From the [Pasteurella] pneumotropica complex to Rodentibacter spp.: an update on [Pasteurella] pneumotropica.

The species [Pasteurella] pneumotropica has been reclassified into the new genus Rodentibacter, within the family Pasteurellaceae. Along with the type species (Rodentibacter pneumotropicus) of the new genus, seven new species have been named. These organisms were formerly mainly known as the [P.] pneumotropica complex and [P.] pneumotropica was considered as the most important Pasteurellaceae species colonizing laboratory rodents. The aim of this review is to update the veterinary relevant aspects of clinical manifestations, pathogenesis, virulence and diagnostics of members of Rodentibacter with a focus on the most important species from a veterinary perspective. The organisms are obligate commensals of the mucous membranes and members of Rodentibacter are not able to persist for long in the environment. Members of Rodentibacter spp. are responsible for the most prevalent bacterial infections in laboratory mice and rats, but are also common in rodents outside laboratory settings. Some Rodentibacter spp. produce mainly localised disease in connection with favouring factors and seldomly act as primary pathogens in healthy immunocompetent animals. The subclinical infection with Rodentibacter spp. can affect the results of certain types of research using contaminated animals thus placing them on a list of microbes which are often not tolerated in experimental rodent facilities. The presences of RTX toxins, YadA-like proteins and a capsule with possible role in the pathogenesis have been described. Some species of Rodentibacter are able to form robust biofilms which might be involved in colonisation and persistence within the host. Current possibilities for diagnostics and differentiation among Rodentibacter spp. are outlined and options for treatment and control are provided.

Exhaust Air Dust Monitoring is Superior to Soiled Bedding Sentinels for the Detection of Pasteurella pneumotropica in Individually Ventilated Cage Systems.

Reliable detection of unwanted organisms is essential for meaningful health monitoring in experimental animal facilities. Currently, most rodents are housed in IVC systems, which prevent the aerogenic transmission of pathogens between cages. Typically soiled-bedding sentinels (SBS) exposed to soiled bedding collected from a population of animals within an IVC rack are tested as representatives, but infectious agents often go undetected due to inefficient transmission. Pasteurellaceae are among the most prevalent bacterial pathogens isolated from experimental mice, and the failure of SBS to detect these bacteria is well established. In this study, we investigated whether analysis of exhaust air dust (EAD) samples by using a sensitive and specific real-time PCR assay is superior to conventional SBS monitoring for the detection of Pasteurella pneumotropica (Pp) infections. In a rack with a known prevalence of Pp-positive mice, weekly EAD sampling was compared with the classic SBS method over 3 mo. In 6 rounds of testing, with a prevalence of 5 infected mice in each of 7 cages in a rack of 63 cages, EAD PCR detected Pp at every weekly time point; SBS failed to detect Pp in all cases. The minimal prevalence of Pp-infected mice required to obtain a reliable positive result by EAD PCR testing was determined to be 1 in 63 cages. Reliable detection of Pp was achieved after only 1 wk of exposure. Analysis of EAD samples by real-time PCR assay provides a sensitive, simple, and reliable approach for Pp identification in laboratory mice.

Elimination of Pasteurella pneumotropica from a mouse barrier facility by using a modified enrofloxacin treatment regimen.

Multiple NOD. Cg-Prkdc(scid)Il2rg(tm1Wjl)Tg(HLA-A2.1)Enge/Sz (NSG/A2) transgenic mice maintained in a mouse barrier facility were submitted for necropsy to determine the cause of facial alopecia, tachypnea, dyspnea, and sudden death. Pneumonia and soft-tissue abscesses were observed, and Pasteurella pneumotropica biotype Jawetz was consistently isolated from the upper respiratory tract, lung, and abscesses. Epidemiologic investigation within the facility revealed presence of this pathogen in mice generated or rederived by the intramural Genetically Engineered Mouse Model (GEMM) Core but not in mice procured from several approved commercial vendors. Epidemiologic data suggested the infection originated from female or vasectomized male ND4 mice obtained from a commercial vendor and then comingled by the GEMM Core to induce pseudopregnancy in female mice for embryo implantation. Enrofloxacin delivered in drinking water (85 mg/kg body weight daily) for 14 d was sufficient to clear bacterial infection in normal, breeding, and immune-deficient mice without the need to change the antibiotic water source. This modified treatment regimen was administered to 2400 cages of mice to eradicate Pasteurella pneumotropica from the facility. Follow-up PCR testing for P. pneumotropica biotype Jawetz remained uniformly negative at 2, 6, 12, and 52 wk after treatment in multiple strains of mice that were originally infected. Together, these data indicate that enrofloxacin can eradicate P. pneumotropica from infected mice in a less labor-intensive approach that does not require breeding cessation and that is easily adaptable to the standard biweekly cage change schedule for individually ventilated cages.

Analysis of 16S-23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents.

