Necessity of Molecular Techniques To Distinguish between Neisseria meningitidis Strains Isolated from Patients with Meningococcal Disease and from Their Healthy Contacts

Serogroup C strains of Neisseria meningitidis were isolated from a Germany patient with severe meningococcal disease after a trip to the Czech Republic. These strains (case isolates) were characterized by classical and molecular techniques, as were other strains (carrier isolates) isolated from healthy contacts. Five of 10 carrier isolates had switched off the expression of capsular polysaccharide, as demonstrated by a serogroup-specific PCR. The two case isolates were indistinguishable by multilocus sequence typing and belonged to the ET-37 complex. The carrier isolates belonged to four different sequence types, all unrelated to that of the case strains. Pulsed-field gel electrophoresis showed that the case isolates differed from reference ET-37 complex strains from the Czech Republic and Canada as well as from all the carrier isolates. The isolate from the patient’s nasopharynx was indistinguishable from the blood isolate except for a 40,000-bp chromosomal deletion that had occurred during systemic spread.Bacterial meningitis due to Neisseria meningitidis (the meningococcus) continues to be of global importance for public health authorities. While pandemics affecting China and Africa are usually caused by meningococci of the A capsular serogroup, sporadic meningitis, outbreaks, and hyperendemic disease in Central Europe and the United States are usually caused by serogroups B and C (2). For sporadic meningococcal meningitis, public health efforts often include bacteriological analysis of throat swabs obtained from close contacts of the patient and the treatment of healthy nasopharyngeal carriers with prophylactic antibiotics and/or vaccines (5).Meningococci isolated from the healthy contacts of a diseased patient (carrier isolates) are not necessarily related to the strain causing disease (case isolates), even if all these isolates express the same capsular serogroup. Various subtyping methods have been used to test relationships among the strains from a cohort, including serotyping and serosubtyping with monoclonal antibodies (MAbs) (11), pulsed-field gel electrophoresis (PFGE) (7), multilocus enzyme electrophoresis (MLEE) (2), ribotyping (22), the randomly amplified polymorphic DNA method (25), and PCR-restriction fragment length polymorphism analysis (16). Recently, a novel portable approach, multilocus sequence typing (MLST), which is based on the DNA sequences of six housekeeping gene fragments, has been developed (18). MLST allows assignment of meningococci to clonal groups within a globally accessible, continuously expanding central database.In outbreak situations, so many bacteria may be isolated that only some of the carrier strains, usually selected on the basis of their capsular serogroup by latex agglutination, are evaluated in detail. However, the expression of capsular polysaccharide by serogroup B meningococci can undergo phase variation, resulting in the isolation of strains from carriers which are not obviously related to the index strain because they are capsule negative and nonserogroupable (6, 14, 15). The siaD gene encodes a polysialyltransferase which is needed for the synthesis of capsular polysialic acid chains. siaD can be amplified from serogroup B and C meningococci by PCRs (6), enabling the determination of the potential serogroups of bacteria which have become phenotypically nonserogroupable. Here we describe a two-step PCR, an improved version of the siaD PCR (6), which distinguishes between serogroup B, C, W135, and Y meningococci.Antigenic variation through horizontal genetic exchange can also lead to capsule switching among highly related bacteria (23). Such capsule-switching variants would be classified as unrelated to the parent strain by classical serogrouping. Similarly, antigenic variation can also lead to the switching of other antigens (13), including those used for serological subtyping, such as PorA (21). Thus, serological methods cannot reliably recognize the relatedness of meningococci and epidemiological analyses should rely primarily on molecular techniques, particularly those based on multiple loci scattered around the chromosome (3).We report here on the molecular investigation of meningococci isolated from individuals who had been in contact with a patient with severe serogroup C meningococcal disease.     

Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

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.)     

Evidence of Nosocomial Infection in Japan Caused by High-Level Gentamicin-Resistant Enterococcus faecalis and Identification of the Pheromone-Responsive Conjugative Plasmid Encoding Gentamicin Resistance

