Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

Nonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the “fuzzy species” strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment.     

Identification of Haemophilus influenzae and Haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244?H. influenzae and 33?H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.     

Comparison of multilocus sequence typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolates

In the United States, multidrug-resistant phenotypes of Salmonella enterica serotype Newport (commonly referred to as MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Although pulsed-field gel electrophoresis (PFGE) is the current “gold standard” typing method for Salmonella, multilocus sequence typing (MLST) may be more relevant to investigations exploring evolutionary and population biology relationships. In this study, 81 Salmonella enterica serotype Newport isolates from humans, food animals, and retail foods were examined for antimicrobial susceptibility and characterized using PFGE and MLST of seven genes, aroC, dnaN, hemD, hisD, purE, sucA, and thrA. Forty-nine percent of the isolates were resistant to nine or more of the tested antimicrobials. Salmonella isolates displayed resistance most often to sulfamethoxazole (57%), streptomycin (56%), tetracycline (56%), ampicillin (52%), and ceftiofur (49%) and, to a lesser extent, to kanamycin (19%), trimethoprim-sulfamethoxazole (17%), and gentamicin (11%). A total of 43 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained isolates from clinically ill swine, cattle, and humans. MLST resulted in 12 sequence types (STs), with one type encompassing 62% of the strains. Ten new sequence types and one novel allele type were identified. Furthermore, MLST typing showed that strains closely related by PFGE clustered in major STs, whereas more distantly related strains were separated into two clusters by PFGE. The results of this study demonstrated that the MLST scheme employed here clustered S. enterica serovar Newport isolates in distinct molecular populations, and strain discrimination was enhanced by combining PFGE, antimicrobial susceptibility, and MLST results.     

Identification of Haemophilus influenzae Clones Associated with Invasive Disease a Decade after Introduction of H. influenzae Serotype b Vaccination in Italy

The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the bla_TEM gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were β-lactamase positive, harboring the bla_TEM gene. Most bla_TEM genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the β-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.     

Molecular Capsular Typing and Multi Locus Sequence Typing of Invasive,Non-invasive and Commensal Streptococcus pneumoniae Isolates from North India

Purpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods: S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer’s disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer’s disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.     

Comparative Analysis of Subtyping Methods against a Whole-Genome-Sequencing Standard for Salmonella enterica Serotype Enteritidis

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.     

Evidence of a clonal expansion of Streptococcus pneumoniae serotype 19A in adults as in children assessed by the DiversiLab® system

Streptococcus pneumoniae serotype 19A was the main serotype responsible for invasive pneumococcal disease (IPD) in the Paris area in 2007 and 2009 in both adults and children. To verify if a particular clone is emerging, we determined the populational structure of S. pneumoniae isolates. Eighty-four S. pneumoniae strains responsible for invasive infections isolated from 52 adults and 32 children hospitalized in Parisian hospitals were analyzed. Capsular typing was performed by polymerase chain reaction (PCR) using the semi-automated repetitive sequence-based (rep-PCR) DiversiLab® System. Multilocus sequence typing (MLST) was also performed on 26 strains (ten selected strains after cluster analysis and 16 control strains). In 2007 and 2009,?S. pneumoniae serotype 19A represented, respectively, 28.6 % and 25 % of the serotypes involved in IPDs in children and 13 % and 13.7 % in adults. The rep-PCR DiversiLab® analysis showed that the 84 S. pneumoniae serotype 19A isolates were distributed in five clusters and four unique rep-PCR types. Overall, 77/84 (91.6 %) S. pneumoniae 19A serotypes grouped into three main genetically related clusters (71/77 belonged to the cluster I). The five other strains exhibited different profiles. Using MLST, we demonstrated that most strains of the main cluster belonged to clonal complex (CC) 230, sequence type (ST) 276. However, for the other strains, the DiversiLab® method cannot be used to predict to which ST a strain belongs. The DiversiLab® method allowed us to identify the clonal expansion of S. pneumoniae serotype 19A strains isolated from both children and adults in 2007 and 2009.     

