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LC-MS/MS-based H-type determination of Escherichia coli
A comparative analysis of serotyping and mass spectrometry (MS) methods for the determination of the flagellar type (H type) of clinically isolated Escherichia coli has been performed. In this analysis, it was shown that determination of the correct H type of a clinical E. coli strain was better achieved by MS than serotyping. Whole genome sequencing was used for the validation of this analysis.
by M. Chan, Dr H. Chui, D. Hernandez, Dr G. Wang and Dr K. Cheng
Background
During outbreaks of disease caused by pathogenic Escherichia coli, it is important to be able to identify the precise E. coli strain involved in order to track and prevent the spread of infection. Typically, the identification of E. coli strains has been based on the serotype of the cell surface antigens such as the lipopolysaccharide O antigen and the flagellar H antigen. Even though serotyping is viewed as the ‘gold standard’ for O- and H-type determination, this technique does have its downfalls. These conventional serotyping methods are based on antisera, which makes procedures costly and laborious to perform because of the variable quality of antibody preparations and the number of antibody agglutination reactions needed to assign a final classification [1, 2, 3]. Also, when bacterial cells do not generate lipopolysaccharide on the surface, the cultured colonies become ‘rough strains’. This makes both O- and H-antigen identification by antibody-based agglutination problematic despite the cellular motility and presence of the flagella H-antigen structure [1, 3, 4]. In addition, flagellar serotyping needs the induction of motility, which can take up to two weeks, and so does not result in the fast identification required in an E. coli outbreak situation [5].
A promising technique for E. coli H-type identification is the use of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, termed ‘MS-H’. In our own analytical assays, we harvest, enrich and digest the flagella proteins. LC-MS/MS uses liquid chromatography to separate flagellar fragments after trypsin digestion, and mass spectrometry to analyse and determine the protein sequences of these fragments. The MS results are then analysed against a curated database of H antigen variants to determine the H type. Compared with serotyping, MS-H has a high throughput, requires less labour, needs less time to perform, and provides sequence-level information.
Comparative H typing of E. coli with serotyping and MS-H
Using reference strains of all the different forms of E. coli flagella, it was shown that all 53 different types of H antigen can be determined through MS-H. Table 1 details the comparisons between H serotyping and MS-H [1]. It is important to note that both methods can reach 100% sensitivity and specificity for H-type determination. However, MS-H also provides sequence-level identification of the H type. This is important because this provides more reliable results, while antisera serotyping only provides a visual conformation, making there more of a chance for subjective and inaccurate determination of the H type.
Flagella motility induction is commonly used in serotyping. This increases the time-length of the procedure and requires more hands-on involvement [5]. In comparison, MS-H uses motility induction much less frequently, which makes MS-H identification of E. coli H type quicker and less labour-intensive.
Clinically isolated strains of E. coli that had already had their H types determined by serotyping were supplied from three provinces within Canada. A preliminary H-type analysis of the same E. coli isolates was performed by MS-H [5]. The workflow that was completed to analyse the H type of these strains is detailed in Figure 1.
Things to note within the workflow are that vortexing the suspended culture in Step 2 was to shear the flagella away from the E. coli. Also in Step 2, the flagella were trapped onto a filter membrane. This serves as an isolation step to separate the flagella from the supernatant as well as an enrichment process to concentrate the flagella. We then compared the flagellar sequence obtained by MS to a curated database formed from NCBI flagella protein sequence entries. A curated database was necessary because a public database can be problematic because of size, specificity and inconsistent annotation of the data for the specific flagellar protein entries. The curated database focuses on the flagellar proteins with fit-for-purpose annotations, thus allowing differentiation between the 53 different H types better than the public database [6].
On comparison of the H types identified by serotyping and MS-H, the majority of the results agreed with one another. However, there were some discrepancies between the two methods. In these cases, whole genome sequencing and polymerase chain reaction (PCR) detection was used to identify the correct H type [5] and these results predominantly agreed with those obtained by MS-H.
