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Cytomegalovirus (CMV) is the most common virus associated with congenital infection worldwide and is a major cause of sensorineural hearing loss (SNHL) and developmental delay. Up to 90% of infants with congenital CMV (cCMV) infection are asymptomatic at birth, making the diagnosis challenging.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

Group A streptococcus (GAS) species cause bacterial pharyngitis in both adults and children. Early and accurate diagnosis of GAS is important for appropriate antibiotic therapy to prevent GAS sequalae. The Revogene Strep A molecular assay (Meridian Bioscience Canada Inc, Quebec City, QC, Canada) is an automated real-time PCR assay for GAS detection from throat swab specimens within approximately 70 min. This multicenter prospective study evaluated the performance of the Revogene Strep A molecular assay compared to that of bacterial culture., Group A streptococcus (GAS) species cause bacterial pharyngitis in both adults and children. Early and accurate diagnosis of GAS is important for appropriate antibiotic therapy to prevent GAS sequalae. The Revogene Strep A molecular assay (Meridian Bioscience Canada Inc, Quebec City, QC, Canada) is an automated real-time PCR assay for GAS detection from throat swab specimens within approximately 70 min. This multicenter prospective study evaluated the performance of the Revogene Strep A molecular assay compared to that of bacterial culture. Dual throat swab specimens in either liquid Amies or Stuart medium were collected from eligible subjects (pediatric population and adults) enrolled across 7 sites (USA and Canada). Revogene Strep A and reference testing was performed within 7 days and 48 h of sample collection, respectively. Of the 604 evaluable specimens, GAS was detected in 154 (25.5%) samples by the reference method and in 175 (29%) samples by the Revogene Strep A assay. Revogene Strep A assay sensitivity and specificity were reported to be 98.1% (95% confidence interval [CI], 94.4 to 99.3) and 94.7% (95% CI, 92.2 to 96.4), respectively. The positive predictive value was 86.3% (95% CI, 80.4 to 90.6), negative predictive value was 99.3% (95% CI, 98.0 to 99.8) with a 1.0% invalid rate. Discrepant analysis with alternative PCR/bidirectional sequencing was performed for 24 false-positive (FP) and 3 false-negative (FN) specimens. Concordant results were reported for 17 (FP only) of 27 discordant specimens. The Revogene Strep A assay had high sensitivity and specificity for GAS detection and provides a faster alternative for GAS diagnosis.

Here, we report 17 nearly complete genome sequences of enterovirus D68 (EV-D68) isolated from Kansas City, MO, in 2018. Phylogenetic analysis suggests that these strains belong to subclade B3, similar to the ones that caused the 2016 epidemics in the United States but different from the 2014 outbreak B1 strains.

Among known parechovirus (PeV) types infecting humans, PeV-A3 (formerly HPeV3) and PeV-A1 (formerly HPeV1) are associated with pediatric central nervous system (CNS) infections. The prevalence of PeV-A3 among hospitalized infants with sepsis-like illness and viral CNS infection is well described; however, the contribution of PeV-A4 to infant CNS infection is relatively unexplored. We report the first 11 U.S. cases of PeV-A4 CNS infections occurring in Kansas City infants during 2010 to 2016 and compare the clinical presentation with that of PeV-A3. PeV-positive cerebrospinal fluid (CSF) specimens from 2010 to 2016 underwent sequencing for genotyping. Among all PeV-CSF positives, PeV-A4 was detected in 11 CSF samples from 2010 to 2016. PeV-A4 was first detected in 2010 (n = 1/4), followed by detections in 2014 (n = 1/39), 2015 (n = 6/9), and 2016 (n = 3/33). The median age of PeV-A4-infected infants in weeks (median, 4; range, 1 to 8) was similar to that of infants infected with PeV-A3 (median, 4; range, 0.25 to 8). Clinical characteristics of PeV-A4 (n = 11) were compared with those of select PeV-A3-infected children (n = 34) with CNS infections and found to be mostly similar, although maximum temperature was higher (P = 0.017) and fever duration was shorter (P = 0.03) for PeV-A4 than for PeV-A3. Laboratory test results were also similar between genotypes, although they showed significantly lower peripheral white blood cell (P = 0.014) and absolute lymphocyte (P = 0.04) counts for PeV-A4 infants. Like PeV-A3, PeV-A4 caused summer-fall seasonal clusters of CNS infections in infants, with mostly similar presentations. Further surveillance is necessary to confirm potential differences in laboratory findings and in fever intensity/duration.