Occurrence, distribution, and origins of Streptococcus pneumoniae Serotype 6C, a recently recognized serotype

The prevalence of Streptococcus pneumoniae serotype 6C, a recently recognized serotype that cross-reacts serologically with serotype 6A, was investigated. Isolates of serotype 6A in various collections were recovered, and serotype 6C was differentiated from 6A by multiplex PCR of DNA extracts by using appropriate primers. Antimicrobial susceptibility was performed by Clinical and Laboratory Standards Institute broth microdilution, and selected isolates were typed by pulsed-field gel electrophoresis, repetitive sequence-based PCR typing, and rapid multilocus sequence typing (MLST) by electrospray ionization mass spectrometry of PCR products. A total of 60 serotype 6C isolates were found: 30 of 122 Cleveland isolates collected from 1979 to 2007, 19 of 39 pediatric isolates collected nationwide in 2005 and 2006, and 11 pediatric isolates from Massachusetts collected in 2006 and 2007. Only four isolates were recovered prior to introduction of the conjugate pneumococcal vaccine in 2000; the earliest isolate was recovered in 1989. The sources of the isolates included blood (n = 5), the lower respiratory tract (n = 27), the sinus (n = 5), the ear (n = 2), and the nasopharynx (n = 18); isolates were recovered from 49 children and 11 adults. Pediatric isolates were found in all six major U.S. geographic regions. Antimicrobial susceptibility showed that 22 isolates were nonsusceptible to penicillin, macrolides, and trimethoprim-sulfamethoxazole, 8 had other resistance patterns, and 30 were fully susceptible. The three typing methods used showed similar clusters of up to eight isolates per cluster. MLST showed five clusters related to serotype 6A, two clusters related to serotype 6B, one cluster related to serotype 3, and one cluster related to serotype 34. This study documents the occurrence, nationwide distribution, diversity, likely origins, and increasing incidence after 2001 of this recently recognized serotype. Serotype 6C warrants consideration for addition to future conjugate pneumococcal vaccines.     

Typing of Haemophilus influenzae by Coagglutination and Conventional Slide Agglutination

Coagglutination was compared with conventional slide agglutination for the typing of 297 clinical isolates of Haemophilus sp. A 100% correlation was found with the H. influenzae type b isolates. Coagglutination showed no false-positive reactions with the nontypable strains of H. influenzae and H. parainfluenzae isolates; however, conventional slide agglutination exhibited many false-positive and non-interpretable reactions.     

Epidemiological characterisation of Streptococcus pneumoniae from India using multilocus sequence typing

Objective: The aim of this study was to utilize the multilocus sequence typing (MLST) technique to characterise Streptococcus pneumoniae among clinical isolates in India. MLST was used to determine clonality, to establish genetic relatedness, to check for correlation between serotypes and sequence types (STs) and its relevance associated with antibiotic resistance. Methods: Forty consecutive invasive S. pneumoniae isolates in children <5 years were characterised. Preliminary identification of serotype and antibiotic susceptible profile was followed with MLST technique to identify the STs of the isolates. STs were then analysed for clonality using an eBURST algorithm and genetic relatedness using Sequence Type Analysis and Recombinational Tests version 2 software. Results: The most common ST was ST63. Among the forty isolates, we identified nine novel STs, six of which had known alleles but in new combinations, three of which had new alleles in their sequence profile. The new STs assigned were 8501–8509. One clonal complex was found among the 40 strains characterised. The most common serotypes in this study were serotype 19F, 14 and 5. Non-susceptibility to penicillin and erythromycin was observed in 2.5% and 30% of the isolates, respectively. Conclusion: This study shows a significant number of novel STs among the 40 isolates characterised (9/40, 22.5%), however, internationally recognised strains were also circulating in India, indicating, there could be greater geographical variation in pneumococcal STs in India. Molecular epidemiology data is essential to understand the population dynamics of S. pneumoniae in India before the introduction of pneumococcal vaccines in NIP in India.     

Multilocus Sequence Analysis of Streptococcus canis Confirms the Zoonotic Origin of Human Infections and Reveals Genetic Exchange with Streptococcus dysgalactiae subsp. equisimilis

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals (n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans (n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet(M)- and tet(O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis. PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm-like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection.     

Genetic diversity of fluoroquinolone-nonsusceptible Streptococcus pneumoniae clinical isolates and the first identification of serotype 20B in China

