The method used to dry washed hands affects the number and type of transient and residential bacteria remaining on the skin.

BACKGROUND: Widespread antibiotic resistance has led to fears that we are entering a post-antibiotic era and the relatively simple premise of hand washing to reduce transfer of bacteria and viruses has never been more important. Much of the emphasis has been on hand-washing technique, type of soap, and maintaining compliance but effective drying of the hands is just as important. AIM: To compare the efficacy of drying washed hands with a jet air dryer or paper towels to remove transient bacterial contamination and to determine the effect on residential flora. METHODS: Eighty volunteers were recruited. The entire surfaces of volunteers' hands were artificially contaminated with Escherichia coli before being washed and dried; then bacteria remaining on the skin were recovered and enumerated. In the second part of the study the number and types of bacteria comprising the natural flora remaining on washed and dried hands were determined. FINDINGS: Significantly fewer transient and residential bacteria remained on the skin if hands were dried with a jet air dryer (P < 0.001). Drying hands with paper towels increased the number of resident bacteria, including potentially pathogenic species, released from the volunteers' skin, compared to a jet air dryer. CONCLUSION: The number and types of bacteria remaining on washed hands were affected by the drying method. Hands dried with a jet air dryer harboured fewer viable bacteria, reducing the risk of infection transmission via touch. This could be especially important for healthcare workers who are constantly in contact with large numbers of vulnerable patients.

[Gastrointestinal infections]. / Gastrointestinale Infektionen.

Infectious diarrhea is one of the most common diseases. This article summarizes the current state of the diagnostics and treatment and includes the most important pathogens, i.e. Norovirus, Rotavirus, Campylobacter, Salmonella, Shigella and pathogenic Escherichia coli. Infections caused by toxin-producing strains of Clostridium difficile are described in more detail due to the increasing importance. Symptomatic therapy is still the most important component of treatment. Empirical antibiotic therapy is reserved for severely ill patients with a high stool frequency, fever, bloody diarrhea, underlying immune deficiency or significant comorbidities. Increasing bacterial resistance (in particular against fluoroquinolones) has to be considered. Motility inhibitors are not recommended for infections due to Shiga toxin-producing E. coli, C. difficile infections (CDI) and severe enterocolitis caused by other pathogens. The macrocyclic antibiotic fidaxomicin can reduce the recurrence rate of CDI. Fecal microbiota transplantation (FMT) currently provides a reserve treatment option for multiple recurrences of CDI and is subject to the Medicines Act (Arzneimittelgesetz, AMG) in Germany.

[Hygiene and infection control measures in intensive care units]. / Hygienemaßnahmen auf der Intensivstation.

The prevention of infections is the purpose of hospital hygiene/infection control using control and preventive measures and strategies to achieve this. Especially in intensive care units with seriously ill patients. The continuous increase of multidrug-resistant organisms (MDRO) and as a consequence difficult to treat infections clearly shows the importance of prevention. Furthermore success and failure of hygienic/infection control activities is one of the few measurable quality and performance characteristics in the era of quality medicine and is therefore crucial for the evaluation of hospitals. Consequently it is recommended to meet the challenges and understand hygienic/infection control measures as vital and essential for the hospital. Central topics of present-day prevention are presented.

Effect of age on gingival crevicular fluid concentrations of MIF and PGE2.

We used an experimental gingivitis study design to compare crevicular fluid concentrations of Migration Inhibitory Factor (MIF) and Prostaglandin E(2) (PGE(2)) in younger (18 to 30 yrs) and older (46 to 77 yrs) healthy adults. PGE(2) increased after 1 wk in younger participants, whereas it decreased in older individuals after 1 wk of plaque accumulation. A significant interaction between age and time was observed for PGE(2) (p = 0.04). High concentrations of MIF were identified in both age groups at baseline. MIF increased in the younger participants, whereas in the older individuals a decrease over time was observed. MIF concentration was positively correlated with plaque index and gingival index in the older age group. Total counts of bacteria, Parvimonas micra and Prevotella intermedia, were significantly correlated with MIF concentration in older participants. In conclusion, MIF and PGE(2) production in response to bacterial accumulation seems to be modified by age.

Quantitative detection of Clostridium difficile in hospital environmental samples by real-time polymerase chain reaction.

