A multilocus sequence typing (MLST) scheme has been developed for Pseudomonas aeruginosa which provides molecular typing data that are highly discriminatory and electronically portable between laboratories. MLST data confirm the data from previous studies that suggest that P. aeruginosa is best described as nonclonal but as having an epidemic population. The index of association was 0.17, indicating a freely recombining population; however, there was evidence of clusters of closely related strains or clonal complexes among the members of this population. It is apparent that the sequence types (STs) from single isolates, representing each of the present epidemic clones in the United Kingdom from Liverpool, Manchester, and the West Midlands, are not closely related to each other. This suggests distinct evolutionary origins for each of these epidemic clones in the United Kingdom. Furthermore, these clones are distinct from European clone C. Comparison of the results of MLST with those of toxA typing and serotyping revealed that strains with identical STs may possess different toxA types and diverse serotypes. Given that recombination is important in the population of P. aeruginosa, the lack of a linkage between toxA type and serotype is not surprising and reveals the strength of the MLST approach for obtaining a better understanding of the epidemiology of P. aeruginosa.

We describe here a method for determining confidence intervals for a commonly used index of diversity. This approach facilitates the comparison of the genetic population structure of microorganisms isolated from different environments and improves the objective assessment of the discriminatory power of typing techniques.The discrimination of organisms on the basis of variable phenotypic or genetic markers is still the mainstay of quantitative microbial ecology and descriptive epidemiology. To determine the diversity of microorganisms in defined environments (ecosystems) or to identify the reproductive success of disease causing organisms, i.e., the spread of particular strains between hosts, genetic typing techniques are deployed which have the ability to distinguish diverse organisms of the same species. Importantly, when one is comparing the diversity of a single species between different ecosystems or comparing the various typing methods used to resolve such differences, a robust statistical approach is required that allows an objective assessment. To this end, indices of diversity have been defined mathematically that are based on the frequency with which organisms of a particular type occur in a population or can be discriminated by a given typing tool (3, 4, 5). Individuals of a population will belong to one of Z types and will occur with frequencies of 1 . . . Z such that ⌺ ϭ 1. For microorganisms that usually have a very large population size, the genetic diversity () can be described as ϭ 1 -⌺ 2 , which will be the probability that two individuals chosen at random will be of a different type.Inferences on the diversity of the population involve a sampling process. The index of diversity D, as defined by Simpson (5) and lately utilized for the assessment of the discriminatory power of typing techniques (2, 6), is an unbiased estimate of the true diversity of a population based on a sample of n individuals. Simply by chance, different samples will give different results, the difference being due to sample variation and by drawing repeated samples, the precision of the mean estimate for D will improve. If repeated samples of a fixed size n are drawn from the sample population, the values of D will be distributed about with the variance 2 (5): 2 ϭ 4 nwhere j is the frequency n j /n, n j is the number of strains belonging to the jth type, and n is the total number of strains in the sample population. An estimate of the standard deviation of is given by the square root of 2 , and we propose the following as approximate 95% confidence interval (CI):We have applied these equations to determine confidence intervals (i) when assessing the genetic diversity of Staphylococcus aureus isolated from healthy carriers in the community as opposed to hospitalised patients and (ii) when comparing the discriminatory power of macrorestriction analysis by using SmaI restriction patterns with that of RAPD [random(ly) amplified polymorphic DNA] typing.By using the same sampling frame, healthy individuals in the community and in...

