- Volume 62, Issue 6, 2013

Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two genes encoding late acyltransferases responsible for lipid A biosynthesis jointly contribute to the biological activities and pathogenicity of M. catarrhalis. The double mutant O35ElpxXL with dramatically reduced toxicity is proposed as a potential vaccine candidate against M. catarrhalis infections for further investigation.

Leptospirosis is recognized as an emerging zoonotic disease generally affecting urban slums in developing countries and tropical regions. A combination of non-specific symptoms, low awareness among the medical community and a lack of readily available diagnostic tests have made leptospirosis an underdiagnosed disease. In this study, we tested an in-house ELISA with formalin-treated and boiled bacteria from the intermediate species Leptospira fainei as an antigen to detect Leptospira-specific IgM antibodies. A total of 819 serum samples, tested by a microscopic agglutination test (MAT) as a reference test, were used to evaluate the ELISA. Compared with positive and negative sera, the ELISA showed 94 % sensitivity and 99 % specificity. Positive and negative likelihood ratios were 94 and 0.06, respectively. No cross-reactivity was observed in sera from subjects with dengue and syphilis infections. The kappa value was 0.92 (95 % confidence interval 0.88–0.96), which indicated excellent agreement between the MAT and ELISA. The overall performance of this in-house ELISA suggests applicability as a rapid screening test for the diagnosis of leptospirosis in resource-limited settings and in hospitals and laboratories where a MAT is not available.

This study reports on the emergence of cfr-harbouring coagulase-negative staphylococci (CoNS) among patients who received linezolid therapy in two hospitals in Hangzhou, China. The mechanisms of resistance and transmission were analysed for these resistant isolates. Eight Staphylococcus capitis isolates, one Staphylococcus epidermidis isolate and one Staphylococcus hominis isolate, obtained from patients who had received linezolid therapy in two hospitals in Hangzhou, China, were confirmed as linezolid resistant, with MICs ranging from 8 to >256 mg l−1. The linezolid usage data of the ten patients before isolation of the linezolid-resistant CoNS were collected. PFGE analysis showed that the eight S. capitis isolates from the two hospitals belonged to the same clone. Nine of the linezolid-resistant CoNS isolates carried the cfr gene, which was located on plasmids of a similar size. A 5.3 kb fragment containing the cfr gene, revealing 99 % identity to the sequence of the cfr-harbouring plasmid pSS-01 reported previously, was determined by PCR mapping for all cfr-positive isolates, and the cfr gene was flanked by two copies of IS256-like elements. Thus, these results document the emergence of linezolid-resistant CoNS isolates carrying the cfr gene in Hangzhou, China. Effective nosocomial infection control strategies and the judicious use of antibiotics will be required to prevent further spread of this resistance mechanism.

Antimicrobial resistance is on the rise, and its early detection and surveillance are critical to implement effective control measures. The aim of this study was to develop a rapid hierarchical clustering bioinformatic tool for application on antibiotic susceptibility testing (AST) results (resistant, intermediate, sensitive) of a series of Klebsiella pneumoniae clinical isolates from Algeria and from France for surveillance of antibiotic-resistance phenotypes. A total of 1011 K. pneumoniae strains were collected from August 2008 to December 2012: 221 clinical isolates from western Algeria and 790 clinical isolates from Marseille, France. AST against a panel of 16 antibiotics was done for all isolates. Results of AST were introduced into MultiExperiment Viewer (MeV) software to perform hierarchical clustering, with resistant, intermediate and sensitive being translated to 1, 0 and −1 values, respectively. Hierarchical clustering results were compared to standard resistance phenotypes to evaluate the accuracy of the method. Based on the AST results, the 221 K. pneumoniae strains from Algeria could be separated into six phenotype groups as regards their resistance to β-lactam compounds: extended spectrum β-lactamase (ESBL) (68.3 %), ESBL associated with cephalosporinase (13.1 %), cephalosporinase (0.9 %), penicillinase (3.6 %) and wild-type (14.0 %). Hierarchical clustering by the MeV software applied to the AST results for all 1011 isolates generated clusters that were significantly representative of phenotypic classification and geographical origin, in less than 1 min. Moreover, adding to the dataset the AST results of a K. pneumoniae NDM-1 positive strain, the only strain resistant to imipenem in the series, immediately generated a new branch in the dendrogram. We have developed a rapid and simple hierarchical clustering tool for application on AST results that was able to survey qualitatively and quantitatively the prevalence of known and unknown phenotypes. This tool could be easily implemented in routine clinical microbiology laboratories.

The prevalence of dental caries continues to increase, and novel strategies to reverse this trend appear necessary. The probiotic Streptococcus salivarius strain M18 offers the potential to confer oral health benefits as it produces bacteriocins targeting the important cariogenic species Streptococcus mutans, as well as the enzymes dextranase and urease, which could help reduce dental plaque accumulation and acidification, respectively. In a randomized double-blind, placebo-controlled study of 100 dental caries-active children, treatment with M18 was administered for 3 months and the participants were assessed for changes to their plaque score and gingival and soft-tissue health and to their salivary levels of S. salivarius, S. mutans, lactobacilli, β-haemolytic streptococci and Candida species. At treatment end, the plaque scores were significantly (P = 0.05) lower for children in the M18-treated group, especially in subjects having high initial plaque scores. The absence of any significant adverse events supported the safety of the probiotic treatment. Cell-culture analyses of sequential saliva samples showed no differences between the probiotic and placebo groups in counts of the specifically enumerated oral micro-organisms, with the exception of the subgroup of the M18-treated children who appeared to have been colonized most effectively with M18. This subgroup exhibited reduced S. mutans counts, indicating that the anti-caries activity of M18 probiotic treatments may be enhanced if the efficiency of colonization is increased. It was concluded that S. salivarius M18 can provide oral health benefits when taken regularly.

There is growing interest in breeding rheas (Rhea americana) in Brazil. However, there are no data on the yeast microbiota of the gastrointestinal tract of this avian species, and the phenotypic characteristics of these yeasts are not known. Therefore, the aim of this work was to isolate Candida species from the digestive tract of rheas and to evaluate the in vitro antifungal susceptibility and secretion of phospholipases of the recovered isolates. For this purpose, 58 rheas from breeding operations in the cities of Fortaleza and Mossoró, north-eastern Brazil, were used. Samples were gathered from the oropharynx and cloaca of the animals using sterile swabs. Stool samples were collected from their pens by scraping with a scalpel blade. For the primary isolation, the material was seeded onto 2 % Sabouraud dextrose agar supplemented with chloramphenicol (0.5 g l−1). The isolates were identified based on morphological and biochemical features. After identification, all the strains were submitted to antifungal susceptibility testing for amphotericin B, itraconazole and fluconazole. The phospholipase activity of the Candida species isolates was also tested by culturing on egg yolk agar. Candida species were isolated from at least one anatomical site in 36/58 birds (14/17 juveniles and 22/41 adults) and in 6/10 faecal samples. Mostly, only a single species was isolated from each collection site (36/56 positive sites), with up to three species being observed only in four cases (4/56). A total of 77 isolates were obtained, belonging to the species Candida parapsilosis sensu lato (19), Candida albicans (18), Candida tropicalis (13), Candida guilliermondii (12), Candida krusei (10) and Candida famata (5). C. albicans was more prevalent in the oropharynx of the juvenile rheas when compared with adult ones (P<0.001). All tested isolates were susceptible to amphotericin B, but 16 isolates were simultaneously resistant to the two azole derivatives (11/18 C. albicans, 1/10 C. krusei, 2/19 C. parapsilosis sensu lato and 2/13 C. tropicalis). C. albicans presented a particularly high resistance rate to fluconazole (15/18) and itraconazole (13/18). Finally, 23/77 strains secreted phospholipases. In summary, healthy rheas carry potentially pathogenic Candida species in their gastrointestinal tract, including azole-resistant strains that secrete phospholipases, and are prone to disseminating them in the environment. Thus, breeding and handling these animals may have some implications for human and animal health.

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a common cause of diarrhoea in healthy, malnourished and immune-deficient adults and children. There is no reproducible non-neonatal animal model for longitudinal studies of disease mechanism or therapy. Using two strains of human-derived EAEC to challenge weaned C57BL/6 mice, we explored an in vivo model of EAEC infection in mice, in which disease was monitored quantitatively as the growth rate, stool shedding and tissue burden of organisms; nutritional status was varied, and a new class of therapeutics was assessed. A single oral challenge of EAEC strain 042 resulted in significant growth shortfalls (5–8 % of body weight in 12 days), persistent shedding of micro-organisms in stools [>103.2 c.f.u. (10 mg stool)−1 for at least 14 days] and intestinal tissue burden [~103 c.f.u. (10 mg tissue)−1 detectable up to 14 days post-challenge]. Moderate malnourishment of mice using a ‘regional basic diet’ containing 7 % protein and reduced fat and micronutrients heightened all parameters of infection. Nitazoxanide in subMIC doses, administered for 3 days at the time of EAEC challenge, lessened growth shortfalls (by >10 % of body weight), stool shedding [by 2–3 logs (10 mg stool)−1] and tissue burden of organisms (by >75 % in the jejunum and colon). Thus, weaned C57BL/6 mice challenged with EAEC is a convenient, readily inducible model of EAEC infection with three highly quantifiable outcomes in which disease severity is dependent on the nutritional status of the host, and which is modifiable in the presence of inhibitors of pyruvate ferredoxin oxidoreductase such as nitazoxanide.

The microbiota of the human oropharynx plays an important role in health through involvement in the aetiology of infection and the carriage of adventitious pathogens. Despite this, there are few models available for the preclinical assessment of novel antimicrobials directed to the human throat. We have profiled bacterial consortia sampled from the palatine tonsil and posterior pharyngeal wall microbiotas of healthy adult volunteers (n = 10) using differential culture and 16S rRNA gene sequencing, together with PCR-denaturing gradient gel electrophoresis. The data generated were used to assess the validity of an oropharyngeal microcosm system based on replicated constant-depth film fermenters (CDFFs; n = 5), which were continuously fed using an artificial airway surface liquid. Developed microcosms exhibited significant homology to ex situ consortia according to principal components analysis, whilst compositional reproducibility was apparent in replicated models for tonsillar and pharyngeal inocula. Differential viable count data and Shannon–Weiner diversity indices indicated that representative tonsil and pharyngeal model systems achieved dynamic compositional stability about 6 days after inoculation which could be maintained for ≥20 days. In conclusion, the CDFF facilitated the continuous maintenance of bacteriologically stable microcosms that were compositionally similar to ex situ inocula.

A 49-year-old male with underlying liver disease presented with fever and signs of sepsis. Non-O1/non-O139 Vibrio cholerae was isolated from his blood culture, which was positive for the hlyA and toxR genes. We report this fatal case of non-O1/non-O139 V. cholera sepsis and review the literature on non-O1/non-O139 V. cholerae sepsis in patients with chronic liver disease.