A total of 1,799 Enterococcus faecalis isolates were isolated from inpatients of Gunma University Hospital, Gunma, Japan, between 1992 and 1996. Four hundred thirty-two (22.3%) of the 1,799 isolates had high-level gentamicin resistance. Eighty-one of the 432 isolates were classified and were placed into four groups (group A through group D) with respect to the EcoRI restriction endonuclease profiles of the plasmid DNAs isolated from these strains. The 81 isolates were isolated from 36 patients. For 35 of the 36 patients, the same gentamicin-resistant isolates were isolated from the same or different specimens isolated from the same patient at different times during the hospitalization. For one other patient, two different groups of the isolates were isolated from the same specimen. Groups A, B, C, and D were isolated from 5, 14, 12, and 6 patients, respectively. The strains had multiple-drug resistance. The restriction endonuclease digestion patterns of the E. faecalis chromosomal DNAs isolated from isolates in the same group were also identical. The patients who had been infected with the gentamicin-resistant isolates from each group were geographically clustered on a ward(s). These results suggest that the isolates in each group were derived from a common source and had spread in the ward. The gentamicin-resistant isolates exhibited a clumping response upon exposure to pheromone (E. faecalis FA2-2 culture filtrate). The gentamicin resistance transferred at a high frequency to the recipient E. faecalis isolates by broth mating, and the pheromone-responsive plasmids encoding the gentamicin resistance were identified in these isolates.Enterococcus strains have become a significant cause of nosocomial infections (15, 17, 18, 22, 27). Of the members of the genus Enterococcus, E. faecalis and E. faecium are commonly isolated from humans. These two organisms account for 85 to 95 and 5 to 10% of the strains isolated from clinical infections, respectively. The Enterococcus strains isolated from clinical infections have multiple-drug resistance. The multiple-drug resistance of the enterococci provides these organisms with a selective advantage in the hospital environment. Outbreaks of nosocomial infections caused by enterococcal strains resistant to various drugs have been reported previously (9, 10, 16–18, 23, 28, 29).In a study of clinical isolates from patients in Gunma University Hospital in Gunma, Japan, enterococci were found to be the second most common among the gram-positive bacteria, after Staphylococcus aureus (unpublished data). Of the clinical E. faecalis isolates, most (about 80%) were resistant to tetracycline. Between 30 and 40% of the isolates were resistant to gentamicin or erythromycin. Ampicillin- or vancomycin-resistant strains were not isolated (14, 24). Certain E. faecalis conjugative plasmids confer a mating response to the small sex pheromones secreted by potential recipient cells (1–4, 8, 11). This mating signal induces the synthesis of a surface aggregation substance that facilitates the formation of mating aggregates and plasmid transfer (2–4, 7, 11, 25). Most (60%) of the drug-resistant strains exhibit a clumping response with a culture filtrate of a plasmid-free E. faecalis recipient strain (24), suggesting that the strains harbor a pheromone-responding plasmid.To our knowledge, there is no report concerning nosocomial infection caused by enterococci in Japan. In this report, we describe nosocomial infections in Gunma University Hospital caused by high-level gentamicin-resistant isolates of E. faecalis and isolation of the pheromone-responsive plasmids from the isolates.     

d-Lactate Production and [14C]Succinic Acid Uptake by Adherent and Nonadherent Escherichia coli

Escherichia coli isolates of different adherence phenotypes produced different amounts of d-lactate. Alterations of culture conditions did not influence the amount of d-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in d-lactate concentration. Very little [¹⁴C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E. coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E. coli.One of the virulence associated properties of enteropathogenic Escherichia coli (5, 13, 14) is the ability to adhere to small intestinal mucosa (3, 11, 12, 21, 24, 26, 27). Although this adherence is an important event in the induction of diarrhea, the mechanism by which adherent E. coli mediates pathogenicity remains uncertain (1, 2, 7, 18, 26, 27).Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28, 29). We used the HeLa assay (20) to detect this virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture medium (17). The pH changes induced by organisms with different adherence phenotypes differed. Since the characteristic end products of E. coli fermentation include lactic acid, succinic acid, and acetic acid, the pH changes could be explained by differences in the production of organic acids. Other plausible explanations are differences in the removal of organic acids from the medium and interactions between bacteria and HeLa cells during adherence.This paper describes two sets of experiments, one based on the production of lactic acid and the other on the removal of succinic acid from the medium. The objectives were to determine (i) whether there is a metabolic difference between localized, diffuse, and nonadherent isolates in the amount of lactate produced or succinate removed from the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to 5 h under different culture conditions, (iii) whether an increase in lactate production or succinate removal coincides with the drop in pH previously observed, and (iv) whether the pH changes can be attributed to differences in bacterial growth rates between isolates with different in vitro adherence patterns and nonadherent strains.     

