Methicillin-Resistant Staphylococcus aureus in Spain: Molecular Epidemiology and Utility of Different Typing Methods

In a point-prevalence study performed in 145 Spanish hospitals in 2006, we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 135 (29.2%) were methicillin (meticillin)-resistant S. aureus (MRSA) isolates. Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal chromosomal cassette mec (SCCmec) typing, agr typing, spa typing with BURP (based-upon-repeat-pattern) analysis, and multilocus sequence typing (MLST). The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% showed the M phenotype. All of the isolates were susceptible to glycopeptides. Twelve resistance patterns were found, of which four accounted for 65% of the isolates. PFGE revealed 36 different patterns, with 13 major clones (including 2 predominant clones with various antibiotypes that accounted for 52.5% of the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for the first time in Spain. SCCmec type IV accounted for 6.7% of the isolates (70.1% were type IVa, 23.9% were type IVc, 0.9% were type IVd, and 5.1% were type IVh), and SCCmec type I and SCCmec type II accounted for 7.4% and 5.2% of the isolates, respectively. One isolate was nontypeable. Only one of the isolates produced the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), type 1 (14.8%), and type 3 (3.0%). spa typing revealed 32 different types, the predominant ones being t067 (48.9%) and t002 (14.8%), as well as clonal complex 067 (78%) by BURP analysis. The MRSA clone of sequence type 125 and SCCmec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCCmec typing, and MLST presented good correlations for the majority of the MRSA strains; we suggest the use of spa typing and PFGE typing for epidemiological surveillance, since this combination is useful for both long-term and short-term studies.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections worldwide (5, 25). The appearance of MRSA in the community and the potential risk of it entering hospitals are also matters of concern (29, 44). Moreover, the increasing prevalence of multidrug resistance and the emergence of isolates with intermediate or high-level vancomycin resistance emphasize the importance of the use of infection control measures (2, 49, 50). Although the rates of isolation of MRSA have been increasing throughout the world for the last few decades and in some areas the rates reach >50%, there are considerable variations in the prevalence of MRSA according to geographic area (3, 18, 21, 39, 44). In Spain, the prevalence of MRSA increased from 1.5% in 1986 to 29.2% in 2006, although it seems to have stabilized (13). Despite the worldwide increase in isolation rates, only a limited number of clones of MRSA have spread in most countries (20).Historically, the dissemination of epidemic clones such as EMRSA type 15 (EMRSA-15), EMRSA-16, the Iberian clone, and the Brazilian clone, as well as the high incidence of the community-acquired MRSA USA300 clone, has led to the increased use of molecular typing methods (11, 38, 42, 47, 53).In recent years, a variety of molecular techniques have been used for the typing of MRSA isolates. Of these, SmaI macrorestriction analysis is the “gold standard” for the analysis of the local epidemiology in the short term, spa typing in combination with BURP (based-upon-repeat-pattern) analysis has become a frontline tool for routine epidemiological typing, and multilocus sequence typing (MLST)-staphylococcal chromosomal cassette mec (SCCmec) typing is the reference method for the definition of MRSA clones (10, 34, 37, 46).The aim of the present study was to determine which clones are circulating in Spain and whether the strains have spread between hospitals by analyzing a representative sample of isolates collected in a point-prevalence study. Isolates were grouped by using pulsed-field gel electrophoresis (PFGE) and spa typing and were assigned to MRSA clones on the basis of MLST and SCCmec typing. The congruence between the different grouping methods was assessed.(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 [O. Cuevas, C. Marcos, P. Trincados, T. Boquete, E. Cercenado, E. Bouza, and A. Vindel; abstr. C2-148].)     

Multilocus Sequence Types of Carbapenem-Resistant Pseudomonas aeruginosa in Singapore Carrying Metallo-β-Lactamase Genes,Including the Novel blaIMP-26 Gene

Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-β-lactamase genes, including bla_IMP-1, bla_IMP-7, bla_VIM-2, bla_VIM-6, and the novel bla_IMP-26. Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.Carbapenem-resistant Pseudomonas aeruginosa is an increasing problem worldwide. While many underlying mechanisms may account for carbapenem resistance in this species, the possession of metallo-β-lactamase (MBL) genes is of particular concern because these enzymes are able to hydrolyze all β-lactam antimicrobials with the exception of aztreonam. In addition, these genes may be mobilized and transferred between different species of bacteria. We conducted a study in 2008 to investigate if there were any changes in the epidemiology of P. aeruginosa isolates containing MBL genes in our hospital compared to results from an earlier survey carried out in 2000 (3).Of 2,552 nonduplicate P. aeruginosa organisms isolated in 2008, 123 isolates were imipenem resistant. Of these, 11 were positive for MBL production by imipenem-EDTA disk diffusion (5). Nine of these yielded a product by multiplex PCR for MBL genes (2). The individual MBL genes were then amplified and sequenced. The clonal relationship between isolates with MBL genes was determined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA restricted with SpeI (3). The PFGE band patterns were analyzed with Bionumerics (Applied Maths NV, Sint-Martens-Latem, Belgium), and all strains with more than 85% similarity were considered to belong to the same clone. All strains were further subjected to multilocus sequence typing (MLST) (1). Because it is a nucleic acid sequence-based method, MLST is able to characterize bacterial types in an unambiguous fashion and establish evolutionary relationships between strains better than band-based methods like PFGE. Representative MBL-producing P. aeruginosa isolates from the 2000 survey were also subjected to PFGE and MLST. MLST profiles were submitted to eBURST V3 (http://eburst.mlst.net/) on 10 March 2010. Isolates sharing six out of seven alleles were assigned to the same BURST group and can be considered to belong to the same clonal complex descended from a common founder genotype. The PFGE, MBL gene sequence, and MLST results are summarized in Fig. ​Fig.11.Open in a separate windowFIG. 1.Dendrogram of PFGE patterns of P. aeruginosa isolates with metallo-β-lactamase genes, showing the year of isolation, MLST sequence type, and BURST group.In our previous study, 21 of 2,094 (1.0%) of all nonduplicate P. aeruginosa isolates in our hospital had MBL genes (3). With the exception of one isolate with bla_IMP-7, all other isolates had bla_IMP-1 and belonged to one of two PFGE clones. Isolates belonging to clone A had sequences identical to that of the original bla_IMP-1 first reported in Japan. Four representatives of clone A isolated from our hospital in 2000 had sequence type 964 (ST964) by MLST. Isolates belonging to clone B isolated in 2000 had sequences for variant bla_IMP-1 (bla_IMP-1v) with four silent mutations. Three representatives of this clone from 2000 had ST233 and one had ST742 based on MLST. All four representatives of clone B belong to the same BURST group, which was different from that of clone A.In contrast, in the 2008 survey, 9 of 2,552 (0.35%) nonduplicate P. aeruginosa isolates had MBL genes. Unlike the earlier study, there were no large clonal outbreaks. Two isolates with bla_IMP-1v had similar PFGE patterns and belonged to the same BURST group as representative isolates from clone B in 2000.Two isolates from 2008 with bla_IMP-7 had similar PFGE patterns and shared the same BURST group. The rest of the isolates from 2008 had distinct PFGE patterns.There was a greater diversity of MBL genes compared to the 2000 survey results. In particular, this is the first time that bla_VIM-2 and bla_VIM-6 have been found in P. aeruginosa in Singapore. bla_IMP-26 is a novel MBL gene that differs from bla_IMP-4 at position 145 (G-to-T change). The translated amino acid sequence differs from IMP-4 at residue 49 (phenylalanine for valine). This sequence has been previously deposited in the GenBank database as IMP-4 from an Acinetobacter calcoaceticus isolate from Malaysia (accession number {"type":"entrez-protein","attrs":{"text":"ABC24668.1","term_id":"83583501"}}ABC24668.1).Three of the isolates in this study (separately containing bla_VIM-2, bla_IMP-1, and bla_IMP-7) belonged to ST235. This sequence type has been described in a VIM-producing P. aeruginosa isolate in Belgrade and is the founder of an international clonal complex of isolates bearing MBL genes found in several countries in Europe (6). Recently, an increasing prevalence of IMP-1-producing P. aeruginosa has been found in Hiroshima, Japan. This was due entirely to the clonal expansion of only two lineages, ST235 (BURST group 3) and ST357 (BURST group 108) (4). This is similar to the situation that existed in Singapore in 2000, where only two lineages (BURST groups 29 and 44) accounted for the majority of MBL-producing P. aeruginosa (3).It is noteworthy that the original fear that a clone of MBL-producing P. aeruginosa would become established in Singapore has not been realized. The BURST group 29 and 44 lineages from 2000 were represented by only one to two isolates in 2008. The two P. aeruginosa isolates with bla_IMP-7 in 2008 are unrelated to the solitary isolate with bla_IMP-7 from 2000. It has been suggested that P. aeruginosa displays an epidemic population structure, with a limited number of clones emerging from a large number of unrelated genotypes (7). Although we did not correlate our study with hospital infection control measures, the Japanese data and our own seem to suggest that controlling the prevalence of MBL-producing P. aeruginosa may be achieved by preventing the transmission of specific epidemic clones.While it is reassuring to note that the prevalence of MBL producers in carbapenem-resistant P. aeruginosa has not increased, the increased diversity of MBL genes represents a new cause for concern. We were unable to characterize the gene responsible for the MBL phenotype in two isolates in this study, and these may represent novel resistance determinants. Although clones of MBL-producing P. aeruginosa have not become established, it seems likely, given the variation of MBL genes and MLST types in this study, that MBL-producing P. aeruginosa continues to be introduced to our hospital from diverse sources.     

