A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 μm width × 150 μm depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.
We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37°C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.
Microfluidic reactors exhibit intrinsic advantages of reduced chemical consumption, safety, high surface-area-to-volume ratios, and improved control over mass and heat transfer superior to the macroscopic reaction setting. In contract to a continuous-flow microfluidic system composed of only a microchannel network, an integrated microfluidic system represents a scalable integration of a microchannel network with functional microfluidic modules, thus enabling the execution and automation of complicated chemical reactions in a single device. In this review, we summarize recent progresses on the development of integrated microfluidics-based chemical reactors for (i) parallel screening of in situ click chemistry libraries, (ii) multistep synthesis of radiolabeled imaging probes for positron emission tomography (PET), (iii) sequential preparation of individually addressable conducting polymer nanowire (CPNW), and (iv) solid-phase synthesis of DNA oligonucleotides. These proof-of-principle demonstrations validate the feasibility and set a solid foundation for exploring a broad application of the integrated microfluidic system.
Integrated microfluidics; Chemical screening, In situ click chemistry; Sequential synthesis, Positron emission tomography probes; Oligonucleotide synthesis; Conducting polymer nanowires
A prospective observational study was carried out to examine antimicrobial resistance patterns of fecal Escherichia coli isolates of calves on arrival at the feedlot, and then evaluate the associations between the total volume of antimicrobial used for disease treatment and changes in antimicrobial resistance, during the feeding period. No macrolides or tetracyclines were administered in the feed during this study. On arrival, at the animal level, all 3 isolates obtained from 36.6% [95% confidence interval (CI): 29.0 to 44.8] of all cattle sampled (n = 153), were susceptible to all antimicrobials, while 5.9% (95% CI: 2.7 to 10.9) of cattle had at least 1 isolate that was resistant to ≥ 3 antimicrobials out of the 7 antimicrobials tested. The most frequent antimicrobials for which resistance was observed were sulphamethoxazole, ampicillin, and tetracycline where, of all cattle, 44.4% (95% CI: 36.4 to 52.7), 20.3% (95% CI: 14.2 to 27.5), and 17.7% (95% CI: 12.0 to 24.6), respectively had at least 1 resistant isolate. All cattle received antimicrobial metaphylaxis on arrival at the feedlot. Antimicrobial use was described for a cohort of 95 cattle. Antimicrobials were given to 42 of the 95 cattle during the feeding period, to treat disease. Amongst the 42 treated cattle, there were a total of 133 animal daily doses (ADDFeedlot), where 1 ADDFeedlot represented 1 day of antimicrobial treatment received by a feedlot animal at the approved dose. Only 1 ADDFeedlot was given in the 100 days immediately prior to slaughter. There were no associations found between antimicrobial use and antimicrobial resistance in this study.
There is great interest in point-of-care antibody testing for the diagnosis of infectious and autoimmune diseases. As a first step in the development of self-contained and miniaturized devices for highly quantitative antibody detection, we demonstrate the application of Luciferase Immunoprecipitation Systems (LIPS) technology in a microfluidic format. Protein A/G was immobilized on the walls of PDMS-glass microchannels of 500 nL volume. The assay proceeds with the simultaneous introduction of plasma and Renilla luciferase–tagged antigens. Following washing, coelenterazine substrate was added and bound antigen-luciferase measured by chemiluminescence. Total assay time, including rinsing and detection, is under ten minutes. Using these stable microfluidic devices, high diagnostic performance (100% sensitivity and 100% specificity) was achieved for the diagnosis of HSV-2 infection. Based on these findings, the LIPS microfluidic format should readily lend itself to automation and the transfer to portable instrumentation.
antibody detection; biosensor; LIPS; microfluidics
A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format.
This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768–772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 ± 250 s−1), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2–4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.
Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, which has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Similar to how blood glucose sensors revolutionized the management of diabetes and pregnancy tests are now conducted at home, biosensors are poised to significantly improve UTI diagnosis. Biosensors are amenable to integration with microfluidic technology for point-of-care applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing and hurdles in the translation of biosensor technology from bench to bedside.
In the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale.
FigureDependence of the linear range on surface-to-volume ratio in microfluidic detection.
