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PubMed2005

Role of real-time molecular typing in the surveillance of Campylobacter enteritis and comparison of pulsed-field gel electrophoresis profiles from chicken and human isolates.

The goal of the present study was to assess the contribution of real-time molecular typing, used alone or with clinical surveillance, to the prompt identification of clusters of Campylobacter enteritis. Potential poultry sources were sought by comparing the pulsed-field gel electrophoresis genotypes of human and fresh whole retail chicken isolates collected during the same study period. Among 183 human isolates, 82 (45%) had unique genotypes, 72 (39%) represented 26 clusters of 2 to 7 isolates each, and 29 (16%) represented three clusters of 8 to 11 isolates each. Molecular typing was useful for the confirmation of outbreaks suspected on the basis of epidemiological surveillance, but for most small clusters, no epidemiological link could be established. Thus, the added value of real-time molecular typing is questionable, since the numerous small clusters identified were of unclear public health significance. Among 177 chickens, 41 (23%) yielded campylobacter isolates; of these, 19 (46%) had genotypes similar to those of 41 (22%) human isolates. However, a temporal association was demonstrated in only a minority of cases, and most genotypes were present only in a single species, suggesting that sources other than chickens are important in human campylobacteriosis. Further investigation with samples from water and other possible environmental sources is needed to define the most efficient strategy for the application of molecular typing and identification of the source(s) of sporadic cases of campylobacteriosis.

Adolescent↗

PubMed2004

Molecular typing of Cryptococcus neoformans by PCR fingerprinting, in comparison with serotyping and Fourier transform infrared-spectroscopy-based phenotyping.

Molecular typing by PCR fingerprinting using the single primer (GACA)4 was performed with 110 isolates of Cryptococcus neoformans. Seventy clinical isolates of C. neoformans var. neoformans from Germany (n = 52) and Africa (n = 18) were included. Of these, serotype A (C. neoformans var. grubii) accounted for 47 isolates, serotype D for 12 and serotype AD for 11. Fourier transform infrared (FT-IR) spectroscopy was evaluated for its discriminatory power in phenotyping. Molecular types, defined by different PCR fingerprinting patterns, were compared to serotypes, and both sets of results were compared with the results of analysis by FT-IR spectroscopy. PCR fingerprinting revealed genotypic diversity within each serotype; it showed three different genotypes (designated VNA1-VNA3) within serotype A, two within serotype D (VND1 and VND2), and three within serotype AD (VNAD1-VNAD3). The nomenclature of molecular types within C. n. var. neoformans, as seen in publications to date, is not uniform. In this study, the name assigned to each genotype was based on the 98.6% concordance of genotypes with serotypes, a correspondence that facilitates interlaboratory comparison. This nomenclature is tentatively recommended as a standard. FT-IR spectroscopy combined with hierarchical cluster analysis successfully distinguished C n. var. neoformans from C. n. var. gattii. For C. n. var. neoformans, FT-IR confirmed three distinct genotypes within serotype A and was able to distinguish isolates derived from particular patients as well as isolates differing at the sub-genotype level. Within C. n. var. gattii, the serotypes B and C did not correlate with the four genotypes VGI-VGIV. However, these serotypes could clearly be separated by FT-IR spectroscopy. The molecular profiles were reproducible, and were more stable and more discriminating than serotyping. In connection with a standardized nomenclature, PCR fingerprinting can be a beneficial tool for global epidemiological studies. FT-IR spectroscopy adds an additional level of resolution.

Bacterial Typing Techniques↗

PubMed2004

Does molecular typing make any contribution to the care of patients with infection?

Molecular typing has been used extensively to study the epidemiology of infection, but there are few studies on the role of typing in the clinical management of infected patients. Examples of this include distinguishing relapse of infection from new infection, determining the significance of colonisation with potential pathogens, assessing the capacity of isolates to spread and cause serious illness, and linking changes in antimicrobial resistance with treatment. Further studies in selected patient groups and greater collaboration between molecular microbiologists, clinical microbiologists and infectious disease physicians are required to determine the impact of molecular typing in these and other scenarios.

