R.B. McClurg

Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

A rapid molecular assay for the detection of antibiotic resistance determinants in causal agents of infective endocarditis

Aims: To develop and employ a PCR amplification system, directly from clinical specimens, for the rapid molecular detection of common antimicrobial resistance genes for streptococci, staphylococci and enterococci organisms causing infective endocarditis (IE).

Prevalence of Gastrointestinal Bacterial Pathogens in a Population of Zoo Animals

Faecal prevalence of gastrointestinal bacterial pathogens, including Campylobacter, Escherichia coli O157:H7, Salmonella, Shigella, Yersinia, as well as Arcobacter, were examined in 317 faecal specimens from 44 animal species in Belfast Zoological Gardens, during July–September 2006. Thermophilic campylobacters including Campylobacter jejuni, Campylobacter coli and Campylobacter lari, were the most frequently isolated pathogens, where members of this genus were isolated from 11 animal species (11 of 44; 25%). Yersinia spp. were isolated from seven animal species (seven of 44; 15.9%) and included, Yersinia enterocolitica (five of seven isolates; 71.4%) and one isolate each of Yersinia frederiksenii and Yersinia kristensenii. Only one isolate of Salmonella was obtained throughout the entire study, which was an isolate of Salmonella dublin (O 1,9,12: H g, p), originating from tiger faeces after enrichment. None of the animal species found in public contact areas of the zoo were positive for any gastrointestinal bacterial pathogens. Also, water from the lake in the centre of the grounds, was examined for the same bacterial pathogens and was found to contain C. jejuni. This study is the first report on the isolation of a number of important bacterial pathogens from a variety of novel host species, C. jejuni from the red kangaroo (Macropus rufus), C. lari from a maned wolf (Chrysocyon brachyurus), Y. kristensenii from a vicugna (Vicugna vicugna) and Y. enterocolitica from a maned wolf and red panda (Ailurus fulgens). In conclusion, this study demonstrated that the faeces of animals in public contact areas of the zoo were not positive for the bacterial gastrointestinal pathogens examined. This is reassuring for the public health of visitors, particularly children, who enjoy this educational and recreational resource.

Potential misidentification of a new Exiguobacterium sp. as Oerskovia xanthineolytica isolated from blood culture

Blood culture of a male patient attending Sligo General Hospital became positive with a coryneform-like Grampositive rod, and phenotypic identification of the organism by the API Coryne kit (bioMerieux, France) yielded Oerskovia xanthineolytica (isolate identifier: 290703/S6645SLIGO). The isolate grew aerobically on blood agar at 37 ̊C. However, given the relatively poor phenotypic identification obtained, the isolate subsequently was forwarded for molecular identification through polymerase chain reaction (PCR) amplification and direct sequencing of a large but partial region of the 16S rRNA gene, as previously described by this group. The resulting sequence obtained (1024 bp) was compared with those stored in the GenBank Data system using FASTA alignment software (www.ebi.ac.uk), and was deposited in GenBank (accession number: AY360351). On BLAST analysis in combination with previously reported criteria used for interpretation of partial 16S rRNA gene sequences, the sequence gave a 99% identification for Exiguobacterium sp. (AY205564), bacterium Str61610 (AF227839), 98% identity with E. aurantiacum (X70316), 96% identity with E. undae (AJ344151) and 92% identity with Bacillus hackensackii (AY148429), as illustrated in Figure 1. Although the isolated organism was not believed to be clinically significant, it presented difficulties in its correct and reliable phenotypic identification and gave the identification of O. xanthineolytica with the API Coryne scheme. At present, there are four species described in the genus Exiguobacterium, including E. acetylicum, E. antarcticum, E. aurantiacum and E. undae. The genus Exiguobacterium was first described by Collins et al. in 1983, with E. aurantiacum as the type strain, isolated from potato processing effluent. Previously, the organism has been misidentified as the former CDC coryneform group A-5; however, cell wall and cellular fatty acid analyses clearly separate this genus from other genera most likely to be misidentified, particularly Microbacterium spp. Until recently, the clinical significance of members of this genus was confined to a few sporadic reports of the organism being found in various clinical sources, including skin, wounds and cerebrospinal fluid. However, a recent report has suggested that Exiguobacterium sp. is an important agent in periodontal disease. This short report highlights the benefits of the integration of a sequence-based typing approach employing partial regions of the 16S rRNA gene in the identification of difficult-to-identify bacterial isolates. Continued routine adoption of such techniques by clinical diagnostic laboratories may prove beneficial for the correct identification of bloodborne infections, as well as for the correct epidemiological characterisation of unusual causal agents of bacteraemia.

Quantitative colorimetric measurement of residual antimicrobials in the urine of patients with suspected urinary tract infection

Abstract A simple microtitre plate assay is used to detect antimicrobial activity in clinical urine specimens and its potential as a screening tool is assessed. The assay is based on a colorimetric substrate, p-nitrophenyl-β-D-glucopyranoside, in combination with a Bacillus subtilis strain to detect antimicrobial residues. The assay identified antimicrobial activity in 31% of the 527 clinical urine samples tested. The majority of the samples (65%) came from the community, with the rest comprising hospital in-patients (19%) and out-patients (16%). The results demonstrated that there is an association between gender and the presence of inhibitory substances, as 40% of males and 27% of females tested positive. Just over two-fifths of hospital patients (46%) tested positive for inhibitory substances, compared to 26% of samples from community patients. Of the 306 samples that were culture-negative (<104 bacteria/mL), 42% were positive for inhibitory substances, compared with 17% among the remaining 221 samples. However, there was no evidence of an association between age and the presence of inhibitory substances. This study demonstrates that the bacteriostatic effect of the bacterial preservative boric acid is sufficient to upset the specificity of the assay. Furthermore, it has been suggested that antimicrobial activity can confuse the interpretation of culture results, as they have been found to play a major role in the occurrence of apparently sterile pyuria.

