Clinical Outcomes and Bacterial Characteristics of Carbapenem-resistant Acinetobacter baumannii Among Patients From Different Global Regions.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAb) is 1 of the most problematic antimicrobial-resistant bacteria. We sought to elucidate the international epidemiology and clinical impact of CRAb. METHODS: In a prospective observational cohort study, 842 hospitalized patients with a clinical CRAb culture were enrolled at 46 hospitals in five global regions between 2017 and 2019. The primary outcome was all-cause mortality at 30 days from the index culture. The strains underwent whole-genome analysis. RESULTS: Of 842 cases, 536 (64%) represented infection. By 30 days, 128 (24%) of the infected patients died, ranging from 1 (6%) of 18 in Australia-Singapore to 54 (25%) of 216 in the United States and 24 (49%) of 49 in South-Central America, whereas 42 (14%) of non-infected patients died. Bacteremia was associated with a higher risk of death compared with other types of infection (40 [42%] of 96 vs 88 [20%] of 440). In a multivariable logistic regression analysis, bloodstream infection and higher age-adjusted Charlson comorbidity index were independently associated with 30-day mortality. Clonal group 2 (CG2) strains predominated except in South-Central America, ranging from 216 (59%) of 369 in the United States to 282 (97%) of 291 in China. Acquired carbapenemase genes were carried by 769 (91%) of the 842 isolates. CG2 strains were significantly associated with higher levels of meropenem resistance, yet non-CG2 cases were over-represented among the deaths compared with CG2 cases. CONCLUSIONS: CRAb infection types and clinical outcomes differed significantly across regions. Although CG2 strains remained predominant, non-CG2 strains were associated with higher mortality. Clinical Trials Registration. NCT03646227.

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Prevalence and Characterization of the Cefazolin Inoculum Effect in North American Methicillin-Susceptible Staphylococcus aureus Isolates.

Antistaphylococcal penicillins and cefazolin remain the primary treatments for infections with methicillin-susceptible Staphylococcus aureus (MSSA). The cefazolin inoculum effect (CzIE) causes the cefazolin MIC to be elevated in proportion to the number of bacteria in the inoculum. The objective of this multicenter study was to evaluate the prevalence of the CzIE in North American MSSA isolates. Clinical MSSA isolates from six microbiology laboratories in the United States and one microbiology laboratory in Canada were screened for the CzIE by broth microdilution at a standard inoculum (~5 × 105 CFU/mL) and a high inoculum (~5 × 107 CFU/mL). Genome sequencing was performed to further characterize the MSSA isolates. The CzIE was present in 57/305 (18.6%) MSSA isolates, ranging from 0% to 27.9% across study sites. More of the CzIE-positive isolates (29.8%) had standard inoculum cefazolin MICs of 1.0 µg/mL than the CzIE-negative isolates did (3.2%) (P < 0.0001). Conversely, more CzIE-negative isolates (39.5%) had standard inoculum MICs of 0.25 µg/mL than the CzIE positive isolates did (5.3%) (P < 0.0001). The most common BlaZ ß-lactamase types found in the CzIE-positive strains were type C (53.7%) and type A (44.4%). ST8 and ST30 were the most common sequence types among CzIE-positive isolates and correlated with BlaZ type C and A, respectively. The CzIE was present in up to a quarter of clinical MSSA isolates from North American clinical laboratories. Further studies to determine the impact of the presence of the CzIE on clinical outcomes are needed.

Multiomics characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates with heterogeneous intermediate resistance to vancomycin (hVISA) in Latin America.

