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The aim of this review was to provide an update on the complex relationship between manure application, altered pathogen levels and antibiotic resistance. This is necessary to protect health and improve the sustainability of this major farming practice in agricultural systems based on high levels of manure production. It is important to consider soil health in relation to environment and land management practices in the context of the soil microflora and the introduction of pathogens on the health of the soil microbiome. Viable pathogens in manure spread on agricultural land may be distributed by leaching, surface run-off, water source contamination and contaminated crop removal. Thus it is important to understand how multiple pathogens can persist in manures and on soil at farm-scale and how crops produced under these conditions could be a potential transfer route for zoonotic pathogens. The management of pathogen load within livestock manure is a potential mechanism for the reduction and prevention of outbreaks infection with Escherichia coli, Listeria Salmonella, and Campylobacter. The ability of Campylobacter, E. coli, Listeria and Salmonella to combat environmental stress coupled with their survival on food crops and vegetables post-harvest emphasizes the need for further study of these pathogens along with the emerging pathogen Providencia given its link to disease in the immunocompromised and its’ high levels of antibiotic resistance. The management of pathogen load within livestock manure has been widely recognized as a potential mechanism for the reduction and prevention of outbreaks infection but any studies undertaken should be considered as region specific due to the variable nature of the factors influencing pathogen content and survival in manures and soil. Mediocre soils that require nutrients could be one template for research on manure inputs and their influence on soil health and on pathogen survival on grassland and in food crops.

Nasal pathogen detection sensitivities can be as low as 70% despite advances in molecular diagnostics. This may be linked to the choice of sampling method. A diagnostic test accuracy review for sensitivity was undertaken to compare sensitivity of swabbing to the nasopharynx and extracting nasal aspirates, using the PRISMA protocol, Cochrane rapid review methodology, and QUADAS-2 risk of bias tools, with meta-analysis of included studies. Sensitivities were calculated by a consensus standard of positivity by either method as the ‘gold standard.’ Insufficient sampling methodology, cross sectional study designs, and studies pooling samples across anatomical sites were excluded. Of 13 subsequently eligible studies, 8 had ‘high’ risk of bias, and 5 had ‘high’ applicability concerns. There were no statistical differences in overall sensitivities between collection methods for eight different viruses, and this did not differ with use of PCR, immunofluorescence, or culture. In one study alone, Influenza H1N1(2009) favored nasopharyngeal swabs, with aspirates having 93.3% of the sensitivity of swabs (p > 0.001). Similarly equivocal sensitivities were noted in reports detecting bacteria. The chain of sampling, from anatomical site to laboratory results, features different potential foci along which sensitivity may be lost. A fair body of evidence exists that use of a different sampling method will not yield more respiratory pathogens.

The nasopharyngeal microbiome is a dynamic microbial interface of the aerodigestive tract, and a diagnostic window in the fight against respiratory infections and antimicrobial resistance. As its constituent bacteria, viruses and mycobacteria become better understood and sampling accuracy improves, diagnostics of the nasopharynx could guide more personalized care of infections of surrounding areas including the lungs, ears and sinuses. This review will summarize the current literature from a clinical perspective and highlight its growing importance in diagnostics and infectious disease management.

Background The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. Results Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. Conclusions This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