Group A Streptococcus (GAS) is one of the leading causes of bacterial pharyngitis. Early GAS diagnosis is critical for appropriate antibiotic administration that reduces the risk of GAS sequelae and limits spread of the infection. The Aries Group A Strep (GAS) assay (Luminex, Austin, TX) is a fully automated PCR assay for direct detection of GAS in throat swab specimens in less than 2 h with minimum hands-on time. This multicenter prospective study evaluated the clinical performance of the Aries GAS assay compared to that of Streptococcus pyogenes culture. Subjects with symptoms consistent with pharyngitis were enrolled across four sites in the United States, and a throat swab in liquid Amies medium was obtained. Aries and reference testing was performed within 72 and 48 h after sample collection, respectively. Of 623 throat swab specimens from patients with pharyngitis (93.6%

The Alere i Influenza A&B assay is a newly developed rapid molecular assay which has the potential to generate results within 15 min from sample collection. In this study, we evaluated the Alere i Influenza A&B assay by using salvaged frozen respiratory specimens that were collected in viral transport medium from children ages 10 months to 19 years. Alere i Influenza A&B assay test results were compared with viral culture and ProFlu + real-time reverse transcription-PCR (RT-PCR) assay results. We found that the overall sensitivity and specificity of the Alere i Influenza A&B assay were 93.3% and 94.5% for the detection of influenza A virus and 100% and 100% for the detection of influenza B virus, respectively, compared to viral culture. In comparison to ProFlu + real-time RT-PCR, overall sensitivity and specificity of the Alere i Influenza A&B assay for the detection of influenza A virus were 88.8% and 98.3% and 100% and 100% for detecting influenza B virus. Overall, the Alere i Influenza A&B assay performed well compared to either virus cell culture or RT-PCR.

The performance characteristics of three real-time influenza A/B virus reverse transcription-PCR (RT-PCR) assays and two real-time 2009 H1N1 RT-PCR assays were evaluated using previously characterized clinical specimens. A total of 150 respiratory specimens from children (30 influenza A/H1 virus-, 30 influenza A/H3 virus-, 30 2009 H1N1-, and 30 influenza B virus-positive specimens and 30 influenza virus-negative specimens) were tested with the CDC influenza A/B PCR (CDC), ProFlu + multiplex real-time RT-PCR assay (ProFlu + ), and MGB Alert Influenza A/B & RSV RUO (MGB) assays. A second set of 157 respiratory specimens (100 2009 H1N1-, 22 seasonal influenza A/H1-, and 15 seasonal influenza A/H3-positive specimens and 20 influenza-negative specimens) were tested with a new laboratory-developed 2009 H1N1 RT-PCR and the CDC 2009 H1N1 assay. The overall sensitivities of the CDC, ProFlu + , and MGB assays for detection of influenza A and B viruses were 100%, 98.3%, and 94%, respectively. The ProFlu + assay failed to detect one influenza A/H1 virus-positive specimen and yielded one unresolved result with another influenza A/H1 virus-positive specimen. The MGB assay detected 84/87 (96.5%) of influenza A and B viruses and 26/30 (86.6%) of 2009 H1N1 viruses. The new laboratory-developed 2009 H1N1 RT-PCR assay detected 100/100 (100%) 2009 H1N1 virus-positive specimens, while the CDC SW Inf A and SW H1 PCR assays failed to detect one and three low-positive 2009 H1N1-positive specimens, respectively. The CDC influenza A/B virus assay and the newly developed 2009 H1N1 RT-PCR assay with an internal control can be set up in two separate reactions in the same assay for routine clinical testing to detect influenza A and B viruses and to specifically identify the 2009 H1N1 influenza virus.