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) compromise the clinical efficacy of vancomycin. The hVISA isolates spontaneously produce vancomycin-intermediate Staphylococcus aureus (VISA) cells generated by diverse and intriguing mechanisms. OBJECTIVE: To characterize the biomolecular profile of clinical hVISA applying genomic, transcriptomic and metabolomic approaches. METHODS: 39 hVISA and 305 VSSA and their genomes were included. Core genome-based Bayesian phylogenetic reconstructions were built and alterations in predicted proteins in VISA/hVISA were interrogated. Linear discriminant analysis and a Genome-Wide Association Study were performed. Differentially expressed genes were identified in hVISA-VSSA by RNA-sequencing. The undirected profiles of metabolites were determined by liquid chromatography and hydrophilic interaction in six CC5-MRSA. RESULTS: Genomic relatedness of MRSA associated to hVISA phenotype was not detected. The change Try38 → His in Atl (autolysin) was identified in 92% of the hVISA. We identified SNPs and k-mers associated to hVISA in 11 coding regions with predicted functions in virulence, transport systems, carbohydrate metabolism and tRNA synthesis. Further, capABCDE, sdrD, esaA, esaD, essA and ssaA genes were overexpressed in hVISA, while lacABCDEFG genes were downregulated. Additionally, valine, threonine, leucine tyrosine, FAD and NADH were more abundant in VSSA, while arginine, glycine and betaine were more abundant in hVISA. Finally, we observed altered metabolic pathways in hVISA, including purine and pyrimidine pathway, CoA biosynthesis, amino acid metabolism and aminoacyl tRNA biosynthesis. CONCLUSIONS: Our results show that the mechanism of hVISA involves major changes in regulatory systems, expression of virulence factors and reduction in glycolysis via TCA cycle. This work contributes to the understanding of the development of this complex resistance mechanism in regional strains.

Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host-pathogen interactions.

A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus.

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-ß-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (107 CFU/ml) was used as a reference standard (cutoff ≥16 µg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 µg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.

The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP-R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR-independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to antibiotics. We adapted E. faecalis OG1RF (a laboratory strain) and S613TM (a clinical strain) lacking liaR to increasing concentrations of DAP, leading to the development of DAP-R and elevated MICs to bacitracin and ceftriaxone. Whole genome sequencing identified changes in the YxdJK two-component regulatory system and a putative fatty acid kinase (dak) in both DAP-R strains. Deletion of the gene encoding the YxdJ response regulator in both the DAP-R mutant and wild-type OG1RF decreased MICs to DAP, even when a functional LiaFSR system was present. Mutations in dak were associated with slower growth, decreased membrane fluidity and alterations of cell morphology. These findings suggest that overlapping stress response pathways can provide protection against antimicrobial peptides in E. faecalis at a significant cost in bacterial fitness.

Novel Insights into the Classification of Staphylococcal ß-Lactamases in Relation to the Cefazolin Inoculum Effect.

Cefazolin has become a prominent therapy for methicillin-susceptible Staphylococcus aureus (MSSA) infections. However, an important concern is the cefazolin inoculum effect (CzIE), a phenomenon mediated by staphylococcal ß-lactamases. Four variants of staphylococcal ß-lactamases have been described based on serological methodologies and limited sequence information. Here, we sought to reassess the classification of staphylococcal ß-lactamases and their correlation with the CzIE. We included a large collection of 690 contemporary bloodstream MSSA isolates recovered from Latin America, a region with a high prevalence of the CzIE. We determined cefazolin MICs at standard and high inoculums by broth microdilution. Whole-genome sequencing was performed to classify the ß-lactamase in each isolate based on the predicted full sequence of BlaZ. We used the classical schemes for ß-lactamase classification and compared it to BlaZ allotypes found in unique sequences using the genomic information. Phylogenetic analyses were performed based on the BlaZ and core-genome sequences. The overall prevalence of the CzIE was 40%. Among 641 genomes, type C was the most predominant ß-lactamase (37%), followed by type A (33%). We found 29 allotypes and 43 different substitutions in BlaZ. A single allotype, designated BlaZ-2, showed a robust and statistically significant association with the CzIE. Two other allotypes (BlaZ-3 and BlaZ-5) were associated with a lack of the CzIE. Three amino acid substitutions (A9V, E112A, and G145E) showed statistically significant association with the CzIE (P = <0.01). CC30 was the predominant clone among isolates displaying the CzIE. Thus, we provide a novel approach to the classification of the staphylococcal ß-lactamases with the potential to more accurately identify MSSA strains exhibiting the CzIE.

