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Review Article
iMedPub Journals ARCHIVES OF CLINICAL MICROBIOLOGY 2015
http://www.imedpub.com/ ISSN 1989-8436 Vol. 7 No. 1: 3
1
Identification and Typing Methods for the Graciela Castro-Escarpulli ,
Study of Bacterial Infections: a Brief Review Nayelli Maribel Alonso-
1
Aguilar ,
and Mycobacterial as Case of Study Gildardo Rivera Sánchez3,
4
Virgilio Bocanegra-Garcia ,
5
Xianwu Guo ,
Abstract 6
Sara R Juárez-Enríquez ,
2
Several techniques based on molecular biology and analytical chemistry has been Julieta Luna-Herrera ,
developed to reduce some of the bacterial characterization limitations. Molecular 5
Cristina Majalca Martínez ,
methods represent the best alternative to identify bacterial strains isolated from Aguilera-Arreola Ma
diverse origins and to improve research in the context of molecular epidemiology. 1
However, these methodologies are laborious and costly compared to phenotypic Guadalupe
or classical techniques, and there are no reliable routine laboratories. This review 1 Laboratorio de Bacteriología Médica,
shall provide basic elements for the understanding of these methodologies and Departamento de Microbiología, Mexico
raise interest in their collaborative use among analytical laboratories where 2 Laboratorio de Inmunoquímica II,
bacterial identification and typing are priorities, because molecular methods Departamento de Inmunología, Escuela
are not universally implemented but are available in research and reference Nacional de Ciencias Biológicas del
laboratories. Instituto Politécnico Nacional, Mexico
Keywords: Identification; Characterization; Bacteria; Infections DF, 11340, Mexico
3 Laboratorio de Biotecnología Ambiental,
Mexico
Received: October 25, 2015; Accepted: December 05, 2015; Published: December 4 Laboratorio de Medicina de
Conservación, Mexico
15, 2015 5 Lab. Biotecnología Genómica Centro de
Biotecnología Genómica del Instituto
Politécnico Nacional, Reynosa, 88710,
Introduction Mexico
6 Laboratorio de pruebas especiales,
One of the fundamental tasks of a microbiology laboratory Centro Médico Nacional 20 de
is to fully identify the microorganisms involved in processes Noviembre del Instituto de Seguridad y
associated to infection or related to humans. This allows knowing Servicios Sociales de los Trabajadores del
their etiopathogenic implications, their clinical evolution, as well Estado, Mexico DF, 03229, Mexico
as applying an efficient antimicrobial therapy [1].
Identification and characterization of bacteria in the past were Corresponding Author:
based on diverse phenotypic and genotypic methods (Table 1) Dr. Ma. Guadalupe Aguilera Arreola
however, in the last decades, it has been observed that the Laboratorio de Bacteriología Médica,
genotypic methods can represent a better alternative to establish Departamento de Microbiología, Escuela
the identity of bacteria and to enrich epidemiological research of Nacional de Ciencias Biológicas, IPN, Esq.
infectious diseases [2]. Prolongación de Carpio y Plan de Ayala s/n,
Bacterial infections cause morbidity and mortality, and are Col. Casco de Santo Tomás, Del. Miguel
responsible for the increase in costs and hospitalization times of Hidalgo, CP. 11340. México City, DF, Mexico.
patients. The time needed to identify a pathogen based on its lupita_aguilera@hotmail.com
phenotypic characteristics is the first challenge, as the sample
has to be seeded and incubated for at least 24 hours and, Tel: (+52-55) 57296300, extension 62374
then, conventional biochemical tests must be performed in at Fax: (+52-55) 57296207
least another 24-hour period, conditions that delay results and
compromise the patient’s health. Citation: Aguilera-Arreola MG. Identification
Currently, in many microbiology laboratories, the use of and Typing Methods for the Study of Bacterial
automated or semi-automated commercial systems for Infections: a Brief Review and Mycobacterial as
bacterial identification is common practice, as for example: API Case of Study. Arch Clin Microbiol. 2015, 7:1.
