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Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients., Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia gladioli, Burkholderia dolosa, Burkholderia vietnamiensis, and B. cepacia. These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro. Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 μg/ml PAAG following a 1-h treatment (P

Summary Here, we evaluated the impact of bacterial growth stage on the effect of chitosan-arginine (Ch-arg) on Escherichia coli O157:H7 cell numbers and metabolic activity within contaminated beef juice held at room temperature. Using a lux-marked metabolic reporter strain of E. coli O157:H7, the results showed that Ch-arg was most bioactive against cells in the lag phase and exponential phase. In comparison, there was a reduced, although still significant, inhibitory effect of Ch-arg on the viability and metabolic activity of E. coli O157 held in stationary phase. Ch-arg reduced, but did not eliminate E. coli O157 growth in the meat juice over 48 h. Based on the evidence presented here and elsewhere, we conclude that Ch-arg can limit the growth and activity of food spoilage bacteria; however, it cannot completely eliminate bacterial contaminants originally present. Ch-arg should therefore be viewed as a potentially protective measure rather than a biocidal agent that completely eliminates the risk of pathogen transfer in the food chain.

The antimicrobial action of chitosan and functionalized chitosan derivatives is well known; however, limited research has been undertaken on its use in meat environments. In this study, the antibacterial activity of a newly-developed arginine-functionalized chitosan was tested against pathogenic Escherichia coli O157 in chicken juice. The chicken juice was representative of the liquid which accumulates in food packaging and which is frequently implicated in food poisoning incidents. Aliquots of chicken juice (50 ml) were inoculated with a lux -marked strain of E. coli O157 to approximately 8.75 log 10 CFU ml −1 . Samples were subsequently mixed with chitosan-arginine of varying concentrations (0–500 mg l −1 ) and incubated at 4 or 20 °C to mimic refrigeration and room temperatures, respectively. Pathogen persistence and metabolic activity (indexed by bioluminescence measurements) were subsequently quantified in the liquor at 0 (immediately after mixing), 3, 12, 24, 48, and 72 h post-incubation. The presence of chitosan-arginine significantly reduced both the numbers and metabolic activity of the pathogen in a dose-dependent manner with greater inhibition seen at higher concentration. In addition, it also suppressed the growth of general food spoilage bacteria, reduced malodor and prevented pathogen re-growth up to 72 h. The results imply that incorporating water soluble chitosan-arginine into packaging may help maintain both product shelf life and freshness as well as minimize the risk of food poisoning in both retail outlets and domestic homes.

In 1994, an outbreak of Enterobacter sakazakii infections occurred in a neonatal intensive care unit in France from 5 May to 11 July. During the outbreak, 13 neonates were infected with E. sakazakii , resulting in 3 deaths. In addition, four symptomless neonates were colonized by E. sakazakii . The strains were subjected to 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis, and phenotyped for a range of enzyme activities. E. sakazakii was isolated from various anatomical sites, reconstituted formula, and an unopened can of powdered infant formula. A fourth neonate died from septic shock, attributed to E. sakazakii infection, during this period. However, 16S rRNA gene sequence analysis revealed that the organism was Enterobacter cloacae . There were three pulsotypes of E. sakazakii associated with infected neonates, and three neonates were infected by more than one genotype. One genotype matched isolates from unused prepared formula and unfinished formula. However, no pulsotypes matched the E. sakazakii strain recovered from an unopened can of powdered infant formula. One pulsotype was associated with the three fatal cases, and two of these isolates had extended-spectrum β-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.

Enterobacter sakazakii is an opportunistic pathogen associated with contaminated powdered infant formula and a rare cause of Gram-negative sepsis that can develop into meningitis and brain abscess formation in neonates. Bacterial pathogenesis remains to be fully elucidated. In this study, the host inflammatory response was evaluated following intracranial inoculation of Ent. sakazakii into infant rats. Infiltrating macrophages and neutrophils composed multiple inflammatory foci and contained phagocytosed bacteria. Several genotypically distinct Ent. sakazakii strains (16S cluster groups 1-4) were shown to invade rat capillary endothelial brain cells (rBCEC4) in vitro. Further, the persistence of Ent. sakazakii in macrophages varied between strains. The presence of putative sod genes and SOD activity may influence the survival of acidic conditions and macrophage oxidase and contribute to Ent. sakazakii intracellular persistence. The influence of macrophage uptake of Ent. sakazakii on immunoregulatory cytokine expression was assessed by ELISA. This demonstrated that the IL-10/IL-12 ratio is high after 24 h. This is suggestive of a type 2 immune response which is inefficient in fighting intracellular infections. These findings may help explain how the diversity in virulence traits among Ent. sakazakii isolates and an unsuccessful immune response contribute to the opportunistic nature of this infection.

