Combination Of Pulsed-field Gel Electrophoresis Research Articles

Use of the quantitative antibiogram method for assessing nosocomial transmission of ESBL-producing Enterobacterales in a French hospital

Extended-spectrum beta-lactamase-producing Enterobacterales (eESBL) have high prevalence in hospitals, but real-time monitoring of nosocomial acquisition through conventional typing methods is challenging. Moreover, patient-to-patient transmission varies between the main species, namely Escherichia coli and Klebsiella pneumoniae, which draws into question the relevance of applying identical preventive measures. To detect eESBL cross-transmission events (CTEs) using the quantitative antibiogram (QA) method in combination with epidemiological data (combined QA method), and to determine the effectiveness of standard or contact precautions for eESBL species. First, a validation set was used to compare the relevance of the combined QA method with a combination of pulsed-field gel electrophoresis and epidemiological data (combined PFGE method). Next, a 4-year retrospective analysis was conducted to detect eESBL CTEs in hospitalized patients. Two species were screened - ESBL E.coli (ESBL-Ec) and ESBL K.pneumoniae (ESBL-Kp). During the study, standard precautions alone were applied to patients with ESBL-Ec, whereas contact precautions were applied for patients with ESBL-Kp. As a proof of concept, use of the combined QA method and the combined PFGE method for the detection of CTEs gave identical results for E.coli, and similar results for at least 75% of K.pneumoniae. Overall, 722 patients with ESBL-Ec isolates and 280 patients with ESBL-Kp isolates were included in the retrospective analysis. Nine and 23 CTEs were identified for E.coli and K.pneumoniae, respectively, involving 20 (2.7%) and 36 (12.8%) patients, respectively. The combined QA method constitutes a rapid tool for epidemiological surveillance to detect CTEs. In the study hospital, standard precautions are sufficient to prevent acquisition of ESBL-Ec, whereas contact precautions must be implemented to prevent acquisition of ESBL-Kp.

Heterogeneity of Staphylococcus epidermidis in prosthetic joint infections: time to reevaluate microbiological criteria?

Prosthetic joint infection (PJI) is a feared and challenging to diagnose complication after arthroplasty, with Staphylococcus epidermidis as the major pathogen. One important criteria to define PJI is the detection of phenotypically indistinguishable microorganisms with identical antibiotic susceptibility pattern in at least two different samples. However, owing to phenotypical variation within genetic clones and clonal variation within a phenotype, the criteria may be ambiguous. We investigated the extent of diversity among coagulase-negative staphylococci (CoNS) in PJI and characterised S. epidermidis isolates from PJI samples, specifically multiple S. epidermidis isolates identified in individual PJI patients. We performed a retrospective cohort study on 62 consecutive patients with PJI caused by CoNS from two hospitals in Northern Sweden. In 16/62 (26%) PJIs, multiple S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in samples from 40/62 (65%) of the patients using a combination of pulsed-field gel electrophoresis and multilocus sequence typing. Whole-genome sequencing showed the presence of multiple sequence types (STs) in 7/16 (44%) PJIs where multiple S. epidermidis isolates were available. Within-patient phenotypical variation in the antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%) among isolates with the same ST. The results highlight the ambiguity of S. epidermidis phenotypic characterisation as a diagnostic method in PJI and call for larger systematic studies for determining the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and the therapeutic outcomes in patients.

Isolation and identification of Listeria monocytogenes utilizing DC insulator-based dielectrophoresis

Foodborne pathogens pose one of the greatest challenges facing public health in the modern day. One important pathogen, Listeria monocytogenes, is known to be challenging to detect and identify. Three serovars cause most of the Listeria related food-borne illnesses, which the Centers for Disease Control currently utilizes a combination of pulsed-field gel electrophoresis and whole genome sequencing for identification and the determination of clusters and outbreaks. There is a potential method for rapid collection of epidemiological information by exploiting the electrokinetic and dielectrophoretic properties of the L. monocytogenes serovars. Using dielectrophoresis, the three most commonly identified serovars of L. monocytogenes can be distinguished from each other. The electrokinetic and dielectrophoretic mobilities of each serovar was determined through a combination of electrokinetic velocity and dielectrophoretic trapping assessments, in conjunction with finite element multi-physics modeling. A mathematical model of the data, which defines the various factors of dielectrophoretic trapping, is utilized and verified based on the behavior of L. monocytogenes in the microchannel. The trapping condition for the serovars were evaluated as 2.8±0.2×109, 2.2±0.2×109, and 2.2±0.3×109Vm−2 and the electrokinetic mobility was assessed to be 19±0.7, 17±0.7, and 9.2±0.3×10−9m2V−1s−1 for the L. monocytogenes serovars 1/2a, 1/2b, and 4b, respectively.

