- Volume 58, Issue 9, 2009

Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide. Nosocomial infections caused by this organism are often hard to treat because of both the intrinsic resistance of the species (it has constitutive expression of AmpC β-lactamase and efflux pumps, combined with a low permeability of the outer membrane), and its remarkable ability to acquire further resistance mechanisms to multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones. P. aeruginosa represents a phenomenon of bacterial resistance, since practically all known mechanisms of antimicrobial resistance can be seen in it: derepression of chromosomal AmpC cephalosporinase; production of plasmid or integron-mediated β-lactamases from different molecular classes (carbenicillinases and extended-spectrum β-lactamases belonging to class A, class D oxacillinases and class B carbapenem-hydrolysing enzymes); diminished outer membrane permeability (loss of OprD proteins); overexpression of active efflux systems with wide substrate profiles; synthesis of aminoglycoside-modifying enzymes (phosphoryltransferases, acetyltransferases and adenylyltransferases); and structural alterations of topoisomerases II and IV determining quinolone resistance. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. This review describes the known resistance mechanisms in P. aeruginosa to the most frequently administrated antipseudomonal antibiotics: β-lactams, aminoglycosides and fluoroquinolones.

Bacteria of the genus Bartonella are emerging zoonotic bacteria recognized in a variety of human diseases. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming, expensive and technically demanding. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for bacterial species identification. This study evaluated the use of MALDI-TOF MS for rapid genus and species identification of Bartonella species. Reference strains representing 17 recognized Bartonella species were studied. For each species, MS spectra for four colonies were analysed. The consensus spectrum obtained for each species was unique among spectra obtained for 2843 bacteria within the Bruker database, including 109 alphaproteobacteria. Thirty-nine additional blind-coded Bartonella strains were correctly identified at the species level, including 36 with a significant score. Altogether, these data demonstrate that MS is an accurate and reproducible tool for rapid and inexpensive identification of Bartonella species.

Vibrio cholerae O1, O139 and occasionally non-O1/non-O139 serogroups are most often responsible for epidemic and pandemic cholera. This study used genotypic patterns of PCR-based detection of virulence-associated and regulatory protein genes, along with phage typing, to characterize 86 V. cholerae strains. Thirty-eight of 53 O1 biotype El Tor strains harboured both tcpA classical and tcpA El Tor genes, and three El Tor strains lacked the V. cholerae O1-specific gene (Vc-O1); three O139 strains contained both Vc-O1 and Vc-O139 genes and seven out of ten non-O1/non-O139 strains possessed the Vc-O1 gene. The latter strains all harboured the virulence-associated genes ctxA, zot, ace, RS1, hlyA, ompU, rtxA and sxt. Two phage types, T27 and T25, were predominant in strains from different geographical regions of India, whereas more variation in phage susceptibility was observed for tetracycline-resistant strains from Kolkata. These results suggest that the pattern and distribution of virulence genes and phage types of V. cholerae are equally useful and discriminatory in tracing the origin of newly emerging strains.

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.

This study describes the development of a novel assay to detect fungal DNA and identify the most clinically relevant invasive human pathogenic fungi to the species level using oligonucleotide probes, labelled with electrochemically active groups, and solid-state electrodes. A panfungal probe designed against the 18S rRNA gene region, capable of detecting all fungal pathogens tested, and species-specific probes, designed against the ITS2 region for detection of the five Candida species most commonly encountered in the clinical setting (Candida albicans, Candida glabrata, Candida parapsilosis species complex, Candida krusei and Candida tropicalis), are described. When tested with PCR-amplified DNA from both type and clinical strains of the relevant species, the probes were able to positively identify the relevant fungi, indicated by production of a current significantly elevated above the background reading. No cross-reactivity was observed with any of the species-specific probes when compared with nine non-target Candida species or in the presence of human DNA equivalent to an equal number of ITS2 targets. The panfungal probe gave results that were similarly positive against 15 other fungal species and also did not cross-react with human DNA. The limit of detection of the assay was shown to be approximately 1 genome equivalent for all probes using extracted genomic DNA.

Our study was aimed at comparing the meticillin-resistant Staphylococcus aureus (MRSA) strains isolated from an anonymous group of health-care workers (HCWs) with those obtained from patient samples during a 3-month time interval. We employed spa typing and virulence gene profiling to characterize the MRSA strains. Our data revealed that a total of 14 discrete spa types were circulating in both patients and HCWs. The t032 spa type, characteristic of EMRSA15 and the Barnim EMRSA ST 22 clones, accounted for over 70 % of isolates, and was equally distributed between patients and HCW groups. In addition, a number of epidemic and sporadic strains were identified, which highlighted the diversity of spa types that can be found within a health-care setting. Virulence profiling for the carriage of 7 genes by the 14 different spa types demonstrated that 10 types carried the fnbA, cna, sdrE, hlg and ica virulence factors. We concluded that there was no significant difference between the MRSA strains found circulating in the patients and the HCWs, and noted that the dominant spa types carried an identical set of virulence genes that included the key adhesins fnbA, cna and sdrE.

