- Volume 58, Issue 4, 2009

This case–control study examined the prevalence of enteroaggregative Escherichia coli (EAEC), its genes and elicited inflammatory response, and the stool characteristics of adult patients with and without acute diarrhoeal illness presenting to an urgent-care clinic in the USA. A total of 1004 individual stool specimens (253 from patients with acute diarrhoeal illness and 751 from patients without diarrhoeal illness) were collected between 1 June 2003 and 30 June 2008. EAEC was identified as the sole cause of acute diarrhoeal illness in 6 % (n=15) of patients and in 2 % (n=15) without diarrhoeal illness. Control patients (n=15) were similar to case patients (n=15) for age, gender and co-morbidities. The EAEC genes aggR, aap, aat, astA and/or set1A were identified more frequently in case patients compared with control patients (P <0.05). aggR-positive EAEC elicited higher levels of interleukin (IL)-1ra, IL-6, IL-8 and tumour necrosis factor-α compared with aggR-negative EAEC during co-incubation with HCT-8 cells. Patients with EAEC diarrhoea and isolates with the genes aggR, aap, aatA, astA or set1A had stools characterized by gross mucus and the presence of faecal leukocytes (P <0.05). These results indicate that EAEC is a potential cause of acute diarrhoeal illness affecting patients presenting to an acute-care clinic in the USA and suggest that aggR, aap, aatA, astA and set1A may be markers for virulence.

Enteroaggregative Escherichia coli (EAEC) strains have been implicated as emerging aetiological agents of diarrhoea worldwide. In the present study, 43 EAEC strains were serotyped and characterized according to random amplification of polymorphic DNA profiles, PFGE, multilocus enzyme electrophoresis (MLEE) and the presence of putative virulence genes (hly, aero, kps, fim, aggA, aafA, aggR, astA, she, aap, shf and pet). The EAEC strains consisted of a diversity of serotypes including eight O-non-typable and 35 O-typable strains arranged into 21 O : H combinations. Amplification of specific genes revealed that all strains carried at least two of the virulence sequences investigated. fim, aggR and aap were the most frequent genes in both groups studied. hly, aero and aggA sequences were more prevalent in the diarrhoeal group. kps occurred exclusively in strains isolated from symptomatic children and showed strong association with diarrhoeal disease. The molecular approaches used to investigate the relatedness among EAEC strains revealed a high degree of polymorphism, suggesting that these micro-organisms have a non-clonal origin. A closer relationship was observed among EAEC strains sharing O : H types. No significant clustering could be identified related to the virulence traits investigated; however, the she locus showed clonal distribution by MLEE typing. These results are in accordance with previous findings in revealing the conservation of particular EAEC factors, despite the high degree of diversity related to both genotypic and phenotypic markers.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that has measurable genetic heterogeneity across its geographical range and reservoir spectrum. In the present study, publicly available sequences of the genes that have prevailingly been used for typing A. phagocytophilum were analysed to identify the segments giving the highest resolution with respect to the predictability of host and geographical provenances of the strains. Selected partial sequences of 16S rRNA, groL, msp4 and ank genes were then employed in a tentative multilocus typing scheme used to characterize the strains causing equine granulocytic anaplasmosis (EGA). We were able to both identify alleles characteristic for equine strains of A. phagocytophilum and distinguish two unique genetic variants infecting horses in the Czech Republic. This resolution far exceeded the discriminatory potential of any of the four sequenced genes when used singly. The two novel A. phagocytophilum variants appeared to be phylogenetically closer to the strains reported as causing human disease in Slovenia than to strains thus far isolated from other European EGA cases. A decline in the quality of recently deposited A. phagocytophilum sequences was also demonstrated.

The possible association between the use of triclosan and the development of antibiotic resistance was examined in triclosan-resistant mutants of Salmonella enterica serovar Typhimurium. These mutants were obtained from a sensitive parental strain and from ciprofloxacin-resistant isogenic strains using spontaneous mutagenesis or selection after one short exposure or continuous exposure to low concentrations of triclosan. The results showed that triclosan in the environment does not increase the mutation frequency but selects bacterial strains with reduced antibiotic susceptibility. This property depended on the multiple antibiotic resistance (Mar) phenotype of bacterial strains and on the triclosan concentration.

Coagulase-negative staphylococci (CoNS) are the main causative agents of bacteraemia in infants managed in neonatal intensive care units (NICUs). Intraluminal colonization of long-term central venous catheters by these bacteria and subsequent biofilm formation are the prerequisites of the bloodstream infections acquired in NICUs. The catheter lock technique has been used to treat catheter colonization; however, the optimum choice of antimicrobial agents and their corresponding concentrations and exposure times have not been determined. The effectiveness of catheter lock solutions (CLSs) was assessed by determining the minimal biofilm eradication concentration of antimicrobial agents against CoNS biofilms. Five conventional antibiotics (oxacillin, gentamicin, vancomycin, ciprofloxacin and rifampicin) alone or in combination, as well as ethanol, were evaluated. Ethanol was found to be superior to all of these conventional antibiotics when used as a CLS. A time–kill study and confocal laser scanning microscopy revealed that exposure to 40 % ethanol for 1 h was sufficient to kill CoNS biofilm cells. To our knowledge, this is the first in vitro study to provide solid evidence to support the rationale of using ethanol at low concentrations for a short time as a CLS, instead of using conventional antibiotics at high concentrations for a long period to treat catheter-related bloodstream infections.

