In vitro and in vivo role of heat shock protein 90 in Amphotericin B resistance of Aspergillus terreus

Aspergillus terreus (A. terreus) is of serious concern because of a high propensity to dissemination and in vitro and in vivo resistance to Amphotericin B (AmB). The underlying molecular mechanism of AmB is not known yet and here we want to explore whether fungal heat shock protein 90 (HSP90) is involved in polyene resistance in A. terreus. AmB-susceptible (ATS) and AmB-resistant (ATR) A. terreus and AmB-susceptible Aspergillus fumigatus (AFS) were investigated in response to AmB with a special focus on HSP90. HSP90 inhibitors resulted in significant improvement of AmB activity against ATR as minimum inhibitory concentrations (MIC) decreased from 32 to 0.38 mg/L. Gene expression profiling showed a greater basal amount of HSP90 levels in ATR and ATS when compared with AFS. HSP90 blockers in combination with AmB were evaluated in a murine model of disseminated aspergillosis. HSP90 inhibitors were not beneficial for mice infected with ATR, and neither mono- nor combination treatment with AmB yielded clinical improvement. HSP90 inhibition with 17-allylamino-17-demethoxygeldanamycin (17-AAG) was harmful. HSP90 seems to play a vital role in antifungal stress response in all aspergilli tested, whereas HSP90 does not substantiate the origin of AmB resistance in ATR.     

Global VGIIa isolates are of comparable virulence to the major fatal Cryptococcus gattii Vancouver Island outbreak genotype

The ongoing cryptococcosis outbreak on Vancouver Island, BC, Canada, is caused by two VGII sub-genotypes of the primary pathogen, Cryptococcus gattii: VGIIa isolates predominate, whereas VGIIb isolates are rare. Although higher virulence of the VGIIa genotype has been proposed, an unresolved key question is whether VGIIa isolates from other regions are also more virulent than VGIIb isolates. We report the relationship between genotype and virulence for a global collection of C. gattii VGIIa and VGIIb isolates (from Australia, Argentina, Brazil, Canada, Thailand and the USA). In vitro and in vivo virulence studies were conducted. At 37°C, growth [at 18 h: 0.2 optical density (OD) difference, p 0.026; at 36 h: 0.6 OD difference, p 0.036) and mean melanin production (OD = 0.25 vs. OD = 0.15, p 0.059] of VGIIa isolates was greater than that of VGIIb isolates. The inhibitory effect of high temperature on melanin production of VGIIa isolates was less than that of VGIIb isolates (OD = 0.36 vs. OD = 0.69; p 0.001). Capsule production at 37°C of VGIIa isolates was less than that of VGIIb isolates. All VGIIa isolates were fertile, whereas only 17% of VGIIb isolates were fertile (p <0.001). In vivo virulence studies using the BALB/c mice nasal inhalation model revealed that VGIIa isolates were more virulent than VGIIb isolates (p <0.001) independent of their clinical (p 0.003) or environmental origin (p <0.001). This study established a clear association between genotype and virulence of the primary fungal pathogen, C. gattii.     

Virulence plasmid of Aeromonas hydrophila induces macrophage apoptosis and helps in developing systemic infection in mice

Pathogenic Aeromonas hydrophila Strain AO1 bears a 21 kb plasmid encoding several virulence determinants. Infection studies revealed that this isolate induced cytotoxicity in BALB/c mice splenic macrophages involving reactive oxygen species generation. DNA gel, Hoechst 33342, annexin-V and TUNEL assay documented macrophage death induced by 21 kb plasmid bearing isolates to be apoptotic in nature. Apoptosis induced by the plasmid bearing isolates involved initiator caspase-8 and caspase-9 and executed by effector caspase-3. ELISA revealed the wild-type isolate as weak inducer of pro-inflammatory cytokine IL-1β. Oral infection with wild-type isolates caused systemic infection in BALB/c mice. With plasmid curing the isolate looses several virulence attributes including cytotoxic potential. The cured isolate induced significant amounts of IL-1β from infected macrophages, disseminated into Peyer's patches, spleen and liver but never attained the bacterial loads recorded with wild-type isolates and were rapidly cleared. Transformation of 21 kb plasmid helped the cured bacteria regain wild-type virulence attributes, apoptotic potential and ability to cause systemic infection in mice. Thus the 21 kb plasmid is a virulence factor in mice. It helps in suppressing the production of pro-inflammatory cytokine IL-1β and induced apoptosis of host macrophages enabling A. hydrophila to evade host immune responses and establish systemic infection in mice.     

