Beverley C. Millar

Three Drinking- Water-Associated Cryptosporidiosis Outbreaks, Northern Ireland

Three recent drinking-water–associated cryptosporidiosis outbreaks in Northern Ireland were investigated by using genotyping and subgenotyping tools. One Cryptosporidium parvum outbreak was caused by the bovine genotype, and two were caused by the human genotype. Subgenotyping analyses indicate that two predominant subgenotypes were associated with these outbreaks and had been circulating in the community.

Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction

Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Postamplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C. muris, C. baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted. The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 10(3)-10(4) -fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10(1)-10(3) oocysts, depending on water turbidity. Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.

Emerging issues in infective endocarditis.

Infective endocarditis, a serious infection of the endocardium of the heart, particularly the heart valves, is associated with a high degree of illness and death. It generally occurs in patients with altered and abnormal heart architecture, in combination with exposure to bacteria through trauma and other potentially high-risk activities involving transient bacteremia. Knowledge about the origins of endocarditis stems from the work of Fernel in the early 1500s, and yet this infection still presents physicians with major diagnostic and management dilemmas. Endocarditis is caused by a variety of bacteria and fungi, as well as emerging infectious agents, including Tropheryma whiplei, Bartonella spp., and Rickettsia spp. We review the evolution of endocarditis and compare its progression with discoveries in microbiology, science, and medicine.

Improved molecular detection ofBurkholderia cepacia genomovar III and Burkholderia multivorans directly from sputum of patients with cystic fibrosis

Optimum detection of the Burkholderia cepacia complex (BCC) from sputum of patients with cystic fibrosis (CF) is essential in preventing patient-to-patient transmission of this organism. The aim of this study was to develop an improved PCR assay with reference to sensitivity for the direct detection of BCC organisms from CF sputum employing the recA locus. The sensitivity results of three recA PCR assays were compared using various combinations of previously published primers. These included (i) a single-round approach using the primer set BCR1/BCR2, yielding a 1036-bp product, (ii) a single-round approach using the primer set BCR1/Mr, yielding a 465-bp product, and (iii) a semi-nested PCR (SN-PCR) approach using the primer set BCR1/BCR2 followed by BCR1/Mr. The sensitivity of these assays were determined by spiking B. cepacia-free sputum with known numbers of four strains of BCC, namely, genomovar II [B. multivorans] (C1576), genomovar IIIa (C5424, C6433) and genomovar IIIb (C1394). Following optimization, the chosen assay was performed on 14 patients. Employment of the single-round assay with BCR1/BCR2 was the least sensitive with a detection threshold of 10(7) cfu/g sputum for GIIIa and GIIIb, and 10(8) cfu/g sputum for GII. Sensitivity was improved by targeting the smaller amplification region of the recA locus (465 bp) employing the BCR1/Mr primer pair, in combination with a single-round approach, whereby the detection threshold was improved by 1 log for each genomovar. Employment of the semi-nested assay demonstrated optimum sensitivity, whereby the detection threshold increased to 10(1) and 10(2) cfu/g sputum for genomovar IIIa/IIIb and genomovar II, respectively. Subsequent genomovar characterisation can be performed by sequencing of the PCR amplicon without the need for culture which may be beneficial in patients in the initial stages of colonisation or who are transiently colonised and who may be culture-negative for BCC.

Pandoraea apista isolated from a patient with cystic fibrosis: problems associated with laboratory identification.

