Stephen H. Richardson

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in non‐mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In‐frame deletion and complementation studies of individual psl genes revealed that 11 psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl‐dependent polysaccharide, which consists of a repeating pentasaccharide containing d‐mannose, d‐glucose and l‐rhamnose:

Divergent Mechanisms for Passive Pneumococcal Resistance to β-Lactam Antibiotics in the Presence of Haemophilus influenzae

BACKGROUND Otitis media, for which antibiotic treatment failure is increasingly common, is a leading pediatric public health problem. METHODS In vitro and in vivo studies using the chinchilla model of otitis media were performed using a β-lactamase-producing strain of nontypeable Haemophilus influenzae (NTHi 86-028NP) and an isogenic mutant deficient in β-lactamase production (NTHi 86-028NP bla) to define the roles of biofilm formation and β-lactamase production in antibiotic resistance. Coinfection studies were done with Streptococcus pneumoniae to determine if NTHi provides passive protection by means of β-lactamase production, biofilm formation, or both. RESULTS NTHi 86-028NP bla was resistant to amoxicillin killing in biofilm studies in vitro; however, it was cleared by amoxicillin treatment in vivo, whereas NTHi 86-028NP was unaffected in either system. NTHi 86-028NP protected pneumococcus in vivo in both the effusion fluid and bullar homogenate. NTHi 86-028NP bla and pneumococcus were both recovered from the surface-associated bacteria of amoxicillin-treated animals; only NTHi 86-028NP bla was recovered from effusion. CONCLUSIONS Based on these studies, we conclude that NTHi provides passive protection for S. pneumoniae in vivo through 2 distinct mechanisms: production of β-lactamase and formation of biofilm communities.

Residence of Streptococcus pneumoniae and Moraxella catarrhalis within polymicrobial biofilm promotes antibiotic resistance and bacterial persistence in vivo

Otitis media (OM) is an extremely common pediatric ailment caused by opportunists that reside within the nasopharynx. Inflammation within the upper airway can promote ascension of these opportunists into the middle ear chamber. OM can be chronic/recurrent in nature, and a wealth of data indicates that in these cases, the bacteria persist within biofilms. Epidemiological data demonstrate that most cases of OM are polymicrobial, which may have significant impact on antibiotic resistance. In this study, we used in vitro biofilm assays and rodent infection models to examine the impact of polymicrobial infection with Moraxella catarrhalis and Streptococcus pneumoniae (pneumococcus) on biofilm resistance to antibiotic treatment and persistence in vivo. Consistent with prior work, M. catarrhalis conferred beta-lactamase-dependent passive protection from beta-lactam killing to pneumococci within polymicrobial biofilms. Moreover, pneumococci increased resistance of M. catarrhalis to macrolide killing in polymicrobial biofilms. However, pneumococci increased colonization in vivo by M. catarrhalis in a quorum signal-dependent manner. We also found that co-infection with M. catarrhalis affects middle ear ascension of pneumococci in both mice and chinchillas. Therefore, we conclude that residence of M. catarrhalis and pneumococci within the same biofilm community significantly impacts resistance to antibiotic treatment and bacterial persistence in vivo.

Nonencapsulated Streptococcus pneumoniae causes otitis media during single-species infection and during polymicrobial infection with nontypeable Haemophilus influenzae

Streptococcus pneumoniae strains lacking capsular polysaccharide have been increasingly reported in carriage and disease contexts. Since most cases of otitis media involve more than one bacterial species, we aimed to determine the capacity of a nonencapsulated S. pneumoniae clinical isolate to induce disease in the context of a single-species infection and as a polymicrobial infection with nontypeable Haemophilus influenzae. Using the chinchilla model of otitis media, we found that nonencapsulated S. pneumoniae colonizes the nasopharynx following intranasal inoculation, but does not readily ascend into the middle ear. However, when we inoculated nonencapsulated S. pneumoniae directly into the middle ear, the bacteria persisted for two weeks post-inoculation and induced symptoms consistent with chronic otitis media. During coinfection with nontypeable H. influenzae, both species persisted for one week and induced polymicrobial otitis media. We also observed that nontypeable H. influenzae conferred passive protection from killing by amoxicillin upon S. pneumoniae from within polymicrobial biofilms in vitro. Therefore, based on these results, we conclude that nonencapsulated pneumococci are a potential causative agent of chronic/recurrent otitis media, and can also cause mutualistic infection with other opportunists, which could complicate treatment outcomes.