Comparison of Typing Results Obtained for Methicillin-Resistant Staphylococcus aureus Isolates with the DiversiLab System and Pulsed-Field Gel Electrophoresis

We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same “unrelated” organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.Although pulsed-field gel electrophoresis (PFGE) is often considered the gold standard for typing methicillin-resistant Staphylococcus aureus (MRSA) isolates for epidemiologic studies (8, 12, 13), PFGE requires several days to complete and the results are often difficult for inexperienced users to interpret. On the other hand, DNA sequence-based methods, such as spa typing, which has also been shown to be useful for epidemiologic studies of MRSA (3), are not practical for many clinical laboratories in the United States, which lack access to DNA sequencing facilities. An alternative strain typing method, which is available commercially, is the DiversiLab typing system (DL) (bioMérieux, Inc., Durham, NC), which uses the presence of DNA repetitive elements present in the organism''s genome to determine the genetic relatedness of bacterial and fungal isolates (4-6, 9, 18). DL has been used successfully in several MRSA typing studies to distinguish sporadic from outbreak-related isolates and is noted to be more rapid to perform and easier to learn than PFGE (14, 15). Agreement between DL clusters of organisms and USA PFGE types, as defined by McDougal et al. (12), was reported for five well-defined U.S. outbreaks, although specific data were not shown (14). However, a recent study of representative MRSA strains from the Harmony collection in Europe concluded that while DL, PFGE, and multilocus sequence typing (MLST) provided concordant classification of strains, PFGE showed a higher level of strain discrimination than either DL or MLST (17). Thus, whether DL can differentiate accurately among USA types remains an open question.The goal of this study was to use DL to characterize a series of MRSA isolates of known PFGE types from U.S. hospitals to determine whether DL could (i) differentiate among PFGE types USA100 through USA1100, (ii) identify DL banding patterns that correlated with specific USA types, and (iii) differentiate contemporary outbreak-related MRSA isolates from sporadic isolates collected from U.S. hospitals.     

Comparison of Fingerprinting Methods for Typing Methicillin-Resistant Staphylococcus aureus Sequence Type 398

This study evaluates the multiple-locus variable-number tandem-repeat assay (MLVA) and pulsed-field gel electrophoresis (PFGE) when using restriction enzymes BstZI, SacII, and ApaI to fingerprint a diverse collection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) isolates. These isolates had been characterized previously by multilocus sequence typing, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed, and the concordance between the various methods was determined. All MRSA ST398 isolates were typeable by the MLVA and PFGE using BstZI, SacII, and ApaI. With each method, the MRSA ST398 isolates formed a separate group from the two non-ST398 MRSA strains. PFGE, performed with any of the three restriction enzymes, had the most discriminatory power, followed by MLVA, spa typing, and SCCmec typing. The MLVA showed the highest concordance with PFGE using ApaI and spa typing. As further expressed by the Wallace coefficient, the MLVA type was poorly predicted by spa typing, whereas the spa type was well predicted by MLVA. PFGE, using a combination of all three restriction enzymes, had the highest concordance with the MLVA but had a low probability of being predicted by MLVA. PFGE, using a combination of all three restriction enzymes, was able to predict SCCmec type and MLVA type completely and had a high probability of predicting spa type. Both the MLVA and PFGE could be used to discriminate among the MRSA ST398 isolates. Although the MLVA is a faster technique, PFGE had more discriminatory power than the MLVA, especially when a combination of restriction enzymes was used.Infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) have been a problem in hospitals and nursing homes for many decades. These MRSA isolates are therefore called health care-associated MRSA (HA-MRSA) isolates (1). Since the early 1990s, MRSA has emerged in healthy persons without risk factors for MRSA infections. These isolates are the so-called community-associated MRSA (CA-MRSA) isolates (20). In the last few years, MRSA has been isolated from livestock animals (pigs in particular) and pig farmers (5, 6, 30). These MRSA strains are called animal-associated MRSA (AA-MRSA) strains. It seems that (livestock) animals form a new, separate reservoir. These AA-MRSA strains all appear to belong to the new clonal complex 398 (CC398), with sequence type 398 (ST398) as the basic type, as determined by multilocus sequence typing (MLST) (29). MRSA ST398 has already been isolated in Europe, Asia, and North America (32). Considering the worldwide spread of MRSA, epidemiological questions arise about its transmission within farms, among farms, and from farms to the population. Fast and inexpensive typing methods with good discriminatory power are necessary to conduct large-scale epidemiological studies.Traditionally, human MRSA isolates have been typed by pulsed-field gel electrophoresis (PFGE), using SmaI as the restriction enzyme (19). The advantages of using PFGE are good discriminatory power and good reproducibility at the interlaboratory level when standardized protocols are used. However, AA-MRSA is not typeable by this method, as the activity of SmaI is blocked due to methylation of the restriction site (2).More recently, methods based on DNA sequencing, such as MLST and spa typing, are increasingly being used to discriminate among different MRSA strains. Given their excellent interlaboratory reproducibility, online databases have been made to collate and harmonize data from various geographic regions. The drawback of MLST, which measures sequence variation at seven housekeeping loci, is its limited use with epidemiological studies due to its weak discriminatory power, time-consuming protocols, and high costs. spa typing, based on the variation in repeats present in the X-region of staphylococcal protein A, has a discriminatory power that lies between those of PFGE and MLST. Within ST398, several spa types have been distinguished, although the number of spa types seems rather limited in most countries.One promising method is the multiple-locus variable-number tandem-repeat assay (MLVA), a PCR-based method, based on the analysis of the number of repeats in the variable-number tandem-repeat regions of various individual genes. This method has proven to be useful for typing both Staphylococcus aureus and clinical MRSA isolates with good reproducibility and good discriminatory power. Because the MLVA is also simple, inexpensive, and easy to interpret, it is useful as a typing method for large-scale epidemiological studies (10, 11, 12, 15, 16, 17, 23, 24, 27).This study aimed to investigate various methods for typing MRSA ST398 isolates. An MLVA, consisting of a selection of primers from three existing MLVA systems, was tested with a collection of MRSA ST398 isolates. In addition, PFGE with restriction enzymes other than SmaI was performed with this set of isolates. These isolates had been previously characterized by MLST, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed for all methods, and the concordance among the different methods was determined.     

