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Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability, optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions.

Although the TEM-1 β-lactamase (Bla TEM-1 ) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to β-lactam/β-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only bla TEM-1 One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher β-lactamase activity and Bla TEM-1 protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in bla TEM-1 copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in bla TEM-1 gene dosage, allowing Bla TEM-1 to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the bla TEM-1 copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the bla TEM-1 gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP. IMPORTANCE Understanding how bacteria acquire resistance to antibiotics is essential for treating infected patients effectively, as well as preventing the spread of resistant organisms. In this study, a clinical isolate of E. coli was identified that dedicated more than 15% of its genome toward tandem amplification of a ~10-kb resistance module, allowing it to escape antibiotic-mediated killing. Our research is significant in that it provides one possible explanation for clinical isolates that exhibit discordant behavior when tested for antibiotic resistance by different phenotypic methods. Our research also shows that GRM amplification is difficult to detect by short-read WGS technologies. Analysis of raw long-read sequence data was required to confirm GRM amplification as a mechanism of antibiotic resistance., (Copyright © 2018 Schechter et al.)

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The capacity of absorbent beads in BacT/ALERT® FA Plus and BACTEC® Aerobic/F Plus blood culture bottles to bind and neutralize antibiotics was compared. Binding was established using reverse-phase HPLC, and inactivation was based on the recovery of susceptible test stains from simulated blood cultures. The FA Plus medium demonstrated more rapid and better overall binding kinetics for each drug tested, resulting in significantly better overall recovery rates. Differences in time to detection favored the FA Plus medium for three drug/organism combinations and Aerobic/F Plus for two.

Population analysis was performed for 42 Escherichia coli isolates to determine whether heterogeneity of resistance was a factor in piperacillin-tazobactam category differences between agar dilution and broth microdilution. Of 20 isolates discordant between methods, 80% were heterogeneous. Of 22 isolates in agreement, 59% were homogeneous. Heterogeneity and homogeneity rates for those in agreement were significantly different from those that were discordant (P value, 0.010). Heterogeneity of resistance expression appears to be an important factor in category differences observed between broth microdilution and agar dilution for piperacillin-tazobactam.

Preventing the dissemination of antimicrobial resistance depends on appropriate antibiotic stewardship and accurate antimicrobial susceptibility testing (AST). We report the international dissemination of Escherichia coli strains, showing discrepancies between reference methods when phenotypically tested for susceptibility to piperacillin/tazobactam (TZP). We demonstrate that these related strains are predisposed to problematic TZP AST interpretations.

Background: Optimal treatment regimens for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are not well-defined., Objectives: This study describes the treatment and outcomes in patients with urinary tract infection (UTI) caused by KPC-producing Enterobacteriaceae., Methods: This retrospective cohort study analyzed data from adult inpatients with bacteriuria caused by KPC-positive organisms treated at Barnes-Jewish Hospital from June 1, 2006, to February 1, 2008. KPC-positive isolates were identified utilizing disk-diffusion susceptibility testing and confirmed to contain bla(KPC) via molecular methods., Results: Twenty-one patients met the inclusion criteria and all were classified as having symptomatic UTI. The majority of patients were female (15/21 [71%]), and the mean (SD) age was 62.4 (15.2) years. Successful clinical and microbiologic responses were observed in 16 patients (76%) for both outcomes. Patients with urinary catheters had them removed or replaced in 9 of 15 cases (60%). Antibiotics active against the isolated pathogen were provided in 14 of 21 cases (67%), often after considerable delay (median, 72.5 hours [range, 4-312 hours]). All 7 patients receiving aminoglycoside therapy had successful clinical and microbiologic responses, and in vitro testing of an extended antibiotic panel revealed high susceptibility rates for tigecycline (28/29 [97%]), minocycline (22/29 [76%]), and fosfomycin (25/29 [86%]) against the KPC-positive isolates., Conclusions: Although receipt of appropriate therapy was delayed in many cases, clinical outcomes investigated revealed [corrected] high rates of successful response in this defined group of patients Aminoglycosides and tetracycline derivatives suggested therapeutic promise in the treatment of KPC-producing Enterobacteriaceae UTI., (Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.)

