Determination of Haemophilus influenzae and Haemophilus parainfluenzae susceptibility to ampicillin and other antibiotics.

A sample comprising 40 H. influenzae and 74 H. parainfluenzae strains was used to verify methods for determining susceptibility to antibiotics. Modified Levinthal agar proved to be suitable for the agar dilution and agar diffusion method, while brain heart infusion with the thermally released components of sheep blood (X and V factor) and lysed horse blood performed well in the dilution micromethod. The iodometric method served well for beta-lactamase production. A substantial proportion of strains was resistant to penicillin, erythromycin, roxitromycin and sulfamethoxazole. Ampicillin susceptibility was of crucial importance. Resistance was largely due to beta-lactamase production. Since there are ampicillin-resistant strains which fail to produce beta-lactamase, it is necessary either to determine the MIC value or use a disk with 2 micrograms ampicillin. A disk containing 10 micrograms ampicillin may yield a false positive result.     

Population genetics and antibiotic susceptibility of invasive Haemophilus influenzae in Manitoba, Canada, from 2000 to 2006

One hundred and twenty-two isolates of Haemophilus influenzae causing invasive disease were collected in Manitoba, Canada, from 2000 to 2006 and examined for serotype, biotype, sequence type (ST) by multilocus sequence typing and antibiotic susceptibility. Nonserotypeable (NST) isolates accounted for over half of the isolates collected (69 isolates, 56.6%). There were 36 serotype a, five serotype b, two serotype c, one serotype d, four serotype e and five serotype f isolates collected. The 69 NST isolates were found to be very diverse, with isolates representing six biotypes and 45 STs. The serotypeable isolates were more clonal, with each of the serotypes showing little diversity in their biotypes and STs. Of the 122 isolates, 17% were resistant to ampicillin due to beta-lactamase production, 10.7% were resistant to trimethoprim-sulfamethoxazole, 1.6% were resistant to clarithromycin, 2.5% were resistant to amoxicillin-clavulanic acid and none was resistant to ciprofloxacin or moxifloxacin. Antibiotic resistance was more common in the NST strains, with 37.7% showing resistance to at least one antibiotic compared to 15% in the serotypeable strains. The results of this study suggest a shift in the epidemiology of invasive H. influenzae infections in the post-Hib vaccine era, and surveillance should include all serotypeable and NST isolates.     

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Haemophilus influenzae type b (Hib), a Gram‐negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl‐ribitol‐phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake‐flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (～500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α‐ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508–1519, 2017     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae

An isolate of Bacillus thuringiensis designated as PG-14 obtained from the Philippines was highly toxic to the mosquitoes Aedes aegypti and Culex molestus but nontoxic to the silkworm, Bombyx mori, and adults of a daphnid. The degree of toxicity to mosquito larvae was the same as that of the reference strain of Bacillus thuringiensis subsp. israelensis (serotype 14). Parasporal inclusion produced by the isolate PG-14 was spherical or irregular in shape and morphologically similar to that produced by the reference strain of subsp. israelensis. The H antigenic structure of the isolate was identical to that of the reference strain of B. thuringiensis subsp. morrisoni (serotype 8a:8b). Differences were shown in the O antigenic structures and in the production of lecithinase. Thermostable exotoxin was not produced by the isolate PG-14. The results indicate the isolation of a B. thuringiensis strain which shows the same toxicity as that of subsp. israelensis.