Lab-on-a-chip; Microfluidics; Optical detection; Fluorescence; Fluorescein; Surface-to-volume ratio
A survey to estimate the prevalence of antimicrobial resistance in Escherichia coli was conducted in 7 Canadian federally inspected processing plants during 2001. Escherichia coli isolates were recovered during routine hazard analysis critical control point sampling from beef carcasses and trim and subsequently tested for their antimicrobial susceptibility by using susceptibility panels.
Of the 2653 isolates analyzed, 68% were sensitive to all 18 antimicrobials tested. For 14 of the 18 antimicrobials evaluated, the percentage of resistant isolates was ≤ 1. Twenty-five percent of the isolates were resistant to tetracycline, 9% to sulfamethoxazole, 7% to streptomycin, and 3% to ampicillin. Multiple resistance was found in 12% of the isolates, with 7% showing resistance to 2 antimicrobials, 2% to 3 antimicrobials, 2% to 4 antimicrobials, and 1% to 5 or more antimicrobials. Forty-five different antimicrobial resistance patterns were observed. The reasons for the development of the antimicrobial resistance were not investigated in this study.
This study was useful as a pilot to help to develop a national antimicrobial resistance surveillance program in Canada. This study indicates that laboratory standardization is possible for consistent results across the country and that the indicator organism, E. coli, is fairly easy to obtain for surveillance but Salmonella are not, due to their low prevalence in beef.
Early targeted antimicrobial therapy helps decrease costs and prevents the spread of antimicrobial resistance, including in Escherichia coli, the most frequent Gram-negative bacterium that causes sepsis. Therefore, rapid susceptibility testing represents the major prerequisite for knowledge-based successful antimicrobial treatment. To accelerate testing for antibiotic susceptibility, we have developed a new mass spectrometry-based assay for antibiotic susceptibility testing (MAAST). For proof of principle, we present an ampicillin susceptibility test for E. coli with a turnaround time of 90 min upon growth detection.
Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents. Antimicrobial peptides (AMPs) are receiving attention as alternatives to antibiotics. In this study, we used phage-display random peptide library to identify peptides binding to the cell surface of E. coli. The peptide with sequence RLLFRKIRRLKR (EC5) bound to the cell surface of E. coli and exhibited certain features common to AMPs and was rich in Arginine and Lysine residues. Antimicrobial activity of the peptide was tested in vitro by growth inhibition assays and the bacterial membrane permeabilization assay. The peptide was highly active against Gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log10 CFU/ml. In homologous plasma and platelets, incubation of EC5 with the bacteria resulted in significant reduction of E. coli and P. aeruginosa, compared to the peptide-free controls. The peptide was non-hemolytic and non-cytotoxic when tested on eukaryotic cells in culture. EC5 was able to permeabilize the outer membrane of E. coli and P. aeruginosa causing rapid depolarization of cytoplasmic membrane resulting in killing of the cells at 5 minutes of exposure. The secondary structure of the peptide showed a α-helical conformation in the presence of aqueous environment. The bacterial lipid interaction with the peptide was also investigated using Molecular Dynamic Simulations. Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.
The establishment of bacterial infections at mucosal epithelial surfaces is determined by the balance of virulence attributes of the pathogen with the activity of innate host defenses. Polymorphonuclear leukocytes (PMN) are key responders in many bacterial infections, but the mechanisms by which pathogens subvert these early responses to establish infection are largely undefined. Here, we model early interactions between human PMN and the primary cause of urinary tract infections, namely uropathogenic Escherichia coli (UPEC). Our objective was to define virulence phenotypes of uropathogens that permit evasion of PMN activity. We show that UPEC strains, as compared with laboratory and commensal E. coli, resist phagocytic killing and dampen the production of antimicrobial reactive oxygen species by PMN. Analysis of the transcriptional responses of PMN to E. coli strains revealed that UPEC exposure downregulates the expression of PMN genes that direct proinflammatory signaling and PMN chemotaxis, adhesion, and migration. Consistent with these data, UPEC attenuated transepithelial neutrophil recruitment in an in vitro model of acute infection and in a murine model of bacterial cystitis. We propose that these UPEC strategies are important in the establishment of epithelial infection, and that the findings are germane to bacterial infections at other epithelial surfaces.