Molecular Characteristics of Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Isolates Causing Urinary Tract Infections

Background: Coagulase-negative Staphylococcus epidermidis is the most prevalent member of the human skin normal biota that plays an important role as a common cause of catheter and prosthetic device-related infections from, for example, indwelling catheters. Objectives: This study aimed to characterize the clonality of biofilm-producing methicillin-resistant S. epidermidis (MRSE) strains isolated from catheterized patients with urinary tract infection at a referral hospital in Tehran, Iran. Methods: Between 2014 and 2016, a total of 56 methicillin-resistant S. epidermidis (MRSE) strains were recovered from catheterized patients. The MRSE isolates were tested for biofilm formation and different genes involved in this process were detected. Clonal dissemination of MRSE isolates was determined using the combination of pulsed-field gel electrophoresis (PFGE) and SCCmec typing. Results: Out of the 56 MRSE strains, 50 (89%) formed biofilm and were positive for icaA and icaD genes, and 73% (n = 41) harbored IS256. The PFGE patterns revealed a total of 32 different pulsotypes consisting of 16 single types (STs), 16 common types (CTs), and 2 SCCmec types (III and IV) were detected. Moreover, all STs carried SCCmec type IV and classified as community-acquired strains. Four CTs were common among biofilm and non-biofilm producing strains. Conclusions: The presence of icaA and icaD genes with a high prevalence of IS256 element in clonal groups of MRSE strains suggests that ica, IS256, and biofilm forming ability occur simultaneously in specific S. epidermidis clones and spread preferentially in hospitals and community.

Post-meiotic DNA double-strand breaks are conserved in fission yeast

In mammals, spermiogenesis is characterized by transient formation of DNA double-strand breaks (DSBs) in the whole population of haploid spermatids. DSB repair in such haploid context may represent a mutational transition. Using a combination of pulsed-field gel electrophoresis and specific labelling of DSBs at 3'OH DNA ends, we showed that post-meiotic, enzyme-induced DSBs are also observed in the synchronizable pat1-114 mutant of Shizosaccharomyces pombe as well as in a wild-type strain, while DNA repair is observed at later stages. This transient DNA fragmentation arises in the whole cell population and is seemingly independent of the caspase apoptotic pathway. Because histones are still present in spores, the transient DSBs do not require a major change in chromatin structure. These observations confirm the highly-conserved nature of the process in eukaryotes and provide a powerful model to study the underlying mechanism and its impact on the genetic landscape and adaptation.

Subtyping of Salmonella isolates on retail raw chicken in China by pulsed-field gel electrophoresis and plasmid analysis

Abstract Salmonellosis is one of the most common bacterial foodborne diseases worldwide. Poultry and poultry products are considered significant vehicles for foodborne salmonellosis transmission. We subtyped 725 Salmonella isolates recovered from 1152 retail raw chickens using pulsed-field gel electrophoresis (PFGE), and 448 ampicillin-resistant ones among 725 isolates using plasmid analysis in this study. Based on 80% similarity, 126 PFGE clusters were identified among 725 Salmonella isolates. Sixty one (8.4%) of 725 isolates belonged to PFGE cluster C96, followed by cluster C5 (38/725, 5.2%), and cluster C18 (38/725, 5.2%). Fifty two distinct PFGE patterns were detected among the isolates in Shaanxi Province, and 67, 64, 82, 67, 55, 88, and 47 patterns were identified in isolates in Henan, Sichuan, Beijing, Guangxi, Shanghai, Guangdong, and Fujian, respectively. Plasmids were detected in 331 (73.9%) of the 448 ampicillin-resistant Salmonella isolates. One hundred and twenty three plasmid profiles were identified, and P3, P11, P30, P12, were defined as predominant ones. Combination of PFGE and plasmid analysis was an effective way to enhance the genotypic discrimination ability to Salmonella , especially for S . Typhimurium isolates. Our results indicated that multiple Salmonella subtypes were prevalent in retail raw chicken in China, and they were potential hazard for food safety.

Re-characterization of an extrachromosomal circular plasmid in the pathogenic Leptospira interrogans serovar Lai strain 56601.