Pseudomonas aeruginosa Genotype Prevalence in Dutch Cystic Fibrosis Patients and Age Dependency of Colonization by Various P. aeruginosa Sequence Types

The patient-to-patient transmission of highly prevalent Pseudomonas aeruginosa clones which are associated with enhanced disease progression has led to strict segregation policies for cystic fibrosis (CF) patients in many countries. However, little is known about the population structure of P. aeruginosa among CF patients. The aim of the present cross-sectional study was to determine the prevalence and genetic relatedness of P. aeruginosa isolates from CF patients who visited two major CF centers in The Netherlands in 2007 and 2008. These patients represented 45% of the Dutch CF population. P. aeruginosa carriage in the respiratory tract was determined by standard microbiological culture techniques, and all phenotypically different isolates in the first specimens recovered in 2007 and 2008 were genotyped by multilocus sequence typing. A total of 313 (57%) of 551 patients whose samples were cultured carried P. aeruginosa. Two sequence types (STs), ST406 and ST497, were found in 15% and 5% of the patients, respectively, and 60% of the patients harbored a strain that was also found in at least two other patients. The risk ratios for carrying ST406 and ST497 were 17.8 (95% confidence interval [CI], 7.2 to 43.6) for those aged between 15 and 24 years and 6 (95% CI, 1.4 to 26.1) for those aged >25 years. ST406 and ST497 were not genetically linked to previously described epidemic clones, which were also not found in this CF population. The population structure of P. aeruginosa in Dutch CF patients is characterized by the presence of two prevalent STs that are associated with certain age groups and that are not genetically linked to previously described epidemic clones.Pseudomonas aeruginosa is a ubiquitous, versatile bacterium that can infect humans as well as plants and animals. The species is infamous for causing nosocomial infections in immunocompromised patients and patients in intensive care units and is a major cause of morbidity and mortality in patients with cystic fibrosis (CF) (26).The widely held belief that CF patients acquire P. aeruginosa strains mainly from their inanimate environment, with most patients being colonized by unique strains, has been challenged by reports indicating that P. aeruginosa clones may frequently be transmitted between CF patients (3, 6, 18, 19, 23, 24). Some of these clones, such as the Liverpool epidemic strain and the Melbourne epidemic strain, have been associated with enhanced disease progression and higher rates of mortality, respectively (1, 13). In The Netherlands, the patient-to-patient transmission of P. aeruginosa was documented during a summer camp (4). These findings have led to strict segregation policies for CF patients in many countries, including The Netherlands. However, despite these studies, there is little information on the population structure of P. aeruginosa within populations of CF patients. We therefore investigated the prevalence and genetic relatedness of P. aeruginosa isolates compared to those of the international known genotypes in an unbiased cohort representing 45% of the CF population in The Netherlands in 2007 and 2008.     

Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis

The aim of this study was to compare two traditional pattern matching techniques, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD), with the more reproducible technique of multilocus sequence typing (MLST) to genotype a blinded sample of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. A blinded sample of 48 well-characterized CF P. aeruginosa isolates was genotyped by PFGE, RAPD, and MLST, each performed in a different laboratory. The discriminatory power and congruence between the methods were compared using the Simpson’s index, Rand index, and Wallace coefficient. PFGE and MLST had the greatest congruence with the highest Rand index (0.697). The discriminatory power of PFGE, RAPD, and MLST were comparable, with high Simpson’s indices (range 0.973–0.980). MLST identified the most clonal relationships. When clonality was defined as agreement between two or more methods, MLST had the greatest predictive value (100?%) in labeling strains as unique, while PFGE had the greatest predictive value (96?%) in labeling strains as clonal. This study demonstrated the highest level of agreement between PFGE and MLST in genotyping P. aeruginosa isolates from CF patients. MLST had the greatest predictive value in identifying strains as unique and, thus, has the potential to be a cost-efficient, high-throughput, first-pass typing method.     

Low Rates of Pseudomonas aeruginosa Misidentification in Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.The accurate identification of Pseudomonas aeruginosa is a critical component of cystic fibrosis (CF) patient management. Once established within CF lungs, P. aeruginosa is rarely eradicated, leading to increased treatment requirements and an accelerated decline in pulmonary function, quality of life, and life expectancy (10, 13, 27). Emerging evidence indicates that aggressive antipseudomonal therapy at the time of initial acquisition may eliminate P. aeruginosa, preventing the development of chronic infection for months or even years (37). Similarly, separating patients with P. aeruginosa from other CF patients may reduce the spread of multiple-antibiotic-resistant strains capable of person-to-person transmission (16). Such strategies are contingent upon the early and correct identification of these organisms (30).While there is much emphasis on misidentifying P. aeruginosa as another species (39), less attention is paid to falsely identifying other species as P. aeruginosa. Nevertheless, accurate identification of P. aeruginosa is important, as this may avoid prolonged and sometimes unnecessary antibiotic treatments, which could select for other antibiotic-resistant pathogens (6). Similarly, in CF clinics where cohort isolation is practiced as an infection control measure, false identification could mean exposure of the CF patient to potentially transmissible bacteria (2, 17, 28, 33).While most clinical strains of P. aeruginosa are easily identified, respiratory isolates from patients with CF can present a taxonomic challenge (15, 24). Phenotypic identification of P. aeruginosa from patients with CF is often complicated by slow growth, auxotrophic metabolic activity, loss of pigment production, multiple antibiotic resistance, atypical colonial morphology, and development of mucoid exopolysaccharide (14, 25). Commercial identification platforms are also considered unreliable (18, 21, 39). Moreover, CF respiratory secretions may contain other nonfermenting gram-negative bacilli, such as Achromobacter, Stenotrophomonas, and Burkholderia species, which can further impede the identification of P. aeruginosa (29, 32, 35, 39).Although several molecular strategies have been developed recently (1, 35, 39), most clinical microbiology laboratories still identify P. aeruginosa by traditional phenotypic techniques. However, there are few published data describing the frequency at which bacterial species in CF sputum are falsely identified as P. aeruginosa by phenotypic methods. In this study, we used P. aeruginosa-specific duplex real-time (PAduplex) PCR assays, phenotypic analysis, amplified rRNA gene restriction analysis (ARDRA), and partial 16S rRNA gene sequence analysis to assess the rate and extent of misidentification of P. aeruginosa isolates in CF sputum by Australian clinical microbiology laboratories.     