Reduced Bacterial Adherence to Silicone Plastic Neurosurgical Prosthesis

Bacteria have been shown to adhere to smooth surfaces, such as shunts, by secreting a complex polysaccharide coat called the glycocalyx. We assume that if bacterial adherence could be reduced to zero, foreign-body-related infections would be essentially eliminated. This study describes a new technique for quantitating bacterial adherence to plastic using radioactive chromium, and demonstrates that presoaking the silicone plastic surgical tubing used for ventriculoperitoneal and ventriculoatrial shunts in bacitracin A solution (50,000 units in 250 ml) reduces the adherence of Staphylococcus epidermidis by 54%. We conclude that pretreatment of a hydrocephalic shunt tubing with an aqueous bacitracin solution before its implantation may help to reduce the postoperative shunt infections due to direct contamination of the shunt at the time it is inserted.

An ion translocase system from rabbit intestinal mucosa. Preparation and properties of the (Na+-K+)-activated ATPase

1. 1. A particulate enzyme complex from rabbit intestinal mucosa exhibiting (Na+ + K+)-stimulated ATPase activity has been isolated and partially purified. 2. 2. The ATPase activity exhibits an asymmetric stimulatory response to Na+ and K+ and is inhibited by ouabain and Ca2+. 3. 3. Specific inhibition of the ATPase activity by mersalyl, phenolphthalein, and culture filtrates from Vibrio cholerae are also reported. 4. 4. It is concluded that the ATPase activity is a moiety of the membrane-associated ion translocase system of rabbit intestinal mucosa and that mucinase activity in the culture filtrates selectively destroys or inactivates this system in vitro.

Enteropathogenicity of non-toxigenic Vibrio cholerae O1 for adult mice

The enteropathogenic potential of 32 Vibrio cholerae O1 isolates that do not produce cholera toxin was examined in the orally inoculated, sealed adult mouse model. Live cultures (2 x 10(10) cfu/ml) of 7/16 clinical and 6/16 environmental isolates produced a positive intestinal fluid accumulation (FA) ratio that reached near maximum at approximately 5 h post-inoculation. Colony hybridization did not detect genes for cholera toxin, Escherichia coli heat-labile and heat-stable toxins, or shiga-like toxins. FA activity did not correlate precisely with cytotoxic activities on Chinese hamster ovary (28/32 positive), Vero (29/32) or HeLa (25/32) cells. Certain clinical and environmental isolates of non-toxigenic V. cholerae O1 appear to be enteropathogenic for the mouse, providing evidence that they may have pathogenic potential for humans through an as yet undefined mechanism(s).

Virulence mechanisms associated with clinical isolates of non-O1 Vibrio cholerae.

Twenty one isolates of non-O1 V. cholerae from patients with diarrheal illness were examined for the presence of potential virulence mechanisms. The motile strains (90%) produced cell-associated mannose-sensitive hemagglutinins which reacted with human group O, chicken, sheep and rabbit erythrocytes. Motile isolates also attached to embryonic intestinal epithelial cells (ATCC 407), and the adherence was not inhibited by the presence of 1% D-mannose. All vibrio isolates hemolyzed sheep erythrocytes. Three vibrio isolates (14%) harbored two or three plasmids which ranged in size between 1.7 and 5.2 megadaltons. The presence of the plasmid did not correlate with the presence of hemolysin, hemagglutinins, adhesions or antibiotic resistance in any of the isolates. Thus, it appears that multiple factors associated with bacterial cell surfaces influence adhesin and apparently pathogenic potential of the non-O1 vibrio isolates in the host intestine.