The effect of amphotericin B treatment on Candida albicans-induced immunosuppression in mice

The data presented shows that C. albicans-infected mice had a reduced capacity to mount a delayed-type hypersensitivity response to sheep red blood cell antigens. During treatment of these mice with amphotericin AmB, the drug caused a further significant reduction of the immune response. However, when AmB treatment was stopped, the immunosuppression caused by C. albicans and also AmB was alleviated. Nevertheless, it seems, from these findings, that the host may experience a combined immunosuppression which may be a problem relevant to cancer patients, since fungal infections are common in such patients.     

High virulent clinical isolates of Mycobacterium abscessus from patients with the upper lobe fibrocavitary form of pulmonary disease

Pulmonary disease caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, can be classified into two distinct types of clinical disease; the upper lobe fibrocavitary (UC) form and nodular bronchiectatic (NB) form. However, the relationship between mycobacterial strain virulence and disease type in the pulmonary M. abscessus diseases has not been reported. To determine the differential virulence between strains causing two forms of disease, we obtained clinical isolates from patients with the UC and NB form of pulmonary disease caused by M. abscessus. In present study, we investigated the intracellular growth of clinical isolates in macrophages and their pathogenicity in C57BL/6 mice. For the isolates from the UC form, intracellular macrophage growth was faster and higher levels of cytokines were induced in macrophages than for those from NB form. Moreover, severe lung inflammation was only observed in mice intranasally infected with the isolate from the UC form with the increase of bacterial load. These findings suggest that M. abscessus isolates from the UC form of pulmonary disease are more virulent than those from NB form. This differential virulence of clinical strains may be one of the important factors involved in the determination of the disease form of pulmonary M. abscessus disease.     

Aerial Prevalence of Aspergillus calidoustus Isolates in and around a Tertiary Care Hospital in Kuwait and Assessment of Their Pathogenicity

Seven Aspergillus calidoustus isolates from 486 Aspergillus spp. isolates (1.4% overall prevalence) from outdoor/indoor air samples and one isolate from the bronchoalveolar lavage fluid of a patient with pneumonia were obtained. These 8 isolates exhibited reduced susceptibility to triazoles. Preliminary pathogenicity data from BALB/c mice suggest that A. calidoustus can persist in tissues for long periods without causing mortality. Further studies using graded doses of inoculum and immunosuppression models are warranted to gain an understanding of the factors associated with its pathogenicity and virulence.     

Haemopathological responses to chronic inflammation in the houbara bustard (Chlamydotis undulata macqueenii)

Blood samples were collected from 12 clinically normal (CN) and 12 clinically abnormal (CA) captive houbara bustards (Chlamydotis undulata macqueenü). Total white blood cell counts for each group were carried out, followed by differential white cell and thrombocyte counts. One hundred thrombocytes from each bird were also measured using a micrometer eye piece and oil immersion light microscopy. The CA birds had significant increases both in the total white blood cell count and in the counts for heterophils, monocytes and basophils. This was associated with a significant thrombocytosis. The thrombocytes from the CA birds were also found to be significantly larger (‘mega’ thrombcytes) than those from the CN birds. These results suggest that the increase in the total white blood cell count in chronic inflammation in the houbara bustard is mainly due to an increase in the number of heterophils, monocytes and basophils. These results also suggest that both the thrombocyte count and the thrombocyte morphometrics may be important components of routine diagnostic haematological investigations in houbara bustards.     

Trends in the Prevalence of Amphotericin B-Resistance (AmBR) among Clinical Isolates of Aspergillus Species

The challenges of the invasive infections caused by the resistant Aspergillus species include the limited access to antifungals for treatment and high mortality. This study aimed to provide a global perspective of the prevalence of amphotericin B resistance (AmBR), geographic distribution, and the trend of AmBR from 2010 to 2020. To analyze the prevalence of in vitro AmBR in clinical Aspergillus species, we reviewed the literature and identified a total of 72 articles. AmBR was observed in 1128 out of 3061 Aspergillus terreus (36.8%), 538 out of 3663 Aspergillus flavus (14.9%), 141 out of 2691 Aspergillus niger (5.2%), and 353 out of 17,494 Aspergillus fumigatus isolates (2.01%). An increasing trend in AmB-resistant isolates of A. fumigatus and a decreasing trend in AmB-resistant A. terreus and A. flavus isolates were observed between 2016 and 2020. AmB-resistant A. terreus and A. niger isolates, accounting for 40.4% and 20.9%, respectively, were the common AmB-resistant Aspergillus species in Asian studies. However, common AmB-resistant Aspergillus species reported by European and American studies were A. terreus and A. flavus isolates, accounting for 40.1% and 14.3% in 31 studies from Europe and 25.1% and 11.7% in 14 studies from America, respectively. The prevalence of AmB-resistant A. niger in Asian isolates was higher than in American and European. We found a low prevalence of A. terreus in American isolates (25.1%) compared to Asian (40.4%) and European (40.1%). Future studies should focus on analyzing the trend of AmBR on a regional basis and using the same methodologies.     

