MLST Genotyping Research Articles

Genotyping, local prevalence and international dissemination of β-lactamase-producing Kingella kingae strains

β-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of β-lactamase production among K. kingae isolates from different geographical origins and genetically characterized β-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for β-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. β-lactamase was identified in invasive strains from Iceland (n = 4/14, 28.6%), the USA (n = 3/15, 20.0%) and Israel (n = 2/190, 1.1%) and in carriage strains in the USA (n = 5/17, 29.4%) and Israel (n = 66/429, 15.4%). No French, Spanish or Canadian isolates were β-lactamase producers. Among β-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic β-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli β-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae β-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of β-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for β-lactamase production.

An investigation of broiler caecalCampylobactercounts at first and second thinning

The objectives of this study were the following: (i) to investigate if Campylobacter negative flocks at first thinning remain negative at second thinning; (ii) to determine if the caecal counts in birds infected during first thinning remain lower than in birds that were positive at first thinning; and (iii) to determine if reducing the time between first and second thinning to a maximum of 4 days would reduce both the incidence and prevalence of broiler caecal contamination. Twenty-two flocks were tested at first and second thinning using ISO methodologies. Of the 14 that had a 4-day duration between first and second thinning, nine flocks were Campylobacter negative at first thinning. By second thinning, all 14 flocks were positive and Campylobacter counts ranged from 5·5 to 6·6 log(10) CFU g(-1) regardless of the status at first thinning. The other eight flocks were all Campylobacter positive at first thinning with counts ranging from 0·8 to 6·1 log(10) CFU g(-1) which increased to 5·1 to 6·9 log(10) CFU g(-1) by second thinning (3-10 days). PCR speciation and MLST genotyping suggested the majority of isolates were Camp. jejuni belonging to STs 257, 814, 6763 and 6764. It was concluded that; thinning introduces Campylobacter into broiler flocks; caecal counts in birds at second thinning are similar, regardless of flock status at first thinning and reducing the time between first and second thinning to a maximum of 4 days is not an effective control strategy. This study informs Campylobacter control strategy at primary production, suggesting all post-first thinning broilers should be treated as 'high risk' regardless of Campylobacter status at first thinning or duration between first and second thinning.

The Clumping factor B gene typing in discriminating the strains of nosocomial-acquired methicillin-resistant Staphylococcus aureus

To evaluate the clfB typing method in discriminating the ST239 methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients under nosocomial infection in Tianjin first central hospital so as to access the clinical risk factors and outcomes of the MRSA nosocomial infection from ICU and non-ICU departments. Forty-two stains of MRSA with known SCCmec type were chosen in both ICU (n = 35) and non-ICU (n = 7) wards, from 2006 to 2012, of which MLST genotype was ST239. Clinical risk factors and rates on drug resistant to MRSA were counted, respectively. All the isolates of MRSA belonged to the same lineage 3 and 6 haplotypes, based on clfB variable-number tandom repeats typing. Thirty-five isolates from ICU belonged to 6 haplotypes, among which clfB3-52, 3-52E, 3-50, 3-52C, 3-50A and 3-50E were accouted for 42.9%, 37.1%, 8.6%, 5.7%, 2.9% and 2.9%, respectively. Seven isolates from non-ICU belonged to 3 haplotypes, in which 3-52, 3-52E and 3-50 were accouted for 42.8%, 28.6%, 28.6%, respectively. When clfB typing was combined with SCCmec typing in use, results showed that the index of discrimination as 0.767, better than clfB (ID = 0.688) or SCCmec (ID = 0.303) used alone. SCCmec III -clfB3-52E seemed as the major clone among the 10 haplotypes of clfB/SCCmec typing, which was accounted for 40.4% . There were significant differences on the length of hospitalization (P < 0.005) and the duration of antibiotics use (P < 0.05) between ICU and non-ICU. The clfB typing method which was based on variable-numbers of tandom repeats showed powerful ability of resolution. It could also be combined with MLST and SCCmec typing to be used in local epidemiological investigations.

