Updating breakpoints in the United States: a summary from the ASM Clinical Microbiology Open 2022.

Accurate antimicrobial susceptibility testing (AST) and reporting are essential for guiding appropriate therapy for patients and direction for public health prevention and control actions. A critical feature of AST reporting is the interpretation of AST results using clinical breakpoints for reporting as susceptible, susceptible-dose dependent, intermediate, or resistant. Breakpoints are subject to continuous adjustment and updating to best reflect current clinical data. These breakpoint changes can benefit patients and public health only if adopted in a timely manner. A recent survey identified that up to 70% of College of American Pathologists (CAP)-accredited U.S. laboratories and 45% of CAP-accredited laboratories outside the U.S. use various obsolete clinical breakpoints to interpret AST results to guide patient care. The reason for the ongoing use of obsolete breakpoints is multifactorial, including barriers encountered by laboratories, commercial AST device manufacturers, standards development organizations, and regulatory bodies alike. To begin to address this important patient safety issue, CAP implemented checklist requirements for CAP-accredited laboratories to ensure up-to-date clinical breakpoint use. Furthermore, the topic was discussed at the June 2022 American Society for Microbiology Clinical Microbiology Open (CMO) with various stakeholders to identify potential solutions. This minireview summarizes the breakpoint setting process in the U.S. and highlights solutions to close the gap between breakpoint revisions and implementation in clinical and public health laboratories. Solutions discussed include clarification of data requirements and minimum inhibitory concentration only reporting for regulatory clearance of AST devices, clinical data generation to close breakpoints gaps, advocacy, education, and greater dialogue between stakeholders.

Assessing the in vitro impact of ceftazidime on aztreonam/avibactam susceptibility testing for highly resistant MBL-producing Enterobacterales.

BACKGROUND: Aztreonam/avibactam is a combination agent that shows promise in treating infections caused by highly antibiotic-resistant MBL-producing Enterobacterales. This combination can be achieved by combining two FDA-approved drugs: ceftazidime/avibactam and aztreonam. It is unknown whether ceftazidime in the combination ceftazidime/aztreonam/avibactam has a synergistic or antagonistic effect on the in vitro activity of aztreonam/avibactam by significantly increasing or decreasing the MIC. OBJECTIVES: To determine whether increasing ceftazidime concentrations affect the MICs of aztreonam/avibactam alone. METHODS: A custom 8 × 8 chequerboard broth microdilution (BMD) panel was made using a digital dispenser (Hewlett-Packard, Corvallis, OR, USA). The panel included orthogonal 2-fold dilution series of aztreonam and ceftazidime ranging from 0.5 to 64 mg/L. Avibactam concentration was kept constant at 4 mg/L throughout the chequerboard. Thirty-seven Enterobacterales isolates from the CDC & FDA Antibiotic Resistance Isolate Bank or CDC's internal collection with intermediate or resistant interpretations to aztreonam and ceftazidime/avibactam were included for testing. All isolates harboured at least one of the following MBL genes: blaIMP, blaNDM or blaVIM. RESULTS: Regardless of the concentration of ceftazidime, aztreonam/avibactam with ceftazidime MICs for all 37 isolates were within one 2-fold doubling dilution of the aztreonam/avibactam MIC. CONCLUSIONS: Ceftazidime, in the combination ceftazidime/avibactam/aztreonam, did not affect the in vitro activity of aztreonam/avibactam in this sample of isolates. These findings can help assure clinical and public health laboratories that testing of aztreonam/avibactam by BMD can act as a reliable surrogate test when the combination of ceftazidime/avibactam and aztreonam is being considered for treatment of highly antibiotic-resistant MBL-producing Enterobacterales.

Validation of Aztreonam-Avibactam Susceptibility Testing Using Digitally Dispensed Custom Panels.