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients'' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.     

Differences in virulence of clinical isolates of Candida tropicalis and Candida albicans in mice.

Prior reports from this institution indicated that Candida tropicalis was more pathogenic than C. albicans in oncology patients. Pairs of clinical isolates of C. tropicalis and C. albicans recovered from similar patients at other institutions were examined to determine their relative virulence. After intravenous inoculation in normal mice, three pairs of isolates had no significant differences in the 50% lethal dose, and one C. tropicalis isolate was less virulent than its companion C. albicans isolate. In contrast, in mice treated with antibiotics and cytarabine, an antineoplastic drug which damages the gastrointestinal mucosa and produces granulocytopenia, oral inoculation of yeast cells produced striking differences in the 50% infective dose: each C. tropicalis isolate was more virulent than the companion C. albicans isolate from the same institution. The increased virulence of the C. tropicalis isolates compared with the C. albicans isolates when given orally to compromised mice parallels clinical observations in compromised patients.     

Stable Phenotypic Resistance of Candida Species to Amphotericin B Conferred by Preexposure to Subinhibitory Levels of Azoles

The fungicidal activity of amphotericin B (AmB) was quantitated for several Candida species. Candida albicans and C. tropicalis were consistently susceptible to AmB, with less than 1% survivors after 6 h of exposure to AmB. C. parapsilosis and variants of C. lusitaniae and C. guilliermondii were the most resistant, demonstrating 50 to 90% survivors in this time period and as high as 1% survival after a 24-h exposure time. All Candida species were killed (<1% survivors) after 24 h of exposure to AmB. In contrast, overnight exposure to either fluconazole or itraconazole resulted in pronounced increases in resistance to subsequent exposures to AmB. Most dramatically, C. albicans was able to grow in AmB cultures after azole preexposure. Several other Candida species did not grow in AmB but showed little or no reduction in viability after up to 24 h in AmB. Depending on the growth conditions, Candida cells preexposed to azoles may retain AmB resistance for days after the azoles have been removed. If this in vitro antagonism applies to the clinical setting, treatment of patients with certain antifungal combinations may not be beneficial. The ability of some Candida isolates to survive transient exposures to AmB was not reflected in the in vitro susceptibility changes as measured by standard MIC assays. This finding should be considered in studies attempting to correlate patient outcome with in vitro susceptibilities of clinical fungal isolates. Patients who fail to respond to AmB may be infected with isolates that are classified as susceptible by standard in vitro assays but that may be resistant to transient antifungal exposures which may be more relevant in the clinical setting.Consideration of the interactions between azoles and amphotericin B (AmB) has become clinically significant in recent years. Fluconazole and, to a lesser extent, itraconazole are widely used and largely effective but are not fungicidal. An additional limitation is that they are not effective against several Candida species, notably Candida krusei and C. glabrata (2, 4, 11, 17). AmB is a potent, fungicidal agent that is effective against most isolates of Candida but that has toxic side effects (1, 39). In addition, several Candida species, including C. lusitaniae, demonstrate intrinsic resistance to AmB (2, 6, 18, 19, 34, 35). Recent reports suggest that antifungal therapy may select for AmB-resistant variants of C. albicans and other susceptible species (5, 10, 14–16, 20, 21, 23, 35). However, mutants verified by in vitro testing to be resistant remain elusive (10). Inadvertent clinical selection for resistance to AmB may be more likely due to prolonged azole use than to AmB therapy. Some mutations in C. albicans that confer resistance to fluconazole act by altering the synthesis of ergosterol, the putative target of AmB action, and thereby confer cross-resistance (19).We previously demonstrated that preexposing C. albicans in vitro to fluconazole or itraconazole conferred resistance to otherwise fungicidal concentrations of AmB (37). Depending on the conditions, up to 100% of the preexposed cells tolerated AmB at 2 μg/ml for up to 24 h. However, simultaneous exposure of C. albicans to azoles and AmB had much less effect, with only a small increase in the Candida population surviving relative to controls exposed to AmB alone. Moreover, several investigators have found synergistic interactions by simultaneous exposure of C. albicans to azoles and AmB (13, 30). One group, on the other hand, described antagonisms with preexposures of Candida to the more lipophilic azoles, such as itraconazole, but not to fluconazole (31, 32).In this paper, we offer new observations describing the complex azole-AmB interactions. First, we compare the fungicidal effects of AmB on representative isolates of six species of Candida. We are able to show differences in AmB killing rates among some of these Candida isolates. Second, and most importantly, preexposure to azoles decreased the susceptibilities of all Candida species that were otherwise found to be susceptible to AmB by standardized in vitro susceptibility studies. C. albicans and, to a lesser extent, C. tropicalis demonstrated the greatest degree of antagonism. C. albicans was unique in that preexposure to azoles routinely allowed growth, not just survival, during subsequent incubations in AmB. Third, we show that fluconazole-mediated AmB tolerance is established by just a few hours of exposure to fluconazole. The protection endures for several days after azoles are removed, but only if the cells are maintained in a nongrowing state or if the exposure to AmB is continuous following azole incubation.     

