Fighting antimicrobial resistance (AMR): Chinese herbal medicine as a source of novel antimicrobials - an update.

Antimicrobial resistance (AMR) has now emerged as a global public health crisis, requiring the discovery of new and novel antimicrobial compounds, that may be precursors of future therapeutic antibiotics. Chinese Herbal Medicine (CHM) comes with a rich pedigree of holistic and empirical usage in Asia for the last 5000 years. Extracts of Anemarrhena asphodeloides Bunge, Angelica sinensis (Oliv.) Diels, Dianthus superbus L. Forsythiae fructus (Lian Qiao), Lonicerae flos (Jin Yin Hua), Naemorhedi cornu, Platycladus orientalis Franco, Polygonum aviculare, Polygonum cuspidatum, Poria cocos (Schw.), Rehmannia glutinosa (Gaertn.) DC, Rheum palmatum, Salvia miltiorrhiza Bunge, Scutellaria barbata, Scutellariae radix (Huang Qin) and Ursi fel (Xiong Dan) have shown to have antimicrobial properties against clinically significant Gram-negative and Gram-positive bacterial pathogens, as well as the mycobacteria (TB and non-tuberculous mycobacteria). Evidence is now beginning to emerge through systematic reviews of the outcomes of clinical studies employing CHM to treat infections. Of the 106 Cochrane systematic reviews on CHM, 16 (ca 15%) reviews examine CHM in the context of treating a specific infection disease or state. This update examines direct antimicrobial effect of CHM on bacterial pathogens, as well as synergistic effects of combining CHM with conventional antibiotics.

Successful disinfection of trumpet mouthpieces using domestic steam disinfection.

There have been numerous reports in the literature describing the diversity of microbial flora isolated from woodwind and brass instruments, with potential infection risks for players, especially when such instruments are shared. Steam disinfection has become established as a trusted method of decontamination; however, there have been no reports on the employment of this technology to disinfect parts of musical instruments, hence it was the aim of this study to examine the fate of bacterial and yeast pathogens on artificially contaminated trumpet mouthpieces and to evaluate whether such disinfection is an effective method of disinfection for such instrument parts. Trumpet mouthpieces were artificially contaminated with 18 microbial strains (17 bacteria from four genera (Enterococcus, Escherichia, Staphylococcus and Streptococcus) and one yeast (Candida)), each at an inoculum density of approximately 1·5 × 107 colony forming units and subjected to a disinfection cycle. The experiment was repeated including 50% (v/v) sterile sputum as soil. No bacteria or yeast organisms were recovered post disinfection, including following recovery and with nonselective cultural enrichment techniques.

Muddy puddles - the microbiology of puddles located outside tertiary university teaching hospitals.

In the British Isles, the frequency of rain results in the formation of puddles on footpaths and roads in/around hospitals. No data are available demonstrating the microbiological composition of such puddles and therefore a study was undertaken to examine the microbiology of puddles in the grounds of two tertiary university-teaching hospitals (18 sites) and compared with control puddles from non-hospital rural environments (eight sites), estimating (i) total viable count; (ii) identification of organisms in puddles; (iii) enumeration of Escherichia coli: (iv) detection of Extended Spectrum ß-Lactamase producing organisms and (v) direct antimicrobial susceptibility testing. A mean count of 2·3 × 103  CFU per ml and 1·0 × 109  CFU per ml was obtained for hospital and non-hospital puddles respectively. Isolates (n = 77; 54 hospital and 23 non-hospital) were isolated comprising of 23 species among 17 genera (hospital sites), where the majority (10/16; 62·5%) of genera identified were Gram-negative approximately, a fifth (20·6%) were shared by hospital and non-hospital rural samples. Escherichia coli was detected in half of the hospital puddles and under-half (37·5%) of the rural puddles extended spectrum ß-lactamase organisms were not detected in any samples examined. Rainwater puddles from the hospital and non-hospital environments contain a diverse range of bacteria, which are capable of causing infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the presence of a wide diversity of bacterial taxa associated with rainwater puddles around hospitals, many of which are capable of causing human disease. Of clinical significance is the presence of Pseudomonas aeruginosa isolated from a hospital puddle, particularly for patients with cystic fibrosis. The presence of potentially disease-causing bacteria in puddles in and around hospitals identifies a new potential environmental reservoir of bacteria. Furthermore work is now needed to define their potential of entering or exiting hospital wards by contaminated footwear.