© Copyright iMedPub | This article is available from: www.acmicrob.com 1
ARCHIVES OF CLINICAL MICROBIOLOGY 2015
ISSN 1989-8436 Vol. 7 No. 1: 3
Table 1 Methods used in clinical laboratories for bacterial identification or typing.
Phenotypic methods Genotypic methods
Biochemical reactions Hybridization
Serological reactions Plasmids profile
Susceptibility to anti-microbial agents Analysis of plasmids polymorphism
Susceptibility to phages Restriction enzymes digestion
Susceptibility to bacteriocins Reaction and separation by Pulsed-Field Gel Electrophoresis (PFGE)
Profile of cell proteins Ribotyping
Polymerase Chain Reaction (PCR) and its variants
Ligase Chain Reaction (LCR)
Transcription-Based Amplification System (TAS)
Multilocus Sequence Typing (MLST)
Spoligotyping and MIRUS-VNTR
ENTEROTUBE, VITEK, PHOENIX, MALDI-TOF MS and the GENOTYPE not yet universally implemented, they are available at research
MYCOBACTERIUM CM system for mycobacteria. Some of the and reference laboratories that could provide the expertise to
characteristics taken into account to choose the identification solve with first level methodologies the health problems of a
system are: the easiness to inoculate samples, characteristics to country.
be determined, the required handling for the sample processing Phenotypic identification
after incubation, and the availability and extent of databases [3].
Phenotypic methods are not always able to identify the For the identification of the causal agent of an infectious process,
microorganism to the species level, and much less to the the following must be considered: 1) sample collection, 2)
strain level. Therefore, if a breakout, in which only one clone determination of microscopic and colonial morphotypes, and
is responsible, is to be determined, more time and the use of 3) identification based on the bacterial metabolism through
genotypic (molecular) or more specific immunological techniques conventional or automated tests [2]. The phenotypic study
are required. Despite their limitation, phenotypic techniques represents the classical point of view for identification, and most
provide an initial identification that allows taking decisions and is identification strategies are based on it [5].
more available at clinical laboratories or hospitals due to their low In most cases, phenotypic identification is based not only on
costs and ample training of the personnel in this health area [4]. one method but rather on the combination of more than one.
Methods for the isolation and identification of organisms The sample must come from the site where the microorganism
from human samples, biological products, or of any other is causing the damage or must be representative of the site or
origin involve the isolation in a pure culture of the organism of product where it is multiplying. Some samples used in clinical
interest, followed by the necessary tests to discern the microbial microbiology are: feces, urine, pharyngeal exudate, cerebrospinal
metabolism and/or by diverse immunological techniques that fluid, tears, semen, vaginal fluid, tissues, and/or biopsies. Some
will facilitate identification. In many aspects, the culture methods methods require a pure isolation of the microorganism from the
and other techniques used for identification are limited in terms sample, whereas others do not need it. Phenotypic bacterial
of sensitivity, specificity, or both. Improvements in sensitivity, identification is based fundamentally on the comparison of
specificity, and required time are based on progresses in molecular phenotypic characteristics of unknown bacteria to those of type
biology that have been integrated in commercial strategies for culture. The reliability of the identification is in direct proportion
fast diagnoses. The use of molecular biology techniques for to the number of similar characteristics. In medical bacteriology,
the identification and follow-up of pathogens is based on the the previous expertise of the analyzer and the association
characteristics of the genome of the particular organism to among the microorganism, the site, and type of infection are
be detected or characterized. However, several aspects still instrumental for the preliminary identification. Hence, in the
complicate their application in the microbiology laboratory: the traditional or classical bacterial identification process, three
difficulty in the isolation, the slow growth, the costs of the tests, levels of processing have been established [1].
