Adaptable microfluidic system for single-cell pathogen classification and antimicrobial susceptibility testing.

Infectious diseases caused by bacterial pathogens remain one of the most common causes of morbidity and mortality worldwide. Rapid microbiological analysis is required for prompt treatment of bacterial infections and to facilitate antibiotic stewardship. This study reports an adaptable microfluidic system for rapid pathogen classification and antimicrobial susceptibility testing (AST) at the single-cell level. By incorporating tunable microfluidic valves along with real-time optical detection, bacteria can be trapped and classified according to their physical shape and size for pathogen classification. By monitoring their growth in the presence of antibiotics at the single-cell level, antimicrobial susceptibility of the bacteria can be determined in as little as 30 minutes compared with days required for standard procedures. The microfluidic system is able to detect bacterial pathogens in urine, blood cultures, and whole blood and can analyze polymicrobial samples. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST.

Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods.

Campylobacter diagnosis is hampered because many laboratories continue to use traditional stool culture, which is slow and suffers false-negative results. This large multi-site study used a composite reference method consisting of a new FDA-cleared immunoassay and four molecular techniques to compare to culture. Prospectively collected patient fecal specimens (1552) were first preliminarily categorized as positive or negative by traditional culture. All specimens were also tested by EIA, and any EIA-positive or culture-discrepant results were further characterized by 16S rRNA qPCR, eight species-specific PCR assays, bidirectional sequencing, and an FDA-cleared multiplex PCR panel. The five non-culture methods showed complete agreement on all positive and discrepant specimens which were then assigned as true-positive or true-negative specimens. Among 47 true-positive specimens, culture incorrectly identified 13 (28%) as negative, and 1 true-negative specimen as positive, for a sensitivity of 72.3%. Unexpectedly, among the true-positive specimens, 4 (8%) were the pathogenic species C. upsaliensis. Culture had a 30% false result rate compared to immunoassay and molecular methods. More accurate results lead to better diagnosis and treatment of suspected campylobacteriosis.

Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing.

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

Antibiotic-tolerant Staphylococcus aureus Biofilm Persists on Arthroplasty Materials.

BACKGROUND: The continued presence of biofilm may be one cause of the high risk of failure observed with irrigation and débridement with component retention in acute periprosthetic joint infection (PJI). There is a poor understanding of the role of biofilm antibiotic tolerance in PJI. QUESTIONS/PURPOSES: (1) Do increasing doses of cefazolin result in decreased viable biofilm mass on arthroplasty materials? (2) Is cefazolin resistance phenotypic or genotypic? (3) Is biofilm viability a function of biofilm depth after treatment with cefazolin? (4) Is the toxin-antitoxin system, yoeB expression, associated with antibiotic stress? METHODS: Methicillin-sensitive Staphylococcus aureus biofilm was cultured on total knee arthroplasty (TKA) materials and exposed to increasing doses of cefazolin (control, 0.5, 1.0, 10.0, 100.0 µg/mL). Quantitative confocal microscopy and quantitative culture were used to measure viable biofilm cell density. To determine if cefazolin resistance was phenotypic or genotypic, we measured minimum inhibitory concentration (MIC) after exposure to different cefazolin concentrations; changes in MIC would suggest genotypic features, whereas unchanged MIC would suggest phenotypic behavior. Finally, quantitative reverse transcription-polymerase chain reaction was used to quantify expression of yoeB levels between biofilm and planktonic bacteria after exposure to 1 µg/mL cefazolin for 3 hours. RESULTS: Although live biofilm mass was reduced by exposure to cefazolin when compared with biofilm mass in controls (39.2 × 10(3) ± 26.4 × 10(3) pixels), where the level after 0.5 µg/mL exposure also showed reduced mass (20.3 × 10(3) ± 11.9 × 10(3) pixels), no further reduction was seen after higher doses (mass at 1.0 µg/mL: 5.0 × 10(3) pixels ± 1.1 × 10(3) pixels; at 10.0 µg/mL: 6.4 × 10(3) ± 9.6 × 10(3) pixels; at 100.0 µg/mL: 6.4 × 10(3) ± 3.9 × 10(3)). At the highest concentration tested (100 µg/mL), residual viable biofilm was present on all three materials, and there were no differences in percent biofilm survival among cobalt-chromium (18.5% ± 15.1%), polymethylmethacrylate (22.8% ± 20.2%), and polyethylene (14.7% ± 10.4%). We found that tolerance was a phenotypic phenomenon, because increasing cefazolin exposure did not result in changes in MIC as compared with controls (MIC in controls: 0.13 ± 0.02; at 0.5 µg/mL: 0.13 ± 0.001, p = 0.96; at 1.0 µg/m: 0.14 ± 0.04, p = 0.95; at 10.0 µg/m: 0.11 ± 0.016, p = 0.47; at 100.0 µg/m: 0.94 ± 0.047, p = 0.47). Expression of yoeB after 1 µg/mL cefazolin for 3 hours in biofilm cells was greater in biofilm but not in planktonic cells (biofilm: 62.3-fold change, planktonic cells: -78.8-fold change, p < 0.001). CONCLUSIONS: Antibiotics are inadequate at complete removal of the biofilm from the surface of TKA materials. Results suggest that bacterial persisters are responsible for this phenotypic behavior allowing biofilm high tolerance to antibiotics. CLINICAL RELEVANCE: Antibiotic-tolerant biofilm suggests a mechanism behind the poor results in irrigation and débridement for acute TKA PJI.