Does ivacaftor interfere with the antimicrobial activity of commonly used antibiotics against Pseudomonas aeruginosa?-Results of an in vitro study.

WHAT IS KNOWN AND OBJECTIVE: Ivacaftor is a novel potentiator of defective cystic fibrosis transmembrane conductance regulator (CFTR) protein, which corrects the gating defect and increases ion-function of activated cell-surface CFTR. Bacteria also regulate their physiology through ion channels. However, little is known about the potential effects of ivacaftor on bacterial ion channels, which, in turn, may have a potential effect on transport across the bacterial cell membrane. Therefore, any change in the ability to transport molecules across cell membranes in bacteria could have an important impact on bacterial transport physiology. One area where this could be particularly important is in the movement of antibiotics, both into and out of the bacterial cell. An in vitro study was therefore performed to examine the influence of ivacaftor at therapeutic concentration on antibiotic susceptibility of 11 commonly used anti-pseudomonal antibiotics against a population of clinical Pseudomonas aeruginosa [PA], from CF and non-CF sources. METHOD: Pseudomonas aeruginosa (n = 80; including 70 ivacaftor-naïve clinical PA from sputa from adult CF patients and 10 control PA from non-CF clinical blood culture sources) were examined. Antibiotic susceptibility was determined by standard disc diffusion assay using CLSI criteria and measuring zone size (mm), against four classes of anti-pseudomonal antibiotics, including beta-lactams (temocillin, ceftazidime, piperacillin/tazobactam, imipenem, meropenem and aztreonam), aminoglycosides (gentamicin, tobramycin, amikacin), fluoroquinolone (ciprofloxacin) and polymyxin (colistin), in the absence and presence of ivacaftor (5 µmol/L), as previously determined. In addition, all CF and non-CF PA were examined phenotypically in vitro, as previously described, for changes linked to bacterial virulence, including (i) growth density (ii) pigmentation, (iii) presence of adhesins and (iv) change to mucoidy, in the presence/absence of ivacaftor at therapeutic concentration. RESULTS AND DISCUSSION: Antibiotic susceptibility did not decrease significantly with any of the antibiotics examined with CF PA isolates or with non-CF PA control organisms. There was a statistically significant increase in zone size (CF PA and amikacin, gentamicin, temocillin and ciprofloxacin; Non-CF PA and amikacin, gentamicin and aztreonam). However, at a population level, this did not translate into a shift in CLSI category to a more susceptible phenotype. None of the PA isolates examined were susceptible to ivacaftor alone, and additionally, no changes were noted with the four phenotypic parameters examined in the presence of ivacaftor. WHAT IS NEW AND CONCLUSION: This study showed that antibiotic susceptibility of commonly used anti-pseudomonal antibiotics was not negatively affected by ivacaftor, in a population of ivacaftor-naive P. aeruginosa.

Pseudomonas aeruginosa in cystic fibrosis patients with c.1652G>A (G551D)-CFTR treated with ivacaftor-Changes in microbiological parameters.