Even though PCR as a form of detection for the flagella gene is popular, the accuracy for determining all forms of flagella types through PCR is low and the use a many different primers had to be implemented, making it not ideal for the detection of an unknown H type [5]. Whereas whole genome sequencing may be more expensive then PCR detection, it was much more accurate at determining the correct H-type allele than PCR detection. Thus moving into the confirmational assays for the comparative analysis of MS-H determination and H serotyping, whole genome sequencing was only used to resolve disputed results between the two methods as it is not exclusively representative of a host’s flagella phenotype. On the other hand serotyping and MS-H is representative of the bacteria’s H type. Whole genome sequencing isn’t ideal for identifying clinical strains of E. coli for it is laborious and a long process compared to MS-H which is faster and contains less labour-intensive.
MS-H in clinical laboratories
Having shown that MS-H determined E. coli H type with high accuracy and in a short time frame, the application of H typing through MS-H could become very useful for the identification of unknown E. coli strains in a clinical laboratory. This is especially useful in the identification of an E. coli strain during a disease outbreak [7]. Faster identification of the pathogenic strain of E. coli would in turn also help combat the pathogenic E. coli more quickly.
Without the use of antibodies for agglutination and procedures for motility induction that are required in serotyping, MS-H is much less laborious in comparison. This would greatly benefit the professionals in the medical microbiology and public health laboratories who perform H typing.
Advantages and disadvantages of MS-H
It is significant that MS-H better determined the H type compared to conventional serotyping. As MS-H is faster, less laborious, provides sequence-level identification and has a higher throughput than serotyping, MS-H can be very useful in rapid and accurate identification of E. coli flagellar antigens. This may be a little over-idealistic as LC-MS/MS machines are very expensive and serotyping has been around for a very long time. It might be difficult currently for clinical laboratories to afford a LC-MS/MS machine or to change their workflow to incorporate MS-H as their method of H determination. However, mass spectrometer platforms are becoming more common in clinical laboratories. The uses of matrix-assisted laser desorption/ionization (MALDI) mass spectrometers have shown varying successes in microbiological identification. Also the use of MS isn’t just limited to H typing. MS could extend to the determination of the lipopolysaccharides (O antigen) of E. coli, toxins, and other relevant molecules within the spectrum of the mass spectrometer. This would not only determine the surface antigens of pathogenic E. coli, it would also give a broader profile of a pathogenic E. coli.
Conclusion and future directions
MS has potential to determine the H antigen type of E. coli better than serotyping, as shown in a comparative study between MS-H and serotyping through the use of a mass LC-MS/MS platform. MS-H provides sequence level identification of the flagellum type, whereas serotyping only provides visual agglutination assays for positive results and is therefore more prone to errors such as false positives and misidentification. Also, without the need for antisera, MS-H is less laborious, requires less time, and has a high throughput.
With the completion of the preliminary assay results of the comparative study between MS-H and serotyping in the determination of the flagella type, we are currently working on a country-wide thorough validation of the platform.
References
1. Cheng K, Drebot M, McCrea J, Peterson L, Lee D, McCorrister S, Nickel R, Gerbasi A, Sloan A, Janella D, Van Domselaar G, Beniac D, Booth T, Chui L, Tabor H, Westmacott G, Gilmour M, Wang G. MS-H: A novel proteomic approach to isolate and type the E. Coli H antigen using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). PLoS One 2013; 8(2): 1–12.
2. Tenover FC, Arbeit RD, Goering RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: A review for healthcare epidemiologists. Molecular Typing Working Group of the Society for Healthcare Epidemiology of America. Infect Control Hosp Epidemiol. 1997; 18(6): 426–439.
3. Machado J, Grimont F, Grimont PA. Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene. Res Microbiol 2000; 151(7): 535–546.
4. Edwards PR, Ewing WH. Edwards and Ewing’s Identification of Enterobacteriaceae, p536. Elsevier 1986. ISBN 978-0444009817.