Objective: To determine whether exposure to antimicrobial agents in the previous 90 days resulted in decreased bacterial susceptibility and increased hospital mortality in patients with severe sepsis or septic shock attributed to Gram-negative bacteremia., Design: A retrospective cohort study of hospitalized patients (January 2002 to December 2007)., Setting: Barnes-Jewish Hospital, a 1200-bed urban teaching hospital., Patients: Seven hundred fifty-four consecutive patients with Gram-negative bacteremia complicated by severe sepsis or septic shock., Interventions: Data abstraction from computerized medical records., Measurements and Main Results: Escherichia coli (30.8%), Klebsiella pneumoniae (23.2%), and Pseudomonas aeruginosa (17.6%) were the most common isolates from blood cultures. Three hundred ten patients (41.1%) had recent antibiotic exposure. Cefepime was the most common agent with previous exposure (50.0%) followed by ciprofloxacin (32.6%) and imipenem or meropenem (28.7%). Patients with prior antibiotic exposure had significantly higher rates of resistance to cefepime (29.0% vs. 7.0%), piperacillin/tazobactam (31.9% vs. 11.5%), carbapenems (20.0% vs. 2.5%), ciprofloxacin (39.7% vs. 17.6%), and gentamicin (26.1% vs. 7.9%) (p < .001 for all comparisons). Patients with recent antibiotic exposure had greater inappropriate initial antimicrobial therapy (45.4% vs. 21.2%; p < .001) and hospital mortality (51.3% vs. 34.0%; p < .001) compared with patients without recent antibiotic exposure. Multivariate logistic regression analysis demonstrated that recent antibiotic exposure was independently associated with hospital mortality (adjusted odds ratio, 1.70; 95% confidence interval, 1.41-2.06; p = .005). Other variables independently associated with hospital mortality included use of vasopressors, infection resulting from P. aeruginosa, inappropriate initial antimicrobial therapy, increasing Acute Physiology and Chronic Health Evaluation II scores, and the number of acquired organ failures., Conclusions: Recent antibiotic exposure is associated with increased hospital mortality in Gram-negative bacteremia complicated by severe sepsis or septic shock. Clinicians caring for patients with severe sepsis or septic shock should consider recent antibiotic exposure when formulating empiric antimicrobial regimens for suspected Gram-negative bacterial infection.

In this study, we tested the abilities of the Vitek 2, BD Phoenix, and Kirby Bauer disk diffusion tests to detect carbapenemase production in a collection of 14 Klebsiella pneumoniae carbapenemase (KPC)-producing non-Klebsiella pneumoniae isolates. In addition, we evaluated 13 KPC-positive K. pneumoniae isolates by using each of these methods and applied both 2009 and 2010 CLSI carbapenem interpretive guidelines.

In January 2006, the Clinical and Laboratory Standards Institute (CLSI) updated breakpoints for vancomycin susceptibility testing for Staphylococcus aureus such that an MIC greater than 2 mg/liter was considered to represent nonsusceptibility to vancomycin. Previously, an MIC of 4 mg/liter had been considered to represent susceptibility. Additionally, in 2009, the CLSI altered the guidelines for staphylococci such that disk diffusion was no longer an acceptable means for testing vancomycin susceptibility in these organisms. To accommodate the change in breakpoints and methodological updates, we designed a medium consisting of brain heart infusion agar with 3 mg/liter vancomycin (BHI-V3) to screen for isolates of S. aureus with reduced susceptibility to vancomycin. We challenged this medium using a previously characterized collection of 100 isolates of S. aureus, including 55 vancomycin-susceptible isolates and 45 isolates representing vancomycin-intermediate strains of S. aureus (VISA) (with vancomycin MICs of 4 mg/liter or greater). All of the VISA isolates grew on the agar, for 100% sensitivity. Nineteen vancomycin-susceptible isolates also grew on the agar, for 65% specificity. We then incorporated BHI-V3 into clinical practice. In the first 60 days postimplementation, we identified 17 potential VISA isolates out of 421 S. aureus isolates tested. Thirteen out of the 17 were confirmed to represent VISA isolates. In light of the excellent sensitivity of this medium, we recommend that clinical laboratories incorporate BHI-V3 to screen for vancomycin-nonsusceptible isolates of S. aureus.

Three reference MIC methods approved by the Clinical and Laboratory Standards Institute were compared by testing 567 staphylococci against trimethoprim-sulfamethoxazole. Category agreement ranged from 94.9% (broth macrodilution versus broth microdilution) to 98.6% (agar dilution versus broth microdilution). Twenty-seven strains resistant by broth macrodilution were susceptible by broth microdilution.

During 6 months, we obtained Enterobacteriaceae isolates from patients with gram-negative bacteremia at a 1,250-bed teaching hospital in St. Louis, MO, and compared carbapenem susceptibilities with the presence of bla(KPC), a transferable carbapenemase gene. Three (1.2%) out of 243 isolates were bla(KPC) positive. Ertapenem nonsusceptibility had a low positive predictive value.