WHAT IS KNOWN AND OBJECTIVE: The CFTR potentiator, ivacaftor (IVA), has been widely used in the treatment of cystic fibrosis (CF) patients with the G551D mutation. To date, there has been limited information on the microbiological status of patients on this therapy and no data on the effect (if any) on the in vivo antibiotic susceptibility of Pseudomonas aeruginosa isolated from patients on therapy. Although IVA intervention is not designed per se as anti-infective, the effect (if any) of this molecule to CF patients' microbiological status merits careful monitoring. Therefore, it was the aim of this observational study to examine the effect in patients, both before and after commencement of IVA therapy, on several commonly reported microbiological markers in CF patients, including (i) bacterial density, (ii) frequency (rate) of isolation of bacterial pathogens, particularly P. aeruginosa, and (iii) antimicrobial susceptibility of these isolates to commonly prescribed oral and iv antibiotics. In addition, we wished to examine the requirements for these antibiotics in CF patients, before and after commencement of IVA therapy. METHODS: Archived data from 15 adult cystic fibrosis patients with the c.1652G>A (G551D) mutation were followed from two years pre-IVA therapy to two years after commencement of IVA therapy. The microbiological parameters examined included (i) oral antibiotic courses taken, (ii) intravenous (iv) antibiotic courses taken, (iii) rate of isolation of non-mucoid Pseudomonas aeruginosa (NM-PA) and mucoid P. aeruginosa (M-PA), (iv) density of NM-PA and M-PA and (v) antimicrobial susceptibility of NM-PA and M-PA to 11 antibiotics [aminoglycosides, beta-lactams, polymyxin and fluoroquinolone]. RESULTS AND DISCUSSION: Following commencement of IVA therapy, patients required less iv antibiotic courses but no change in number of oral antibiotics courses. There was significant reduction in both the rate of isolation and density of M-PA (P = .02; P = .006, respectively). In contrast, there was no significant reduction in both the rate of isolation and density of NM-PA (P = .90; P = .07, respectively). Antimicrobial susceptibility in NM-PA and M-PA was not significantly reduced within any of the antibiotics classes or individual antibiotics examined. Increased susceptibility was noted in the beta-lactam class for NM-PA and M-PA, in particular with ceftazidime. WHAT IS NEW AND CONCLUSION: Overall, (i) the requirement for less iv antibiotic therapy, (ii) a reduction in the rate and density of M-PA and (iii) no reduction in antibiotic susceptibility indicate that microbiological parameters with patients on IVA therapy were not detrimentally affected.

Molecular identification and characterisation of catalase and catalase-like protein genes in urease-positive thermophilic Campylobacter (UPTC).

BACKGROUND: Thermophilic Campylobacter are important bacterial pathogens of foodborne diseases worldwide. These organisms' physiology requires a microaerophilic atmosphere. To date, little is known about the protective catalase mechanism in urease-positive thermophilic campylobacters (UPTC); hence, it was the aim of this study to identify and characterise catalase and catalase-like protein genes in these organisms. MATERIALS AND METHODS: Catalase (katA) and catalase (Kat)-like protein genes from the Japanese UPTC CF89-12 strain were molecularly analysed and compared with C. lari RM2100 and other C. lari and thermophilic Campylobacter reference isolates. RESULTS: A possible open reading frame of 1,422 base pairs, predicted to encode a peptide of 474 amino acid residues, with calculated molecular weight of 52.7 kilo Daltons for katA, was identified within UPTC CF89-12. A probable ribosome binding site, two putative promoters and a putative ρ-independent transcription terminator were also identified within katA. A similar katA cluster also existed in the C. lari RM2100 strain, except that this strain carries no DcuB genes. However, the Kat-like protein gene or any other homologue(s) were never identified in the C. lari RM2100 strain, or in C. jejuni and C. upsaliensis. CONCLUSIONS: This study demonstrates the presence of catalase/catalase-like protein genes in UPTC organisms. These findings are significant in that they suggest that UPTC organisms have the protective genetic capability of helping protect the organisms from toxic oxygen stress, which may help them to survive in physiologically harsh environments, both within human and animal hosts, as well as in the natural environment.

Reliability of self-reporting of antibiotic consumption in the community - Index of Reliability.

WHAT IS KNOWN AND OBJECTIVE: To date, there is no evidence to indicate the reliability of how patients self-report their own antibiotic usage in the community. Such data are fundamental in supporting antimicrobial stewardship practices, and so there is a need to determine its accuracy and reliability. COMMENT: Patients in the community (n = 476) were required to recollect their antibiotic usage in the past three months. Simultaneously, similar information was obtained by careful extraction from their respective medical notes, which was qualitatively compared with the patient's recollection. Overall, concordance was high (88·1%), but age (<20 and >80 years) and sex (female) were significant factors of reliability. WHAT IS NEW AND CONCLUSION: This study suggests that basic self-reporting of antibiotic usage amongst patients is relatively reliable, with increasing accuracy with years until 80 years. Where such information is critical, the current study can help decide who to interview and whose notes to interrogate, in the quest to obtain reliable and accurate information.

