- Volume 55, Issue 4, 2006

BALB/cA mice were used to study the interaction of diabetes and meticillin-resistant Staphylococcus aureus (MRSA) infection on pathogen distribution, cytokine profile and inflammatory and endothelial-injury markers, as well as coagulation and anticoagulation factors. Meticillin-susceptible S. aureus (MSSA) infection did not cause death within the experimental period. MRSA-infected nondiabetic and diabetic mice died on 19·1±1·4 and 10·6±0·7 days post-infection (p.i.), respectively. MRSA and MSSA infection in diabetic mice did not result in symptomatic bacteraemia; however, MRSA infection in diabetic mice significantly reduced glucose levels (P<0·05). Diabetic mice showed significantly higher levels of C-reactive protein, fibrinogen, fibronectin and von Willebrand factor than nondiabetic mice (P<0·05), and MRSA infection further elevated the plasma levels of these inflammatory and endothelial markers (P<0·05). Before infection, diabetic mice had significantly higher plasminogen activator inhibitor-1 (PAI-1) activity, lower antithrombin III (AT-III) and protein C activities (P<0·05), and MRSA infection significantly increased PAI-1 activity further and reduced the activity of AT-III and protein C (P<0·05). MRSA infection increased the production of three Th1 cytokines, interleukin 2 (IL-2), tumour necrosis factor alpha and gamma interferon, in diabetic mice (P<0·05); however, three Th2 cytokines, IL-4, IL-6, IL-10, were elevated at 2 and 4 days p.i., and then dropped gradually. MRSA infection in diabetic mice accelerated the inflammation process, endothelial injury and blood coagulation in diabetic mice. Therefore, the development of proper infection diagnosis and timely use of effective treatments for MRSA-infected diabetic individuals is important and necessary.

A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains. Pairwise cpn60 sequence identities between Campylobacter spp. ranged from 71 to 92 %, with most between 71 and 79 %, making discrimination of these species obvious. The method described overcomes limitations of existing PCR-based methods, which require time-consuming and complex post-amplification steps such as the cloning of amplification products. The results of this study demonstrate the potential for use of the reference chaperonin sequence database, cpnDB, as a tool for identification of bacterial isolates based on cpn60 sequences amplified with universal primers.

The main objective of this study was to determine the prevalence of filarial infection among migrant workers in Kuwait. The study was conducted from April 2000 to November 2003. A total of 1050 migrant workers (>90 % from the Indian subcontinent) from filarial endemic countries and 260 individuals residing in Kuwait as controls (50 healthy Kuwaiti blood donors, 50 microfilaria-negative individuals from endemic areas and 160 patients with other parasitic infections) were screened for filarial infection. All specimens were tested for microfilaraemia by microscopy of nucleopore-filtered blood (NFB) and detection of circulating filarial antigen (CFA) by an immunochromatographic test (ICT) and the TropBio assay. The overall prevalence of filarial antigenaemia was 18·3 % (192 individuals) using the ICT and 20·3 % (213 individuals) using the TropBio assay. Thirty-two cases (3 %) of Wuchereria bancrofti were detected by microscopy and the mean microfilaria count in these cases was 816 microfilariae ml−1. CFA was detected only in two of the 260 control subjects. Statistical analysis to calculate the sensitivity, specificity and prevalence of infection was carried out using maximum-likelihood statistical methods. The overall sensitivity and specificity of the ICT and TropBio assay to detect CFA were comparable. Compared with NFB microscopy, the sensitivity of the ICT was 93·8 % and specificity ranged from 84 to 100 %. The sensitivity and specificity of the TropBio assay were 90·1 and 100 %, respectively. However, the ICT failed to detect CFA in two cases with a microfilarial load of <20 microfilariae ml−1. In conclusion, the prevalence of filarial infection among the migrant workers in Kuwait was 18·3 % as determined by the ICT.

