Raymond T. Cursons

The use of polymerase chain reaction to detect septicemia in critically ill patients

OBJECTIVE To describe the use of bacterial DNA amplification of conserved bacterial 16S ribosomal DNA nucleotide sequences by polymerase chain reaction (PCR) to detect the presence of septicemia in critically ill septic patients. DESIGN Case series of blood samples from septic patients comparing the PCR results with conventional blood culture results. SETTING A general intensive care unit in a tertiary referral hospital. PATIENTS Two sets of samples (n = 101 and n = 55) from patients diagnosed as clinically septic and requiring blood cultures. They were classified by internationally accepted criteria into systemic inflammatory response syndrome, severe sepsis, and septic shock groups. INTERVENTIONS Blood samples taken in a sterile fashion concurrently for blood culture, and PCR of the bacterial 16S ribosomal RNA gene in leukocytes and plasma. Two different DNA extraction techniques for PCR were tried sequentially. MEASUREMENTS AND MAIN RESULTS Blood culture and PCR positivity were measured in relation to the clinical classification of severity of sepsis. Using the initial extraction method (n = 101), ten patients were positive by both PCR and blood culture, eight patients were PCR positive and blood culture negative, and seven patients were blood culture positive and PCR negative. From the clinical criteria, PCR detected at least six true positives that had been missed on blood culture and missed four true Gram-positive bacteremias. When the initial code was broken, this deficiency was rectified using the improved extraction technique (n = 55), in which ten patients were positive by PCR and blood culture, 29 patients were PCR positive and blood culture negative, and two patients were PCR negative and PCR positive. CONCLUSIONS We conclude that the use of PCR (for the 16S ribosomal DNA in the plasma) was significantly more sensitive than the use of conventional blood culturing techniques for the detection of bacteremia in seriously ill patients. This could prove to be a valuable adjunct to conventional blood cultures.

Somatotropic axis components and nutrient partitioning in genetically diverse dairy cows managed under different feed allowances in a pasture system

The somatotropic axis [including growth hormone (GH), GH receptor, and insulin-like growth factor (IGF)-I] is uncoupled in high-producing cows in early lactation so that the liver fails to respond to GH and produces less IGF-I. This uncoupling was implicated in the process of nutrient partitioning, enabling high milk production. Different genetic selection goals may affect functional components of the somatotropic axis. Thus, the somatotropic axis was examined in diverse genetic strains of dairy cows [North American Holstein 1990 (NA90), New Zealand Holstein-Friesian 1990 (NZ90), and New Zealand Holstein-Friesian 1970 (NZ70)] that were managed similarly within a pasture-based system but were offered feed allowances commensurate with their genetic ability to produce milk. The NA90 cows produced more milk (26.2 +/- 0.3, 24.1 +/- 0.3, and 20.1 +/- 0.4 kg/d, for NA90, NZ90, and NZ70, respectively), but had lower milk fat percentages (4.28 +/- 0.03, 4.69 +/- 0.03, and 4.58 +/- 0.04 kg/d for NA90, NZ90, and NZ70, respectively) compared with both NZ strains. Milk protein percentages (3.38 +/- 0.02, 3.52 +/- 0.02, and 3.29 +/- 0.03 kg/d for NA90, NZ90, and NZ70, respectively) were greater for NZ90 cows. During early lactation (wk 2 to 6), the total net energy produced in milk was greater in NA90 compared with NZ90 or NZ70 cows, but total net energy in milk after wk 6 was equivalent for NA90 and NZ90 cows. The greater milk production in early lactation in NA90 cows was associated with lower body condition scores (BCS; 1 to 10 scale; 4.0 +/- 0.1) elevated blood GH concentrations (1.6 +/- 0.1 ng/mL), and low blood IGF-I concentrations (14.8 +/- 1.1 ng/mL), indicating an uncoupled somatotropic axis. In comparison, the NZ70 cows retained a coupled somatotropic axis during early lactation, maintaining greater BCS (4.6 +/- 0.1), lower blood GH (0.7 +/- 0.1 ng/mL), and greater blood IGF-I (21.9 +/- 1.2 ng/mL). The degree of uncoupling in NZ90 cows was intermediate between the other 2 strains. Additional feed allowance failed to change blood IGF-I concentrations in NA90 cows but increased IGF-I concentrations in NZ90 cows (20.9 +/- 1.4 and 13.2 +/- 1.4 ng/mL for the high and low feed allowance, respectively). Furthermore, additional feed allowance in NZ90 cows lessened BCS loss in early lactation, but did not affect BCS loss in NA90 cows. Functional components of the somatotropic axis differed for the respective strains and were consistent with strain differences in milk production, BCS, and feed allowance.

