Characterization of Virulence Plasmid Diversity among Clostridium perfringens Type B Isolates

The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to ∼90-kb plasmids in most type B isolates, although a few isolates carried a ∼65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located ∼3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.Isolates of the Gram-positive, spore-forming anaerobe Clostridium perfringens are classified (31) into five different types (A to E), depending upon their production of four (alpha, beta, epsilon, and iota) lethal typing toxins. All C. perfringens types produce alpha-toxin; in addition, type B isolates produce both beta- and epsilon-toxins, type C isolates produce beta-toxin, type D isolates produce epsilon-toxin and type E isolates produce iota-toxin. Except for the chromosomal alpha-toxin gene (plc), all C. perfringens typing toxins are encoded by genes resident on large plasmids (11, 22, 23, 32, 33). Large plasmids can also encode other C. perfringens toxins, such as the enterotoxin (CPE) or beta2-toxin (8, 9, 14, 35), as well as other potential virulence factors such as urease (12, 23).The large virulence plasmids of C. perfringens are only now being characterized (23, 28, 29, 33). The first analyzed, and still most studied, C. perfringens toxin plasmids are the CPE-encoding plasmids of type A isolates (14, 28). In type A isolates, most plasmids carrying the enterotoxin gene (cpe) belong to one of two families: (i) a 75.3-kb plasmid with a cpe locus containing an IS1151 element and the cpb2 gene encoding beta2-toxin or (ii) a 70.5-kb plasmid that lacks the cpb2 gene and carries a cpe locus with an IS1470-like sequence instead of an IS1151 element. Sequence comparisons (28) revealed that these two cpe plasmid families of type A isolates share a conserved region of ∼35 kb that includes the transfer of clostridial plasmid (tcp) locus, which is related to the conjugative transposon Tn916. Confirming that cpe plasmids can be conjugative, mixed mating studies have directly demonstrated transfer of the cpe plasmid from type A isolate F4969 to other C. perfringens isolates (5). A similar tcp locus is also shared by the tetracycline resistance plasmid pCW3 and several other toxin plasmids (2, 23, 28, 29, 33), as discussed below. Mutagenesis analyses demonstrated the importance of several genes in the tcp locus for conjugative transfer of pCW3 (2) and, by extension, presumably the tcp-carrying, conjugative toxin plasmids, such as the cpe plasmid of isolate F4969 (5) and some etx plasmids of type D isolates (19).Although the sequence of an iota-toxin-encoding plasmid has not yet been published, pulsed-field gel electrophoresis (PFGE) and PCR analyses determined that these plasmids are typically larger than the cpe plasmids of type A isolates (23). Specifically, iota-toxin plasmids are often ≥100 kb in size, reaching up to a size of ∼135 kb. These plasmids of type E isolates often encode, in addition to the iota-toxin, other potential virulence factors such as lambda-toxin and urease. These plasmids also carry a tcp locus, suggesting that they may be capable of conjugative transfer. Interestingly, many iota-toxin plasmids appear to be related, sometimes extensively, to the cpe plasmids of type A isolates. Consequently, it has been suggested (3, 23) that many iota-toxin plasmids arose from insertion of an iota-toxin gene-carrying mobile genetic element near the cpe gene on a tcp-carrying type A plasmid. This insertional event apparently inactivated the cpe gene, so most or all type E isolates now carry silent cpe genes (3, 23).The epsilon-toxin-encoding plasmids of type D isolates show considerable size variations (33), ranging from ∼48 kb to ∼110 kb. These size variations in type D etx plasmids reflect, in part, differences among their toxin gene carriage. The small 48-kb etx plasmids present in some type D isolates typically lack either the cpe gene or the cpb2 gene (encoding beta2-toxin), while the larger (>75-kb) etx plasmids found in other type D isolates can also carry the cpe gene, the cpb2 gene, or both the cpe and cpb2 genes. Consequently, some type D isolates carry a toxin plasmid encoding only etx, other type D isolates carry a toxin plasmid with up to three different functional toxin genes (etx, cpb2, and cpe), and the remaining type D isolates carry their etx, cpe, and cpb2 genes on up to three distinct plasmids.C. perfringens type B isolates uniquely produce both beta- and epsilon-toxins, the two most lethal C. perfringens toxins (13). These bacteria are important pathogens of sheep but also cause disease in goats, calves, and foals (26). For unknown reasons, diseases caused by C. perfringens type B isolates apparently are restricted to certain geographic regions (24, 25, 26). C. perfringens type B enterotoxemias initiate when these bacteria proliferate in the gut, accompanied by toxin production. Those toxins initially affect the intestines but later are absorbed and act systemically. Studies from our group (13) showed that beta- and epsilon-toxins each contribute to lethality in a mouse model involving intravenous injection of type B culture supernatants.There has been characterization of only one type B virulence plasmid to date. Our recent study (29) showed that most, if not all, type B isolates carry a common etx plasmid of ∼65 kb that also possesses a tcp locus and a cpb2 gene, although not the cpb gene encoding beta-toxin. Interestingly, the type B etx plasmid is highly (80%) related to the ∼75-kb cpe- and cpb2-carrying plasmid found in some type A isolates (28). The ∼65-kb etx plasmid present in most, if not all, type B isolates is also carried by a minority of type D isolates (29).The absence of the cpb gene from their etx plasmids suggested that most type B isolates might carry additional virulence plasmids. Therefore, the current study was performed to better address virulence plasmid carriage and diversity among type B disease isolates.     

Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.Avian pathogenic Escherichia coli (APEC) strains cause respiratory disease and septicemia in poultry and are economically important worldwide, causing significant mortality (13). The carriage of large plasmids is considered characteristic of APEC isolates (8), and pathogenicity is thought to be determined by virulence-associated factors encoded by them (15). These factors include serum resistance, encoded by the iss gene (14), temperature-sensitive hemagglutination, encoded by tsh (10), adhesins, the production of colicin V (ColV) and the possession of iron-scavenging mechanisms, such as aerobactin production (encoded by the iucABCD operon), and the more recently identified putative iron transport system encoded by the etsABC operon (18).Another iron acquisition system found in APEC utilizes salmochelin, a catecholate siderophore. The chromosomal iroA gene cluster that encodes this system was first found in Salmonella enterica (2) and is absent from the corresponding region of the E. coli chromosome (32), although it has been found on a transmissible plasmid from a uropathogenic E. coli isolate (34). The iroA gene cluster has been found on multiple APEC virulence plasmids (9, 17, 18, 37), and deletion studies have shown that the iroA gene cluster is required for full virulence (9).A further iron transport system, designated the sitABCD system, was first identified on a pathogenicity island in Salmonella enterica serovar Typhimurium (39), and it has been shown that sitABCD is required for full virulence of Salmonella serovar Typhimurium (16). Genomic subtraction identified the plasmid-located sitA gene from the sitABCD operon as unique to an APEC strain (32), and the sitA gene was found to be more prevalent in APEC than in commensal E. coli (18, 29, 32).The sitABCD operon occurs on APEC virulence plasmids (17, 18, 30, 37), but a sitABCD deletion mutant was still pathogenic for birds, suggesting that other iron transport systems are able to compensate for the loss of sitABCD (30).The carriage of ColV plasmids has previously been thought to be essential for virulence (3, 33, 38). However, other studies have suggested it is not the presence of the ColV gene itself but other genes that these plasmids carry that are responsible for virulence (28, 35). The well-characterized APEC virulence plasmids pAPEC-O2-ColV (18) and pAPEC-1 (9) encode ColV, while carriage of the Australian APEC virulence plasmid pVM01 does not confer production of ColV (12). Despite various ColV statuses, all three of these virulence plasmids are F-type plasmids, and hence this is potentially another way to characterize APEC virulence plasmids.SopA and SopB, which have similarity to the ParA and ParB proteins of the P1 plasmid, are thought to be essential for F-plasmid partitioning (22, 24). Detection of the genes of the sopABC locus could thus indicate the presence of a putative virulence plasmid.Strain E3 is an O-nontypeable:H28 APEC field isolate (11) that carries the 151-kb virulence plasmid pVM01 (12), which contains a virulence region with the virulence-associated genes iucA, tsh, iss, iroN, and sitA, as well as hlyF, ompT, and the etsABC operon (37). The arrangement of the virulence-associated genes around pVM01 (37) is similar to that in the plasmids pAPEC-O2-ColV from APEC strain O2 (18), pAPEC-O1-ColBM from APEC strain O1 (17), and pAPEC-1 from APEC strain χ7122 (23). Identifying a specific region that is conserved in highly virulent APEC strains will facilitate diagnosis of colibacillosis by differentiation of pathogenic strains from commensal E. coli and will also enable surveillance for pathogenic isolates in the environment of poultry.This study examined six E. coli strains, some of which were isolated from diseased birds and some of which were recovered from healthy birds (11, 36). The pathogenicity of these strains has been determined using aerosol exposure (11, 36), making this the largest known collection of APEC strains fulfilling Koch''s postulates. The series of strains includes the highly virulent strains E3, E30, and E956 and the less-virulent strains E133, E1043, and E1292. The presence of the virulence-associated genes iucA, tsh, and iss in these strains has previously been elucidated by PCR amplification (36). However, while previous studies have found many of these virulence factors to be encoded by APEC strains associated with disease (29) and have suggested that they are encoded on virulence plasmids (18), they have not conclusively determined whether they are encoded on virulence plasmids or are chromosomally encoded. Similarly, although previous studies suggest that these virulence-associated genes are consistently present in isolates from diseased birds (1, 6, 18, 21, 26, 29), no study has yet determined if these genes are consistently associated with each other.The aim of this study was to examine a series of strains of known pathogenicities for the possession of large plasmids and to determine if known virulence-associated genes from the putative virulence region were carried on them. The second objective was to investigate any association between the virulence-associated genes.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).     

Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias

Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.Clostridium perfringens, an anaerobic, spore-forming, gram-positive rod, is a pathogen of humans and domestic or wild animals (9). The virulence of C. perfringens is mostly due to toxin secretion, which varies from strain to strain (9). This variability allows classification of C. perfringens isolates into five types (A to E), depending upon their production of four typing toxins (9, 11, 12). All five types produce alpha toxin (CPA), type B and C isolates produce beta toxin (CPB), type B and D isolates produce epsilon toxin (ETX), and type E isolates produce iota toxin (20).C. perfringens type C isolates cause highly lethal diseases, mostly in newborn animals of many mammalian species (20). These diseases originate when type C isolates proliferate and produce toxins in the intestine. Although often involving intestinal damage, death in affected animals is thought to result primarily from a toxemia following absorption of toxins from the intestine into the circulation (17, 18). C. perfringens bacteremia is not usually observed in cases of type C infection (17). In humans, C. perfringens type C isolates cause enteritis necroticans (also known as Darmbrand or Pigbel) (13). Enteritis necroticans is a highly lethal and endemic disease throughout much of Southeast Asia, but particularly in Papua New Guinea, where this disease was the leading cause of mortality in children during the 1960s (3, 5). Less frequently, this disease also occurs in diabetic patients elsewhere (8).Type C isolates produce CPB, which is a 35-kDa protein that forms pores in the membranes of susceptible cells, leading to swelling and lysis (10, 15, 19). CPB is lethal for mice, with a calculated 50% lethal dose of 310 ng per kg when administered intravenously (i.v.) (14). In addition, CPB has been shown to produce acute intestinal necrosis when inoculated into ligated intestinal loops of rabbits, the effects of which were inhibited when the toxin was mixed with a CPB monoclonal antibody (MAb) before inoculation (21). We recently constructed a series of C. perfringens type C toxin null mutants, which demonstrated that CPB, but not perfringolysin O (PFO) or CPA, is necessary and sufficient for the type C isolate CN3685 to cause intestinal damage in a rabbit ileal loop model (16). However, it was notable that none of the rabbits challenged with this potent type C isolate died during the 6-h course of those ileal loop studies (16).Despite these recent advances, the systemic lethal effects of CPB or C. perfringens type C isolates remain poorly characterized. In part, this is due to the lack of a laboratory animal model that reproduces the lethality of natural C. perfringens type C enterotoxemia (16, 21). Mice have been used to study the lethal effects of i.v. administered C. perfringens type C vegetative culture supernatants or pure CPB (2). The mouse i.v. injection model is useful for studying the systemic lethal effects of CPB and indicates the sensitivity of this species to type C toxins. However, this model differs significantly from natural type C enterotoxemias in human and animals, where toxins are produced in the gastrointestinal tract, act locally, and are then absorbed into the circulation (20). We now present the development and application of infectious intragastric (i.g.) and intraduodenal (i.d.) challenge mouse models to investigate the lethal enterotoxemias induced by C. perfringens type C infections, including a virulence evaluation of C. perfringens type C toxin mutants.     

Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the bla_KPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.Since the beginning of the last decade, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) isolates have been increasingly detected in the United States and subsequently in several regions worldwide (3, 4, 13, 17, 21). KPC enzymes efficiently hydrolyze all β-lactam molecules (1, 22), conferring various levels of resistance to all β-lactam compounds, including carbapenems (13). However, KPC-producing K. pneumoniae may appear susceptible to carbapenems, mainly meropenem (2, 13), by reference CLSI agar dilution or broth microdilution methods as well as by automated systems (6, 15, 17). Characteristically, it has been reported that automated systems may identify as many as 87% of KPC-KP isolates to be susceptible to meropenem (13). The detection of the susceptibility level of KPC-KP isolates to carbapenems has been shown to be difficult due to the phenotypic heterogeneity that they commonly exhibit (3, 10, 13). For instance, in agar diffusion methods such as disk diffusion or Etest, the heterogeneous growth to carbapenems of KPC-KP results in the appearance of scattered colonies within the inhibition zones (9, 13).These issues raise the need for cautious evaluation of susceptibility testing in KPC-KP isolates that are recovered in clinical laboratories. In our clinical laboratories, several KPC-KP isolates that appear susceptible by automated susceptibility assays or reference dilution assays contain heterogeneous subpopulations (D. Sofianou and K. Themeli-Digalaki, personal communications). It has been also shown that among Greek KPC-KP isolates, meropenem tends to exhibit lower MICs than imipenem or ertapenem (17, 20). In that respect, the aim of the present study was to characterize the heterogeneous mode of growth of apparently meropenem-susceptible KPC-KP clinical isolates by population analyses and bactericidal assays.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