A 17-year-old male patient with cystic fibrosis (CF), with CF mutation homozygous ∆F508, was admitted to the Royal Belfast Hospital for Sick Children in August 2001 for a course of intravenous antibiotics to treat a chronic pulmonary infection. He had an 11-year history of pulmonary infection with both a mucoid and a non-mucoid Pseudomonas aeruginosa, which had remained relatively sensitive to several antibiotics used routinely in the treatment of P. aeruginosa CF chest infections, including gentamicin, tobramycin, aztreonam, ceftazidime, ciprofloxacin, piperacillin/tazobactam, imipenim and meropenem. During that time he was not reported to have been colonised/infected with any other bacterial organism in his respiratory tract. On admission, an unidentified Gram-negative rod was isolated in addition to his chronic strains of mucoid and nonmucoid P. aeruginosa. This new organism was isolated from freshly expectorated sputum as the sole organism type on Burkholderia cepacia selective agar (BCSA; MAST Diagnostics Ltd, Merseyside, UK) following incubation at 37 ̊C for 48 h. As this was a new isolate growing on BCSA, it was sent for molecular identification and characterisation. Subsequently, this organism was isolated from the patient’s sputum on five occasions over a two-month period, and presently remains a part of the established resident bacterial flora. Semi-quantitative determination of organism numbers demonstrated that it was consistently present at the ++ level, approximating to 10 10 colony forming units(cfu)/g sputum. Clinically, since the first isolation of this organism from the patient’s sputum, there has been deterioration in his clinical status, including increased cough, chest tightness, wheeze, shortness of breath, production of purulent sputum, fatigue and weight loss of 1.8 kg over this two-month period. In addition, his forced vital capacity (FVC) fell from 89% to 77% predicted. Drug therapy during this period included a twoweek course of intravenous (iv) antibiotics (ceftazidime and tobramycin), as well as an oral course of ciprofloxacin and nebulised colomycin. He also received a course of oral steroids and itraconazole during this period, as there was some evidence of allergic bronchopulmonary aspergillosis (ABPA). Microbiologically, this organism was poorly identified (48% identification) phenotypically by the API 20NE scheme as Alcaligenes faecalis, with the profile 0000457. The organism was resistant in vitro by standard NCCLS disc diffusion assay to gentamicin, temocillin, ceftazidime, azlocillin, meropenem, aztreonam and colistin, and was sensitive to tobramycin, piperacillin/tazobactam, imipenem and ciprofloxacin. Subsequently, in order to aid identification, the organism was examined using molecular techniques. All DNA isolation procedures were carried out in a Class II biological safety cabinet in a room geographically separate from that used to set up reaction mixes and also from the ‘post PCR’ room, in order to minimise false-positive results and in accordance with good molecular diagnostic practice (GMDP), as detailed in the guidelines of Millar et al. DNA was extracted from a single colony using the Roche High Purity PCR Template kit (Roche Diagnostics Ltd, UK), following the manufacturer’s instructions. All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA, and from the amplification and post-PCR room, in order to minimise contamination. Reaction mixes (50 μL) were set up as follows: 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl2, 200 μmol/L (each) dATP, dCTP, dGTP and dTTP, 1.25 units Thermus aquaticus (Taq) DNA polymerase (Amplitaq; Perkin Elmer), 0.2 μmol/L (each) of the 16S rRNA primers P11P (forward) 5’ GAG GAA GGT GGG GAT GAC GT -3’ and P13P (reverse) 5’ AGG CCC GGG AAC GTA TTC AC -3’, as previously described, and 4 μL DNA template. Following a hot start, the reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 96 ̊C for 3 min, followed by 40 cycles of 96 ̊C for 1 min, 55 ̊C for 1 min, 72 ̊C for 1 min, and a final extension at 72 ̊C for 10 min. During each run, molecular-grade water was included randomly as a negative control and appropriate DNA template from Staphylococcus aureus was included as a positive control. Following amplification, samples (15 μL) were removed from each reaction mixture and examined by electrophoresis (80 V, 45 min) in gels composed of 2% (w/v) agarose (Gibco, UK) in TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L EDTA [pH 8.3]), stained with ethidium bromide (5 μg/100 mL). Gels were visualised under ultraviolet illumination, using a gel image analysis system (UVP Products, England), and all images archived as digital (*.bmp) graphic files. Subsequently, amplicons were purified (particularly to remove dNTPs, polymerases, salts and primers) using a QIAquick PCR purification kit (Qiagen Ltd., UK) and eluted in Tris-HCl (10 mmol/L [pH 8.5]) prior to sequencing. Cy-5’labelled primer (P11P) was prepared and used for sequencing in the forward direction with the ALF Express II (Amersham-Pharmacia Ltd., Bucks, UK) employing the Thermo Sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, UK; cat no: RPN 2438). Thermal cycling parameters were: 96 ̊C for 1 min, followed by 25 cycles of 96 ̊C for 10 sec, 50 ̊C for 5 sec, 60 ̊C for 5 sec, followed by a 4 ̊C hold. Sequences obtained were compared with those stored in the GenBank data system, using BLAST alignment software