Comparative Genomics Reveal Extensive Transposon-Mediated Genomic Plasticity and Diversity among Potential Effector Proteins within the Genus Coxiella

Genetically distinct isolates of Coxiella burnetii, the cause of human Q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogenic potential, we deciphered the whole-genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structures of other bacterial pathogens that have recently emerged from nonpathogens with expanded niches. The observation that the attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests that this isolate's lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.     

Strain and Virulence Diversity in the Mouse Pathogen Chlamydia muridarum

The mouse chlamydial pathogen Chlamydia muridarum has been used as a model organism for the study of human Chlamydia trachomatis urogenital and respiratory tract infections. To date, two commonly used C. muridarum isolates have been used interchangeably and are essentially taken to be identical. Herein, we present data that indicate that this is not the case. The C. muridarum Weiss isolate and C. muridarum Nigg isolate varied significantly in their virulences in vivo and possessed different growth characteristics in vitro. Distinct differences were observed in intravaginal 50% infectious doses and in challenge infections, with the Weiss isolate displaying greater virulence. Respiratory infection by the intranasal route also indicated a greater virulence of the Weiss isolate. In vitro, morphometric analysis revealed that the Weiss isolate produced consistently smaller inclusions in human cervical adenocarcinoma cells (HeLa 229) and smaller plaques in monolayers of mouse fibroblasts (L929) than did the Nigg isolate. In addition, the Weiss isolate possessed significantly higher replicative yields in vitro than did the Nigg isolate. In plaque-purified isolates derived from our stocks of these two strains, total genomic sequencing identified several unique nonsynonymous single nucleotide polymorphisms and insertion/deletion mutations when our Weiss (n = 4) and Nigg (n = 5) isolates were compared with the published Nigg sequence. In addition, the two isolates shared 11 mutations compared to the published Nigg sequence. These results prove that there is genotypic and virulence diversity among C. muridarum isolates. These findings can be exploited to determine factors related to chlamydial virulence and immunity.Until quite recently, there has been no means to consistently and predictably mutate chlamydiae in order to study virulence and immunity (2). Until such a method is available for routine use, one could potentially study closely related chlamydial strain variants in which one isolate is more virulent than the other and then employ a readily available animal model with which to assess the putative differences, as was done previously by Kari et al. (14). In this report, we will present evidence to demonstrate that the natural mouse chlamydial pathogen Chlamydia muridarum could prove to be such a model but has gone unrecognized for the 60 years that it has been used by chlamydial researchers.While studying human influenza virus and certain mouse respiratory pathogens, several investigators in the late 1930s and early 1940s identified a separate and distinct pathogen that caused pneumonitis in mice but was native to the mouse colonies of the day (10, 11, 20). Each investigator individually concluded that they had isolated a new “virus” from mice that produced disease similar to, but distinct from, human influenza virus and other viral respiratory tract infections. This “virus” was found to create inclusion bodies in infected host cells and was termed the agent of mouse pneumonitis (MoPn). It was also noted to be serologically related to the causative agent of human lymphogranuloma venereum and avian psittacosis (10, 21). Eventually, MoPn was accepted as a distinct biovar of the bacterium Chlamydia trachomatis (19). Subsequently, it was asserted through phylogenetic analyses of the 16S and 23S rRNA genes that although closely related to C. trachomatis, MoPn actually should be assigned to a separate species (8). As such, it was reclassified as Chlamydia muridarum. It is still referred to by most investigators by the original term, MoPn.In 1981, Barron et al. first reported a successful MoPn infection in the urogenital tract of mice and proposed this as a model for human urogenital chlamydial infection (1). Since that time, MoPn urogenital and respiratory tract infections in mice have become popular animal models for the study of immune responses and pathological host responses to chlamydiae (18, 26, 27).To date, two isolates of MoPn have been commonly employed in this model, MoPn Nigg and MoPn Weiss. It has been generally assumed that both MoPn isolates were essentially identical and derived from a single original strain. Each laboratory using these strains has maintained their own lineage from the original stocks, which have been passed from one laboratory to another, or they obtained them from the culture depository at the American Type Culture Collection (ATCC). Initially passed through embryonated hen''s homogenized yolk sacs, each line has been variously maintained in mouse fibroblasts (L929 or McCoy cells) or in human adenocarcinoma lines (HeLa 229 cells).Through some initial coincidental observations, serendipity, and subsequent systematic study, we have concluded that these two isolates, while closely related, possess distinct growth characteristics in vitro and vary significantly in virulence in vivo. We believe that the findings described herein not only will be important to those employing this model but ultimately will contribute significantly to an understanding of the basis of chlamydial pathogenesis and immunity, chlamydia population biology, as well as chlamydia-host cell interactions and biology.     