Shiga Toxin,Cytolethal Distending Toxin,and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliC_H14). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx_2dact, encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx₁. Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients'' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause diarrhea and a life-threatening hemolytic-uremic syndrome (HUS) worldwide (23, 44). STEC strains isolated from patients usually possess, in addition to one or more stx genes, the eae gene, encoding adhesin intimin (7, 11, 16, 25, 26, 41, 49). However, a subset of STEC strains associated with human disease lack eae, and among these, strains of serogroup O91 are the most common (2, 7, 35, 37, 47, 48). In Germany during the last 5 years, serogroup O91 accounted for 6.4% to 11.0% of all STEC strains reported from human infections and was therefore the fourth-most-common STEC serogroup (after O157, O26, and O103) isolated (47, 48; http://www.rki.de). However, in contrast to eae-positive STEC strains of the three leading serogroups, which cause disease mostly in young children (47), STEC O91 is the most common serogroup isolated from adult patients (48).Despite their association with human diseases worldwide (7, 9, 11, 13, 14, 30, 35, 37, 38, 40, 47, 48), the spectrum of serotypes of STEC O91 isolates from patients and the pathogenic traits of such strains are poorly understood. Moreover, characteristics of STEC O91 strains which could assist with their isolation from human stools and further subtyping in clinical microbiological laboratories have not been systematically investigated or reported. To gain insight into the serotype composition and putative virulence factors of STEC O91 strains causing human disease and to identify characteristics which can facilitate laboratory diagnosis of these organisms, we determined the motility and flagellar phenotypes, fliC types, stx genotypes, non-stx putative virulence loci, and diagnostically useful phenotypes of 100 clinical STEC O91 isolates. Moreover, we investigated possible associations between bacterial characteristics and clinical infection phenotypes.     