Several imipenem-susceptible and -resistant Aeromonas veronii biovar sobria isolates with different morphologies and antimicrobial susceptibilities recovered from bile samples of a patient with cholangitis were analysed. These isolates belonged to the same clone and the imipenem-resistant strains showed overexpression of the imiS gene, encoding a chromosomal carbapenemase. These results should make clinicians aware of the possible emergence of multidrug-resistant A. veronii biovar sobria, perhaps as a consequence of previous treatment of a urinary tract infection with amoxicillin plus clavulanic acid.

Transmission of Streptococcus mutans, a major dental caries pathogen, occurs mainly during the first 2.5 years of age. Children appear to acquire S. mutans mostly from their mothers, but few studies have investigated non-familial sources of S. mutans transmission. This study prospectively analysed initial S. mutans oral colonization in 119 children from nursery schools during a 1.5-year period and tracked the transmission from child to child, day-care caregiver to child and mother to child. Children were examined at baseline, when they were 5–13 months of age, and at 6-month intervals for determination of oral levels of S. mutans and development of caries lesions. Levels of S. mutans were also determined in caregivers and mothers. A total of 1392 S. mutans isolates (obtained from children, caregivers and mothers) were genotyped by arbitrarily primed PCR and chromosomal RFLP. Overall, 40.3 % of children were detectably colonized during the study, and levels of S. mutans were significantly associated with the development of caries lesions. Identical S. mutans genotypes were found in four nursery cohorts. No familial relationship existed in three of these cohorts, indicating horizontal transmission. Despite high oral levels of S. mutans identified in most of the caregivers, none of their genotypes matched those identified in the respective children. Only 50 % of children with high levels of S. mutans carried genotypes identified in their mothers. The results support previous evidence indicating that non-familial sources of S. mutans transmission exist, and indicate that this bacterium may be transmitted horizontally between children during the initial phases of S. mutans colonization in nursery environments.

The plaque-control potential of commercially available amylase, lipase and protease was evaluated by observing their effects on coaggregation and on bacterial viability within various plaque microcosms. A quantitative coaggregation assay indicated that protease significantly inhibited the extent of coaggregation of Actinomyces naeslundii and Streptococcus oralis (P <0.05) and of Porphyromonas gingivalis and S. oralis. Amylase significantly (P <0.05) increased the coaggregation of A. naeslundii versus Fusobacterium nucleatum and A. naeslundii versus P. gingivalis. Concomitant challenge of constant-depth film fermenter-grown plaques with the enzymes did not result in detectable ecological perturbations (assessed by differential culture and denaturing gradient gel electrophoresis). Similar dosing and analysis of multiple Sorbarod devices did not reveal increases in bacterial dispersion which could result from disaggregation of extant plaques. A short-term hydroxyapatite colonization model was therefore used to investigate possible enzyme effects on early-stage plaque development. Whilst culture did not indicate significant reductions in adhesion or plaque accumulation, a vital visual assay revealed significantly increased aggregation frequency following enzyme exposure. In summary, although hydrolytic enzymes negatively influenced binary coaggregation, they did not cause statistically significant changes in bacterial viability within plaque microcosms. In contrast, enzyme exposure increased aggregation within extant plaques.

In this study it was shown for what is believed to be the first time that the African migratory locust can be used as a model for the study of Acanthamoeba pathogenesis. Mature adult locusts were injected intra-abdominally with 10 μl suspension of 106 Acanthamoeba (a clinical isolate of the T4 genotype) in culture medium, or with the same volume of sterile culture medium. Locusts injected with Acanthamoeba showed significant weight loss and reduced production of faeces compared with control locusts. Furthermore, injection of amoebae killed all of the locusts within 17 days at room temperature, although the speed of kill was temperature and dose dependent. When samples of faecal pellets and various tissues of infected locusts were cultured on non-nutrient agar plates containing bacterial lawns, live amoebae were recovered from haemolymph, flight muscle and fat body samples, but not from faeces. When brains dissected from locusts were incubated with an anti-amoebic drug (100 μM chlorhexidine) to kill extracellular amoebae, and then washed, homogenized and cultured on bacteria-seeded non-nutrient agar plates, only lysates from amoebae-infected locusts were positive for Acanthamoeba. This strongly suggests that amoebae invade the locust brain and, indeed, trophozoites of Acanthamoeba could be identified within the brain in histological sections of brains from infected locusts, but not from uninfected locusts. These findings support the view that locusts can be used as a model for the study of Acanthamoeba pathogenesis in vivo.

Rickettsia conorii infection is endemic in the Mediterranean basin, where it is known as Mediterranean spotted fever, also known as Boutonneuse fever and Marseilles fever. We report the case of a 66-year-old diabetic man who presented a severe form of the disease, complicated by acute renal failure, thrombocytopenia and encephalitis. Diagnosis was confirmed by indirect immunofluorescence assay. Despite appropriate treatment, severe neurological sequelae have remained. Medical literature on encephalitis caused by R. conorii is also reviewed.