de la rigidité (p = 0,003) au travers les différents temps de mesure. Les analyses post hoc ont révélé une importante diminution significative au niveau du tonus et de la raideur entre les temps de mesure T1 et T2 (p ≤ 0,004) et aucun changement entre T2 et T3 (p p ≥ 0,10). À T3, seule la raideur est demeurée significativement plus basse qu'au temps de mesureT1 (p = 0,013). Conclusions : la présente étude apporte de nouvelles évidences sur les effets mécaniques immédiats de l'AS sur le tonus et la raideur des PG. Les prochaines études devront vérifier si ces effets sont associés à l'amélioration des symptômes et sur des effets à plus long terme.

Neurophysiological and psychophysical effects of dry versus sham needling of the infraspinatus muscle in patients with chronic shoulder pain: a randomized feasibility study.

BACKGROUND: Dry needling (DN) is increasingly used for treating myofascial trigger points (MTrPs) and has shown significant effects on pain and function. This study aimed to assess feasibility of conducting a randomized sham-controlled trial and to collect preliminary data on the effects of infraspinatus DN on corticospinal excitability and mechanical pain sensitivity. METHOD: This randomized feasibility study included adults with chronic non-traumatic shoulder pain and a infraspinatus MTrP. Participants were randomized to receive real DN or sham DN in the infraspinatus MTrP. Feasibility outcomes included data pertaining to recruitment, retention of participants, completeness and safety of assessment procedures. Neurophysiological and psychophysical outcomes included corticospinal excitability and mechanical pain sensitivity measured by active motor threshold (aMT) and pressure pain threshold (PPT), respectively. They were assessed at baseline, immediately after and 24 h post-intervention. RESULTS: Twenty-one participants were recruited over a 6-month period. Nineteen participants completed the treatment and follow-up assessment. Motor evoked potential responses were discernible in all but 1 participant. Only 1 minor adverse event related to transcranial magnetic stimulation (mild headache) affected the measurements. No DN adverse effects were recorded in both groups. An overall completeness rate of 81% was reached, with 70% completeness in the DN group and 91% in the sham group. Data analysis revealed that real DN increased corticospinal excitability (reduced aMT) 24 h post-intervention (Mdn = - 5.96% MSO, IQR = 5.17, p = 0.04) and that sham DN triggered similar responses immediately after the intervention (Mdn = - 1.93% MSO, IQR = 1.11, p = 0.03). Increased mechanical pain sensitivity (reduced PPT) was significant only in the sham group, both immediately (Mdn = - 0.44 kg/cm2, IQR = 0.49, p = 0.01) and 24 h post-intervention (Mdn = - 0.52 kg/cm2, IQR = 1.02, p = 0.02). Changes in corticospinal excitability was positively correlated with changes in mechanical pain sensitivity in the DN group, both immediately (r = 0.77, p = 0.02) and 24 h post-intervention (r = 0.75, p = 0.05). CONCLUSION: The present study demonstrates the feasibility of quantifying the neurophysiological and psychophysical effects of DN, and provides recommendations and guidelines for future studies. Moreover, it provides preliminary evidence that DN may increase corticospinal excitability of the infraspinatus muscle in patients with chronic shoulder pain and that the relationship of neurophysiological and psychophysical effects is promising to better understand its mechanisms of action. TRIAL REGISTRATION: NCT04316793 ; retrospectively registered November 3, 2020.

The Female Pelvic Floor Fascia Anatomy: A Systematic Search and Review.

The female pelvis is a complex anatomical region comprising the pelvic organs, muscles, neurovascular supplies, and fasciae. The anatomy of the pelvic floor and its fascial components are currently poorly described and misunderstood. This systematic search and review aimed to explore and summarize the current state of knowledge on the fascial anatomy of the pelvic floor in women. METHODS: A systematic search was performed using Medline and Scopus databases. A synthesis of the findings with a critical appraisal was subsequently carried out. The risk of bias was assessed with the Anatomical Quality Assurance Tool. RESULTS: A total of 39 articles, involving 1192 women, were included in the review. Although the perineal membrane, tendinous arch of pelvic fascia, pubourethral ligaments, rectovaginal fascia, and perineal body were the most frequently described structures, uncertainties were identified in micro- and macro-anatomy. The risk of bias was scored as low in 16 studies (41%), unclear in 3 studies (8%), and high in 20 studies (51%). CONCLUSIONS: This review provides the best available evidence on the female anatomy of the pelvic floor fasciae. Future studies should be conducted to clarify the discrepancies highlighted and accurately describe the pelvic floor fasciae.