Characterization of Epidemiologically Unrelated Acinetobacter baumannii Isolates from Four Continents by Use of Multilocus Sequence Typing,Pulsed-Field Gel Electrophoresis,and Sequence-Based Typing of blaOXA-51-like Genes

This study used a diverse collection of epidemiologically unrelated Acinetobacter baumannii isolates to compare the robustness of a multilocus sequence typing (MLST) scheme, based on conserved regions of seven housekeeping genes, gltA, gdhB, recA, cpn60, rpoD, gyrB, and gpi, with that of sequence-based typing of bla_OXA-51-like genes (SBT-bla_OXA-51-like genes). The data obtained by analysis of MLST and SBT-bla_OXA-51-like genes were compared to the data generated by pulsed-field gel electrophoresis (PFGE). The topologies of the phylogenetic trees generated for the gyrB and gpi genes showed evidence of recombination and were inconsistent with those of the trees generated for the other five genes. MLST identified 24 sequence types (STs), of which 19 were novel, and 5 novel alleles. Clonality was demonstrated by eBURST analysis and standardized index of association values of >1 (P < 0.001). MLST data revealed that all isolates harboring the major bla_OXA-51-like alleles OXA-66, OXA-69, and OXA-71 fell within the three major European clonal lineages. However, the MLST data were not always in concordance with the PFGE data, and some isolates containing the same bla_OXA-51-like allele demonstrated <50% relatedness by PFGE. It was concluded that the gyrB and gpi genes are not good candidates for use in MLST analysis and that a SBT-bla_OXA-51-like gene scheme produced results comparable to those produced by MLST for the identification of the major epidemic lineages, with the advantage of having a significantly reduced sequencing cost and time. It is proposed that studies of A. baumannii epidemiology could involve initial screening of bla_OXA-51-like alleles to identify isolates belonging to major epidemic lineages, followed by MLST analysis to categorize isolates from common lineages, with PFGE being reserved for fine-scale typing.Acinetobacter baumannii is a Gram-negative bacterium that causes serious nosocomial infections, especially in critical care units (13, 14, 42). Several outbreaks have been caused by multidrug-resistant (MDR) strains of A. baumannii (23, 33, 43), and the rate of resistance to carbapenems, which have been the antibiotics of choice to treat infections caused by this pathogen, has increased considerably over the last decade (4, 32, 42, 46). In addition, the prevalence of A. baumannii in hospitals has increased worldwide (3, 25, 28, 29, 47), and, therefore, finding suitable molecular typing methods for A. baumannii is essential for epidemiological investigations and infection control studies. Many genomic typing methods have been used, including ribotyping (36), infrequent-restriction-site analysis (48), repetitive extragenic palindromic sequence-based PCR (rep-PCR) (18), random amplified polymorphic DNA (RAPD) analysis (21), amplified fragment length polymorphism (AFLP) analysis (39), and multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) (6). Pulsed-field gel electrophoresis (PFGE) is still considered the “gold standard” for the typing of bacterial isolates (36), but it has drawbacks when its comes to interchanging data among laboratories for comparison purposes (35) and may lose its discriminatory power when isolates from geographically diverse areas are analyzed.Multilocus sequence typing (MLST) schemes, which use several housekeeping genes, have already been used to type many pathogenic bacteria (15, 17, 20, 40), including A. baumannii (1, 31, 44), and MLST is emerging as an alternative to PFGE. MLST is used mainly for global epidemiology studies, but it has also been used successfully for short-term investigation of an outbreak of meningococcal disease (11). Although MLST has many advantages over other molecular typing methods, many questions remain to be answered, including whether several loci are required to obtain a robust scheme and whether the criteria for the selection of the housekeeping genes are sufficiently reliable to reveal the population structure of the strains analyzed.bla_OXA-51-like genes are unique to A. baumannii and may be used as markers for identification of this species (16). They have also successfully been used as one of three loci in a PCR-based typing scheme that is able to assign isolates of A. baumannii to sequence groups (SGs) that appear to correlate with the major epidemic lineages within the species (41). This raises the question of whether the bla_OXA-51-like genes themselves could be utilized in a typing scheme. Accordingly, the aim of this work was to investigate the robustness of MLST in categorizing epidemiologically unrelated A. baumannii isolates from four continents and to evaluate the use of variations within the intrinsic bla_OXA-51-like gene as a typing tool comparable to MLST.     

Impact of Pseudomonas aeruginosa Genomic Instability on the Application of Typing Methods for Chronic Cystic Fibrosis Infections

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients in the United Kingdom and has emerged recently in North America. In this study, we report the analysis of 24 “anomalous” CF isolates of P. aeruginosa that produced inconsistent results with regard to either pulsed-field gel electrophoresis (PFGE) or PCR tests for the LES. We used a new typing method, the ArrayTube genotyping system, to determine that of the 24 anomalous isolates tested, 13 were confirmed as the LES. LES isolates could not be clearly distinguished from non-LES isolates by two other commonly used genetic fingerprinting tests, randomly amplified polymorphic DNA (RAPD) analysis and BOX-PCR, and varied considerably in their carriage of LES genomic islands and prophages. The genomic instability of the LES suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.Chronic Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients are associated with a decline in lung function and increased mortality (11). Infection usually occurs during adolescence and, once established, the pathogen is impossible to eradicate (17). In recent years, the view of P. aeruginosa infection in CF patients has been altered because of the emergence of transmissible strains. Originally, it was thought that patients acquired their own unique strains from the environment and only in specific circumstances, such as siblings with CF, were patients found to share the same strain (35). However, in 1996 Cheng et al. (6) described the use of molecular typing to demonstrate the spread of a drug-resistant strain of P. aeruginosa (named the Liverpool epidemic strain [LES]) among patients in a children''s CF center in Liverpool, United Kingdom (6). Seven years later, an analysis of patient samples taken after the year 2000 identified the LES in 79% of 80 P. aeruginosa-colonized CF patients in the Liverpool adult CF center, confirming the spread and longevity of LES infections (29). In a survey of 31 CF centers in England and Wales in 2004, involving more than 1,200 isolates of P. aeruginosa, the LES was identified as the most common clone (33). Furthermore, the LES has also been identified in Scotland (10). Recently, cases of CF patients infected with the LES have also emerged in Canada (1).In addition to its transmissibility, the LES appears to be more aggressive than other P. aeruginosa strains. It has been shown to replace previously established strains of P. aeruginosa (superinfection) (24), has infected both non-CF parents of a CF patient (25), and has infected a pet cat, causing significant morbidity (26). Furthermore, the LES is associated with greater morbidity (2) and increased renal failure (3) and appears to have enhanced survival in air (30).Patient segregation based on LES status requires simple but effective strain typing methods with adequate discriminatory powers to achieve both the initial identification of the LES and subsequent epidemiological surveillance. The genome of P. aeruginosa is a mosaic structure consisting of a core genome and a variable accessory genome (23). The accessory genome includes large insertions, such as prophages and genomic islands, contributing to genome size variations between 5.2 and 7 Mb (38). The earliest archived isolate of the LES (LESB58, from 1988) has recently been genome sequenced (40), revealing the presence of six prophages and five genomic islands.Specific PCR assays have been developed for detection of the LES (31, 34). Following reports of false positives for the original marker PS21 (20), a second marker (LES-F9) was identified (34). Neither PS21 nor LES-F9, which map to separate genomic islands (LESGI-3 and LESGI-1, respectively), is 100% specific to the LES, but the combination of the two has not been reported previously in any non-LES strain. The use of PCR assays in routine diagnostic tests in Liverpool and elsewhere in the United Kingdom has led to the implementation of successful segregation measures. However, we have a number of putative LES CF isolates that do not give concordant results with respect to the presence of PS21, the presence of LES-F9, and the molecular typing method pulsed-field gel electrophoresis (PFGE). The Clondiag ArrayTube (AT) system is a portable method for interrogating both the conserved and accessory genomes of P. aeruginosa isolates. The process is rapid, relatively inexpensive, and robust and has been used to type P. aeruginosa isolates from diverse habitats (39).Here, we report the use of the AT system to resolve the strain status of a collection of suspected LES isolates of P. aeruginosa from CF patients. We provide evidence that the genomic instability exhibited by the LES could impact the validity of routine typing schemes, causing both false-positive and false-negative identification, and suggest that normal paradigms for bacterial typing may be limited in the context of chronic infections in CF.     