Enterococci and biofilm-associated infections are a growing problem worldwide, given the rise in antibiotic resistance in environmental and clinical settings. The increasing incidence of antibiotic resistance and its propagation potential within enterococcal biofilm is a concern. This requires a deeper understanding of how enterococcal biofilm develops, and how antibiotic resistance transfer takes place in these biofilms. Enterococcal biofilm assays, incorporating the study of antibiotic resistance transfer, require a system which can accommodate non-destructive, real-time experimentation. We adapted a Gene Frame® combined with fluorescence microscopy as a novel non-destructive platform to study the conjugal transfer of vancomycin resistance in an established enterococcal biofilm.A multi-purpose fluorescent in situ hybridisation (FISH) probe, in a novel application, allowed the identification of low copy number mobile elements in the biofilm. Furthermore, a Hoechst stain and ENU 1470 FISH probe identified Enterococcus faecium transconjugants by excluding Enterococcus faecalis MF06036 donors. Biofilm created with a rifampicin resistant E. faecalis (MW01105Rif) recipient had a transfer efficiency of 2.01 × 10−3, double that of the biofilm primarily created by the donor (E. faecalis MF06036). Conjugation in the mixed enterococcal biofilm was triple the efficiency of donor biofilm. Double antibiotic treatment plus lysozyme combined with live/dead imaging provided fluorescent micrographs identifying de novo enterococcal vancomycin resistant transconjugants inside the biofilm. This is a model system for the further study of antibiotic resistance transfer events in enterococci. Biofilms promote the survival of enterococci and reduce the effectiveness of drug treatment in clinical settings, hence giving enterococci an advantage. Enterococci growing in biofilms exchange traits by means of horizontal gene transfer, but currently available models make study difficult. This work goes some way to providing a non-destructive, molecular imaging-based model system for the detection of antibiotic resistance gene transfer in enterococci.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water. HIGHLIGHTS The high cost of current protozoa detection methods limits their widespread use in limited settings.; Immunomagnetic separation improves detection by cleaning the sample.; Recovery efficiency is maintained for Giardia duodenalis with 50% less beads.; Organisms adhering to beads after dissociation may impact recovery levels.

Combating antimicrobial resistance is one of the most serious public health challenges facing society today. The development of new antibiotics or alternative techniques that can help combat antimicrobial resistance is being prioritised by many governments and stakeholders across the globe. Antimicrobial photodynamic therapy is one such technique that has received considerable attention but is limited by the inability of light to penetrate through human tissue, reducing its effectiveness when used to treat deep-seated infections. The related technique sonodynamic therapy (SDT) has the potential to overcome this limitation given the ability of low-intensity ultrasound to penetrate human tissue. In this study, a Rose Bengal–antimicrobial peptide conjugate was prepared for use in antimicrobial SDT (ASDT). When Staphylococcus aureus and Pseudomonas aeruginosa planktonic cultures were treated with the conjugate and subsequently exposed to ultrasound, 5 log and 7 log reductions, respectively, in bacterial numbers were observed. The conjugate also displayed improved uptake by bacterial cells compared with a mammalian cell line ( P ≤ 0.01), whilst pre-treatment of a P. aeruginosa biofilm with ultrasound resulted in a 2.6-fold improvement in sensitiser diffusion ( P ≤ 0.01). A preliminary in vivo experiment involving ASDT treatment of P. aeruginosa -infected wounds in mice demonstrated that ultrasound irradiation of conjugate-treated wounds affects a substantial reduction in bacterial burden. Combined, the results obtained from this study highlight ASDT as a targeted broad-spectrum novel modality with potential for the treatment of deep-seated bacterial infections.

(2007). Environmental persistence of Pseudomonas aeruginosa and Burkholderia multivorans in sea water: preliminary evidence of a viable but non-culturable state. British Journal of Biomedical Science: Vol. 64, No. 3, pp. 129-131.

Background Enterococci are now well recognised for their ability to transfer antibiotic resistance and for their association with nosocomial infections, but less is known regarding their relevance in the wider environment. Enterococcus faecalis and Enterococcus faecium were isolated from a range of agrarian associated sources (low-flow water, septic tank, poultry litter, high flow water, slurry/soil) and were assessed for latent ability to transfer antimicrobial resistance. Results The isolates were tested for phenotypic clumping in the presence of cell-free supernatant from other isolates. Some isolates were identified which demonstrated clumping, indicating that they possessed peptide sex pheromone conjugal machinery. All isolates were also tested for antibiotic resistance phenotypes using both disc diffusion and minimum inhibitory concentration (MIC) assays. These tests revealed that the enterococci demonstrated both phenotypic clumping and antibiotic resistance phenotypes. Based on these selection criteria, the isolates were identified as having the potential for horizontal gene transfer and were used to investigate the transfer of multiple antibiotic resistance phenotypes. Conjugal transfer of antibiotic resistance phenotypes was determined using a solid agar mating method followed by a standard antibiotic selection test resulting in different transfer patterns. An interspecies conjugal transfer of vancomycin resistance from E. faecalis to E. faecium was identified while the remaining reactions were within the same species. Transfer efficiencies ranging from 2 × 10−1 to 2.3 × 10−5 were determined based on the reactions of three donor isolates (MF06036, MF0410 and MF06035) and two recipient isolates (MW01105Rif and ST01109Rif), with the transfer of vancomycin, erythromycin and tetracycline resistance genes. Conclusions The conjugation reactions and selection conditions used in this study resulted in a variety of co-transferred resistance phenotypes suggesting the presence of different mobile elements in the set of natural isolates. This study highlights the potential for extensive horizontal gene transfer in a previously neglected reservoir for enterococci.