Occurrence of Cryptosporidium parvum and bacterial pathogens in faecal material in the red fox (Vulpes Vulpes) population

Y. Nagano1, M.B. Finn2, C.J. Lowery2, T. Murphy3, J. Moriarty4, E. Power5, D. Toolan6, A. O’Loughlin7, M. Watabe1, K.A. McCorry1, E. Crothers1, J.S.G. Dooley2, J.R. Rao8, P.J. Rooney1, B.C. Millar1, M. Matsuda9, J.S. Elborn1 and J.E. Moore1∗ 1Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast; 2School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK; 3Department of Parasitology, Department of Agriculture and Food, Central State Veterinary Laboratory, Abbottstown, Castleknock, Dublin; 4Regional Veterinary Laboratory, Department of Agriculture and Food, Dublin; 5Regional Veterinary Laboratory, Department of Agriculture and Food, Cork; 6Regional Veterinary Laboratory, Department of Agriculture and Food, Kilkenny; 7Regional Veterinary Laboratory, Department of Agriculture and Food, Limerick, Ireland; 8Applied Plant Science Research Division, Agri-Food and Bioscience Institute (AFBI), Belfast, Northern Ireland; 9Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Kanagawa, Japan ∗Correspondence: E-mail: jemoore@niphl.dnet.co.uk

High diversity of bacterial pathogens and antibiotic resistance in salmonid fish farm pond water as determined by molecular identification employing 16S rDNA PCR, gene sequencing and total antibiotic susceptibility techniques

The aim of this study was to examine the microbiological and related parameters (antibiotic resistance and pathogen identification) of water at two salmonid fish farms in Northern Ireland. Total Bacterial Counts at the Movanagher Fish Farm was 1730 colony forming units (cfu)/ml water (log10 3.24cfu/ml) and 3260cfu/ml (log10 3.51cfu/ml) at the Bushmills Salmon Station. Examination of resulting organisms revealed 10 morphological phenotypes, which were subsequently sequenced to determine their identification. All these organisms were Gram-negative and no Gram-positive organisms were isolated from any water sample. From these phenotypes, eight different genera were identified including Acinetobacter, Aeromonas, Chryseobacterium, Erwinia, Flavobacterium, Pseudomonas and Rheinheimera. One unnamed novel taxon was identified from water at the Movanagher Fish Farm, belonging to the genus Acinetobacter and has been tentatively named Acinetobacter movanagherensis. No other novel taxa were observed. All but one of these environmental organisms (Erwinia) are potential pathogens of fish disease. Total antibiotic resistance was observed to varying degrees in water specimens. The most resistant populations were observed in water taken from the Bushmills Salmon Station inlet, followed by water from the Movanagher Fish Farm. No resistance was observed against tetracycline and there was only one occurrence of resistance against ciprofloxacin. Overall, this study indicates that potential fish pathogens made up the majority of environmental organisms identified, even in the absence of recorded fish disease. There was also relatively high levels of total antibiotic resistance in the bacterial water populations examined, where tetracycline was the only antibiotic with zero resistance. These data indicate that the threat of bacterial disease is relatively close due to the indigenous colonization of farm water and that husbandry standards should be maintained at a high standard to avert bacterial disease outbreaks, rather than relying on the absence of specific pathogens in the immediate farm environment.

Identification and characterization of intervening sequences within 23S rRNA genes from more than 200 Campylobacter isolates from seven species including atypical campylobacters