and their poor detection sensitivity for the identification of some a) Primary tests are considered in the first level. These are fast and
bacterial species coming from complex samples, among others. easy tests to perform, such as uptake of dyes and stains as Gram
This review covers the different phenotypic, also called classical, or Ziehl-Neelsen, microscopic determination of the bacterial
methodologies, as well as different molecular biology methods morphotype revealed by the stains, growth characteristics at
that are applied to bacterial characterization. Likewise, it is different incubation atmospheres, different temperatures, and
aimed at raising the interest in the collaborative use of these in diverse culture media, production of oxidase and catalase
methodologies among laboratories where bacterial identification enzymes, oxidation-fermentation, glucose fermentation,
and typing are priorities, since, although molecular methods are productions of spores, and mobility. Through these tests, it is
This article is available from: www.acmicrob.com
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ARCHIVES OF CLINICAL MICROBIOLOGY 2015
ISSN 1989-8436 Vol. 7 No. 1: 3
generally possible to place the pathogen, provisionally, in some of are: MALDI-TOF for microbial identification; MicrobeLynxTM of
the main groups of clinical relevance. Afterwards, other methods Waters Corporation, MALDI BiotyperTM of Bruker Daltonics, and
with greater discriminatory power can be used, to be able to MS-ID of BioMérieux [12]. The last one allows the mycobacteria
discern among microorganisms that present a very similar aspect identification [13].
in the macro and microscopic analyses [6]. Genotypic (molecular) identification
b) The second level of identification must specify the genus In recent years and with the advent of new methodologies based
of the microorganism. In both this and the former level, the on molecular methods great advances have been made in the
hypothesis on the probably identity of microorganisms is based on diagnosis of clinically relevant bacteria. Among them, stand
the characteristics of the culture and on the primary tests, which will out the ribosomal RNA detection through hybridization with a
allow determining the genus, group of genera, or, in some cases, the DNA probe and that of nucleic acids amplification from clinical
family of the isolate. Clinical data must also be taken into account. samples. These techniques improve the sensitivity and diagnostic
This will depend to a great extent on a stable pattern of phenotypic specificity with respect to other detection techniques, including
features and on the expertise of the microbiologist [7]. culture, and, in some cases, have allowed for the simultaneous
c) Finally, the third identification level is at the species level. detection of several microbial agents from the same sample [14].
Some biochemical tests allow identifying accurately most of The first step in the development of methodologies based on
the clinically significant bacteria. If this is not possible, a more molecular biology techniques was supported by the detection
ample battery of tests can be used, like those found in different of nucleic acids of microorganisms by means of a probe through
commercial systems. hybridization. The genetic probe is a nucleic acid molecule, in a
Numerous multi-test systems or equipments are available in the monocatenated state and marked, that can be used to detect
market to make bacterial identification fast and reliable. These a complementary DNA sequence. Oligonucleotide probes
techniques require a precise control of the inoculum, its purity, are obtained from natural DNA by cloning DNA fragment into
and way of inoculation, incubation, and reading of the tests, appropriate plasmid vectors and then isolating the cloned DNA
because not following these criteria may lead to errors. These or through direct synthesis by means of combinatory chemistry.
systems can be manual, semi-automated, or automated. The Probes can be labeled with substances that produce colorful
result is compared to standardized tests or to the database of reactions under adequate conditions [15].
numerical profiles that the commercial methods have developed DNA hybridization techniques are relatively easy to perform and
for this purpose. A limitation is the appearance of mutating strains interpret. Amplification techniques based on the detection of DNA
and the acquisition of plasmids that can give origin to strains of using Polymerase Chain Reaction (PCR) and Ligase Chain Reaction
different characteristics [5,8]. (LCR) or transcription-mediated specific rRNA amplification is
In contrast to the laboratories of clinical biochemistry or already available both to be performed in house or commercially
hematology that have benefitted from the technology to obtained. These techniques provide faster results with better
simplify sample processing and thus obtain results in a short sensitivity and specificity than conventional techniques.