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 incr...

The viridans group streptococci (VGS) are an extremely diverse range of organisms within the genus Streptococcus, which are characterized by a greening of the blood in blood agar, on which they are normally grown. Oral streptococci largely comprise members of the VGS, which currently include 20 species, which are commensal inhabitants of the oropharyngeal cavity, and the gastrointestinal and genital tracts of mammals. On the basis of 16S rRNA sequence homology, these bacteria are categorised into four groups: the salivarius rRNA homology group, including Streptococcus thermophilus, Streptococcus vestibularis and Streptococcus salivarius; the mitis group, including Streptococcus cristatus, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus sanguinis and Streptococcus parasanguinis; the anginosus group, including Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius; and the mutans group, including Streptococcus mutans, Streptococcus criceti, Streptococcus downei, Streptococcus ferus, Streptococcus macacae, Streptococcus ratti and Streptococcus sobrinus. This review aims at comparing conventional and molecular detection methods and the emergence of antibiotic resistance within the VGS. (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Control of waterborne gastrointestinal parasites represents a major concern to water industries worldwide. In developed countries, pathogens in drinking water supplies are normally removed by sand filtration followed by chemical disinfection. Cryptosporidium spp. are generally resistant to common disinfection techniques and alternative control strategies are being sought. In the current study, the photocatalytic inactivation of C. parvum oocysts was shown to occur in buffer solution (78.4% after 180 min) and surface water (73.7% after 180 min). Viability was assessed by dye exclusion, excystation, direct examination of oocysts and a novel gene expression assay based on lactate dehydrogenase 1 (LDH1) expression levels. Collectively, this confirmed the inactivation of oocysts and scanning electron microscopy (SEM) confirmed cleavage at the suture line of oocyst cell walls, revealing large numbers of empty (ghost) cells after exposure to photocatalytic treatment.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP). Currently, empirical treatment with quinolones is being used due to the emergence of beta-lactam and macrolide resistance in S. pneumonaie. Although the prevalence of quinolone-resistant S. pneumoniae remains low, increasing numbers of resistant isolates are being seen. Genetic mechanisms leading to fluoroquinolone resistance in pneumococci are complex. This study aims to use molecular methods to characterise all isolates through sequence analysis of their QRDR regions. Thirty-two S. pneumoniae isolates were obtained from nasal swabs from adult and paediatric patients attending local general practices in Northern Ireland. Phenotypic minimum inhibitory concentration (MIC) was determined for Clinical and Laboratory Standards Institute (CLSI) broth microdilution against ciprofloxacin, levofloxacin and norfloxacin. Simultaneously, the QRDR regions of gyrA, gyrB, parC and parE were analysed by sequence typing for all pneumococci obtained. Only one isolate (3.1%) showed reduced susceptibility to ciprofloxacin and levofloxacin. Two amino acid positions were discordant in the S. pneumoniae R6 strain and eight (25%) and 23 (71.9%) isolates contained the mutations Ile460Val in gyrA and Lys137Asn in parC (deposited in GenBank, accession numbers GQ999587-GQ999589), respectively. No mutations were found in either the gyrB or parE loci. In conclusion, the study demonstrated increased fluoroquinolone resistance which could not be accounted for simply through QRDR mutations, and, reciprocally, that mutations in the QRDR region do not necessarily result in overt phenotypic resistance.

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.

Clinical isolates of Aeromonas were grown at eight different temperatures from 10 degrees C to 40 degrees C. Whole cell lysates were examined by SDS-PAGE and major temperature-dependent changes to both protein and lipopolysaccharide (LPS) profiles were identified. Cells grown at the higher temperatures (37 degrees C and 40 degrees C) produced abundantly a protein of c. 60 kDa which was not detected at the lower temperatures. Temperature-dependent expressions of other proteins were also noted but these were more variable among the isolates. An effect of temperature on expression of lipopolysaccharides was also noted in that some strains produced significantly less O-polysaccharides at the higher temperatures. After fractionation of cells, major differences in the expression of cell envelope and outer-membrane proteins between cells grown at low and high temperatures were noted although no unifying patterns could be discerned. Such growth temperature-induced changes in the cell envelope constituents have not been described previously for Aeromonas isolates from man.