Evaluation of ompK36 allele groups on clinical characteristics and virulence features of Klebsiella pneumoniae from bacteremia

Background/purposeThis study investigated the implications of ompK36 allele groups on clinical and microbiological features of patients with Klebsiella pneumoniae bacteremia.MethodsA total of 80 K. pneumoniae bloodstream isolates were collected and then divided into four ompK36 allele groups. Clinical characteristics, bacterial antibiotic resistance and virulence determinants were analyzed, including resistance and virulence genes, hypermucoviscosity phenotype, K capsule serotypes, biofilm formation, serum killing, neutrophil phagocytosis, and mouse lethality studies.Results78 isolates were classified into four ompK36 variants, designated groups A (34), B (6), C (26), and D (12), respectively; 2 isolate was untypeable. OmpK36 group C isolates carried higher frequencies of K1/K2 capsule serotypes, hypermucoviscosity phenotype, rmpA gene, allS gene, iroB gene, aerobactin gene, or rmpA2 gene than non-C group isolates. OmpK36 group C isolates were significantly more virulent, as higher serum resistance, higher anti-phagocytosis and higher mouse lethality, than OmpK36 non-C group isolates, except for similar biofilm formation capability. The K20 isolates probably has low expression rates of rmpA and rmpA2 for hypermucoviscosity phenotype. The biofilm formation was significantly associated with ESBL production. OmpK36 group C isolates were more frequently detected in patients with community-acquired bloodstream infection. However, significant underlying diseases and prior use of carbapenem were highly prevalent in patients with OmpK36 non-C group isolates infection. ESBL production was apparently higher in non-C group but did not reach statistical significance.ConclusionOur results suggest that the OmpK36 group C K.pneumoniae is more associated with community-acquired infection with a lower frequency of underlying illness, but with significantly more virulence in bloodstream infection. This would give a remind that clinicians should be aware of such clinical impacts of the ompK36 allele group.     

Development of polyion complex micelles for encapsulating and delivering amphotericin B

A block copolymer poly(2-ethyl-2-oxazoline)-block-poly(aspartic acid) (PEOz-b-PAsp) was synthesized and investigated as the carrier of antifungal drug amphotericin B (AmB). Polyion complex (PIC) micelles with clear core–shell structures were identified by TEM, which revealed that the PAsp segment became hydrophobic after it interacted with AmB. PEOz-b-PAsp increased not only the solubility of AmB but also simultaneously the drug potency. The prolonged release of AmB from micelles effectively inhibited the growth of Candida albicans even after three days of administration. Moreover, the in vitro cytotoxicity of AmB-loaded micelles was less than that of Fungizone^®, which is a powerful antifungal antibiotic that is adopted to treat various fungal infections. The PEOz-b-PAsp PIC micelles with lower cytotoxicity and higher potency than Fungizone^® represent a potential means of encapsulating basic/amphoteric drugs.     