The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, "Hemophilus influenzaemurium" and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species-specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of "H. influenzaemurium", a larger ITS(ile+ala) which contained the genes for tRNA(Ile(GAU)) and tRNA(Ala(UGC)) and a smaller ITS(glu) with the tRNA(Glu(UUC)) gene. "H. influenzaemurium" revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITS(ile+ala) and 56.5 to 86.8% for ITS(glu). Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level.

Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice.

Pasteurella pneumotropica is an opportunistic pathogen in rodents. Natural infection in immunodeficient animals suggests that immunodeficiency is a major factor in P. pneumotropica pathogenesis. To understand this process, we performed clinical, pathological and bacteriological studies of immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice experimentally infected with P. pneumotropica ATCC 35149. From 14 days postinoculation, some of P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of weight loss. Three of 10 P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of depression, ruffled coat, and weight loss and died at 27, 34, and 59 days postinoculation. At 35 days postinoculation, almost all P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice had lung abscesses. The bacteria were isolated from the upper and lower respiratory tracts, including the lungs, and blood. In contrast, P. pneumotropica-infected ICR mice exhibited no clinical signs or lesions. The bacteria were isolated from the upper, but not the lower respiratory tracts. We developed an animal model for understanding host interactions with P. pneumotropica.

Implementation of a PCR assay of Pasteurella pneumotropica to accurately screen for contaminated laboratory mice.

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica.

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.

Enterobacteriaceae and pseudomonadaceae on the dorsum of the human tongue.

OBJECTIVE: The aim of this study was to correlate the presence of Enterobacteriaceae, Pseudomonadaceae, Moraxellaceae and Xanthomonadaceae on the posterior dorsum of the human tongue with the presence of tongue coating, gender, age, smoking habit and denture use. MATERIAL AND METHODS: Bacteria were isolated from the posterior tongue dorsum of 100 individuals in MacConkey agar medium and were identified by the API 20E system (Biolab-Mérieux). RESULTS: 43% of the individuals, presented the target microorganisms on the tongue dorsum, with greater prevalence among individuals between 40 and 50 years of age (p = 0.001) and non-smokers (p=0.0485). CONCLUSIONS: A higher prevalence of Enterobacteriaceae and Pseudomonadaceae was observed on the tongue dorsum of the individuals evaluated. There was no correlation between these species and the presence and thickness of tongue coating, gender and presence of dentures.

Enterobacteriaceae and pseudomonadaceae on the dorsum of the human tongue

OBJECTIVE: The aim of this study was to correlate the presence of Enterobacteriaceae, Pseudomonadaceae, Moraxellaceae and Xanthomonadaceae on the posterior dorsum of the human tongue with the presence of tongue coating, gender, age, smoking habit and denture use. MATERIAL AND METHODS: Bacteria were isolated from the posterior tongue dorsum of 100 individuals in MacConkey agar medium and were identified by the API 20E system (Biolab-Mérieux). RESULTS: 43 percent of the individuals, presented the target microorganisms on the tongue dorsum, with greater prevalence among individuals between 40 and 50 years of age (p = 0.001) and non-smokers (p=0.0485). CONCLUSIONS: A higher prevalence of Enterobacteriaceae and Pseudomonadaceae was observed on the tongue dorsum of the individuals evaluated. There was no correlation between these species and the presence and thickness of tongue coating, gender and presence of dentures.

Comparative analysis of Pasteurella pneumotropica isolates from laboratory mice and rats.

Selected biochemical and genetic characteristics of the wild-type strains of Pasteurella pneumotropica isolated from mice and rats were investigated and compared in order to determine the significant differences among the isolates. The isolates were divided into six groups on the basis of the patterns of carbon source utilization in the host rodents. The genome sizes were determined by electrophoretic analysis, and the mean genome size of the isolates from mice was larger than that of the isolates from rats (P < 0.05). Cluster analysis of the rpoB sequences discriminated five clusters; the differences might have correlated with the host associations. Principal component analysis (PCA) based on both the biochemical and genetic characteristics revealed total 44 strains discriminated into three groups comprising the host-dependent and host-independent groups. Although the P. pneumotropica isolates were mainly classified on the basis of the host rodents by the examinations, the existence of isolates that could not be discriminated on the basis of the host rodents alone was confirmed by the PCA. These results indicated that the P. pneumotropica isolates could be further classified by taxonomic analysis and also suggested the existence of a host-independent group in addition to the host-dependent groups.

Comparison of the in vitro susceptibility of rodent isolates of Pseudomonas aeruginosa and Pasteurella pneumotropica to enrofloxacin.