Viridans-group streptococci (VGS) consist of several taxa which historically have been highly diverse. However, at times it may become necessary to have a reliable scheme for the identification of these organisms to the species level. The aim of this study is to compare the ability of five gene loci, namely rnpB, 16S rRNA, 16S-23S rRNA, sodA and dnaJ, to speciate such organisms through a sequence typing-based approach. Reference organisms consisting of six VGS species were compared based on sequence typing, followed by comparison of 31 wild-type respiratory isolates, and showed that employment of sequence typing using the rnpB gene locus was the most specific and reliable. Therefore, the use of rnpB sequencing for the identification of VGS to species level is a reliable and feasible option, based on a single gene target.

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.

The aim of this study was to investigate the reliability of disc diffusion testing with penicillin, erythromycin and ciprofloxacin within the viridans group streptococci (VGS). In total, the antibiotic susceptibilities of 167 VGS isolates were compared by standard disc diffusion and broth microdilution methods, and these phenotypic data were compared to the carriage of the respective gene resistance determinants [ermB and mefA/E (macrolides); QRDR, gyrA, gyrB, parC and parE (quinolones)]. Overall, there were 35 discrepancies [resistant by MIC and susceptible by zone diameter (21.0%)] between MIC and disc diameter when penicillin susceptibility was interpreted by Clinical and Laboratory Standards Institute criteria. Scattergrams showed a bimodal distribution between non-susceptible and susceptible strains when erythromycin susceptibility was tested by both methods. Thirty-four (20.4%) isolates were categorized as resistant by MIC breakpoints, while disc diameter defined these as having intermediate resistance. With ciprofloxacin, three isolates (1.8%) showed minor discrepancies between MIC breakpoints and disc diameter. Isolates non-susceptible to all three antimicrobial agents tested were reliably distinguished from susceptible isolates by disc diffusion testing, except for the detection of low-level resistance to penicillin, where broth microdilution or an alternative quantitative MIC method should be used. Otherwise, we conclude that disc diffusion testing is a reliable method to detect strains of VGS non-susceptible to penicillin, erythromycin and ciprofloxacin, as demonstrated with their concordance to their gene resistance characteristics.

Clustered regulatory interspaced short palindromic repeats (CRISPRs) have been discovered in many bacteria and archaea. Many CRISPR-like sequences have been identified in an increasing number of studies on the function of CRISPRs. One CRISPR-like sequence of approximately 240 base pairs has been found to be highly conserved within 11 genome sequences of Streptococcus pneumoniae. A specific CRISPR-like polymerase chain reaction (PCR) assay was designed with the novel primers CRISPR 5F (forward primer) 5'-CTA ATY TCA TAA CCA TAR GAA TC-3' and CRISPR 3R (reverse primer) 5'-GAT AAR ATC CTY TAA WCT TCT AG-3' to detect the presence of this CRISPR-like sequence in pneumococci, as well as in viridans-group streptococci (VGS). This study investigates the prevalence of this CRISPR-like sequence in S. pneumoniae and 12 viridans-group streptococcal species and shows its existence to be shared by the majority of S. pneumoniae and, to a lesser extent, S. mitis. This CRISPR-like sequence was also found in S. australis and it is highly conserved among these strains, suggesting possible biological functional differences from true CRISPR because this CRISPR-like sequence has relatively few repeat numbers, and adjacent homology of CRISPR-associated (cas) genes was absent. The sharing of this CRISPR-like sequence between pneumococci, the mitis group and other VGS, as well as its high sequence homology, may suggest close evolutionary emergence of this sequence between these species.