Lipopolysaccharide (LPS) is a heat stable endotoxin that persists during the processing of powdered infant formula milk (IFM). Upon ingestion it may increase the permeability of the neonatal intestinal epithelium and consequently bacterial translocation from the gut. To determine the level of endotoxin present in IFM, 75 samples were collected from seven countries (representing 31 brands) and analysed for endotoxin using the kinetic colorimetric Limulus amoebocyte lysate (LAL) assay. The endotoxin levels ranged from 40 to 5.5×104 endotoxin units (EU) per gram and did not correlate with the number of viable bacteria. The neonate rat model was used to address the risk of endotoxin-induced bacterial translocation from the gut. Purified Escherichia coli LPS was administered to rat pups followed by inoculation with Enterobacter sakazakii ATCC 12868. Bacteria were isolated from the mesentery, spleen, blood and cerebral spinal fluid (CSF) of endotoxin-treated rats due to enhanced gut and blood brain barrier penetration. Histological analysis of the colon showed marked distension of the mucosal and muscular layers. It is plausible that the risk of neonatal bacteraemia and endotoxemia, especially in neonates with immature innate immune systems, may be raised due to ingestion of IFM with high endotoxin levels.

Citrobacter koseri causes neonatal meningitis frequently complicated with multiple brain abscesses. During C. koseri central nervous system infection in the neonatal rat model, previous studies have documented many bacteria-filled macrophages within the neonatal rat brain and abscesses. Previous studies have also shown that C. koseri is taken up by, survives phagolysosomal fusion and replicates in macrophages in vitro and in vivo. In this study, in order to elucidate genetic and cellular factors contributing to C. koseri persistence, a combinatory technique of differential fluorescence induction and transposon mutagenesis was employed to isolate C. koseri genes induced while inside macrophages. Several banks of mutants were subjected to a series of enrichments to select for gfp : : transposon fusion into genes that are turned off in vitro but expressed when intracellular within macrophages. Further screening identified several mutants attenuated in their recovery from macrophages compared with the wild-type. A mutation within an Escherichia coli fliP homologue caused significant attenuation in uptake and hypervirulence in vivo, resulting in death within 24 h. Furthermore, analysis of the immunoregulatory interleukin (IL)-10/IL-12 cytokine response during infection suggested that C. koseri fliP expression may alter this response. A better understanding of the bacteria–macrophage interaction at the molecular level and its contribution to brain abscess formation will assist in developing preventative and therapeutic strategies.

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 μg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 μg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

This chapter summarizes the understanding in the neonatal intestinal microbial flora, immunity, and infections, with particular emphasis on the preterm intestinal flora and immunity development. It aims at widening the general microbiologist’s knowledge of the issue of Enterobacter sakazakii and other Enterobacteriaceae as infectious agents of neonates in the context of the neonate having a complex intestinal flora, which includes Enterobacter species and where endogenous infections occur. Infants, particularly premature infants, are made more susceptible to disease by reduced natural epidermal and epithelial barriers and secreted products that comprise part of the innate immune system. The majority of studies have been of duodenal and stool samples, which represent the small and large intestines, respectively. The study also included the postmortem flora of a 3-month-old infant who had died of toxic dyspepsia and endocardial fibrosis. From 1986 to 1989, Escherichia coli was the most common isolate reported to the U.S. National Nosocomial Infections Surveillance System, followed by enterococci, Pseudomonas aeruginosa, Staphylococcus aureus, and coagulasenegative staphylococci (CoNS). Contaminated enteral nutrition can cause nosocomial bloodstream infections and may contribute to necrotizing enterocolitis (NEC). A section focuses on the general topic of infections through contaminated enteral feeding. The major species involved in neonatal infection is Staphylococcus epidermidis, which causes 60 to 93% of CoNS bacteremias. Occasionally, CoNS are acquired from the mother at birth, but the majority of CoNS colonization is acquired nosocomially, predominantly from the hands of health care workers.

The establishment of a phylogenetic tree is a prerequisite for studying the evolution of virulence. The current nomenclature of the genus Salmonella is based on this phylogenetic tree and distinguishes only two species: Salmonella enterica and Salmonella bongori. If acquisition of SPI-1 introduced a virulence factor required for the pathogenesis of diarrheal disease, then mutational inactivation of this determinant should attenuate Salmonella serotypes in animal models of gastroenteritis. The contribution of the invasion-associated type III secretion system to serotype Typhimurium pathogenesis in this animal model has recently been investigated using strains carrying mutations in hilA and prgH. The majority of antibodies elicited by immunization with heat-killed serotype Typhimurium or with a live-attenuated serotype Typhimurium aroA vaccine is directed against the immunodominant O-antigen. Mathematical models predict that in this between-serotype competition, the serotype with higher transmissibility will dominate and eventually eliminate its competitor. The generation of O-antigen polymorphism through horizontal gene transfer was therefore a likely mechanism that allowed Salmonella serotypes to adapt to the enhanced immune memory encountered in warm-blooded hosts. The fljB gene is present in biphasic S. enterica subspecies but absent from monophasic S. enterica subspecies and Escherichia coli, suggesting its acquisition by horizontal gene transfer. A primary pathogen can be defined as an organism capable of entering a host and finding a unique niche to multiply and avoid or subvert the host defenses, the outcome of which may be clinical disease manifestations.