Staphylococcal Interspersed Repeat Unit Typing of Staphylococcus aureus: Evaluation of a New Multilocus Variable-Number Tandem-Repeat Analysis Typing Method

The present study evaluates the performance of the staphylococcal interspersed repeat unit (SIRU) method applied to a diverse collection of 104 Staphylococcus aureus isolates previously characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec typing for methicillin-resistant S. aureus. The SIRU method distributed the 104 strains into 81 SIRU profiles that could be clustered into 12 groups and 29 singletons. The discriminatory power of the method at the profile level, translated by Simpson''s index of diversity (SID), was similar to that of PFGE subtyping (SID = 99.23% versus 99.85%) and slightly higher than that of spa typing (SID = 97.61%). At the group level, the SIRU SID (93.24%) was lower than that of PFGE typing (95.41%) but higher than that of MLST (SID = 91.77%). The adjusted Rand (AR) coefficient showed that SIRU typing at the group level had the highest congruence with MLST (AR = 0.5736) and with clonal complex (CC) (AR = 0.4963) but the lowest congruence with PFGE subtype (AR = 0.0242). The Wallace coefficient indicated that in the present collection, two strains with the same SIRU profile have a 100% probability of belonging to the same CC, a 90% probability of sharing the same spa type, and an 83% probability of being classified in the same sequence type. The high discriminatory power of the SIRU method, along with its apparent concordance with MLST results, makes it potentially valuable for S. aureus short-term epidemiological investigations and population dynamics as well.Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), continues to be a major cause of health care-associated and, more recently, community-associated infections (40, 62). It is critical to have access to an accurate typing method to design cost-effective intervention and prevention strategies (45, 48, 61). A large number of molecular typing methods have been developed to assess strain relatedness for outbreak control, surveillance programs, and population structure and evolution studies (58, 61). The three most used typing methods for S. aureus have advantages and disadvantages, as follows. (i) Pulsed-field gel electrophoresis (PFGE), which is the “gold standard” typing method, has high discriminatory power and accuracy, but it is time-consuming and expensive, and the interlaboratory exchange of results is challenging. (ii) Sequence-based multilocus sequence typing (MLST) is easy to perform, and the results, given as an allelic profile, are portable and easy to exchange due to a public database available on the Internet (http://www.mlst.net), but it is expensive and not useful for local outbreak investigations. MLST is frequently combined with staphylococcal cassette chromosome mec (SCCmec) typing in order to define clonal types of MRSA (24). (iii) spa typing, a single-locus sequence typing method, is being used more frequently for S. aureus typing, and the development of a public database on the Internet (http://spaserver.ridom.de), as with MLST, ensured an international typing nomenclature and thus a great facility in exchanging typing data. By calculation of Simpson''s index of diversity (SID), it was shown that spa typing is nearly as discriminatory as PFGE (1, 25), although it takes into account a single variable region of the protein A gene.In choosing a new method, it is worth taking into consideration that PCR-based methods are commonly used in typing laboratories because of their accuracy, ease of use, low cost, and speed in retrieving results (in a few hours).Many bacterial genomes carry loci of repetitive DNA, which may contain variable repeated units among strains (43, 60). Systems based on a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) have been used extensively for typing of clinical isolates of several bacterial species and were shown to perform well compared to other genotyping methods (43, 61). S. aureus harbors a diverse population of DNA repeats, which allowed the design of various MLVA schemes (28, 30, 39, 53, 60). Hardy et al. (36, 38) developed a MLVA scheme for S. aureus where seven novel multiple tandem repeats with a high degree of similarity in the flanking regions were identified based on the alignment of seven S. aureus sequenced genomes (strains N315, MW2, Mu50, MSSA476, MRSA252, NCTC8325, and COL). Six of these seven loci were located on intergenic regions scattered around the S. aureus genome; the remaining locus corresponds to the protein A gene, spa. The method, designated staphylococcal interspersed repeat unit (SIRU) typing, relies on PCR amplification of the seven loci of repetitive DNA, using primers specific for the flanking regions of each locus, and on the determination of the size of each amplicon, which reflects the number of repeated units present on the targeted SIRU. To each of the seven loci is attributed the respective number of DNA repeats, generating a combination of seven numbers that characterizes each strain and corresponds to the allelic profile. This allelic profile makes the SIRU method amenable to interlaboratory comparisons and database management, comparable to MLST. So far, the SIRU method has been applied to S. aureus isolates from nosocomial outbreaks in the United Kingdom and Germany, mainly MRSA isolates, and therefore to highly related strains (29, 35-37). Very recently, a single study evaluated a MLVA scheme including the SIRU typing loci and the sspA gene, using a European collection of contemporary S. aureus isolates (39).The aim of the present study was to evaluate the SIRU method with a more diverse collection of S. aureus isolates, including MRSA and methicillin-susceptible S. aureus (MSSA) isolates, from different continents, isolated throughout several decades and previously characterized by well-established typing methods (PFGE, spa typing, MLST, and SCCmec for MRSA).     

Multiplex Strategy for Multilocus Sequence Typing,fla Typing,and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.Infection with Campylobacter has become the major cause of bacterial enteritis in Europe and other parts of the developed world, overtaking Salmonella infection (8). Campylobacter jejuni accounts for approximately 90% of all Campylobacter infection cases, whereas C. coli is responsible for approximately 10% of infections. Other Campylobacter species, such as C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus, are sporadically found (24). Due to the fact that Campylobacter is mostly commensal in the enteron of many warm-blooded animals used for meat production, campylobacteriosis is a zoonotic disease. Quality control, monitoring, and eventually tracing of contaminated food products is therefore important for public health reasons. Campylobacter typing by applying various, mostly genetic, methods is used for this purpose. Classical pulsed-field gel electrophoresis and amplified fragment length polymorphism, as well as flaA typing based on the restriction analysis of PCR-amplified fragments or sequencing of the flagellin-encoding gene, have been described for Campylobacter (20, 37). Recently, multilocus sequence typing (MLST) has been established as a highly reproducible method allowing precise and simple worldwide comparison of types, and it is becoming the gold standard in this field (4-6,13,17-19, 22, 23, 30, 33). Despite its many advantages, MLST is still time-consuming and expensive and therefore not feasible for routine testing. For example, the scheme for C. jejuni typing recommended by the PubMLST database hosted by the University of Oxford, Oxford, United Kingdom (http://pubmlst.org/campylobacter) includes a total of 51 different primers to be used for PCR amplification and sequencing of the seven target gene sequences. Another 14 primers are described for MLST of C. coli. With problematic isolates, optimal primer combinations have to be determined, and reactions have to be repeated in order to obtain all seven allele sequences needed for sequence type (ST) determination.MLST alone provides excellent information about the global epidemiology and population structure of Campylobacter, but it appears to be less discriminative in short-term epidemiological studies (28). The addition of more variable targets, such as flagellin-encoding genes, increases the discriminatory power of sequence-based typing. The most frequently used gene for this purpose is flaA (2, 5, 7, 17, 20, 26, 29), although flaB is also used, and as a more stable gene, flaB might become more important (21). Other important factors to consider are the time and effort needed to perform the appropriate data analysis, especially in the context of internationally standardized approaches and the use of publicly available typing tools, such as http://pubmlst.org.Since the 1990s, the prevalence of antibiotic resistance has increased dramatically in both animal and human Campylobacter isolates. This is especially the case for quinolone resistance, the emergence of which is correlated with the introduction of quinolones in the treatment of food-producing animals. The emergence of macrolide-resistant Campylobacter isolates has also been observed but until recently was less pronounced than quinolone resistance (41). Quinolone resistance is mainly based on a point mutation in the gyrase gene, gyrA (C257T or, less frequently, A256G) (1). In the case of macrolide resistance, it is caused by a point mutation (A2075G or A2074C) in the loop in domain V of the 23S rRNA gene (34).In order to optimize and simplify the amplification and sequencing strategy for MLST and combine it with sequence-based fla typing, as well as with antibiotic resistance determination, we established a modular and adaptable multiplex PCR and sequencing protocol using the minimum number of primers, which can be used equally well for C. jejuni and C. coli. About 95% of human Campylobacter infections can be covered with our typing scheme. Proper identification of Campylobacter isolates is not always trivial, and misidentification might hamper downstream typing, especially genotyping. 16S rRNA and rpoB genes were included in the multiplex approach as a basic genetic identification module for the genus Campylobacter, and the discriminatory power at the species level was examined. Through this approach, enteritis-causing Campylobacter species other than C. jejuni and C. coli are dealt with by proper identification.The robustness of the multiplex approach was tested on more than 300 C. jejuni and C. coli strains. Data analysis was performed using a newly developed Internet-based Integrated Database Network System (IDNS) (SmartGene, Zug, Switzerland) platform for genotyping Campylobacter.     

