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Clostridium difficile infection (CDI) remains a major healthcare burden despite recent global falls in its prevalence. The risk of recurrence is high when using antibiotics such as vancomycin, particularly in already recurrent disease. In light of this, new therapy options are being perused, including novel antibiotics such as fidaxomicin, probiotics, intravenous immunoglobulin and faecal transplantation. Faecal transplantation, referred to here as human probiotic infusion (HPI), is attracting an increasing amount of interest from physicians and patients. Its use has been documented in ca. 500 cases for the treatment of CDI, with overall efficacy rates reported to be ca. 91%. The first randomised controlled trial (RCT) demonstrated that HPI was superior to a 14-day course of vancomycin (89% vs. 31%; P<0.001) and reported no deaths or serious adverse events. Safety and patient acceptability are often cited as limitations to the widespread use of this technique. However, data suggest that the short-term safety profile is encouraging, and concerns over patient acceptability are not warranted in the majority of cases. It seems appropriate to treat an infection which is caused by a major disturbance in the gut microbiota with a treatment that reverses this disturbance, rather than antibiotics that may exacerbate the problem. However, to fully understand the role of HPI in the management of CDI, further RCTs are needed with comparator antibiotics such as fidaxomicin and to establish the most efficacious HPI protocol for administration and preparation., (Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Capsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes - cap8E (type 8) and tts (type 37) - were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF-ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001). Mol Microbiol 42, 1223-1232]. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates.

The molecular genetic basis of high-frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated. Pneumococci were grown in sorbarod biofilms at 34 degrees C to mimic nasopharyngeal carriage. Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11-239 bp segments within the cap3A gene of the type 3 capsule locus. These duplications alone were found to be responsible for high-frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wild-type cap3A genes, indicating the precise excision of the duplication. Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication. This apparently random duplication giving rise to phase variation is in stark contrast to the 'preprogrammed' contingency genes in many Gram-negative organisms that possess homopolymeric sequence repeats or motifs for site-specific recombination.

The classical method for determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of an antibiotic is the tube test. Details of this test are well known and recognized methods are published (1). The recent development of the E-test (AB Biotest, Solna, Sweden), has been a significant development in the determination of MIC. This system is straightforward to perform and is suitable for larger scale work.

Background: The clinical interactive role of medical microbiologists has been underestimated and the discipline is perceived as being confined to the laboratory. Previous studies have shown that most microbiology interaction takes place over the telephone., Aim: To determine the proportion of clinical ward based and laboratory based telephone interactions and specialties using a microbiology service., Methods: Clinical microbiology activity that took place during November 1996 was prospectively analysed to determine the distribution of interactions and specialties using the service., Results: In all, 1177 interactions were recorded, of which nearly one third (29%) took place at the bedside and 23% took place on call. Interactions involving the intensive treatment unit, general ward visits, and communication of positive blood cultures and antibiotic assays were the main areas of activity identified. There were 147 visits to 86 patients on the general wards during the study, with the number of visits to each individual varying from one to eight. The need for repeated visits reflected the severity of the underlying condition of the patients. Ward visits were regarded as essential to obtain missing clinical information, to assess response to treatment, and to make an appropriate entry in a patient's notes., Conclusions: Ward visits comprise a significant proportion of clinical microbiology interactions and have potential benefits for patient management, service utilisation, and education.

The cell-wall characteristics of Gardnerella vaginalis grown in conventional and biofilm systems were studied by electron microscopy. The gram-positive nature of the cell wall was confirmed. Novel cell-wall particles which appeared to be associated with cell division were also identified, particularly in organisms of biofilm origin.