The purpose of this study was to investigate the genetic characteristics of fluoroquinolone-nonsusceptible Streptococcus pneumoniae clinical isolates in China. A total of 377 S. pneumoniae clinical isolates, including 307 pediatric strains and 70 adult strains, were collected from eight centers in China. The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents were determined by agar dilution. Multilocus sequence typing (MLST), serotyping, and quinolone resistance-determining region (QRDR) variations were conducted in levofloxacin-nonsusceptible isolates by polymerase chain reaction (PCR)-based methods. Seven levofloxacin-nonsusceptible isolates were found, with an overall resistance rate of 1.9 % (7/377) and 8.6 % (6/70) in adults. Sequence analyses of parC, gyrA, and parE QRDRs in levofloxacin-resistant isolates demonstrated mutations in dual target sites at the hot spots. These seven strains represented multiple clones: two strains were serotype 19F (Taiwan^19F-14) and MLST clonal complex (CC) 271/320, two were typed as 23F (Spain^23F-1) and CC81, two were determined as serotype 20B and a new sequence type of ST6935, and one non-serotypeable pediatric strain belonged to a new sequence type of ST6946. Two serotype 19F strains possessed a variety of characteristic alterations of viridans group streptococci in gyrA (Ser114Gly) or parC (Ser52Gly, Asn91Asp). Fluoroquinolone-nonsusceptible S. pneumoniae isolates showed a substantial degree of genetic diversity and belonged to pre-existing epidemic clones together with native clones. S. pneumoniae strains with serotype 20B was recovered for the first time to be associated with levofloxacin resistance in China.     

Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002–2010)

We aimed to characterize Haemophilus influenzae invasive isolates recovered in Portugal over a 9-year period (2002–2010) following the inclusion of H. influenzae serotype b (Hib) conjugate vaccination in the National Immunization Program (NIP) in the year 2000 and compare the results with those obtained in a similar study from the pre-vaccination era (1989–2001) previously described by us. As part of a laboratory-based passive surveillance system, 144 invasive isolates obtained in 28 Portuguese hospitals were received at the National Reference Laboratory for Bacterial Respiratory Infections and were characterized. Capsular types and antibiotic susceptibility patterns were determined. The ftsI gene encoding PBP3 was sequenced for β-lactamase-negative ampicillin-resistant (BLNAR) isolates. Genetic relatedness among isolates was examined by multilocus sequencing typing (MLST). Most isolates (77.1 %) were non-capsulated, a significant increase compared to the pre-vaccination era (19.0 %, p?<?0.001). Serotype b strains decreased significantly (from 81.0 to 13.2 %, p?<?0.001) and serotype f increased significantly (from 0.8 to 6.9 %, p?=?0.03). Ten percent of the isolates were β-lactamase producers, a value lower than that previously observed (26.9 %, p?=?0.005). Eight percent of all isolates were BLNAR. A high genetic diversity among non-capsulated isolates was found. By contrast, capsulated isolates were clonal. The implementation of Hib vaccination has resulted in a significant decline in the proportion of serotype b H. influenzae invasive disease isolates. Most episodes of invasive disease occurring in Portugal are now due to fully susceptible, highly diverse, non-capsulated strains. Given the evolving dynamics of this pathogen and the increase in non-type b capsulated isolates, continuous surveillance is needed.     

Rapid identification of uropathogenic Escherichia coli of the O25:H4-ST131 clonal lineage using the Diversi-Lab repetitive sequence-based PCR system

Several recent studies have highlighted the emergence of a globally disseminated clone of uropathogenic and invasive Escherichia coli isolates of serotype O25:H4 and sequence type 131. The ability to characterize rapidly E. coli isolates of this lineage would facilitate enhanced surveillance for this pathogen. We have used the semi-automated Diversi-Lab repetitive PCR-based system to analyse a collection of 35 clinical isolates of uropathogenic E. coli from across the UK, with particular focus on the O25:H4-ST131 lineage. All isolates had been characterized using multilocus sequence typing (MLST), and 14 had previously been typed using pulsed-field gel electrophoresis (PFGE). The Diversi-Lab system allowed discrimination of O25:H4-ST131 isolates from those of other E. coli lineages. It was slightly more discriminatory than MLST, but was less discriminatory than PFGE. With an analysis time of <4 h between receipt of a cultured organism and provision of a typing result, the system offers information on a real-time basis, a major advantage over current practice. We suggest that introduction of the Diversi-Lab system would be useful for rapid exclusion of E. coli isolates during outbreak investigations, and that the approach could be employed for surveillance for pathogenic or antibiotic-resistant clones of this organism.     

Population structure analyses of Staphylococcus aureus at Tygerberg Hospital,South Africa,reveals a diverse population,a high prevalence of Panton–Valentine leukocidin genes,and unique local methicillin-resistant S. aureus clones