A new mutation in mgrb mediating polymyxin resistance in Klebsiella variicola.

Polymyxin resistance is a public health concern - present in humans, animals and the environment - caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola (K. variicola). This study performed microbiological and genomic characterisation of three polymyxin-resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing using Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial-resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.

The Role of ArlRS and VraSR in Regulating Ceftaroline Hypersusceptibility in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus infections are a global health problem. New control strategies, including fifth-generation cephalosporins such as ceftaroline, have been developed, however rare sporadic resistance has been reported. Our study aimed to determine whether disruption of two-component environmental signal systems detectably led to enhanced susceptibility to ceftaroline in S. aureus CA-MRSA strain MW2 at sub-MIC concentrations where cells normally continue to grow. A collection of sequential mutants in all fifteen S. aureus non-essential two-component systems (TCS) was first screened for ceftaroline sub-MIC susceptibility, using the spot population analysis profile method. We discovered a role for both ArlRS and VraSR TCS as determinants responsible for MW2 survival in the presence of sub-MIC ceftaroline. Subsequent analysis showed that dual disruption of both arlRS and vraSR resulted in a very strong ceftaroline hypersensitivity phenotype. Genetic complementation analysis confirmed these results and further revealed that arlRS and vraSR likely regulate some common pathway(s) yet to be determined. Our study shows that S. aureus uses particular TCS environmental sensing systems for this type of defense and illustrates the proof of principle that if these TCS were inhibited, the efficacy of certain antibiotics might be considerably enhanced.

Vertical and horizontal dissemination of an IncC plasmid harbouring rmtB 16S rRNA methylase gene, conferring resistance to plazomicin, among invasive ST258 and ST16 KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Carbapenem resistance in Klebsiella pneumoniae is a major clinical challenge. Aminoglycosides remain an important asset in the current therapeutic arsenal to treat these infections. We examined aminoglycoside resistance phenotypes and genomics in a collection of 100 invasive KPC-producing K. pneumoniae isolates sequentially collected in a Brazilian tertiary hospital between 2014 and 2016. METHODS: Aminoglycoside susceptibility testing was performed. We used a combined long-read (MinION) and short-read (Illumina) whole-genome sequencing strategy to provide a genomic picture of aminoglycoside resistance genes, with particular emphasis on 16S rRNA methyltransferases and related plasmids. RESULTS: 68% of the strains were resistant to gentamicin and 42% to amikacin, with 35% resistant to both of these commonly used aminoglycosides. We identified the 16S rRNA methyltransferase gene rmtB in 30% of these isolates: 97% (29/30) belonged to sequence type 258 (ST258) and a single isolate to the emergent ST16 clone. In ST258 and ST16 the rmtB gene was located on large IncC plasmids of 177 kb and 174 kb, respectively, highly similar to a plasmid previously identified in Proteus mirabilis in the same hospital. Moreover, 99% of the isolates remained susceptible to the veterinary-approved drug apramycin, currently under clinical development for human medicine. CONCLUSION: Such findings in geographically and temporally related isolates suggest a combination of vertical clonal spread as well as horizontal interspecies and intraspecies plasmid transfer. This broad rmtB dissemination in an endemic setting for KPC-producing clones is worrisome since it provides resistance to most clinically available aminoglycosides, including the novel aminoglycoside-modifying enzyme-resistant plazomicin.

The MyotonPRO: A reliable tool for quantifying the viscoelastic properties of a trigger point on the infraspinatus in non-traumatic chronic shoulder pain.