The majority of clinical isolates of Moraxella catarrhalis produce beta-lactamase. The role of this enzyme in the phenomenon of indirect pathogenicity, in which a true pathogen such as Streptococcus pneumoniae is protected from the action of certain beta-lactam antibiotics, is well recognized. By using a simple continuous-culture biofilm system, it has been shown that the pneumococcus attains high titers in excess of 10(12) CFU/biofilm; furthermore, the penicillin-sensitive pneumococcus used remained susceptible to a range of beta-lactam antibiotics in these biofilms (R. K. Budhani and J. K. Struthers, J. Antimicrob. Chemother. 40:601-602, 1997). This system was used to characterize the antibiotic susceptibility of this isolate when grown with beta-lactamase-negative or -positive moraxellae. When grown with beta-lactamase-producing moraxellae in the presence of either benzylpenicillin or amoxicillin, the pneumococcus was protected in the range of the antibiotic concentrations to which it would be considered resistant. With amoxicillin-clavulanic acid the titers of the two organisms collapsed at the antibiotic concentration at which moraxellae became susceptible. The levels of beta-lactamase activity in cell-free supernatants of broth culture, in biofilm, and in biofilm effluent revealed distinct differences in this activity; levels in biofilm were significantly lower than those in broth culture supernatants. The system appears suitable for studying organisms under antibiotic stress and for investigating the interactions of bacteria under such conditions.

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 microgram of [14C-U-ring]atrazine ml-1 in 72 h with a concurrent increase in the population size from 7.9 x 10(5) to 5.0 x 10(7) cells ml-1. Under these conditions cells mineralized the [ethyl-14C]atrazine and incorporated approximately 30% of the 14C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 10(5) J14a cells g-1 into soil with a low indigenous population of atrazine degraders treated with 50 and 200 microgram of atrazine g-1 soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (10(5) cells g-1) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.

Gardnerella vaginalis and Lactobacillus acidophilus have been shown to grow to high titers in a simple biofilm system. This system was used in the present investigation to compare the biofilm-eradicating concentrations (BECs) of amoxicillin, clindamycin, erythromycin, and metronidazole to standard tube MIC and minimum bactericidal concentration (MBC) results. With the lactobacillus, the BEC/tube MBC ratio was at least 16:1, while for G. vaginalis the ratio varied from 2:1 to 512:1. The simple continuous-culture system used in the present investigation is ideal for investigating the BEC for bacteria involved in complex ecological situations such as bacterial vaginosis and may be useful for the identification of the most effective and selective antibiotic therapy.

Sorbarod biofilms were investigated for their suitability in establishing continuous culture biofilms for the study of bacterial vaginosis. Two important organisms in the condition, Gardnerella vaginalis and Lactobacillus acidophilus, were studied. In contrast to growth in broth culture, both organisms were maintained for at least 96 h in a steady state on the biofilms. With G. vaginalis, the haemolytic activity was consistently maintained in the biofilms in contrast to short-term activity in broth culture which matched the bacterial titre. The simple Sorbarod system appears to be suitable for studying the growth conditions of bacteria in continuous culture and has potential for investigating interactions between micro-organisms.

We evaluated Cobas Amplicor, a highly automated polymerase chain reaction (PCR) system, to test first-void urine (FVU) and urethral swab specimens for Chlamydia trachomatis and Neisseria gonorrhoeae in men attending a sexually transmitted infection (STI) clinic. Results were compared against an in-house radioimmune dot blot (DB) test for C. trachomatis and selective culture for N. gonorrhoeae. Three hundred and ninety sets of specimens were obtained from 378 consecutive new and returned-new patients. Gonorrhoea prevalence was 9.49%, with no significant difference in sensitivity or specificity between culture and PCR. Chlamydia prevalence was 15.4%, with sensitivities of: DB 55%, PCR of FVU 86.7%, urethral swab PCR 90%. The specificity of PCR on FVU and urethral swabs was 100%. We have shown that Cobas Amplicor PCR is highly sensitive and specific in the diagnosis of chlamydia and gonorrhoea in men attending an STI clinic. Further economic and scientific studies are needed to determine the cost-effectiveness of this technique for screening in primary care settings.

A 48-yr-old female on immunosuppressive therapy for fibrosing alveolitis and polymyositis developed a septic arthritis of the left middle finger proximal interphalangeal joint, tenosynovitis of the left palm and osteomyelitis of the right hindfoot due to infection with Mycobacterium marinum. Such widespread and severe bone and joint involvement has not been described previously with this organism.

