- Volume 55, Issue 12, 2006

Different definitions of the terms multidrug-resistant (MDR) and pandrug-resistant (PDR) Acinetobacter baumannii and Pseudomonas aeruginosa have been used in the biomedical literature. The authors searched for relevant studies indexed in the PubMed database (01/2000–09/2005) to systematically examine the various definitions of MDR and PDR for these bacteria. Initially 107 retrieved relevant studies were reviewed. Ninety-two studies were further analysed, 50 of which focused on A. baumannii and 42 on P. aeruginosa. A considerable diversity of definitions of the terms MDR and PDR A. baumannii and P. aeruginosa was found. Of note, the term PDR was inappropriately used in all five studies that used it. The review reveals that various definitions have been used for the terms MDR and PDR A. baumannii and P. aeruginosa, a fact that causes confusion to researchers and clinicians. The authors believe that at least a widely accepted definition for PDR A. baumannii and P. aeruginosa should be uniformly used worldwide.

Citrobacter koseri causes neonatal meningitis frequently complicated with multiple brain abscesses. During C. koseri central nervous system infection in the neonatal rat model, previous studies have documented many bacteria-filled macrophages within the neonatal rat brain and abscesses. Previous studies have also shown that C. koseri is taken up by, survives phagolysosomal fusion and replicates in macrophages in vitro and in vivo. In this study, in order to elucidate genetic and cellular factors contributing to C. koseri persistence, a combinatory technique of differential fluorescence induction and transposon mutagenesis was employed to isolate C. koseri genes induced while inside macrophages. Several banks of mutants were subjected to a series of enrichments to select for gfp : : transposon fusion into genes that are turned off in vitro but expressed when intracellular within macrophages. Further screening identified several mutants attenuated in their recovery from macrophages compared with the wild-type. A mutation within an Escherichia coli fliP homologue caused significant attenuation in uptake and hypervirulence in vivo, resulting in death within 24 h. Furthermore, analysis of the immunoregulatory interleukin (IL)-10/IL-12 cytokine response during infection suggested that C. koseri fliP expression may alter this response. A better understanding of the bacteria–macrophage interaction at the molecular level and its contribution to brain abscess formation will assist in developing preventative and therapeutic strategies.

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1–2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6–6.9, P<0.001]. Nine of the ten strains negative by both Western blotting and zymography were non-clonal, and all but one of these was positive for the protease IV gene. There was a marked strain-to-strain variation in the amount of protease IV produced. Secretion of protease IV by clonal strains may enhance their infectivity and ability to adapt to the changing CF lung environment. Overall the findings suggest that protease IV plays an important role in the pathogenesis of P. aeruginosa infection in the CF lung.

This study reports the detection of an extracellular staphylococcal product, designated secretory inhibitor of platelet microbicidal protein (SIPMP), that causes local inhibition of the bactericidal action of platelet microbicidal protein (PMP) in the fluid phase. Urethral isolates of Staphylococcus aureus (n=24) and coagulase-negative staphylococci (CNS) (n=47) from patients with or without chronic bacterial prostatitis (CBP) were tested. SIPMP production was tested by inhibition of PMP bioactivity against Bacillus subtilis and was expressed as percentage inhibition of PMP bactericidal activity. The PMP susceptibility of staphylococcal strains was determined by exposing bacterial cells to serial dilutions of PMP. Staphylococci from patients without CBP produced SIPMP at levels of 10.3±1.2 and 13.25±1.72 % for S. aureus and CNS, respectively. Strains isolated from men with CBP inhibited PMP-induced killing of B. subtilis by 23.38±4.2 % (P<0.05) and 23.69±1.87 % (P<0.01) for S. aureus and CNS, respectively. SIPMP production correlated with staphylococcal resistance to PMP (r 2=0.6082 and 0.7264 for S. aureus and CNS, respectively). SIPMP represents a hitherto unrecognized determinant of staphylococcal pathogenicity. These results suggest that SIPMP production is associated with the CBP source. Data from this study may have significant implications for the understanding of the pathogenesis of CBP.