time, automatization of the microbiology laboratory is more Depending on the type of sample these techniques detect from
complex given the large variety of clinical samples to be analyzed 15 to 20% more infectious agents than the conventional ones and
and the inherent characteristics of different microorganisms. 25 to 70% more than through immunofluorescence or Enzyme
Recently, mass spectrophotometry (MS) has become part of the Immunoanalysis (EIA) [14,16].
microbiology laboratory offering a fast and reliable alternative for Construction of probes to detect virulence markers, as those
the identification of microorganisms, including one of the most directed to genes encoding toxins, allows identifying those
difficult identifiable bacterial groups, mycobacteria [9,10]. organisms that carry these genes in the clinical samples, without
MS is an analytical technique that allows analyzing with great having to cultivate the samples. Examples of the later are the
accuracy the composition of different chemical elements by probes for Escherichia coli enterotoxins, for Vibrio cholerae toxin,
permitting the measurement of ions derived from molecules or for toxins of Clostridium difficile, which can be applied directly
and separating them in function of their mass/load (m/z) ratio to fecal samples [17].
[11]. The mass spectrum of each compound is named “chemical Different target genes are used for the detection of
trace” and is a graphical representation of the fragments microorganisms, for example those causing Sexual Transmission
obtained, by an increasing order of mass in terms of its relative Infections (STI), which have been used in PCR assays; among
abundance. Bacterial identification based on the proteins profile them are genes omp1 and omp2 of the Main Membrane Proteins
obtained by MALDI-TOF mass spectrometry was proposed (MOMP) to study the main etiological agents; the cryptic plasmid
several decades ago. However, it has been used only recently pCT and genes 16S rRNA and 23S rRNA, for assays addressed at
as a fast and reliable method for bacterial identification [9]. The identifying C. trachomatis [14,18,19]. Focusing on genes 16S rRNA
currently commercialized platforms use MS for the identification and 23S rRNA increases sensitivity of the assay, as normally there
of microorganisms through different approaches: identification are multiple copies in microorganisms. However, some authors
based on the specific protein profile of each microorganism suggest that the crossed reactions with other bacteria could
(proteomic approach) or on the analysis of its nucleic acids pose a problem; whereas others have demonstrated that the
(genomic approach). Some of the commercial systems that use MS use of conserved regions of gene 16S rRNA in the amplification
© Copyright iMedPub
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ISSN 1989-8436 Vol. 7 No. 1: 3
reactions allows for species-specific differentiation [19, 20]. Use In the amplification-pyrosequencing platforms, bacterial
of genes and target regions for the detection of mycobacteria is identification is achieved by PCR of three variable regions of
a well studied area, particularly due to the difficulty posed for the 16S rRNA (V1-V3, or V1, V2 and V6). Lower amplicons of
the isolation of these microorganisms from biological samples 500 bp are obtained, their nucleotides composition can be
and furthermore because of the current hardships in handling determined by means of the emission of light by the release
these very virulent microorganisms. Several sequences, genes, of pyrophosphates (extension byproducts by polymerization of
and intergenic regions have been used for the identification the DNA chain). This platform has been increasingly innovated
of this bacterial genus, among them, the rRNA 16-23S region, based on the type of clinical sample and on the determination of
genes 16S rRNA, gyrB and rpoB, the insertion element IS6110, different genic fragments corresponding to the different virulence
and the eliminate differentiation regions RD1 and RD4 [21]. The factors and the resistance to antimicrobials, which has improved
study of these genes or genic sequences by means of PCR will the versatility of this platform [2,25]
eventually allow comparative sequence analysis of the obtained Another innovating platform for bacterial identification is the
product with the sequences of reference isolates. Several conjunction of amplification (by PCR) and mass spectrophotometry
commercial probes for the diagnosis of infectious diseases have (PCR/ESI-TOF). The latter allows for the universal detection of
been designed, but the capacity of detecting a small number of one or more pathogens encountered in a wide variety of samples
organisms or few copies of the gene in the clinical sample is still (environmental, clinical, foodborne, o in cultures). It consists
a limiting factor of this technique. However, combination of PCR in the extraction of nucleic acids and PCR amplification with
with probes hybridization can become the method of choice, primer pairs of ample range; one or several PCR products are
particularly, for microorganisms whose culture in the laboratory obtained that correspond to genomic identification regions of the
is slow and difficult [15]. different microbial domains in relation to the complexity of the
PCR is an in vitro method of the DNA synthesis with which a problem sample. The products are desalted and then ionized and
particular segment of DNA is amplified by being delimited with aerosolized towards the mass spectrometer, generating signals
a pair of flanking primers. Copying is achieved exponentially that are processed to determine their mass and composition.