Peripheral blood leucocytes from patients who had recently had measles infection were examined for their ability to produce alpha- or gamma-interferon, their antiviral state and the level of E enzyme. These results were compared with peripheral blood leucocytes from healthy control subjects. The results show that while peripheral blood leucocytes from control patients produced alpha- and gamma-interferon, those from the measles patients produced only alpha-interferon. The peripheral blood leucocytes from all the measles patients were in an antiviral state whereas those from only 20% of the controls were in this state. Since gamma-interferon is mainly produced by T lymphocytes, the lack of gamma-interferon production by peripheral blood leucocytes from patients with measles correlates with previously reported depressed T-cell function in patients after measles infection.

Persistent infections with Sendai and SSPE virus were established in Vero cells. Sequential passages of these cells were monitored by immunofluorescence and for their sensitivity to the antiviral and antiproliferative effects of interferon (IFN). The cells rapidly developed resistance to the antiviral effect of IFN as judged by the inability of IFN to inhibit the replication of exogenous Sindbis virus. This decrease was accompanied by a reduction in the induction of the 2'-5' oligo A synthetase. Both cell lines were resistant to the antiproliferative effect of IFN. A decrease or absence of IFN receptors on the surface of the cells was not found to be the cause of their resistance to IFN.

Reversed passive hemagglutination (RPHA) tests were performed by coating glutaraldehyde-fixed and tannic acid-treated sheep erthrocytes with antibodies to Rift Valley fever (RVF) and Crimean-Congo hemorrhagic fever (CCHF) viruses. Cells coated with crude antibody, in the form of mouse immune ascitic fluid, reacted with high specificity and sensitivity in RPHA tests with live or inactivated virus antigens. In inhibition (RPHI) tests, RVF antibody titers in human and sheep sera were similar to those determined by hemagglutination inhibition test. RVF virus antigen could be detected in viremic sheep sera by the RPHA technique. RPHI antibody titers to CCHF virus in human and hare sera were similar to indirect immunofluorescence (IF) titers, but sheep and cattle sera with RPHI titers of 1:32 or less were negative in IF tests.

Crimean-Congo hemorrhagic fever virus was isolated for the first time in South Africa in February 1981, from the blood of a 13-year-old boy who died in Johannesburg after attending a camp in a nature reserve in the western Transvaal. Virus was isolated from 21/120 pools of questing ticks from the nature reserve, the infected species being Hyalomma marginatum rufipes and H. truncatum. Virus was also isolated from 4/38 pools of partially engorged ticks and other ectoparasites collected off hosts, the infected species being H.m. rufipes, H. truncatum and Rhipicephalus evertsi. Antibodies were found in the sera of 5/74 humans, 8/26 wild vertebrates, 74/270 sheep, and 109/170 cattle from the reserve and surrounding farms. Antibodies were also found in 28/200 hares from various locations in the country. It was concluded that the virus is widely prevalent in South Africa, but the full medical and veterinary significance of its presence has yet to be determined.

Homologous and heterologous haemagglutination-inhibition (HAI), complement-fixation (CF), immunodiffusion (ID) and mouse neutralization tests were performed with the Lunyo (LUN) and a Zimbabwean strain of Rift Valley fever (RVF) virus, the prototype and a South African strain of Arumowot (AMT) virus and prototype strains of Gordil (GOR), Saint-Floris (SAF) and Gabek Forest (GF) viruses, using immune mouse ascitic fluids prepared against these viruses. Reactions of identity occurred in all tests between LUN and the Zimbabwean strains of RVF and between the two strains of AMT virus. Otherwise, cross-reactions occurred between all the phleboviruses in HAI tests, while reactions in CF, ID and neutralization tests were monospecific for virus serotypes, except that weak cross-reaction occurred between GOR and SAF viruses in CF and ID tests. Four sheep infected subcutaneously with the Zimbabwean strain of RVF virus developed transient fever, viraemia, leucopaenia, relative thrombocytopaenia, haemoconcentration and raised serum enzyme levels, which indicated that the sheep had developed necrotic hepatitis. Disseminated focal necrotic hepatitis was confirmed in a sheep killed for examination on day 4 post-infection. The other three sheep recovered uneventfully after only mild depression and anorexia. Groups of three sheep infected with SAF, GOR, AMT and GF viruses had no demonstrable viraemia or other sign of infection or illness, except that the sheep infected with AMT developed mild fever lasting less than 24 h. Antibody responses were monitored at intervals over a period of 24 weeks in all sheep by homologous and heterologous HAI, CF and cell culture neutralization (CPENT) tests. Homologous antibody responses were marked in the RVF-infected sheep and their sera cross-reacted strongly in HAI tests with antigens of the other viruses. The sera of the RVF-infected sheep cross-reacted less markedly in CF and CPENT tests. Homologous antibody responses were poor in all the sheep infected with phleboviruses other than RVF, and the cross-reactivity of their sera for RVF antigen or virus was negligible. All sheep were challenged with RVF virus 48 weeks after their initial infection. The sheep which had originally been infected with RVF virus were immune and developed neither fever nor viraemia. All other sheep developed fever, viraemia and antibodies to RVF virus. It was concluded that the African phleboviruses, other than RVF, are unlikely to cause disease in livestock or to induce antibodies which could cause confusion in the diagnosis of RVF.

A rifampicin-resistant Achromobacter mutant with an altered RNA polymerase was isolated. The mutant supports phage alpha3a growth in both log and stationary phase cells. Phage growth on stationary phase cells is sensitive to aeration and growth only occurs at oxygen concentrations of less than 5-2 p.p.m. The rifampicin-resistant mutant is similar to the spontaneous mutant strain 14 reported by Woods (1976) in that both mutants support stationary-phase phage growth under micro-aerophilic conditions. The isolation of the rifampicin-resistant mutant with an altered RNA polymerase suggests that the phenomenon of stationary phase phage growth could be due to a change in the template specificity of the Achromobacter RNA polymerase. Plaque morphology mutants which grow on log and/or stationary phase cells of the Achromobacter wild type, strain 14 and rifampicin-resistant strains are also described.

Rift Valley fever virus-induced protein synthesis was examined by polyacrylamide gel electrophoresis and fluorography. Five virus-induced polypeptides were detected, the nucleocapsid protein N, the nucleus-associated nonstructural protein NS1, the glycoproteins G1 and G2, and a protein of molecular weight 80K. The N, G1, G2, and 80K proteins were present in virion preparations. Sequential studies showed that NS1 accumulated in the nucleus as soon as it was formed and readily associated with nuclei partitioned from noninfected cells. The G1 and G2 proteins labelled with [3H]glucosamine and [3H]mannose. NS1 was shown to be the only virus-induced protein which was phosphorylated.

A non-structural protein of mol. wt. 34 X 10(3) was demonstrated in the nuclei of Rift Valley fever virus-infected Vero cells by SDS-polyacrylamide gel electro-phoresis. The protein appears to correspond to the virus-induced antigen demonstrated by indirect immunofluorescence in intranuclear inclusions.

Nine serological techniques were compared by monitoring the response to infection with Rift Valley fever (RVF) virus in three sheep. Antibodies were monitored daily for the first 14 days after infection, then weekly and later fortnightly up to week 24. The earliest antibody response was detected in one sheep on day 3 by a plaque reduction neutralization test, and by day 6 antibodies were demonstrable in all three sheep by haemagglutination-inhibition, reversed passive haemagglutination-inhibition, immunodiffusion, indirect immunofluorescence (IF), enzyme-linked immunosorbent assay and neutralization of cytopathic effect in cell cultures. Antibodies were demonstrable by complement fixation on day 8 at the earliest. IF and the two neutralization techniques produced the highest titres, but all tests could be used satisfactorily for the serological diagnosis of RVF. Inactivated antigen could be used for all except the neutralization tests. A radioimmunoassay technique using 125I-labelled staphylococcal protein A detected antibodies on day 8 at the earliest and produced lower mean titres than some of the other techniques. This was probably because sheep immunoglobulins bind protein A poorly.