Predominance and Emergence of Clones of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus in Malaysia

We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.Methicillin-resistant Staphylococcus aureus (MRSA) is an established human pathogen that causes both health care-associated (HA) and community-acquired (CA) infections. Modern MRSA has evolved from several successful clonal lineages of methicillin-susceptible S. aureus strains via acquisition of a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). This element contains the mecA gene, which encodes penicillin-binding protein 2′ (PBP2′) with significantly reduced affinity for β-lactams (36).Increased emergence of multidrug resistance among MRSA strains has become a major concern in the hospital environment, as it invokes a tremendous financial burden and enhanced morbidity and mortality due to hard-to-treat systemic infections (7). Genotyping data from large international studies have shown that a limited number of major clones of MRSA display an enhanced propensity to spread and cause opportunistic human infections in various parts of the world (5, 13, 33). MRSA was introduced into Malaysia in the early 1970s (26), and a review of the records of the microbiology laboratories of all state hospitals in Malaysia showed that the proportion of MRSA isolates from S. aureus-infected individuals had been approximately 21% throughout the last few years.Phenotypic MRSA typing methods are not suited to detailed epidemiological surveillance (20, 40). Several superior molecular typing methods can be used for supporting infection control and for tracing the nosocomial sources and transmission routes of bacterial pathogens. Among these methods, multilocus sequence typing (MLST) has recently been proven to be the best for long-term global epidemiological and bacterial population genetics studies. It is a discriminatory genotypic method where isolates are typed by sequencing variable regions of housekeeping genes. A combination of alleles from the seven loci forms a sequence type (ST). Similar STs are grouped into clonal complexes (13). spa typing has been shown to be as discriminatory as the current gold standard method, pulsed-field gel electrophoresis (PFGE) (10). spa types can be determined via amplification and sequencing of the X region of the protein A gene (spa), which contains polymorphic direct repeats. StaphType (version 1.4; Ridom GmbH, Würzburg, Germany) provides a software tool enabling straightforward sequence analysis and designation of spa types by synchronization via a central server (42). spa typing is useful in analyzing hospital outbreaks and in identifying genetic changes that occur over a relatively short time span (14, 43). Molecular typing data on HA MRSA isolates in Malaysia are sparse in comparison with those on strains deriving from Europe, the United States, or Japan. This is notwithstanding the steadily increasing but already high rates of MRSA infections in Malaysia (38). Pilot data hinted at the predominance of a single MRSA genotype in Malaysia. This is similar to the situation in many other Asian countries (1, 23, 31).For the present study, clinical MRSA isolates associated with various infections were collected from the largest public tertiary referral hospital in Kuala Lumpur, the capital of Malaysia. The epidemiology of MRSA in this hospital (HKL) will most likely reflect the nation''s epidemiology as it is the major government referral hospital for patients from all states in Malaysia. Each year at least 1 million people (∼3.8% of the total Malaysian population) are treated here. HKL records an annual MRSA prevalence over 40%. Many of these MRSA strains are multidrug resistant. Additional epidemiological studies are clearly warranted in order to increase the insight into the dynamics of MRSA epidemiology in Malaysia.The aims of the present study were to characterize the current Malaysian MRSA isolates and determine their molecular epidemiology by MLST, spa typing, SCCmec typing, and virulence gene profiling.     

Population Structure of Staphylococcus aureus Strains Isolated from Intensive Care Unit Patients in The Netherlands over an 11-Year Period (1996 to 2006)

The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.Around 20% of all patients in intensive case units (ICUs) acquire an ICU-related infection as a consequence of frequent use of antibiotics and intensive treatment procedures (1, 31). Of all ICU-related infections, 25% are caused by Staphylococcus aureus (31). Knowledge of the S. aureus population structure and of the prevalence of virulence factors has been proven crucial for the investigation of the epidemiology of S. aureus throughout the world (34).Methicillin-resistant S. aureus (MRSA) clones can emerge by horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) between methicillin-resistant coagulase-negative Staphylococcus or MRSA and methicillin-susceptible S. aureus (MSSA) (51). In the event of antibiotic pressure, the MSSA isolates have a high risk of SCCmec transfer and survive. As shown in the literature, MSSA lineages with a MRSA-unrelated background may not provide a stable genomic environment for the integration of SCCmec (4, 23, 30, 32, 36, 43). SCCmec transfer has been found to be stable in MSSA with a MRSA-related genetic background, i.e., multilocus sequence typing (MLST) clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45 (39). The MSSA lineages with a MRSA background possess certain characteristics that favor their persistence in the host as well as the transfer between hosts.As the highest antibiotic pressure in hospitals is found in ICUs, changes in the genetic background will be the most obvious among isolates from ICU patients. However, little is known about the genetic backgrounds of ICU isolates over time, and, therefore, this study investigates the genetic background and the virulence of S. aureus isolates obtained from 1996 to 2006 from ICU patients from 14 hospitals in The Netherlands.     

Population Analysis and Epidemiological Features of Inhibitor-Resistant-TEM-β-Lactamase-Producing Escherichia coli Isolates from both Community and Hospital Settings in Madrid,Spain

Punctual mutations in the TEM-1 or TEM-2 gene may lead to inhibitor-resistant-TEM (IRT) β-lactamases with resistance to β-lactam-β-lactamase inhibitor combinations and susceptibility to cephalosporins. The aim of this work was to analyze the current epidemiology of IRT β-lactamases in contemporary clinical Escherichia coli. Isolates were prospectively collected in our hospital (2007 and 2008) from both outpatients (59.8%) and hospitalized patients (40.2%). The genetic relationships of the isolates were determined by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and phylogenetic group analysis. IRT genes were sequenced and located by hybridization, and the incompatibility group of the plasmids was determined. From a total of 3,556 E. coli isolates recovered during the study period, 152 (4.3%) showed reduced susceptibility to amoxicillin-clavulanate, with 18 of them producing IRT enzymes (0.5%). These were mostly recovered from urine (77.8%). A high degree of IRT diversity was detected (TEM-30, -32, -33, -34, -36, -37, -40, and -54), and the isolates were clonally unrelated but were mostly associated with phylogenetic group B2 (55.5%). In 12 out of 16 (75%) isolates, the bla_IRT gene was plasmid located and transferred by conjugation in 9 of them, whereas chromosomal localization was demonstrated in 4 isolates (25%). The sizes of the plasmids ranged from 40 kb (IncN) to 100 kb (IncFII, IncFI/FIIA), and they showed different restriction patterns by restriction fragment length polymorphism analysis. Unlike extended-spectrum β-lactamase producers, the frequency of IRT producers remains low in both community and hospital settings, with most of them causing urinary tract infections. Although bla_IRT genes are mainly associated with plasmids, they can be also located in the chromosome. Despite this situation, clonal expansion and/or gene dispersion was not observed, denoting the independent emergence of these enzymes.Resistance to β-lactam-β-lactamase inhibitor combinations in Escherichia coli may be due to different mechanisms, including TEM-1 penicillinase hyperproduction, constitutive AmpC overproduction or plasmid AmpC production, OXA-type β-lactamase production, permeability deficiencies involving OmpF and/or OmpC porins, inhibitor-resistant TEM (IRT)- and complex mutant TEM (CMT)-like ß-lactamase production, and more recently, carbapenemase production (4).IRT enzymes comprise a group of plasmid-encoding variants of TEM-1 and TEM-2 with decreased affinities for amino-, carboxy-, and ureidopenicillins and altered interaction with class A ß-lactamase inhibitors (6). IRT-producing isolates remain susceptible to cephalosporins, cephamycins, carbapenems, and in most cases, piperacillin-tazobactam. They are usually resistant to ampicillin-sulbactam and intermediate or resistant to amoxicillin-clavulanate combinations. IRT enzymes have previously been reported in different organisms, such as E. coli, Klebsiella spp., Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii, and Shigella sonnei (4); but there are only a few recent epidemiological studies concerning these enzymes. Moreover, the population structure of IRT-producing E. coli isolates has not been addressed using a multilocus sequence typing (MLST) technique.The aim of the present work was to analyze the current epidemiology of IRT β-lactamases in contemporary E. coli isolates with reduced susceptibility to amoxicillin-clavulanate recovered in our hospital in 2007 and 2008.     