Rapid Cytopathic Effects of Clostridium perfringens Beta-Toxin on Porcine Endothelial Cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C β-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.The anaerobic, spore-forming bacterium Clostridium perfringens is an important Gram-positive pathogen of humans and animals (18, 42). It causes diverse gastrointestinal diseases, such as food poisoning, enterotoxemia, and enteritis, as well as wound infections and septicemias (37). The virulence of different C. perfringens strains is related to the production of a large array of exotoxins (34). C. perfringens type C isolates are defined by production of two major toxins, α-toxin (CPA) and β-toxin (CPB). In addition, type C isolates may secrete other toxins, such as β2-toxin (CPB2), perfringolysin (PFO), enterotoxin (CPE), and TpeL (2, 34, 36) C. perfringens type C strains cause severe, acute, necrotizing enteritis in livestock and humans (18, 42). Outbreaks of human type C enteritis were recorded after the Second World War in Germany (20), but this disease has been reported only sporadically in developed countries (25, 27, 39, 51). A similar disease has been diagnosed more frequently in parts of Southeast Asia (7, 17, 26), particularly in the highlands of Papua New Guinea (23), where it was a frequent cause of childhood mortality until vaccination programs were initiated (24). C. perfringens type C causes enteritis more frequently in animals, such as calves, sheep, goats, and particularly pigs (42, 43). Typically, neonatal piglets are affected from the first day of life until they are approximately 3 weeks old. The peracute to acute type of the disease affects piglets within the first few days postpartum (12, 14, 43). Macroscopic lesions at necropsy are pathognomonic, with deep, segmental mucosal necrosis and massive hemorrhage in the small intestine. In most cases the lesions are confined to the proximal jejunum; however, they can extend into the distal small intestine and the colon. This suggests that lesions are initiated in the upper small intestine and can spread rapidly throughout the intestine. In addition to these marked necro-hemorrhagic lesions, thrombosis of small vessels in the lamina propria and submucosa is a consistent finding (12, 14). A more protracted clinical course of type C enteritis is seen mainly in piglets that die when they are 1 to 3 weeks old (12, 14, 43). The pathological lesion is a segmental to diffuse fibrino-necrotizing enteritis. Histopathologically, such cases are characterized by demarcation of the deeply necrotic mucosa by marked infiltration of neutrophilic granulocytes. Similar acute and subacute forms of type C enteritis also occur in humans (6, 18, 21). In humans, however, subacute lesions are more often described as multifocal patchy necrosis of the small intestine. Again, mucosal and submucosal vascular thrombosis is a frequent finding, especially in acute lesions (20, 21).Besides the clear epidemiological evidence for the importance of CPB in type C enteritis (41, 42), recent experimental studies using a rabbit intestinal loop model and a mouse infection model clearly demonstrated that CPB is the essential virulence factor of type C strains (38, 47, 49). In rabbit ileal loops, application of purified CPB and infection with C. perfringens type C strains caused villous tip necrosis, which indicated that there was initial intestinal epithelial damage. Vascular thrombosis in mucosal and submucosal vessels was also observed in this model. In general, the vascular damage observed in naturally occurring and experimentally induced type C enteritis is considered a secondary effect due to massive epithelial and mucosal necrosis (22, 43). However, the potential direct effects of exotoxins on vascular endothelia during type C enteritis have never been investigated.CPB is a beta-barrel-pore-forming toxin (9) that has been shown to form oligomers in the membrane of human endothelial cells (44) and the human HL 60 cell line (31). So far, cytotoxic and cytopathic effects of CPB have been demonstrated only for HL 60 cells. HeLa, Vero, CHO, MDCK, Cos-7, P-815, and PC12 cells were not sensitive to this toxin (31, 40). These findings indicate that CPB toxicity is cell type specific and most likely occurs via binding to specific membrane receptors. Recently, we localized CPB at vascular endothelial cells in acute type C enteritis lesions in piglets and a human patient (28, 29). As a result of this, we hypothesized that direct targeting of endothelial cells and induction of local vascular damage could contribute to the rapid tissue necrosis observed in the acute form of type C enteritis. To validate our initial reports, we performed additional immunohistochemical studies with naturally diseased piglets and subsequently studied the direct cytopathic effects of CPB on cultured primary porcine endothelial cells. The objectives of this study were (i) to evaluate the susceptibility of porcine endothelial cells to CPB in vitro, (ii) to characterize early morphological changes induced by CPB in these cells, and (iii) to relate the findings obtained to pathological lesions observed in acute type C enteritis in piglets. Our results reveal for the first time that porcine aortic endothelial cell (PAEC) cultures are highly sensitive to CPB, which results in rapid disruption of the actin cytoskeleton and retraction of the cell borders progressing to marked cell shrinkage.     

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.)     

Examination of Type IV Pilus Expression and Pilus-Associated Phenotypes in Kingella kingae Clinical Isolates

Kingella kingae is a gram-negative bacterium that is being recognized increasingly as a cause of septic arthritis and osteomyelitis in young children. Previous work established that K. kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells. PilA1 is the major pilus subunit in K. kingae type IV pili and is essential for pilus assembly. To develop a better understanding of the role of K. kingae type IV pili during colonization and invasive disease, we examined a collection of clinical isolates for pilus expression and in vitro adherence. In addition, in a subset of isolates we performed nucleotide sequencing to assess the level of conservation of PilA1. The majority of respiratory and nonendocarditis blood isolates were piliated, while the majority of joint fluid, bone, and endocarditis blood isolates were nonpiliated. The piliated isolates formed either spreading/corroding or nonspreading/noncorroding colonies and were uniformly adherent, while the nonpiliated isolates formed domed colonies and were nonadherent. PilA1 sequence varied significantly from strain to strain, resulting in substantial variability in antibody reactivity. These results suggest that type IV pili may confer a selective advantage on K. kingae early in infection and a selective disadvantage on K. kingae at later stages in the pathogenic process. We speculate that PilA1 is immunogenic during natural infection and undergoes antigenic variation to escape the immune response.Kingella kingae is a gram-negative bacterium that is a member of the Neisseriaceae family and is being recognized increasingly as a cause of pediatric diseases, including septic arthritis, osteomyelitis, and endocarditis. K. kingae was originally identified by Henriksen and Bovre in 1968 (10) but was dismissed early on as an important pathogen due to its infrequent recovery from infected sites. Recent improvements in cultivation techniques and the application of PCR-based assays have led to increased detection of K. kingae in association with invasive disease (3, 6, 17, 25, 27, 28, 31). A recent study identified K. kingae as a major cause of pediatric joint and bone infections and the leading etiology of these infections in children under 36 months of age (3).Invasive disease due to K. kingae is believed to begin with colonization of the upper respiratory tract (32). A sizeable percentage of children are colonized with K. kingae at least once per year during the first 2 years of life and appear to acquire the organism by person-to-person transmission (1, 14, 22, 27, 29-31). Following colonization, the organism must breach the respiratory epithelium, enter the bloodstream, and then disseminate to deeper tissues. An essential step in both colonization of the respiratory tract and seeding of remote sites is adherence to host tissues. Recent work demonstrated that K. kingae expresses type IV pili that are necessary for in vitro adherence to both respiratory epithelial and synovial cells (11). The major pilin subunit in K. kingae type IV pili is called PilA1 and is essential for pilus assembly (11, 12).Type IV pili have been shown to be necessary for adherence and colonization in a variety of organisms, including the pathogenic Neisseria species (2, 4, 15, 16, 19, 20, 23, 24, 26). In this work, we examined a collection of clinical isolates of K. kingae for pilus expression, adherence, and antigenic diversity of PilA1. Our results revealed that K. kingae has three naturally occurring colony types that correlate with density of piliation, including high-density piliation, low-density piliation, and nonpiliation. Further analysis demonstrated that respiratory isolates and nonendocarditis blood isolates were generally piliated and that joint fluid, bone, and endocarditis blood isolates were usually nonpiliated. Only piliated isolates were capable of adherence to cultured respiratory epithelial and synovial cells in vitro. The PilA1 subunit in piliated isolates exhibited significant strain-to-strain variation in sequence and antibody reactivity.     