Aztreonam-avibactam is a combination antimicrobial agent with activity against carbapenemase-producing Enterobacteriaceae (CPE) with metallo-ß-lactamases (MßLs). Although aztreonam-avibactam is not yet approved by the U.S. Food and Drug Administration (FDA), clinicians can administer this combination by using two FDA-approved drugs: aztreonam and ceftazidime-avibactam. This combination of drugs is recommended by multiple experts for treatment of serious infections caused by MßL-producing CPE. At present, in vitro antimicrobial susceptibility testing (AST) of aztreonam-avibactam is not commercially available; thus, most clinicians receive no laboratory-based guidance that can support consideration of aztreonam-avibactam for serious CPE infections. Here, we report our internal validation for aztreonam-avibactam AST by reference broth microdilution (BMD) according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The validation was performed using custom frozen reference BMD panels prepared in-house at the Centers for Disease Control and Prevention (CDC). In addition, we took this opportunity to evaluate a new panel-making method using a digital dispenser, the Hewlett Packard (HP) D300e. Our studies demonstrate that the performance characteristics of digitally dispensed panels were equivalent to those of conventionally prepared frozen reference BMD panels for a number of drugs, including aztreonam-avibactam. We found the HP D300e digital dispenser to be easy to use and to provide the capacity to prepare complex drug panels. Our findings will help other clinical and public health laboratories implement susceptibility testing for aztreonam-avibactam.

Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing Position Statements on Polymyxin B and Colistin Clinical Breakpoints.

Recent data on polymyxin pharmacokinetics, pharmacodynamics, toxicity, and clinical outcomes suggest these agents have limited clinical utility. Pharmacokinetics-pharmacodynamics data show a steady-state concentration of 2 µg/mL is required for killing bacteria with colistin minimum inhibitory concentrations of 2 µg/mL. Less than 50% of patients with normal renal function achieve this exposure, and it is associated with high risk of nephrotoxicity. This exposure does not achieve bacterial stasis in pneumonia models. Randomized and observational studies consistently demonstrate increased mortality for polymyxins compared with alternative agents. The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) are 2 global organizations that establish interpretive criteria for in vitro susceptibility data. CLSI has recently taken the step to eliminate the "susceptible" interpretive category for the polymyxins, whereas EUCAST maintains this interpretive category. This viewpoint describes the opinions of these organizations and the data that were used to inform their perspectives.

Rationale for a Neisseria gonorrhoeae Susceptible-only Interpretive Breakpoint for Azithromycin.

BACKGROUND: Azithromycin (AZI) is recommended with ceftriaxone (CRO) for treatment of uncomplicated gonococcal urethritis and cervicitis in the United States, and an AZI-susceptibility breakpoint is needed. Neither the Food and Drug Administration (FDA) nor the Clinical and Laboratory Standards Institute (CLSI) has set interpretive breakpoints for AZI susceptibility. As a result, AZI antimicrobial susceptibility testing (AST) cannot be interpreted using recognized standards. This has contributed to increasingly unavailable clinical laboratory AST, although gonorrhea is on the rise with >550 000 US gonorrhea cases reported to the Centers for Disease Control and Prevention in 2017, the highest number of cases since 1991. METHODS: This article summarizes the rationale data reviewed by the CLSI in June 2018. RESULTS: The CLSI decided to set a susceptible-only interpretive breakpoint at the minimum inhibitory concentration of ≤1 µg/mL. This is also the epidemiological cutoff value (ECV) (ie, the end of the wild-type susceptibility distribution). This breakpoint presumes that AZI (1-g single dose) is used in an approved regimen that includes an additional antimicrobial agent (ie, CRO 250 mg, intramuscular single dose). CONCLUSIONS: Having a breakpoint can improve patient care and surveillance and allow future development and FDA regulatory approval of modernized AST to guide treatment. The breakpoint coincides with a European Committee on AST decision to remove previously established, differing AZI breakpoints and use the ECV as guidance for testing. The CLSI breakpoint is now the recognized standard that defines AZI susceptibility for gonococcal infections.

Harnessing alternative sources of antimicrobial resistance data to support surveillance in low-resource settings.

One of the most pressing challenges facing the global surveillance of antimicrobial resistance (AMR) is the generation, sharing, systematic analysis and dissemination of data in low-resource settings. Numerous agencies and initiatives are working to support the development of globally distributed microbiology capacity, but the routine generation of a sustainable flow of reliable data will take time to establish before it can deliver a clinical and public health impact. By contrast, there are a large number of pharma- and academia-led initiatives that have generated a wealth of data on AMR and drug-resistant infections in low-resource settings, together with high-volume data generation by private laboratories. Here, we explore how untapped sources of data could provide a short-term solution that bridges the gap between now and the time when routine surveillance capacity will have been established and how this could continue to support surveillance efforts in the future. We discuss the benefits and limitations of data generated by these sources, the mechanisms and barriers to making this accessible and how academia and pharma might support the development of laboratory and analytical capacity. We provide key actions that will be required to harness these data, including: a mapping exercise; creating mechanisms for data sharing; use of data to support national action plans; facilitating access to and use of data by the WHO Global Antimicrobial Resistance Surveillance System; and innovation in data capture, analysis and sharing.