Antimicrobial resistance determinants in Acinetobacter baumannii isolates taken from military treatment facilities.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.

Bioterrorism: a laboratory who does it?

In October 2001, the first disseminated biological warfare attack was perpetrated on American soil. Initially, a few clinical microbiology laboratories were testing specimens from acutely ill patients and also being asked to test nasal swabs from the potentially exposed. Soon after, a significant number of clinical microbiology and public health laboratories received similar requests to test the worried well or evaluate potentially contaminated mail or environmental materials, sometimes from their own break rooms. The role of the clinical and public health microbiology laboratory in response to a select agent event or act of bioterrorism is reviewed.

Pulse lavage is inadequate at removal of biofilm from the surface of total knee arthroplasty materials.

In acute periprosthetic infection, irrigation and debridement with component retention has a high failure rate in some studies. We hypothesize that pulse lavage irrigation is ineffective at removing biofilm from total knee arthroplasty (TKA) components. Staphylococcus aureus biofilm mass and location was directly visualized on arthroplasty materials with a photon collection camera and laser scanning confocal microscopy. There was a substantial reduction in biofilm signal intensity, but the reduction was less than a ten-fold decrease. This suggests that irrigation needs to be further improved for the removal of biofilm mass below the necessary bioburden level to prevent recurrence of acute infection in total knee arthroplasty.

Genome sequences of four divergent multidrug-resistant Acinetobacter baumannii strains isolated from patients with sepsis or osteomyelitis.

Acinetobacter baumannii is a Gram-negative bacterium that causes nosocomial infections worldwide, with recent prevalence and higher frequency in wounded military personnel. Four A. baumannii strains from the Walter Reed Army Medical Center (WRAMC) isolated between 2008 and 2009 were sequenced, representing diverse, multidrug-resistant isolates from osteomyelitis or septic patients.

Antibacterial activities of iron chelators against common nosocomial pathogens.

The activities of iron chelators (deferoxamine, deferiprone, Apo6619, and VK28) were evaluated against type strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. Deferiprone, Apo6619, and VK28 each inhibited growth in standard and RPMI tissue culture medium, while deferoxamine had no effect. Additionally, time-kill assays revealed that VK28 had a bacteriostatic effect against S. aureus. Therefore, these newly developed iron chelators might provide a nontraditional approach for treatment of bacterial infections.

Impact of removing ESBL designation from culture reports on the selection of antibiotics for the treatment of infections associated with ESBL-producing organisms.

We characterized the impact of removal of the ESBL designation from microbiology reports on inpatient antibiotic prescribing. Definitive prescribing of carbapenems decreased from 48.4% to 16.1% (P = .01) and ß-lactam-ß-lactamase inhibitor combination increased from 19.4% to 61.3% (P = .002). Our findings confirm the importance of collaboration between microbiology and antimicrobial stewardship programs.

In vitro activity of tigecycline against multidrug-resistant Acinetobacter baumannii and selection of tigecycline-amikacin synergy.

Polymyxin B, minocycline, and tigecycline were the most potent of 10 antibiotics against 170 isolates of multidrug-resistant Acinetobacter baumannii. In time-kill studies, the exposure of a highly tigecycline-resistant isolate to tigecycline resulted in enhanced susceptibility to amikacin and synergistic bactericidal activities of the two drugs.