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Clinically Important Yeast Species

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid identification of yeast species. Using Bruker Daltonics MALDI BioTyper software, we created a spectral database library with m/z ratios of 2,000 to 20,000 Da for 109 type and reference strains of yeast (44 species in 8 genera). The database was tested for accuracy by use of 194 clinical isolates (23 species in 6 genera). A total of 192 (99.0%) of the clinical isolates were identified accurately by MALDI-TOF MS. The MALDI-TOF MS-based method was found to be reproducible and accurate, with low consumable costs and minimal preparation time.Invasive fungal infections due to opportunistic pathogens are a significant cause of morbidity and mortality (2, 5, 8). The current rise in fungal infections correlates with the widespread use of broad-spectrum antibacterial agents, prolonged hospitalization of critically ill patients, and the increased number of immunocompromised patients. Candida species comprise the fourth most common cause of nosocomial bloodstream infections, and Cryptococcus neoformans is the most common cause of fungus-related mortality in HIV-infected patients (15, 19). While Candida albicans is still involved in more than half of all Candida-related bloodstream infections, an increase in recovery of non-C. albicans Candida spp., Rhodotorula spp., Trichosporon spp., and Malassezia spp. has occurred (2, 29). Treatment with amphotericin B may be useful for these organisms and inefficient for those belonging to other genera (5, 8). While many Candida species remain susceptible to fluconazole, it is important to differentiate the more resistant organisms, namely, Candida glabrata, Candida krusei, Rhodotorula spp., and some members of the genus Trichosporon. Additionally, Rhodotorula spp. have an innate resistance to voriconazole, and Trichosporon, Cryptococcus, and Rhodotorula are intrinsically resistant to the echinocandins (1, 15). These organisms present new challenges not only to treatment but also to standard identification methods used in the clinical laboratory (4, 8, 28).Commercially available biochemical test systems identify most of the commonly isolated species of yeast accurately but may result in no identification or misidentification of more-unusual isolates (4, 21, 28). Additionally, samples for these tests must be incubated for 1 to 3 days before results are obtained. To overcome the inaccuracies of biochemical identification methods, nucleic acid-based tests have been developed. These tests amplify and then sequence a target gene, such as the rRNA genes or the internal transcribed spacer (ITS) region (9, 10, 14, 17). While these assays are highly accurate, they require considerable processing time and costly reagents.As an alternative to biochemical and genome-based identification schemes, proteomic profiling by mass spectral analysis was recently evaluated for use in species differentiation of a variety of microorganisms. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is emerging as a rapid and accurate tool for identifying pathogens, including Gram-positive and Gram-negative bacteria, mycobacteria, molds, and yeast species (3, 6, 11-13, 16, 18, 22, 23, 27). The technique can be performed rapidly, with minimal consumable expenses, and produces reproducible, species-specific spectral patterns that are not dependent upon the age of culture, growth conditions, or medium selection (7, 13, 20, 26).In this work, we present the development of a yeast database library consisting of 109 type and reference strains (44 species in 8 genera), and we tested the robustness and accuracy of this library by using 194 well-characterized clinical isolates (23 species in 6 genera).     