Characterization of the Roles of Hemolysin and Other Toxins in Enteropathy Caused by Alpha-Hemolytic Escherichia coli Linked to Human Diarrhea

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.Escherichia coli is one of the major causes of human infectious diseases, partly because of the wide variety of virulence mechanisms and pathotypes (15), and new pathotypes continue to be described. A new pathotype was proposed by Gunzburg et al. after examining diarrheal pathogens in a prospective community-based study among Australian Aboriginal children (22). One group of isolates was significantly (P < 0.05) associated with diarrhea, and these isolates were particularly common among children younger than 18 months. The isolates did not produce any recognized enterotoxin or classic enteric virulence factor, although they exhibited diffuse or aggregative adhesion in a modified adhesion assay (15). All isolates were able to detach HEp-2 cell monolayers and were termed “cell-detaching E. coli.” This property was shown to be mediated by alpha-hemolysin, and we demonstrate below that all cell-detaching E. coli strains produce alpha-hemolysin and that some may also produce cytotoxic necrotizing factor 1 (CNF1) and other toxins. However, neither alpha-hemolysin nor CNF1 has been clearly demonstrated to be an enteric virulence factor, and the role of hemolysin in particular is controversial. We will refer to these isolates as diarrhea-associated hemolytic E. coli (DHEC) isolates.Alpha-hemolytic E. coli strains have been associated with human enteric disease, especially among young children (8, 10–12, 20–22), and the related enterohemolysin of E. coli O157 (35) appears to be involved in enteric disease. There has, however, been no large prospective case-controlled epidemiologic study of the association of alpha-hemolysin with human diarrhea. Alpha-hemolytic bacteria are also associated with enteric disease and diarrhea in pigs, cattle, and dogs (9, 13, 33, 36, 44, 45). Porcine diarrheal strains are almost universally hemolytic (23a), and alpha-hemolysin in these isolates enhanced virulence and colonization (37) but was not itself diarrheagenic. More recent studies have found that Hly⁺ CNF1⁺ strains caused fluid accumulation in piglets (33) and that neonatal pigs were susceptible to challenge with Hly⁺ CNF⁺ strains, which caused bloody diarrhea, enterocolitis, and systemic disease (45).In contrast, some earlier studies were unable to demonstrate a role for hemolysin in enteric disease, since neither hemolytic bacteria nor their supernatants caused fluid accumulation in ileal loops (10, 14, 37). Hemolytic strains may be isolated from the feces of asymptomatic people (26), and, among humans, hemolysin is more commonly associated with strains causing extraintestinal infections (5, 26).The genetics and in vitro mechanisms of alpha-hemolysin are well known. The hlyCABD operon encodes the structural 110-kDa hemolysin protein (HlyA) and proteins involved in processing and export (42). Once secreted, hemolytic activity is short-lived, and this has complicated studies of hemolysin toxigenicity (42). Hemolysin does not require a receptor to bind to target cells, inserting instead into the target cell membrane to form a pore that allows the free flow of cations, sugars, and water. This leads to leakage of intracellular contents and affects the cytoskeleton and metabolism (4, 9, 42, 43). In extraintestinal infections, hemolysin has multiple effects and roles, including resistance to host defense, tissue damage, and lethality, either by direct action or by stimulation of inflammatory mediators and signal transduction pathways (7, 9, 16, 42).CNF is a 114-kDa protein with homology to a family of dermonecrotic toxins (18) and is encoded by the monocistronic cnf gene, which lies just downstream of hly. The CNF1 toxin causes HeLa cells to become large and multinucleated as a result of actin disassembly, which results from activation of Rho (10, 19, 31). Similar to alpha-hemolysin, the role of CNF1 in diarrhea remains unclear. CNF1-producing strains have been isolated from diarrheal stools and have been associated with several outbreaks in humans (8, 10) and animals (13, 33, 44). Unfortunately, no large, prospective, case-controlled studies have been performed, and the best evidence for the pathogenicity of CNF1-toxigenic isolates is the marked virulence in piglet challenge experiments (45), outlined above. Purified CNF1 did not show enterotoxic potential in the suckling mouse or induce fluid accumulation in the rabbit ileal loop (10, 14), in contrast to the related CNF2, which is linked to enteric disease in animals (13, 14, 30). Both CNF toxins are extremely lethal, and have a variety of in vivo effects including tissue necrosis and edema (12–14).In this paper, we characterize DHEC isolates that were obtained from a study where alpha-hemolysin was significantly associated with disease (22) and show that they are able to cause disease in rabbits. Using molecular genetics, we attempt to analyze the role of each gene in pathogenesis.     

