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Streptococcus agalactiae, also known as group B Streptococcus, is an aetiological agent of urinary tract infection (UTI) in adults, including cystitis, pyelonephritis and asymptomatic bacteriuria (ABU). Whereas ABU-causing S. agalactiae (ABSA) have been shown to grow and achieve higher culture denstity in human urine compared to uropathogenic S. agalactiae (UPSA) other phenotypic distinctions between S. agalactiae isolated from different forms of UTI are not known. Here, we define the hemolytic activities and biofilm-formation of a collection of clinical isolates of UPSA, ABSA and recurrent S. agalactiae bacteriuria (rSAB) strains to explore these phenotypes in the context of clinical history of isolates. A total of 61 UPSA, 184 ABSA, and 47 rSAB isolates were analyzed for relative hemolytic activity by spot assay on blood agar, which was validated using a erythrocyte lysis suspension assay. Biofilm formation was determined by microtiter plate assay with Lysogeny and Todd-Hewitt broths supplemented with 1% glucose to induce biofilm formation. We also used multiplex PCR to analyze isolates for the presence of genes encoding adhesive pili, which contribute to biofilm formation. Comparing the hemolytic activities of 292 isolates showed, surprisingly, that ABSA strains were significantly more likely to be highly hemolytic compared to other strains. In contrast, there were no differences between the relative abilities of strains from the different clinical history groups to form biofilms. Taken together, these findings demonstrate a propensity of S. agalactiae causing ABU to be highly hemolytic but no link between clinical history of UTI strains and ability to form biofilm.

Urinary tract infections (UTI) are among the most common infectious diseases of humans and are the most common nosocomial infections in the developed world. It is estimated that 40-50% of women and 5% of men will develop a UTI in their lifetime, and UTI accounts for more than 1 million hospitalizations and $1.6 billion in medical expenses each year in the USA. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI. This review presents an overview of recent discoveries related to the primary virulence factors of UPEC and major innate immune responses to infection of the lower urinary tract. New and emerging themes in UPEC research are discussed in the context of the interface between host and pathogen.

Uropathogenic Escherichia coli is the primary cause of urinary tract infections, which affects over 60% of women during their lifetime. UPEC exhibits a number of virulence traits that facilitate colonization of the bladder, including inhibition of cytokine production by bladder epithelial cells. The goal of this study was to identify the mechanism of this inhibition. We observed that cytokine suppression was associated with rapid cytotoxicity toward epithelial cells. We found that cytotoxicity, cytokine suppression and alpha-hemolysin production were all tightly linked in clinical isolates. We screened a UPEC fosmid library and identified clones that gained the cytotoxicity and cytokine-suppression phenotypes. Both clones contained fosmids encoding a PAI II(J96)-like domain and expressed the alpha-hemolysin (hlyA) encoded therein. Mutation of the fosmid-encoded hly operon abolished cytotoxicity and cytokine suppression. Similarly, mutation of the chromosomal hlyCABD operon of UPEC isolate F11 also abolished these phenotypes, and they could be restored by introducing the PAI II(J96)-like domain-encoding fosmid. We also examined the role of alpha-hemolysin in cytokine production both in the murine UTI model as well as patient specimens. We conclude that E. coli utilizes alpha-hemolysin to inhibit epithelial cytokine production in vitro. Its contribution to inflammation during infection requires further study.

The study established the virulence potential of multidrug-resistant Escherichia coli (MDREC) isolates from nosocomial infections in hospitalised dogs. The isolates were resistant to fluoroquinolones, belonged to two distinct clonal groups (CG1 and CG2) and contained a plasmid-mediated AmpC (CMY-7) beta-lactamase. CG1 isolates (n=14) possessed two of 36 assayed extraintestinal virulence genes (iutA and traT) and belonged to phylogenetic group A, whereas CG2 isolates (n=19) contained four such genes (iutA, ibeA, fimH and kpsMT K5) and belonged to group D. In a mouse gastrointestinal tract colonisation model, colonisation by index CG1 strain C1 was transient, in contrast to the index CG2 strain C2b, which persisted up to 40days post-inoculation. In a mouse subcutaneous challenge model, both strains were less virulent than archetypal group B2 extraintestinal pathogenic E. coli (ExPEC) strain CFT073; strain C1 caused no systemic signs and strain C2b was lethal to only one of six mice. In a mouse urinary tract infection model, strain C2b colonised the mouse bladder over 2 logs higher compared to strain C1. Whilst both groups of canine MDREC appear less virulent than a reference human ExPEC strain, CG2 strains have greater capacity for colonisation and virulence.

Group B Streptococcus agalactiae (GBS) is a major cause of neonatal lower respiratory tract infection. Following non-opsonic phagocytosis, GBS trigger programmed cell death of macrophages, early effector cells in the lung. The elimination of macrophages may be an important strategy used by the bacteria to suppress host immune responses and to persist at sites of infection. To better understand GBS-induced programmed cell death, we evaluated mechanisms by which GBS induces apoptosis. Compared to uninfected macrophages, caspase-3 activity of GBS-infected macrophages increased 4.4 ± 2.4 fold (p = 0.009) at 24 h and 1.7 ± 0.7 fold at 48 h following infection and caspase-9 activity increased 1.9 ± 0.01 fold (p = 0.009) at 48 h. GBS-infected macrophages incubated with a caspase-3 specific inhibitor had significantly increased survival compared to infected untreated macrophages. GBS also induced the release of cytochrome c from mitochondria, and depolarization of mitochondrial membrane potentials. This process was associated with increases in the pro-apoptotic protein Bad, and alterations in the regulation and localization of 14-3-3, Bcl-XL and Omi/HtrA2. We conclude GBS induces programmed cell death of macrophages by creating an imbalance in pro- and anti-apoptotic factors that ultimately activates the intrinsic pathway of apoptosis. This mechanism has not been previously associated with microbial infections.

Burkholderia pseudomallei, the etiological agent of melioidosis, causes significant mortality in endemic regions, but little is known regarding the immune mechanisms required for successful protective immunity. To establish a model of immunization that could be used to study this we screened a library of B. pseudomallei strains for immunogenicity in mice. BALB/c mice were immunized with test strains, and 2 weeks later were given a lethal challenge (LC) of virulent B. pseudomallei. Among 49 strains tested, a single strain, CL04, exhibited strong immunoprotective capacity. Interestingly, CL04 had been cultured from a patient with chronic colonization of B. pseudomallei, which is a rare phenomenon. Mice immunized with 0.1 × LD50 (5 × 103 CFU) of CL04 had significantly better survival and lower bacterial loads after LC compared to naive controls. Dose–response analysis demonstrated more robust immunity after higher immunizing doses, and bacterial inactivation by gamma irradiation diminished the protective effect, indicating a requirement for viable organism for immunity. CL04-induced immunity was demonstrated both in B. pseudomallei-susceptible BALB/c and -resistant C57BL/6 mice. We investigated the gene profile of CL04-induced immunity by analyzing responses to immunization using cDNA microarray. Unique responses involving granulocyte macrophage colony stimulating factor (GM-CSF), the proapoptotic regulator Bad and cyclin-dependent kinase (CDK5) were detected in immunized mice, but these responses were absent in naive-LC mice. Further, responses differed between mouse strains, indicating dependence on host genetic background. This model will be useful in identifying elements of the immune response required for successful adaptive immunity against B. pseudomallei.

Melioidosis is a potentially fatal disease of both human and animals caused by the bacterium Burkholderia pseudomallei. Disease is endemic in tropical and subtropical regions of Southeast Asia and Northern Australia. The pathogenesis of melioidosis is poorly understood. In particular, the host responses that occur following infection, and the specific host-pathogen interactions that result in the development of either acute or chronic infection are unclear. Using an established murine model, we investigated early proinflammatory cytokine responses believed to be critical in the development of acute and chronic B. pseudomallei infection. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to assess levels of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in the liver of mice following infection. We demonstrate that the level of mRNA for these cytokines increase moderately in chronic infection in C57BL/6 mice. However, in acute infection in BALB/c mice, mRNA responses for these cytokines were shown to be comparatively greater. These results demonstrate that early proinflammatory cytokine responses are important in the immunopathogenesis of melioidosis.