- Volume 56, Issue 10, 2007

The Gram-negative enteric bacterium Proteus mirabilis is a frequent cause of urinary tract infections (UTIs) in individuals with long-term indwelling catheters or with complicated urinary tracts. The recent release of the P. mirabilis strain HI4320 genome sequence has facilitated identification of potential virulence factors in this organism. Genes appearing to encode a type III secretion system (TTSS) were found in a low GC-content pathogenicity island in the P. mirabilis chromosome. This island contains 24 intact genes that appear to encode all components necessary to assemble a TTSS needle complex, plus at least two putative secreted effector proteins and their chaperones. The genetic organization of the TTSS genes is very similar to that of the TTSS of Shigella flexneri. RT-PCR analysis indicated that these genes are expressed at low levels in vitro. However, insertional mutation of two putative TTSS genes, encoding the requisite ATPase and a possible negative regulator, resulted in no change in either the growth rate of the mutant or the secreted protein profile compared to wild-type. Furthermore, there was no difference in quantitative cultures of urine, bladder and kidney between the ATPase mutant and the wild-type strain in the mouse model of ascending UTI in either independent challenge or co-challenge experiments. The role of the P. mirabilis TTSS, if any, is yet to be determined.

Campylobacter jejuni is one of the leading causes of food-borne gastroenteritis. Because of the high prevalence of C. jejuni in poultry, poultry meat is considered a major source of C. jejuni infections for humans. However, it is not known whether all poultry-associated C. jejuni strains are capable of causing disease in humans. Four different virulence properties of C. jejuni strains were compared between 20 poultry isolates and 24 human isolates. Strains were chosen based on their PFGE pattern to represent a heterogeneous population. The isolates were compared for their ability to invade and induce interleukin-8 (IL-8) production in T84 cells, their production of functional cytolethal distending toxin (CDT) using HEp-2 cells, and their sodium deoxycholate resistance. All four virulence factors were present among strains of human and poultry origin, with strong differences observed among strains. For invasion and IL-8 induction, no difference was observed between the two populations. However, on average, human isolates arrested more HEp-2 cells in their cell cycle than did the poultry isolates (P=0.041), suggesting higher CDT production by the former. The ability to survive 16 000 μg sodium deoxycholate ml−1 was significantly more pronounced (P=0.006) among human isolates than poultry isolates, although all strains possessed the cmeABC operon. These data suggest that all four virulence properties are widespread among C. jejuni isolates, but that a higher degree of bile-salt resistance and more pronounced CDT production are associated with strains causing enteritis in humans.

Invasion of vascular endothelial cells is thought to be a critical step in the development of metastatic infections in patients with Staphylococcus aureus bacteraemia. This study was designed to evaluate the association between the ability to invade endothelial cells and metastatic infection by S. aureus. Patients with metastatic infection were identified among those with community-acquired S. aureus bacteraemia in a tertiary referral hospital. Patients with simple bacteraemia caused by S. aureus over the same period served as the control group. The ability of each clinical isolate to invade endothelial cells was evaluated by counting the number of intracellular organisms 1 h after inoculation onto human umbilical vein endothelial cells in vitro. The cytotoxic activity of intracellular S. aureus was determined 24 h after internalization, and expressed as the percentage of cells killed. The clinical isolates varied in invasiveness and cytotoxicity. The median invasiveness, relative to S. aureus reference strain ATCC 29213, was 145  % in the cases (n=10) [interquartile range (IQR) 103–160] and 153  % (IQR 111–173) in the controls (n=11; P=0.44). The median cytotoxicity was 59.4  % (IQR 47–68) in the cases and 65.2  % (IQR 50–74) in the controls (P=0.44). Differences in the ability of S. aureus to invade and destroy vascular endothelial cells in vitro were not associated with the development of metastatic complications in patients with S. aureus bacteraemia. This implies that the invasiveness and toxicity of S. aureus for endothelial cells may not be major determinants of metastatic infection.

The present study was designed to determine the slime production of coagulase-negative staphylococci (CoNS) and the enterotoxigenic properties of Staphylococcus aureus strains, and to evaluate the clinical importance of slime-producing CoNS and enterotoxigenic S. aureus strains isolated from various human clinical specimens. For this purpose, a total of 120 Staphylococcus strains were isolated and identified, and further characterized for their slime production and enterotoxigenicity. Of the clinical isolates, 55 (45.8 %) were found to be S. aureus, and the others (54.2 %) were identified as CoNS. Of the CoNS, 20 (16.7 %) were further identified as Staphylococcus hominis, 18 (15 %) as Staphylococcus epidermidis, six (5 %) as Staphylococcus xylosus, six (5 %) as Staphylococcus warneri, five (4.2 %) as Staphylococcus sciuri, four (3.3 %) as Staphylococcus haemolyticus, and two each (1.7 %) as Staphylococcus simulans, Staphylococcus capitis and Staphylococcus saprophyticus, respectively. Thirty-nine (60 %) of 65 CoNS were found to be slime producers. Slime production was observed in all CoNS, except S. capitis, mostly from blood (38.5 %), tracheal aspiration (20.5 %) and urine (12.8 %) specimens. In addition, of the 55 S. aureus isolates, 46 (83.6 %) were found to be enterotoxigenic, and of these S. aureus strains, 39 (84.7 %) were positive for staphylococcal enterotoxin (SE)A. The results of this study showed that the slime-producing CoNS were mostly found in clinical specimens of blood, tracheal aspirate and urine. SEA was the predominant enterotoxin type detected in S. aureus strains from human clinical specimens.

Farmers' lung disease (FLD) is a pulmonary disease that results from repeated inhalation of antigens from mouldy hay or straw. The objective of this prospective study was to assess the reliability of four serological techniques in FLD diagnosis. Sera from 15 consecutive patients with FLD, 15 healthy control farmers and 30 urban controls were analysed using four serological techniques [electrosyneresis (ES), Ouchterlony double diffusion (DD), ELISA and Western blot (WB)] with four antigens (Absidia corymbifera, Eurotium amstelodami, Wallemia sebi and Saccharopolyspora rectivirgula). In the authors' region, ES on cellulose acetate with A. corymbifera antigen was the most relevant diagnostic tool for discriminating FLD patients from healthy exposed farmers (sensitivity 87 %, specificity 100 %). DD tests were in accordance with ES, but their discriminatory power was lower. No threshold indicating both good sensitivity and specificity could be established with ELISA. WB analysis failed to identify specific bands for FLD. This study demonstrates the efficacy of determining precipitin levels with an appropriate technique, using a panel of antigens consistent with the specific exposure of a given area.

A proportion of patients with chronic rhinosinusitis, especially if nasal polyps are present, have a diagnosis of fungal rhinosinusitis. The diagnosis is difficult to establish because the symptoms and clinical and radiological signs are non-specific. Also current diagnostic methods, i.e. histology, fungal staining and culture, are insensitive. The performance of the Aspergillus galactomannan (GM) ELISA and real-time PCR for Aspergillus fumigatus mitochondrial DNA was evaluated for the detection of Aspergillus in sinus mucus samples from 25 patients with chronic rhinosinusitis with nasal polyposis. The results were compared with those from nasal lavage fluid from 19 healthy volunteers. Seven patients (28 %) were diagnosed as having fungal rhinosinusitis according to the presence of filaments in histology or direct microscopy using Calcofluor white. All fungal rhinosinusitis patients were negative in the GM ELISA. GM ELISA was positive in five patients whose samples were negative using conventional methods and A. fumigatus PCR. Two out of seven patients with fungal rhinosinusitis were positive by A. fumigatus PCR: one also had a positive A. fumigatus culture, and one had hyphae consistent with Aspergillus in histology. One additional patient had a weak positive PCR result, but other fungal tests were negative. In control subjects, the GM ELISA was positive in 21 %, whereas direct microscopy, culture and A. fumigatus PCR were negative in all samples. Direct microscopy and culture together with histology remain pivotal in defining fungal rhinosinusitis diagnosis. A. fumigatus PCR may have additional value in allowing the diagnosis to be made sooner, whereas the GM ELISA is not reliable in diagnosing Aspergillus infection of the paranasal sinuses.

Coagulase-negative staphylococci (CoNS) are now recognized as the aetiological agents of an important range of infections in humans. Most developed countries have reported an increase in CoNS infections in hospitalized patients that are resistant to meticillin and other antibiotics. Staphylococcal cassette chromosome mec (SCCmec) typing is essential for understanding the molecular epidemiology of meticillin-resistant Staphylococcus strains. SCCmec elements are currently classified into types I to VI based on the characteristics of the mec and ccr gene complexes and are further classified into subtypes according to their ‘junkyard DNA’ region. We evaluated the distribution of SCCmec types in CoNS from patients attending the Hospital de Clínicas de Porto Alegre over the period August 2004–December 2005. Among the 129 bloodstream isolates, 36 (27.9 %) harboured SCCmec type I, 4 (3.0 %) harboured SCCmec type II, 67 (52 %) harboured SCCmec type III, 1 (0.8 %) harboured SCCmec type IV and 4 (3.0 %) harboured SCCmec types I and III. Seventeen isolates were not typable. Identification of CoNS at the species level indicated that Staphylococcus epidermidis was the most common species, with 87 isolates, followed by Staphylococcus haemolyticus (15), Staphylococcus hominis (13), Staphylococcus capitis (12) and Staphylococcus sciuri (1). SCCmec type III was the most prevalent among isolates of S. epidermidis (52 %). Among these strains, 30 (23 %) harboured a modified SCCmec type III which contained an additional dcs region in comparison with regular type III. SCCmec type III was also highly prevalent (75 %) among S. capitis isolates. The predominant SCCmec type found among S. haemolyticus isolates was type I. However, all four isolates harbouring SCCmec type II belonged to S. haemolyticus. Our results indicate that SCCmec type III was the most prevalent among the CoNS. Isolates with SCCmec type III were more resistant to non-β-lactam antimicrobials than isolates harbouring SCCmec types I, II and IV, although the increase in resistance was statistically significant only for clindamycin (P=0.021), rifampicin (P=0.010) and levofloxacin (P=0.005).

A prospective study was conducted of the rapid FASTPlaque-Response test for determination of rifampicin resistance in Mycobacterium tuberculosis with and without the addition of an antimicrobial supplement containing nystatin, oxacillin and aztreonam (NOA) to control specimen-related contamination. A total of 631 smear-positive sputum specimens was tested. The age of specimens ranged from 0 to 21 days. The NOA antimicrobial was effective at controlling contamination, with 4.1 % of specimens contaminated when the NOA antimicrobial supplement was used compared with 13.9 % contamination without NOA. Overall levels of interpretability of the test with NOA were 87.8 % with specimens of ≤3 days and 79.0 % for all specimens. This compared with 70.1 and 73.8 % readable results, respectively, from conventional culture-based drug susceptibility testing (DST). Sensitivity, specificity and overall accuracy of the FASTPlaque-Response test for rifampicin resistance were 98.1, 96.3 and 96.6 %, respectively, for all specimens with NOA, and 93.2, 96.3 and 95.9 % without NOA, when compared with resolved conventional DST results. Inclusion of the NOA supplement reduced contamination, increased the number of interpretable results and did not adversely affect the performance of the FASTPlaque-Response test. Thus, the use of NOA improves the robustness of the test, facilitating its wider implementation.

A mAb-based simple, specific and rapid two-tip dipstick ELISA was developed for simultaneous detection of toxin- and non-toxin-producing strains of Vibrio cholerae, and for direct detection of V. cholerae from rectal swabs of patients and from environmental water samples. Rabbit polyclonal antibodies and murine mAbs were raised against recombinant protein (r-protein) antigens of cholera toxin B (CtxB) and outer membrane protein W (OmpW). Rabbit polyclonal antibodies to both r-proteins were coated individually onto the tips of nitrocellulose (NC) membranes of a two-tipped NC dipstick as capture antibodies and a mixture of two mAbs was used for the detecting antibodies. The test was found to be specific for V. cholerae strains O1, O139, non-O1 and non-O139, and did not show any cross-reaction to closely related bacterial strains. The test was evaluated on rectal swabs collected at the bedside of 75 hospitalized diarrhoeal patients and on 50 environmental water samples after enrichment for 4 h in alkaline peptone water. The mAb two-tip dipstick ELISA detected V. cholerae in 52/75 rectal swabs and 2/50 environmental water samples for CtxB antigen, and in 1/50 environmental water samples for the non-toxin OmpW antigen of V. cholerae within 1.5 h. These findings were identical to those observed using PCR and conventional culture methods. Thus, this mAb-based two-tip dipstick ELISA could be used for early and reliable simultaneous detection of toxigenic and non-toxigenic strains of V. cholerae from clinical and environmental water samples.

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3′ end of the primer (3′ mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (101–107 cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015–0.016 ng C. jejuni and C. coli DNA μl−1 in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the ‘gold’ standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner.

The application of genotyping techniques for subtyping uropathogenic Escherichia coli has contributed to better understanding of the epidemiology of community-acquired urinary tract infection (UTI). However, the current techniques are hampered by limited reproducibility, poor discriminatory power, labour-intensive performance or high cost. A screening test that is sequence-based would provide an inexpensive, reproducible way to subtype E. coli isolates. Such a test, if also discriminatory, would be highly useful for epidemiological studies. The discriminatory ability of 12 putative virulence genes (fimH, fliD, fliM, iha, motA, papA/H, kpsMTII, fepE, fimA, flgA, malG, purD) was evaluated based on single nucleotide polymorphisms (SNPs) in nine uropathogenic E. coli isolates, all previously found to belong to a single multilocus sequence type (MLST) complex (ST69). An additional 25 epidemiologically well-characterized E. coli isolates belonging to 12 distinct MLST clonal complexes were analysed for fimH SNP. None of the 12 genes except fimH were able to further discriminate the nine ST69-complex strains. Isolates belonging to the 12 non-ST69 MLST groups were separated into 10 fimH SNP subgroups. While fimH SNP analysis may not be an appropriate phylogenetic method, it offers discriminatory power similar to that of MLST and could be used as a simple, inexpensive screening test for epidemiological studies of uropathogenic E. coli.

Enteroaggregative Escherichia coli (EAEC) is associated with diarrhoea among travellers to developing countries. EAEC virulence properties predisposing to illness are not clear. Sixty-four EAEC strains identified by a HEp-2 cell assay and isolated from faecal samples from US and European travellers to developing countries were studied for the prevalence of 11 putative virulence genes by PCR: 49 EAEC strains from adults with acute diarrhoea and 15 EAEC strains from adults without diarrhoea. E. coli strains from the stools of healthy travellers to the same region were used as controls. EAEC carrying aggR, aap, astA and set1A were identified individually more often in the stools of subjects with diarrhoea compared with those without diarrhoea (P<0.05). EAEC isolates with two or three of these genes were associated with diarrhoea compared with EAEC isolates without the presence of these genes (P<0.05). Subjects with diarrhoea who shed EAEC isolates positive for these genes were more likely than subjects shedding EAEC negative for these genes to pass stools with gross mucus (57 vs 14 %) and faecal leukocytes (40 vs 7 %) (P<0.05). This study shows the heterogeneity of gene profiles of EAEC strains found in the stools of international travellers and suggests that the presence of aggR, aap, astA or set1A, the number of genes present and stool characteristics may be markers for more virulent EAEC strains.