In China, Leptospira interrogans serovar Lai strain 56601 (str.56601) is one of main pathogenic strains that cause severe leptospirosis in both human and animals. The genome of this organism was completely sequenced in 2003. However, in 2011, we identified and corrected some assembly errors in the str.56601 genome due to the repeat sequences widely distributed in the Leptospira genome. In this study, we re-analyzed the previously reported mobile, phage-related genomic island in the chromosome and rectified detailed sequence information in both the plasmid and chromosome using various experimental methods. The presence of a separate circular extrachromosomal plasmid was also confirmed, and its location in the genomic region was determined relative to the genomic island reported in L. interrogans serovar Lai by a combination of pulsed-field gel electrophoresis -based and plasmid extraction-based Southern blot analysis. This report confirmed that the separate extrachromosomal circular plasmid is not integrated into the chromosome of L. interrogans str.56601 and markedly improved our understanding of the genomic organization, evolution, and pathogenesis of L. interrogans. In particular, characterization of this extrachromosomal circular plasmid will contribute to the development of genetic manipulation systems in pathogenic Leptospira species.

Twenty-Five-Year Outbreak of Pseudomonas aeruginosa Infecting Individuals with Cystic Fibrosis: Identification of the Prairie Epidemic Strain

Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enrollment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epidemic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique isolates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection.

Clonal dissemination of methicillin-resistant Staphylococcus aureus in patients and the hospital environment

Methicillin is the drug of choice to treat infections caused by resistant strains of Staphylococcus aureus. However, methicillin-resistant S. aureus (MRSA) is now becoming endemic in many hospitals worldwide and is the cause of nosocomial outbreaks. To assess clonality and dissemination of MRSA strains in the hospitals of Tehran, a total of 60 MRSA strains were isolated from hospitalized patients (n=44) and hospital equipment and environment (n=16) of three metropolitan hospitals in Tehran between July 2009 and March 2010. These strains were subjected to antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and biochemical fingerprinting using the PhPlate system. Results showed the presence of between one and three dominant clonal groups within each hospital, with most equipment and environmental strains being identical to the dominant clones of hospitalized patient strains. The rate of resistance of these strains to the 13 antibiotics tested ranging from 2% to 100%, with resistance being highest for penicillin, ciprofloxacin, and tetracycline (>98% of the isolates). Comparison of the strains isolated from the three hospitals using a combination of PFGE and PhP types showed the presence of 11 clonal groups of MRSA among these hospitals; of these, three common clonal groups also had identical antibiotic resistance patterns and were found in more than one hospital. These data suggest dissemination of a few dominant clonal groups of MRSA strains in hospitals in Tehran, with high level resistance to other commonly used antibiotics.

Molecular typing of Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates from cattle by multiple-locus variable-number tandem-repeats analysis

To evaluate the usefulness of multiple-locus variable-number tandem-repeats analysis (MLVA) as a tool for the epidemiological analysis of bovine Salmonellosis, Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates (544 and 18, respectively) obtained from cattle in Hokkaido, Japan, between 1977 and 2009, were characterised by MLVA. MLVA identified 184 profiles versus 121 profiles identified by pulsed-field gel electrophoresis (PFGE). Cluster analysis of the MLVA profiles demonstrated 3 major clusters (A, B, and C) and 3 minor clusters (D, E, and F). Cluster A was associated with PFGE cluster I, which included isolates of definitive phage type 104 (DT104), while cluster C was associated with PFGE cluster VII, which has been disseminating among cattle since 2002. An isolate of serotype Typhimurium belonging to MLVA cluster F, in which 10 serotype 4,5,12:i:- isolates were included, was found to have an MLVA profile closely related to those of serotype 4,5,12:i:- isolates, suggesting that such a strain may be an ancestral candidate for serotype 4,5,12:i:-. Overall, the discriminatory power of MLVA was higher than that of PFGE, and MLVA differentiated between the isolates of the DT104 family, which appeared to be clonal by PFGE. However, this depended on PFGE clusters because PFGE allowed greater discrimination between isolates within PFGE cluster IV and VI than MLVA. The combination of PFGE and MLVA data allowed for improved subtype discrimination and enabled the identification of recently disseminated clones. Hence, MLVA can be used in combination with PFGE to effectively accelerate the molecular epidemiologic investigation of Salmonella.

Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones

Staphylococcus aureus infections continue to pose a global public health problem. Frequently, this epidemic is driven by the successful spread of single S. aureus clones within a geographic region, but international travel has been recognized as a potential risk factor for S. aureus infections. To study the molecular epidemiology of S. aureus infections in the Caribbean, a major international tourist destination, we collected methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates from community-onset infections in the Dominican Republic (n = 112) and Martinique (n = 143). Isolates were characterized by a combination of pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) typing. In Martinique, MRSA infections (n = 56) were mainly caused by t304-ST8 strains (n = 44), whereas MSSA isolates were derived from genetically diverse backgrounds. Among MRSA strains (n = 22) from the Dominican Republic, ST5, ST30, and ST72 predominated, while ST30 t665-PVL+ (30/90) accounted for a substantial number of MSSA infections. Despite epidemiological differences in sample collections from both countries, a considerable number of MSSA infections (~10%) were caused by ST5 and ST398 isolates at each site. Further phylogenetic analysis suggests the presence of lineages shared by the two countries, followed by recent genetic diversification unique to each site. Our findings also imply the frequent import and exchange of international S. aureus strains in the Caribbean.

Group B streptococci causing neonatal infections in barcelona are a stable clonal population: 18-year surveillance.

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years.

A molecular study of the Salmonella enterica serovars Abortusovis, Typhimurium, and Enteritidis

This study presents a molecular analysis of 3 important human and animal serovars of Salmonella: Typhimurium, Enteritidis, and Abortusovis. We also provide information that can be applied in the surveillance of salmonellosis. Over the course of 3 decades, 90 isolates were collected, with 30 isolates representing each type of serovar. In addition to conventional serotyping, serovars were confirmed by polymerase chain reaction (multiplex PCR and IS200 PCR). Pulsed-field gel electrophoresis (PFGE) of the serovars confirmed 12, 15, and 20 patterns, respectively. In an analysis of the results, 5 pulsotypes (80% of isolates) of Salmonella Typhimurium and 4 pulsotypes (64% of isolates) of Salmonella Enteritidis were confirmed to be prevalent, and Salmonella Abortusovis showed unexpectedly high diversity, with just 2 prevalent pulsotypes (40% of isolates). S. Enteritidis and S. Abortusovis were identified as polyphyletic (possibly the first report), while S. Typhimurium was identified as monophyletic. The combination of PFGE, random amplified polymorphic DNA-PCR (RAPD-PCR) with 2 primers, and antibiotic susceptibility tests showed 29, 23, and 21 distinct patterns in the serovars, respectively; for this combination of methods, the value of the discrimination index was established at more than 0/95 in each serovar. The results of this study indicate that this combination of methods can increase the discrimination index. It is therefore suitable and applicable for use in a Salmonella surveillance system, especially in endemic regions in which there is no active surveillance system or information.

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.

Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

BackgroundThe soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined.MethodsFixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10×10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).Principal Findings B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations.ConclusionsGenetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.

A molecular survey on virulence associated genotypes of non-O1 non-O139 Vibrio cholerae in aquatic environment of Tehran, Iran

Thirty-seven Vibrio cholerae strains were isolated from surface water sources at 5 different locations in Tehran, Iran during 2006 and were identified as non-O1 and non-O139 isolates. PCR for SXT element and class 1 integron was positive for 19% and 5.4% of isolates, respectively. PCR for virulence associated-genes within the vibrio pathogenicity island (VPI) gene cluster showed the presence of LJ, int and RJ in 8, 59 and 30% of the isolates, respectively. None of the V. cholerae isolates contained the toxin encoding genes ( ace, zot, ctx) in the CTX genetic element. Biochemical fingerprinting using PhPlate system (PhP-RV) was able to type all strains and resulted in 8 common types (containing 78% of the isolates) and 8 single types (22%). Out of 37 isolates, only 26 isolates were typeable with pulsed-field gel electrophoresis (PFGE) producing banding patterns. The results presented in this study showed no genotyping correlation between the V. cholerae isolated from surface water and the clinical setting which had been reported previously by this laboratory. Furthermore, combination of PFGE and PhP-RV methods was proved beneficial for non-typeable V. cholerae isolates.