The objectives of this study were to determine and compare the in vitro enrofloxacin susceptibility of 94 Pseudomonas aeruginosa isolates obtained from enrofloxacin-treated and untreated mice and that of 40 Pasteurella pneumotropica strains and also to assess the efficacy and effects of enrofloxacin treatment of laboratory mice. The minimum inhibitory concentrations (MICs) of enrofloxacin against all the Ps. aeruginosa isolates were in the range of 1 to 4 microg/ml, whereas those against all the P. pneumotropica strains were less than 0.5 microg/ml. The mutation frequency in 54% of the Ps. aeruginosa isolates on treatment with enrofloxacin ranged from 10(-6) to 10(-8); however, none of the P. pneumotropica strains could grow on medium containing more than 3 microg/ml enrofloxacin. Comparison of in vitro enrofloxacin susceptibilities suggested that enrofloxacin was effective for eliminating P. pneumotropica but not for eliminating Ps. aeruginosa for which the MIC of enrofloxacin was more than 1 microg/ml. These results indicated that the enrofloxacin susceptibility of P. pneumotropica was higher than that of Ps. aeruginosa, and that the enrofloxacin treatment might not affect the susceptibility of Ps. aeruginosa.

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits.

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.

A specific polymerase chain reaction based on the gyrB gene sequence and subsequent restriction fragment length polymorphism analysis of Pasteurella pneumotropica isolates from laboratory mice.

For a molecular identification and typing tool, we developed a specific polymerase chain reaction (PCR) based on the gyrB gene sequence and a subsequent restriction fragment length polymorphism (RFLP) analysis using the products amplified from the specific PCR to facilitate discrimination of biotypes of Pasteurella pneumotropica from laboratory mice. Appropriate PCR products, a 1039-basepair fragment for biotype Jawetz and a 1033-basepair fragment for biotype Heyl, were amplified by use of the primers CZO-1 and NJO-2 from all 105 P. pneumotropica isolates tested and the 2 reference strains but not from other bacterial species tested. MspI digestion of PCR-generated products showed 3 RFLP patterns among the 105 isolates, and these patterns correlated with the biotype of the isolate (RFLP pattern 1, biotype Jawetz; RFLP pattern 2, biotype Heyl; and RFLP pattern 3, biotype Jawetz with Beta-hemolytic activity). Our procedure identifies and biotypes isolates of P. pneumotropica from laboratory mice, using simple PCR and enzymatic restriction techniques. Therefore, this procedure is useful as a rapid identification and biotyping tool for isolates of P. pneumotropica from laboratory mice.

Phylogenetic analysis of isolates of Pasteurella pneumotropica from laboratory animals based on the gyrB gene sequence.

Phylogenetic analysis using the gyrB sequence was performed to investigate the genetic relevance among 49 isolates of P. pneumotropica. In the phylogeny, the isolates were clearly classified into three groups as follows: group A for the isolates of biotype Jawetz derived from mice, group B for the isolates of biotype Jawetz derived from rats, and group C for the isolates of biotype Heyl. These results suggest that the gyrB sequence of P. pneumotropica differs between the isolates of two biotypes, and also between the isolates derived from mice and rats in the biotype Jawetz.

Phylogenetic relationship of Pasteurella pneumotropica isolates from laboratory rodents based on 16S rDNA sequence.

A 1344 bp fragment of the 16S ribosomal DNA (rDNA) sequence was used to determine the genetic relationship of Pasteurella pneumotropica isolates from laboratory rodents. A total of 30 nucleotide sequences of P. pneumotropica, including 24 wild strains, 3 reference strains, and 3 nucleotide sequences deposited in GenBank, were examined for heterogeneity of their 16S rDNA sequences. Phylogenetic analysis based on 16S rDNA sequence discriminated 5 types of branching lineages. Of these 5 types, 3 types had significant associations with mice or rats, and 2 had significant associations with the beta-hemolytic phenotype. These results suggest that 16S rDNA sequencing of P. pneumotropica isolates demonstrates genetic heterogeneity and phylogenetic discrimination in terms of their hemolytic phenotype and host associations.

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis.

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with Hae III revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, Apa I and Not I digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with Hae III.

Identification procedure for Pasteurella pneumotropica in microbiologic monitoring of laboratory animals.

Discrepancies have been recognized in the identification of Pasteurella pneumotropica between testing laboratories. To determine the causes of the differences and to propose a reliable identification procedure for P. pneumotropica, a working group was organized and 69 isolates identified or suspected as P. pneumotropica were collected from 8 laboratories in Japan. These isolates were examined by colony morphology, Gram-staining, the slide agglutination test using two antisera (ATCC35149 and MaR), two commercially available biochemical test kits (ID test, API20NE) and two primer sets of PCR tests (Wang PCR, CIEA PCR). The 69 isolates and two reference strains were divided into 10 groups by test results. No single procedure for P. pneumotropica identification was found. Among tested isolates, large differences were not observed by colony morphology and Gram-straining except for colony colors that depended on their biotypes. Sixty-eight out of 69 isolates were positive by the slide agglutination test using two antisera except for one isolate that tested with one antiserum. The ID test identified 61 out of 69 isolates as P. pneumotropica and there was no large difference from the results of CIEA PCR. From these results, we recommend the combination of colony observation, Gram-straining, the slide agglutination tests with two antisera and biochemical test using the ID test for practical and reliable identification of this organism.