Use of Ribotyping and Hemolysin Activity To Identify Highly Virulent Streptococcus suis Type 2 Isolates

Nineteen Streptococcus suis type 2 isolates were evaluated for their virulence in pigs and mice. Of these, seven were determined to be highly virulent in pigs on the basis of clinical sign scores and gross pathology and histopathology results. Clinical sign scores correlated with gross pathology and histopathology scores at P equal to 0.004 and P equal to 0.009, respectively. The virulence of highly virulent isolates in pigs compared somewhat with virulence in mice, but the correlation was not significant. No correlation of virulence was noted among the moderately virulent and avirulent isolates in pigs and mice. Chromosomal DNAs from all S. suis isolates were evaluated by PstI, PvuII, EcoRI, and HaeIII restriction enzyme digestion followed by hybridization with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from 16S and 23S rRNAs from Escherichia coli. The hybridization patterns (ribotypes) varied depending upon the enzyme used, but a significant number of isolates determined to be highly virulent in pigs had unique hybridization patterns compared with those of the moderately virulent and avirulent isolates (P = 0.002). In addition, hemolysin activity showed a high correlation to virulence (P = 0.00008) and ribotype (P = 0.002).     

Comparison of Candida parapsilosis,Candida orthopsilosis,and Candida metapsilosis adhesive properties and pathogenicity

Retrospective studies indicate that Candida metapsilosis and Candida orthopsilosis each represents 1–10% of the infections/colonisations attributed to C. parapsilosis by conventional biochemical tests. Little is known on the virulence properties of these fungi and on their role in the establishment/progression of the infection. In this study, the adhesive properties of clinical isolates belonging to the ‘psilosis’ species were assessed in an in vitro model of co-incubation with human buccal epithelial cells (HBECs). Ectophosphatase activity was also measured for all isolates, since the activity of this enzyme has previously been linked to adhesion properties in C. parapsilosis. The results indicate that whilst C. parapsilosis and C. orthopsilosis strains showed similar adhesion abilities, C. metapsilosis isolates displayed a significantly lower ability to adhere to HBECs (P < 0.05). No evidence of a correlation between ectophosphatase activity and adhesion was observed, and this finding was also confirmed by phosphatase inhibition experiments. Experimental vaginal candidiasis induced in oestrogen-treated mice with representative isolates of the 3 species indicated that mice infected with C. metapsilosis displayed a reduced vaginal fungal burden, especially in the early stages of the infection. The overall findings confirm that C. orthopsilosis has a comparable behaviour to C. parapsilosis, whilst C. metapsilosis seems to possess a reduced virulence potential.     

Virulence versus fitness determinants in Escherichia coli isolated from asymptomatic bacteriuria in healthy nonpregnant women

Purpose: Escherichia coli isolated from asymptomatic bacteriuria (ABU) correlated genotypically and phenotypically with cystitis isolates may help in distinguishing urovirulence determinants from ‘fitness factors’, latter necessary only for survival of E. coli in urinary tract; for gaining insight into the pathogenesis of urinary tract infection. Materials and Methods: In this cross-sectional study, we compared genotypic (phylogroups and 15 putative virulence genes), and phenotypic profiles of ABU E. coli strains with our previously genotyped collection of cystitis isolates. Virulence score was calculated for each isolate as a number of virulence genes detected. Results: Significant differences were observed in the proportion of four phylogenetic groups (P = 0.009) amongst cystitis and ABU isolates. Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2); and hlyA (P = 0.001), cytotoxic necrotising factor 1 (P = 0.00), fyuA (P = 0.00), ibeA (P = 0.00), kpsMII (P = 0.01), and malX/pathogenicity-associated island (P = 0.01) were more frequently present in ABU strains. Conclusions: The expression of adhesins, haemolysin, aerobactin, and capsule synthesis gene were similar in two groups suggesting their role as fitness factors. ABU isolates were better biofilm producers, reflecting its importance in silent persistence. Serum resistance gene which was more expressed in cystitis isolates may represent virulence determinant. Genetic makeup of E. coli does not change much rather genes helping in survival and colonisation are expressed equally in ABU and cystitis isolates as opposed to phenotypic attenuation of those that helps in invasion or inflammation in ABU isolates.     

Nontoxigenic Vibrio cholerae Non-O1/O139 Isolate from a Case of Human Gastroenteritis in the U.S. Gulf Coast

An occurrence of Vibrio cholerae non-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. cholerae infection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae, suggesting that there might be additional virulence factors in non-O1/O139 V. cholerae yet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. cholerae isolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. cholerae in the interest of public health.