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium ( S. typhimurium ) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10 10 CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP , with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis . These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.

Enterobacter hormaechei is a Gram-negative bacterium within the Enterobacter cloacae complex, and has been shown to be of clinical significance by causing nosocomial infections, including sepsis. Ent. hormaechei is spread via horizontal transfer and is often associated with extended-spectrum beta-lactamase production, which increases the challenges associated with treatment by limiting therapeutic options. This report considers 10 strains of Ent. hormaechei (identified by 16S rDNA sequencing) that had originally been identified by phenotyping as Cronobacter (Enterobacter) sakazakii. Seven strains were from different neonates during a nosocomial outbreak in a California hospital. PFGE analysis revealed a clonal relationship among six of the seven isolates and therefore a previously unrecognized Ent. hormaechei outbreak had occurred over a three-month period. Antibiotic-resistance profiles were determined and extended-spectrum beta-lactamase activity was detected. The association of the organism with powdered infant formula, neonatal hosts and Cr. sakazakii suggested that the virulence of these organisms may be similar. Virulence traits were tested and all strains were shown to invade both gut epithelial (Caco-2) and blood-brain barrier endothelial cells (rBCEC4), and to persist in macrophages (U937). Due to misidentification we suggest that Ent. hormaechei may be an under-reported cause of bacterial infection, especially in neonates. Also, its isolation from various sources, including powdered infant milk formula, makes it a cause for concern and merits further investigation.

Background In 1994, an outbreak of Enterobacter sakazakii infections in France occurred in a neonatal intensive care unit during which 17 neonates were infected. More than half of the infected neonates had severe clinical symptoms; 7 cases of necrotising enterocolitis (one with abdominal perforation), one case of septicemia, and one case of meningitis. The other 8 neonates were shown to be colonized but remained asymptomatic. There were three deaths. Four distinguishable pulsotypes of E. sakazakii were isolated during the outbreak, and the deaths were attributable to one pulsotype. This paper compares strains, from the four pulsotypes, for attachment and invasion of mammalian intestinal cells, macrophage survival and blood-brain barrier invasion. A fourth death from septic shock also occurred during the E. sakazakii outbreak. This was due to E. cloacae which at the time of the outbreak had been misidentified as E. sakazakii. This isolate has been included in this study. Results All E. sakazakii strains attached and invaded Caco-2 human epithelial cells, and invaded rat brain capillary endothelial cells. The majority of strains persisted in macrophage cells for 48 h. Two strains from fatal NEC and meningitis cases showed the highest invasion rate of Caco-2 intestinal cells. Their invasion of brain capillary endothelial cells was equivalent or greater than that of the neonatal E. coli meningitis strain K1. These strains also had extended spectrum β-lactamase activities. E. cloacae differed from E. sakazakii due to the greater attachment and less invasion of epithelial cells, no survival in macrophages, and less invasion of capillary endothelial brain cells. Conclusion While variables such as host factors and treatment strategies determine the outcome of infection, our in vitro studies evaluated the virulence of the isolates associated with this outbreak. It was not possible to directly correlate clinical symptoms and outcomes with in vitro studies. Nevertheless, we have shown the variation in invasive potential of E. sakazakii with intestinal and blood-brain barrier cells between and within pulsotypes from a neonatal intensive care unit outbreak. E. sakazakii strains were able to persist and even replicate for a period within macrophage cells. These traits appear to facilitate host immune evasion and dissemination.

Escherichia coliis a major cause of neonatal bacterial sepsis and meningitis. We recently identified a gene,traJ, which contributes to the ability ofE. coliK1 to penetrate the blood-brain barrier in the neonatal rat. Because very little is known regarding the most critical step in disease progression, translocation to the gut and dissemination to the lymphoid tissues after a natural route of infection, we assessed the ability of atraJmutant to cause systemic disease in the neonatal rat. Our studies determined that thetraJmutant is significantly less virulent than the wild type in the neonatal rat due to a decreased ability to disseminate from the mesenteric lymph nodes to the deeper tissues of the liver and spleen and to the blood during the early stages of systemic disease. Histopathologic studies determined that although significantly less or no mutant bacteria were recovered from the spleen and livers of infected neonatal rats, the inflammatory response was considerably greater than that in wild-type-colonized tissues. In vitro studies revealed that macrophages internalize thetraJmutant less frequently than they do the wild type and by a morphologically distinct process. Furthermore, we determined that tissue macrophages and dendritic cells within the liver and spleen are the major cellular targets ofE. coliK1 and that TraJ significantly contributes to the predominantly intracellular nature ofE. coliK1 within these professional phagocytes exclusively during the early stages of systemic disease. These data indicate that, contrary to earlier indications,E. coliK1 resides within professional phagocytes, and this is essential for the efficient progression of systemic disease.