C. difficile-associated diarrhoea occurs commonly in hospitals and is a significant cause of morbidity and mortality. Hospital surfaces are often contaminated with nosocomial pathogens and may be responsible for cross-transmission, especially if hardy Gram-positive and spore-forming organisms are involved. The aim of this study was to quantify C. difficile in the hospital environment near C. difficile-positive and -negative patients using a quantitative real-time polymerase chain reaction. A total of 531 samples was collected from the clinical environment and classified into three groups according to patient and ward status for C. difficile. As expected, there were significantly higher counts of C. difficile on the floor and in the near environment of C. difficile patients. However, a significant correlation was found between C. difficile counts on the floor and on the hands of patients and healthcare workers (HCWs) in wards without evidence of C. difficile. This suggests that asymptomatic carriage among patients and HCWs can also contribute towards C. difficile transmission in hospitals. In conclusion, C. difficile can be transmitted via personal contact or via contaminated areas of the hospital environment.

Microbiological risk of anaesthetic breathing circuits after extended use.

INTRODUCTION: Daily change of breathing circuits in the operating theatre requires a lot of resources and is time and labour consuming. The extended use of breathing circuits could reduce the workload of the staff and health care costs. The aim of the present study was to evaluate the contamination rate of anaesthesia breathing circuits changed after 24, 48 or 72 h of use. MATERIALS: The study was performed as an experimental observational study. Microbiological samples were taken from 112 breathing systems including both parts of the ventilator circuit (inspiration and expiration) and analysed using microbiological standard techniques. Breathing circuits were changed according to three different schedules. In the 24-h group, breathing circuits were changed every day, whereas in the 48-h group changing of the circuits took place on Mondays, Wednesdays and Fridays. A period of 72 h operating use was tested on weekends. RESULTS: A total of 112 breathing systems comprised of 224 samples from the ventilator circuit were tested for bacteria and yeast contamination. A non-significant increase in the contamination rate was observed with the extended use for breathing circuits (24 h: 3.33%, 48 h: 4.35% and 72 h: 5.56%; P for trend=0.66). Similarly, no significant increase in contamination rate could be observed at the sample level (24 h: 1.67%, 48 h: 3.26% and 72 h: 2.78%; P for trend=0.71). CONCLUSION: The extended use of breathing circuits for 48 and 72 h does not increase significantly the risk of contamination, provided that HME filters are changed separately for every patient.

The stage of native biofilm formation determines the gene expression of human beta-defensin-2, psoriasin, ribonuclease 7 and inflammatory mediators: a novel approach for stimulation of keratinocytes with in situ formed biofilms.

BACKGROUND/AIMS: Antimicrobial peptides such as human beta-defensin-2 (hBD-2), psoriasin (PSO), and ribonuclease 7 (RNase 7) play an important role in innate immunity. The aim of the present study was to test the hypothesis that epithelial cells show a differential gene expression pattern of antimicrobial peptides (hBD-2, PSO, RNase 7) and inflammatory mediators such as interleukin-8 (IL-8) and 5-lipoxygenase (5-LO) in response to different stages of naturally formed biofilms. METHODS: Epithelial cells were cultured from biopsies obtained from five healthy individuals. Native bacterial biofilms were taken from the same subjects that donated the gingival biopsies. To obtain different stages of biofilm formation, polymer disks were attached to prostheses and carried intraorally for 1, 3, 5, and 9 days. The expression of genes for hBD-2, PSO, RNase 7, 5-LO, and IL-8 was examined using semi-quantitative reverse transcription-polymerase chain reaction. The bacterial composition of the individual biofilms was defined using a microarray system (Parocheck), which showed the presence of 20 different bacterial species that are associated with plaque formation. RESULTS: The expression of the messenger RNAs of hBD-2, RNase 7, and 5-LO was upregulated as a result of the exposure to early biofilm stages, whereas the gene expression of IL-8 was increased in response to matured biofilms. Inter-individual differences in the innate immune response were observed. CONCLUSION: The results of the present study showed a time-dependent messenger RNA expression of antimicrobial peptides (hBD-2, RNase 7), 5-LO, and IL-8 in oral epithelial cells responding to different stages of biofilm formation.

[Recommendations for the treatment of respiratory complications in case of a viruspandemic]. / Empfehlung zur Behandlung respiratorischer Komplikationen bei einer Viruspandemie.