P2.086 Origins of Repeat Infections with Chlamydia Trachomatis (Ct) Among Heterosexual Men in Two Southern Cities in the United States

Background Recent clinical trials have reported high repeat infection rates (12%–14%) following 1 g azithromycin. These data suggest that single-dose azithromycin may be inadequate, but high repeat infections rates could also be explained by exposure to an original or new partner or retesting before DNA clearance. The purpose of this study was to examine the origins of repeat CT infections among men. Methods Men diagnosed with Ct by Gen-Probe Aptima Combo 2 at STD clinics in New Orleans, and Jackson, Mississippi were re-tested an average of 6 weeks after treatment with single-dose azithromycin. Detailed sexual behaviour histories were collected at baseline and follow-up via computer-assisted/self-administered interview and MLST genotyping was performed. Results Of 367 men with Ct, 222 returned for a f/u visit [mean of 45 days post-baseline (s.d. 13)] and 14/217 (6.5%) were positive. Of the 14, 36% reported sexual re-exposure to a baseline partner, 14% reported sexual exposure to a new partner, 7% reported sexual exposure to both, and 43% denied sexual re-exposure. Thus far MLST genotyping completed for 3 baseline-f/up positive pairs. Two pairs with the same genotype (E/39) reported sexual re-exposure to a baseline partner and the pair with a new genotype reported sexual exposure to a new partner (D/19 to C/15). Conclusion Early repeat infection rate among men with Ct in this study was lower than recently reported and about half could be explained by sexual re-exposure. Rates in the other two studies may have been inflated by high re-exposure rates or premature testing using NAAT since many of the participants were tested before 3 weeks. Studies that examine repeat infections should consider re-exposure/new exposure and retest when DNA clearance is assured. Our data does not support high treatment failure rates for 1 g azithromycin treatment of Ct.

Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78

BackgroundAcinetobacter baumannii is responsible for large epidemics in hospitals, where it can persist for long time on abiotic surfaces. This study investigated some virulence-related traits of epidemic A. baumannii strains assigned to distinct MLST genotypes, including those corresponding to the international clones I-III as well as emerging genotypes responsible for recent epidemics.MethodsGenotyping of bacteria was performed by PFGE analysis and MLST according to the Pasteur’s scheme. Biofilm formation on polystyrene plates was assessed by crystal violet staining; resistance to desiccation was evaluated on glass cover-slips when kept at room-temperature and 31% relative humidity; adherence to and invasion of A549 human alveolar epithelial cells were determined by the analysis of viable bacteria associated with or internalized by A549 human alveolar epithelial cells; Galleria mellonella killing assays were used to analyze the virulence of A. baumannii in vivo.ResultsThe ability to form biofilm was significantly higher for A. baumannnii strains assigned to ST2 (international clone II), ST25 and ST78 compared to other STs. All A. baumannii strains survived on dry surfaces for over 16 days, and strains assigned to ST1 (international clone I) and ST78 survived for up to 89 and 96 days, respectively. Adherence to A549 pneumocytes was higher for strains assigned to ST2, ST25 and ST78 than other genotypes; a positive correlation exists between adherence and biofilm formation. Strains assigned to ST78 also showed significantly higher ability to invade A549 cells. No significant differences in the killing of G. mellonella worms were found among strains.ConclusionsElevated resistance to desiccation, high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells might have favoured the spread and persistence in the hospital environment of A. baumannii strains assigned to the international clones I and II and to the emerging genotypes ST25 and ST78.