The prevalence of opportunistic fungal infections has increased dramatically among the aged population in recent years. This work investigated the effect of ageing on murine defences against Candida albicans. Aged C57BL/6 mice that were experimentally infected intravenously had a significantly impaired survival and a higher tissue fungal burden compared with young mice. In vitro production of tumour necrosis factor (TNF)-α by macrophages from aged mice in response to yeast cells and hyphae of C. albicans was significantly lower than production by macrophages from young mice. In vitro production of cytokines, such as TNF-α and gamma interferon (IFN-γ), by antigen-stimulated splenocytes from mice intravenously infected with C. albicans cells was also diminished in old mice. This decrease in production of T helper 1 cytokines in old mice correlated with a diminished frequency of IFN-γ-producing CD4+ T lymphocytes, although the ability to develop an acquired resistance upon vaccination (primary sublethal infection) of mice with the low-virulence PCA2 strain was not affected in aged mice. The diversity of antigens recognized by C. albicans-specific antibodies in sera from infected aged mice was clearly diminished when compared with that from infected young mice. Taken together, these data show that aged mice develop an altered innate and adaptive immune response to C. albicans and are more susceptible to systemic primary candidiasis.

A rapid and inexpensive method for the detection of drug resistance in Mycobacterium tuberculosis is essential for the effective control of tuberculosis. The aim of this study was to evaluate a colorimetric method using 2,3,5-triphenyltetrazolium chloride (TTC) for antibiotic susceptibility testing of M. tuberculosis isolates. Eleven multidrug-resistant (MDR) isolates of M. tuberculosis and 12 isolates which were susceptible to rifampicin (RIF) and isoniazid (INH) were used. The test was performed with a critical concentration of 0.2 μg ml−1 for INH and 2.0 μg ml−1 for RIF in 7H9GC broth with 0.625 μg TTC ml−1. Each isolate was inoculated under these conditions and inspected daily for colour changes; the results were obtained after a mean of 4.9 days. The sensitivity and specificity of this method were 100 % and 92 %, respectively, for both antibiotics. Considering the speed, technical ease and cost-effectiveness of this method, the TTC assay is a good alternative method for drug susceptibility testing of M. tuberculosis isolates.

Salmonella enterica subspecies enterica serovar Paratyphi B [O1,4,(5),12 : Hb : 1,2] can cause either an enteric fever (paratyphoid fever) or self-limiting gastroenteritis in humans. The d-tartrate non-fermenting variant S. enterica subsp. enterica serovar Paratyphi B dT− (S. Paratyphi B) is the causative agent of paratyphoid fever, and the d-tartrate fermenting variant S. enterica subsp. enterica serovar Paratyphi B dT+ (S. Paratyphi B dT+; formerly called Salmonella Java) causes gastroenteritis. S. Java is currently recognized as an emerging problem worldwide. Twelve dT+ S. Java isolates were collected in Indonesia between 2000 and 2002. One-third of them contained Salmonella genomic island 1 (SGI1), which gives the multidrug-resistant phenotype to the bacteria. In this study, a PCR-based method to detect a single nucleotide difference responsible for the inability to ferment d-tartrate, reported elsewhere, was validated. The d-tartrate fermenting phenotype of S. Java was converted to the non-fermenting phenotype by the disruption of the ORF STM 3356, and the d-tartrate non-fermenting phenotype of the ORF STM 3356-disrupted strain and the dT− reference strain was changed to the dT+ phenotype by complementing ORF STM 3356 in trans. The results show that the dT+ phenotype requires a functional product encoded by STM 3356, and support the use of the PCR-based discrimination method for S. Paratyphi B and S. Java as the standard differentiation method.

Screening for Chlamydia trachomatis infections can be performed on urine samples and genital swabs using molecular techniques. A novel approach was developed that combined an automated extraction procedure, an automated liquid-handling system and real-time PCR to detect C. trachomatis from urine or swabs. This novel real-time PCR approach was compared to the commercial Cobas Amplicor system on 628 specimens. In a retrospective analysis, 51 samples that tested positive using the Cobas assay were also positive with the real-time PCR, whereas the 49 samples negative with Cobas were also negative with the real-time PCR, for an overall agreement of 100 %. Among 528 prospective samples consecutively received at the authors' laboratory with a request for C. trachomatis PCR, five PCR reactions were inhibited when tested with Cobas. These five inhibited samples were found negative with the real-time PCR. Among the remaining 523 samples, 45 (8.6 %) were positive with both methods, 476 (91 %) were negative with both methods, and 2 (0.4 %) were positive with Cobas but negative with the real-time PCR. Thus, when considering Cobas as the gold standard, the overall agreement was 99.6 %, the sensitivity of the real-time PCR was 95.7 % and the specificity was 100 %. The two discrepant samples were retested in parallel and were found negative with both methods. When testing a batch of 25 samples, both reagent costs and laboratory technician time were reduced with the new technique (7.30 euros per sample and 134 min) compared to Cobas (11.20 euros per sample and 232 min). Moreover, due to reduced organizational constraints, the median time from sample reception to result was only 24 h using the automated platform. Overall, this novel real-time PCR approach exhibited an excellent specificity and a sensitivity similar to that of Cobas Amplicor PCR for the detection of C. trachomatis. Given its high throughput potential and low costs/laboratory technician time requirement, it may be useful for future use in large C. trachomatis screening programs.