Enterotoxigenicity of Clinical Isolates of Non-O1 Vibrio Cholerae

Whole cultures, but not culture supernatant fluids, of 21 isolates of non-O1 V. cholerae from patients with diarrhea were shown to induce positive fluid accumulation in infant mice. CHO cell assays demonstrated the elaboration of heat-labile cytotonic, cytotoxic or both factors from most isolates when grown under optimal culture conditions. These factors were not neutralized by anti-cholera toxin serum. Also genetic studies performed on 9 vibrio isolates using a DNA hybridization probe failed to detect gene sequences homologous with cholera toxin. ELISA assays recognized six isolates which produced a cell-associated substance which immunologically cross-reacted with cholera toxin. Enzymatic profiles of the vibrio isolates did not correlate with the production of any toxic factor. The results indicate that mainly heat-labile and cell-associated cytotonic and cytotoxic factors appear to influence the enterotoxigenic potential of this heterogenous group of non-O1 vibrios.

What Causes Mental Retardation and Cerebral Palsy

Mental retardation is present in approximately 780,000 school age children. Cerebral palsy affects 750,000 Americans, and nearly 2,000,000 individuals have epilepsy. Countless individuals suffer combinations of these neurologic disabilities. In an effort to assess our current state of knowledge about the pre-and perinatal factors associated with these brain disorders and to set research goals for the coming decade, the National Institute of Child Health and Human Development (NICHD) and the National Institute of Neurologic and Communicative Disorders and Stroke (NINCDS), appointed a task force to survey current knowledge about pregnancyand birth-related events that account for the continued incidence of neurologic handicap among infants and children. This report, titled Prenatal and Perinatal Factors Associated with Brain Disorders, represents a watershed in our thinking about the causes of mental retardation and cerebral palsy and epilepsy. Despite major advances in obstetric and neonatal medicine, physicians, patients, and attorneys still believe that the major causes of these brain disorders are related to birth trauma and the problems of labor. The committee found that only cerebral palsy could be related to perinatal insults. Only when mental retardation or epilepsy were found in association with cerebral palsy, could ischemia or hypoxia be causally related. The committee stated ‘although it was once simple to say that a specific event such as birth trauma or asphyxia caused these brain disorders, it is not usually possible to pinpoint a single cause and its effect’. Cerebral Palsy Cerebral palsy was found to be associated with prematurity, growth retardation and asphyxia at birth. The asphyxiated infant, documented by an Apgar score of less than 3 at 10–15 min, neurologic abnormalities in the newborn period and neonatal seizures, had a dramatically increased risk of later cerebral palsy. However, the majority of children with these risk factors had no evidence of neurologic dysfunction. More than 75% of children who had later cerebral palsy did not demonstrate these risk factors in the neonatal period. Mental Retardation Severe mental retardation is primarily genetic, biochemical, viral and developmental and not related to birth events. Only when it was associated with cerebral palsy, did severe mental retardation possibly link to asphyxia. Mild retardation, the most common degree of retardation, appears not to be related to pregnancy or birth events, but rather to social and environmental conditions. Associated factors, including maternal life styles, poor nutrition, cigarette smoking, alcohol and drug abuse, increase the likelihood of mild retardation. Prematurity and intrauterine growth retardation are commonly linked to these life styles. The risk of these problems was also correlated with the risk of being raised in an impoverished environment with less opportunity for optimal neurologic and intellectual development. Epilepsy Epilepsy did not appear to be related to preor perinatal events except when associated with cerebral palsy. Current Challenges and Directions for Research The committee noted that major challenges for the future included identifying women at high risk for bearing a neurologically handicapped child. For some such women, lowering the risk may begin before pregnancy, by management of conditions leading to risk and changes in life style. During pregnancy identification of the fetus with growth retardation may be an important area for reducing risk. The amount of damage due to hypoxia or ischemia depends both on its duration and severity, and on the infant’s biologic reserve and repair capabilities. Most infants who experience difficult labors do not develop later neurologic handicaps; most infants who demonstrate later brain disorders had no evidence of problems during the perinatal period. Premature infants are subject to the additional stress of hypoxia, respiratory distress and intraventricular hemorrhage. Prematurity was found to be the salient birth-related risk factor for later cerebral palsy. Understanding premature labor, and prevention of prematurity are the most important challenges in preventing later brain disorders. Major challenges now lie in detecting asphyxia earlier and knowing both its severity and duration. Neurologic damage to the fetus from asphyxia depends on the duration of the asphyxia, its severity, and on the gestational age of the fetus. Criteria which take into account these factors need to be developed for intervention. 176 Richardson