We describe here the characterization of five isolates of Mycobacterium simiae -like organisms representing a novel group based on whole-cell fatty acid analysis and genotypic evaluation. Two of the five isolates in this study, W55 and W58, were previously considered to belong to M. simiae serotype 2. Analysis of cellular fatty acids by gas-liquid chromatography indicated a close clustering of this group, which was well differentiated from the other M. simiae -like species. Molecular characterization was performed by nucleic acid sequencing of the small subunit rRNA gene and the gene encoding the 65-kDa heat shock protein and genomic DNA hybridization. Sequence analysis of the entire 16S rRNA gene showed a unique sequence most closely related to those of M. triplex and M. simiae . The hsp65 partial gene sequence was identical for the five isolates, with 97% identity to the M. simiae type strain. However, qualitative whole genomic DNA hybridization analysis confirmed that this group is genetically distinct from M. simiae and M. triplex . Antimicrobial susceptibilities for this group resemble those of M. simiae and M. lentiflavum . We conclude that this group represents a unique Mycobacterium species for which we propose the name Mycobacterium sherrisii sp. nov.

We report a rapid-cycle, real-time PCR method for identifying six Candida spp. directly from BACTEC blood culture bottles. Target sequences in the noncoding internal transcribed spacer regions of the rRNA operon were simultaneously amplified and interrogated with fluorescent probes to identify Candida albicans , C. glabrata , C. parapsilosis , C. tropicalis , C. krusei , and C. lusitaniae ; these account for 88% of the yeast species isolated from positive blood cultures in our laboratory. Any of the first four species can be identified in a single reaction using two fluorescent hybridization probe sets. The antifungal-resistant species C. krusei and C. lusitaniae are detected in a second reaction, also with two probe sets. The assay was validated with DNA extracted from BACTEC blood culture bottles positive for yeasts ( n = 62) and was 100% concordant with culture identification based on biochemical and morphological features of C. albicans ( n = 22), C. parapsilosis ( n = 10), C. tropicalis ( n = 1) C. glabrata ( n = 22), C. krusei ( n = 2), and C. lusitaniae ( n = 1). No cross-reactivity was observed in blood culture samples growing yeasts other than the above-mentioned species ( n = 4), in those growing bacteria ( n = 12), or in the absence of microbial growth. Our assay allows rapid (≤2 h) and specific detection of the most common Candida spp. directly from positive blood cultures and may facilitate delivery of optimal antifungal therapy.

The performance characteristics of two commercially available rapid tests for influenza, the BD Veritor System for Flu A+B (BD) and the Alere BinaxNOW influenza A&B card (BN), were evaluated using 200 frozen clinical specimens collected from January 2011 to June 2012 from pediatric patients. Real-time reverse transcriptase PCR (RT-PCR) was used as the gold standard to evaluate the results obtained by the two different assays. Of the 200 specimens tested, real-time RT-PCR assay detected influenza A or B virus in 116 samples, while BD detected 104 samples and BN detected 84 samples as positive. The overall sensitivity and specificity for detection of both influenza A and B virus in comparison to those of real-time RT-PCR were 89.6% (95% confidence interval [CI], 82.2 to 94.3) and 98.8% (95% CI, 92.6 to 99.9) for BD Veritor and 72.4% (95% CI, 63.2 to 80.0) and 100% (95% CI, 94.5 to 100.0) for BinaxNOW. Workflow analysis indicated that overall processing times for a batch size of 10 specimens were virtually identical between both systems. Overall, these results indicate that the BD Veritor assay was more sensitive than the BinaxNOW assay in detection of influenza A and B viruses in respiratory specimens from pediatric patients.

Infections caused by Escherichia coli isolates expressing adhesins of the Dr family are associated with diarrhea and urinary tract infections, and these E. coli strains recognize the complement regulatory protein decay-accelerating factor (DAF) as their receptor. Clustering of the DAF receptor at the sites of bacterial adherence to epithelial cells is proposed as an alternative to PCR assay for rapid detection of Dr-positive E. coli .

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048–5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr + adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr − adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr − adhesin) was found to predominate in afa -positive isolates from sepsis patients (75%); it was frequent in afa -positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr + strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa -positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa -positive strains.