INTRODUCTION: Clinicians rely on palpation for locating and diagnosing trigger points in muscles. Measuring a trigger point with clinical palpation remains a challenge. There are currently no validated tools available in clinical practice to objectively measure a trigger point. METHOD: The presence of a trigger point within the infraspinatus muscle was identified on thirty-five individuals with non-traumatic chronic shoulder pain via palpation according to Travell and Simons criteria. Trigger and non-trigger points were marked within the same muscle and the viscoelastic properties of both points were independently measured twice with the MyotonPRO by two evaluators on two days. RESULTS: Significant differences were observed when the trigger and non-trigger point (discriminant validity) were compared. The trigger points showed greater tone and stiffness compared to the non-trigger points (tone: 15.30 ± 1.99 Hz vs 13.57 ± 1.76 Hz; stiffness: 270.20 ± 46.96 N/m vs 227.86 ± 43.44 N/m; p < 0.05) and less elasticity (decrement of 1.13 ± 0.21 vs 1.06 ± 0.27; p < 0.05). The reliability of the three viscoelastic properties was found to be excellent for intra- and inter-evaluator reliability (ICC: 0.925-0.984 and 0.918-0.972, respectively) and good to excellent for test-retest reliability (between days) (ICC: 0.770-0.875). CONCLUSION: The MyotonPRO can differentiate the viscoelastic properties of a trigger point from a non-trigger point. Our findings support the reliability of this myotonometer. This affordable and portable tool can be used to objectively measure viscoelastic properties of trigger points in the infraspinatus.

Carbapenems drive the collateral resistance to ceftaroline in cystic fibrosis patients with MRSA.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015-2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2, targets of carbapenems; no effects were achieved by other ß-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population.

Preparation for an Half-Ironmantm Triathlon amongst Amateur Athletes: Finishing Rate and Physiological Adaptation.

Long distance triathlon has gained in popularity amongst the general population. Coaches establish training programs based upon their knowledge, personal experience and on current training principles. The goal was to observe the effect of a triathlon training program for a half Ironman event in neophyte amateur athletes. A specific triathlon training program was followed from February to June 2016 by a group preparing for their first half ironman. Out of the 32 participants (19 Males and 13 Females; mean age of 39 ± 9.9 years old; body weight of 72.7 ± 13.4 kg and a height of 171.5 ± 10.2 cm), only one did not complete the event. A mean training volume of 410 ± 201 min per week led to a mean finishing time of 6 hours 28 minutes. The training program significantly increased the maximal oxygen consumption (45.9 ± 8.2 to 48.6 ± 7.5 ml/kg/min, p =0.002) and the maximal power output (293.1 ± 63.7 to 307.8 ± 58.7 W, p < 0.001). The absolute oxygen consumption and power output at both ventilatory thresholds also significantly increased (VT1: 2.2 ± 0.4 to 2.5 ± 0.5 L, p = 0.001; 157.8 ± 41.8 to 176.7 ± 41.1 W p = 0.009 and VT2: 2.9 ± 0.4 to 3.0 ± 0.4 L, p = 0.017; 229.3 ± 62.0 to 244.8± 55.2 W, p = 0.022 ). A significant diminution of waist circumference was observed (83.2 ± 10.0 to 81.8 ± 9.5 cm, p = 0.032) with no significant changes in body weight. Thus, a 24-week specific training program appears to be safe and efficient for amateur athletes aiming to finish their first half- Ironman event.

Tedizolid is a promising antimicrobial option for the treatment of Staphylococcus aureus infections in cystic fibrosis patients.