5. Cheng K, Sloan A, Peterson L, McCorrister S, Robinson A, Walker M, Drew T, McCrea J, Chui L, Wylie J, Bekal S, Reimer A, Westmacott G, Drebot M, Nadon C. Knox D, Wang G. Comparative study of traditional flagellum serotyping and liquid chromatography-tandem mass spectrometry-based flagellum typing with clinical Escherichia coli isolates. J Clin Microbiol. 2014; 52(6): 2275-2278.
6. Cheng K, Sloan A, McCorrister S, Babiuk S, Bowden TR, Wang G, Knox D. Fit-for-purpose curated database application in mass spectrometry-based targeted protein identification and validation. BMC Res Notes 2014; 7: 444.
7. Cheng K, Sloan A, McCorrister S, Peterson L, Chui H, Drebot M, Nadon Celine, Knox D, Wang G. Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup. Proteomics Clin Appl. 2014; 8: 963–970.
The authors
Michael Chan*1,2, Huixia Chui1,3 MD, Drexler Hernandez1,2, Gehua Wang*1 MD and Keding Cheng*1,4 MD
1National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada
2University of Manitoba, Winipeg, MB, Canada
3Centre of Disease Control and Prevention, Henan Province, PR China
4Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada
*Corresponding authors
E-mail: M. Chan, umchanm@myumanitoba.ca; G. Wang, gehua.wang@phac-aspc.gc.ca; K. Cheng, keding.cheng@phac-aspc.gc.ca.
Rapid and simultaneous analysis of multiple drugs in hair samples using dynamic multiple reaction monitoring
Hair analysis for forensic diagnostics is gaining popularity in both research and applied settings. Commercially available dynamic multiple reaction monitoring (Dyn-MRM) software applied to hair samples can provide drug-use history for several months. The cost-effective drug test using Dyn-MRM software facilitates analysis of over 200 analytes in a 10-minute chromatographic run.
by Professor D. P. Naughton and Professor A. Petróczi
Background
Considerable global efforts are expended to address substance abuse which has major effects on public health and quality of life, as well as on economic and societal prosperity. This grand challenge impacts on a wide range of healthcare, regulatory and research endeavours. Key examples include destruction of lives through abuse of class A drugs, efforts to reduce doping in sport, attempts to address alcohol abuse and the lethal dangers of ‘legal highs’ (novel psychoactive substances).
Healthcare and regulatory officials engage in a wide range of activities to combat substance abuse. These include criminalization, banning substances in sport, education programs to prevent and efforts to understand and alter drug-related behaviour. However, in many cases there is an unmitigated failure to address the issues around substance misuse or abuse.
For example, for doping in sports, where vast efforts and resources are expended, indirect assessment of doping produces prevalence figures some 10-fold higher than positive doping test rates [1]. These frequent prevalence reports, at odds with figures from analytical tests, corroborate the belief that current anti-doping testing regimes are far from adequate [1]. Using doping in sport as an exemplar, major improvements in approaches to test for prohibited substances are needed. The advent of more advanced instrumentation aids testing in a number of ways. Increased sensitivity and affordability are very important but so are software developments that provide capability to monitor several hundred substances in one liquid chromatography-tandem mass spectrometry (LC-MS/MS) cycle of less than 10 minutes. These advances bring opportunities that require both instrumentation updates and frequent training updates for staff.
Hair analysis
Despite major advances in instrumentation and software, there are still obstacles to performing successful drug tests that benefit the drug taker. In sport, doping practices are frequently highly advanced, with some athletes taking heed of in-depth knowledge about most key parameters including generic testing methods, masking drugs and advances in detection for specific substances. The burden of proof has shifted to acquiring samples both in and out of competition as well as ensuring that appropriate tests are performed on each sample [2]. Owing to varied pharmacokinetics, analyte distribution and analytical procedures used, testing for a wide range of drugs in all samples is prohibitive [3]. Current testing approaches using biofluids impart considerable practical and financial consequences for the testing regime. Furthermore, cases of microdosing, masking and using novel substances make life challenging for the anti-doping officials.