Bacteria↗

PubMed1994

Molecular typing of Actinomyces pyogenes isolates.

The molecular characterization of 28 clinical Actinomyces pyogenes strains was attempted. SDS-PAGE protein profiles did not allow to distinguish isolates. Restriction endonuclease analysis of total DNA gave the finest differentiation between strains but the profiles were difficult to read. Ribotypes after DNA digestion by Bst EII or Sma I have a high discriminatory power and are more helpful epidemiological markers. No relationship could be demonstrated between molecular types and clinical sources.

Actinomyces↗

PubMed1992

Characterization of methicillin-susceptible and methicillin-resistant Staphylococcus aureus isolates by molecular typing methods.

Two groups of clinical Staphylococcus aureus isolates were examined by molecular typing methods. Detailed protocols for the technical procedures are described. By means of plasmid DNA profiles, plasmid DNA restriction enzyme digestion, as well as whole-cell DNA endonuclease digestions subjected to conventional agarose gel electrophoresis and pulsed-field gel electrophoresis, eight methicillin-susceptible S. aureus strains from three patients segregated into three different groups. Consecutive isolates from the same patient revealed identical fingerprints. Generally, good agreement between different molecular typing methods also was achieved in the investigation of 28 methicillin-resistant isolates. However, compared to whole-cell DNA restriction endonuclease analysis, plasmid DNA profiling (all but one strain had detectable plasmid DNA) showed a somewhat greater discriminatory ability. The techniques evaluated were reproducible and relatively easy to perform and provided valuable tools for studying the epidemiology of S. aureus in hospitals.

Bacterial Typing Techniques↗

PubMed1995

Changes in the molecular types of connectin and nebulin during development of chicken skeletal muscle.

Changes in the molecular types of connectin and nebulin during development of chicken breast and leg muscles were determined by an improved SDS-polyacrylamide gel electrophoresis (PAGE) using 2% polyacrylamide slab gel. The adult leg-type alpha-connectin (alpha L-connectin) and nebulin (L-nebulin) appeared in embryonic breast muscle, and changed into the adult breast-type ones (alpha B-connectin, B-nebulin) specific for adult breast muscle after hatching. In leg muscle, alpha L-connectin and L-nebulin appeared in an embryonic stage, and remained unchanged in molecular types throughout the entire process of development. alpha-Connectin and nebulin seemed to be regulated by a similar mechanism during development. On the other hand, beta-connectin appeared in an earlier stage of development of the embryonic breast muscle, independently of alpha-connectin.

Animals↗

PubMed2006

Isothermal amplification and molecular typing of the obligate intracellular pathogen Mycobacterium leprae isolated from tissues of unknown origins.

Molecular diagnostic and epidemiology studies require appreciable amounts of high-quality DNA. Molecular epidemiologic methods have not been routinely applied to the obligate intracellular organism Mycobacterium leprae because of the difficulty of obtaining a genomic DNA template from clinical material. Accordingly, we have developed a method based on isothermic multiple-displacement amplification to allow access to a high-quality DNA template. In the study described in this report, we evaluated the usefulness of this method for error-sensitive, multiple-feature molecular analyses. Using test samples isolated from lepromatous tissue, we also evaluated amplification fidelity, genome coverage, and regional amplification bias. The fidelity of amplified genomic material was unaltered; and while regional differences in global amplification efficiency were seen by using comparative microarray analysis, a high degree of concordance of amplified genomic DNA was observed. This method was also applied directly to archived tissue specimens from leprosy patients for the purpose of molecular typing by using short tandem repeats; the success rate was increased from 25% to 92% without the introduction of errors. This is the first study to demonstrate that serial whole-genome amplification can be coupled with error-sensitive molecular typing methods with low-copy-number sequences from tissues containing an obligate intracellular pathogen.

Bacterial Typing Techniques↗

PubMed2003

Isolation of two molecular types of Cryptococcus neoformans var. gattii from insect frass.