Effect of Prior Experimental Human Enteropathogenic Escherichia coli Infection on Illness following Homologous and Heterologous Rechallenge

Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

Enteropathogenic Escherichia coli (EPEC) strains are one of several categories of pathogenic E. coli strains that cause diarrhea. EPEC infections are prevalent on six continents (5, 22–24, 28, 43). In many parts of the world, EPEC strains are the most common bacterial cause of diarrhea in infants (7, 21, 43). Disease due to EPEC can be severe, refractory to oral rehydration, protracted, and lethal (3, 14, 21, 45, 48).The pathogenesis of EPEC infection involves three distinct stages, initial adherence, signal transduction, and intimate attachment (12). Initial adherence is associated with the production of a type IV fimbria, the bundle-forming pilus (BFP) (20), that is encoded on the large EPEC adherence factor (EAF) plasmid (50). EPEC uses a type III secretion apparatus to export several proteins, including EspA, EspB, and EspD, that are required for tyrosine kinase-mediated signal transduction within the host cell (17, 25, 30, 31). This signaling leads to phosphorylation and activation of a 90-kDa protein that is a putative receptor for the bacterial outer membrane protein intimin (44). Intimin, the product of the eae gene, is required for intimate attachment of bacteria to the host cell membrane and for full virulence in volunteers (13, 26, 27). The interaction between EPEC and host cells results in the loss of microvilli and the formation of adhesion pedestals containing numerous cytoskeletal proteins (16, 33, 34, 39, 46). This interaction between bacteria and host cells is known as the attaching and effacing effect (40).One of the most striking clinical features of EPEC infections is the remarkable propensity of these strains to cause disease in very young infants. Rare reports of disease in older children and adults usually reflect common-source outbreaks that probably involve large inocula (47, 53). In contrast, in nosocomial outbreaks among neonates, EPEC spreads rapidly by person-to-person contact, apparently involving low inocula (54). The incidence of community-acquired EPEC infection is highest in the first 6 months after birth (4, 7, 21). EPEC infection is also more severe in younger children (8). Infants are more likely to develop diarrhea during the first episode of colonization with EPEC than they are during subsequent encounters (8). Whether the low incidence of EPEC diarrhea in older children and adults is due to acquired immunity or decreased inherent susceptibility is not known.The immune response to EPEC infection remains poorly characterized. It has previously been demonstrated that volunteers convalescing from experimental EPEC infection develop antibodies to the O antigen component of lipopolysaccharide (LPS) of the infecting strain, to intimin, and to type I-like fimbriae (13, 15, 29, 38). Antibodies to common EPEC O antigens are found more often in children of greater than 1 year in age than they are in younger children (42). Breast-feeding is protective against EPEC infection (2, 19, 43, 52). Breast milk contains antibodies against EPEC O antigens and outer membrane proteins and inhibits EPEC adherence to tissue culture cells (6, 9, 49).In an earlier study, it was reported that volunteers infected with EPEC developed antibodies to a 94-kDa outer membrane protein (38). Subsequently, it was determined that this antigen was intimin (26). Interestingly, the lone volunteer in that earlier study who did not have diarrhea after challenge with a wild-type EPEC strain had prechallenge serum antibodies to intimin. This led to the hypothesis that antibodies to intimin are protective against EPEC infection. To test this hypothesis and to test the more general hypothesis that EPEC infection induces protective immunity, two volunteer studies were performed. The first was a heterologous-challenge study performed in 1986, in which volunteers were infected with an O55:H6 EPEC strain and challenged, along with a naive cohort, with an O127:H6 EPEC strain. The second was a homologous-challenge study performed in 1991, in which veterans of a study comparing the virulence of a wild-type EPEC O127:H6 strain with that of an isogenic eae mutant (13) were rechallenged, along with a naive cohort, with the homologous wild-type strain. The availability of new purified antigens allowed us to analyze data from these studies in the context of humoral immune responses.     