through repeated cycles of different incubation periods and Results are interpreted with the TIGER (Triangulation
temperatures in the presence of a thermostable DNA polymerase Identification for the Genetic Evaluation of Risks) strategy, and
enzyme. In this way, millions of copies of the desired DNA accessing the information into a genomic database that assigns
sequence can be obtained in a couple of hours. This is a highly the species. The advantages of this platform are that it does not
specific, fast, sensitive, and versatile molecular biology technique require culture, is efficient in polymicrobial samples, and, in the
to detect the smallest amounts of a specific DNA, fostering its case of non-characterized new pathogens, it allows assigning
easy identification and avoiding the use of radioisotopes [22]. them to bacterial genera or families. In addition, it also permits
Despite the benefits that the PCR technique offers in comparison to detect some virulence and resistance genes, and typing of the
to culture for the detection of some microorganisms, the identified microorganism [2,12].
commercially available techniques are scarce and are limited Typing of microorganisms
to research laboratories or to reference laboratories specialized
in molecular diagnoses, among other causes, due to their high After bacterial identification, microorganisms have to be typed
cost. An alternative to make the use of molecular diagnoses for epidemiological studies; hence, molecular typing systems
feasible as routine techniques could be the acquisition of constitute one of the molecular techniques contributions to
reagents in a separate manner and standardization of nucleic microbiology widely used in the last years. These systems involve
acids extraction and amplification protocols designed in each a large variety of techniques aimed at comparing the structure of
diagnostic laboratory; this would lead to a significant reduction genomes of highly inter-related organisms.
in technological dependence and to an increase in the sensitivity
and specificity of the used diagnostic techniques [23]. Typing methods (phenotypic and genotypic) allow differentiating
The multiple amplification for the concomitant detections of one bacterial strain from another. Before using a typing technique
some microorganisms enhances, in some cases, the sensitivity it is important to ensure that the method can differentiate among
and specificity of those addressed at a single microorganism. This non-related isolations, that it is able to detect the same strain in
PCR variant is called multiple PCR (mPCR), in which more than one different samples, and that it reflects the gene relations among
target sequence can be amplified simultaneously by the inclusion isolations with epidemiological relation [26].
of more than a pair of primers in the reaction [24]. This technique From a practical point of view, a typing system should be
has been applied successfully in many diagnostic areas, like the reproducible, have a high discrimination capacity, and be easy to
study of infectious diseases, species genotyping, diagnosis of use and to interpret the results [26]. Notwithstanding, election
hereditary diseases, identification of mutations, paleontology, of the molecular method depends also on other factors, such as
anthropology, and forensic sciences, among others; here, the the microorganism to be studied, the clinical sample, the target
technique has shown the potential to save considerable time, to be studied (a single gene or the whole genome), the area of
without compromising the usefulness and efficiency of the test application, the infrastructure available at the clinical laboratory,
[24]. On the other hand, plataforms for pathogen identification and the speed needed to reach a result. Once the microorganism
are becoming available like pyroseguencing and spectroscopy has been identified, it is important to know whether it is
[25]. responsible for a breakout; therefore, the corresponding
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