Cryptococcus neoformans var. gattii has regularly been the cause of serious human disease. However, the environmental sources of these infections often remain unclear. During an environmental sampling study, two different strains of C. neoformans var. gattii were isolated from fresh insect frass (order Lepidoptera; family Oecophoridae) in a shallow cavity in the bark of a living Eucalyptus tereticornis tree, one molecular type VGI and the other VGII. This is the first published report of the isolation of two different molecular types of C. neoformans var. gattii from a single source, and the third of isolation of molecular type VGII from an environmental source. The potential association with insect frass is consistent with categorising C. neoformans var. gattii within the Tremellales, containing mycoparasitic fungi.

Animals↗

PubMed2007

Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae.

Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.

Autoanalysis↗

PubMed1997

Molecular typing of methicillin-resistant Staphylococcus aureus by polymerase chain reaction: distribution of the typed strains in hospitals.

Three methods for molecular typing of methicillin-resistant Staphylococcus aureus using polymerase chain reaction (PCR) were compared. The method which amplified the variable number of tandem repeat of dru sequences grouped the isolates into six types. Whereas, the method examining restriction fragment length polymorphism of coagulase gene and the method using arbitrarily primed-PCR showed poor diversity in typing. We investigated the distribution of dru types in two hospitals. Obvious concentration of a type in one ward was not recognized in our hospital. In the other hospital, a rare type was detected from the inpatients in the pediatrics ward. It suggested that the infection was an epidemic. We also found that some patients were infected with more than two strains. Even if two isolates show the same type, it does not necessarily mean that they originated from one clone. However, this method brings meaningful information on nosocomial infection, more easily than other genotyping.

Bacterial Typing Techniques↗

PubMedDate not supplied

Molecular typing for investigating an outbreak of Candida krusei.

Invasive infections due to Candida krusei are often observed in immunocompromised patients who have received prior therapy with fluconazole, although infection has also occurred in patients in the absence of this antifungal agent. From August 25 through September 19, 1995, we identified four patients with C. krusei fungemia on our hematology/oncology unit. Molecular typing of all the isolates was performed by restriction endonuclease analysis of genomic DNA using HinfI. A total of 7 patients found to be colonized or infected with C. krusei were matched with 14 controls. There was no difference between the cases and controls with respect to underlying disease, duration of hospitalization, or neutropenia. The numbers of days of hyperalimentation, corticosteroids, and antibiotics were similar between both groups. The mean number of antibiotics was greater in the cases versus controls (5.0 versus 2.5; p = .003). There was no difference with respect to total dose or duration of fluconazole administration. Molecular typing of the isolates revealed that four had identical DNA banding patterns, plus another two that differed by one band and were considered related. Three historical strains were unrelated. In conclusion, this report demonstrates that molecular typing can be used to define clonality and, thereby, support increased infection control practices to eliminate such outbreaks when evidence of clonal spread is present.

Amphotericin B↗

PubMed2004

The use of molecular typing to evaluate the dissemination of antimicrobial resistance among Gram-negative rods in Brazilian hospitals.

Antimicrobial resistance has increased rapidly in Brazil and worldwide during the past few years, giving rise to a growing necessity for antimicrobial resistance surveillance programs. These programs have been instituted in order to monitor bacterial resistance in various regions, and to guide empirical antimicrobial therapy. We evaluated the use of molecular typing in multicenter surveillance programs. We also studied the dissemination modes of selected resistance profiles. Antimicrobial susceptibility to various antimicrobial agents was evaluated by the reference broth microdilution method. Bacterial isolates with selected susceptibility patterns were characterized by pulsed field-gel electrophoresis (PFGE). A total of 119 Gram-negative bacteria were molecularly typed, including 22 imipenem-resistant Pseudomonas aeruginosa, 26 ESBL-producing Escherichia coli, 27 cefoxitin-resistant-ESBL-producing Klebsiella pneumoniae, 33 Enterobacter spp., 8 Citrobacter spp., and 3 S. marcescens isolates resistant to ceftazidime. The isolates were from clinically apparent bacteremia of patients hospitalized in medical centers located in 13 cities of 11 Brazilian states. Our molecular typing results revealed a great genetic diversity among isolates of the same species. However, some major PFGE patterns were found in more than one isolate. All repeated PFGE patterns were detected in only 2 isolates, which were isolated within the same institutions or in different medical centers. We conclude that the ability to characterize organisms phenotypically and genotypically is a powerful epidemiologic tool and it provides unique information that is very important for multicenter surveillance programs.