Borderline oxacillin-resistant Staphylococcus aureus (BORSA) exhibit oxacillin MIC values of 1–8 μg ml−1, but lack mecA, which encodes the low-affinity penicillin-binding protein (PBP)2a. The relationship of the BORSA phenotype with specific genetic backgrounds was assessed, as well as amino acid sequence variation in the normal PBP2. Among 38 BORSA, 26 had a common PFGE profile of genomic DNA, and were multilocus sequence type (ST)25. The other isolates were genetically diverse. Complete pbp2 sequences were determined for three BORSA, corresponding to ST25, ST1 and ST47, which were selected on the basis of lacking blaZ-encoded β-lactamase. The essential transpeptidase-domain-encoding segment of pbp2 was also sequenced from seven additional ST25 isolates. Amino acid substitutions occurred in the transpeptidase domain of all BORSA, irrespective of clonal type. A Gln629→Pro substitution was common to all ST25 BORSA, but most could be distinguished from one another by additional unique substitutions in the transpeptidase domain. The ST1 and ST47 isolates also possessed unique substitutions in the transpeptidase domain. Plasmid-mediated expression of pbp2 from an ST25 or ST1 isolate in S. aureus RN6390 increased its oxacillin MIC from 0.25 to 4 μg ml−1, while pbp2 from a susceptible strain, ATCC 25923, had no effect. Therefore, different amino acid substitutions in PBP2 of diverse BORSA lineages contribute to borderline resistance. The predominant ST25 lineage was not related to any of the five clonal complexes that contain meticillin-resistant S. aureus (MRSA), suggesting that ST25 cannot readily acquire mecA-mediated resistance.

This study characterized the genetic basis of antimicrobial resistance of a number of Shigella spp. isolated from humans from 2000 to 2004 in Hiroshima prefecture, Japan. A total of 26 isolates of Shigella spp. were included in this study. Antimicrobial susceptibility tests revealed high levels of resistance, especially to ampicillin, streptomycin, trimethoprim, tetracycline, nalidixic acid and ciprofloxacin. PCR and DNA sequencing were used for screening and characterization of antibiotic-resistance determinants. PCR sequencing analysis revealed the presence of only one type of class 1 integron in one isolate of Shigella sonnei. This class 1 integron was 1904 bp and contained two gene cassettes: a probable esterase/lipase (estX) and aadA1, which confers resistance to streptomycin and spectinomycin. Two types of class 2 integron were identified in this study. One was the classic type (2158 bp) and carried the three conserved resistance gene cassettes of the class 2 integron, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively. This type was detected in both Shigella sonnei (14 isolates) and Shigella flexneri (five isolates). The other type was shorter (1313 bp) and carried only two gene cassettes, dfrA1 and sat1. This integron was detected in a single isolate of Shigella sonnei. PFGE patterns showed limited diversity within clusters of the same species. Furthermore, an extended-spectrum β-lactamase gene, bla OXA-30, which confers resistance to ampicillin, was characterized in all isolates of Shigella flexneri except the oldest strain, which was isolated in 2000. Southern blot hybridization and conjugation experiments showed that bla OXA-30 was located in the chromosome.

A prospective study was performed to evaluate the correlation between the proposed standard of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) (document 7.1) and the commercial system Etest for determining the MICs of flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole for a collection of 100 clinical and environmental isolates of Cryptococcus neoformans. The agreements among Etest MICs within ±2 log2 dilutions of AFST-EUCAST standard MICs were greater for flucytosine, fluconazole and voriconazole (76, 78 and 88 %, respectively) than for amphotericin B (5 %), the lowest agreement, and itraconazole (67 %). Overall, the correlation coefficients were statistically significant (P<0.05), and it is suggested that the Etest and AFST-EUCAST method are reliable alternatives and present good correlation for all drugs evaluated except amphotericin B. However, the observed differences related to MICs for susceptible, susceptible dose dependent and resistant strains between the methods suggest that it will be necessary to carry out further studies, including assessment of interlaboratory agreement and correlation of MICs by different methods with in vivo response.

A total of 985 group A streptococcus (GAS) bacteraemia isolates collected in Finland during 1995–2004 were T-serotyped, and of these, 336 isolates of serotype T28 were subjected to further emm typing. The total number of isolates referred per year showed an increase within the study period, from 43 in 1995 to 130 in 2004. The annual incidence of invasive GAS (iGAS) bacteraemia showed a general increase during the study period, from 1.1 to 2.5 per 100 000 population. Serotype T28 remained among the most common serotypes, in addition to serotypes TB3264 and T1. The serotype T28 isolates were found to be distributed across six distinct emm types: emm28, emm77, emm53 (including subtypes 53.2 and 53.4), emm87, emm2 and emm4. The serotype distribution and the emm type distribution of serotype T28 fluctuated over time. Within the study period, the proportion of T28/emm28 isolates became the most prominent. During periods of low emm28 incidence, emm types 77 and 53 seemed to show a resurgence. emm typing revealed T28 isolates to be a genetically heterogeneous group harbouring a variety of distinct M proteins. This study confirms that T serotyping alone is not a sufficient method for epidemiological surveillance of iGAS.

A fatal case of nosocomial legionellosis in a low prevalence region (Calgary, Alberta, Canada) prompted investigation into the source of infection. Hospital water systems contaminated with Legionella pneumophila have been shown to pose a risk to compromised patients. Typing of an L. pneumophila serogroup 1 strain isolated from the patient using sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) analysis linked it to a persistent and widespread strain isolated from the hospital water system establishing a nosocomial mode of acquisition. Different SBT and AFLP patterns were determined for non-epidemiologically linked cases and isolates from different hospitals.

The objective of this study was to develop and evaluate a rapid new method of identifying clinically relevant Nocardia species. DNA extracted from different Nocardia strains was used in a real-time PCR assay with SYBR Green and melting-curve analysis to identify Nocardia species. Ten control strains and four bacterial strains of closely related genera were employed, and samples from 28 patients were used. All Nocardia strains were identified correctly, and there was no cross-reaction with strains from genera closely related to Nocardia. The sensitivity and specificity of the method were 90 and 100 %, respectively. This method can be used to rapidly detect Nocardia species in culture, without cross-reaction with other closely related genera.

Ruminants are fed forage which is often contaminated with Listeria, and frequently shed Listeria monocytogenes with their faeces. The present study was designed to localize the sites of infection in the digestive tract concomitant with Listeria faecal excretion in a sheep model. Ten Listeria-free sheep were inoculated per os with a dose of 1010 c.f.u. of a pathogenic L. monocytogenes strain. Listeria received by two of the ten animals were radiolabelled with 111indium oxine. The dissemination of the Listeria was assessed by in vivo imaging, by culture of bacteria in the faeces, organs and digesta samples taken at slaughter on days 1, 2, 6, 10 and 14 post-inoculation, and by measuring gamma radioactivity of samples on day 6. It was shown that Listeria spread through the entire volume of the forestomachs within 4 h, and through the whole gastrointestinal tract (GIT) within 24 h. Faecal shedding of Listeria lasted 10 days. Rumen, duodenum, jejunum, ileum, caecum and colon walls and digesta, mesenteric lymph nodes, liver and spleen were temporarily infected. However, Listeria persisted for at least 14 days in rumen digesta and retropharyngeal lymph nodes, and at a relatively high level (1×104 c.f.u. g−1) in palatine tonsils. These findings suggest that L. monocytogenes can translocate from all parts of the GIT, with the rumen digesta, but not the gallbladder, serving as a reservoir. The results indicate that brief and low-level faecal excretion of L. monocytogenes is concomitant with a transitory asymptomatic infection in sheep.