Surveillance and Epidemiology of Drug Resistant Infections Consortium (SEDRIC): Supporting the transition from strategy to action.

In recognition of the central importance of surveillance and epidemiology in the control of antimicrobial resistance and the need to strengthen surveillance at all levels, Wellcome has brought together a new international expert group SEDRIC (Surveillance and Epidemiology of Drug Resistant Infections Consortium). SEDRIC aims to advance and transform the ways of tracking, sharing and analysing rates of infection and drug resistance, burden of disease, information on antibiotic use, opportunities for preventative measures such as vaccines, and contamination of the environment. SEDRIC will strengthen the availability of information needed to monitor and track risks, including an evaluation of access to, and utility of data generated by pharma and research activities, and will support the translation of surveillance data into interventions, changes in policy and more effective practices. Ways of working will include the provision of independent scientific analysis, advocacy and expert advice to groups, such as the Wellcome Drug Resistant Infection Priority Programme. A priority for SEDRIC's first Working Group is to review mechanisms to strengthen the generation, collection, collation and dissemination of high quality data, together with the need for creativity in the use of existing data and proxy measures, and linking to existing in-country networking infrastructure. SEDRIC will also promote the translation of technological innovations into public health solutions.

Vital Signs: Containment of Novel Multidrug-Resistant Organisms and Resistance Mechanisms - United States, 2006-2017.

BACKGROUND: Approaches to controlling emerging antibiotic resistance in health care settings have evolved over time. When resistance to broad-spectrum antimicrobials mediated by extended-spectrum ß-lactamases (ESBLs) arose in the 1980s, targeted interventions to slow spread were not widely promoted. However, when Enterobacteriaceae with carbapenemases that confer resistance to carbapenem antibiotics emerged, directed control efforts were recommended. These distinct approaches could have resulted in differences in spread of these two pathogens. CDC evaluated these possible changes along with initial findings of an enhanced antibiotic resistance detection and control strategy that builds on interventions developed to control carbapenem resistance. METHODS: Infection data from the National Healthcare Safety Network from 2006-2015 were analyzed to calculate changes in the annual proportion of selected pathogens that were nonsusceptible to extended-spectrum cephalosporins (ESBL phenotype) or resistant to carbapenems (carbapenem-resistant Enterobacteriaceae [CRE]). Testing results for CRE and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are also reported. RESULTS: The percentage of ESBL phenotype Enterobacteriaceae decreased by 2% per year (risk ratio [RR] = 0.98, p<0.001); by comparison, the CRE percentage decreased by 15% per year (RR = 0.85, p<0.01). From January to September 2017, carbapenemase testing was performed for 4,442 CRE and 1,334 CRPA isolates; 32% and 1.9%, respectively, were carbapenemase producers. In response, 1,489 screening tests were performed to identify asymptomatic carriers; 171 (11%) were positive. CONCLUSIONS: The proportion of Enterobacteriaceae infections that were CRE remained lower and decreased more over time than the proportion that were ESBL phenotype. This difference might be explained by the more directed control efforts implemented to slow transmission of CRE than those applied for ESBL-producing strains. Increased detection and aggressive early response to emerging antibiotic resistance threats have the potential to slow further spread.

Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis.