Breakthrough contamination of tuberculosis (TB) cultures is a problem in that it allows the overgrowth of another bacterium present in the sputum specimen, which can potentially mask the presence of Mycobacterium tuberculosis. The aim of this study was to isolate and characterize the bacterial organisms responsible for such overgrowth and contamination, and to examine their susceptibility to (i) various chemical selective decontamination steps and (ii) antibiotics in liquid culture media, in an attempt to develop a method to help alleviate contamination problems associated with the conventional isolation of M. tuberculosis from routine patient sputum specimens. Bacterial contaminants from 102 routine sputum cultures were identified molecularly by 16S rRNA gene PCR and direct sequencing from contaminated Lowenstein–Jensen (LJ) slopes and BacT/Alert liquid medium. It was found that the contaminants from LJ slopes belonged to 11 different genera and were composed largely of Gram-negative organisms (84.9 %; 45/53), whereas the liquid culture contaminants belonged to 13 different genera, with 37/66 isolates (56.1 %) being Gram-negative. Pseudomonas aeruginosa was the dominant contaminant in both media. The effect of six different selective decontamination protocols was examined. Four of the six methods were effective at eliminating all culturable organisms present; these were 5 % oxalic acid, 5 % oxalic acid/2 % NaOH, 5 % oxalic acid/4 % NaOH and 1 % chlorhexidine. NaOH at a concentration of 2 or 4 % was less effective as it was unable to eliminate all organisms of each species tested, with the exception of P. aeruginosa. In conclusion, breakthrough contamination of TB cultures is due to a diverse range of at least 17 different bacterial genera, with P. aeruginosa and Staphylococcus epidermidis accounting for the dominant contaminating flora. Employment of chemical decontaminating protocols solely involving NaOH may lead to higher rates of contamination. Where such contamination is encountered, TB laboratories should consider the reprocessing of such sputum samples with an alternative decontamination method such as 1 % chlorhexidine.

Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs >= 8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to > 64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.

In this study we report on the development and application of a novel method for efficiently extracting and detecting single Cryptosporidium oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of Cryptosporidium oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of Cryptosporidium and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type Cryptosporidium and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control.