Viable bacteria persist on antibiotic spacers following two-stage revision for periprosthetic joint infection.

Treatment in periprosthetic joint infection (PJI) remains challenging. The failure rate of two-stage revision and irrigation and debridement with component retention in PJI suggests that biofilm cells have a high tolerance to antibiotic chemotherapy. Previous work has demonstrated that biofilm cells have high antibiotic tolerance in vitro, but there is little clinical evidence to support these observations. The aim of this study was to determine if retrieved antibiotic spacers from two-stage revision total knee arthroplasty for PJI have evidence of remaining viable bacteria. Antibiotic poly (methyl methacrylate) (PMMA) spacers from two-stage revision total knee arthroplasty for PJI were prospectively collected and analyzed for bacterial 16s rRNA using polymerase chain reaction (PCR), reverse transcription (RT)-PCR, quantitative RT-PCR (qRT-PCR), and single genome analysis (SGA). PCR and RT-PCR identified bacterial species on 53.8% (7/13) of these samples. When initial culture negative cases are excluded, 68% (6/9) samples were identified with bacterial species. A more rigorous qRT-PCR analysis showed a strong positive signal for bacterial contamination in 30.7% (4/13) of cases. These patients did not show any clinical evidence of PJI recurrence after 15 months of follow-up. Because the half-life of bacterial rRNA is approximately a few days, the identification of bacteria rRNA on antibiotic PMMA spacers suggests that viable bacteria were present after conclusion of antibiotic therapy. This study provides evidence for the high tolerance of biofilm cells to antibiotics in vivo and the important role of bacterial persisters in PJI. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:452-458, 2018.

A Multiplex Electrochemical Biosensor for Bloodstream Infection Diagnosis.

Accurate and timely detection of bacterial pathogens will improve the clinical management of infections. Herein, we demonstrate an electrochemical biosensor that directly detects bacteria in human blood samples, resulting in the rapid diagnosis of a bloodstream infection. The multiplex biosensor detects the species-specific sequences of the 16S ribosomal RNA of bacteria for pathogen identification in physiological samples without preamplification. The analytical performance characteristics of the biosensor, including the limit of detection and probe cross-reactivity, are evaluated systematically. The feasibility of the biosensor for a diagnosis of a bloodstream infection is demonstrated by identifying bacterial clinical isolates spiked in whole blood and blood culture samples that were tested positive for bacteria. The electrochemical biosensor correctly identifies all the species in the samples with 100% concordance to microbiological analysis.

Severe Babesia microti Infection in an Immunocompetent Host in Pennsylvania.

Babesiosis, due to infection by a tick-borne protozoan (predominantly Babesia microti in North America), is an emerging health risk that is expanding into new areas and may be unfamiliar to clinicians in locations not previously considered endemic. Manifestations of infection can range from asymptomatic to life threatening, with severe disease more likely in those who have had a splenectomy, are immunocompromised, have chronic medical conditions, or are over 50 years of age. In this article, we describe an elderly but otherwise healthy man from an area not generally considered endemic for babesiosis who presented with severe hemolysis, acute renal failure, and high-level Babesia microti parasitemia; serological results suggestive of possible coinfection by Borrelia burgdorferi (the agent of Lyme disease, which is carried by the same tick as is Babesia microti) also was found. This report highlights that severe babesiosis can occur in an apparently normal host and underscores the continued geographic expansion of this pathogen and the need for early recognition and therapy.

Building an Antimicrobial Stewardship Program: Cooperative Roles for Pharmacists, Infectious Diseases Specialists, and Clinical Microbiologists.

Efforts to optimize the use of antimicrobial agents, referred to as antimicrobial stewardship programs (ASPs), are increasingly becoming part of the clinical enterprise at big and small hospitals. Such programs aim to achieve the synergistic goals of improving patient outcomes, limiting the unintended consequences of drug resistance and superinfections, and reducing health care expenditures. This article will review the need for antimicrobial stewardship and the key components of setting up a program; then, it will describe the ASP at one medical center to underscore how attention to acceptance by the clinical staff is crucial to changing the culture of antimicrobial use. Although the details may differ for each institution, the foundation of a successful stewardship program is support from hospital leadership and the cooperative interaction among the pharmacy, infectious diseases specialists, and clinical microbiologists.

AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments.