Anti-Bacterial Agents↗

PubMed2000

Molecular typing demonstrating transmission of gram-negative rods in a neonatal intensive care unit in the absence of a recognized epidemic.

Molecular typing techniques have been used in outbreak investigations. In this study, molecular typing techniques were used to track the spread of gram-negative rods (GNRs) in a neonatal intensive care unit (NICU) in the absence of an outbreak. Stool or rectal swab cultures for GNRs were obtained from all infants on admission, weekly, and on discharge. GNRs were tested for gentamicin susceptibility and were typed by contour-clamped homogeneous electric field electrophoresis. Transmission of identical strains of GNRs among infants was noted. Shared strains were more gentamicin resistant compared with unique strains (53% vs. 10%; P=.0001). Infants first colonized when they were >1 week of age had more total days of antibiotic treatment and had a higher rate of acquiring a shared and gentamicin-resistant strain, compared with infants colonized earlier. Antibiotic use increases colonization of infants in the NICU with resistant and shared strains of GNRs.

Anti-Bacterial Agents↗

PubMed1996

Quantitative antibiogram as a typing method for the prospective epidemiological surveillance and control of MRSA: comparison with molecular typing.

OBJECTIVE: Evaluation of the quantitative antibiogram as an epidemiological tool for the prospective typing of methicillin-resistant Staphylococcus aureus (MRSA), and comparison with ribotyping. METHODS: The method is based on the multivariate analysis of inhibition zone diameters of antibiotics in disk diffusion tests. Five antibiotics were used (erythromycin, clindamycin, cotrimoxazole, gentamicin, and ciprofloxacin). Ribotyping was performed using seven restriction enzymes (EcoRV, HindIII, KpnI, PstI, EcoRI, SfuI, and BamHI). SETTING: 1,000-bed tertiary university medical center. RESULTS: During a 1-year period, 31 patients were found to be infected or colonized with MRSA. Cluster analysis of antibiogram data showed nine distinct antibiotypes. Four antibiotypes were isolated from multiple patients (2, 4, 7, and 13, respectively). Five additional antibiotypes were isolated from the remaining five patients. When analyzed with respect to the epidemiological data, the method was found to be equivalent to ribotyping. Among 206 staff members who were screened, six were carriers of MRSA. Both typing methods identified concordant of MRSA types in staff members and in the patients under their care. CONCLUSIONS: The quantitative antibiogram was found to be equivalent to ribotyping as an epidemiological tool for typing of MRSA in our setting. Thus, this simple, rapid, and readily available method appears to be suitable for the prospective surveillance and control of MRSA for hospitals that do not have molecular typing facilities and in which MRSA isolates are not uniformly resistant or susceptible to the antibiotics tested.

Bacterial Typing Techniques↗

PubMed2002

Antibiotic resistance and molecular typing of Pseudomonas aeruginosa: focus on imipenem.

Susceptibility tests by disk diffusion and by E-test and molecular typing by macrorestriction analysis were performed to determine the relatedness of Pseudomonas aeruginosa isolates from three distinct hospitals. The resistance profile of 124 isolates to 8 antimicrobial agents was determined in three different hospitals, in Porto Alegre, Brazil. Frequencies of susceptibility ranged from 43.9% for carbenicillin to 87.7% for ceftazidime. Cross-resistance data of imipenem-resistant isolates indicated that most (70%) were also resistant to carbenicillin, although 30% remained susceptible to ceftazidime and cefepime. In general, susceptibility profiles were not able to determine relatedness among isolates of P. aeruginosa. On the other hand, molecular typing by macrorestriction analysis demonstrated high discriminatory power and identified 66 strains among 72 isolates of P. aeruginosa. Imipenem-susceptible isolates were all different. However, identical clones of imipenem-resistant isolates were found in two of the hospitals, despite variable response to other antibiotics. No clustering of infection among the different medical centers was observed. In conclusion, clones of P. aeruginosa did not spread among the different hospitals in our city even though related isolates of imipenem-resistant P. aeruginosa were found.

