Pulsed-field Gel Electrophoresis Typing Methods Research Articles

Comparative study on three molecular typing methods of Vibrio parahaemolyticus

To evaluate the correlation among the three molecular typing method of pulsed field gel electrophoresis(PFGE), repetitive extragenic palindromic(REP)-PCR and en-terobacterial repetitive intergenic consensus(ERIC)-PCR, and to explore the genetic relationship among strains, and to further understand the distribution and epidemic trend of Vibrio parahaemolyticus in Liaoning Province by combining Serotype analysis. Serum typing, PFGE, REP-PCR, and ERIC-PCR molecular typing and cluster analysis were performed on 150 VP isolates from Liaoning Province in 2018. 118 isolates could be divided into 14 Serotype, and 32 isolates could not be classified. The main serotypes were O3, O1 and O2. The resolution(DI) of PFGE is 0.969, the resolution(DI) of REP-PCR is 0.948, and the resolution(DI) of ERIC-PCR is 0.927. The Serotype O3 group strains are highly similar to the molecular types of O1 group strains. In 2018, the epidemic Serotype of clinical VP isolates in Liaoning Province is still O3: K6, and the epidemic serotype of food VP isolates is still O2. The result of PFGE, REP-PCR, and ERIC-PCR typing method are consistent, and the resolution and reproducibility of PFGE typing method are superior to the other two method. The Serotype O3 group is closely related to O1 group.

Genetic Diversity of Food-Isolated Salmonella Strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR)

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

Outbreak analysis and typing of MRSA isolates by automated repetitive-sequence-based PCR in a region with multiple strain types causing epidemics

The usefulness and performance of repetitive-sequence-based polymerase chain reaction (rep-PCR), the DiversiLab system, in the epidemiological surveillance for methicillin-resistant Staphylococcus aureus (MRSA) strain typing was assessed. MRSA isolates from five distinct outbreaks with precise epidemiological data (n = 69) and from the culture collection of well-characterized MRSA strains (n = 132) consisting of 35 spa and 23 pulsed-field gel electrophoresis (PFGE) types were analyzed. The typing results of the DiversiLab system in outbreak analysis were compared to the spa and PFGE typing methods. The DiversiLab system proved to be a reliable tool for the rapid first-line typing of MRSA isolates, showing a good reliability in distinguishing MRSA strains in an area where several MRSA types were causing epidemics. This, however, required that the automatic clustering was combined with manual interpretation using the pattern overlay function when the strain types showing high similarity were clustered together. All outbreaks were distinguished with the DiversiLab system and the PFGE method, but not with the spa typing method. The overall discriminatory power of the DiversiLab system in differentiating diverse MRSA strains proved to be good. We also demonstrated that, in addition to the genetic relatedness analysis of MRSA strains, it is important to obtain accurate epidemiological information in order to perform reliable epidemiological surveillance studies.

Enhanced Discrimination of Highly Clonal ST22-Methicillin-Resistant Staphylococcus aureus IV Isolates Achieved by Combining spa , dru , and Pulsed-Field Gel Electrophoresis Typing Data

ST22-methicillin-resistant Staphylococcus aureus type IV (ST22-MRSA-IV) is endemic in Irish hospitals and is designated antibiogram-resistogram type-pulsed-field group (AR-PFG) 06-01. Isolates of this highly clonal strain exhibit limited numbers of pulsed-field gel electrophoresis (PFGE) patterns and spa types. This study investigated whether combining PFGE and spa typing with DNA sequencing of the staphylococcal cassette chromosome mec element (SCCmec)-associated direct repeat unit (dru typing) would improve isolate discrimination. A total of 173 MRSA isolates recovered in one Irish hospital during periods in 2007 and 2008 were investigated using antibiogram-resistogram (AR), PFGE, spa, dru, and SCCmec typing. Isolates representative of each of the 17 pulsed-field group 01 (PFG-01) spa types identified underwent multilocus sequence typing, and all isolates were ST22. Ninety-seven percent of isolates (168 of 173) exhibited AR-PFG 06-01 or closely related AR patterns, and 163 of these isolates harbored SCCmec type IVh. The combination of PFGE, spa, and dru typing methods significantly improved discrimination of the 168 PFG-01 isolates, yielding 65 type combinations with a Simpson's index of diversity (SID) of 96.53, compared to (i) pairwise combinations of spa and dru typing, spa and PFGE typing, and dru and PFGE typing, which yielded 37, 44, and 43 type combinations with SIDs of 90.84, 91.00, and 93.57, respectively, or (ii) individual spa, dru, and PFGE typing methods, which yielded 17, 17, and 21 types with SIDs of 66.9, 77.83, and 81.34, respectively. Analysis of epidemiological information for a subset of PFG-01 isolates validated the relationships inferred using combined PFGE, spa, and dru typing data. This approach significantly enhances discrimination of ST22-MRSA-IV isolates and could be applied to epidemiological investigations of other highly clonal MRSA strains.