Molecular Epidemiology of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Global Clinical Trials

Determining the genetic characteristics of Staphylococcus aureus is important for better understanding of the global and dynamic epidemiology of this organism as we witness the emergence and spread of virulent and antibiotic-resistant clones. We genotyped 292 S. aureus isolates (105 methicillin resistant and 187 methicillin susceptible) using a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and SCCmec typing. In addition, S. aureus isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes. Isolates were recovered from patients with uncomplicated skin infections in 10 different countries during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. The most common methicillin-resistant clone had multilocus sequence type 8, pulsed-field type USA300, and SCCmec type IV and possessed the PVL genes. This clone was isolated exclusively in the United States. The most common PVL-positive, methicillin-susceptible clone had multilocus sequence type 121 and pulsed-field type USA1200. This clone was found primarily in South Africa and the Russian Federation. Other clones were found at lower frequencies and were limited in their geographic distribution. Overall, considerable genetic diversity was observed within multilocus sequence type clonal complexes and pulsed-field types.

Evaluation of pulsed-field gel electrophoresis and multi-locus sequence typing for the analysis of clonal relatedness among Bartonella henselae isolates

Pulsed-field gel electrophoresis (PFGE) represents the gold standard among band-based methods for the molecular typing of Bartonella henselae. SmaI and NotI have been frequently used for typing B. henselae by PFGE. However, their appropriateness for the analysis of genetic relatedness among B. henselae isolates has not been assessed systematically hitherto. Aim of the present study was to evaluate SmaI, NotI, and three additional endonucleases for typing B. henselae isolates by PFGE and to compare the PFGE results with multi-locus sequence typing (MLST) data. Twenty B. henselae isolates from different sources and geographic regions were analysed. PFGE analysis upon restriction with SmaI, ApaI, Eco52I, and XmaJI revealed six, five, four, and five different PFGE types, respectively, whereas restriction with NotI revealed 13 PFGE types. Five sequence types (STs) were obtained by MLST. The overall concordance between PFGE types obtained with SmaI, ApaI, Eco52I, XmaJI and STs was high. In contrast, NotI-derived types did not correlate with other PFGE types or STs, indicating that NotI is not an appropriate enzyme for PFGE typing of B. henselae. By combining PFGE results obtained with SmaI, ApaI, Eco52I, XmaJI with STs, the isolates could be assigned to five distinct clonal lineages, including the clones Houston-1, Marseille, CAL-1, and Berlin-2. These data indicate that PFGE and MLST are discriminatory and reliable for molecular typing of B. henselae isolates to the strain level. Combination of PFGE and MLST may be useful for further epidemiological studies on B. henselae.

Identification of macrolide-resistant clones of Streptococcus pyogenes in Portugal

Although the overall level of macrolide resistance (27%) has remained stable in Portugal, a rapid inversion in the dominant phenotypes has been noted, with a sharp decrease in the MLS(B) phenotype paralleled by an increase in the M phenotype. To gain further insight into these changes, 325 macrolide-resistant isolates were characterised using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The use of Cfr9I, an isoschizomer of SmaI, to digest M phenotype isolates that were refractory to SmaI digestion allowed direct comparison of MLS(B) and M isolates. The results from PFGE and MLST were highly concordant and identified eight major clones, accounting for 92% of the isolates, each of which was associated exclusively with a single macrolide resistance phenotype. Two major clones were found among MLS(B) isolates, characterised by sequence types (ST) 46 (T12/emm22) and ST52 (T28/emm28), whereas clones characterised by ST39 (T4/emm4) and ST28 (T1/emm1) dominated among M isolates. The clone defined by ST52 corresponded to a bacitracin-resistant clone circulating in Europe, and a novel variant expressing other surface antigens (T12/emm22) was detected. The presence of the four major clones has been reported previously in other European countries, suggesting Europe-wide dissemination of a few macrolide-resistant lineages.

Antigenic Divergence of Bordetella pertussis Isolates in Taiwan

In recent studies, antigenic divergence has been observed in Bordetella pertussis circulating isolates. We collected 80 Bordetella pertussis isolates in Taiwan from 1998 to 2004 and analyzed them using a combination of pulsed-field gel electrophoresis (PFGE) and sequencing of the ptxS1 and prn genes. The incidence of pertussis increases every 3 years, and most of the isolates prevalent since 1998 have expressed nonvaccine ptxS1A and prn2 alleles. Through PFGE analysis, all isolates could be classified into four major groups, and the incidence of these groups exhibited a correlation with the prn allele expressed by the isolates. We found that PFGE is more discriminative than gene sequencing, since it could divide the isolates expressing the prn2 allele into two groups: one group circulating from 1998 to 2001 and another group circulating from 2001 to 2004. The transition between the two groups in 2000 coincided with an outbreak of 326 cases. This research indicates that the antigenic divergence of B. pertussis circulating isolates has evolved over time in Taiwan. Such information will have implications for vaccine policy in Taiwan.