High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.     

Phenotypic and genotypic differences of the vancomycin-resistant Enterococcus faecium isolates from humans and poultry in Korea

A total of 98 vancomycin-resistant Enterococcus faecium (VREF) isolates (58 isolates from patients and 40 isolates from poultry) were compared based on their antimicrobial susceptibility, Tn1546 element organization, and pulsed-field gel electrophoresis (PFGE) patterns. This comparison aided in determining the relationships between the groups of isolates. All the VREF isolates harbored the vanA gene; however, 29 (29.6%) of the isolates exhibited the VanB phenotype-vanA genotype. Furthermore, the VREF isolates from humans and poultry exhibited distinct antimicrobial resistance patterns. The PCR mapping of the Tn1546 elements exhibited 12 different transposon types (A to L). The VREF isolates of poultry were classified into types A to D, whereas the human isolates were classified into types E to L. A PFGE analysis demonstrated a high degree of clonal heterogeneity in both groups of isolates; however, the distinct VREF clones appeared in each group of isolates. The deletion of the vanX-vanY genes or insertion of IS1216V in the intergenic region from the vanX-vanY genes is directly associated with the incongruence of the VanB phenotype-vanA genotype in human VREF isolates. These data suggest that the VREF isolates exhibit distinct phenotypic and genotypic traits according to their origins, which suggests that no evidence exists to substantiate the clonal spread or transfer of vancomycin resistance determinants between humans and poultry.     

The first molecular analysis of clinical isolates of VanA-type vancomycin-resistant Enterococcus faecium strains in Mainland China

AIMS: The aim of this study was to examine two VanA-type vancomycin-resistant Enterococcus faecium (VRE) strains that had been isolated from patients resident in mainland China. This is the first molecular analysis of clinical VRE strains being isolated in mainland China. METHODS AND RESULTS: Two VanA-type VRE isolates were isolated from in-patients at hospitals located in the Chinese cities Beijing and Dalian and were designated C264 and I125. The plasmids pC264V (40 kbp) and pI125V (370 kbp) that were isolated from C264 and I125, respectively, carried a Tn1546-like element encoding VanA resistance. The vancomycin-resistant plasmids pC264V and pI125V were transferred by filter mating at frequencies of 10(-7) and 10(-4) respectively. Sequence analysis of pC264V revealed that two IS1216V sequences and an IS1542 sequence were present within the Tn1546-like element. pI125V had two IS1216V insertions in the Tn1546-like element. CONCLUSIONS: The two VanA-type vancomycin-resistant E. faecium (VRE) strains C264 and I125 were isolated from in-patients in Chinese hospitals. The vancomycin-resistant conjugative plasmids pC264V and pI125V plasmids isolated from these strains carried the Tn1546-like element. The Tn1546-like element was found to contain the insertion sequences IS1216V and IS1542. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first molecular analysis of VanA-type VRE strains from patients resident in mainland China.     

Molecular Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Diarrheal Patients in Korea during 2003–2011

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of infectious diarrhea in developing countries. In order to characterize the molecular features of human ETEC isolates from Korea, we investigated the profiles of enterotoxin and colonization factor (CF) genes by polymerase chain reaction (PCR) and performed multilocus sequence typing (MLST) with a total of 291 ETEC strains. The specimens comprised 258 domestic strains isolated from patients who had diarrhea and were from widely separated geographic regions in Korea and 33 inflow strains isolated from travelers visiting other Asian countries. Heat-stable toxin (STh)-possessing ETEC strains were more frequent than heat-labile toxin (LT)-possessing ETEC strains in the domestic isolates, while the detection rates of both enterotoxin genes were similar in the inflow isolates. The profile of CF genes of domestic isolates was similar to that of inflow isolates and the major CF types of the strains were CS3-CS21-CS1/PCF071 and CS2-CS3-CS21. Most of these 2 CF types were detected in ETEC strains that possess both lt and sth genes. The major MLSTST types of domestic isolates were ST171 and ST955. Moreover, the 2 major CF types were usually found concomitantly with the 2 major MLST STs, ST171 and ST955. In conclusion, our genotyping results may provide useful information for guiding the development of geographically specific vaccines against human ETEC isolates.     

VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants

VanA-type human (n=69), animal (n=49), and food (n=36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n=4) and M49 (n=13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P>0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P<0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.     

Dogs leaving the ICU carry a very large multi-drug resistant enterococcal population with capacity for biofilm formation and horizontal gene transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2-9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×10(8) CFU gram(-1) of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks.     

Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark

AIMS: To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations. METHODS AND RESULTS: Multilocus sequence typing (MLST) was applied to 160 C. coli isolates from animal origin, food products and human cases of gastroenteritis. The isolates were collected in Denmark over a 2-year period. In total, 84 sequence types (STs) were obtained and 57 of these STs were novel to this study. Ten per cent of the isolates possessed STs that were found in both human, poultry and pig isolates. Only 10% of the isolates from pigs shared ST with isolates from humans, and these shared STs were found in poultry isolates as well. CONCLUSIONS: Great genetic diversity was seen within the Danish C. coli population. Furthermore, we found that the C. coli types isolated from Danish pigs constitute a small fraction of the C. coli causing human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: C. coli isolates from pigs is presumably not a significant source of human campylobacteriosis in Denmark. The Danish C. coli isolates include 68% STs novel to this study, showing a great diversity compared with studies from other countries.     

Multilocus sequence typing of Campylobacter jejuni isolates from New South Wales, Australia

AIMS: Multilocus sequence typing (MLST) was used to examine the diversity and population structure of Campylobacter jejuni isolates associated with sporadic cases of gastroenteritis in Australia, and to compare these isolates with those from elsewhere. METHODS AND RESULTS: A total of 153 Camp. jejuni isolates were genotyped. Forty sequence types (STs) were found, 19 of which were previously undescribed and 21 identified in other countries. The 19 newly described STs accounted for 43% of isolates, 16 of which were assigned to known clonal complexes. Eighty-eight percent of isolates were assigned to a total of 15 clonal complexes. Of these, four clonal complexes accounted for 60% of isolates. Three STs accounted for nearly 40% of all isolates and appeared to be endemic, while 21 STs were represented by more than one isolate. Seven infections were acquired during international travel, and the associated isolates all had different STs, three of which were exclusive to the travel-acquired cases. Comparison of serotypes among isolates from clonal complexes revealed further diversity. Eight serotypes were identified among isolates from more than one clonal complex, while isolates from six clonal complexes displayed serotypes not previously associated with those clonal complexes. CONCLUSIONS: Multilocus sequence typing is a useful tool for the discrimination of subtypes and examination of the population structure of Camp. jejuni associated with sporadic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the genotypic diversity of Camp. jejuni in Australia, demonstrating that STs causing disease have both a global and a local distribution evident from the typing of domestically and internationally acquired Camp. jejuni isolates.     

Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal enterococcus faecium population

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that "plasmid addiction systems" may contribute to the persistence of GREF in nonselective environments.     

Multilocus sequence typing and virulence factors analysis of Escherichia coli O157 strains in China

Escherichia coli O157:H7, an important food-borne pathogen, has become a major public health concern worldwide. The aim of this study was to investigate the molecular epidemiologic feature of E. coli O157:H7 strains in China. 105 E. coli O157:H7 isolates were collected from various hosts and places over 9 years. A multilocus sequence typing scheme (MLST) was applied for bacteria genotyping and polymerase chain reaction (PCR) was used for virulence factor identification. Seven new MLST sequence types (STs), namely ST836, ST837, ST838, ST839, ST840, ST841, and ST842 were identified, which grouped into two lineages. Phylogenetic analysis suggested that the most two frequent STs in China, ST837 and ST836, may be the derivatives of E. coli O157:H7 Sakai or E. coli O157:H7 EDL933. Geographical diversity and host variety of E. coli O157:H7 were observed in China. In addition, the different distribution of tccp was detected. The data presented herein provide new insights into the molecular epidemiologic feature of E. coli O157:H7, and aid in the investigation of the transmission regularity and evolutionary mechanism of E. coli O157:H7.     

Occurrence, structure, and mobility of Tn1546-like elements in environmental isolates of vancomycin-resistant enterococci

The occurrence, structure, and mobility of Tn1546-like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored vanA. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as Enterococcus faecium (n = 13), E. casseliflavus (n = 6), E. mundtii (n = 3), E. faecalis (n = 3), and E. durans (n = 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn1546-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn1546 in the prototype strain E. faecium BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for E. casseliflavus strains. Indistinguishable Tn1546-like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to E. faecium BM4105 was demonstrated for E. faecalis, E. mundtii, and E. durans. This study indicates that VRE, including species other than E. faecium and E. faecalis, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn1546-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.     