Identification of Virulence-Associated Characteristics in Clinical Isolates of Yersinia enterocolitica Lacking Classical Virulence Markers

Yersinia enterocolitica is an important enteric pathogen which has well-defined virulence determinants that allow the bacteria to become established in their hosts and overcome host defenses. A number of strains obtained from patients with diarrhea, however, lack these genes. Accordingly, the mechanisms by which they cause disease are uncertain. Most of these isolates belong to biotype 1A. Strains of this biotype are also frequently isolated from a variety of nonclinical sources, such as food, soil, water, and healthy animals, and there is evidence that some of these strains are avirulent. In this study we investigated 111 strains of Y. enterocolitica biotype 1A, 79 from symptomatic humans and 32 from nonclinical sources, for virulence-associated characteristics. DNA hybridization studies showed that none of the strains carried sequences homologous with pYV, the ∼70-kb Yersinia virulence plasmid. Some strains hybridized with DNA probes for one of the following chromosomal virulence-associated genes: ail (7.2%), myfA (11.7%), ystA (0.9%), and ystB (85%). In addition, 33 strains (29.7%) produced an enterotoxin that was reactive in infant mice. However, the frequencies of these virulence-associated properties in clinical and nonclinical isolates were similar. Clinical isolates invaded HEp-2 cells and Chinese hamster ovary cells to a significantly greater extent than nonclinical strains (P ≤ 0.002). In addition, clinical strains colonized the intestinal tracts of perorally inoculated mice for significantly longer periods than nonclinical isolates (P ≤ 0.01). Light and electron microscopic examination of tissue culture cells incubated with invasive yersiniae revealed that the bacteria invaded selected cells in large numbers but spared others, suggesting that biotype-1A strains of Y. enterocolitica may invade cells by a novel mechanism. These results indicate that some clinical isolates of Y. enterocolitica which lack classical virulence markers may be able to cause disease via virulence mechanisms which differ from those previously characterized in enteropathogenic Yersinia species.Yersinia enterocolitica is an important human pathogen which causes a variety of disorders, ranging from nonspecific diarrhea to invasive disease such as mesenteric lymphadenitis, hepatosplenic abscesses, and septicemia (5, 10, 37). The heterogenous nature of Y. enterocolitica, including differences in virulence, has led to the division of the species into subgroups based upon biochemical behavior and lipopolysaccharide O antigens (5, 37). At present, six biotypes are recognized, of which biotype 1B and biotypes 2 through 5 are regarded as including primary pathogens (5, 22, 26, 37, 40). The primary pathogenic strains of Y. enterocolitica are recognized in part by their ability to invade tissue culture cells in large numbers (7, 27, 29, 35). Genes which contribute this ability include the inv and ail genes on the bacterial chromosome and yadA, which is borne on a ca. 70-kb virulence plasmid known as pYV (4, 7, 27). Interestingly, however, other pYV-borne genes impede bacterial penetration of epithelial cells and macrophages, with the result that Y. enterocolitica is located extracellularly in infected animals (9, 15). Chromosomal genes other than inv and ail which may also contribute to virulence include yst (also known as ystA), which encodes a heat-stable enterotoxin (YST-a), myf, which encodes the production of fibrillae (Myf), and the urease gene complex (11, 12, 21).Biotype-1A strains of Y. enterocolitica, which generally are considered to be avirulent, are highly heterogenous, and include a large number of O serogroups (5, 37). They occur throughout the world in a wide range of environments and generally lack the genotypic and phenotypic markers associated with virulence of classical invasive strains of Y. enterocolitica, such as pYV, ail, myf, ystA, or a functional inv gene (12, 21, 28, 35, 40, 41, 45). Moreover, biotype-1A strains of environmental origin do not colonize the gastrointestinal tracts of experimentally inoculated animals (33, 42, 48). Despite these observations, some biotype-1A strains have been implicated as a cause of gastrointestinal disease. For example, a nosocomial outbreak of gastroenteritis in Canada involving nine patients was attributed to a strain of Y. enterocolitica biotype 1A, serogroup O:5 (36). In several countries, moreover, including Australia, Canada, The Netherlands, New Zealand, the Republic of Georgia, South Africa, Switzerland, and the United States of America, a significant proportion of Y. enterocolitica isolates obtained from patients with diarrhea belong to biotype 1A (3, 6, 17, 32, 34, 39, 46, 47). In addition, a prospective case control study with Chilean children showed that biotype-1A strains were significantly associated with diarrhea (30), and a clinical study in Switzerland demonstrated that the illness associated with biotype-1A strains of Y. enterocolitica was indistinguishable from that due to classical virulent biotypes (6).If biotype-1A strains of Y. enterocolitica are able to cause disease, their pathogenic mechanisms are not clear because they lack the well-established virulence markers of primary pathogenic strains of Y. enterocolitica. Some Y. enterocolitica strains produce variants of YST-a, known as YST-b and YST-c (20, 52, 53), but their prevalence and contribution to disease are not known. In addition, a biotype-1A strain of serogroup O:6 was reported to produce a novel heat-stable enterotoxin, termed YST-II. This toxin differs from YST-a in a number of respects, including its mechanism of action, which does not appear to involve activation of guanylate cyclase (41). Other putative virulence determinants in this or other biotype-1A strains have not been investigated or reported.As biotype-1A strains of Y. enterocolitica are so heterogenous and occupy such a diverse range of environmental niches, we hypothesized that there may be a subset of these bacteria which are capable of causing disease but which lack the classical virulence markers of Yersinia species and therefore cannot be identified by assays for these markers. The aim of this study was to test this hypothesis by examining a collection of biotype-1A strains of clinical and nonclinical origins for virulence-associated determinants.     