The vapBC Operon from Mycobacterium smegmatis Is An Autoregulated Toxin-Antitoxin Module That Controls Growth via Inhibition of Translation

The largest family of bacterial toxin-antitoxin (TA) modules is formed by the vapBC operons, and these are grouped together by virtue of their toxin components belonging to the PilT N-terminal domain family of proteins that are thought to function as ribonucleases. We have identified a single vapBC operon in the genome of Mycobacterium smegmatis and herein report the molecular and biochemical characterisation of this TA module. In M. smegmatis, the vapBC genes are transcribed as a leaderless mRNA that is constitutively synthesised throughout the growth cycle. The vapBC operon is autoregulated by the VapBC protein complex as demonstrated by a threefold increase in vapBC expression (promoter-vapB-lacZ) in a DeltavapBC mutant. Electrophoretic mobility shift assays using purified VapBC protein complex show that the complex binds to inverted repeat DNA sequences in the vapBC promoter region that overlap the -35 and -10 promoter elements, thus explaining the autoregulation and the low-level constitutive expression of this operon in M. smegmatis. Neither a DeltavapBC nor a DeltavapB mutant strain exhibited any phenotypic deviation to that of the isogenic wild-type parent strain under normal laboratory growth conditions, but conditional overexpression of VapC in M. smegmatis inhibited growth by a bacteriostatic mechanism and this phenotype is exacerbated in a DeltavapBC mutant. This effect is mediated through VapC-dependent inhibition of translation, not inhibition of DNA replication or transcription. The growth inhibitory effect of VapC was neutralised when co-expressed with its cognate antitoxin VapB. Western blot analysis revealed the overproduction of VapC under inducing conditions and that the VapC protein is not produced in the DeltavapB mutant despite the presence of mRNA transcript. Taken together, these data demonstrate that VapBC from M. smegmatis has all the hallmarks of a TA module with the capacity to cause growth inhibition by regulating translation.

Detection of bacteraemia in critically ill patients using 16S rDNA polymerase chain reaction and DNA sequencing

Abstract. Objective: To confirm the sensitivity of the polymerase chain reaction (PCR) technique (versus blood cultures) and to gain a better understanding of the incidence of true- and false-positive results when using this technique. Design: Observational study. Setting: Fourteen-bed, level 3 intensive care unit. Patients: Hundred twenty-six critically ill adult patients. Hundred ninety-seven blood culture and PCR samples taken as clinically indicated for suspected sepsis, according to routine ICU protocol. Measurements and results: The PCR product (16SrDNA: 341F–1195R) was sequenced and compared with a database of known species (Genebank) to identify the bacterial nucleic acid. The PCR or blood culture result was classified as a true-positive if there was other microbiological or clinical supporting evidence.

Whole genome sequencing of Mycobacterium tuberculosis reveals slow growth and low mutation rates during latent infections in humans.

Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mutation analysis of M. tuberculosis isolates from a multi-decade tuberculosis outbreak of the New Zealand Rangipo strain. We used epidemiological and phylogenetic analysis to identify four cases of tuberculosis acquired from the same index case. Two of the tuberculosis cases occurred within two years of exposure and were classified as recently transmitted tuberculosis. Two other cases occurred more than 20 years after exposure and were classified as reactivation of latent M. tuberculosis infections. Mutation rates were compared between the two recently transmitted pairs versus the two latent pairs. Mean mutation rates assuming 20 hour generation times were 5.5X10−10 mutations/bp/generation for recently transmitted tuberculosis and 7.3X10−11 mutations/bp/generation for latent tuberculosis. Generation time versus mutation rate curves were also significantly higher for recently transmitted tuberculosis across all replication rates (p = 0.006). Assuming identical replication and mutation rates among all isolates in the final two years before disease reactivation, the u20hr mutation rate attributable to the remaining latent period was 1.6×10−11 mutations/bp/generation, or approximately 30 fold less than that calculated during the two years immediately before disease. Mutations attributable to oxidative stress as might be caused by bacterial exposure to the host immune system were not increased in latent infections. In conclusion, we did not find any evidence to suggest elevated mutation rates during tuberculosis latency in humans, unlike the situation in rhesus macaques.

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage I/serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages.

The controlled in vitro susceptibility of gastrointestinal pathogens to the antibacterial effect of manuka honey

The susceptibility of common gastrointestinal bacteria against manuka honey with median level non-peroxide antibacterial activity (equivalent to that of 16.5% phenol) was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a standardized manuka honey with the broth microdilution method. The measured sensitivity of bacteria showed that manuka honey is significantly more effective than artificial honey (a mixture of sugars as in honey), indicating that osmolarity is not the only factor that is responsible for the antibacterial activity of the honey. Most tested gastrointestinal pathogens have MIC and MBC values in the range of 5–10% of honey, other than Enterobacter spp. which was in the range of 10–17%. The difference in efficacy between the honey with and without hydrogen peroxide removed was also studied, and it was found that both hydrogen peroxide and the non-peroxide components contribute to the bacteriostatic and bactericidal activity of the honey. It was also found that treatment against multi-antibiotic resistant microorganisms such as Salmonella typhimurium DT104 and ESBL-producing organisms with manuka honey may be promising.

Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom.

ABSTRACT We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.

The in vitro susceptibility of Campylobacter spp. to the antibacterial effect of manuka honey

We report the antimicrobial effect of manuka honey against Campylobacter spp. isolated by a diagnostic laboratory from specimens from a community in New Zealand. The isolates were differentiated according to species level using multiplex PCR. C. jejuni (20 strains) and C. coli (7 strains) were identified. The clinical isolates identified and type culture collection strains of these species were subjected to testing to determine the minimum inhibitory concentration (MIC) of manuka honey using a microdilution technique. The MIC of the manuka honey against all of the Campylobacter tested was found to be around 1% (v/v) honey. The low MIC values suggest that honey might still inhibit the growth of campylobacteria after dilution by fluid in the gut, but the actual concentration of honey that can be achieved in the intestine is unknown. Therefore, clinical investigation is required to establish the efficacy of honey against Campylobacter spp. in the gut environment.