Detection of heterogeneous vancomycin intermediate resistance in MRSA isolates from Latin America.

BACKGROUND: Vancomycin is a common first-line option for MRSA infections. The heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) phenotype is associated with therapeutic failure. However, hVISA isolates are usually reported as vancomycin susceptible by routine susceptibility testing procedures. OBJECTIVES: To detect and characterize the hVISA phenotype in MRSA isolates causing infections in nine Latin American countries. METHODS: We evaluated a total of 1189 vancomycin-susceptible MRSA isolates recovered during 2006-08 and 2011-14. After an initial screening of hVISA using glycopeptide-supplemented agar strategies, the detection of hVISA was performed by Etest (GRD) and Macro-method (MET). Isolates deemed to be hVISA were subjected to population analysis profile/AUC (PAP/AUC) and WGS for further characterization. Finally, we interrogated alterations in predicted proteins associated with the development of the VISA phenotype in both hVISA and vancomycin-susceptible S. aureus (VSSA) genomes. RESULTS: A total of 39 MRSA isolates (3.3%) were classified as hVISA (1.4% and 5.6% in MRSA recovered from 2006-08 and 2011-14, respectively). Most of the hVISA strains (95%) belonged to clonal complex (CC) 5. Only 6/39 hVISA isolates were categorized as hVISA by PAP/AUC, with 6 other isolates close (0.87-0.89) to the cut-off (0.9). The majority of the 39 hVISA isolates exhibited the Leu-14→Ile (90%) and VraT Glu-156→Gly (90%) amino acid substitutions in WalK. Additionally, we identified 10 substitutions present only in hVISA isolates, involving WalK, VraS, RpoB and RpoC proteins. CONCLUSIONS: The hVISA phenotype exhibits low frequency in Latin America. Amino acid substitutions in proteins involved in cell envelope homeostasis and RNA synthesis were commonly identified. Our results suggest that Etest-based methods are an important alternative for the detection of hVISA clinical isolates.

Disrupting Membrane Adaptation Restores In Vivo Efficacy of Antibiotics Against Multidrug-Resistant Enterococci and Potentiates Killing by Human Neutrophils.

Daptomycin resistance in enterococci is often mediated by the LiaFSR system, which orchestrates the cell membrane stress response. Activation of LiaFSR through the response regulator LiaR generates major changes in cell membrane function and architecture (membrane adaptive response), permitting the organism to survive the antibiotic attack. Here, using a laboratory strain of Enterococcus faecalis, we developed a novel Caenorhabditis elegans model of daptomycin therapy and showed that disrupting LiaR-mediated cell membrane adaptation restores the in vivo activity of daptomycin. The LiaR effect was also seen in a clinical strain of daptomycin-resistant Enterococcus faecium, using a murine model of peritonitis. Furthermore, alteration of the cell membrane response increased the ability of human polymorphonuclear neutrophils to readily clear both E. faecalis and multidrug-resistant E. faecium. Our results provide proof of concept that targeting the cell membrane adaptive response restores the in vivo activity of antibiotics, prevents resistance, and enhances the ability of the innate immune system to kill infecting bacteria.

Linezolid- and Vancomycin-resistant Enterococcus faecium in Solid Organ Transplant Recipients: Infection Control and Antimicrobial Stewardship Using Whole Genome Sequencing.

BACKGROUND: Vancomycin-resistant enterococci are an important cause of healthcare-associated infections and are inherently resistant to many commonly used antibiotics. Linezolid is the only drug currently approved by the US Food and Drug Administration to treat vancomycin-resistant enterococci; however, resistance to this antibiotic appears to be increasing. Although outbreaks of linezolid- and vancomycin-resistant Enterococcus faecium (LR-VRE) in solid organ transplant recipients remain uncommon, they represent a major challenge for infection control and hospital epidemiology. METHODS: We describe a cluster of 4 LR-VRE infections among a group of liver and multivisceral transplant recipients in a single intensive care unit. Failure of treatment with linezolid in 2 cases led to a review of standard clinical laboratory methods for susceptibility determination. Testing by alternative methods including whole genome sequencing (WGS) and a comprehensive outbreak investigation including sampling of staff members and surfaces was performed. RESULTS: Review of laboratory testing methods revealed a limitation in the VITEK 2 system with regard to reporting resistance to linezolid. Linezolid resistance in all cases was confirmed by E-test method. The use of WGS identified a resistant subpopulation with the G2376C mutation in the 23S ribosomal RNA. Sampling of staff members' dominant hands as well as sampling of surfaces in the unit identified no contaminated sources for transmission. CONCLUSIONS: This cluster of LR-VRE in transplant recipients highlights the possible shortcomings of standard microbiology laboratory methods and underscores the importance of WGS to identify resistance mechanisms that can inform patient care, as well as infection control and antibiotic stewardship measures.