Escherichia coli; Neutrophil; Migration
Urinary tract infections (UTI) are the most commonly encountered infections in obstetric patients. Although a variety of etiology is involved, Escherichia coli and other coliforms account for a large majority of these naturally acquired infections. The estimation of local etiology and susceptibility profile could support the most effective empirical treatment.
The current study was undertaken to find the spectrum of micro-organisms responsible for causing UTI in obstetric patients and to find out the most appropriate antibiotic.
Materials and Methods:
Consecutive patients in different stages of pregnancy with or without symptoms of UTI attending the antenatal clinic during November 2011 to March 2012 were screened for significant bacteriuria. The bacterial uropathogens isolated were then subjected to antimicrobial susceptibility testing and screened for ESBL production and methicillin resistance.
During the 5-month study period, out of the 250 samples screened, a total of 60 (24%) samples of urine from pregnant females, in different stages of pregnancy were found to be positive on culture. The Enterobacteriaceae accounted for nearly two-thirds of the isolates and E. coli alone accounted for 63% of the urinary isolates followed by Klebsiella pneumonia 8%. Among the Gram-positive cocci, coagulase-negative Staphylococcus (15%) were more frequently isolated than Staphylococcus aureus (8.3%). A significantly high resistance was noted to the beta-lactam group of antimicrobials, fluoroquinolones and cotrimoxazole, both by the Gram-negative bacilli as well as Gram-positive cocci. Resistance was quite low against the aminoglycosides and nitrofurantoin and virtually absent against imipenem.
The susceptibility patterns seen in our study seem to suggest that it is absolutely necessary to obtain sensitivity reports before initiation of antibiotic therapy in cases of suspected UTI.
ESBL; Pregnancy; Urinary tract infection; Uropathogens
Between January 2002 and June 2007, uropathogens were isolated from 473 of 1557 canine urine samples submitted to Prairie Diagnostic Services from the Western College of Veterinary Medicine Veterinary Teaching Hospital. Culture and susceptibility results were analyzed, retrospectively, to estimate the prevalence of common bacterial uropathogens in dogs with urinary tract infections and to identify changes in antimicrobial resistance. The most common pathogens identified were Escherichia coli, Staphylococcus intermedius, Enterococcus spp., and Proteus spp. Antimicrobial resistance increased during the study period, particularly among recurrent E. coli isolates. Using the formula to help select rational antimicrobial therapy (FRAT), bacterial isolates were most likely to be susceptible to gentamicin, fluoroquinolones, amoxicillin-clavulanic acid, and groups 4 and 5 (third generation) cephalosporins.
The goals of this study were to (i) identify issues that affect the ability of discriminant function analysis (DA) of antimicrobial resistance profiles to differentiate sources of fecal contamination, (ii) test the accuracy of DA from a known-source library of fecal Escherichia coli isolates with isolates from environmental samples, and (iii) apply this DA to classify E. coli from surface water. A repeated cross-sectional study was used to collect fecal and environmental samples from Michigan livestock, wild geese, and surface water for bacterial isolation, identification, and antimicrobial susceptibility testing using disk diffusion for 12 agents chosen for their importance in treating E. coli infections or for their use as animal feed additives. Nonparametric DA was used to classify E. coli by source species individually and by groups according to antimicrobial exposure. A modified backwards model-building approach was applied to create the best decision rules for isolate differentiation with the smallest number of antimicrobial agents. Decision rules were generated from fecal isolates and applied to environmental isolates to determine the effectiveness of DA for identifying sources of contamination. Principal component analysis was applied to describe differences in resistance patterns between species groups. The average rate of correct classification by DA was improved by reducing the numbers of species classifications and antimicrobial agents. DA was able to correctly classify environmental isolates when fewer than four classifications were used. Water sample isolates were classified by livestock type. An evaluation of the performance of DA must take into consideration relative contributions of random chance and the true discriminatory power of the decision rules.
Campylobacter jejuni is a leading foodborne pathogen worldwide and its resistance to antimicrobials is a major concern for public health. The cmeG (Cj1375) gene in C. jejuni encodes a putative efflux transporter of the major facilitator family, but its function in antimicrobial resistance has not been determined. This study aimed to characterize the function of CmeG in conferring resistance to antibiotics and oxidative stress.