Multiple-Locus Variable-Number Tandem-Repeat Analysis of the Swine Dysentery Pathogen,Brachyspira hyodysenteriae

The spirochete Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe colonic infection of pigs that has a considerable economic impact in many swine-producing countries. In spite of its importance, knowledge about the global epidemiology and population structure of B. hyodysenteriae is limited. Progress in this area has been hampered by the lack of a low-cost, portable, and discriminatory method for strain typing. The aim of the current study was to develop and test a multiple-locus variable-number tandem-repeat analysis (MLVA) method that could be used in basic veterinary diagnostic microbiology laboratories equipped with PCR technology or in more advanced laboratories with access to capillary electrophoresis. Based on eight loci, and when performed on isolates from different farms in different countries, as well as type and reference strains, the MLVA technique developed was highly discriminatory (Hunter and Gaston discriminatory index, 0.938 [95% confidence interval, 0.9175 to 0.9584]) while retaining a high phylogenetic value. Using the technique, the species was shown to be diverse (44 MLVA types from 172 isolates and strains), although isolates were stable in herds over time. The population structure appeared to be clonal. The finding of B. hyodysenteriae MLVA type 3 in piggeries in three European countries, as well as other, related, strains in different countries, suggests that spreading of the pathogen via carrier pigs is likely. MLVA overcame drawbacks associated with previous typing techniques for B. hyodysenteriae and was a powerful method for epidemiologic and population structure studies on this important pathogenic spirochete.Brachyspira hyodysenteriae is a Gram-negative, oxygen-tolerant, anaerobic spirochete that colonizes the porcine large intestine to cause swine dysentery. This condition is characterized by a severe mucohemorrhagic diarrhea that primarily affects animals during the growing-finishing period and has been reported from all major pig-rearing countries. The enzootic nature of swine dysentery increases the economic impact of the disease, which arises from decreased rates of growth, poor feed conversion, deaths, costs of medication and treatments, preventive measures, and restrictions on movements of stock (16, 17).Carrier pigs play a main role in the epidemiology of swine dysentery and are considered the major source of transmission between herds (16). Moreover, B. hyodysenteriae survives in the environment for long periods, especially in liquid feces contained in pits and lagoons, where it may remain infective for up to 60 days (16). This spirochete also can naturally colonize mice, rheas, chickens, and mallards (9, 30), and together with mechanical vectors or fomites, this increases the ways in which B. hyodysenteriae may be spread within and between herds.Different typing tools have been developed to discriminate between B. hyodysenteriae field isolates and provide a better understanding of the molecular epidemiology of the pathogen. The methods used have included serotyping (3), DNA restriction endonuclease analysis (REA) (6), random amplification of polymorphic DNA (RAPD) (8), restriction fragment length polymorphism of DNA (21), pulsed-field gel electrophoresis (PFGE) (2), multilocus enzyme electrophoresis (MLEE) (25), and multilocus sequence typing (MLST) (24). These techniques provide different levels of discrimination between isolates, and those that are highly discriminating present associated drawbacks such as difficulties in comparing results between laboratories (for example, RAPD and PFGE). On the other hand, MLEE is extremely time-consuming while MLST has high associated costs that are not compatible with routine use in veterinary clinical diagnostic laboratories. Hence, a highly discriminatory method that is time- and cost-effective and yields portable results which allow interlaboratory comparison is still lacking for the typing of B. hyodysenteriae isolates.In the last few years, multiple-locus variable-number tandem-repeat analysis (MLVA) has been developed as an important epidemiologic tool for strain typing of pathogenic microorganism (26). MLVA is based on PCR amplification of multiple loci of minisatellites called variable numbers of tandem repeats (VNTRs). This sort of minisatellite consists of unique direct head-to-tail DNA repeats which can be found in all bacterial genomes and can be used to define specific isolates of bacterial species (35). In addition, VNTRs have been used to infer the bacterial population structure and phylogeny of diverse bacteria species (22, 29, 33). MLVA has the potential to be a highly discriminatory typing technique, being fast, cost-effective, and easy to implement in laboratories with PCR technology. Moreover, multiplexing the PCR in combination with capillary electrophoresis of fluorescently labeled primers may allow a higher sample throughput.In this study, we aimed to develop a simple and reproducible MLVA typing method for use in veterinary clinical microbiology laboratories equipped with either basic PCR technology or more sophisticated capillary electrophoresis equipment. We then applied the method to analyze an international collection of isolates to provide new information about the molecular epidemiology and population structure of this important pathogenic spirochete.     