Escherichia coli strains expressing Dr fimbriae are able to enter epithelial cells by interacting with a complement-regulatory protein, decay-accelerating factor. This model of bacterial internalization, with a well-characterized bacterial ligand and host receptor, provides a unique opportunity to investigate the early stages of invasion. We used immunofluorescence staining techniques to examine the distribution of receptor and cytoskeletal proteins in HeLa cells infected with E. coli recombinant strains that expressed Dr family of adhesins: Dr, Dr-II, F1845, AFA-I, and AFA-III. A major rearrangement of decay-accelerating factor was found at the adherence sites of recombinant strains expressing Dr, Dr-II, and F1845 adhesins. The changes in the distribution of receptor were significantly smaller on HeLa cells infected with E. coli bearing AFA-I or AFA-III afimbrial adhesins. Receptor aggregation was associated with the redistribution of cytoskeleton-associated proteins such as actin, α-actinin, ezrin, and occasionally tropomyosin. Purified Dr fimbriae coated on polystyrene beads were capable of triggering clustering of receptor and accumulating actin at the adhesion sites of beads to HeLa cells. Using scanning and transmission electron microscopic techniques, we have shown that beads coated with Dr fimbriae, as opposed to beads coated with bovine serum albumin, were enwrapped by cellular microvilli and ultimately internalized into HeLa cells. This indicates that interaction of Dr fimbriae with decay-accelerating factor is associated with redistribution of receptor and is sufficient to promote bacterial internalization.