Drug testing based on biofluids presents a number of issues that add considerably to cost but also are restrictive in terms of the number of tests required to cover a suitable duration owing to the pharmacokinetic profiles of many drugs. Where drugs or their metabolites are washed out efficiently after cessation of use, detection is less viable. The relatively short half-lives of many substances means the window for detection can be limited, which affects the success of occasional testing. The cost of supervised sampling along with the requirement for biofluid storage and handling to avoid sample corruption or infection is prohibitive for major levels of testing. Focusing on doping in sport, further complexities arise through variations in the lists of prohibited substances for testing in and out of competition [4]. The advantage of a longer window of detection via hair-analysis is suited to out of competition testing where a cumbersome system currently exists for sampling which is intrusive and controversial [5]. Thus, new approaches that allow a single test to be conducted simultaneously for (i) a wide range of substances and (ii) covering a prolonged period such as a 3-month window, would be valuable in sport for out of competition testing but also, beyond sport, for social drugs and new psychoactive substances.
In contrast to drug tests based on biofluids, hair analysis provides a range of advantages including: ease of sampling, ability to conduct multiple tests on one cut-hair sample to cover a prolonged duration (a typical 3-cm hair sample is equivalent to approximately 3 months’ growth), lack of issues with infection risk, facile storage at room temperature, lack of requirement to process tissue containing genetic data, and good stability of many drugs and metabolites in the hair matrix.
Instrumentation advances
We recently reported a hair-based method, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), for the analysis of substances of forensic nature [6]. The multi-drug/metabolite assay employs a dynamic multiple reaction monitoring (Dyn-MRM) method using proprietary software [7, 8]. It allows both screening and validated confirmatory analysis depending on the focus of the investigation. This approach has several benefits: (a) the Dyn-MRM software is suited to screen over 200 compounds on a single chromatographic run of under 10 minutes, (b) full validated methods for compounds can be incorporated into the software, (c) hair samples provide the opportunity to cover longer windows for detection in one test (e.g. approximately 3-month history covered in a 3-cm sample), and (d) the software is designed to allow ready adoption of new compounds of interest. The advantage of Dyn-MRM is that multiple reaction monitoring is employed with a focus on scanning for specific peaks at their selected elution times. This efficient method allows the analysis of large numbers of analytes simultaneously in a short run (Fig. 1). In our report, the proprietary software has been extended and applied to cover a range of drugs and metabolites of interest to forensic investigations including cognitive enhancers, amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opioids, steroids and sedatives. The chromatographic run is calibrated by a test mixture containing approximately 20 substances and further tailoring would be required to match a specific remit such as the WADA (World Anti-Doping Agency) prohibited list more closely [4].
Conclusion and future perspectives
The application of Dyn-MRM software to screen for a large range of drugs brings considerable advantages to laboratories involved in drug testing. The ease of use and ability to add new compounds to the screening database are noteworthy. Coupling this commercially available software to hair analysis adds the extra dimension of being able to screen for drug use over several months in one hair sample. This advance will add considerably to the efficiency of drug testing but will remain as an adjunct to other testing methods for out of competition testing in sport as it will not cover all analytes of interest to anti-doping officials [4]. Some substances are unlikely to be found in hair (e.g. performance enhancing peptides) and for other substances there will be issues with establishing a threshold – either for endogenous substances (e.g. testosterone) or for substances consumed through diet (e.g. drugs used in farming). Further limitations are that (i) a single use of a drug may be undetectable owing to the low levels deposited in hair, and (ii) more research is warranted to ascertain the effects of hair type and colour on analyte uptake and stability. In spite of these limitations, hair analysis coupled to modern advances in instrumentation sensitivity and software capabilities is promising in many scenarios especially to obtain a prolonged history of abuse and where ‘zero tolerance’ is applied (e.g. for synthetic steroids). In addition, hair analysis may have a role in support of the Athlete Biological Passport through analysis of indirect biomarkers of doping [9].
References
1. de Hon O, Kuipers H, van Bottenburg M. Prevalence of doping use in elite sports: A review of numbers and methods. Sports Med. 2015; 45(1): 47–69.
2. World Anti-Doping Agency (WADA). International Standards. 2015; https://www.wada-ama.org/en/what-we-do/international-standards.
3. Maennig W. Inefficiency of the anti-doping system: Cost reduction proposals. Subst Use Misuse 2014; 49(9): 1201–1205.
4. WADA. List of prohibited substances and methods. 2015; http://list.wada-ama.org/.
5. Hanstad DV, Loland S. Elite athletes’ duty to provide information on their whereabouts: Justifiable anti-doping work or an indefensible surveillance regime? Eur J Sport Sci. 2009; 9(1): 3–10.
6. Shah I, Petroczi A, Uvacsek M, Ranky M, Naughton DP. Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS. Chem Cent J. 2014; 8(1): 73.
7. Agilent Technical Overview. Ion optics innovations for increased sensitivity in hybrid MS systems. Agilent Technologies USA 5989-7408EN. 2007; http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-7408EN_HI.pdf.
8. Stone P, Glauner T, Kuhlmann F, Schlabach Tim, Miller K. New dynamic MRM mode improves data quality and triple quad quantification in complex analyses. Agilent Technologies USA 5990-3595EN. 2009; http://www.chem.agilent.com/Library/technicaloverviews/Public/5990-3595en_lo%20CMS.pdf.
9. Vernec AR. The athlete biological passport: an integral element of innovative strategies in antidoping. Br J Sports Med. 2014; 48(10):817–819.
The authors
Declan P. Naughton* PhD, Andrea Petróczi PhD
School of Life Sciences, Kingston University, London, UK
*Corresponding author
E-mail: D.Naughton@kingston.ac.uk
Myocardial infarction outcomes: redressing sex
In spite of major medical advances in diagnosis and treatment, cardiovascular disease (CVD) is still the leading cause of mortality in the Western world accounting for 51 percent of female and 42 percent of male deaths. Around half of these deaths are due to coronary heart disease, and it has been recognized for more than two decades that the outcome for women with acute coronary disease (ACD) is worse than it is for men. Quite apart from the fact that surveys show older women are less aware of their risk of myocardial infarction (MI) than men, women presenting with MI are less likely to be appropriately diagnosed.
Various explanations have been given for this disparity. Clinical symptoms of ACD in women may not be the ‘typical’ sudden severe chest pain; physicians have even attributed female symptoms of more diffuse pain, dyspnea and fatigue to falling levels of estrogen and progesterone. And although sex differences in electrocardiography (ECG) were first reported around 90 years ago, with recent studies emphasizing that normal values of the adult ECG should be both age- and sex-specific, the use of sex-specific diagnostic criteria is still not routine in many hospitals. In addition clinical research into ACD was biased towards men in the past, resulting in predictive values for analytes that are not necessarily appropriate for women; results of diagnostic tests should of course take sex-related differences into consideration. Given that the diagnosis of MI relies on a combination of clinical examination, suggestive ECG abnormalities and a rise and fall of key cardiac biomarkers, it is not surprising that myocardial infarction in women is still under-diagnosed.
However, the results of a recently published study in the BMJ should be a step towards more effective diagnosis. The troponin I level of 1126 consecutive patients presenting at a regional cardiac centre with suspected MI, 46% of whom were women, was measured using a high sensitivity assay and sex-specific diagnostic thresholds (men 34 ng/L, women 16 ng/L) in place of the current recommended threshold of 50 ng/L for both sexes. There was a significant increase in the number of women diagnosed with MI (from 11% to 22%) but the increase in men (19% to 21%) was not significant. Although studies continue to show that even when diagnosed women are less likely to undergo percutaneous coronary interventions or bypass surgery, receive prompt thrombolytic therapy or even be prescribed statins on discharge from hospital, more accurate diagnosis should go a long way towards redressing sex inequalities.