In case of a viral pandemic without availability of effective vaccination, one can expect to be faced with additional 250 to 300 new admissions per hospital per week given the worst case scenario. Major complications are expected to occur in the respiratory system with the focus on viral pneumonia often complicated by bacterial superinfection. Frequently these patients will require artificial ventilation. The present infrastructure will not be capable of dealing sufficiently with such high numbers of casualties. These recommendations of the German Society for Pneumonology are based on the successful application of non-invasive ventilation for acute respiratory failure in recent years. It is of importance to achieve effective treatment by the use of relative simple means. The recommendation proposes to use a separate building in order to realize quarantine. In terms of diagnostic tools, a simple x-ray apparatus should be available. To monitor patients pulsoxymetry and ECG devices should be sufficient in most cases. For the treatment of acute respiratory insufficiency a sufficient number of ventilators, masks, tubing systems and filters should be kept in stock. In terms of medical treatment antibiotics to treat superinfections are of major importance. Analgesics, sedatives and intravenous fluids will also be needed. Oxygen should be available for every single patient. The recommendation gives detailed advise for the enforcement of hygiene control, diagnostic as well as therapeutic steps for in hospital treatment of high numbers of casualties of a viral pandemic.

Influence of O(3)/O(2)-pneumoperitoneum as an oxidative stressor on duration of anaesthesia, loss of different reflexes and cytokine mRNA expression.

We analysed the effect of intraperitoneal insufflated ozonized oxygen on the anaesthetic strength generated by tribromoethanol, ketamine/xylazine, chloral hydrate, pentobarbital, and urethane in male Wistar rats. High dosages of anaesthetic drugs normally used for deep surgical anaesthesia were injected. The ozonized oxygen gas mixture was given five times daily on five consecutive days at 0.8 mg ozone/kg body weight before anaesthesia. The reflexes were measured 15, 30, 60, 90, 120, 180, and 240 min after injection of the anaesthetic drug. The sleeping time and the loss and regain of six different reflexes on noxious and non-aversive stimuli were recorded during the 4 h of observation. O(3)/O(2)-pneumoperitoneum (O(3)/O(2)-PP) reduced the sleeping time induced by tribromoethanol and ketamine/xylazine and increased it for chloral hydrate and pentobarbital. In accordance to the changes in the duration of anaesthesia, the O(3)/O(2)-PP induced significant changes in the loss of different reflexes. Additionally, the modulatory effect of the anaesthetic drugs on splenic cytokine mRNA expression was further influenced by O(3)/O(2)-PP. Thus, the influence of an oxidative stressor on anaesthetic potency and on the resting immune system has to be taken into account for experimental designs in which surgical anaesthesia is necessary for small laboratory animals.

Influence of different anaesthetics on pro-inflammatory cytokine expression in rat spleen.

We examined the effect of five anaesthetic drugs commonly used in laboratory animal research (tribromoethanol, ketamine/xylazine, chloral hydrate, pentobarbital, and urethane) on the expression of four pro-inflammatory cytokines. The anaesthetic agents were applied at dosages normally used for deep surgical anaesthesia. Semiquantitative image analysis of interleukin (IL)-1beta, IL-2, IL-6, and tumour necrosis factor alpha (TNFalpha) mRNA expression in the spleen of male Wistar rats 4 h after application of the anaesthetic drugs showed that these had moderate immunomodulatory effects. Ketamine/xylazine, chloral hydrate, and pentobarbital enhanced the basal expression of IL-1beta and IL-6 mRNA in rat spleen, while urethane reduced splenic IL-1beta mRNA expression. Tribromoethanol, ketamine/xylazine, and urethane reduced the basal TNFalpha mRNA levels, whereas TNFalpha mRNA expression was unaffected by chloral hydrate and by pentobarbital. The data demonstrate that these anaesthetics have slight, but significant, effects on the basal immune status of rats.

Repetitive pneumoperitoneum with ozonized oxygen as a preventive in lethal polymicrobial sepsis in rats.