Identification of a novel α-proteobacterium causing bacteraemia in an immunocompetent patient

Abstract An 89-year male with pyrexia and suspected bacteremia was admitted to hospital, where a Gram-negative rod was identified from blood culture. The organism was difficult to identify phenotypically and the resulting sequencing of a 559 bp section of the 16S rRNA gene did not have a high homology score (>97.0%) with any deposited GenBank accession numbers and hence was not able to be assigned to a species within any genus. Given that the isolate was a member of the alpha subclass of the Proteobacteria but did not fall into any of the known genera with more than 93.7% homology ( Brucella, Rhizobium, Ochrobactrum, Agrobacterium ), we believe this isolate to represent a novel α-proteobacterium, which was the cause of bacteraemia in this patient.

Comparison of the identification of Acinetobacter spp. with API20NE and 16S rRNA gene sequencing techniques.

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Mucoid Staphylococcus aureus isolated from a patient with a suspected urinary tract infection.

We report the isolation of an unusual morphological mucoid variant of Staphylococcus aureus. An 82-year-old male presented to the Urology Department at Belfast City Hospital with a suspected urinary tract infection, for which he submitted a midstream urine (MSU) sample. Bacteriological culture was performed on a commercial chromogenic culture medium for the culture of urinary pathogens (CPS ID 3 agar; bioMérieux) at 37 uC for 24 h, and revealed the presence of a mucoid organism, with the typical appearance of a coliform. Subsequent Gram-staining revealed the presence of a Gram-positive coccus, which was further characterized by standard phenotypic assays (Brown et al., 2005) as S. aureus. The identification of this isolate was confirmed by employment of the BBL Crystal Gram-positive identification system, which gave a profile of 0064673045, indicating S. aureus (85 % identity). Given this relatively low percentage identity, the isolate was characterized molecularly through employment of broad range 16S rDNA PCR and automated sequence-based analysis, as described previously (Xu et al., 2004). This confirmed the isolate was S. aureus (100 % identity in 940 bases); the sequence was subsequently submitted to GenBank with accession number EU305717 and the isolate was archived in the culture collection of the Northern Ireland Public Health Laboratory. On routine antibiotic disc susceptibility testing, in accordance with the Clinical and Laboratory Standards Institute criteria, the isolate was resistant to penicillin and trimethoprim, but was sensitive to all other antibiotics tested, including augmentin, ciprofloxacin, clindamycin, erythromycin, fusidic acid, gentamicin, rifampicin, teicoplanin, tetracycline and vancomycin. The organism had an oxacillin MIC of 1.0 mg ml. The mucoid nature of the organism was present following multiple passages on Columbia blood agar supplemented with 5 % (v/v) horse blood, as shown in Fig. 1. Although rare in everyday clinical microbiology, the existence of wild-type mucoid variants of S. aureus has been described by other authors (Lee et al., 1987), where the mucoid nature was attributed to the production of capsular polysaccharides, which also has been described as being an important virulence factor in organisms with capsulated polysaccharides. (For a comprehensive review see O’Riordan & Lee, 2004.) The enhanced virulence of encapsulated bacteria is generally attributed to resistance to phagocytosis, and published studies have demonstrated that encapsulated strains of S. aureus are more virulent in mice than strains lacking capsules (Sompolinsky et al., 1985; Yoshida & Ekstedt, 1968). However, in the patient described above, no further MSU sample or other clinical specimens were submitted for bacteriological examination, suggesting the complete resolution of his clinical condition. Therefore, although extremely rare, clinical bacteriologists should be aware of the existence of mucoid variants of S. aureus originating from clinical specimens and be careful colonies of not to confuse colonies of such organisms with coliforms.

Cross contamination of hospital ophthalmic slit lamps by ocular bacteria.

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Lack of horizontal gene transfer of methicillin-resitance genetic determinants from PBP2a-positive, coagulase-negative Staphylococci to methicillin-sensitive Staphylococcus aureus using transcutaneous electric nerve stimulation (TENS),

Abstract Previous research shows that approximately half of the coagulase-negative staphylococci (CNS) isolated from patients in the intensive care unit (ICU) at Belfast City Hospital were resistant to methicillin. The presence of this relatively high proportion of methicillin-resistance genetic material gives rise to speculation that these organisms may act as potential reservoirs of methicillin-resistance genetic material to methicillin-sensitive Staphylococcus aureus (MSSA). Mechanisms of horizontal gene transfer from PBP2a-positive CNS to MSSA, potentially transforming MSSA to MRSA, aided by electroporation-type activities such as transcutaneous electrical nerve stimulation (TENS), should be considered. Methicillin-resistant CNS (MR-CNS) isolates are collected over a two-month period from a variety of clinical specimen types, particularly wound swabs. The species of all isolates are confirmed, as well as their resistance to oxacillin by standard disc diffusion assays. In addition, MSSA isolates are collected over the same period and confirmed as PBP2a-negative. Electroporation experiments are designed to mimic the time/voltage combinations used commonly in the clinical application of TENS. No transformed MRSA were isolated and all viable S. aureus cells remained susceptible to oxacillin and PBP2a-negative. Experiments using MSSA pre-exposed to sublethal concentrations of oxacillin (0.25 µg/mL) showed no evidence of methicillin gene transfer and the generation of an MRSA. The study showed no evidence of horizontal transfer of methicillin resistance genetic material from MR-CNS to MSSA. These data support the belief that TENS and the associated time/voltage combinations used do not increase conjugational transposons or facilitate horizontal gene transfer from MR-CNS to MSSA.