Background: Nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) can be a precursor to serious infection, and decolonization with topical mupirocin has been studied as a means of preventing clinical infection. Mupirocin resistance in patients with MRSA has been reported, usually in the context of widespread mupirocin use., Methods: Patients admitted to a surgical intensive care unit (SICU) had nasal swab cultures for MRSA performed at admission, weekly, and at discharge in an active surveillance program. Collected MRSA isolates were tested for mupirocin resistance, and molecular analysis was performed. Clinical data on the characteristics and outcomes of the patients who stayed in the SICU for >48 h were collected prospectively., Results: Of the 302 MRSA isolates available for testing, 13.2% were resistant to mupirocin, with 8.6% having high-level resistance (minimum inhibitory concentration, >or=512 microg/mL) and 4.6% having low-level resistance (minimum inhibitory concentration, 8-256 microg/mL). Patients admitted to the SICU for >48 h who were colonized with mupirocin-resistant MRSA were more likely to have been admitted to our hospital during the previous year (P=.016), were older (P=.009), and had higher in-hospital mortality (16% vs. 33%; P=.027), compared with patients colonized with mupirocin-susceptible MRSA. Molecular analysis of the mupirocin-resistant isolates revealed that 72.5% of isolates contained staphylococcal cassette chromosome mec II. Repetitive sequence polymerase chain reaction typing revealed that high-level mupirocin resistance was present in multiple clonal groups. The rate of mupirocin use hospital-wide during the study period was 6.08 treatment-days per 1000 patient-days., Conclusions: We documented a high rate of mupirocin resistance in MRSA isolates from SICU patients, despite low levels of in-hospital mupirocin use.

Strains of Klebsiella pneumoniae that produce one of three possible carbapenemases--KPC--have recently been identified with increasing frequency among isolates recovered from patients residing along the East Coast of the United States, particularly within the New York City metropolitan region. These strains have exhibited resistance to multiple antibiotic classes, including carbapenem agents. We report a case of nosocomial pneumonia and empyema caused by a KPC-producing isolate of K. pneumoniae at a large midwestern U.S. tertiary care facility in which the patient was treated with tigecycline. Although the pneumonia was treated successfully, the empyema recurred in association with a treatment-emergent tigecycline minimum inhibitory concentration (MIC) increase from 0.75 to 2 microg/ml. Clinicians should be aware of the potential occurrence of this treatment-emergent MIC increase, especially in the setting of sustained tigecycline therapy. In addition, the emergence of carbapenem-resistant Enterobacteriaceae reinforces the importance of antibiotic stewardship and strict infection control practices.

Two-hundred consecutive, single patient isolates of Enterobacter spp., Serratia spp., Citrobacter spp., and Pseudomonas aeruginosa were evaluated for AmpC production using a variety of inducer-substrate antibiotic combinations in a disk approximation format. The combinations examined included cefoxitin-piperacillin, imipenem-cefotaxime, imipenem-ceftazidime, imipenem-piperacillin-tazobactam, and imipenem-cefoxitin. All isolates were also screened for the presence of extended-spectrum beta-lactamase (ESBL) activity. In total, 85.5% of isolates were shown to be inducible for the production of AmpC by one or more inducer/substrate combinations and 11% of all isolates were stably derepressed for the expression of AmpC. Of all of the combinations, imipenem/piperacillin-tazobactam provided the greatest sensitivity (97.1%). All combinations were 100% specific when a positive test was observed. Given this background among these organisms in our institution, it is reasonable to develop an antibiotic reporting strategy that favors the selection of agents for therapy of these organisms that do not serve as labile substrates of AmpC.

Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.

Thirty-two isolates of vancomycin-resistant Enterococcus faecium (VRE) recovered from 25 patients hospitalized at six hospitals in the metropolitan Detroit, Mich., area over a 32-month period were examined for relatedness by repetitive-sequence PCR (rep-PCR). All isolates were shown to carry the vanA gene by PCR. The rep-PCR patterns generated from each isolate showed that the first three VRE isolates obtained from hospital A between June 1992 and February 1994 were distinct strains. Thereafter, all VRE isolates originating from hospital A and those collected from five other area hospitals had identical rep-PCR patterns. On detailed examination, subcultures of 25 of the 32 VRE isolates produced two distinct colony types characterized phenotypically by a rough and a smooth appearance, respectively. Both colony types retained the vanA locus and the rep-PCR pattern of the primary isolate. These data suggest that a single strain of VRE with the capacity to produce two colonial variants has been disseminated to several Detroit-area hospitals. The clinical significance of the colonial morphotypes is unclear.

Disk diffusion antibiograms were determined for mixtures of Pseudomonas aeruginosa morphotypes isolated from the sputum of patients with cystic fibrosis (CF). The results were compared with the predicted susceptibility patterns derived from the antibiograms of individual morphotypes within the mixture. Fifty separate cultures, each yielding two, three, or four distinct morphotypes of Pseudomonas aeruginosa were evaluated. Overall, the correlation between observed and predicted results was 92.2% with only 2.9% of all observations leading to major disagreements in susceptibility. These data suggest that mixed morphotype susceptibility is potentially a useful method to monitor the collective resistance of colonizing strains of Pseudomonas aeruginosa from the respiratory tract of patients with CF.