Multilocus Sequence Typing of Uropathogenic ESBL-Producing <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated in a Brazilian Community

In the present study, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction detection of three resistance genes were combined to characterize seven uropathogenic E. coli isolated from outpatients. Selected portions of seven housekeeping and three antibiotic-resistance genes of the isolates were sequenced. The seven isolates were classified into four different sequence types (STs) by MLST and five PGFE types. Three isolates had a novel allelic profile representing a new ST designated as ST528 and showed the same PFGE and resistance genes. Two isolates, both characterized as ST359, were differentiated by PFGE and shared only one of the antibiotic-resistance genes studied. Comparison of MLST results with those of PFGE and resistance genes demonstrated that Escherichia coli had acquired different antibiotic-resistance genes and DNA rearrangements, causing alterations in PFGE patterns but maintaining the same ST. Furthermore, this article also reports the first detection of a CTX-M-2 ESBL E. coli and SHV-5 in a Brazilian community.     

Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.     

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis,reactive arthritis and Guillain–Barré syndrome

Aims: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain–Barré syndrome (GBS) and reactive arthritis (RA). Methods and Results: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002–2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST‐21, ST‐45 and ST‐22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST‐22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. Conclusions: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST‐22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA‐associated genes may vary, by recombination or expression differences, independent of MLST loci. Significance and Impact of the Study: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.     

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%.     

National Surveillance on Vancomycin-Resistant Enterococcus faecium in Taiwan: Emergence and Widespread of ST414 and a Tn1546-Like Element with Simultaneous Insertion of IS1251-like and IS1678

Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.     

Genotypic characteristics of Haemophilus influenzae isolates from pediatric pneumonia patients in Chengdu city, Sichuan, China

Two hundred and seventy-three Haemophilus influenzae strains isolated from pediatric pneumonia patients in China were studied. We used Multilocus Sequence Typing (MLST) to analyze genotypic characteristics. All strains were biotyped and serotyped. Relatedness and patterns of genes among isolates were determined by the analysis of MLST and eBURST. H. influenzae primarily causes acute pneumonia in children under 1 year old. Nontypeable H. influenzae was responsible for most cases of pediatric pneumonia. All 273 strains were classified into eight biotypes. They mostly belonged to the I, II, and III biotypes (17.6%, 43.6%, and 22.7%, respectively). 62 strains (22.7%) produced b-lactamase. We found 28 novel alleles. Fifty different STs were found by MLST, of which 39 were novel. These were ST477 through ST508 and ST521 through ST527. Group 17 and predicted founders 503 were new groups in this study. No STs correlated with strains from Korea, which is adjacent to China. The H. influenzae strains from China appeared to have heterogeneous ST types patterns which may be the reason no outbreaks or epidemics of H. influenzae infections have occurred in Chengdu city, Sichuan, China.     

199株猪链球菌临床分离株血清型、毒力基因及多位点序列分型分析

【背景】猪链球菌（Streptococcus suis,SS）血清型、基因型众多,毒力因子复杂。【目的】了解SS临床分离株血清型、毒力基因分布、分子分型特征及其之间的相关性。【方法】针对199株SS临床分离株,应用PCR技术进行血清分型和毒力基因检测,采用多位点序列分型方法（multilocus sequence typing,MLST）进行基因分型,并分析SS血清型、毒力基因型和序列型（sequence type,ST型）的流行特点及其关联性。【结果】199株SS临床分离株分属于16种血清型（1、2、3、4、6、7、8、9、10、12、15、16、21、24、29和30型）,主要以2、4、3型为主,分别占26.13%（52/199）、14.57%（29/199）和12.06%（24/199）,未定型（NT）菌株占21.61%（43/199）。共鉴定出72种ST型,其中ST1、ST94、ST117、ST7、ST28和ST87为主要ST型,分别占12.56%（25/199）、11.56%（23/199）、9.56%（19/199）、9.04%（18/199）、6.03%（12/199）和3.01%（6/199）,另有24种新发现的ST型（ST1224—ST1227,ST1229—ST1235,ST1241—ST1242,ST1300—ST1310）;分为12个克隆群（cloning complexes,CC）和32个单个ST型。199株SS分离株中毒力基因fbps的检出率最高,为96.98%（193/199）;共有19种毒力基因型,其中66株（33.17%）epf-/mrp-/sly-/gapdh+/fbps+/orf2+型SS为优势毒力基因型。【结论】近年来SS的优势血清型为2、4和3型;ST型具有明显的遗传异质性,种内分化程度较高且与ST型存在一定交叉性;毒力基因分布情况存在差异,毒力基因型呈现多样化。本研究对SS临床分离株的流行特征进行探究,为猪SS病诊断、治疗和制定防控措施提供科学依据。