The cmeG gene (Cj1375) in C. jejuni was inactivated by insertional mutagenesis and overexpressed by cloning with a shuttle vector. These constructs were compared with the wild-type strain using antimicrobial susceptibility tests and drug accumulation assays.
The cmeG mutation reduced bacterial growth and rendered C. jejuni more susceptible to ciprofloxacin, erythromycin, gentamicin, tetracycline, rifampicin, ethidium bromide and cholic acid as well as hydrogen peroxide, and in trans complementation restored the susceptibility to near wild-type level. RT–PCR showed that cmeG is co-transcribed with its downstream gene cmeH (Cj1376) encoding a putative periplasmic protein, but mutation of cmeH alone did not affect the susceptibility to antibiotics. Notably, overexpression of the cmeGH operon in C. jejuni NCTC 11168 significantly increased its resistance to fluoroquinolones. In addition, the cmeG mutant accumulated more EtBr and ciprofloxacin than the wild-type strain.
These results indicate that CmeG functions as a multidrug efflux transporter contributing to antibiotic resistance and oxidative defence in Campylobacter.
antibiotic resistance; efflux pumps; C. jejuni
Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only.
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface.
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.
While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3–10 min) with low sample volume requirements (10 μL) and appreciable sensitivity (nM–pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip-based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.
microfluidics; periodontal disease; diagnostics; point-of-care; POC; immunoassay; lab-on-a-chip; saliva
Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.
Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.
In Sweden, knowledge about the role of enteropathogenic Escherichia coli in neonatal calf diarrhea and the occurrence of antimicrobial resistance in E. coli from young calves is largely unknown. This has therapeutic concern and such knowledge is also required for prudent use of antimicrobials.
In a case control study Esherichia coli isolated from faecal samples from dairy calves were phenotyped by biochemical fingerprinting and analyzed for virulence genes by PCR. Antimicrobial susceptibility was tested by determination of minimum inhibitory concentration (MIC). Farm management data were collected and Fisher's exact test and univariable and multivariable logistic regression analysis were performed.
Of 95 E. coli tested for antimicrobial susceptibility 61% were resistant to one or more substances and 28% were multi-resistant. The virulence gene F5 (K99) was not found in any isolate. In total, 21 out of 40 of the investigated virulence genes were not detected or rarely detected. The virulence genes espP, irp, and fyuA were more common in resistant E. coli than in fully susceptible isolates (P < 0.05). The virulence gene terZ was associated with calf diarrhea (P ≤ 0.01).
The participating 85 herds had a median herd size of 80 lactating cows. Herds with calf diarrhea problems were larger (> 55 cows; P < 0.001), had higher calf mortality (P ≤ 0.01) and calf group feeders were more in use (P < 0.05), compared to herds without calf diarrhea problems.
There was no association between calf diarrhea and diversity of enteric E. coli.
Antimicrobial resistance was common in E. coli from pre-weaned dairy calves, occurring particularly in calves from herds experiencing calf diarrhea problems. The results indicate that more factors than use of antimicrobials influence the epidemiology of resistant E. coli.
Enteropathogenic E. coli seems to be an uncommon cause of neonatal calf diarrhea in Swedish dairy herds. In practice, calf diarrhea should be regarded holistically in a context of infectious agents, calf immunity, management practices etc. We therefore advice against routine antimicrobial treatment and recommend that bacteriological cultures, followed by testing for antimicrobial susceptibility and for virulence factors, are used to guide decisions on such treatment.
During the past decade, a variety of instrument-assisted bacterial identification and antimicrobial susceptibility test systems have been developed which permit provision of test results in a matter of hours rather than days, as has been the case with traditional overnight procedures. These newer rapid techniques are much more expensive than older methods. It has been presumed but not proven that the clinical benefits of rapid testing to patients with infection offset the added cost. The intent of this study was to objectively define the clinical impact of rapid bacterial identification and antimicrobial susceptibility testing. A 1-year study was performed in which infected, hospitalized patients in a tertiary-care, teaching, medical center were randomly assigned to one of two groups: patients for whom identification and susceptibility testing was performed by using a semi-automated, rapid, same-day procedure and those for whom testing was accomplished by using traditional overnight techniques. The two groups were compared with respect to numerous demographic descriptors, and then patients were monitored prospectively through the end of their hospitalization with the aim of determining whether there existed objectively defineable differences in management and outcome between the two groups. The mean lengths of time to provision of susceptibility and identification test results in the rapid test group were 11.3 and 9.6 h, respectively. In the overnight test group, these values were 19.6 and 25.9 h, respectively (P < 0.0005). There were 273 evaluable patients in the first group and 300 in the second group. Other than the length of time required to provide susceptibility and identification test results, no significant differences were noted between the two groups with respect to > 100 demographic descriptors. With regard to measures of outcome, the mean lengths of hospitalization were also the same in both groups. Mortality rates were however, lower in the rapid test group (i.e., 8.8% versus 15.3%). Similarly, statistically significantly fewer laboratory studies, imaging procedures, days of intubation, and days in an intensive or intermediate-care area were observed with patients in the rapid test group. Rapid testing was also associated with significantly shortened lengths of elapsed time prior to alterations in antimicrobial therapy. Lastly, patient costs for hospitalization were significantly lower in the rapid test group. The results of this study indicate the rapid same-day bacterial identification and susceptibility testing in the microbiology laboratory can have a major impact on the care and outcome of hospitalized patients with infection.
Mycoplasmosis is a common infection in human and veterinary medicine, and is associated with chronic inflammation and high morbidity. Mycoplasma species are often intrinsically resistant to many conventional antimicrobial therapies, and the resistance patterns of pathogenic mycoplasmas to commonly used medicinal (antimicrobial) plant extracts are currently unknown.
Aqueous extracts, ethanol extracts, or oils of the targeted plant species and colloidal silver were prepared or purchased. Activity against the wall-less bacterial pathogen Mycoplasma mycoides subsp. capri was determined and compared to activities measured against Escherichia coli and Bacillus subtilis. Antimicrobial susceptibility testing was performed by broth microdilution assays. The lethal or inhibitory nature of each extract was determined by subculture into neat growth medium.
Growth of M. mycoides capri, E. coli, and B. subtilis was inhibited by elderberry extract, oregano oil, ethanol extract of oregano leaves, and ethanol extract of goldenseal root. No inhibition was seen with aqueous extract of astragalus or calendula oil. Growth of M. mycoides capri and B. subtilis was inhibited by ethanol extract of astragalus, whereas growth of E. coli was not. Similarly, M. mycoides capri and E. coli were inhibited by aqueous extract of thyme, but B. subtilis was unaffected. Only B. subtilis was inhibited by colloidal silver. Measured MICs ranged from 0.0003 mg/mL to 3.8 mg/mL. Bacteriostatic and bactericidal effects differed by species and extract.
The atypical pathogen M. mycoides capri was sensitive to extracts from many medicinal plants commonly used as antimicrobials in states of preparation and concentrations currently available for purchase in the United States and Europe. Variation in bacteriostatic and bactericidal activities between species and extracts indicates that multiple effecter compounds are present in these plant species.
Four isolates of an unclassified microaerophilic bacterium resembling Campylobacter species were characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, and protein profile analysis. The unclassified isolates were differentiated from Campylobacter jejuni, Campylobacter coli, Campylobacter fetus subsp. fetus, Campylobacter laridis, Campylobacter pylori, and an ovine isolate. The bacterium was fusiform shaped with a corrugated surface due to the presence of periplasmic fibers and had multiple bipolar flagella. Biochemically, the bacterium was separated from the Campylobacter controls by its negative catalase reaction, negative nitrate reduction, and no growth in 1% glycine. It was also resistant to ampicillin. Protein profile analysis demonstrated nine major protein bands present in the unclassified isolates that were absent in the Campylobacter controls. The bacterium also differed from the ovine isolate by its negative catalase reaction, rapid urea hydrolysis, and susceptibility to clindamycin, erythromycin, and tetracycline. Our results showed that the unclassified bacterium was distinct from the recognized Campylobacter species.