Determination of accessory gene patterns predicts the same relatedness among strains of Streptococcus pneumoniae as sequencing of housekeeping genes does and represents a novel approach in molecular epidemiology

Relatedness between isolates of Streptococcus pneumoniae can be determined from sequences of multiple genes belonging to the core genome (multilocus sequence typing [MLST]), but these do not provide information on gene content that may affect the potential of isolates to cause invasive pneumococcal disease. Gene content data, obtained using microarrays, were gathered for 40 clinical isolates of 12 serotypes belonging to 30 multilocus sequence types. We found that sequence variations in housekeeping genes assessed by MLST correlated well with whole-genome microarray analyses identifying the presence/absence of accessory genes/regions. However, isolates belonging to the same clonal complex, as determined by MLST, may not have identical gene contents, potentially affecting virulence. We found fewer intraclonal (same MLST sequence type) differences associated with pneumococcal serotypes of high invasive disease potential, i.e., serotypes rarely found among carriers compared to serotypes frequently found in carriage. Molecular typing of pneumococci based on the presence/absence of 25 genes localized to accessory regions shows the same relatedness among pneumococcal strains as MLST does. We conclude that molecular typing of pneumococci based on variation in the nucleotide sequences of parts of housekeeping genes (MLST) correlates with the presence/absence of genes in the accessory part of the genome. This covariation is likely due to the fact that both sequence variations and gene content variations are created primarily by recombination events in pneumococci.     

Occurrence,Population Structure,and Antimicrobial Resistance of Enterococci in Marginal and Apical Periodontitis

Subgingival plaque samples and root canal samples were collected from 2,839 marginal periodontitis (MP) patients and 21 apical periodontitis (AP) patients. Enterococcus species were identified by a series of phenotypic and genotypic tests. Antimicrobial susceptibility assays were performed by an agar disk diffusion test. Multilocus sequence typing (MLST), eBURST, and minimum spanning tree were used for enterococcal genetic clustering and population analysis. Enterococcus faecalis was recovered from 3.7% MP patients and 9.5% AP patients, and Enterococcus faecium was recovered from 0.04% MP patients. Enterococci were detected more often in older male patients. E. faecalis isolates of MP were found resistant to tetracycline (49.1%), erythromycin (8.5%), trimethoprim (2.8%), and gentamicin (1.9%), while one AP isolate was resistant to tetracycline. A total of 40 sequence types (STs) were resolved in 108 E. faecalis isolates. Comparison with E. faecalis international MLST database revealed that 27 STs were previously found, 13 STs were novel, and several major clonal complexes in the database were also found in MP isolates. The tetracycline-resistant isolates distributed mainly in the major clonal complexes and singletons, whereas the erythromycin-resistant isolates were more dispersed. Although the rate of occurrence of enterococci recovered in the MP and AP samples was low, 50% of these isolates are resistant to at least one antimicrobial agent, which is most often tetracycline. This implies that subgingival E. faecalis might represent a reservoir of resistance to tetracycline and erythromycin. The subgingival E. faecalis isolates show high genetic diversity but are grouped, in general, with the known isolates from the international database.Enterococci are commensal organisms well adapted to survive in human and animal intestinal tracts (15). Occasionally they become opportunistic pathogens causing urinary tract infections, septicemia, and endocarditis (16). In the United States, Enterococcus faecalis has been found to account for approximately 60% of health care-associated enterococcal infections and Enterococcus faecium for most of the remaining part (13).Enterococci display intrinsic resistance to a number of antimicrobial agents and have a propensity for acquiring resistance to currently prescribed antimicrobials, which poses serious problems in the treatment of invasive enterococcal infections (3, 7, 18). Molecular typing of E. faecalis and E. faecium, e.g., multilocus sequence typing (MLST), has shown the emergence of specific genetic lineages of enterococci, i.e., hospital-adapted clones of antimicrobial-resistant E. faecalis and E. faecium (11, 40). E. faecalis clonal complexes CC2, CC9, and CC87 seem to be widely distributed in hospital patients in Europe and the United States (17, 31).Enterococci have been isolated at low rates and in small numbers from the oral cavities of healthy individuals (10, 28, 33). However, in patients with posttreatment apical periodontitis (AP), enterococci are predominant bacteria, with reported prevalence ranging from 29% to 77% (37). Moreover, enterococcal biofilms have been observed in root canals of patients with AP after treatment for AP (8, 39). Enterococci recovered in root canals of AP patients often express resistance toward commonly used antimicrobials (6, 26).Only a few studies have reported on the occurrence of enterococci in marginal periodontitis (MP) patients, but with a variation from 1% to 47.8% (29, 36). There is a considerable gap in our knowledge concerning the prevalence of enterococci in MP, their genetic population structure, antimicrobial susceptibility profile, and their role in pathogenesis.The aims of the present study were as follows: (i) to investigate the occurrence and species distribution of enterococci in subgingival and root canal samples from MP and AP patients, respectively; (ii) to examine their antimicrobial susceptibility profile; (iii) to analyze the genetic relatedness of oral E. faecalis isolates and compare their population structure with E. faecalis international MLST database; and (iv) to assess the association, if any, between sequence type (ST) and specific antimicrobial-resistant phenotypes.     

Multilocus sequence typing of Campylobacter jejuni isolates from humans, chickens, raw milk, and environmental water in Quebec, Canada

Molecular strain typing is essential for deciphering the epidemiology of Campylobacter jejuni infections. We applied two different methods, multilocus sequence typing (MLST) and analysis of the flaA short variable repeat (SVR), to 289 isolates (163 human, 56 chicken, 34 raw milk, and 36 environmental water isolates) collected in the province of Québec, Canada, over 3 years; in addition, the analysis included the pulsed-field gel electrophoresis (PFGE) typing results for a subset of 131 isolates studied previously. MLST defined 96 sequence types (STs) and 20 clonal complexes (CCs), including 49 STs (73 isolates, 25%) and 39 alleles not previously documented in an international database. The frequency of new STs was significantly higher among water isolates than among isolates from other sources (18/36 [50%] and 55/253 [22%], respectively; P < 0.001). Nine of the 10 most prevalent CCs included isolates from humans and at least one other source; five CCs comprised exclusively or mostly human and chicken isolates. However, water and milk were the predominant nonhuman sources among the remaining CCs, suggesting that sporadic C. jejuni infections in humans may frequently arise from sources other than chickens. All three typing systems were discriminatory (discriminatory index > 0.9). Among 131 isolates analyzed by PFGE, each of the 20 types represented by two or more isolates corresponded to a single CC. In contrast, among the 14 most prevalent types detected by analysis of the flaA SVR (5 to 27 isolates each), 8 (57%) included isolates that represented multiple different CCs. The basis for these discordant results was uncertain. Antimicrobial resistance was randomly distributed among the CCs and appeared to be more closely related to the source of an isolate than its genotype. Although MLST is labor-intensive and expensive, it remains the single best method for the genotyping of C. jejuni isolates and deciphering the epidemiologic relationships among isolates.     