The aim of this study was to test whether repetitive pretreatments of rats with ozonized oxygen at relatively low gas volumes into the abdomen (20 ml per rat per day) have any beneficial or detrimental effects on the course of a polymicrobial-induced lethal peritonitis. Peritonitis was induced in a surgical or a nonsurgical model by usage of fecal material from the cecum. As the biological read out we used the mortality analysis. To include possible mechanisms by which ozone might influence the septic outcome, we characterized the gene expression of the pro-inflammatory cytokines IL-1beta, IL-2, and TNF-alpha mRNA in lymphoid organs. In both models, we found a significant beneficial influence of a dose-dependent O(2)/O(3 )pneumoperitoneum on the survival rate when compared to control animals or to room air. The ozone-enhanced survival seems to be independent from altered cytokine expression because there were no differences noticed in the levels of bacterial-induced gene expression of IL-1beta and TNF-alpha in septic animals pretreated with ozonized oxygen when compared to control animals.

Characterisation of a novel Mannheimia sp from Australian feedlot cattle.

OBJECTIVE: To characterise eight isolates of a Gram-negative organism obtained from the upper respiratory tract of cattle showing evidence of mild upper respiratory tract disease. DESIGN: The isolates were compared with the five recognised species within the genus Mannheimia - M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena--using a range of phenotypic and genotypic methods. RESULTS: Phenotypic characterisation indicated that the isolates belonged to the trehalose-negative [Pasteurella] haemolytica complex. This complex has recently been reorganised into five species within the new genus Mannheimia. Ribotyping performed using HindIII and a computerised analysis system indicated that the eight Australian isolates formed a distinct cluster that was related to, but different from, the five recognised species of Mannheimia. The 16S rRNA sequence of one isolate (BNO311) was determined and a phylogenetic analysis performed. Isolate BNO311 was distinct from the five named Mannheimia spp but did join a larger cluster consisting of rRNA cluster IV (M varigena) and the unnamed rRNA cluster V of Mannheimia. DNA:DNA hybridisation between isolate BNO311 and M haemolytica NCTC 9380T, M granulomatis P411 and Actinobacillus ligniersii NCTC 4189T all suggested similarities of approximately 30%. CONCLUSIONS: These phenotypic and genotypic characterisation studies suggest that the eight Australian isolates represent a new species of Mannheimia. Until further characterisation studies are performed, we are unwilling to propose a name for this taxon, preferring to refer to this possible new species as Bisgaard taxon 39 of cluster V of Mannheimia.

Microbiological characteristics of subgingival microbiota in adult periodontitis, localized juvenile periodontitis and rapidly progressive periodontitis subjects.

OBJECTIVE: To describe the prevalence of the cultivable subgingival microbiota in periodontal diseases and to draw attention to the polymicrobial nature of periodontic infections. METHODS: The study population consisted of 95 patients, 51 females and 44 males, aged 14-62 years. Twenty-nine patients exhibited adult periodontitis (AP), six localized juvenile periodontitis (LJP), and 60 rapidly progressive periodontitis (RPP). Two to four pooled bacterial samples were obtained from each patient. Samples were collected with sterile paper points from the deepest periodontal pockets. The samples were cultured under anaerobic and microaerophilic conditions using selective and non-selective media. Isolates were characterized to species level by conventional biochemical tests and by a commercial rapid test system. RESULTS: Prevotella intermedia and Capnocytophaga spp. were the most frequently detected microorganisms in all diagnostic groups. Porphyromonas gingivalis and Peptostreptococcus micros were found more frequently in AP and RPP patients, while Actinobacillus actinomycetemcomitans and Eikenella corrodens were associated with AP, LJP and RPP patients. The other bacterial species, including Actinomyces spp., Streptococcus spp. and Eubacterium spp., were detected at different levels in the three disease groups. CONCLUSIONS: The data show the complexity of the subgingival microbiota associated with different periodontal disease groups, indicating that the detection frequency and levels of recovery of some periodontal pathogens are different in teeth affected by different forms of periodontal disease.

Phenotypic and genetic characterization of NAD-dependent Pasteurellaceae from the respiratory tract of pigs and their possible pathogenetic importance.