Staphylococcus aureus bloodstream infections in Latin America: results of a multinational prospective cohort study.

BACKGROUND: Substantial heterogeneity in the epidemiology and management of Staphylococcus aureus bacteraemia (SAB) occurs in Latin America. We conducted a prospective cohort study in 24 hospitals from nine Latin American countries. OBJECTIVES: To assess the clinical impact of SAB in Latin America. PATIENTS AND METHODS: We evaluated differences in the 30 day attributable mortality among patients with SAB due to MRSA compared with MSSA involving 84 days of follow-up. Adjusted relative risks were calculated using a generalized linear model. RESULTS: A total of 1030 patients were included. MRSA accounted for 44.7% of cases with a heterogeneous geographical distribution. MRSA infection was associated with higher 30 day attributable mortality [25% (78 of 312) versus 13.2% (48 of 363), adjusted RR: 1.94, 95% CI: 1.38-2.73, P < 0.001] compared with MSSA in the multivariable analysis based on investigators' assessment, but not in a per-protocol analysis [13% (35 of 270) versus 8.1% (28 of 347), adjusted RR: 1.10, 95% CI: 0.75-1.60, P = 0.616] or in a sensitivity analysis using 30 day all-cause mortality [36% (132 of 367) versus 27.8% (123 of 442), adjusted RR: 1.09, 95% CI: 0.96-1.23, P = 0.179]. MRSA infection was not associated with increased length of hospital stay. Only 49% of MSSA bloodstream infections (BSI) received treatment with ß-lactams, but appropriate definitive treatment was not associated with lower mortality (adjusted RR: 0.93, 95% CI: 0.70-1.23, P = 0.602). CONCLUSIONS: MRSA-BSIs in Latin America are not associated with higher 30 day mortality or longer length of stay compared with MSSA. Management of MSSA-BSIs was not optimal, but appropriate definitive therapy did not appear to influence mortality.

A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci.

Substitutions in the LiaFSR membrane stress pathway are frequently associated with the emergence of antimicrobial peptide resistance in both Enterococcus faecalis and Enterococcus faecium Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology, including stress responses. We have previously identified a mutation in a gene (designated yybT) in E. faecalis that was associated with the development of daptomycin resistance, resulting in a change at position 440 (yybTI440S) in the predicted protein. Here, we show that intracellular c-di-AMP signaling is present in enterococci, and on the basis of in vitro physicochemical characterization, we show that E. faecalisyybT encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5'pApA. The E. faecalis GdpPI440S substitution reduces c-di-AMP phosphodiesterase activity more than 11-fold, leading to further increases in c-di-AMP levels. Additionally, deletions of liaR (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both E. faecalis and E. faecium also resulted in increased c-di-AMP levels, suggesting that changes in the LiaFSR stress response pathway are linked to broader physiological changes. Taken together, our data show that modulation of c-di-AMP pools is strongly associated with antibiotic-induced cell membrane stress responses via changes in GdpP activity or signaling through the LiaFSR system.

Genomic and Molecular Characterization of Clinical Isolates of Enterobacteriaceae Harboring mcr-1 in Colombia, 2002 to 2016.

Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1-positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1, including 8 Escherichia coli, 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate. E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10-5 to 2.07 × 10-1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids.

A Prospective Cohort Multicenter Study of Molecular Epidemiology and Phylogenomics of Staphylococcus aureus Bacteremia in Nine Latin American Countries.

Staphylococcus aureus is an important pathogen causing a spectrum of diseases ranging from mild skin and soft tissue infections to life-threatening conditions. Bloodstream infections are particularly important, and the treatment approach is complicated by the presence of methicillin-resistant S. aureus (MRSA) isolates. The emergence of new genetic lineages of MRSA has occurred in Latin America (LA) with the rise and dissemination of the community-associated USA300 Latin American variant (USA300-LV). Here, we prospectively characterized bloodstream MRSA recovered from selected hospitals in 9 Latin American countries. All isolates were typed by pulsed-field gel electrophoresis (PFGE) and subjected to antibiotic susceptibility testing. Whole-genome sequencing was performed on 96 MRSA representatives. MRSA represented 45% of all (1,185 S. aureus) isolates. The majority of MRSA isolates belonged to clonal cluster (CC) 5. In Colombia and Ecuador, most isolates (≥72%) belonged to the USA300-LV lineage (CC8). Phylogenetic reconstructions indicated that MRSA isolates from participating hospitals belonged to three major clades. Clade A grouped isolates with sequence type 5 (ST5), ST105, and ST1011 (mostly staphylococcal chromosomal cassette mec [SCCmec] I and II). Clade B included ST8, ST88, ST97, and ST72 strains (SCCmec IV, subtypes a, b, and c/E), and clade C grouped mostly Argentinian MRSA belonging to ST30. In summary, CC5 MRSA was prevalent in bloodstream infections in LA with the exception of Colombia and Ecuador, where USA300-LV is now the dominant lineage. Clonal replacement appears to be a common phenomenon, and continuous surveillance is crucial to identify changes in the molecular epidemiology of MRSA.

Transferable vancomycin resistance in a community-associated MRSA lineage.

We report the case of a patient from Brazil with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that was susceptible to vancomycin (designated BR-VSSA) but that acquired the vanA gene cluster during antibiotic therapy and became resistant to vancomycin (designated BR-VRSA). Both strains belong to the sequence type (ST) 8 community-associated genetic lineage that carries the staphylococcal chromosomal cassette mec (SCCmec) type IVa and the S. aureus protein A gene (spa) type t292 and are phylogenetically related to MRSA lineage USA300. A conjugative plasmid of 55,706 bp (pBRZ01) carrying the vanA cluster was identified and readily transferred to other staphylococci. The pBRZ01 plasmid harbors DNA sequences that are typical of the plasmid-associated replication genes rep24 or rep21 described in community-associated MRSA strains from Australia (pWBG745). The presence and dissemination of community-associated MRSA containing vanA could become a serious public health concern.

Influence of Minimum Inhibitory Concentration in Clinical Outcomes of Enterococcus faecium Bacteremia Treated With Daptomycin: Is it Time to Change the Breakpoint?

BACKGROUND: Daptomycin has become a front-line antibiotic for multidrug-resistant Enterococcus faecium bloodstream infections (BSIs). We previously showed that E. faecium strains with daptomycin minimum inhibitory concentrations (MICs) in the higher end of susceptibility frequently harbor mutations associated with daptomycin resistance. We postulate that patients with E. faecium BSIs exhibiting daptomycin MICs of 3-4 µg/mL treated with daptomycin are more likely to have worse clinical outcomes than those exhibiting daptomycin MICs ≤2 µg/mL. METHODS: We conducted a multicenter retrospective cohort study that included adult patients with E. faecium BSI for whom initial isolates, follow-up blood culture data, and daptomycin administration data were available. A central laboratory performed standardized daptomycin MIC testing for all isolates. The primary outcome was microbiologic failure, defined as clearance of bacteremia ≥4 days after the index blood culture. The secondary outcome was all-cause in-hospital mortality. RESULTS: A total of 62 patients were included. Thirty-one patients were infected with isolates that exhibited daptomycin MICs of 3-4 µg/mL. Overall, 34 patients had microbiologic failure and 25 died during hospitalization. In a multivariate logistic regression model, daptomycin MICs of 3-4 µg/mL (odds ratio [OR], 4.7 [1.37-16.12]; P = .014) and immunosuppression (OR, 5.32 [1.20-23.54]; P = .028) were significantly associated with microbiologic failure. Initial daptomycin dose of ≥8 mg/kg was not significantly associated with evaluated outcomes. CONCLUSIONS: Daptomycin MICs of 3-4 µg/mL in the initial E. faecium blood isolate predicted microbiological failure of daptomycin therapy, suggesting that modification in the daptomycin breakpoint for enterococci should be considered.