Bacterial Typing Techniques↗

PubMedDate not supplied

[Molecular typing by amplified fragment length polymorphism and PCR-restriction fragment length polymorphism , biotyping and antimicrobial susceptibility of Campylobacter jejuni].

Molecular typing systems have provided invaluable information for tracking infectious agents through the food chain. These tools have been essential for understanding the epidemiology of gastrointestinal infectious diseases, therefore providing essential and evidence-based information for appropriate interventions and preventative measures. Two such molecular typing techniques based on the polymerase chain reaction (PCR) that have been applied to the epidemiology of foodborne pathogens are, amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) analysis. Campylobacter is responsible for one of the most common bacteria foodborne gastrointestinal infections affecting humans, especially in developed countries. The object of this paper is to apply AFLP and RFLP analysis of the flagellin (flaA) gene to 18 isolates of Campylobacter jejuni from human sporadic cases in Italy. Results of these analyses were compared to the phenotypes of these isolates based on biotyping and antimicrobial resistance determinations. All isolates were typable by the four methods. The RFLP procedure was performed with DdeI and HinfI enzymes, and 12 and 8 distinct profiles respectively were recognised. AFLP analysis was more discriminatory, and recognised 16 different profiles. Results from AFLP were reproducible and applicable for definitive characterisation of C. jejuni isolated from different outbreaks. PCR-RFLP of the flaA gene represents a useful tool only to compare isolates within a single outbreak.

Anti-Bacterial Agents↗

PubMed1995

Analysis of methicillin-resistant and methicillin-susceptible Staphylococcus aureus by a molecular typing method based on coagulase gene polymorphisms.

A molecular typing method for Staphylococcus aureus based on coagulase gene polymorphisms (coagulase gene typing) was evaluated by examining a total of 240 isolates which comprised 210 methicillin-resistant S. aureus (MRSA) and 30 methicillin-susceptible S. aureus (MSSA) collected from a single hospital. By AluI restriction enzyme digestion of the PCR-amplified 3'-end region of the coagulase gene including 81-bp repeated units, the MRSA and MSSA isolates examined were divided into 6 and 12 restriction fragment length polymorphism (RFLP) patterns, respectively, whereas five patterns were commonly detected in MRSA and MSSA. MRSA isolates that showed a particular RFLP pattern were considered to be predominant in the hospital. Coagulase typing with type-specific antisera was also performed for all S. aureus isolates for comparison. Coagulase types II and VII were most frequently detected and included isolates with four and five different AluI RFLP patterns, respectively, whereas each of the other coagulase types corresponded to a single RFLP pattern. These results indicated that RFLP typing was more discriminatory than serological typing, for typing S. aureus and demonstrated its utility in epidemiologic investigation of S. aureus infection in hospitals.

Bacterial Typing Techniques↗

PubMed2003

Molecular typing of Mannheimia (Pasteurella) haemolytica serotype A1 isolates from cattle in Japan.

Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods were applied for molecular typing of 130 Mannheimia (Pasteurella) haemolytica serotype A1 isolates obtained from 13 prefectures in Japan. These isolates were divided into 15 ApaI PFGE profiles that formed six distinct clusters (clusters A-F). Fifty-three (40.7%) isolates were classified in cluster B, and 20.0, 13.8, 12.3, 6.9 and 6.1% of isolates were in clusters E, A, F, D and C, respectively. The isolates of cluster B were differentiated into seven subtypes (B1-B7) and subtype B5 contained 63% (34/53) of isolates. RAPD revealed four banding patterns (types I-IV), and among 130 isolates 60.7% (79/130) of isolates were RAPD type I. All of the RAPD type I isolates were grouped into clusters A-C by PFGE. There was no relationship between molecular typing and geographic origin of these isolates. These results indicate that isolates of M. haemolytica A1 strain with various molecular profiles have already spread in Japan and may have caused sporadic infections.

Animals↗