Gonorrhea affects 150 million people worldwide annually. One of the most serious clinical forms of gonococcal infections is disseminated gonococcal infection (DGI) (36). DGI affects 1 to 3% of patients with gonorrhea (2, 7, 12, 18, 25, 31). Further, women infected with human immunodeficiency virus (HIV) are at increased risk of contracting DGI (3, 5, 17, 23, 32). Gonorrhea during pregnancy increases the risk of DGI (9, 33). Gestational DGI increases the risk of perinatal infant morbidity and mortality (6, 24, 27). Infected mothers may transmit Neisseria gonorrhoeae to their newborns during pregnancy or delivery or in the postpartum period (1, 11, 13, 27). The prevalence of prenatal gonococcal infection has been estimated at about 7.5% among high-risk populations in the United States (1). Newborns exposed to gonorrhea may develop systemic illnesses, including septicemia and arthritis. Neonatal sepsis is a life-threatening disease (1). The factors involved in the transmission of N. gonorrhoeae from the mother to the fetus during pregnancy are not characterized. The absence of an adequate experimental model hampers understanding of the mechanisms of transmission of N. gonorrhoeae to the fetus during pregnancy. Identification of the host and bacterial factors that increase risk for neonatal gonococcal sepsis would be important to understand the mechanism of gestational infection and to develop novel preventive approaches. A nonprimate animal model of gonococcal bacteremia in rat pups was recently developed (28). In this model, intraperitoneal (i.p.) inoculation of serum-resistant (serr) N. gonorrhoeae JC1, preincubated with human complement C1q, resulted in bacteremia that lasted 6 to 7 days (28). Preincubation of strain JC1 with C1q, instead of the initiation of killing via activation of the classical complement pathway, protected N. gonorrhoeae from the bactericidal effect of rat pup serum both in vitro and in vivo (28). The purpose of this investigation was to evaluate (i) whether serr strains of N. gonorrhoeae that express C1q-mediated virulence may be transmitted from a pregnant mother to the fetus and (ii) whether human complement C1q may enhance transmission. In the first set of experiments, we investigated whether pregnant rats are susceptible to N. gonorrhoeae infection. Three strains of N. gonorrhoeae, JC1 from DGI, 2005 from pelvic inflammatory disease (PID), and 1658 from local infection (LI), were used to infect pregnant rats. Selection of these three strains was based on significantly different clinical virulence to humans, which has been shown to correlate with C1q-mediated peritonitis to rat pups (Table ​(Table1).1). Eighty-six Sprague-Dawley rats were infected on day 20 of their pregnancy by i.p. inoculation with three different N. gonorrhoeae strains originating from patients with PID, DGI, and LI. Each group was divided into two subgroups consisting of those inoculated with 5 × 108 CFU of N. gonorrhoeae preincubated for 15 min at 37°C with 80 μg of C1q/ml or bovine serum albumin (BSA) (control). Human C1q isolated from human serum was shown to be pure by sodium dodecyl sulfate–5.6% polyacrylamide gel electrophoresis and by double immunodiffusion against a goat anti-C1q monospecific antiserum (Cytotech, San Diego, Calif.) (20). The C1q was shown to be active by standard hemolytic assay. BSA (catalog no. A3156) and nonimmune goat serum (catalog no. S2007) were purchased from Sigma (St. Louis, Mo.). TABLE 1 Relevant phenotype and genotype of strains used for experimental infection Bacterial suspension in buffered saline was prepared from piliated (P+) and opaque (OP+) colonies (75% P+, OP+). Twenty-four hours postinoculation, blood samples were collected from pregnant rats and cultured. Pregnant rats were sacrificed 24 h postinoculation, the uterus was opened immediately, and the number of live and/or infected fetuses was evaluated. Ten-microliter blood samples were collected from fetuses by heart puncture without anticoagulant, and dilutions were made immediately and subcultured on modified Thayer-Martin agar plates in triplicate and incubated for 48 h in 5% CO2. Bacterial infection was evaluated by counting CFU per milliliter of blood. Five pregnant control rats received 200 μl of buffer with C1q (80 μg/ml) i.p. to determine whether C1q alone influenced pregnancy outcome. Results showed that none of the three N. gonorrhoeae strains JC1, 2005, or 1658, pretreated with BSA, were recovered from the blood of the pregnant rats. Pretreatment of tested strains of N. gonorrhoeae with C1q did not result in infection of the pregnant rats themselves (Table ​(Table2);2); however, the fetuses of these pregnant rats were infected. It is not clear why adult rats are resistant to infection, but one possible explanation is that they have bactericidal antibody to N. gonorrhoeae, while pups do not. TABLE 2 Transmission of N. gonorrhoeae from pregnant rats to fetusese Table ​Table22 shows that 100% of live fetuses of pregnant rats inoculated with N. gonorrhoeae JC1 and 2005 pretreated with C1q had positive blood cultures, while none inoculated with strain 1658 pretreated with C1q had positive blood cultures. Blood cultures of dead fetuses were negative. Furthermore, the fetuses of pregnant rats inoculated with BSA-pretreated strain JC1, 2005, or 1658 showed negative blood cultures. Figure ​Figure11 shows quantitative blood cultures of fetuses obtained from one litter. The mean values, expressed as CFU/ml, of fetal blood for strains JC1 (DGI), 2005 (PID), and 1658 (LI) were 106, 104, and 0, respectively. FIG. 1 Recovery of N. gonorrhoeae from fetuses’ blood 24 h after i.p. inoculation of rats on day 20 of pregnancy with the following three types of N. gonorrhoeae strains: DGI (JC1), PID (2005), and LI (1658). These results demonstrate that N. gonorrhoeae strains associated with DGI or PID were able to spread from the pregnant rats to the fetuses. These two strains possess a 344-bp DNA fragment of sac-4, conferring resistance to N. gonorrhoeae to human and rat pup sera and virulence to rat pups in the presence of C1q (29). The fetuses were not infected in utero when the pregnant rat was challenged with sers LI strain missing the 344-bp DNA fragment of sac-4 or with BSA-pretreated N. gonorrhoeae (DGI, PID, and LI). The next set of experiments was designed to investigate the effects of C1q concentration on bacteremia and fetal death. There was an 18-fold increase in CFU/ml in the blood of fetuses infected with JC1 and a 7.6-fold increase in the blood of fetuses infected with strain 2005 treated with a higher concentration of C1q (120 μg/ml) and reached statistical significance for both strains. For statistical evaluation of the associations between two factors, the Fisher exact test was utilized to determine the significance level. P values less than 0.05 implied the existence of a statistically significant association between the tested factors. When pregnant rats were inoculated with C1q (80 μg/ml)-pretreated DGI and PID strains, 38.4 and 33.3% of the fetuses, respectively, died (P > 0.75). With an increase in the C1q concentration (120 μg/ml), the death rate among the fetuses increased to 45.3% (DGI) and 33.7% (PID), respectively, but did not reach statistical significance (P > 0.75). Incidentally, human gonococcal infection during pregnancy is also associated with 35% infant mortality (27). It is important that blood cultures from the dead fetuses were negative. Although the reason for this result is not clear at this time, it is possible that N. gonorrhoeae cannot survive in dead animals. Alternatively, fetal death may have been caused by complement and/or proinflammatory cytokines that could be induced by lipooligosaccharide released from killed N. gonorrhoeae in the maternal bloodstream. This hypothesis is supported by previous findings that systemic administration of bacterial lipopolysaccharide to pregnant rats induced fetal resorption or fetal death (4, 10, 37). The rate of fetal bacteremia correlated with C1q concentration. The results suggest that C1q not only supported the spread of N. gonorrhoeae strains carrying the 344-bp DNA region from the peritoneal cavity of the pregnant rat to the fetus but also increased morbidity and mortality. Human C1q is known to activate rat complement (28) and also protected serr N. gonorrhoeae from the killing effect of rat pup complement (28). It is, therefore, conceivable that serr strains inoculated with human C1q activate complement on the surface of bacteria in vivo without bacterial death. Further activation of complement in infected fetuses (15) may contribute to the proinflammatory responses via cytokines, activation of neutrophils, and the enhancement of vasopermeability and, therefore, increase virulence to the fetus (9, 12, 22). Although the precise role of the complement in the pathogenesis of gonococcal sepsis is not clear (8, 21, 27, 34, 35, 40), in human bacteremia, complement played an essential role in septic shock (14, 16, 20, 26, 30, 34, 38, 39). It is also known that inhibition of the biologic effects of complement in baboons with sepsis attenuates the lethal complications (38). It remains to be investigated whether inhibition of the biologic effects of complement C1q in pregnant rats may successfully prevent neonatal sepsis. Overall, we propose that the model showing C1q-dependent transmission of serr N. gonorrhoeae from mother to fetus may be relevant for the evaluation of pathogenesis of gestational gonococcal infection and fetal morbidity. Future studies are planned to evaluate the molecular mechanisms responsible for the C1q-dependent mother-fetus transmission of N. gonorrhoeae and the inhibition of this process before the onset of neonatal sepsis and stillbirth.

Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa + E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE + afaD and afaE afaD + mutants. The clinical E. coli strain ( afaE + afaD + ) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE + afaD + strain, followed by the group infected with the afaE + afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE + strains were the most invasive ( afaE + afaD strain > afaE + afaD + strain), while the afaE mutant strains ( afaE afaD + and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE + E. coli strains in vitro.

The pathogenic mechanism of recurrent or chronic urinary tract infection is poorly understood. Escherichia coli cells bearing Dr fimbriae display unique tropism to the basement membrane (BM)-renal interstitium that enables the bacteria to cause chronic pyelonephritis in experimental mice. The renal receptors for Dr-fimbriated E. coli are type IV collagen and decay-accelerating factor (DAF). We hypothesized that type IV collagen receptor-mediated BM-interstitial tropism is essential for E. coli to cause chronic pyelonephritis. To test the role of the type IV collagen tropism of Dr-fimbriated E. coli in renal persistence, we constructed an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to DAF and examined its virulence in the mouse model. The collagen-binding mutant DrI113T was eliminated from the mouse renal tissues in 6 to 8 weeks, while the parent strain caused persistent renal infection that lasted at least 14 weeks ( P ≤ 0.02). Transcomplementation with the intact Dr operon restored collagen-binding activity, BM-interstitial tropism, and the ability to cause persistent renal infection. We conclude that type IV collagen binding mediated by DraE adhesin is a critical step for the development of persistent renal infection in a murine model of E. coli pyelonephritis.

Dr-fimbriatedEscherichia colicapable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr+E. coliwas characterized in a cell-cell interaction model. Binding of Dr+E. colito the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser165by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr+E. colithrough a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-β-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr+E. coliwere morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr+E. coliand demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.