BACKGROUND: The spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide. Due to its large public health and societal implications, multidrug-resistant tuberculosis has been long regarded by WHO as a global priority for investment in new drugs. In 2016, WHO was requested by member states to create a priority list of other antibiotic-resistant bacteria to support research and development of effective drugs. METHODS: We used a multicriteria decision analysis method to prioritise antibiotic-resistant bacteria; this method involved the identification of relevant criteria to assess priority against which each antibiotic-resistant bacterium was rated. The final priority ranking of the antibiotic-resistant bacteria was established after a preference-based survey was used to obtain expert weighting of criteria. FINDINGS: We selected 20 bacterial species with 25 patterns of acquired resistance and ten criteria to assess priority: mortality, health-care burden, community burden, prevalence of resistance, 10-year trend of resistance, transmissibility, preventability in the community setting, preventability in the health-care setting, treatability, and pipeline. We stratified the priority list into three tiers (critical, high, and medium priority), using the 33rd percentile of the bacterium's total scores as the cutoff. Critical-priority bacteria included carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, and carbapenem-resistant and third-generation cephalosporin-resistant Enterobacteriaceae. The highest ranked Gram-positive bacteria (high priority) were vancomycin-resistant Enterococcus faecium and meticillin-resistant Staphylococcus aureus. Of the bacteria typically responsible for community-acquired infections, clarithromycin-resistant Helicobacter pylori, and fluoroquinolone-resistant Campylobacter spp, Neisseria gonorrhoeae, and Salmonella typhi were included in the high-priority tier. INTERPRETATION: Future development strategies should focus on antibiotics that are active against multidrug-resistant tuberculosis and Gram-negative bacteria. The global strategy should include antibiotic-resistant bacteria responsible for community-acquired infections such as Salmonella spp, Campylobacter spp, N gonorrhoeae, and H pylori. FUNDING: World Health Organization.

FDA-CDC Antimicrobial Resistance Isolate Bank: a Publicly Available Resource To Support Research, Development, and Regulatory Requirements.

The FDA-CDC Antimicrobial Resistance Isolate Bank was created in July 2015 as a publicly available resource to combat antimicrobial resistance. It is a curated repository of bacterial isolates with an assortment of clinically important resistance mechanisms that have been phenotypically and genotypically characterized. In the first 2 years of operation, the bank offered 14 panels comprising 496 unique isolates and had filled 486 orders from 394 institutions throughout the United States. New panels are being added.

Diagnosing antimicrobial resistance.

Antimicrobial resistance constitutes a global burden and is one of the major threats to public health. Although the emergence of resistant microorganisms is a natural phenomenon, the overuse or inappropriate use of antimicrobials has had a great effect on resistance evolution. Rapid diagnostic tests that identify drug-resistant bacteria, determine antimicrobial susceptibility and distinguish viral from bacterial infections can guide effective treatment strategies. Moreover, rapid diagnostic tests could facilitate epidemiological surveillance, as emerging resistant infectious agents and transmission can be monitored. In this Viewpoint article, several experts in the field discuss the drawbacks of current diagnostic methods that are used to identify antimicrobial resistance, novel diagnostic strategies and how such rapid tests can inform drug development and the surveillance of resistance evolution.

Antimicrobial-Resistant Pathogens Associated With Healthcare-Associated Infections: Summary of Data Reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2011-2014.

OBJECTIVE To describe antimicrobial resistance patterns for healthcare-associated infections (HAIs) that occurred in 2011-2014 and were reported to the Centers for Disease Control and Prevention's National Healthcare Safety Network. METHODS Data from central line-associated bloodstream infections, catheter-associated urinary tract infections, ventilator-associated pneumonias, and surgical site infections were analyzed. These HAIs were reported from acute care hospitals, long-term acute care hospitals, and inpatient rehabilitation facilities. Pooled mean proportions of pathogens that tested resistant (or nonsusceptible) to selected antimicrobials were calculated by year and HAI type. RESULTS Overall, 4,515 hospitals reported that at least 1 HAI occurred in 2011-2014. There were 408,151 pathogens from 365,490 HAIs reported to the National Healthcare Safety Network, most of which were reported from acute care hospitals with greater than 200 beds. Fifteen pathogen groups accounted for 87% of reported pathogens; the most common included Escherichia coli (15%), Staphylococcus aureus (12%), Klebsiella species (8%), and coagulase-negative staphylococci (8%). In general, the proportion of isolates with common resistance phenotypes was higher among device-associated HAIs compared with surgical site infections. Although the percent resistance for most phenotypes was similar to earlier reports, an increase in the magnitude of the resistance percentages among E. coli pathogens was noted, especially related to fluoroquinolone resistance. CONCLUSION This report represents a national summary of antimicrobial resistance among select HAIs and phenotypes. The distribution of frequent pathogens and some resistance patterns appear to have changed from 2009-2010, highlighting the need for continual, careful monitoring of these data across the spectrum of HAI types. Infect Control Hosp Epidemiol 2016;1-14.