Genotypic Comparison of Invasive Neisseria meningitidis Serogroup Y Isolates from the United States,South Africa,and Israel,Isolated from 1999 through 2002

The proportion of meningococcal disease in the United States, South Africa, and Israel caused by Neisseria meningitidis serogroup Y (NmY) was greater than the worldwide average during the period 1999-2002. Genotypic characterization of 300 NmY isolates by multilocus sequence typing, 16S rRNA gene sequencing, and PorA variable region typing was conducted to determine the relationships of the isolates from these three countries. Seventy different genotypes were found. Two groups of ST-23 clonal complex isolates accounted for 88% of the U.S. isolates, 12% of the South African isolates, and 96% of the isolates from Israel. The single common clone (ST-23/16S-19/P1.5-2,10-1) represented 57, 5, and 35% of the NmY isolates from the United States, South Africa, and Israel. The predominant clone in South Africa (ST-175/16S-21/P1.5-1,2-2), and 11 other closely related clones made up 77% of the South African study isolates and were not found among the isolates from the United States or Israel. ST-175 was the predicted founder of the ST-175 clonal complex, and isolates of ST-175 and related sequence types have been described previously in other African countries. Continued active surveillance and genetic characterization of NmY isolates causing disease in the United States, South Africa, and Israel will provide valuable data for local and global epidemiology and allow monitoring for any expansion of existing clonal complexes and detection of the emergence of new virulent clones in the population.Neisseria meningitidis is an important cause of morbidity and mortality worldwide and a leading cause of bacterial meningitis and septicemia in children and young adults. Serogroups A, B, and C have caused ca. 90% of the cases of sporadic and epidemic meningococcal disease globally, and N. meningitidis serogroup Y (NmY) has, historically, been responsible for only a minor proportion of meningococcal disease worldwide (9, 14). In the United States, the proportion of meningococcal disease caused by NmY increased from 10% in 1992 to 34% in 1996 with an estimated increase in incidence from 0.1/100,000 population in 1992 to 0.2/100,000 in 1996 (3, 15).In South Africa, meningococcal disease is endemic but, unlike the patterns of disease in the African meningitis belt, the incidence rates are low, and increases of sporadic disease occur during the winter and spring months (1). Serogroup Y has caused little disease historically, although comprehensive national surveillance data on meningococcal serogroups have been lacking. Recent national laboratory-based surveillance data (1999 to 2002) in South Africa have shown a high proportion (∼21%) of meningococcal disease caused by NmY (5). In Israel, the incidence of NmY meningococcal disease rose from 0.05/100,000 population in 1990 to 1991 to 0.16/100,000 in 1998, and the proportion of NmY among all isolates increased from 3% in 1991 to 21% in 1998 (data from the National Center for Meningococci [Israel]).The goals of the present study were to determine whether a particular genetic lineage of NmY had caused the majority of NmY invasive disease in the United States, South Africa, and Israel and to compare the genotypes of the NmYs found in these countries to the genotypes of NmY found globally. We characterized 300 NmY isolates collected during 1999 through 2002 from the United States, South Africa, and Israel by 16S rRNA gene sequence typing, multilocus sequence typing (MLST), and PorA variable region (VR) typing.     

A Trilocus Sequence Typing Scheme for Hospital Epidemiology and Subspecies Differentiation of an Important Nosocomial Pathogen,Enterococcus faecalis

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST^M; based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.Enterococci are commensal members of the gastrointestinal tract flora of humans and animals. Within the last 2 decades, enterococci have emerged as the second to third most frequent cause of nosocomial infections, including endocarditis and bloodstream, urinary tract, and wound infections, among others (8, 15, 19, 24, 39). These organisms are also known to have the ability to acquire and transfer antibiotic resistance genes and virulence-associated genes (37). Although there are more than 30 species of the genus Enterococcus, two species, Enterococcus faecalis and Enterococcus faecium, account for a vast majority of enterococcal clinical and nosocomial infections (15, 21, 35). In the past, several molecular typing studies have shown that specific lineages of pathogenic bacteria arise periodically, proliferate, and spread in the presence of selective pressures (34). Therefore, accurate typing of enterococcal strains is crucial for the identification of particular clones capable of causing infections and with the ability to spread in the hospital environment.A number of phenotypic and genotypic typing methods have been applied to the subspecies differentiation of enterococcal isolates. Phenotypic methods which have been used in the past include serotyping (17, 22, 26) and multilocus enzyme electrophoresis (50). Genotypic methods include, among others (3, 52, 53), ribotyping (14, 38), repetitive sequence-based PCR (25, 35), multilocus variable-number tandem-repeat analysis (49, 54), pulsed-field gel electrophoresis (PFGE) (10, 12, 49), and multilocus sequence typing (MLST) (10, 26, 31, 41). Among these methods, PFGE, based on chromosomal restriction endonuclease digestion patterns, is widely used for the study of hospital outbreaks and is considered by many to be the “gold standard” molecular typing technique (48). However, this methodology has several limitations due to the facts that it is labor-intensive and the results have poor interlaboratory transportability. This technique is also unsuitable for long-term epidemiology and population studies due to changes in restriction sites, genomic rearrangements, and/or acquisition of DNA by a clonal lineage, which may markedly change the restriction pattern (41). A more appropriate typing technique for long-term epidemiology, which is currently also widely used for subspecies differentiation, is MLST. MLST, based on the allelic variations in sequences of multiple loci, unambiguously types strains (23) and offers an advantage over other techniques used for typing, such as PFGE, since the data are objective and easily stored, compared, and shared via the Internet.Two different MLST schemes have been used successfully for differentiation of E. faecalis strains (31, 41). The first scheme, which assessed three antigenic genes and one housekeeping gene, found that the allelic profile of two antigenic genes (ace and salA) was sufficient to discriminate the 22 E. faecalis isolates included therein (31). The second MLST scheme, based on the allelic profiles of seven housekeeping genes, was used to type 110 isolates and provided insight into the population structure as well as long-term epidemiological relationships of E. faecalis strains (41). However, typing studies on other organisms, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus, have suggested that MLST based on housekeeping genes may not provide enough discriminatory power to study hospital outbreaks or to accurately determine short-term genetic relationships, which can be crucial for hospital epidemiology and infection control purposes (9, 13, 27).Our hypothesis for this work was that a sequence-based methodology applied to genes encoding antigenic or cell surface proteins (rather than housekeeping genes) may potentially be more useful to establish short-term epidemiologic relationships in E. faecalis, since these genes would be more susceptible to evolutionary selective pressures and potentially could identify and discriminate isolates from hospital outbreaks, similar to PFGE.In the present work, the trilocus sequence types (ST^T; sequence type based on three genes) of 50 isolates were compared to their multilocus sequence types (ST^M; sequence type based on seven housekeeping genes). To determine the applicability of trilocus sequence typing (TLST) for a clinical setting, the scheme was also used to type sets of predetermined (by PFGE) clones and was then applied to a collection of hospital isolates from Bogota, Colombia, recently reported by Arias et al. to belong to an ST-2 clonal lineage (1).(Part of this work was presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007.)     

Validation of Vitek 2 Nonfermenting Gram-Negative Cards and Vitek 2 Version 4.02 Software for Identification and Antimicrobial Susceptibility Testing of Nonfermenting Gram-Negative Rods from Patients with Cystic Fibrosis

Accurate identification and antimicrobial susceptibility testing (AST) of nonfermenters from cystic fibrosis patients are essential for appropriate antimicrobial treatment. This study examined the ability of the newly designed Vitek 2 nonfermenting gram-negative card (NGNC) (new gram-negative identification card; bioMérieux, Marcy-l''Ètoile, France) to identify nonfermenting gram-negative rods from cystic fibrosis patients in comparison to reference methods and the accuracy of the new Vitek 2 version 4.02 software for AST compared to the broth microdilution method. Two hundred twenty-four strains for identification and 138 strains for AST were investigated. The Vitek 2 NGNC identified 211 (94.1%) of the nonfermenters correctly. Among morphologically atypical microorganisms, five strains were misidentified and eight strains were determined with low discrimination, requiring additional tests which raised the correct identification rate to 97.8%. Regarding AST, the overall essential agreement of Vitek 2 was 97.6%, and the overall categorical agreement was 92.9%. Minor errors were found in 5.1% of strains, and major and very major errors were found in 1.6% and 0.3% of strains, respectively. In conclusion, the Vitek NGNC appears to be a reliable method for identification of morphologically typical nonfermenters and is an improvement over the API NE system and the Vitek 2 GNC database version 4.01. However, classification in morphologically atypical nonfermenters must be interpreted with care to avoid misidentification. Moreover, the new Vitek 2 version 4.02 software showed good results for AST and is suitable for routine clinical use. More work is needed for the reliable testing of strains whose MICs are close to the breakpoints.Pseudomonas aeruginosa is the most important cause of lung infections in patients with cystic fibrosis (CF) (14). In our hospital, 50% of sputum-producing CF patients are colonized in their lower airways with P. aeruginosa or other nonfermenting bacteria. Accurate identification and antimicrobial susceptibility testing (AST) are essential for appropriate antimicrobial therapy.A variety of automated commercial systems for identification and susceptibility testing of nonfermenting bacteria are available (2, 3, 12, 19, 20). They are widely used because of the increasing volumes of clinical specimens processed by clinical laboratories and perceived cost effectiveness. The automated systems decrease the in-laboratory turnaround time and enable a faster targeted antimicrobial therapy. Unfortunately, errors in classification and AST by any test system can have serious implications for the clinical outcome of patients. The most frequently reported errors have involved the inaccurate identification of nonfermenters due to their phenotypic variations and slower growth rates and the inconsistencies between the tested broad-spectrum β-lactam antibiotics. Because of the perceived inaccuracies of AST from CF isolates, a consensus conference on CF microbiology recommended the use of the disk diffusion method for testing P. aeruginosa and other nonfermenters (13, 22).To improve the identification rate of nonfermenting gram-negative bacilli, a new colorimetric Vitek 2 card (nonfermenting gram-negative card [NGNC]) with an enlarged database was recently introduced. To advance the accuracy of the AST results, a new software (version 4.02) was developed.The aim of the present study was to evaluate the performance of the new NGNC for identification and the new software version 4.02 for AST of nonfermenters isolated from CF patients.     

Molecular Surveillance of Clinical Neisseria gonorrhoeae Isolates in Russia

The choice of adequate methods for epidemiological purposes remains a challenging problem in Neisseria gonorrhoeae molecular monitoring. In this study, the collection of geographically unrelated gonococci (n = 103) isolated in Russian clinics was comparably tested by (i) a traditional serotyping scheme, (ii) por typing, (iii) Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST), and (iv) multilocus sequence typing (MLST). It is shown that, according to sequencing data, a third of the strains carried new porB1 alleles, as well as tbpB ones, and more than half of the samples had new sequence types (STs) as determined by NG-MAST or MLST. The discriminatory power for each typing method was calculated by using the Hunter-Gaston discriminatory index, D. Commonly, modern nucleic acid-based typing methods (por typing, NG-MAST, and MLST) appeared to be more efficient than the classical serotyping scheme. While the traditional serotyping gave a D value of 0.82, the por typing, NG-MAST, and MLST approaches yielded D values of 0.97, 0.98, and 0.91, respectively. Each typing technique revealed the distribution of gonococci slightly correlated with their geographical sources. However, only the MLST method STs were highly associated with certain phenotypes. Although ST1594, ST1892, and ST6720 were typical for susceptible gonococci, ST1901 and ST6716 were undoubtedly associated with a multidrug-resistant phenotype. We conclude that every tested nucleic acid-based typing method is suitable for N. gonorrhoeae molecular surveillance. However, the MLST method seems to serve large-scale epidemiological purposes, whereas the NG-MAST and por typing approaches are more appropriate for the investigation of local outbreaks.The choice of an optimal epidemiological approach for Neisseria gonorrhoeae typing remains a problem for public health control strategies. The worldwide spread of drug-resistant strains requires typing methods to be introduced into a national surveillance programs that are already realized within the Australian Gonococcal Surveillance Programme (33). In Russia, strains that are resistant to penicillin, tetracycline, and fluoroquinolones are common, reaching up to 60% prevalence in some regions (14). As is generally known, most resistance mechanisms in N. gonorrhoeae are linked to mutations in genomic DNA, and a wide dissemination of such mutations has been demonstrated in subjects with gonorrhea in the Russian population (11). In fact, chromosomally mediated drug resistance expands clonally in a bacterial population. Thus, tracing the spread of gonorrhea by using strain differentiation methods is of great importance. In addition, population genetics are important in understanding the evolutionary history, epidemiology, and population dynamics of pathogens.Widespread adoption of the molecular diagnosis of gonorrhea could compromise traditional bacteriological cultivation in routine practice and highlights the need for the development of molecular tools for N. gonorrhoeae typing. Several promising approaches exist in this field. One of them is por typing (7, 8, 27), based on comparative analysis of porB gene nucleotide sequences. It possesses rather high discriminatory power (39) and presents data in a format comparable with serotyping data. It should be mentioned that PorB1 protein plays a key role in the adhesion of gonococci to epitheliocytes and reflects the pathogenicity of isolates. Moreover, in earlier studies the multidrug-resistant phenotype of N. gonorrhoeae was bound to certain serotypes determined in accordance with the antigenic properties of PorB1 proteins (7, 21, 29).Today, the N. gonorrhoeae multiantigen sequence typing (NG-MAST) (19) appears to be a leading method for N. gonorrhoeae typing. It is based on the analysis of internal fragments of two hypervariable genes, porB and tbpB, encoding superficial gonococcus antigens and therefore under positive selection (19). An online database (www.ng-mast.net) collects current information on the sequence types of gonococcus isolates in different regions of the world, and it can be an efficient instrument for objective estimation of the genetic variability of the microbial population and tracing the spread of infection (2). Moreover, an association of the NG-MAST sequence type with the antibiotic resistance profile has been demonstrated by some investigators (20, 26).In addition, NG-MAST, which is usually carried out on DNA extracted from a pure bacterial culture, can be performed directly from noncultured samples such as a piece of clothing (18) or clinical specimens (40). Although some researchers have reported a successful application of NG-MAST to urogenital specimens (urine specimens and swabs from the cervix, urethra, and vagina) and to rectal swabs, this typing method was found to be less suitable for throat swabs due to cross-reaction with commensal Neisseria species. It seems that direct typing schemes utilizing probe hybridization methods (16, 17) for a broad spectrum of clinical samples are more convenient than sequencing.The opposite approach—analysis of conservative, presumably selective neutral housekeeping genes—is taken by multilocus sequence typing (MLST) (1, 38). Possessing a sufficient number of allelic variants and characterized by the slow accumulation of mutations, these genes reflect the natural evolution of the microbial population and common trends in the spread of gonorrhea.Often, the effectiveness of N. gonorrhoeae surveillance depends on the methods used for species identification and the typing of clinical isolates. However, considering the variety of typing systems but the lack of world experience in their application to monitoring gonococci, it has not yet been determined whether there is a unique objective method for revealing the relationships between clinical isolates. The goal of the present study was to evaluate a number of approaches, including por typing, NG-MAST, MLST, and serotyping for typing geographically unrelated gonococcal isolates in Russia.(This study was partially presented during the 16th International Pathogenic Neisseria Conference in 2008 in the Netherlands.)     

Development of a Multilocus Sequence Typing Scheme for Molecular Typing of Mycoplasma pneumoniae

Mycoplasma pneumoniae is a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme for M. pneumoniae was developed based on the sequences of eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) and applied to 55 M. pneumoniae clinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57 M. pneumoniae isolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for the M. pneumoniae isolates examined, providing a method for further and more detailed analysis of observed epidemic peaks of M. pneumoniae infection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).