Historical Zoonoses and Other Changes in Host Tropism of Staphylococcus aureus, Identified by Phylogenetic Analysis of a Population Dataset

Background Staphylococcus aureus exhibits tropisms to many distinct animal hosts. While spillover events can occur wherever there is an interface between host species, changes in host tropism only occur with the establishment of sustained transmission in the new host species, leading to clonal expansion. Although the genomic variation underpinning adaptation in S. aureus genotypes infecting bovids and poultry has been well characterized the frequency of switches from one host to another remains obscure. We sought to identify sustained switches in host tropism in the S. aureus population, both anthroponotic and zoonotic, and their distribution over the species phylogeny.Methodologies/ResultsWe have used a sample of 3042 isolates, representing 696 distinct MLST genotypes, from a well-established database (www.mlst.net). Using an empirical parsimony approach (AdaptML) we have investigated the distribution of switches in host association between both human and non-human (henceforth referred to as animal) hosts. We reconstructed a credible description of past events in the form of a phylogenetic tree; the nodes and leaves of which are statistically associated with either human or animal habitats, estimated from extant host-association and the degree of sequence divergence between genotypes. We identified 15 likely historical switching events; 13 anthroponoses and two zoonoses. Importantly, we identified two human-associated clade candidates (CC25 and CC59) that have arisen from animal-associated ancestors; this demonstrates that a human-specific lineage can emerge from an animal host. We also highlight novel rabbit-associated genotypes arising from a human ancestor.Conclusions S. aureus is an organism with the capacity to switch into and adapt to novel hosts, even after long periods of isolation in a single host species. Based on this evidence, animal-adapted S. aureus lineages exhibiting resistance to antibiotics must be considered a major threat to public health, as they can adapt to the human population.

Sharing of Bacterial Strains Between Breast Milk and Infant Feces

In previous years, it has been shown that human milk is a potential source of bacteria for the infant gut. The results of this work confirm the presence of the same specific bacterial strains of Bifidobacterium, Lactobacillus, and Staphylococcus in breast milk and infant fecal samples. The identity of bacteria isolated from breast milk and infant feces from 20 mother-infant pairs was investigated at the strain level. DNA from Staphylococcus, Lactobacillus, and Bifidobacterium was detected by qRTi-PCR in nearly all samples analyzed. These samples were cultured on different agar media. One colony representative of each morphology was selected and identified at the species level combining classical tests and molecular techniques (PCR, RAPD, PFGE, and/or MLST genotyping). Breast milk and infant feces from 19 mother-infant pairs shared different Staphylococcus, Lactobacillus, and/or Bifidobacterium species and strains. Significantly, 2 mother-infant pairs shared 4 bacterial strains although most pairs shared 2. These results confirm that breast milk and infant feces from mother-infant pairs share the same strain(s), indicating that breastfeeding could contribute to the bacterial transfer from the mother to the infant and, therefore, to the infant gut colonization.

Development of an extended multilocus sequence typing for genotyping of Brucella isolates

By amplifying and sequencing longer sequences, an extended multi locus sequence typing (EMLST) theme was developed for Brucella. 61 isolates were genotyped by the EMLST with increased resolution. This strategy could be extended to other bacteria to improve MLST genotyping resolution without additional loci.

Impact of Prevnar vaccination on nasopharyngeal carriage of Streptococcus pneumoniae in healthy children in New Caledonia

We assessed the impact of the heptavalent pneumococcal conjugate vaccine (PCV7) on the nasopharyngeal carriage of Streptococcus pneumoniae in healthy children aged 2 to 24 months, four years after its implementation in New Caledonia. The data were compared with those obtained, before the introduction of the vaccine, in the same target population From February to October 2008, 592 children were enrolled prospectively, regardless of their vaccinal status. Between 2002 and 2008, the prevalence of the overall pneumococcal carriage and of vaccine type carriage decreased significantly, respectively 52.3% to 42.1% (p < 10-3) and 46.9% to 22.2% (p < 10-3). This reduction was offset by an increase (20.8% to 29.0%, p = 0.013) of the carriage of non-vaccine type pneumococci with reduced susceptibility to penicillin (PRSP), notably the serotypes 15B (p=0.027) and 19A (p=0.001). This increase in PRSP carriage was marked in the Northern Province (p = 0.005) and among Melanesian children (p = 0.009). Surprisingly this increase was mainly attributed to the vaccine type 19F (p < 10-3). In conclusion, as expected, the PCV7 vaccine led to a decrease of the pneumococcal carriage and the replacement of vaccine strains by non vaccine strains, however increasingly resistant to penicillin. In the Northern Province, the increasing carriage of penicillin resistant 19F strains escaping the vaccine is of concern and justifies a further comprehensive analysis using MLST genotyping.