UNLABELLED: Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE: The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

Development and validation of a multiplex TaqMan real-time PCR for rapid detection of genes encoding four types of class D carbapenemase in Acinetobacter baumannii.

A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D ß-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87 %) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2 %) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.

Extracellular stress and lipopolysaccharide modulate Acinetobacter baumannii surface-associated motility.

Acinetobacter baumannii is a nosocomial bacterial pathogen, and infections attributed to this species are further complicated by a remarkable ability to acquire antimicrobial resistance genes and to survive in a desiccated state. While the antibiotic resistance and biofilm formation of A. baumannii is well-documented, less is known about the virulence attributes of this organism. Recent studies reported A. baumannii strains display a motility phenotype, which appears to be partially dependent upon Type IV pili, autoinducer molecules, and the response to blue light. In this study, we wanted to determine the prevalence of this trait in genetically diverse clinical isolates, and any additional required factors, and environmental cues that regulate motility. When strains are subjected to a wide array of stress conditions, A. baumannii motility is significantly reduced. In contrast, when extracellular iron is provided or salinity is reduced, motility is significantly enhanced. We further investigated whether the genes required for the production of lipopolysaccharide (lpsB) and K1 capsule (epsA/ptk) are required for motility as demonstrated in other Gram-negative bacteria. Transposon mutagenesis resulted in reduced motility by the insertion derivatives of each of these genes. The presence of the parental allele provided in trans, in the insertion mutant background, could only restore motility in the lpsB mutant. The production of core LPS directly contributes to the motility phenotype, while capsular polysaccharide may have an indirect effect. Further, the data suggest motility is regulated by extracellular conditions, indicating that A. baumannii is actively sensing the environment and responding accordingly.

The majority of US combat casualty soft-tissue wounds are not infected or colonized upon arrival or during treatment at a continental US military medical facility.

BACKGROUND: The microbiology of war wounds has changed as medicine and warfare have evolved. This study was designed to determine the microbial flora and bacterial quantification of present-day war wounds in US troops from Iraq and Afghanistan upon arrival at the National Naval Medical Center (NNMC). METHODS: Patients with extremity combat wounds treated with a vacuum-assisted wound closure device were enrolled in study. Wounds were biopsied every 48 to 72 hours with quantitative microbiology performed on all biopsies. RESULTS: Two hundred forty-two wound biopsies from 34 patients; 167 (69%) showed no growth, and 75 (31%) showed positive growth. The incidence of any bacterial isolation from biopsies weekly from the time of injury was 28% (first), 31% (second), and 37% (≥third). Acinetobacter baumannii was the most prevalent isolate. CONCLUSIONS: Most soft-tissue wounds from Iraq and Afghanistan do not have significant bacterial burden upon arrival to and during initial treatment at NNMC. Improved evaluation of combat wound microbiology at all levels of care is warranted to determine shifts in microbiology and to impact care practices.

Analysis of antibiotic resistance genes in multidrug-resistant Acinetobacter sp. isolates from military and civilian patients treated at the Walter Reed Army Medical Center.

Military medical facilities treating patients injured in Iraq and Afghanistan have identified a large number of multidrug-resistant (MDR) Acinetobacter baumannii isolates. In order to anticipate the impact of these pathogens on patient care, we analyzed the antibiotic resistance genes responsible for the MDR phenotype in Acinetobacter sp. isolates collected from patients at the Walter Reed Army Medical Center (WRAMC). Susceptibility testing, PCR amplification of the genetic determinants of resistance, and clonality were determined. Seventy-five unique patient isolates were included in this study: 53% were from bloodstream infections, 89% were resistant to at least three classes of antibiotics, and 15% were resistant to all nine antibiotics tested. Thirty-seven percent of the isolates were recovered from patients nosocomially infected or colonized at the WRAMC. Sixteen unique resistance genes or gene families and four mobile genetic elements were detected. In addition, this is the first report of bla(OXA-58)-like and bla(PER)-like genes in the U.S. MDR A. baumannii isolates with at least eight identified resistance determinants were recovered from 49 of the 75 patients. Molecular typing revealed multiple clones, with eight major clonal types being nosocomially acquired and with more than 60% of the isolates being related to three pan-European types. This report gives a "snapshot" of the complex genetic background responsible for antimicrobial resistance in Acinetobacter spp. from the WRAMC. Identifying genes associated with the MDR phenotype and defining patterns of transmission serve as a starting point for devising strategies to limit the clinical impact of these serious infections.