Nicotinamide adenine dinucleotide (NAD)-dependent Pasteurellaceae other than Actinobacillus pleuropneumoniae and Haemophilus parasuis are frequently isolated from the respiratory tract of pigs. The taxonomic classification and relevance for pathogenicity of these bacteria deserves further attention. In the present study, 107 of these NAD-dependent isolates from the porcine respiratory tract, primarily from lungs with pathological changes, were investigated. On the basis of phenotypic criteria, such as haemolysis, urease, catalase, and indole formation as well as other fermentative activities, 50 of the isolates were assigned to Actinobacillus minor, 36 isolates to Actinobacillus porcinus and 21 isolates to Actinobacillus indolicus. However, many isolates among the three species showed fermentative activities differing from those of the respective type strain of the species. Serotyping on the basis of heat-stable polysaccharide antigens and 16 rDNA sequencing also revealed substantial heterogeneity within each of the three species although they clustered together in three distinct groups in the phylogenetic analysis. These three groups of NAD-dependent bacteria are different from, or in a borderline position, to the existing species or genera within the family Pasteurellaceae. A considerable number of isolates of these three groups were isolated in pure cultures from pneumonic lungs. Consequently, it will be necessary to critically review the opinion, that these NAD-dependent Pasteurellaceae are only "agents colonizing the mucosa". Further, taxonomic examinations of the strains within these three groups are indispensable to testing isolates for their virulence in gnotobiotic pigs.

Chronic osteomyelitis in a new rabbit model.

Dogs, rats, and rabbits are the most suitable species to induce chronic osteomyelitis and to study different methods of treatment. In rabbits, the incidence of and mortality from Staphylococcus aureus-induced osteomyelitis of the tibia depends on the method of prelesions and the amount and virulence of species-specific bacteria used. In this study two different lesions were combined simultaneously in the medullary canal of the femurs by aspiration of bone marrow, leaving the insertion needle in situ. A sclerosing agent was then inoculated followed by 300,000 bacteria of a rabbit-derived S. aureus strain to initiate infection. With this method, the incidence of chronic progressive osteomyelitis of the femur was increased to 100%. A relatively low mortality was observed, probably due to a lower number of inoculated bacteria as compared to other rabbit models described. The incidence of acute to chronic osteomyelitis was diagnosed by local signs, x-rays, microbiological recovery, and gross pathology of the femur. Initial fever, weight loss, abscess formation in soft tissues, and pain on palpation characterize the clinical features in the course of development of this chronic disease.

Is characterization of a single isolate sufficient for valid publication of a new genus or species? Proposal to modify recommendation 30b of the Bacteriological Code (1990 Revision).

From 1990 to 2000, the number of published named taxa based upon new isolates at species and genus levels in International Journal of Systematic and Evolutionary Microbiology, formerly International Journal of Systematic Bacteriology, have increased by approximately four- and sevenfold, respectively. New taxa based upon characterization of only a single isolate remained at around 40% for both categories. The Bacteriological Code (1990 Revision) has no recommendations on the number of strains required for definition of new taxa. For a few groups, a minimum number of 5-10 strains has been suggested in minimal standards. Since an exponential increase in new taxa can be expected in the future, the authors discuss problems related to naming new species and genera based upon descriptions of a single isolate and suggest that this practice is re-evaluated. It is proposed that the following should be added to Recommendation 30b of the Bacteriological Code: 'Descriptions should be based on as many strains as possible (minimum five), representing different sources with respect to geography and ecology in order to be well characterized both phenotypically and genotypically, to establish the centre (from which the type strain could be chosen) and the extent of the cluster to be named. In addition, comparative studies should be performed, including reference strains that represent neighbouring species and/or genera, in order to give descriptions that are sufficiently detailed to allow differentiation from these neighbours.'

DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA.

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae. Optimal conditions were obtained with 300 ng DNA added per well and bound by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the micro-well method (y) and by the spectrophotometric method (x) was y = 0.534x+30.6, when these criteria had been applied to 23 pairs of strains of Actinobacillus species, avian [Pasteurella] haemolytica-like bacteria and Mannheimia species. The correlation (Pearson) between DNA similarities obtained by interchange of strains used for covalent binding and hybridization was 0.794. Significantly lower DNA similarities were observed by the spectrophotometric compared with the micro-well method for three pairs of hybridizations. After removal of these data, the relationship between DNA similarities obtained by the micro-well and spectrophotometric methods improved to y = 0.855x + 11.0. It was found that the accuracy and precision of the micro-well method was at the same level as that of the spectrophotometric method, but the labour and analysis time were reduced significantly. The use of hybridization in the micro-well format will allow DNA-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition.

Evaluation of ozonated oxygen in an experimental animal model of osteomyelitis as a further treatment option for skull-base osteomyelitis.