T he global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) has become an urgent public health concern (1,2). In 2016, the World Health Organization included CRE in a list of antimicrobial-resistant priority pathogens on which to concentrate future drug development strategies. Of note, carbapenem-resistant Klebsiella pneumoniae (CRKP) account for 60%-90% of clinical CRE infections in the United States, Europe, and China (1-3), resulting in an increased mortality rate of up to 40%-50% in nosocomial settings (4). The dissemination of CRKP is mostly clonal, and the population structure is geographically specific. Since its emergence during the early to mid-2000s, sequence type (ST) 258 has become the most prevalent CRKP clone in North America, Latin America, and Europe (5). However, in Asia, especially China, ST11 is the predominant clone, accounting for up to 60% of CRKP (3). ST11 is a single-locus (tonB) variant of ST258, and both types belong to the clonal group 258. A recombination event is thought to have occurred between a recipient ST11 and a donor ST442like strain, giving rise to ST258 during 1985-1997 (6,7). A phylogenomic study revealed that the ST258 population consists of >2 clades, resulting from an ≈215-kb recombination event that includes the capsule polysaccharide (cps) synthesis locus (6). The genetic differences generated by the resulting capsular switch are supposed to be primarily responsible for the ST258 diversification (8). Likewise, a segregation was identified in the ST11 population, resulting in >3 clades with different capsular loci (KL) (9-11). These studies consistently indicate that cps is a recombination hotspot in K. pneumoniae. However, the K-type distribution within ST11 in clinical settings is unclear. More important, the biological, epidemiologic, and

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (IA S). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS −/exoU +. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48–like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC. Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

A PCR identification of methicillin-resistant Staphylococcus aureus (MRSA), obviating the need for subculture on agar media, was investigated. The combination of MRSA detection by mecA femB PCR with prior enrichment in selective broth was tested for 439 swabs. PCR identified 36 MRSA-positive samples, in concordance with conventional methods.Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) in clinical specimens is essential for timely decisions on isolation procedures and effective antimicrobial chemotherapy. Numerous approaches that improve turnaround time for the identification of MRSA have been described. Fluorescence tests (14), PCR assays (4), or penicillin-binding protein 2a (PBP2a) antibody agglutination tests have been described elsewhere (12). Yet, these require subculture on solid media, and many are unable to determine species and methicillin susceptibility at the same time. A simultaneous test of methicillin resistance and species confirmation by a mecA femB duplex PCR has been proposed elsewhere (16). The mecA gene encodes the extra PBP2a, which is unique to methicillin-resistant staphylococci. The femB gene codes for an enzyme important in cross-linking peptidoglycan in various different Staphylococcus spp. The specificity of the femB PCR primers used for DNA amplification of the species S. aureus has been demonstrated previously (8).This study describes the performance of this technique in a clinical setting of moderate MRSA endemicity where large numbers of screens need to be processed on a daily basis. Moreover, the robustness of the test was investigated by determining the number of false-positive readings due to coamplification of femB and mecA from methicillin-susceptible S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (R-CNS) coexisting at the sample site (C. M.

We assessed the capacity of three DNA typing techniques to discriminate between 81 geographically, temporally, and epidemiologically unrelated strains of Pseudomonas aeruginosa. The methods, representing powerful tools for hospital molecular epidemiology, included hybridization of restricted chromosomal DNA with toxA and genes coding for rRNA (rDNA) used as probes and macrorestriction analysis of SpeI-digested DNA by pulsed-field gel electrophoresis. The probe typing techniques were able to classify all strains into a limited number of types, and the discriminatory powers were 97.7 and 95.6% for toxA and rDNA typing, respectively. Strains that were indistinguishable on the basis of both toxA and rDNA types defined 12 probe type homology groups. Of these, one contained five strains, three contained three strains each, and eight groups were represented by two strains each. Strains in 10 of the homology groups had the same O serotype. SpeI macrorestriction patterns discriminated between all strains with at least four band differences, which corresponded to a similarity level of 85%. Fifteen pairs of strains were similar at a level of >75% and differed by only four to seven bands. Of these pairs, 11 belonged to the same probe type homology group, indicating their clonal relatedness. We conclude that macrorestriction analysis of P. aeruginosa with SpeI provides the best means of discrimination between epidemiologically unrelated strains. However, DNA probe typing with either toxA or rDNA reveals information on the strain population structure and evolutionary relationships.

This study demonstrates that multimodal prevention strategies aiming at improving CVC insertion practice and HH reduce CRBSI in diverse European ICUs. Compliance explained CRBSI reduction and future quality improvement studies should encourage measuring process indicators.