Two hundred and fifty-one unique patient isolates of Klebsiella pneumoniae (123), Escherichia coli (114), Klebsiella oxytoca (7), Enterobacter cloacae (5) and Citrobacter freundii (2), flagged as extended-spectrum β-lactamase (ESBL) positive by the Vitek system (GNS-526 card), were collected. These strains were isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of the Royal Infirmary of Edinburgh (RIE), Edinburgh, UK (and associated GP practices), Hairmyers Hospital, Glasgow, UK, and the Amiri and Farwania Hospitals, Kuwait. Of the 101 RIE strains tested, 15 E. coli strains were found to be ESBL negative by Etest ESBL strips. On retesting the 15 E. coli strains with the Vitek GNS-532 card, 14 were found to be ESBL negative, despite being originally flagged as ESBL positive. The remaining 236 ESBL-producing strains were also subjected to the double disc-diffusion (DDD) technique for the detection of ESBLs. Of these, two were false negatives by Etest ESBL test strips (using both cefotaxime and ceftazidime strips), and 38 were false negatives by the DDD method. The Etest false-negative ESBL-producing strains of K. pneumoniae were positive by DDD. Technically, the Vitek method was the least demanding method to perform, as it was an integral part of the routine susceptibility test card. Etest strips were reliable, but were the most expensive of all the techniques used. The DDD test, while relatively inexpensive, was technically subjective, and in our hands, seven of the ESBL-positive strains that were confirmed by the other two techniques were not detected. Despite the false-positive ESBL-producing E. coli strains, the Vitek susceptibility card with its integral ESBL test offers the clinical laboratory a valuable and quick option to screen for ESBL-producing Klebsiella spp. and E. coli as part of the routine laboratory methodology.

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T. rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5′-d[GGTGCGGGAA]-3′ and 5′-d[CCCGTCAGCA]-3′, as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPD products showed a high degree of similarity (>90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60 % similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPD analysis is especially suitable for molecular typing in T. rubrum.

The IS6110 repetitive element is present in multiple copies in most Mycobacterium tuberculosis complex bacteria, except for Mycobacterium bovis strains, which usually contain a single copy of IS6110 located on a 1·9 kb PvuII fragment of the direct repeat region. IS6110 transposition can disrupt coding regions and is a major force of genomic variation. In a previous work it was demonstrated that phospholipase C genes are preferential loci for IS6110 transposition in M. tuberculosis clinical strains. Bacterial phospholipase C enzymes participate in pathogenic mechanisms used by different organisms, and have been implicated in intracellular survival, cytolysis and cell-to-cell spread. Four phospholipase C genes (plcA, plcB, plcC and plcD) were detected in the genomes of M. tuberculosis, Mycobacterium africanum, Mycobacterium microti and ‘Mycobacterium canettii’. M. bovis and the vaccine strain M. bovis Bacillus Calmette–Guérin contain only the plcD gene. In the present work, the existence of IS6110 insertions within plcD, the unique phospholipase C gene of M. bovis, has been investigated by PCR, Southern blot hybridization and sequencing analysis. In 18 (7·3 %) of 245 isolates analysed, the plcD gene was interrupted by the insertion of one copy of IS6110, which in all cases was transposed in the same orientation and at the same position, 1 972 894, relative to the genome of M. bovis AF2122/97. These 18 isolates were distributed in 6 different spoligotype patterns and contained 4 to 8 IS6110 copies. In contrast, strains showing an intact plcD gene contained one (87 %), two (9·4 %) or three (2·4 %) IS6110 copies, and only a single isolate (1·2 %) had four IS6110 copies. The implications of plcD gene disruption in M. bovis have not been fully investigated, but no differences in the organ distribution of the disease were detected when animals infected with strains from the same spoligotype patterns bearing plcD : : IS6110 and intact plcD were compared.

Parotid gland infection as a source of meticillin-resistant Staphylococcus aureus bacteraemia has been rarely reported. It is predominantly a disease of the elderly and is associated with significant mortality. Two cases are described here that presented over a 6 month history at a district general hospital. Many cases may be preventable with adequate hydration and good oral hygiene, combined with effective infection control.