Construction and expression of a recombinant urease gene cluster from Campylobacter sputorum biovar paraureolyticus.

Recombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureE and ureF) were constructed in vitro from the Campylobacter sputorum biovar paraureolyticus LMG17591 strain and expressed in Escherichia coli JM109 cells. A urease-positive reaction (1.885 micromol/min/mg protein) in the log-phase cultured E. coli cells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE- and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. coli cell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureE gene in the transformed and log-phase cultured E. coli cells from the C. sputorum showed positively accelerated urease activities when cultured in the medium containing NiCl2 (750 micromol/L), but no activity was accelerated in the C. sputorum cultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorum examined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease alpha (A) and beta (B), raised against Helicobacter pylori.

Molecular characterisation of a type III restriction-modification system in Campylobacter upsaliensis.

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.

Molecular structural analysis of major outer membrane protein (MOMP) gene clusters in Campylobacter lari.

Southern hybridisation shows that urease-negative (UN) Campylobacter lari JCM2530(T) carries two putative major outer membrane protein (MOMP) genes. Sequences of approximately 2.1 kbp, encoding non-coding (NC) regions, with possible open reading frames (ORFs) for MOMP (porA1 or porA2) of approximately 1.2 kbp, NC regions and partial and putative Cla_0435 or Cla_1109 ORFs were identified in all five UN C. lari isolates examined, following polymerase chain reaction (PCR) cloning and sequencing. Each putative MOMP structural gene carried start and stop codons and ribosome binding sites of 1236-1278 bp in length. The putative sigma70 transcriptional promoter and the hypothetical rho-independent transcription terminator structures were also seen. Using Northern hybridisation, there was in vivo monocistronic MOMP gene transcription. In addition, in a Japanese urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain, the porA1 gene locus, including an extra gene (approximately 2000 bp in length) was identified. The extra gene may occur within the porA1 gene locus in the eight UPTC isolates of the 23 C. lari isolates examined. Thus, a genetic heterogeneity occurred within the porA1 gene locus from some of the C. lari organisms including the UPTC CF89-12.

Construction, expression and characterisation of recombinant molecules of the urease gene operon from a urease-positive thermophilic Campylobacter (UPTC) isolate.

A recombinant molecule of the full-length urease gene operon was constructed in vitro from the Japanese urease-positive thermophilic Campylobacter (UPTC) CF89-12 isolate and expressed in Escherichia coli cells. Several large deletion recombinant variants of urease subunit genes were also constructed and expressed in E. coli cells. A positive urease reaction with the log-phase cultured E. coli JM109 cells in the NiCl2-containing medium transformed with pGEM-T vector carrying the recombinant molecule of the full-length operon was detected with isopropyl-beta-D-thiogalactoside. Among the several deletion recombinant variants, each ureA-, ureB-, ureE-, ureF-, ureG- and ureH-large deficient, only ureE-large deletion variant (63% deficient) showed a positive urease reaction (approximately 15-fold). In addition, a ureE-complete deletion recombinant variant (100% deficient) constructed also showed a positive reaction of urease (approximately 18-fold). Recombinant urease subunits A and B were immunologically identified by Western blot analysis with anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

Biochemical characterisation of urease from urease-positive thermophilic campylobacter (UPTC).

This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

Antibacterial effects on acinetobacter species of commonly employed antineoplastic agents used in the treatment of haematological malignancies: an in vitro laboratory evaluation.