Use of a Clinical Decision Support System Alert to Prevent Supratherapeutic Vancomycin Concentrations.

Background: Alerts issued by clinical decision support systems (CDSS) may be useful to identify and prevent the occurrence of acute kidney injury among patients on nephrotoxic drugs, particularly vancomycin. Objective: The purpose of this instructive study was to determine the effectiveness of using a pharmacist-run CDSS alert of early serum creatinine increases in patients receiving intravenous vancomycin to decrease the proportion of severely elevated vancomycin concentrations. Methods: This was a retrospective study of a prospectively reviewed CDSS alert that triggered in patients with an increase in serum creatinine by 25% from baseline within 24 hours. Severely elevated vancomycin concentrations were divided into a control group (before alert implementation) and a study group (after alert implementation) and considered for study inclusion. The proportion of severely elevated vancomycin concentrations (ie, >30 mg/L) were collected in the control and study groups. Results: There were 1290 and 1501 vancomycin concentrations in the control group and the study group, respectively. A total of 696 CDSS alerts triggered during the study period. The proportion of severely elevated vancomycin troughs decreased from 5.3% (n = 68, median = 36.6 mg/L, interquartile range = 33.75-43.2 mg/L) in the control group to 3.7% (n = 55, median = 34.7 mg/L, interquartile range = 31.3-39.3 mg/L) in the study group. This reflects a statistically significant decrease in the proportion of severely elevated vancomycin concentrations (P = .04). Conclusion: Overall, this instructive analysis on a novel use of CDSS software suggests that the implementation of an alert based on early detection of serum creatinine changes led to a significant decrease in the proportion of severely elevated serum vancomycin concentrations.

Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases.

Klebsiella pneumoniae carbapenemases (KPCs) were originally identified in the USA in 1996. Since then, these versatile ß-lactamases have spread internationally among Gram-negative bacteria, especially K pneumoniae, although their precise epidemiology is diverse across countries and regions. The mortality described among patients infected with organisms positive for KPC is high, perhaps as a result of the limited antibiotic options remaining (often colistin, tigecycline, or aminoglycosides). Triple drug combinations using colistin, tigecycline, and imipenem have recently been associated with improved survival among patients with bacteraemia. In this Review, we summarise the epidemiology of KPCs across continents, and discuss issues around detection, present antibiotic options and those in development, treatment outcome and mortality, and infection control. In view of the limitations of present treatments and the paucity of new drugs in the pipeline, infection control must be our primary defence for now.

Development of doxycycline MIC and disk diffusion interpretive breakpoints and revision of tetracycline breakpoints for Streptococcus pneumoniae.

A study was performed to derive susceptibility testing interpretive breakpoints for doxycycline with Streptococcus pneumoniae and to reassess breakpoints for tetracycline using the requirements defined in Clinical and Laboratory Standards Institute (CLSI) document M23-A3. Tetracycline and doxycycline MICs and disk diffusion zone sizes were determined on 189 isolates selected from the 2009-2010 CDC Active Bacterial Core surveillance strain collection according to the testing methods described in CLSI documents M07-A8 and M02-A10. Tetracycline and doxycycline MICs and zones were compared to each other directly, and the reproducibility of MICs and zone diameters for both drugs was determined. Scattergrams of tetracycline MICs versus corresponding zone diameters and doxycycline MICs versus zones were prepared, and analysis indicated that the present CLSI tetracycline MIC and disk breakpoints did not fit the susceptibility data for doxycycline. Doxycycline was 1 to 3 dilutions more potent than tetracycline, especially in strains harboring the tetM resistance determinant. tetM was detected in ≥ 90% of isolates having tetracycline MICs of ≥ 4 µg/ml and in ≥ 90% with doxycycline MICs of ≥ 1. Limited pharmacokinetic/pharmacodynamic (PK/PD) data coupled with application of the error-rate bounded method of analysis suggested doxycycline-susceptible breakpoints of either ≤ 0.25 µg/ml or ≤ 0.5 µg/ml, with intermediate and resistant breakpoints 1 and 2 dilutions higher, respectively. The disk diffusion zone diameter correlates were susceptible at ≥ 28 mm, intermediate at 25 to 27 mm, and resistant at ≤ 24 mm. Revised lower tetracycline MIC breakpoints were suggested as susceptible at ≤ 1 µg/ml, intermediate at 2 µg/ml, and resistant at ≥ 4 µg/ml. Suggested tetracycline disk diffusion zones were identical to those of doxycycline.

Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010.

OBJECTIVE: To describe antimicrobial resistance patterns for healthcare-associated infections (HAIs) reported to the National Healthcare Safety Network (NHSN) during 2009-2010. METHODS: Central line-associated bloodstream infections, catheter-associated urinary tract infections, ventilator-associated pneumonia, and surgical site infections were included. Pooled mean proportions of isolates interpreted as resistant (or, in some cases, nonsusceptible) to selected antimicrobial agents were calculated by type of HAI and compared to historical data. RESULTS: Overall, 2,039 hospitals reported 1 or more HAIs; 1,749 (86%) were general acute care hospitals, and 1,143 (56%) had fewer than 200 beds. There were 69,475 HAIs and 81,139 pathogens reported. Eight pathogen groups accounted for about 80% of reported pathogens: Staphylococcus aureus (16%), Enterococcus spp. (14%), Escherichia coli (12%), coagulase-negative staphylococci (11%), Candida spp. (9%), Klebsiella pneumoniae (and Klebsiella oxytoca; 8%), Pseudomonas aeruginosa (8%), and Enterobacter spp. (5%). The percentage of resistance was similar to that reported in the previous 2-year period, with a slight decrease in the percentage of S. aureus resistant to oxacillins (MRSA). Nearly 20% of pathogens reported from all HAIs were the following multidrug-resistant phenotypes: MRSA (8.5%); vancomycin-resistant Enterococcus (3%); extended-spectrum cephalosporin-resistant K. pneumoniae and K. oxytoca (2%), E. coli (2%), and Enterobacter spp. (2%); and carbapenem-resistant P. aeruginosa (2%), K. pneumoniae/oxytoca (<1%), E. coli (<1%), and Enterobacter spp. (<1%). Among facilities reporting HAIs with 1 of the above gram-negative bacteria, 20%-40% reported at least 1 with the resistant phenotype. CONCLUSION: While the proportion of resistant isolates did not substantially change from that in the previous 2 years, multidrug-resistant gram-negative phenotypes were reported from a moderate proportion of facilities.

pSK41-like plasmid is necessary for Inc18-like vanA plasmid transfer from Enterococcus faecalis to Staphylococcus aureus in vitro.

Vancomycin-resistant Staphylococcus aureus (VRSA) is thought to result from the in vivo conjugative transfer of a vanA plasmid from an Enterococcus sp. to S. aureus. We studied bacterial isolates from VRSA cases that occurred in the United States to identify microbiological factors which may contribute to this plasmid transfer. First, vancomycin-susceptible, methicillin-resistant S. aureus (MRSA) isolates from five VRSA cases were tested for their ability to accept foreign DNA by conjugation in mating experiments with Enterococcus faecalis JH2-2 containing pAM378, a pheromone-response conjugative plasmid. All of the MRSA isolates accepted the plasmid DNA with similar transfer efficiencies (∼10(-7)/donor CFU) except for one isolate, MRSA8, for which conjugation was not successful. The MRSA isolates were also tested as recipients in mating experiments between an E. faecalis isolate with an Inc18-like vanA plasmid that was isolated from a VRSA case patient. Conjugative transfer was successful for 3/5 MRSA isolates. Successful MRSA recipients carried a pSK41-like plasmid, a staphylococcal conjugative plasmid, whereas the two unsuccessful MRSA recipients did not carry pSK41. The transfer of a pSK41-like plasmid from a successful MRSA recipient to the two unsuccessful recipients resulted in conjugal transfer of the Inc18-like vanA plasmid from E. faecalis at a frequency of 10(-7)/recipient CFU. In addition, conjugal transfer could be achieved for pSK41-negative MRSA in the presence of a cell-free culture filtrate from S. aureus carrying a pSK41-like plasmid at a frequency of 10(-8)/recipient CFU. These results indicated that a pSK41-like plasmid can facilitate the transfer of an Inc18-like vanA plasmid from E. faecalis to S. aureus, possibly via an extracellular factor produced by pSK41-carrying isolates.