Dissemination of multiple MRSA clones among community-associated methicillin-resistant Staphylococcus aureus infections from Japanese children with impetigo

The proportion of MRSA strains that cause skin and soft infections has recently increased. In 3months we have characterized 17 MRSA strains isolated from children with impetigo at a Japanese hospital. Seventeen MRSA strains belonged to 7 clones defined by clonal complex (CC) in MLST genotype and type of SCCmec, which were rarely identified among healthcare-associated MRSA: CC 91-SCCmecIIb (4 strains); CC91-SCCmecIIn (2 strains); CC91-SCCmecIVa (2 strains); CC91-SCCmecV (4 strains); CC88-SCCmecIVg (3 strains); CC1-SCCmecIVc (1 strain); and CC5-SCCmecIVn (1 strain). Although one strain belonged to CC5, which has been commonly identified in healthcare-associated MRSA, it did not carry type II SCCmec, but carried type IV SCCmec. Fourteen of the 17 strains carried exfoliative toxin a or b gene, and none carried Panton-Valentine leukocidine gene. Furthermore, we determined the entire nucleotide sequences of two type V SCCmec elements carried by strains JCSC5952, a CC91 strain, and TSGH17, a Taiwanese CC59 strain. The structure of SCCmecJCSC5952 was more than 99% homologous in nucleotide identity with those of Taiwanese PVL-positive ST59 MRSA strains TSGH17 and PM1, which were designated as type V (5C2&5). Identification of multiple MRSA clones distinct from those disseminating at the hospital suggests that MRSA strains might be emerging in the community from MSSA strains by acquiring SCCmec elements on various occasions. Carriage of the similar type V(5C2&5) SCCmec element by strains of distinct genetic backgrounds, CC91 and CC59, suggested horizontal transfer of the SCCmec element.

Extended and global phylogenetic view of the Bacillus cereus group population by combination of MLST, AFLP, and MLEE genotyping data

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database ( http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing ( Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.

Putative household outbreaks of campylobacteriosis typically comprise single MLST genotypes

During a 15-month period in Scotland a small but important number of human Campylobacter cases (3·2%) arose from 91 putative household outbreaks. Of the 26 outbreaks with known strain composition, 89% were composed of the same MLST which supports the potential use of MLST in public health epidemiology. The number of cases associated with household outbreaks is much larger than general outbreaks and there is some evidence to indicate that there may be secondary transmission, although this is relatively rare.

Biogeography and Virulence of Staphylococcus aureus

Background Staphylococcus aureus is commonly carried asymptomatically in the human anterior nares and occasionally enters the bloodstream to cause invasive disease. Much of the global diversity of S. aureus remains uncharacterised, and is not clear how disease propensity varies between strains, and between host populations.MethodologyWe compared 147 isolates recovered from five kindergartens in Chengdu, China, with 51 isolates contemporaneously recovered from cases of pediatric infection from the main hospital serving this community. The samples were characterised by MLST, the presence/absence of PVL, and antibiotic resistance profiling.Principal FindingsGenotype frequencies within individual kindergartens differ, but the sample recovered from cases of disease shows a general enrichment of certain MLST genotypes and PVL positive isolates. Genotypes under-represented in the disease sample tend to correspond to a single sequence cluster, and this cluster is more common in China than in other parts of the world.Conclusions/SignificanceVirulence propensity likely reflects a synergy between variation in the core genome (MLST) and accessory genome (PVL). By combining evidence form biogeography and virulence we demonstrate the existence of a “native” clade in West China which has lowered virulence, possibility due to acquired host immunity.

Purification of embryogenesis stimulating activity from bovine oviduct epithelial cell conditioned medium and effect on the development of bovine embryos

Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures.