Showing papers in "Journal of Medical Microbiology in 2006"

Antimicrobial activities of silver dressings: an in vitro comparison.

Abstract: A range of silver-coated or -impregnated dressings are now commercially available for use but comparative data on their antimicrobial efficacies are limited. The antibacterial activities of five commercially available silver-coated/impregnated dressings were compared against nine common burn-wound pathogens, namely methicillin-sensitive and -resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Proteus vulgaris, Acinetobacter baumannii and a multi-drug-efflux-positive Acinetobacter baumannii (BM4454), using a broth culture method. The rapidity and extent of killing of these pathogens under in vitro conditions were evaluated. All five silver-impregnated dressings investigated exerted bactericidal activity, particularly against Gram-negative bacteria, including Enterobacter species, Proteus species and E. coli. The spectrum and rapidity of action, however, ranged widely for different dressings. Acticoat and Contreet had a broad spectrum of bactericidal activities against both Gram-positive and -negative bacteria. Contreet was characterized by a very rapid bactericidal action and achieved a reduction of > or =10,000 c.f.u. ml(-1) in the first 30 min for Enterobacter cloacae, Proteus vulgaris, Pseudomonas aeruginosa and Acinetobacter baumanii. Other dressings demonstrated a narrower range of bactericidal activities. Understanding the characteristics of these dressings may enable them to be targeted more appropriately according to the specific requirements for use of a particular dressing, as in for prophylaxis in skin grafting or for an infected wound with MRSA.

Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance.

Abstract: Matrix material was extracted from biofilms of Candida albicans and Candida tropicalis and analysed chemically Both preparations contained carbohydrate, protein, hexosamine, phosphorus and uronic acid However, the major component in C albicans matrix was glucose (32 %), whereas in C tropicalis matrix it was hexosamine (27 %) Biofilms of C albicans were more easily detached from plastic surfaces by treatment with the enzyme lyticase (β-1,3-glucanase) than were those of C tropicalis Biofilms of C albicans were also partially detached by treatment with proteinase K, chitinase, DNase I, or β-N-acetylglucosaminidase, whereas C tropicalis biofilms were only affected by lipase type VII or chitinase To investigate a possible role for the matrix in biofilm resistance to antifungal agents, biofilms of C albicans were grown under conditions of continuous flow in a modified Robbins device (MRD) These biofilms produced more matrix material than those grown statically, and were significantly more resistant to amphotericin B Biofilms of C tropicalis synthesized large amounts of matrix material even when grown statically, and such biofilms were completely resistant to both amphotericin B and fluconazole Mixed-species biofilms of C albicans and a slime-producing strain of Staphylococcus epidermidis (RP62A), when grown statically or in the MRD, were also completely resistant to amphotericin B and fluconazole Mixed-species biofilms of C albicans and a slime-negative mutant of S epidermidis (M7), on the other hand, were completely drug resistant only when grown under flow conditions These results demonstrate that the matrix can make a significant contribution to drug resistance in Candida biofilms, especially under conditions similar to those found in catheter infections in vivo, and that the composition of the matrix material is an important determinant in resistance

Invasive fungal infections: a review of epidemiology and management options.

Abstract: Fungi are increasingly recognised as major pathogens in critically ill patients. Candida spp. and Cryptococcus spp. are the yeasts most frequently isolated in clinical practice. The most frequent filamentous fungi (moulds) isolated are Aspergillus spp., but Fusarium spp., Scedosporium spp., Penicillium spp., and Zygomycetes are increasingly seen. Several reasons have been proposed for the increase in invasive fungal infections, including the use of antineoplastic and immunosuppressive agents, broad-spectrum antibiotics, and prosthetic devices and grafts, and more aggressive surgery. Patients with burns, neutropenia, HIV infection and pancreatitis are also predisposed to fungal infection. The epidemiology and clinical features of fungal infections are reviewed, together with antifungal agents currently or soon to be available.

The diversity of definitions of multidrug-resistant (MDR) and pandrug-resistant (PDR) Acinetobacter baumannii and Pseudomonas aeruginosa.

Abstract: 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.

Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen

Abstract: Listeria monocytogenes is an opportunistic intracellular pathogen that has become an important cause of human foodborne infections worldwide. Given its close relationship to other Listeria species and its tendency to produce non-specific clinical symptoms, the availability of rapid, sensitive and specific diagnostic tests for the differentiation of L. monocytogenes from other Listeria species is helpful for selecting appropriate treatment regimens. In addition, with L. monocytogenes comprising a diversity of strains of varying pathogenicity, the ability to precisely track the strains involved in listeriosis outbreaks and speedily determine their pathogenic potential is critical for the control and prevention of further occurrences of this deadly disease. Extensive research in recent decades has revealed significant insights regarding the molecular mechanisms of L. monocytogenes infection. This in turn has facilitated the development of laboratory procedures for enhanced detection and identification of L. monocytogenes, and has also contributed to the implementation of improved control and prevention strategies against listeriosis. The purpose of this review is to summarize recent progress in the species-specific identification, subtyping and virulence determination of L. monocytogenes strains, and to discuss future research needs pertaining to these important areas of listeriosis.

Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species.

Abstract: The composition of the essential oil of Thymus pulegioides and its antifungal activity on Candida, Aspergillus and dermatophyte fungal strains were studied. Essential oil from the aerial parts of the plant was obtained by hydrodistillation and analysed by GC and GC-MS. The oil showed high contents of carvacrol and thymol. The MIC and minimal lethal concentration were used to evaluate the antifungal activity against Candida (seven clinical isolates and four ATCC type strains), Aspergillus [five clinical isolates, and two Coleccion Espanola de Cultivos Tipo (CECT) and two ATCC type strains] and five clinical dermatophyte strains. Antifungal activity was evaluated for the essential oil and for its main components. To clarify its mechanism of action on yeasts and filamentous fungi, flow-cytometric studies of cytoplasmic membrane integrity were performed, and the effect on the amount of ergosterol was investigated. Results showed that T. pulegioides essential oil exhibited a significant activity against clinically relevant fungi, mainly due to lesion formation in the cytoplasmic membrane and a considerable reduction of the ergosterol content. The present study indicates that T. pulegioides essential oil has considerable antifungal activity, deserving further investigation for clinical applications.

Genetic analysis of Cryptosporidium from 2414 humans with diarrhoea in England between 1985 and 2000.

Abstract: The characterization of Cryptosporidium using DNA extracted from whole faecal samples collected from 2414 humans with diarrhoea in England between 1985 and 2000 where cryptosporidial oocysts were detected using conventional methods is described. Characterization was achieved by PCR/RFLP and DNA sequencing of fragments of the Cryptosporidium oocyst wall protein and the 18S rDNA genes. Cryptosporidium parvum was detected in 56.1 % of cases, Cryptosporidium hominis in 41.7 % and a mixture of C. parvum and C. hominis in 0.9 %. In the remainder of cases, Cryptosporidium meleagridis (0.9 %), Cryptosporidium felis (0.2 %), Cryptosporidium andersoni (0.1 %), Cryptosporidium canis (0.04 %), Cryptosporidium suis (0.04 %) and the Cryptosporidium cervine type (0.04 %) were detected.

The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn's disease and ulcerative colitis patients.

Abstract: The bacterial community (microbiota) that inhabits the gut of humans appears to be an important source of antigens that drive the chronic immunological processes characteristic of Crohn's disease (CD) and ulcerative colitis (UC). Most of the members of the microbiota have not yet been cultured, but nucleic-acid-based methods of detection and enumeration can provide information about the community. This investigation used these methods to obtain information about the bacteria associated with mucosal surfaces in the gut of 20 CD and 15 UC patients. Biopsies were collected from inflamed and non-inflamed sites in the intestines of newly diagnosed, untreated patients. Biopsies were also collected from several intestinal sites of 14 healthy subjects. The bacterial collections associated with the biopsies were analysed by generating PCR/denaturing gradient gel electrophoresis (DGGE) profiles, the preparation of 16S rRNA gene clone libraries, and qualitative PCR to detect specific groups of bacteria. The total numbers of bacteria associated with the biopsies were determined by real-time quantitative PCR. DGGE profiles generated from the terminal ileum and various colonic regions were characteristic of each individual but differed between subjects. DGGE profiles and 16S rRNA gene libraries showed that the bacteria associated with inflamed and non-inflamed tissues did not differ. UC patients had more bacteria associated with biopsies than did CD patients (P<0.01). Statistical analysis of the composition of 16S rRNA gene libraries showed that the bacterial collections in UC and CD patients differed (P<0.05). Unclassified members of the phylum Bacteroidetes were more prevalent in CD than in UC patients. Therefore, the types and numbers of bacteria associated with biopsy samples were distinctly different for UC and CD patients. The observations made in this study should permit targeting of specific bacteriological abnormalities in investigations of the pathogenesis of inflammatory bowel diseases and provide targets for medical interventions.

Epidemiology of meningococcal disease in England and Wales 1993/94 to 2003/04: contribution and experiences of the Meningococcal Reference Unit.

Abstract: The laboratory confirmation of meningococcal disease and characterization of Neisseria meningitidis isolates was improved considerably in England and Wales by the Meningococcal Reference Unit between epidemiological years 1993/94 and 2003/04 to meet the challenge of increasing numbers of cases of clinical disease and the requirement for enhanced surveillance. Improved case ascertainment was made possible by the rapid introduction of an innovative centralized reference service for non-culture PCR-based DNA detection of meningococci utilizing the ctrA and siaD PCR assays, complemented by consistent phenotypic characterization of submitted isolates from culture-proven cases. This allowed the increased prevalence of serogroup C disease in specific age groups and the apparent associated increase in mortality from 1995/96 to 1999/00 to be defined, thereby prompting accelerated intervention with the newly licensed meningococcal serogroup C conjugate (MCC) vaccines into the under-25-year UK population (in November 1999). The continued increase in and predominance of serogroup B cases (1993/94 to 2000/01) were observed in conjunction with their diverse and changing phenotypic characteristics. Trends observed to be associated with the predominant phenotypic combinations of serogroup, serotype and sero-subtype were: a decline of both C : 2b and B : 2b meningococci, and a decline of B : 15 : P1.7,16 with a concomitant increase of B : 4 : P1.4 over the 11-year period. Detailed routine surveillance rapidly confirmed the introduction of W135 : 2a : P1.5,2 meningococci into the UK during 2000 and 2001. The importance of continued detailed surveillance of this important pathogen cannot be overestimated, both to monitor the effectiveness of the MCC vaccine and to identify changes within the meningococcal population that can inform the design of anti-serogroup B vaccines.

Prevalence of Bacteroides and Prevotella spp. in ulcerative colitis

Abstract: The resident bacterial flora of the large intestine has become increasingly recognized as an essential component in the pathogenesis of ulcerative colitis (UC). However, it is still not known whether the bacterial flora in general or certain bacterial species of the intestinal microbial flora contribute to the pathogenesis of the disease. In order to investigate the composition of the mucosa-associated microbial flora in UC, mucosal tissue samples from patients with active UC and from control subjects with non-inflammatory conditions were analysed and compared. To cover the whole spectrum of intestinal bacteria and to circumvent the known bias introduced by culture-based techniques, comparative 16S rRNA gene sequence analysis was used to determine the bacterial composition in the mucosal tissue samples. The investigation revealed an abundance of sequences from Bacteroides spp. and Prevotella spp. in the mucosal tissue of patients with UC compared with individuals showing no signs of disease. The higher incidence of populations of members of the Bacteroidetes in UC suggests that these may have an influence on the pathogenesis of the disease.

Invasive multidrug-resistant non-typhoidal Salmonella infections in Africa: zoonotic or anthroponotic transmission?

Abstract: In Africa, multidrug-resistant non-typhoidal salmonellae (NTS) are one of the leading causes of morbidity and high mortality in children under 5 years of age, second in importance only to pneumococcal disease. The authors studied NTS isolates from paediatric admissions at two hospitals in Nairobi, Kenya, and followed the index cases to their homes, where rectal swabs and stools from parents and siblings, and from animals in close contact, were obtained. The majority of NTS obtained from cases were Salmonella enterica serotype Typhimurium (106 out of 193; 54·9 %) and Salmonella enterica serotype Enteritidis (64; 33·2 %), a significant proportion (34·2 %) of which were multiply resistant to three or more antibiotics, including ampicillin, tetracycline, cotrimoxazole and chloramphenicol. Only 23·4 % of NTS were fully susceptible to all 10 antibiotics tested. Of the 32 NTS obtained from contacts (nine adults and 23 children) at the homes of index cases, 21 (65·6 %) isolates were similar by antibiotic-susceptibility profiles and plasmid content, and their XbaI- and SpeI-digested chromosomal DNA patterns were indistinguishable from those of the corresponding index cases. Only three out of 180 (1·7 %) samples from environmental sources, including animals, soil, sewers and food, contained NTS matching those from corresponding index cases. The carriage of NTS in an asymptomatic population was represented by 6·9 % of human contacts from 27 out of 127 homes sampled. This population of carriers may represent an important reservoir of NTS that would play a significant role in the epidemiology of community-acquired NTS bacteraemia in children.

Protean clinical manifestations and diagnostic challenges of human brucellosis in adults: 16 years' experience in an endemic area

Abstract: A prospective study was carried out to elucidate the clinical, epidemiological and laboratory features of human brucellosis. A total of 26 948 blood samples (from adults aged 15 years and above) were screened for serological evidence of brucellosis over a period of 16 years. The slide agglutination/Rose Bengal plate agglutination test gave positive results in 517 patients, of which 509 had detectable titres by the standard tube agglutination test (SAT). The diagnosis of brucellosis was documented in 495 (1.8 %) patients based on diagnostic titres (> or = 1 : 160, 490 cases) and rising titres from insignificant titres (four cases) by serology and for one case by blood-culture isolation alone. Blood cultures were carried out in 345 cases, of which 191 cases (55.3 %) yielded Brucella melitensis. In 77/79 cases undertaken for follow up, there was a steady fall in 2-mercaptoethanol (2ME) agglutination titres along with clinical improvement (P < 0.01). SAT titres remained detectable in most cases for a longer period in spite of an effective antimicrobial therapy and clinical recovery. A substantial number of patients (84.2 %) presented with fever, this being the only complaint in 51.1 % of the cases. Complications were present in 8.8 % of the patients (arthritis excluded): this included the unusual complications of hydrocele (two cases), Stevens-Johnson syndrome (one case) and urinary tract infection (one case). Brucella agglutinins were demonstrated in synovial, testicular, hydrocele and cerebrospinal fluids. There was no clinical suspicion of brucellosis in 439 cases (88.7 %) and the diagnosis was made only by routine serology. A two-drug regimen for 42-84 days with a follow-up 2ME test resulted in lower levels of relapse. These results suggest that, in endemic areas of the world, it should be mandatory to screen routinely for brucellosis due to protean clinical manifestations.

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria.

Abstract: The differences in faecal bacterial population between irritable bowel syndrome (IBS) and control subjects have been reported in several studies. The aim of the present study was to compare the predominant and clostridial faecal microbiota of IBS subjects and healthy controls by applying denaturing gradient gel electrophoresis (DGGE) and a recently developed multiplexed and quantitative hybridization-based technique, transcript analysis with the aid of affinity capture (TRAC). According to the results, the studied clostridial groups (Clostridium histolyticum, Clostridium coccoides-Eubacterium rectale, Clostridium lituseburense and Clostridium leptum) represented the dominant faecal microbiota of most of the studied subjects, comprising altogether 29–87 % of the total bacteria as determined by the hybridized 16S rRNA. The C. coccoides-E. rectale group was the dominant subgroup of clostridia, contributing a mean of 43 % of the total bacteria in control subjects and 30 % (constipation type) to 50 % (diarrhoea type) in different IBS symptom category subjects. The proportion of the C. coccoides-E. rectale group was found to be significantly lower in the constipation-type IBS subjects than in the control subjects. DNA-based PCR-DGGE and RNA-based RT-PCR-DGGE analyses targeted to the predominant bacterial population showed considerable biodiversity as well as uniqueness of the microbiota in each subject, in both control and IBS subject groups. The RT-PCR-DGGE profiles of the IBS subjects further indicated higher instability of the bacterial population compared to the control subjects. The observations suggest that clostridial microbiota, in addition to the instability of the active predominant faecal bacterial population, may be involved in IBS.

A review of an emerging enteric pathogen: enteroaggregative Escherichia coli.

Abstract: Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized enteric pathogen. It is a cause of both acute and persistent diarrhoea among children, adults and HIV-infected persons, in both developing and developed countries. In challenge studies, EAEC has caused diarrhoeal illness with the ingestion of 10(10) c.f.u. Outbreaks of diarrhoeal illness due to EAEC have been reported, and linked to the ingestion of contaminated food. Diarrhoeal illness due to EAEC is the result of a complex pathogen-host interaction. Some infections due to EAEC result in diarrhoeal illness and elicit an inflammatory response, whereas other infections do not result in a symptomatic infection. Many putative virulence genes and EAEC strains that produce biofilm have been identified; however, the clinical significance of these genes and of biofilm production has yet to be defined. A -251 AA single nucleotide polymorphism (SNP) in the interleukin (IL)-8 promoter region is reported to increase host susceptibility to EAEC diarrhoea. Ciprofloxacin and rifaximin continue to be an effective treatment in persons infected with EAEC. This review is intended to provide an updated review for healthcare workers on EAEC, an emerging enteric pathogen.

Natural resistance, iron and infection: a challenge for clinical medicine.

Abstract: Natural resistance to infection, which does not depend on antibiotics, is a powerful protective mechanism common to all mankind that has been responsible for the survival of our species during countless millennia in the past The normal functioning of this complex system of phagocytic cells and tissue fluids is entirely dependent on an extremely low level of free ionic iron (10(-18) M) in tissue fluids This low-iron environment is maintained by the unsaturated iron-binding proteins transferrin and lactoferrin, which depend on well-oxygenated tissues, where a relatively high oxidation-reduction potential (Eh) and pH are essential for the binding of ferric iron Freely available iron is derived from iron overload, free haem compounds, or hypoxia in injured tissue leading to a fall in Eh and pH This can severely damage or abolish normal bactericidal mechanisms in tissue fluids leading to overwhelming growth of bacteria or fungi The challenge for clinical medicine is to reduce or eliminate the presence of freely available iron in clinical disease In injured or hypoxic tissue, treatment with hyperbaric oxygen might prove very useful by increasing tissue oxygenation and restoring normal bactericidal mechanisms in tissue fluids, which would be of huge benefit to the patient

Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol.

Abstract: Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a phytoalexin compound with anti-inflammatory and antioxidant activities. The effect of resveratrol on swarming and virulence factor expression of Proteus mirabilis, an important pathogen infecting the urinary tract, was determined on swarming agar plates with and without the compound. Bacteria harvested at different times were assayed for cell length and the production of flagella, haemolysin and urease. Resveratrol inhibited P. mirabilis swarming and virulence factor expression in a dose-dependent manner. Resveratrol significantly inhibited swarming at 15 μg ml−1, and completely inhibited swarming at 60 μg ml−1. Inhibition of swarming and virulence factor expression was mediated through RsbA, a His-containing phosphotransmitter of the bacterial two-component signalling system possibly involved in quorum sensing. Complementation of an rsbA-defective mutant with the rsbA gene restored its responsiveness to resveratrol. The compound also inhibited the ability of P. mirabilis to invade human urothelial cells. These findings suggest that resveratrol has potential to be developed as an antimicrobial agent against P. mirabilis infection.

Prevalence of human immunodeficiency virus, hepatitis B virus, hepatitis C virus and syphilis among prison inmates and officers at Nsawam and Accra, Ghana.

Abstract: Although the high prevalence of blood-borne viral infections and syphilis in correctional facilities has been well documented globally, such data are sparse from Africa, and there has been no such data from Ghana. This study sought to estimate the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis among prison inmates and officers at prisons in Nsawan and Accra, Ghana. Prisoners and officers in 3 of the 46 prisons in Ghana were surveyed from May 2004 to May 2005. Subjects voluntarily completed a risk-factor questionnaire and provided blood specimens for unlinked anonymous testing for the presence of antibodies to HIV, HCV and Treponema pallidum, the causative agent of syphilis, and the surface antigen of hepatitis B virus (HBsAg). Almost 16% (3770) of the total of 23,980 prison inmates in Ghana were eligible, and 281 (7.5%) of those eligible took part, whilst almost 23% (1120) of the total of 4910 prison officers were eligible, and 82 (7.3%) of those eligible took part. For the 281 inmates tested, HIV seroprevalence was 19.2%, 17.4% had HBsAg, HCV seroprevalence was 19.2% and reactive syphilis serology was noted in 11%. For the 82 officers tested, HIV seroprevalence was 8.5%, 3.7% had HBsAg, HCV seroprevalence was 23.2% and reactive syphilis serology was noted in 4.9%. The data indicate a higher prevalence of HIV and HCV in correctional facilities (both prison inmates and officers) than in the general population in Ghana, suggesting their probable transmission in prisons in Ghana through intravenous drug use, unsafe sexual behaviour and tattooing as pertains to prisons worldwide.

Molecular characterization of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from the USA

Abstract: Drug-resistant tuberculosis poses a significant problem for treatment. The mechanisms of resistance to the front-line drug isoniazid (INH) are complex and can be mediated by katG, inhA and other unknown genes. To identify the percentage of INH-resistant strains with no katG or inhA mutation, this study characterized a panel of 28 clinical isolates of Mycobacterium tuberculosis and five mutants derived from H37Rv resistant to INH. Seventeen of 33 resistant strains (51 %) had katG mutations with 12 of the 17 strains having the most common KatG Ser315Thr mutation. Three of the 17 strains with the KatG 315 mutation had an additional mutation in the inhA promoter and were resistant to a high level of INH. Seventeen of the 33 INH-resistant strains (51 %) had inhA mutations. The most common inhA promoter mutation was -15C-->T and was present in 13 of the 17 inhA mutations. This promoter mutation occurred alone without katG mutations and was associated with a low level of INH and ethionamide resistance. However, other inhA mutations were associated with katG mutations. No mutations were found in the ndh gene. Three of 33 strains (9 %) had no mutations in katG, inhA or ndh, indicating that their resistance was due to a new mechanism of resistance. Detection of the KatG Ser315Thr mutation and the -15C-->T inhA mutation accounted for 76 % (25/33) of the INH-resistant strains and should be useful for rapid detection of INH-resistant strains by molecular tests.

Sporadic human Yersinia enterocolitica infections caused by bioserotype 4/O : 3 originate mainly from pigs.

Abstract: Yersinia enterocolitica 4/O : 3 is the most frequent cause of sporadic human yersiniosis in Finland and Germany. To investigate the possible link between pigs and humans, 282 human and 534 porcine strains from Finland and Germany were characterized with PFGE using NotI, ApaI and XhoI enzymes. Most of the human strains (>80 %) were indistinguishable from the porcine strains in both countries and most of the genotypes (178/182) were different in Finland and Germany. The indistinguishable genotypes among human and porcine strains together with different genotypes in Finland and Germany indicate that pigs are an important source of sporadic yersiniosis in both countries.

Characterization of group A streptococci (Streptococcus pyogenes): correlation of M-protein and emm-gene type with T-protein agglutination pattern and serum opacity factor.

Abstract: Strain characterization of group A streptococci (GAS) has traditionally been based on serological identification of M protein. Additional tests to determine T-protein serotype and production of streptococcal serum opacity factor (SOF) provide important information both to aid in and to supplement M-protein serotyping. Advances in DNA-sequencing technology in the late twentieth century resulted in the development of a method for determining the M type of GAS from the sequence of the gene encoding M protein, the emm gene. Although emm-sequence typing has largely replaced M typing in many laboratories, information provided by T typing and SOF determination continues to provide valuable supplementary information for strain characterization. A comprehensive summary of the correlation of T pattern and SOF production with M type was last published in 1993, several years before emm typing became widely available. Since then, the ease of M-type identification afforded by emm typing has resulted in an increase in the number of confirmed M/emm types of more than 50 %. However, comprehensive information about T-protein serotype and the correlation of SOF production with these new M/emm types is not widely available. This report presents a comprehensive summary of this information, not only for newly described types, but also updated information for previously described types. This information was extracted from combined records from streptococcal reference laboratories at the University of Minnesota and at the Centers for Disease Control and Prevention in Atlanta. Data from more than 40,000 strains (representing uncomplicated GAS infections, systemic invasive infections and strains associated with non-suppurative sequelae, collected from the US and diverse locations worldwide) were analysed.

Immunization with 3-oxododecanoyl-l-homoserine lactone–protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection

Abstract: Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-l-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-l-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL–carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL–BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3×106 c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-α in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.

Cytokine secretion profiles of human keratinocytes during Trichophyton tonsurans and Arthroderma benhamiae infections

Abstract: Dermatophytes cause intractable superficial infections in humans. Arthroderma benhamiae, a zoophilic dermatophyte, triggers severe inflammatory responses in humans, while Trichophyton tonsurans, an anthropophilic dermatophyte, triggers minimal ones. Cytokines and other factors derived from keratinocytes play important roles in inflammatory and immune responses in the skin. The authors performed an in vitro investigation to determine the human keratinocyte cytokine profiles during dermatophyte infection. The human keratinocyte cell line PHK16-0b was infected with A. benhamiae or T. tonsurans for 24 h, and the cytokines secreted were analysed using a human cytokine antibody array. Marked differences were observed in the cytokine profiles of the cells infected with the two dermatophytes. A. benhamiae infection resulted in the secretion of a broad spectrum of cytokines, including proinflammatory cytokines, chemokines, and immunomodulatory cytokines. In contrast, T. tonsurans-infected keratinocytes secreted only limited cytokines, including eotaxin-2, interleukin (IL)-8 and IL-16. cDNA microarray analysis confirmed that A. benhamiae infection upregulated genes encoding IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-13, IL-15, IL-16, IL-17 and interferon (IFN)-gamma, while T. tonsurans infection upregulated only a few genes, such as those encoding IL-1beta and IL-16. RT-PCR demonstrated that infection by both dermatophytes enhanced IL-8 mRNA expression in keratinocytes. These results suggest that A. benhamiae-induced secretion of several cytokines from keratinocytes may be involved in a severe inflammatory response, and that the limited cytokine secretion from keratinocytes in response to T. tonsurans infection may result in a minimal inflammatory response in the skin. These cytokine profiles may aid in proving the clinical features of dermatophytosis.

Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications

Abstract: The ability of Acanthamoeba to feed on Gram-negative bacteria, as well as to harbour potential pathogens, such as Legionella pneumophila, Coxiella burnetii, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes and Mycobacterium avium, suggest that both amoebae and bacteria are involved in complex interactions, which may play important roles in the environment and in human health. In this study, Acanthamoeba castellanii (a keratitis isolate belonging to the T4 genotype) was used and its interactions with Escherichia coli (strain K1, a cerebrospinal fluid isolate from a meningitis patient, O18 : K1 : H7, and a K-12 laboratory strain, HB101) were studied. The invasive K1 isolate exhibited a significantly higher association with A. castellanii than the non-invasive K-12 isolate. Similarly, K1 showed significantly increased invasion and/or uptake by A. castellanii in gentamicin protection assays than the non-invasive K-12. Using several mutants derived from K1, it was observed that outer-membrane protein A (OmpA) and LPS were crucial bacterial determinants responsible for E. coli K1 interactions with A. castellanii. Once inside the cell, E. coli K1 remained viable and multiplied within A. castellanii, while E. coli K-12 was killed. Again, OmpA and LPS were crucial for E. coli K1 intracellular survival in A. castellanii. In conclusion, these findings suggest that E. coli K1 interactions with A. castellanii are carefully regulated by the virulence of E. coli.

Role of the ABC transporter TruMDR2 in terbinafine, 4-nitroquinoline N-oxide and ethidium bromide susceptibility in Trichophyton rubrum.

Abstract: A single-copy gene, designated TruMDR2, encoding an ATP-binding cassette (ABC) transporter was cloned and sequenced from the dermatophyte Trichophyton rubrum. The ORF of TruMDR2 was 4048 nt and the deduced amino acid sequence showed high homology with ABC transporters involved in drug efflux in other fungi. The encoded ABC protein predicted 12 transmembrane segments (TMSs) and two almost identical nucleotide-binding domains (NBDs) arranged in two halves in a (TMS(6)-NBD)(2) configuration and could be classified as a member of the multidrug-resistance (MDR) class of ABC transporters. Northern blot analyses revealed an increased level of transcription of the TruMDR2 gene when mycelium was exposed to acriflavine, benomyl, ethidium bromide, ketoconazole, chloramphenicol, griseofulvin, fluconazole, imazalil, itraconazole, methotrexate, 4-nitroquinoline N-oxide (4NQO) or tioconazole. Disruption of the TruMDR2 gene rendered the mutant more sensitive to terbinafine, 4NQO and ethidium bromide than the control strain, suggesting that this transporter plays a role in modulating drug susceptibility in T. rubrum.

Bacterial killing in gastric juice – effect of pH and pepsin on Escherichia coli and Helicobacter pylori

Abstract: The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5–7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1×106 E. coli ml−1 and 1×108 H. pylori ml−1) were pre-incubated with test solutions at 37 °C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml−1 at pH 3.5 reduced viable counts by 100 % for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50 % of the control after 20 min incubation in 1 mg pepsin ml−1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions.

Abstract: Bacterial invasion of host epithelial cells plays an important role in the pathogenesis of periodontal diseases; however, the interactions between subgingival species and the gingival crevice cells are not fully understood. This study determined the prevalence of a group of oral bacterial species on or in epithelial cells derived from periodontal pockets and the gingival crevice of subjects with periodontitis. Samples of epithelial cells were obtained from 120 sites with periodontal pockets > or =4 mm and 92 periodontally healthy sites from 49 patients (mean age 46.3+/-1.4 years; 43% males) with chronic periodontitis. Bacteria in or on epithelial cells were separated from unattached bacteria by Percoll density-gradient centrifugation. The presence and levels of 33 oral species were determined in epithelial cell samples by whole genomic DNA probes and the checkerboard method. The most frequently detected species were Porphyromonas gingivalis (42%), Treponema denticola (38%), Prevotella intermedia (37%), Streptococcus intermedius (36%), Campylobacter rectus (35%), Streptococcus sanguinis (35%) and Streptococcus oralis (34%). Species of Actinomyces were found in low prevalence and levels. The data indicated that there were more micro-organisms on or in epithelial cells obtained from periodontal pockets than from healthy sulci; however, no significant differences regarding the percentage and level of any specific species were found between these sites. Veillonella parvula, S. oralis, Streptococcus gordonii and Streptococcus mitis tended to be more prevalent in sites without disease. These findings demonstrated that a wide range of oral species may be detected on or in crevicular epithelial cells from sites with periodontitis and from periodontally healthy sulci.

Molecular typing and virulence markers of Yersinia enterocolitica strains from human, animal and food origins isolated between 1968 and 2000 in Brazil.

Abstract: Molecular typing and virulence markers were used to evaluate the genetic profiles and virulence potential of 106 Yersinia enterocolitica strains. Of these strains, 71 were bio-serotype 4/O : 3, isolated from human and animal clinical material, and 35 were of biotype 1A or 2 and of diverse serotypes, isolated from food in Brazil between 1968 and 2000. Drug resistance was also investigated. All the strains were resistant to three or more drugs. The isolates showed a virulence-related phenotype in the aesculin, pyrazinamidase and salicin tests, except for the food isolates, only two of which were positive for these tests. For the other phenotypic virulence determinants (autoagglutination, Ca++ dependence and Congo red absorption), the strains showed a diverse behaviour. The inv, ail and ystA genes were detected in all human and animal strains, while all the food isolates were positive for inv, and 3 % of them positive for ail and ystA. The presence of virF was variable in the three groups of strains. The strains were better discriminated by PFGE than by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A higher genomic similarity was observed among the 4/O : 3 strains, isolated from human and animal isolates, than among the food strains, with the exception of two food strains possessing the virulence genes and grouped close to the 4/O : 3 strains by ERIC-PCR. Unusually, the results revealed the virulence potential of a bio-serotype 1A/O : 10 strain, suggesting that food contaminated with Y. enterocolitica biotype 1A may cause infection. This also suggests that ERIC-PCR may be used as a tool to reveal clues about the virulence potential of Y. enterocolitica strains. Furthermore, the results also support the hypothesis that animals may act as reservoirs of Y. enterocolitica for human infections in Brazil, an epidemiological aspect that has not been investigated in this country, confirming data from other parts of the world.

Isolation and characterization of Streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis.

Abstract: Streptococcus mutans, known to be an aetiologic agent of dental caries, also causes infective endocarditis (IE), although a comparison of isolates from the oral cavity and infected heart valve of the same patient has not been reported. In the present study, infected heart valve and dental plaque samples from a patient with IE were analysed. Broad-range PCR with DNA sequencing revealed that 50 clones from the dental plaque isolates were composed of oral streptococci and periodontopathic bacteria, whereas only Streptococcus mutans was detected in 50 clones from the heart valve. Eighteen strains of Streptococcus mutans were isolated from dental plaque and seven from the heart valve, and the biochemical properties of each were in accordance with those of Streptococcus mutans. DNA fingerprinting analysis revealed that all the oral isolates of Streptococcus mutans had similar patterns, which were different from those of the isolates from the infected heart valve. Western blotting using glucosyltransferase (GTF)-specific antiserum showed that the seven strains from the heart valve lacked the three types of intact GTF. In addition, the sucrose-dependent adhesion rates of these isolates were significantly lower than those of the oral isolates (P<0.001). Furthermore, the isolates from the heart valve were less susceptible to erythromycin and kanamycin. These results indicate that the properties of the Streptococcus mutans strains isolated from the infected valve were different from those of typical oral strains, which may be related to the effects of IE.

Detection of typical and atypical enteropathogenic Escherichia coli (EPEC) in Iranian children with and without diarrhoea.

Abstract: The present study was performed to investigate the contribution of typical and atypical enteropathogenic Escherichia coli (EPEC) as a cause of infectious diarrhoea among children less than 10 years old in Iran. During the summer months, 247 specimens from children with diarrhoea and 1108 from asymptomatic children were analysed for the presence of EPEC and other bacterial pathogens. Potential enteric pathogens were identified in 140 cases of children with diarrhoea (56.7%). EPEC was the most frequently identified agent (111 cases), followed by Shiga toxin-producing E. coli (13), Shigella (9), Salmonella (6) and Aeromonas sp. (1). EPEC isolates were examined for the presence of eaeA, bfpA and stx genes by PCR. EPEC isolates were classified as typical (eaeA+ bfpA+) or atypical (eaeA+ bfpA-). Typical EPEC was diagnosed in 35 cases (11.8%), compared with 8 (0.4%) in the asymptomatic group (P<0.05). Atypical EPEC strains were isolated from 23 cases (9.3%), compared with 13 (1.2%) of the healthy control group (P<0.05). In conclusion, the data suggest that typical and atypical EPEC are an important cause of diarrhoea in Iranian children.

Candida albicans HWP1 gene expression and host antibody responses in colonization and disease.

Abstract: In vivo expression of the developmentally regulated Candida albicans hyphal wall protein 1 (HWP1) gene was analysed in human subjects who were culture positive for C. albicans and had oral symptoms (n=40) or were asymptomatic (n=29), or had vaginal symptoms (n=40) or were asymptomatic (n=29). HWP1 mRNA was present regardless of symptoms, implicating hyphal and possibly pseudohyphal forms in mucosal carriage as well as disease. As expected, in control subjects without oral symptoms (n=10) and without vaginal symptoms (n=10) who were culture negative in oral and vaginal samples, HWP1 mRNA was not detected. However, exposure to Hwp1 in healthy culture-negative controls, as well as in oral candidiasis and asymptomatic mucosal infections, was shown by the existence of local salivary and systemic adaptive antibody responses to Hwp1. The results are consistent with a role for Hwp1 in gastrointestinal colonization as well as in mucosal symptomatic and asymptomatic infections. Overall, Hwp1 and hyphal growth forms appear to be important factors in benign and invasive interactions of C. albicans with human hosts.