Avian-Pathogenic Escherichia coli Strains Are Similar to Neonatal Meningitis E. coli Strains and Are Able To Cause Meningitis in the Rat Model of Human Disease

Escherichia coli strains causing avian colibacillosis and human neonatal meningitis, urinary tract infections, and septicemia are collectively known as extraintestinal pathogenic E. coli (ExPEC). Characterization of ExPEC strains using various typing techniques has shown that they harbor many similarities, despite their isolation from different host species, leading to the hypothesis that ExPEC may have zoonotic potential. The present study examined a subset of ExPEC strains: neonatal meningitis E. coli (NMEC) strains and avian-pathogenic E. coli (APEC) strains belonging to the O18 serogroup. The study found that they were not easily differentiated on the basis of multilocus sequence typing, phylogenetic typing, or carriage of large virulence plasmids. Among the APEC strains examined, one strain was found to be an outlier, based on the results of these typing methods, and demonstrated reduced virulence in murine and avian pathogenicity models. Some of the APEC strains tested in a rat model of human neonatal meningitis were able to cause meningitis, demonstrating APEC''s ability to cause disease in mammals, lending support to the hypothesis that APEC strains have zoonotic potential. In addition, some NMEC strains were able to cause avian colisepticemia, providing further support for this hypothesis. However, not all of the NMEC and APEC strains tested were able to cause disease in avian and murine hosts, despite the apparent similarities in their known virulence attributes. Thus, it appears that a subset of NMEC and APEC strains harbors zoonotic potential, while other strains do not, suggesting that unknown mechanisms underlie host specificity in some ExPEC strains.Escherichia coli strains causing extraintestinal disease are known as extraintestinal pathogenic E. coli (ExPEC) and include the uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and avian-pathogenic E. coli (APEC) subpathotypes. Recent studies have shown that members of various ExPEC subpathotypes harbor similar virulence-associated genes, despite their isolation from varied hosts and tissues (3, 8, 10, 20, 25, 27, 30, 32), and genomic sequencing of APEC O1 revealed that only 4.5% of the genome was not found in the other ExPEC strains sequenced (17). More recently, a cluster of isolates from human and avian hosts thought to represent potential zoonotic pathogens has been identified (20).Common among the isolates of this mixed cluster are genes associated with the conserved region of large virulence plasmids, which are a defining trait of the APEC subpathotype (15, 19, 24, 36, 37) and which are essential for APEC virulence (5, 23). Interestingly, a closely related plasmid that was associated with high-level bacteremia in a neonatal rat meningitis model has also been described in an NMEC isolate (30).Other virulence traits are also shared among ExPEC subpathotypes. Indeed, few traits, if any, appear to be exclusive to a particular ExPEC subpathotype, and in fact, some traits that were thought to be exclusive have been shown to contribute to the pathogenesis of more than one condition (8).Such similarities in the virulence traits found among APEC and other ExPEC subpathotypes have led to speculation that APEC has zoonotic potential (20, 25, 27) and may be a food-borne source of ExPEC causing disease in humans (10, 14, 18, 22). Indeed, ExPEC strains have been identified in retail foods and poultry products (7, 11, 12, 18), and at least one study has found avian isolates to be indistinguishable from human isolates (10). However, other studies showed that human ExPEC strains were clearly distinct from avian strains (6) and that the consumption of poultry or contact with poultry did not correlate with the colonization of antimicrobial-resistant E. coli (34).Here, we seek to further test the hypothesis that APEC strains have zoonotic potential. Of particular interest are O18 strains, which are common among human NMEC strains but which are also found among APEC strains (20, 26). In fact, it has been suggested that APEC O18:K1:H7 strains are potential human pathogens (27). Though it has been shown that human ExPEC strains can cause avian colibacillosis similar to that caused by APEC, suggesting that these ExPEC strains are not host specific (26), it has also been reported that E. coli strains from avian septicemia are more virulent to chicks than NMEC strains (33). However, the ability of APEC to cause disease in mammals has not yet been established.The aim of the present study was to explore the zoonotic potential of NMEC and APEC O18 strains by comparing their plasmid contents, genotypes, phylogenetic group assignments, pulsed-field gel electrophoresis (PFGE) patterns, and sequence types (ST), determined by multilocus sequence typing (MLST), and their abilities to cause disease in the rat model of human neonatal meningitis and chicken models of avian colisepticemia.     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C. difficile in Stool Specimens

Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.Clostridium difficile is one of the leading causes of infectious antibiotic-associated diarrhea and pseudomembranous colitis worldwide (2, 16). This is illustrated by the increased incidence and severity of C. difficile infection, suggesting the emergence of a new hypervirulent strain (5, 13-15, 17, 25, 32).While TcdB, a cytotoxin, is the known established virulence factor of C. difficile, toxin A (TcdA), a cytotoxic enterotoxin, works synergistically with TcdB, causing damage to the intestinal mucosa in cases of C. difficile infection (17). The genes that encode these toxins are located on the pathogenicity locus of C. difficile (4, 10, 24). Additionally, several deletions in the tcdC gene, a putative negative regulator of the expression of the toxin A (tcdA) and the toxin B (tcdB) genes, have been identified, and these deletions result in higher levels of cytotoxin expression (11). Furthermore, research has shown that some C. difficile strains produce another toxin, known as the binary toxin (19, 22, 28). The genes that encode this toxin, cdtA and cdtB, together produce an actin-specific ADP-ribosyltransferase that induces damage to the actin skeleton, leading to cytopathic effects in cell lines (1). It has been suggested that the binary toxin genes and deletions in the tcdC gene are potential virulence factors in the recent emerging hypervirulent strain (22, 29).The “gold standard” for the detection of C. difficile toxin production is a cytotoxin assay with stool specimens or isolates from anaerobic culture. The cytotoxin assay is difficult to perform and time-consuming, and it is often less sensitive than molecular assays (20, 23, 26). Enzyme immunoassays (EIAs) are used most often, and recent reports suggest that manufacturers have improved the performance of EIA kits since their introduction; however, the disadvantages of EIAs include the lower levels of sensitivity and specificity compared to those of the gold standard methods. More importantly, culture is not specific for the identification of toxigenic strains. The laboratory at the Wadsworth Center has developed a multiplex real-time PCR assay and a tcdC gene pyrosequencing assay that rapidly identify potential virulence factors of C. difficile strains and that can be used to directly test patient stool specimens for C. difficile.(Part of this report was presented at the 107th American Society for Microbiology General Meeting in 2007 [Toronto, Canada].)     

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb₃) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb₃ and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.Shiga toxin-producing Escherichia coli (STEC) is the principal etiologic agent of diarrhea-associated hemolytic-uremic syndrome (HUS) (42, 60, 66). Renal disease is thought to be due to the combined action of Shiga toxins (Shiga toxin 1 [Stx1] and Stx2), the primary virulence factors of STEC, and bacterial lipopolysaccharide (LPS) on the renal glomeruli and tubules (6, 42, 60, 66). Of these, Stx2 is most frequently associated with the development of HUS (45). Shiga toxin enters susceptible cell types after binding to the cell surface receptor glycosphingolipid globotriaosylceramide (Gb₃) and specifically depurinates the 28S rRNA, thereby inhibiting protein synthesis (42, 60, 66). The damage initiates a ribotoxic stress response consisting of mitogen-activated protein (MAP) kinase activation, and this response can be associated with cytokine release and cell death (21, 22, 25-27, 61, 69, 73). This cell death is often caspase-dependent apoptosis (18, 61). Gb₃ is expressed by human glomerular endothelial cells, podocytes, and multiple tubular epithelial cell types, and damage markers for these cells can be detected in urine samples from HUS patients (10-12, 15, 49, 73). Shiga toxin binds to these cells in renal sections from HUS patients, and along with the typical fibrin-rich glomerular microangiopathy, biopsy sections demonstrate apoptosis of both glomerular and tubular cell types (9, 29, 31).Concomitant development of the most prominent features of HUS: anemia, thrombocytopenia, and renal failure, requires both Shiga toxin and LPS in the murine model (30, 33). Nevertheless, our previous work demonstrated that renal failure is mediated exclusively by Stx2 (33). While it is established that Gb₃ is the unique Shiga toxin receptor (46), the current literature regarding the mechanism by which Shiga toxin causes renal dysfunction in mice is inconsistent. Even though Gb₃ has been localized to some murine renal tubules and tubular damage has been observed (19, 23, 46, 53, 65, 68, 72, 74), the specific types of tubules affected have been incompletely characterized. Although multiple groups have been unable to locate the Shiga toxin receptor Gb₃ in glomeruli in murine renal sections (19, 53), one group has reported that murine glomerular podocytes possess Gb₃ and respond to Stx2 in vitro (40), and another group has reported that renal tubular capillaries express the Gb₃ receptor (46). Furthermore, murine glomerular abnormalities, including platelet and fibrin deposition, occur in some murine HUS models (28, 30, 33, 46, 59, 63). We demonstrate here that murine glomerular endothelial cells and podocytes are unresponsive to Stx2 because they do not produce the glycosphingolipid receptor Gb₃ in vitro or in vivo. Further, murine renal tubules, including collecting ducts, express Gb₃ and undergo Stx2-induced apoptosis, resulting in dysfunctional urine production and dehydration.