BACKGROUND: Tedizolid is a protein synthesis inhibitor in clinical use for the treatment of Gram-positive infections. Pulmonary MRSA infections are a growing problem in patients with cystic fibrosis (CF) and the efficacy of tedizolid-based therapy in CF pulmonary infections is unknown. OBJECTIVES: To evaluate the in vitro and in vivo activity of tedizolid and predict the likelihood of tedizolid resistance selection in CF-background Staphylococcus aureus strains. METHODS: A collection of 330 S. aureus strains (from adult and paediatric patients), either of normal or small colony variant (SCV) phenotypes, gathered at three CF centres in the USA was used. Tedizolid activity was assessed by broth microdilution, Etest and time-kill analysis. In vivo tedizolid efficacy was tested in a murine pneumonia model. Tedizolid in vitro mutants were obtained by 40 days of exposure and progressive passages. Whole genome sequencing of clinical S. aureus strains with reduced susceptibility to tedizolid was performed. RESULTS: MRSA strain MIC90s were tedizolid 0.12-0.25 mg/L and linezolid 1-2 mg/L; for MSSA strains, MIC90s were tedizolid 0.12 mg/L and linezolid 1-2 mg/L. Two strains, WIS 441 and Seattle 106, with tedizolid MICs of 2 mg/L and 1 mg/L, respectively, had MICs above the FDA tedizolid breakpoint (0.5 mg/L). Tedizolid at free serum concentrations exhibited a bacteriostatic effect. Mean bacterial burdens in lungs (log10 cfu/g) for WIS 423-infected mice were: control, 11.2±0.5; tedizolid-treated (10 mg/kg), 3.40±1.87; linezolid-treated (40 mg/kg), 4.51±2.1; and vancomycin-treated (30 mg/kg), 5.21±1.93. For WIS 441-infected mice the (log10 cfu/g) values were: control, 9.66±0.8; tedizolid-treated, 3.18±1.35; linezolid-treated 5.94±2.19; and vancomycin-treated, 4.35±1.7. CONCLUSIONS: These results suggest that tedizolid represents a promising therapeutic option for the treatment of CF-associated MRSA/MSSA infections, having potent in vivo activity and low resistance potential.

Efficacy of newly generated short antimicrobial cationic lipopeptides against methicillin-resistant Staphylococcus aureus (MRSA).

INTRODUCTION: Infection caused by methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) is a serious clinical challenge and research to develop new antimicrobials is imperative. METHODS: This study investigated the in vitro and in vivo efficacy of the short cationic dialkyl lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2. The antibacterial efficacy of (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 was evaluated in representative clinical methicillin-susceptible S. aureus and MRSA strains by both in vitro (MIC, time-kill curve) and in vivo (wax worms model) approaches. RESULTS: These studies revealed that both (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 have rapid bactericidal activity, with a decrease of > 3 log10 colony forming units (CFU)/mL achieved in the first 6 hours of treatment. Furthermore, (C10)2-KKKK-NH2 performed similarly to daptomycin, with a sustained bacterial killing after 24 hours. Wax worms infected and treated with these lipopeptides showed a decreased survival rate of 90% to 50% within the first day of treatment. Scanning electron microscopy determined that the effect of the short lipopeptides in S. aureus was associated with important morphological structural changes that may suggest cell membrane perturbation. CONCLUSION: These findings suggest that the short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 may be potential new options for treating MRSA infections.

Thermosensitive PBP2a requires extracellular folding factors PrsA and HtrA1 for Staphylococcus aureus MRSA ß-lactam resistance.

Staphylococcus aureus is a major human pathogen and represents a clinical challenge because of widespread antibiotic resistance. Methicillin resistant Staphylococcus aureus (MRSA) is particularly problematic and originates by the horizontal acquisition of mecA encoding PBP2a, an extracellular membrane anchored transpeptidase, which confers resistance to ß-lactam antibiotics by allosteric gating of its active site channel. Herein, we show that dual disruption of PrsA, a lipoprotein chaperone displaying anti-aggregation activity, together with HtrA1, a membrane anchored chaperone/serine protease, resulted in severe and synergistic attenuation of PBP2a folding that restores sensitivity to ß-lactams such as oxacillin. Purified PBP2a has a pronounced unfolding transition initiating at physiological temperatures that leads to irreversible precipitation and complete loss of activity. The concordance of genetic and biochemical data highlights the necessity for extracellular protein folding factors governing MRSA ß-lactam resistance. Targeting the PBP2a folding pathway represents a particularly attractive adjuvant strategy to combat antibiotic resistance.

Activity of Telavancin against Staphylococcus aureus Isolates, Including Those with Decreased Susceptibility to Ceftaroline, from Cystic Fibrosis Patients.