Although about 75-80% of neutropenic fevers are thought to be caused by infections, a causal organism can be confirmed microbiologically or suspected clinically in only 30-50%, and even fewer of these cases (16%) have a documented bacteraemia. The cause of neutropenic fever in the remaining cases remains elusive. The reasons for this failure may be due to the difficulty in recovering low numbers of organisms, fastidious organisms which fail to grow using conventional culture media, the presence of non-culturable organisms, or the presence of inhibitory substances in specimens. Previously, the authors showed the presence of Acinetobacter in peripheral blood of febrile neutropenic patients with a haematological malignancy, using 16S rDNA polymerase chain reaction (PCR) and sequencing techniques. However, conventional culture was unable to detect these organisms. Hence, it was felt necessary to examine the antibacterial properties of four antineoplastic agents used in the treatment of haematological malignancy, namely bleomycin, cisplatin, doxorubicin and vincristine. A total of 56 wild-type Acinetobacter including seven species (A. calcoaceticus [n=17], A. septicus [n=11], A. baumannii [n=10], A. johnsonii [n=7], A. lwoffii [n=8] A. haemolyticus [n=2] and A. radioresistens [n=1]) were examined for their susceptibility to the four antineoplastic agents at therapeutic concentration. No inhibition was observed, but inhibition was seen at higher concentrations of both bleomycin and doxorubicin. Time to detection of blood culture bottles containing separate antineoplastic agents (i.e., bleomycin and doxorubicin) was compared to that containing saline using a paired t-test. Samples containing doxorubicin at 1 pg/mL were shown to have a mean time to detection of 21.8 h (range: 15.6-31.4 h). Bottles containing saline had a mean time to detection of 22.9 h (range: 18.2-31.3 h). Statistical analysis showed no significant difference (P=0.3361) between time to detection for blood culture bottles containing doxorubicin at achievable plasma concentration and corresponding negative controls. With regard to bleomycin (300 miu/mL), the mean time to detection was 27.29 h (range: 20.2-38.4 h) in the test bottles, with mean time to detection in the saline negative controls of 22.56 h (range: 17.0-30.1 h). Paired t-test gave P=0.000451, hence a significant difference in time to detection for blood cultures containing therapeutic levels of bleomycin. Overall, the antineoplastic agents vincristine, cisplatin or doxorubicin did not have any inhibitory effects on the Acinetobacter organisms examined. At worst, therapeutic concentrations of bleomycin may delay automated detection of an Acinetobacter bacteraemia by a mean time of 5.9 h.

Expression and analysis of a cytolethal distending toxin (cdt) gene operon in Campylobacter lari.

The present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari (n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter [UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lari isolates using a primer pair to amplify the cdtB gene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtB and cdtC gene operon was identified to be polycistronicly transcribed in the C. lari cells. The cdtB gene translation in the C. lari cells was also confirmed by Western blot analysis. Thus, the cdt gene operon in C. lari organisms, including UN C. lari and UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdt genes may be functional in the cells.

Exposure to clinical X-ray radiation does not alter antibiotic susceptibility or genotype profile in gram-negative and gram-positive clinical pathogens.

Inadvertent exposure of bacterial pathogens to X-ray radiation may be an environmental stress, where the bacterium may respond by increasing mutational events, thereby potentially resulting in increased antibiotic resistance and alteration to genotypic profile. In order to examine this, four clinical pathogens, including the Gram-negative organisms Escherichia coli O157:H7 NCTC12900 and Pseudomonas aeruginosa NCTC10662, as well as the Gram-positive organisms Staphylococcus aureus NCTC6571 and Enterococcus faecium were exposed to X-rays (35,495 cGy/cm2) over a seven-day period. Antibiotic susceptibility was assessed before, during and after exposure by examining susceptibility, as quantified by E-test with six antibiotics, as well as to a further 11 antibiotics by measurement of susceptibility zone sizes (mm). Additionally, the DNA profile of each organism was compared before, during and after exposure employing the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC PCR). Results indicated that exposure of these organisms to this amount of X-ray radiation did not alter their antibiotic susceptibility, nor their genomic DNA profile. Overall, these data indicate that exposure of bacteria to X-ray radiation does not alter the test organisms' antibiotic susceptibility profiles, nor alter genomic DNA profiles of bacteria, which therefore does not compromise molecular epidemiological tracking of bacteria within healthcare environments in which patients have been exposed to X-ray radiation.