BackgroundIdentification and characterization of intervening sequences (IVSs) within 23S rRNA genes from Campylobacter organisms including atypical campylobacters were carried out using two PCR primer pairs, designed to generate helix 25 and 45 regions.ResultsOnly C. sputorum biovar sputorum LMG7975 and fecalis LMG8531, LMG8534 and LMG6728 of a total of 204 Campylobacter isolates (n = 56 C. jejuni; n = 11 C. coli; n = 33 C. fetus; n = 43 C. upsaliensis; n = 30 C. hyointestinalis; n = 4 C. sputorum biovar sputorum; n = 5 C. sputorum biovar fecalis; n = 5 C. sputorum biovar paraureolyticus; n = 10 C. concisus; n = 7 C. curvus) were shown to carry IVSs in helix 25 region. C. sputorum biovar fecalis LMG8531 and LMG8534, interestingly, carried two different kinds of the 23S rRNA genes with and without the IVS, respectively. Consequently, in a total of 265 isolates of 269, including 65 C. lari isolates examined previously, the absence of IVSs was identified in the helix 25 region. In the helix 45 region, all the C. hyointestinalis, C. sputorum and C. concisus isolates were shown not to carry any IVSs. However, the 30 of 56 C. jejuni isolates (54%), 5 of 11 C. coli (45%), 25 of 33 C. fetus (76%), 30 of 43 C. upsaliensis (70%) and 6 of 7 C. curvus (90%) were shown to carry IVSs. In C. jejuni and C. upsaliensis isolates, two different kinds of the 23S rRNA genes were also identified to occur with and without IVSs in the helix 45 region, respectively.ConclusionsSecondary structure models were also constructed with all the IVSs identified in the present study. In the purified RNA fractions from the isolates which carried the 16S or 23S rRNA genes with the IVSs, no 16S or 23S rRNA was evident, respectively.

Molecular Characterization of Intervening Sequences in 23S rRNA Genes and 23S rRNA Fragmentation in Taylorella equigenitalis

Using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) of the 23S rRNA gene sequences of the genus Taylorella, none of the three representative T. equigenitalis strains NCTC11184T, Kentucky 188 and EQ59 was shown to contain any IVSs in the first quarter region. In the central region, all three strains possessed one ≈70 bp IVS (TeIVS2) different from any IVSs found in T. asinigenitalis. The predicted secondary structure model of the IVSs contained stem and loop structures. The central region of the IVS-stem structure contains an identical double-stranded consensus 15-bp sequence. The purified RNA fraction from the three strains contained 16S and 4-5S RNA species but no 23S rRNA species. Thus, the primary 23S rRNA transcripts from the three strains would be cleaved into approximately 1.2- and 1.6-kb rRNA fragments and ≈70-bp IVS. In addition, 16 other T. equigenitalis isolates were found to carry a similar 70-bp IVS in the central region and to produce fragmented 23S rRNA.

Investigation of infection with Campylobacter jejuni in a man with hypogammaglobulinaemia using PCR-single-strandedconformational polymorphism (PCR-SSCP) typing

This study investigated several episodes of infection of Campylobacter jejuni in an immunocompromised male with hypogammaglobulinaemia, presenting with diarrhoea and bacteraemia over a 16-month period, by employing three phenotyping and four genotyping schemes, including the single-stranded conformational polymorphism (SSCP) technique to establish if infection was reinfection or persistent infection. Four isolates from blood culture and two faecal isolates of Campylobacter jejuni were obtained from the patient by direct selective plating on Skirrow Selective agar. Isolates were characterised at the sub-species level by Penner serology, Preston biotyping, Preston phage-typing, as well as E3CJC2 restriction fragment length polymorphism (RFLP), 16S ribotyping, flagellin (flaA) RFLP and single-stranded conformational polymorphism (SSCP) analyses. Phenotyping and genotyping sub-species analyses demonstrated that the patient was infected with at least two different strains of Campylobacter jejuni, i. e. one strain that persisted throughout the 16-month period and another strain that was transient suggesting reinfection from a different source. SSCP analysis was the most discriminatory of all the typing schemes examined and demonstrated an altered genotype of the persistent strain, whereby there were subtle modifications to the hypervariable regions of the flaA gene. Overall, as SSCP examines the hypervariable region of the flaA gene and as this technique can detect point mutations, differences between SSCP banding patterns may represent markers and thus examine mutations that occur under immune selection, thereby permitting the C. jejuni to evade the host immune response. In conclusion, this study describes the novel use of SSCP genotyping of C. jejuni and demonstrated that this method is a highly discriminatory technique which may be beneficial in outbreak characterisation, but which is not suitable to examine the clonal patterns of C. jejuni over a long period of time.