The standard treatment of chromic skull-base osteomyelitis is antibiotics and surgical removal of sequestrums. Hyperbaric oxygen therapy has been found to be a useful method for managing refractory cases of chronic osteomyelitis. Since a minimal blood supply is needed for hyperbaric oxygen therapy, chronic osteomyelitis can produce necrotic infected areas that are not nutrified and therefore not assessable for hyperbaric oxygen therapy. Ozone is known to be an oxidizing medium with a strong bactericidal effect. We investigated the influence of locally applied ozonated oxygen on the development of chronic osteomyelitis in an experimental animal model using the femur of the rabbit. The proximal sides of the femurs of 40 rabbits were prepared and a needle inserted into the intramedullary cavity. Osteomyelitis was induced with an infusion of Staphylococcus aureus and sodium morrhuate into the bone. The needle was left in a intramedular location. After a 4-week delay animals were randomly separated into treatment and control groups. The infected femur of treated animals was flushed three times a day with 20 ml of ozonated oxygen at an ozone concentration of 107 micrograms/ml O2 over periods of 2 or 4 weeks. Clinical, radiographic and microbiological findings were documented. Chronic osteomyelitis occurred in all animals. Ten rabbits were excluded from further study during the investigation because of excessive weight loss (> 15% of the original weight). Bacterial cultures showed no significant reduction of S. aureus concentrations in the ozone-treated group, although comparison of radiographic results revealed less serious osteomyelitis-related bone damage in these animals (P < 0.01). These findings indicate that refractory osteomyelitis in the head and neck may benefit from locally applied "flush" therapy with ozonated oxygen in addition to treatment with antibiotics, surgery and hyperbaric oxygen.

Taxonomic relationships of the [Pasteurella] haemolytica complex as evaluated by DNA-DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov., Mannheimia granulomatis comb. nov., Mannheimia glucosida sp. nov., Mannheimia ruminalis sp. nov. and Mannheimia varigena sp. nov.

The present paper presents the conclusions of a polyphasic investigation of the taxonomy of the trehalose-negative [Pasteurella] haemolytica complex. Clusters previously identified by ribotyping and multilocus enzyme electrophoresis (MEE) have been evaluated by 16S rRNA sequencing and DNA-DNA hybridizations. Results obtained by the different techniques were highly related and indicated that the [P.] haemolytica complex contains distinct genetic and phenotypic groups. At least seven species were outlined, five of which were named. We refrained in formal naming of more groups until additional strains are characterized. Five 16S rRNA clusters were identified corresponding to distinct lineages previously outlined by MEE. Within 16S rRNA cluster I two distinct genotypic groups have been outlined in addition to [P.] haemolytica sensu stricto (biogroup 1). Each of the clusters II, III, IV and V represent at least one new species. The investigations underline that [P.] haemolytica sensu stricto only contains strains that do not ferment L-arabinose even though they are referred to as 'biotype A' of [P.] haemolytica. The five 16S rRNA clusters identified had a common root relative to the other species within the family Pasteurellaceae, and the overall sequence similarity among these five clusters was higher than what is observed within the existing genera of the family. The allocation of the trehalose-negative [P.] haemolytica complex to a new genus seems to be indicated. Based on the polyphasic investigation performed a new genus Mannheimia is proposed for the trehalose-negative [P.] haemolytica complex. At the present stage two previously named species are transferred to this new genus and three new species are described. [P.] haemolytica is reclassified as Mannheimia haemolytica comb. nov., whereas Pasteurella granulomatis, Bisgaard taxon 20 and [P.] haemolytica biovar 3J are reclassified and combined in the species Mannheimia granulomatis comb. nov. Mannheimia glucosida sp. nov. corresponds to [P.] haemolytica biogroups 3A-3H and the beta-glucosidase and meso-inositol-positive strains of [P.] haemolytica biogroup 9. All typable strains within M. glucosida belong to serotype 11. Mannheimia ruminalis sp. nov. consists of strains previously classified as Bisgaard taxon 18 and [P.] haemolytica biogroup 8D. Finally, Mannheimia varigena sp. nov. includes [P.] haemolytica biogroup 6 as well as Bisgaard taxon 15 and Bisgaard taxon 36. The type strains are NCTC 9380T (M. haemolytica), ATCC 49244T (M. granulomatis), CCUG 38457T = P925T (M. glucosida), CCUG 38470T = HPA92T (M. ruminalis) and CCUG 38462T = 177T (M. varigena).