Previous epidemiological studies have demonstrated a potential link between the serotypes of Yersinia enterocolitica recovered from cattle, sheep and pigs and those isolated from human disease cases. Further studies utilizing amplified fragment length polymorphisms have shown a relationship at the genetic level between strains of biotypes 3 and 4 from humans and livestock, and also suggested that some biotype 1A isolates, classically defined as non-pathogenic, are closely related to biotype 3 and 4 isolates. This study sought to understand further the pathogenic potential of Y. enterocolitica isolates from livestock in Great Britain. A range of surrogate in vitro models, such as invasion of epithelial tissue cultures, survival in cultured macrophages and cytokine secretion response, was employed to assess the pathogenicity of 88 strains. The results suggested that all isolates examined were capable of adhering to and invading epithelial cells and of surviving within macrophages. However, the inflammatory response of the infected macrophages differed with the infecting Y. enterocolitica subtype, with the response to pathogenic biotype 3 and 4 isolates different to that observed with biotype 1A isolates, and with the biotype 3 O : 5,27 isolates recovered exclusively from animals. Infections of porcine tissue also suggested the possibility of host-tissue tropism within Y. enterocolitica subtypes.

An immunogenic 22 kilodalton exported Mycobacterium avium subspecies paratuberculosis (MAP) lipoprotein (P22) was previously identified, and found to belong to the LppX/LprAFG family of mycobacterial lipoproteins. N-terminal polyhistidine-tagged P22 was produced and purified from Escherichia coli. Antibody recognition of P22, and interferon-gamma (IFN-γ) responses in vitro using blood from a sheep vaccinated with Neoparasec, confirmed its immunogenicity. To evaluate the immunogenicity of P22 in vivo, five sheep were immunized with a single dose containing 0.8 mg recombinant P22 protein in adjuvant. Blood was collected at 4, 13 and 29 weeks post-immunization (p.i.) and tested for anti-P22 antibodies and P22-specific IFN-γ production. P22-specific antibodies were detected by Western blot analysis in all five Neoparasec-immunized sheep at the three time points. Three out of five P22-immunized sheep produced P22-specific antibodies for up to 13 weeks p.i., and two gave a response at 29 weeks p.i. Recombinant P22 was able to stimulate significant IFN-γ production in blood of P22-immunized sheep at 13 and 29 weeks p.i. Recombinant P22 also elicited an IFN-γ response in blood of sheep immunized with Neoparasec.

The purpose of this study was to compare the frequency of recovery of aerobic and anaerobic organisms with interfering capability against group A β-haemolytic streptococci (GABHS) and β-lactamase-producing bacteria (BLPB) from the tonsils of GABHS carriers and non-carriers. The presence of aerobic and anaerobic bacteria capable of such interference in vitro was evaluated in cultures obtained from the tonsils of 20 healthy children who were non-GABHS carriers and 20 who were GABHS carriers, and also from 20 children who were asymptomatic after completing a course of penicillin for acute GABHS pharyngo-tonsillitis (PT) and were non-GABHS carriers and 20 who were GABHS carriers. In healthy children, 32 interfering isolates were recovered from 16 non-GABHS carriers (1.6 per child) and 13 were isolated from 7 GABHS carriers (0.65 per child) (P<0.001). In children who had suffered acute GABHS PT, 26 interfering organisms were recovered from 15 non-GABHS carriers (1.3 per child) and 8 were isolated from 5 GABHS carriers (0.4 per child) (P<0.005). In healthy children, 13 BLPB were recovered from 5 non-GABHS carriers and 13 were isolated from 6 GABHS carriers. In children who had suffered acute GABHS PT, 14 BLPB were recovered from 5 (25 %) non-GABHS carriers and 32 were isolated from 17 (85 %) GABHS carriers (P<0.05). It was demonstrated in this study that there was a higher rate of recovery of aerobic and anaerobic organisms capable of interfering with GABHS in non-GABHS carriers than in GABHS carriers. This was observed in all GABHS non-carriers and included healthy children as well as those recently treated for symptomatic GABHS PT with penicillin that failed to eradicate GABHS. A higher rate of recovery of BLPB was observed only in GABHS carriers who were treated with penicillin for GABHS PT.

A case is reported of a post-traumatic olecranon bursitis caused by Mycobacterium kansasii following an injury sustained in a public swimming pool. It responded to surgical debridement and combined rifampicin, isoniazid, pyrazinamide and ethambutol antimicrobial therapy. A literature search was performed and a treatment regimen for this uncommon condition is suggested.