Comparison of an Automated Repetitive-Sequence-Based PCR Microbial Typing System with Pulsed-Field Gel Electrophoresis for Molecular Typing of Vancomycin-Resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VRE) has become an important health care-associated pathogen because of its rapid spread, limited therapeutic options, and possible transfer of vancomycin resistance to more-virulent pathogens. In this study, we compared the ability to detect clonal relationships among VRE isolates by an automated repetitive-sequence-based PCR (Rep-PCR) system (DiversiLab system) to pulsed-field gel electrophoresis (PFGE), the reference method for molecular typing of VRE. Two sets of VRE isolates evaluated in this study were collected by active microbial surveillance at a large teaching hospital in Taiwan during 2008. The first set included 90 isolates randomly selected from the surveillance cohort. The first set consisted of 34 pulsotypes and 10 Rep-PCR types. There was good correlation between the two methods (P < 0.001). The second set included 68 VRE isolates collected from eight clusters of colonization. A dominant clone was detected in five out of eight clusters by both methods. Two clusters were characterized by Rep-PCR as being caused by a dominant clone, whereas PFGE showed polyclonal origins. One cluster was shown to be polyclonal by both methods. A single Rep-PCR clone type was detected among 12 of 14 vancomycin-intermediate enterococci, whereas PFGE detected six pulsotypes. In conclusion, the Rep-PCR method correlated well with PFGE typing but was less discriminative than PFGE in defining clonal relationships. The ease of use and more rapid turnaround time of Rep-PCR compared to PFGE offers a rapid screening method to detect outbreaks of VRE and more rapidly implement control measures. PFGE remains the preferred method to confirm clonal spread.Vancomycin-resistant Enterococcus faecium (VRE) was first isolated in England in 1986 (26). It has become an important health care-associated pathogen because of its rapid spread, significant attributable mortality, limited options for therapy, and the possible transfer of vancomycin resistance to more-virulent pathogens (3, 4, 14). VRE organisms are now commonly isolated in hospitals around the world. Molecular typing methods are needed to distinguish prevalent strains from epidemic strains. Pulsed-field gel electrophoresis (PFGE) is the accepted reference method for molecular typing of VRE chromosomes because of its high discriminative power and established interpretative guidelines (25, 29). PFGE is labor-intensive and skill dependent, and the turnaround time is 2 to 3 days (7, 27). VRE isolates collected during outbreak investigations at our hospital during the past 2 years consisted of a wide variety of PFGE types. Polyclonal outbreaks have also been reported by other investigators (8, 12). It is not clear whether this represents multiple introductions of different strains or microevolutionary changes in native VRE or whether PFGE is overly discriminatory.Repetitive-sequence-based PCR (Rep-PCR) methods are rapid typing procedures that amplify the regions between the noncoding repetitive sequences in bacterial genomes (28). Recently, Healy et al. described an automated Rep-PCR technology and its performance characteristics (10). Rep-PCR has been used in VRE outbreak investigations (20, 23), but we are unaware of any comparative field tests of Rep-PCR and PFGE. This study was designed to compare the ability of the two methods to distinguish clonal relationships among a panel of epidemiologically well-defined isolates of VRE. We also evaluated the association of specific drug resistance patterns with selected genotypes.