Methicillin-resistant Staphylococcus aureus (MRSA) acquisition in cystic fibrosis (CF) patients confers a clinical outcome worse than that in non-CF patients with an increased rate of declined lung function. Telavancin, an approved lipoglycopeptide used to treat infections due to S. aureus, has a dual mode of action causing inhibition of peptidoglycan synthesis and membrane depolarization. MRSA infections in CF patients remain an important problem with no foreseeable decline in prevalence rates. Although telavancin is currently in clinical use for the treatment of complicated skin infections and hospital-acquired pneumonia, the activity against S. aureus infections in CF patients has not been investigated. In this work, we studied the activity of telavancin against CF patient-derived S. aureus strains collected from geographically diverse CF centers in the United States. We found that the telavancin MIC90 was 0.06 µg/ml, 8-fold lower than the ceftaroline or daptomycin MIC90 and 25-fold lower than the linezolid and vancomycin MIC90 We demonstrate that telavancin at serum free concentrations has rapid bactericidal activity, with a decrease of more than 3 log10 CFU/ml being achieved during the first 4 to 6 h of treatment, performing better in this assay than vancomycin and ceftaroline, including against S. aureus strains resistant to ceftaroline. Telavancin resistance was infrequent (0.3%), although we found that it can occur in vitro in both CF- and non-CF patient-derived S. aureus strains by progressive passages with subinhibitory concentrations. Genetic analysis of telavancin-resistant in vitro mutants showed gene polymorphisms in cell wall and virulence genes and increased survival in a Galleria mellonella infection model. Thus, we conclude that telavancin represents a promising therapeutic option for infections in CF patients with potent in vitro activity and a low resistance development potential.

Combination Antibiotic Exposure Selectively Alters the Development of Vancomycin Intermediate Resistance in Staphylococcus aureus.

Invasive methicillin-resistant Staphylococcus aureus (MRSA) treated with vancomycin (VAN) is associated with reduced VAN susceptibility and treatment failure. VAN combination therapy is one strategy to improve response, but comprehensive assessments of combinations to prevent resistance are limited. This study identifies optimal combinations to prevent the emergence of VAN-intermediate Staphylococcus aureus (VISA). Two standard MRSA and two heterogeneous VISA (hVISA) strains were exposed for 28 days in vitro to VAN alone, VAN with cefazolin (CFZ), fosfomycin, gentamicin, meropenem, rifampin, piperacillin-tazobactam (TZP), or trimethoprim-sulfamethoxazole. In addition to VAN susceptibility testing, cell wall thickness (CWT), carotenoid content, and membrane fluidity were determined for Mu3. VAN plus any ß-lactam limited the VAN MIC increase to 1 to 4 mg/liter throughout the 28-day exposure, with CFZ and TZP being the most effective agents (VAN MIC = 1 to 2 mg/liter). Similar MIC trends occurred with the lipo-/glycopeptide agents daptomycin and telavancin, where ß-lactam combinations with VAN prevented MIC increases to these agents as well. Combinations with non-ß-lactams were ineffective in preventing VAN MIC increases with VAN MICs of 4 to 16 mg/liter emerging during weeks 2 to 4 of treatment. VAN plus ß-lactam decreased CWT significantly, whereas VAN plus other antibiotics significantly increased the CWT. No correlation was observed between carotenoid content or membrane fluidity and antibiotic exposure. Only the combination exposures of VAN plus ß-lactam suppress the development of VISA. Rational selection of VAN plus ß-lactam should be further explored as a long-term combination treatment of MRSA infections due to their ability to suppress VAN resistance.

Daptomycin Resistance in Clinical MRSA Strains Is Associated with a High Biological Fitness Cost.

Daptomycin remains as one of the main treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA). Sporadic resistance cases reported in patients treated with either daptomycin or glycopeptides are a growing concern. In a previous study, we described a clinical case of a patient with a community-acquired MRSA infection resistant to daptomycin and with intermediate resistance to vancomycin who developed a recurrent infection with a susceptible isogenic strain. In the present work, we further investigated the sequential events to determine whether the switch from a daptomycin resistance to a susceptible phenotype was due to a phenomenon of resistance reversion or recurrent infection with a susceptible strain. Pairwise competition experiments showed that the susceptible clinical recurrent SA6850 strain had increased fitness when compared to the resistant counterpart SA6820 strain. In fact, although we have demonstrated that reversion of daptomycin resistance to daptomycin susceptible can occur in vitro after serial passages in drug-free media, phylogenetic analysis suggested that the in vivo process was the result of a recurrent infection with a previous susceptible isolate carried by the patient rather than a resistance reversion of the strain. Whole genome sequence of evolved strains showed that daptomycin resistance in MRSA is associated with a high fitness cost mediated by mutations in mprF gene, revealed as a key element of the biological cost. Moreover, we determined that daptomycin resistance-associated fitness cost was independent of vancomycin intermediate resistance phenotype, as demonstrated in additional clinical MRSA vancomycin susceptible strains. This study highlights important observations as, despite daptomycin offers a useful treatment option for the patients with persistent infections, it has to be carefully monitored. The high fitness cost associated to daptomycin resistance may explain the reduced dissemination of daptomycin resistance and the absence of daptomycin reported outbreaks.

Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to ß-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial resistance is recognized as one of the principal threats to public health worldwide, yet the problem is increasing. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most difficult to treat in clinical settings due to the resistance of MRSA to nearly all available antibiotics. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) is the clinical mainstay of anti-MRSA therapy. The decreased susceptibility to DAP (DAP resistance [DAPr]) reported in MRSA is frequently accompanied by a paradoxical decrease in ß-lactam resistance, a process known as the "seesaw effect." Despite the observed discordance in resistance phenotypes, the combination of DAP and ß-lactams has been proven to be clinically effective for the prevention and treatment of infections due to DAPr MRSA strains. However, the mechanisms underlying the interactions between DAP and ß-lactams are largely unknown. In the study described here, we studied the role of mprF with DAP-induced mutations in ß-lactam sensitization and its involvement in the effective killing by the DAP-oxacillin (OXA) combination. DAP-OXA-mediated effects resulted in cell wall perturbations, including changes in peptidoglycan insertion, penicillin-binding protein 2 (PBP 2) delocalization, and reduced membrane amounts of PBP 2a, despite the increased transcription of mecA through mec regulatory elements. We have found that the VraSR sensor-regulator is a key component of DAP resistance, triggering mutated mprF-mediated cell membrane (CM) modifications that result in impairment of PrsA location and chaperone functions, both of which are essential for PBP 2a maturation, the key determinant of ß-lactam resistance. These observations provide for the first time evidence that synergistic effects between DAP and ß-lactams involve PrsA posttranscriptional regulation of CM-associated PBP 2a.

Exposure of Staphylococcus aureus to subinhibitory concentrations of ß-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus.

Glycopeptides are known to select for heterogeneous vancomycin-intermediate Staphylococcus aureus (h-VISA) from susceptible strains. In certain clinical situations, h-VISA strains have been isolated from patients without previous exposure to glycopeptides, such as cystic fibrosis patients, who frequently receive repeated treatments with beta-lactam antibiotics. Our objective was to determine whether prolonged exposure to beta-lactam antibiotics can induce h-VISA. We exposed 3 clinical vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) strains to ceftazidime, ceftriaxone, imipenem, and vancomycin (as a control) at subinhibitory concentrations for 18 days in vitro. Population analyses showed progressive increases in vancomycin resistance; seven of the 12 derived strains obtained after induction were classified as h-VISA according to the following criteria: area under the curve (AUC) on day 18/AUC of Mu3 of ≥90% and/or growth on brain heart infusion (BHI) agar with 4 mg/liter vancomycin. The derived isolates had thickened cell walls proportional to the level of glycopeptide resistance. Genes known to be associated with glycopeptide resistance (vraSR, yvqF, SA1703, graRS, walKR, and rpoB) were PCR sequenced; no de novo mutations were observed upon beta-lactam exposure. To determine whether trfA, a gene encoding a glycopeptide resistance factor, was essential in the selection of h-VISA upon beta-lactam pressure, a trfA-knockout strain was generated by allelic replacement. Indeed, beta-lactam exposure of this mutated strain showed no capacity to induce vancomycin resistance. In conclusion, these results showed that beta-lactam antibiotics at subinhibitory concentrations can induce intermediate vancomycin resistance in vitro. This induction required an intact trfA locus. Our results suggest that prior use of beta-lactam antibiotics can compromise vancomycin efficacy in the treatment of MRSA infections.