Molecular typing of methicillin-resistant Staphylococcus aureus isolated from bloodstream infections in a university hospital

Aim: Bloodstream infections due to methicillin-resistant Staphyococcus aureus (MRSA) strains are one of the major problems in many hospitals. Molecular typing provides very useful information about the origin and the spreading ways of strains. The aim of the present study is to assess the clonal relationship amongst MRSA strains isolated from bloodstream infections of patients in Turgut Özal Medical Center, İnönü University in Turkey. Materials and methods: A total of 37 consecutive MRSA strains were identified from the blood cultures from January to December 2003. Methicillin resistance was confirmed with amplification of the mecA gene by polymerase chain reaction (PCR). Clonal relatedness of the strains was investigated by arbitrarily primed polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE). Results: Of the 37 mecA positive S. aureus strains identified in a 1-year period, 29 (78.3%) were from intensive care units (ICUs) and the remaining 6 from other wards. The MRSA strains were resistant to most clinically useful anti-staphylococcal agents. AP-PCR and PFGE typing methods indicated that 67.6% and 60.7% of the typed strains were clonally related, respectively. Clonally related strains were not restricted in a specific clinic and period. Conclusion: Our findings indicated that MRSA bloodstream infections in our hospital were not originated from any predominant clone and AP-PCR typing can be used to screen clonal relatedness of these strains. The present data showed that there was no predominant MRSA clone in our hospital. However, because of the high rates of MRSA and clonally related strains, the infection control practices were reconsidered and more strict rules were proposed to the infection control committee to eliminate the spread of these strains between wards in our hospital.

Clinical, microbiologic, and epidemiologic characteristics of Pseudomonas aeruginosa infections in a University Hospital, Malatya, Turkey

Pseudomonas aeruginosa strains are generally resistant to many antibiotics, and nosocomial infections because of this species are one of the major problems in many hospitals. Molecular typing provides very useful information about origin and transmission of the strains. The aims of the present study were to investigate clinical and microbiologic characteristics of the nosocomial infections caused by P aeruginosa strains in a medical center and to bring up the cross-transmission level of this opportunistic pathogen in a university hospital by analyzing the clonal relationship among the isolates. A total of 105 P aeruginosa strains had been identified among the 80 inpatients in a 1-year period from August 2003 to August 2004. Demographic, clinical, and epidemiologic data of the patients were prospectively recorded. The standardized disk-diffusion method was used to determine resistance of the strains to imipenem, ceftazidime, aztreonam, amikacin, gentamicin, mezlocillin, cefepime, tobramycin, meropenem, ceftriaxone, and ciprofloxacin. Clonal relatedness of the strains was investigated by pulsed-field gel electrophoresis (PFGE). Of the 105 P aeruginosa strains identified, 45 (43%) were isolated from the patients hospitalized in intensive care units. Thirteen patients had repeated pseudomonas infection (total 38 infections/13 patients); 26 of these repeated infections in 9 patients showed the same localization. Half of the patients had at least 1 underlying disease such as burn (48%), chronic illness (32%), and malignancy (20%). Fifty-seven patients (71%) had urinary and/or other catheterization. Urinary tract infection (35%) was the most frequent infection encountered, followed by respiratory tract infection (34%) and sepsis (13%). Resistance to the antibiotics tested was in the 12% to 88% range; amikacin was the most effective and ceftriaxone was the least effective antibiotic. The PFGE typing method showed that 28 of the 80 patients' isolates were clonally related, including 23 indistinguishable or closely related strains (29%), and 5 possibly related strains (6%). Epidemiologic data of the 16 patients (20% of the patients) confirmed a clonal relationship among the strains. Of the 26 isolates of the 9 patients having repeated infection in the same location, 18 (69%) were in the clonally related groups, whereas 11 of the 12 strains isolated from repeated infections on different body sites were clonally different. Our results indicated that P aeruginosa infections in our hospital mainly affected the patients hospitalized in intensive care units and those having catheterization, burn, and/or chronic illness. Amikacin was the best antibiotic as far as bacterial resistance was considered. Although lack of major PFGE type confirmed no P aeruginosa outbreak, typing results showed that cross transmission and treatment failure are the 2 main problems, which should be consider together to prevent this bacterial infection in medical centers.