Human cryptosporidiosis has emerged as an important gastrointestinal infection in the 1990s due to the ingestion of contaminated water and foodstuffs containing the protozoan parasite Cryptosporidium spp. (4, 9). This pathogen has particular clinical significance for immunocompromised persons, including AIDS patients and cancer patients receiving toxic chemotherapeutic drug regimens (4, 9). Concern relating to this parasite is founded on three parameters: (i) the causal agent of this infectious disease can be transmitted through contaminated water and food, (ii) when ingested, the causal agent is capable of causing a high degree of morbidity in healthy populations and mortality in vulnerable populations, and (iii) there is no effective antimicrobial treatment to eradicate this agent from the gastrointestinal tract in symptomatic individuals. To date, there have been very limited data describing the frequency and microbiological etiology of mixed enteric infections in the developed world, where an additional enteric pathogen has been isolated along with Cryptosporidium parvum. Hence, it was the aim of our investigation to retrospectively determine the frequency of mixed enteric infections with C. parvum and to determine the coinfecting organism in both sporadic and outbreak-related cryptosporidiosis. In 2000 and 2001, the Northern Ireland Public Health Laboratory processed 9,165 and 10,832 fecal specimens, respectively, which were from symptomatic patients and had been submitted mainly by general practitioners as well as from hospital wards. From these, Cryptosporidium sp. was detected by employment of the modified Ziehl-Neelsen acid-fast staining technique in 255 (2.8%) and 191 (1.8%) cases in 2000 and 2001, respectively, with the majority of cases involving children. The age distribution of cryptosporidial cases in 2000 was 46.7% for patients of 20 years. In 2001, the age distribution was 34.0% for patients of 20 years. During these 2 years, there were three waterborne outbreaks of cryptosporidiosis, as previously described (6). Correcting for the outbreaks, the prevalence of sporadic cases during this time was 2.0% (183 of 9,165) and 1.4% (147 of 10,832) for 2000 and 2001, respectively. In addition to undergoing conventional cryptosporidial microscopic analysis, all fecal specimens were concurrently examined for Campylobacter spp., serotypes of Salmonella enterica, Shigella spp., Escherichia coli O157:H7, and Yersinia enterocolitica, in accordance with previously published methodologies (2). In addition, fecal specimens were analyzed for Clostridium difficile by employment of the Oxoid Toxin-A kit in accordance with the manufacturer's instructions (Oxoid Ltd., Basingstoke, England) and for other parasitic ova and cysts on request by the attending physician. The frequency and distribution of concurrent enteric infections with Cryptosporidium spp. are shown in Table ​Table1,1, while the prevalence of bacterial fecal pathogens in stool specimens which were negative for Cryptosporidium spp. is shown in Table ​Table2.2. Overall, in 446 episodes of cryptosporidiosis in the same number of patients over this 2-year period, there was 2.0% coinfection with another enteric pathogen and C. parvum, where Campylobacter sp. accounted for the most frequently isolated copathogen (1.1%; 5 of 446), followed by Salmonella (0.45%; 2 of 446) and others (0.45%; 2 of 446). Similarly, the Intestinal Infectious Disease study in England demonstrated 2.6% (1 of 39) coinfection with Campylobacter sp. and 2.6% (1 of 39) coinfection with Giardia lamblia (Giardia duodenalis) (2). This study also demonstrated a significantly higher incidence of cryptosporidiosis in rural areas than in urban areas and a higher incidence in the north of England than in the Midlands and the south of England. In addition, there has been a previous association between Cryptosporidium and Campylobacter infection in a waterborne outbreak in the north of England (5). TABLE 1. Prevalence and etiology of mixed enteric infections associated with sporadic and outbreak-related cases of human cryptosporidiosis TABLE 2. Prevalence of bacterial fecal pathogens isolated in stool specimens which were negative for Cryptosporidium spp. The occurrence of mainly single-pathogen infection (98%) is of interest in trying to elucidate the potential reservoirs of this parasite in the local context. The three waterborne outbreaks produced only two cases of concurrent infection with an additional pathogen (Table ​(Table1).1). Furthermore, in all coinfection cases, with the exception of the patient with the positive Clostridium difficile toxin assay, none of the patients presented previously with a positive fecal specimen of the coinfecting organism. Although these outbreaks were associated with the contamination of final drinking water with animal and human feces, no other enteric pathogen was associated with the outbreaks. As animal feces and human sewage have previously been shown to be heavily contaminated with enteric pathogens (3, 7), as water has been shown to be an important vehicle in the transmission of viable oocysts to humans (11), and as chlorination has been shown to be inefficient as an effective disinfection process against C. parvum (8), enteric pathogens in animal and human fecal material contaminating water may be eliminated by residual chlorine, with the exception of cryptosporidial oocysts. Our coinfection data are in marked contrast to those for cryptosporidial infection in underdeveloped and developing countries, where coinfection with other enteric pathogens is common. In Nepal, Sherchand and Shrestha (12) reported a coinfection rate of 13% with other enteric pathogens, whereas Okafor and Okunji (10) reported a coinfection rate of 16.7% in Nigeria. In Egypt, Abaza et al. (1) demonstrated a significant association between mixed infections with G. lamblia and C. parvum in immunocompromised patients. In conclusion, individuals with symptomatic cryptosporidiosis have a low probability (2%) of being coinfected with a secondary enteric pathogen locally. This probably reflects the fact that in an urban western European population with a chlorinated water supply, there is less overlap between the risk factors of Cryptosporidium and those of the other enteric pathogens.

(2003). Sequence analysis of partial regions of the 5.8S rRNA internal transcribed region 2 and 28S rRNA of Isospora belli. British Journal of Biomedical Science: Vol. 60, No. 2, pp. 115-116.

Background There is limited data available on the environmental and public health impact of the microbiological hazards associated with sputa from patients with cystic fibrosis [CF]. Pseudomonas aeruginosa, Burkholderia cenocepacia (formerly Burkholderia cepacia genomovar III), Staphylococcus aureus and Stenotrophomonas maltophilia are bacterial pathogens which are commonly found in the sputum of CF patients. A study was performed to ascertain the amount of sputum produced relating to microbial loading, as well as the diversity of bacteria present in a population of adult patients, with particular attention to pathogenic organisms.

Isolates (n = 51) of Pseudomonas aeruginosa obtained from the sputa of 29 adult patients attending the Regional Cystic Fibrosis Centre in Northern Ireland were compared using an enterobacterial repetitive intergenic consensus sequence (ERIC2) primer in a random amplification of polymorphic DNA (RAPD) polymerase chain reaction (PCR) method. Resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients, when employing this genotyping technique.

Background Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. Results Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. Conclusions Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung.