- Volume 54, Issue 6, 2005

The role of quorum-sensing systems in a mouse model of chronic Pseudomonas aeruginosa infection was studied. A chronic P. aeruginosa respiratory infection model was established by placement of a tube pre-coated with strain PAO1 (wild-type) or a quorum-sensing mutant, namely PAO-JP1 (ΔlasI), PDO100 (ΔrhlI) or PAO-JP2 (ΔlasI/ΔrhlI), in the bronchus. At day 14 after infection, the numbers of viable bacteria in the quorum-sensing-mutant groups were lower than in the wild-type group. Histopathological examination showed milder inflammatory changes in the lungs infected with the mutant groups compared with the wild-type group. In the bronchoalveolar lavage fluid from the quorum-sensing-system-mutant groups the proportion of neutrophils was lower than in wild-type group. These findings indicate that the quorum-sensing system plays an important role in chronic P. aeruginosa respiratory infection.

A collection of 63 isolates of Pseudomonas aeruginosa associated with ulcerative keratitis, collected from six centres in England, were typed using serotyping and random amplified polymorphic DNA-PCR, and screened for several variable virulence-related genotypes and phenotypes. Sixty-one percent of the isolates were of either serotype O1 or serotype O11, but there was no evidence for a common clone. The majority of isolates (59 %) were PCR-positive for exoU rather than for exoS (38 %), and carried a-type fliC genes (76 %) rather than b-type (24 %). Isolates were PCR-positive for pyoverdine-receptor types at a prevalence of 38 % for type I, 46 % for type II and 8 % for type III. All but one of the isolates exhibited twitching activity. There was a correlation between the presence of exoS and twitching activity (P = 0.04), suggesting that a combination of exoS genotype and good twitching activity may have a role to play in ExoU-independent corneal virulence.

Th1 immune responses in which gamma interferon (IFN-γ) production predominates are associated with protective immunity against intracellular bacteria. Following infection, interleukin-18 (IL-18) may contribute, in association with IL-12, to optimal IFN-γ production. In this study, the secretion of IL-18 following intracellular infection with virulent Brucella abortus 2308 in CD-1 cultured peritoneal macrophages and splenocyte cultures was investigated. The production of IL-18 was reduced in both CD-1 mouse peritoneal macrophages infected with B. abortus 2308 and splenocyte cultures obtained from B. abortus 2308-infected mice at 3, 6 and 10 days post-infection (p.i.). In contrast, splenocyte cultures obtained from B. abortus 2308-infected mice at 3 days p.i. secreted significant amounts of IFN-γ. Stimulation of these cells with recombinant IL-18 (rIL-18) and/or rIL-12 did not significantly increase IFN-γ secretion at the splenocyte level. These data suggest that once the infection has been established, B. abortus 2308 selectively limits IL-18 secretion without affecting endogenous IFN-γ production.

Human brucellosis is a zoonotic disease which is endemic in Saudi Arabia. The aim of this study was to investigate the humoral immune responses and identify the target antigens that persist at different stages in human brucellosis during antibiotic therapy. To do this, an acute case of accidental nosocomial infection was studied experimentally. Blood was collected from the patient at the time of diagnosis, and at weekly intervals during therapy until remission. IgG and IgM immunoblotting was used to characterize specific antigenic determinants, and ELISA antibody titration was performed to quantify the circulating antibodies. Results indicated that protein bands of 12–13.5 kDa bound IgG in the patient's sera but did not bind IgM on immunoblots and are probably not specific for, or important in, early stage infections. However, an 18 kDa band persisted during infection through remission. The pivotal and most important findings were that the number of protein bands seen on immunoblots, the magnitude of ELISA antibody titres and the concomitant changes in the intensity of the polypeptide bands of 42–43 kDa were positively correlated with the stage of infection. High numbers of anti-IgG and -IgM immunoblot bands coupled with high ELISA antibody titres and a concomitant increase in intensity of the 42–43 kDa bands were positively correlated with acute and severe infection. Conversely, a reduction in the number of polypeptide bands as well as a decrease in the intensity, until the complete disappearance of the 42–43 kDa bands, coupled with low (baseline) ELISA antibody titration values indicated successful treatment and remission. The routine use of the methods described here to ascertain the stage of the disease, assess the progress of antimicrobial therapy and monitor cases of relapse in human brucellosis is suggested.

Cerebrospinal fluid (CSF) obtained from patients with partially treated and culture-negative meningitis was subjected to PCR using 16S rDNA universal primers followed by restriction endonuclease digestion. In all, 43 patients and 7 controls were enrolled in this study. Twenty-one meningitic samples were positive by PCR. Mycobacterium tuberculosis was the causative agent in seven cases followed by Haemophilus influenzae (four), Streptococcus pneumoniae (two), Listeria monocytogenes (one), Escherichia coli (one), Pseudomonas aeruginosa (one) and Staphylococcus aureus (one). Only two meningitic CSF samples were culture-positive. In this study, PCR using bacterial 16S rDNA specific universal primers was found to be superior to conventional methods in the diagnosis of partially treated meningitis.

The REP-PCR (repetitive sequence-based PCR using repetitive extragenic palindromic primers) typing method and a modified PFGE method were applied to isolates of Clostridium difficile PCR ribotype 001 with the aim of comparing their performance as methods of subtyping this organism. Of 200 isolates from 60 hospitals tested by REP-PCR, eight subtypes were identified and labelled as REP-PCR subtypes 001–008. The predominant subtype, REP-PCR subtype 003, accounted for 47 % of the total. Fifty-two of the 200 isolates were analysed by a modified PFGE method and seven subtypes were identified, labelled as PF-A–PF-G. There was excellent correlation between REP-PCR subtypes and PFGE subtypes with both methods displaying broadly similar discriminatory powers. However, REP-PCR subtyping proved to be a much easier, cheaper and more rapid method suitable for application for routine subtyping of C. difficile ribotype 001. Application of REP-PCR subtyping to UK isolates of C. difficile PCR ribotype 001 from 60 different centres revealed a wide distribution of REP-PCR subtype 003 throughout England and Wales, with a regional clustering of REP-PCR subtype 001 around Northwest England and North Wales. Analysis of isolates from a single hospital over a 4-year period revealed a change in predominant subtype over time.

A novel compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate was isolated from the plant Datura metel L. The in vitro activity of this dihydropyrrole derivative against Aspergillus and Candida species was evaluated by using standard methods approved by the National Committee for Clinical Laboratory Standards. The compound was found to be active against all the species tested, namely Candida albicans, Candida tropicalis, Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. The MIC at which more than 90 % of growth was inhibited (MIC90) by the compound ranged from 21.87 to 43.75 μg ml−1 against various fungal species by microbroth dilution assay. Since the compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate has antifungal activity it can be explored further to develop new antimycotic drugs.

The effect on the nasopharyngeal bacterial flora of 10 days of amoxycillin-clavulanate or cefdinir antimicrobial therapy was studied in 50 children with acute otitis media. Before therapy, 17 potential pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis) were isolated from the nasopharynx of 14 (56 %) of those treated with amoxycillin-clavulanate, and 20 potential pathogens were recovered from 15 (60 %) of those treated with cefdinir. Following therapy, at days 12–15, the number of potential pathogens was reduced to a similar extent with both therapies, to three in those treated with amoxycillin-clavulanate and two in those treated with cefdinir. However, the number of potential pathogens rebounded faster in those treated with amoxycillin-clavulanate as compared with cefdinir in the two subsequent specimens taken at days 30–35 and 60–65 (12 and 18 in the amoxycillin-clavulanate group, and six and nine in the cefdinir group, P < 0.01 and P < 0.001, respectively). Differences between the groups were also noted in the recovery of organisms with interfering capability. Immediately following amoxycillin-clavulanate therapy, the number of interfering organisms declined from 54 to 13, while following cefdinir treatment their number was reduced from 59 to 39 (P < 0.001). The differences between the two therapy groups persisted in the two later specimens taken at days 30–35 and 60–65 (25 and 38 in the amoxycillin-clavulanate group, and 52 and 51 in the cefdinir group, P < 0.001 and P < 0.05, respectively). This study illustrates the potential beneficial effect of using a narrow-spectrum antimicrobial that selectively spares the interfering organisms while eliminating pathogens. The benefit of such therapy is the prevention of reacquisition of pathogenic bacteria in the nasopharynx. In contrast, utilization of a broad-spectrum antimicrobial is associated with prolonged absence of inhibitory organisms and rapid recolonization with pathogens.

Many antiseptic agents are used in hygienic handwashes in the prevention of nosocomial infections by methicillin-resistant Staphylococcus aureus (MRSA). The plasmid-borne genes qacA/B and smr confer resistance to cationic antiseptic agents in S. aureus. In this study, the susceptibilities for dyes and antiseptic agents (e.g. acriflavine, acrinol, benzalkonium chloride, benzethonium chloride, chlorhexidine digluconate and alkyldiaminoethylglycine hydrochloride) of 894 isolates of MRSA collected from 11 Asian countries (South Korea, China, the Philippines, Singapore, Vietnam, Thailand, Indonesia, India, Sri Lanka, Saudi Arabia and Japan) between 1998 and 1999 were examined. In addition, the distributions of the antiseptic-resistance genes qacA/B and smr were studied by PCR. Among the Asian MRSA isolates 57.7 % (516/894) were acriflavine-resistant. The smr gene was detected in 31.6 % (12/38) of MRSA isolates from India but only in 1.9 % (16/856) of all the isolates from other Asian countries. MRSA with qacA/B comprised 41.6 % (372/894) of the isolates across Asia. In addition, PFGE was performed to type the MRSA and grouped the tested 30 MRSA isolates with qacA/B into 21 PFGE types. The results indicated that qacA/B is functionally the most important gene mediating antiseptic resistance in the MRSA strains of Asia and that a specific MRSA with qacA/B was not prevalent in Asia but qacA/B were widely spread among MRSA of Asia, while the geographical distribution of smr is more limited.

Surveillance of Helicobacter pylori antibiotic susceptibility from patients in London, the largest metropolitan area in the UK, is limited, despite resistance being a key factor in treatment failure. A two-centre survey was performed over 12 months (1999–2000) to determine antibiotic-resistance rates of isolates from dyspeptic patients attending endoscopy clinics serving two ethnically diverse central and south London communities. The in vitro antibiotic susceptibilities were determined from disc diffusion and epsilometer (E) tests on 101 H. pylori isolates. Overall resistance rates were 59 % for metronidazole and 11 % for clarithromycin, with 8 % resistance to both antibiotics. Corresponding primary resistance rates were 50 % and 7 %, respectively. High-level-resistance was a feature of 82 % of the metronidazole (MIC ⩾ 256 mg l−1) -resistant and 55 % of the clarithromycin (MIC ⩾ 32 mg l−1) -resistant strains. All isolates were susceptible to amoxycillin and tetracycline. No associations between resistance and either the gender or the age of the patients were detected. The main risk for resistance to metronidazole was non-UK birth as comparative rates were 68 % for non-UK vs. 40 % for UK-born individuals. Resistant isolates were genotypically diverse with respect to cagA/vacA type. Four 23S rDNA nucleotide polymorphisms were associated with clarithromycin resistance, mostly (9/11) at A2143G. In conclusion, the high overall metronidazole-resistance rate of 59 % for H. pylori from inner London was twice the rate found in other UK-based studies and was attributed to the higher risk of resistant strains infecting individuals born outside the UK. The need for continued resistance surveillance is indicated to monitor the effects of the ‘test and treat’ strategy for H. pylori eradication, particularly of isolates from at-risk individuals

Using similarities of IS6110 banding patterns, isolates of Mycobacterium tuberculosis from a population-based study in London were assigned to 12 large groups termed ‘superfamilies’ (sfams). Analysis of patient data showed a marked geographical association in the distribution of these sfams. In particular, isolates from patients born in Europe were from different sfams than those born elsewhere, indicating that there had been relatively little transmission of tuberculosis in London from immigrant communities into the endogenous population. Multivariate analysis showed that certain sfams were significantly associated with pulmonary rather than extrapulmonary disease, or with sputum smear negativity, independently of country of birth or ethnicity, suggesting that the properties of the infecting organism play a role in the nature of the disease process.

The extracellular phospholipases of Candida albicans are considered to play a significant role in the pathogenesis of human infections. Therefore 30 clinical isolates of C. albicans from human immunodeficiency virus (HIV)-infected individuals were screened for phospholipase production in vitro (using an egg-yolk-agar medium). Two groups of six isolates with positive (group A) or deficient (group B) phospholipase activity were then analysed for phospholipase B1 (PLB1) gene expression both in egg-yolk-agar and yeast extract/peptone/dextrose (YPD) broth media. A total of four virulence attributes of these two groups were in turn characterized, namely their germ-tube formation, cell-surface-hydrophobicity (CSH), adhesion to buccal epithelial cells (ABEC) and haemolysin production, and these factors were subsequently correlated with PLB1 expression. In the phospholipase-producing isolates (group A) a positive correlation was demonstrated between phospholipase production and the degree of PLB1 expression in YPD medium (r = 0.96, P < 0.01). No such association was observed in group A isolates for PLB1 expression in egg-yolk-agar medium. Further, PLB1 expression in egg-yolk agar was less than that in YPD medium, although a positive correlation was seen between the expression levels on regression analysis (r = 0.86, P = 0.026). Surprisingly, however, no significant associations were observed in either growth media between PLB1 expression and any of the four pathogenic attributes examined (P < 0.001). A significant correlation was seen between CSH and ABEC (r = 0.74) in group A isolates. The phospholipase-deficient group B, however, demonstrated a significant correlation between the latter parameters (r=+0^h.50) and also between germ-tube formation and ABEC (r=−0^h.59), and germ-tube formation and haemolysin production (r=+0^h.31). It appears that in oral C. albicans isolates in HIV infection there may be no significant association between the degree of PLB1 expression and other widely recognized major virulence attributes.

Aspirates from 48 chronically inflamed maxillary sinuses from patients who had nasal polyposis were processed for aerobic and anaerobic bacteria. Bacterial growth was present in 46 (96 %) specimens. Aerobic or facultative bacteria were present in 6 (13 %) specimens, anaerobic bacteria alone in 18 (39 %), and mixed aerobic and anaerobic bacteria in 22 (48 %). There were 110 bacterial isolates (2.4 per specimen). Thirty-nine of the isolates were aerobic or facultative organisms (0.85 per specimen). The predominant aerobic or facultative organisms were: Staphylococcus aureus, microaerophilic streptococci, Haemophilus influenzae and Moraxella catarrhalis. Seventy-one anaerobes were isolated (1.5 per specimen), Peptostreptococcus spp., Prevotella spp., Porphyromonas asaccharolytica and Fusobacterium spp. being predominant. These findings illustrate for the first time the presence of polymicrobial aerobic–anaerobic flora in patients with chronic maxillary sinusitis who had nasal polyposis.

The ability of Streptococcus mutans to produce mutacins, combined with the production of other virulence factors such as lactic acid, may contribute to the pathogenesis of this bacterium. In the present study, the detection of genes encoding mutacin types I/III, II and IV was performed by PCR with specific primers to each type in a total of 63 S. mutans genotypes isolated from caries-active and caries-free individuals. In the caries-free group, PCR screening for mutacin IV revealed that 31.8 % of strains were positive for this mutacin. PCR for the other three mutacins tested (I/III and II) did not yield amplicons in any S. mutans strains in this group. The PCR with primers of mutacin IV showed 68.3 % positive genotypes in the caries-active group, on the other hand, the amplicons of mutacins I/III revealed 41.5 % positive strains that carried these genes. The chi square test showed significant differences in the number of positive strains to mutacin IV when comparing the caries-free and caries-active genotypes of S. mutans (P = 0.01). All tested S. mutans strains were negative by PCR for mutacin II. The low frequencies of detection of some mutacin genes suggest the existence of high diversity and polymorphism in the production of genetic determinants of mutacin-like substances. In addition, the production of a wide spectrum of mutacins can play an important biological role in colonization by S. mutans strains, mainly in the niche of high-complexity microbial communities.

Two typical coliforms from an intestinal biopsy from an adult case of bloody diarrhoea carried genes encoding intimin-β, stx 1 and ehxA, and produced verocytotoxin 1 and enterohaemolysin in culture. Both were biochemically typical Escherichia coli O5 : H−, apart from producing urease. Such O5 isolates represent a human pathogenic E. coli lineage.

Cellulitis of the orbit is a common cause of proptosis in children, and also frequently arises in the elderly and the immunocompromised. The condition is characterized by infection and swelling of the soft tissues lining the eye socket, pushing the eye ball outwards and causing severe pain, redness, discharge of pus and some degree of blurred vision. There is a small risk of infection spreading to the meninges of the brain and causing meningitis. This paper reports the case of an adult in whom polymicrobial bilateral orbital cellulitis had developed due to Staphylococcus aureus and Neisseria gonorrhoeae infection. N. gonorrhoeae infections are acquired by sexual contact. Although the infection may disseminate to a variety of tissues, it usually affects the mucous membranes of the urethra in males and the endocervix and urethra in females. To the authors’ knowledge this is the first report of polymicrobial bilateral orbital cellulitis due to S. aureus and N. gonorrhoeae in medical literature.