A unique feature ofCitrobacter koseriis the extremely high propensity to initiate brain abscesses during neonatal meningitis. Previous clinical reports and studies on infant rats have documented manyCitrobacter-filled macrophages within the ventricles and brain abscesses. It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by whichC. koserisubverts the host response and elicits chronic infection, resulting in brain abscess formation. In this study, we showed thatC. kosericauses meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation. Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-termC. koseriinfection. To better understandC. koseripathogenesis, we have characterized the interactions ofC. koseriwith human macrophages. We found thatC. koserisurvives and replicates within macrophages in vitro and that uptake ofC. koseriincreases in the presence of human pooled serum in a dose-dependent manner. EM studies lend support to the hypothesis thatC. koseriuses morphologically different methods of uptake to enter macrophages. FcγRI blocking experiments show that this receptor primarily facilitates the entry of opsonizedC. koseriinto macrophages. Further, confocal fluorescence microscopy demonstrates thatC. koserisurvives phagolysosomal fusion and that more than 90% of intracellularC. koseriorganisms are colocalized within phagolysosomes. The ability ofC. koserito survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.

The two most important human disease syndromes caused by Salmonella serovars are typhoid fever and enterocolitis. Typhoid fever is a systemic infection caused by Salmonella serovars which are strictly adapted to humans or higher primates, including Salmonella enterica serovars Typhi and paratyphi A, B, and C. Human enterocolitis is an infection localized to the intestine and mesenteric lymph nodes which can be caused by any of more than 2000 Salmonella serovars, however, S. enterica serovars Typhimurium and Enteritidis together account for approximately 50% of cases in the USA. The animal model most frequently used to study Salmonella pathogenesis is the typhoid fever-like disease caused by serovar Typhimurium in mice. The pathology in the intestine of mice infected with serovar Typhimurium is characterized by a predominantly mononuclear leukocyte infiltrate, as well as edematous villi which become shortened in height [1]. Capillary thrombosis, hemorrhage, and ulcerations may be present at areas of Peyer's patches. The intestinal pathology caused by serovar Typhimurium in mice is similar to that observed in typhoid fever patients. That is, intestinal biopsies taken from volunteers infected with serovar Typhi or from typhoid fever patients reveal a predominantly mononuclear leukocyte infiltrate [2,3]. Furthermore, colonization of human Peyer's patches by serovar Typhi commonly produces capillary thrombosis, which can result in hemorrhage, necrosis and ulceration [4]. In humans, these lesions can progress to intestinal perforation, a potentially fatal complication which usually occurs in the third week of infection [4]. In contrast, rapid bacterial multiplication in the liver and other internal organs of mice causes death within 6–10 days post serovar Typhimurium infection, while intestinal perforation has not been reported in this model. Since intestinal perforation is an important cause of death during typhoid fever [4], it is likely that …

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to sef, fim, saf , and tcf , which had been described previously in serotype Typhi, we identified eight new putative chaperone-usher-dependent fimbrial operons, which were termed bcf, sta, stb, ste, std, stc, stg , and sth . Hybridization analysis performed with 16 strains of Salmonella reference collection C and 22 strains of Salmonella reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal Salmonella serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other Salmonella serotypes investigated in that it contained a unique combination of putative fimbrial operons.

Numerous Salmonella typhimurium virulence factors have been identified and characterized using experimental infection of mice. While the murine typhoid model has been used successfully for Salmonella typhi vaccine development and to infer virulence mechanisms important during typhoid fever, information derived from infection of mice has been of limited value in elucidating the mechanism by which S. typhimurium causes enteritis in humans. Progress in our understanding of virulence mechanisms contributing to diarrheal disease comes from recent studies of bovine enteritis, a S. typhimurium infection, which manifests as acute gastroenteritis. This review compares virulence genes and mechanisms required during murine typhoid, typhoid fever, and bovine enteritis. Comparison of illnesses caused in different animal hosts identifies virulence mechanisms involved in species specific disease manifestations. The determination of the relative importance of virulence factors for disease manifestations in different host species provides an important link between the in vitro characterization of genes and their role during host pathogen interaction.