Comparative analysis of Campylobacter lari cytolethal distending toxin (CDT) effect on HeLa cells.

We aimed to clarify if Campylobacter lari exerts a cytolethal distending toxin (CDT) effect on HeLa cells. Campylobacter cell lysates (CCLys) from C. jejuni 81-176 and urease-positive thermophilic Campylobacter (UPTC) CF89-12 and UPTC NCTC12893 isolates were shown to exert a CDT effect on HeLa cells with morphological changes examined by Giemsa staining and microscopy. However, Campylobacter lari JCM2530(T) isolate showed no effect. In addition, Campylobacter cell culture supernatant wash gave low or absent toxic effects with both C. jejuni and C. lari organisms. When western blot analysis was carried out to clarify if there was a CDTB effect in the CCLys and soluble fractions from Campylobacter isolates, which had a CDT effect on HeLa cells or did not have any effect, anti-recombinant CjCDTB antibodies identified an immunoreactively positive signal at around approximately 25 kDa on all the C. lari isolates examined, as well as the C. jejuni 81116 strain. Thus, all the Campylobacter isolates including those without any CDT effect were shown to express CDTB at the translational level.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

Molecular analysis and characterisation of the full-length flagellin C gene (flaC) from Campylobacter lari.

A degenerate polymerase chain reaction (PCR) primer pair (f-ClflaC/r-ClflaC) was constructed in silico to amplify flaC and its adjacent genetic loci from Campylobacter lari isolates. Approximately 1.45 kbp amplicons, including the sequences encoding the flaC structural gene of 750 bp, putative promoter, rho-independent intrinsic terminator regions and partial sequences of two putative open reading frames (ORFs), immediately upstream and downstream of the gene, were identified in 16 C. lari isolates (four urease-negative [UN] C. lari; 12 urease-positive thermophilic campylobacters [UPTC)]). All 16 flaC structural genes commenced with an ATG start codon and terminated with a TAA stop codon and probable ribosome-binding sites were identified in all 16 isolates. These probably indicate a monocistronic operon structure for the flaC gene in C. lari isolates. In addition, the putative flaC gene ORFs were deduced to be similar in 747 bp among all 26 thermophilic Campylobacter isolates examined, resulting in a similar calculated molecular weight of approximately 26.6-26.9 kDa. The flaC from C. lari was different from the flaA-like sequence and the shorter flaA of UPTC isolates found previously. Reverse transcription PCR and Northern blot hybridisation analyses identified flaC transcription in C. lari cells. The transcription initiation site for the flaC gene was also determined by primer extension analysis. A dendrogram constructed, based on the nucleotide sequence information of flaC from 17 C. lari isolates, demonstrated that the C. lari isolates were genetically variable and formed two minor clusters for UN C. lari and UPTC.

Reliability of a multiplex PCR assay for the identification of the major Campylobacter taxa.

The primer pair (C412F/C1228R) constructed previously for the polymerase chain reaction (PCR) identification of the genus Campylobacter using an approximate 800 base pair (bp) 16S rRNA gene target segment proved to be useful for the identification of a total of 49 Campylobacter lari isolates including urease-positive thermophilic Campylobacter (UPTC) organisms (n=25). When the primer pair (CLF/R) developed previously for the PCR identification of C. lari species using an approximate 250 bp glyA segment was employed, 27 C. lari isolates, including all the UPTC isolates, were identified to be PCR-negative (55%). Therefore, this PCR procedure developed for the molecular identification of C. lari was shown to be unreliable for C. lari identification. Nucleotide sequencing analysis clarified the reason(s) why PCR-negative examples occurred in many C. lari isolates, including UPTC isolates. The primer pair target sequences in the C. lari-specific PCR-negative isolates apparently varied at the 3' end region, as compared with C. lari-specific PCR-positive isolates. Thus, the multiplex PCR assay developed previously was shown to be unreliable for the molecular identification of C. lari subspecies organisms.