Exogenous Fatty Acids Protect Enterococcus faecalis from Daptomycin-Induced Membrane Stress Independently of the Response Regulator LiaR.

UNLABELLED: Enterococcus faecalis is a commensal bacterium of the gastrointestinal tract that can cause nosocomial infections in immunocompromised humans. The hallmarks of this organism are its ability to survive in a variety of stressful habitats and, in particular, its ability to withstand membrane damage. One strategy used by E. faecalis to protect itself from membrane-damaging agents, including the antibiotic daptomycin, involves incorporation of exogenous fatty acids from bile or serum into the cell membrane. Additionally, the response regulator LiaR (a member of the LiaFSR [lipid II-interacting antibiotic response regulator and sensor] system associated with cell envelope stress responses) is required for the basal level of resistance E. faecalis has to daptomycin-induced membrane damage. This study aimed to determine if membrane fatty acid changes could provide protection against membrane stressors in a LiaR-deficient strain of E. faecalis We noted that despite the loss of LiaR, the organism readily incorporated exogenous fatty acids into its membrane, and indeed growth in the presence of exogenous fatty acids increased the survival of LiaR-deficient cells when challenged with a variety of membrane stressors, including daptomycin. Combined, our results suggest that E. faecalis can utilize both LiaR-dependent and -independent mechanisms to protect itself from membrane damage. IMPORTANCE: Enterococcus faecalis is responsible for a significant number of nosocomial infections. Worse, many of the antibiotics used to treat E. faecalis infection are no longer effective, as this organism has developed resistance to them. The drug daptomycin has been successfully used to treat some of these resistant strains; however, daptomycin-resistant isolates have been identified in hospitals. Many daptomycin-resistant isolates are found to harbor mutations in the genetic locus liaFSR, which is involved in membrane stress responses. Another mechanism shown to increase tolerance to daptomycin involves the incorporation of exogenous fatty acids from host fluids like serum or bile. This improved tolerance was found to be independent of liaFSR and suggests that there are additional ways to impact sensitivity to daptomycin. Thus, further studies are needed to understand how host fatty acid sources can influence antibiotic susceptibility.

A liaR deletion restores susceptibility to daptomycin and antimicrobial peptides in multidrug-resistant Enterococcus faecalis.

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.

Parallel Epidemics of Community-Associated Methicillin-Resistant Staphylococcus aureus USA300 Infection in North and South America.

BACKGROUND: The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the United States is attributed to the spread of the USA300 clone. An epidemic of CA-MRSA closely related to USA300 has occurred in northern South America (USA300 Latin-American variant, USA300-LV). Using phylogenomic analysis, we aimed to understand the relationships between these 2 epidemics. METHODS: We sequenced the genomes of 51 MRSA clinical isolates collected between 1999 and 2012 from the United States, Colombia, Venezuela, and Ecuador. Phylogenetic analysis was used to infer the relationships and times since the divergence of the major clades. RESULTS: Phylogenetic analyses revealed 2 dominant clades that segregated by geographical region, had a putative common ancestor in 1975, and originated in 1989, in North America, and in 1985, in South America. Emergence of these parallel epidemics coincides with the independent acquisition of the arginine catabolic mobile element (ACME) in North American isolates and a novel copper and mercury resistance (COMER) mobile element in South American isolates. CONCLUSIONS: Our results reveal the existence of 2 parallel USA300 epidemics that shared a recent common ancestor. The simultaneous rapid dissemination of these 2 epidemic clades suggests the presence of shared, potentially convergent adaptations that enhance fitness and ability to spread.