Studies reporting on the population structure of Staphylococcus aureus in South Africa have focused only on methicillin-resistant S. aureus (MRSA). This study describes the population structure of S. aureus, including methicillin-susceptible S. aureus (MSSA) isolated from patients at Tygerberg Academic Hospital, Western Cape province. Pulsed-field gel electrophoresis (PFGE), detection of Panton–Valentine leukocidin (PVL), spa typing, multilocus sequence typing (MLST), agr typing and SCCmec typing were used to characterize strains. Of 367 non-repetitive S. aureus isolates collected over a period of 1 year, 56 (15.3%) were MRSA. Skin and soft tissue infections were the most frequent source (54.8%), followed by bone and joint (15.3%) and respiratory tract infections (7.7%). For strain typing, PFGE was the most discriminative method, and resulted in 31 pulsotypes (n = 345, 94.0%), as compared with 16 spa clonal complexes (CCs) (n = 344, 93.4%). Four MLST CCs were identified after eBURST of sequence types (STs) of selected isolates. One hundred and sixty isolates (MSSA, n = 155, 42.2%) were PVL-positive, and agr types I–IV and SCCmec types I–V were identified. Our S. aureus population consisted of genotypically diverse strains, with PVL being a common characteristic of MSSA. MSSA and MRSA isolates clustered in different clones. However, the dominant MRSA clone (ST612) also contained an MSSA isolate, and had a unique genotype. Common global epidemic MRSA clones, such as ST239-MRSA-III and ST36-MRSA-II, were identified. A local clone, ST612-MRSA-IV, was found to be the dominant MRSA clone.     

DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes

While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA, aceK, mdh, icdA, manB, and spaN). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB, fimA, and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This "three-gene sequence typing scheme" allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.     

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales,Australia

Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST.     

Rapid and Specific Detection,Molecular Epidemiology,and Experimental Virulence of the O16 Subgroup within Escherichia coli Sequence Type 131

Escherichia coli sequence type 131 (ST131), a widely disseminated multidrug-resistant extraintestinal pathogen, typically exhibits serotype O25b:H4. However, certain ST131 isolates exhibit serotype O16:H5 and derive from a phylogenetic clade that is distinct from the classic O25b:H4 ST131 clade. Both clades are assigned to ST131 by the Achtman multilocus sequence typing (MLST) system and a screening PCR assay that targets ST131-specific sequence polymorphisms in the mdh and gyrB genes. However, they are classified as separate STs by the Pasteur Institute MLST system, and an ST131 PCR method that targets the O25b rfb region and an ST131-specific polymorphism in pabB detects only the O25b-associated clade. Here, we describe a novel PCR-based method that allows for rapid and specific detection of the O16-associated ST131 clade. The clade members uniformly contained allele 41 of fimH (type 1 fimbrial adhesin) and a narrow range of alleles of gyrA and parC (fluoroquinolone target genes). The virulence genotypes of the clade members resembled those of classic O25b:H4 ST131 isolates; representative isolates were variably lethal in a mouse subcutaneous sepsis model. Several pulsotypes spanned multiple sources (adults, children, pets, and human fecal samples) and locales. An analysis of recent clinical E. coli collections showed that the O16 ST131 clade is globally distributed, accounts for 1 to 5% of E. coli isolates overall, and, when compared with other ST131 isolates, it is associated with resistance to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole and with susceptibility to fluoroquinolones and extended-spectrum cephalosporins. Attention to this O16-associated ST131 clade, which is facilitated by our novel PCR-based assay, is warranted in future epidemiological studies of ST131 and, conceivably, in clinical applications.     

Extended-spectrum β-lactamase producing Klebsiella spp. in chicken meat and humans: a comparison of typing methods

Recently, chicken meat was identified as a plausible source of extended-spectrum β-lactamase (ESBL) -producing Escherichia coli in humans. We investigated the relatedness of ESBL-producing Klebsiella spp. in chicken meat and humans. Furthermore, we tested the performance of SpectraCell RA^® (River Diagnostics), a new typing method based on Raman spectroscopy, in comparison with multilocus sequence typing (MLST) for Klebsiella pneumoniae. Twenty-seven phenotypically and genotypically confirmed ESBL-producing Klebsiella spp. isolates were typed with MLST and SpectraCell RA. The isolates derived from chicken meat, human rectal swabs and clinical blood cultures. In the 22 ESBL-producing K. pneumoniae isolates, CTX-M15 was the predominant genotype, found in five isolates of human origin and in one chicken meat isolate. With MLST, 16 different STs were found, including five new STs. Comparing the results of SpectraCell RA with MLST, we found a sensitivity of 70.0% and a specificity of 81.8% for the new SpectraCell RA typing method. Therefore, we conclude that SpectraCell RA is not a suitable typing method when evaluating relationships of ESBL-producing Klebsiella spp. at the population level. Although no clustering was found with isolates of chicken meat and human origin containing the same ESBL genes, MLST showed no clustering into distinctive clones of isolates from chicken meat and human origin. More studies are needed to elucidate the role of chicken meat in the rise of ESBL-producing Klebsiella spp. in humans.     

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.