The alarming spread of multiple drug resistance in Staphylococcus aureus, combined with the frequent occurrence of S. aureus and Staphylococcus epidermidis in biofilm-type infections, indicates a growing need for new therapies. The experimental steroidal amide anprocide [3β-acetoxy-17β-(l-prolyl)amino-5α-androstane] significantly reduced c.f.u. ml−1 per suture (P <0.0001) in a murine model of topical S. aureus infection. In chequerboard assays with planktonic-grown S. aureus and S. epidermidis, anprocide was synergistic with bacitracin, oxacillin, clindamycin or ceftriaxone. Anprocide was also synergistic in combination with bacitracin or oxacillin against some isolates of biofilm-grown S. aureus and S. epidermidis.

Community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) occurring among hospital isolates in Malaysia has not been reported previously. As CA-MRSA reported worldwide has been shown to carry SCCmec types IV and V, the aim of this study was to determine the SCCmec types of MRSA strains collected in Malaysia from November 2006 to June 2008. From a total of 628 MRSA isolates, 20 were SCCmec type IV, whilst the rest were type III. Further characterization of SCCmec type IV strains revealed 11 sequence types (STs), including ST22, with the majority being ST30/Panton–Valentine leukocidin positive. Eight out of nine CA-MRSA were ST30, one was ST80, and all were sensitive to co-trimoxazole and gentamicin. Five new STs designated ST1284, ST1285, ST1286, ST1287 and ST1288 were discovered, suggesting the emergence of novel clones of MRSA circulating in Malaysian hospitals. The discovery of the ST22 strain is a cause for concern because of its ability to replace existing predominant clones in certain geographical regions.

Epidemiological data based on phenotypic and molecular characterization of meticillin-resistant Staphylococcus aureus (MRSA) in sub-Saharan Africa are limited. This investigation studied 61 MRSA isolates obtained from 13 health-care institutions in KwaZulu-Natal (KZN) province, South Africa, from March 2001 to August 2003. More than 80 % of the isolates were resistant to at least four classes of antibiotics and six isolates were resistant to the aminoglycoside, macrolide-lincosamide and tetracycline groups of antibiotics, heavy metals and nucleic acid-binding compounds. PFGE of SmaI-digested genomic DNA revealed seven types, designated A–G. Type A was the main pulsotype (62.3 %) and was identified in 11 of the 13 health-care institutions, suggesting that it represented a major clone in health-care institutions in KZN province. Analysis of representative members of the three major pulsotypes by spa, multilocus sequence typing and SCCmec typing revealed the types t064-ST1173-SCCmec IV and t064-ST1338-SCCmec IV (PFGE type A, single-locus and double-locus variants of ST8), t037-ST239-SCCmec III (PFGE type F) and t045-ST5-SCCmec III (PFGE type G). The combination of various typing methods provided useful information on the geographical dissemination of MRSA clones in health-care institutions in KZN province. The observation of major clones circulating in health-care facilities in KZN province indicates that adequate infection control measures are urgently needed.

Thirteen patients with leptospirosis were identified, as confirmed by laboratory analysis during the last 5 years in our laboratory, who came from urban areas of Tokyo, Japan. All of the patients came into contact with rats before the onset of illness. Seventeen per cent of Norway rats captured in the inner cities of Tokyo carried leptospires in their kidneys. Most of these rat isolates were Leptospira interrogans serovar Copenhageni/Icterohaemorrhagiae. Antibodies against these serovars and their DNA were detected in the patients. This suggests that rats are important reservoirs of leptospirosis, and that rat-borne leptospires occur in urban areas of Tokyo.

Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica subsp. enterica serovar Typhimurium LT2. DNA encoding an epitope from Sendai virus, SV9 (Sendai virus nucleoprotein peptide 324–332, FAPGNYPAL), which is known to induce cytotoxic T lymphocytes, was incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length DNA segment. To improve cytotoxic T lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) or arginine (RR) residues. Western blotting and immunofluorescence microscopy using AgfA-specific antiserum verified the expression of chimeric AgfA; expression was also proved by a Congo red binding assay. Oral immunizations of C57BL/6 mice with the four strains induced an epitope-specific T-cell response (detected by enzyme-linked immunosorbent spot assay). When the mice were challenged with the Sendai virus, the magnitude of the infection was significantly reduced in the immunized groups compared with the controls. The Salmonella fimbrial display system efficiently induces a cellular immune response and anti-infection immunity in vivo, providing a new strategy for the development of efficient peptide vaccination.

We report a case of community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) bacteraemia with cavernous sinus thrombosis, meningitis and brain abscess in a previously healthy American, who was employed in Belgium. We consecutively reviewed all published cases of CA-MRSA with central nervous system (CNS) involvement. A total of 12 similar cases were found, of which 11 were published in the last 4 years. Predominantly, young previously healthy subjects were affected (median age 28 years). The cases involved brain abscesses (5/12), disseminated disease (4/12), cavernous sinus thrombosis (2/12) and other (1/12). Infection origins were superficial skin infections (5/12), mostly of the face, sinusitis (1/12), otitis media (1/12), other or unknown (5/12). Although, in our review of the literature patients treated with linezolid had a better outcome compared to patients treated with vancomycin, the latter is still the mainstay of therapy for CNS infections associated with MRSA.