Pasteurella pneumotropica and the prevalence of the AHP (Actinobacillus, Haemophilus, Pasteurella)-group in laboratory animals

134 bacterial isolates originally identified as Pasteurella pneumotropica were cultured from healthy, ill or dead mice, rats, hamsters, guinea pigs, rabbits and cats originating from 7 conventional laboratory animal facilities. They occurred seldom in pure culture and were found in a variety of organs. Thorough identification (41 criteria) revealed that only 83 isolates (62%) were P. pneumotropica and could be subdivided into 3 biotypes. 3 isolates were P. aerogenes, 1 was P. ureae, 11 (8%) were qualified as Actinobacillus spp. and 13 (10%) as Haemophilus spp. Close relationship of the 3 genera--the 'AHP-group' --made the differentiation difficult. 23 atypical cultures were discarded at the beginning of the study as not belonging to the 'AHP-group'. Two-thirds of isolates were associated with inflammation or suppuration; Haemophilus spp. seemed to be more pathogenic than Pasteurella and Actinobacillus species.     

Survival and transmissibility of Pasteurella pneumotropica

Survival has been determined for Pasteurella pneumotropica on various surfaces found in an animal room at 23+/-1 degrees C and 50+/-10% relative humidity. Longest survival (120 min) was found on mouse hair, shortest (< 30 min) on laboratory coat fabric. Transmission experiments were performed using sentinel animals in order to evaluate the efficiency of their use for the detection of P. pneumotropica in quarantined mice. In sentinels exposed to infected mice by close contact, P. pneumotropica was detected by culture 2 weeks post-exposure and seroconversion 3 weeks after contact. Transfer of soiled bedding from Pasteurella-infected mice did not infect sentinels within a period of 12 weeks as tested by cultivation or serum antibodies.     

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.     

Role of Pasteurella pneumotropica and Mycoplasma pulmonis in Murine Pneumonia

Pneumonia caused by Mycoplasma pulmonis and Pasteurella pneumotropica was studied in conventional, specific pathogen-free (SPF), and germ-free mice. When P. pneumotropica was serially passed in conventional mice, M. pulmonis, as well as P. pneumotropica, was recovered from mice with gross lesions. When M. pulmonis was serially passed in conventional mice, both organisms were recovered. SPF mice given a nasal instillation of M. pulmonis alone, P. pneumotropica alone, or a combination of the two developed pneumonia when both organisms were present. These findings suggested that both organisms contribute to typical murine pneumonia. That M. pulmonis might be an L form of P. pneumotropica was suggested because some SPF mice inoculated with either organism yielded both on culture. This possibility was investigated with mole per cent guanine plus cytosine (GC) content and nucleic acid hybridization techniques. The GC content of P. pneumotropica is 42.2 mole per cent and that of M. pulmonis is 28.6 mole per cent. No specific hybrids between deoxyribonucleic acid (DNA) from M. pulmonis and DNA from P. pneumotropica were detected. This and the wide disparity in GC content showed that M. pulmonis is not an L form of P. pneumotropica. In germ-free mice, intranasal instillation with either organism alone produced pneumonia. The lesions produced when each organism was inoculated independently were characterized by areas of consolidation with perivascular and peribronchial lymphocytic infiltration. Qualitatively, the lesions produced when both organisms were inoculated simultaneously more closely resembled those seen in naturally occurring murine pneumonia. Statistical analysis indicated that the quantitative effect of the two organisms was additive. The indirect fluorescent antibody technique was used to locate organisms in lung tissue sections. M. pulmonis localized in the bronchial epithelium and P. pneumotropica localized in the alveolar lesions.     

Effect of oestrogen on Pasteurella pneumotropica in rat vagina

The effects of ovarian hormones on the vaginal population of Pasteurella pneumotropica in rats were investigated. Specified-pathogen-free adult female Wistar-Imamichi rats with a 4 day oestrous cycle were inoculated with P. pneumotropica in the vagina. Cyclic changes in the vaginal population of P. pneumotropica were not observed in ovariectomized rats and the bacterial population was at a similar level to that at normal dioestrus. Administration of oestrogen to ovariectomized rats caused an increase in the numbers of P. pneumotropica and total bacteria in the vagina nearly equal to that at oestrus in intact rats. The numbers of these organisms in the vagina of ovariectomized rats treated with progesterone did not change and were similar to those of control ovariectomized rats treated with sesame oil. Vaginal smears of ovariectomized rats treated with oestrogen were characterized by abundant cornified non-nucleated epithelial cells with many adherent Gram-negative coccobacilli and were similar to smears from intact rats at oestrus. These findings suggest that the proliferation of P. pneumotropica at oestrus in rat vagina may be primarily due to the environment provided by the degeneration of vaginal epithelial cells promoted by oestrogen secretion from the ovaries.     

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens

Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography–mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C₄-HSL] and N-hexanoyl-l-homoserine lactone [C₆-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C₈-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC₆-HSL]) and long-acyl-chain (C₁₀, C₁₂, and C₁₄) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.     

Biofilm Formation by and Antifungal Susceptibility of Candida Isolates from Urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC₅₀ of <1 μg/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性研究

目的观察不同来源的嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性.方法取源于野鼠、实验大鼠和小鼠的嗜肺巴氏杆菌3株,对30只受试大鼠和小鼠进行交叉人工感染,并于感染后不同时期取咽拭子分离培养,对感染前后菌株,应用RAPD-PCR、SDS-PAGE和Western blot进行基因型、蛋白和抗原成份比较,以及生物学特性的比较.结果受试实验动物对3株嗜肺巴氏杆菌均易感,被接种的动物能稳定携带嗜肺巴氏杆菌直到试验结束,重新分离的嗜肺巴氏杆菌在生物学特性、蛋白成份、抗原性和基因型方面无明显改变.结论同一株嗜肺巴氏杆菌能在实验大鼠和小鼠中相互传染.     

Proliferation of Pasteurella pneumotropica at oestrus in the vagina of rats

Using a colony of Wistar-Imamichi rats contaminated with P. pneumotropica, the vaginal microflora was qualitatively and quantitatively investigated by swabbing. P. pneumotropica was the most dominant organism in the majority of rats examined. The population of P. pneumotropica and indigenous bacteria increased significantly higher at oestrus than in other oestrous stages. By the vaginal flushing technique changes in the population of P. pneumotropica and total bacteria, and changes in vaginal cell type and bacterial counts adhering to vaginal epithelial cells were consecutively investigated. The populations of P. pneumotropica and total bacteria were maximal at oestrus. The increase was correlated with an increase in cornified non-nucleated cells, with large numbers of adherent Gram-negative coccobacilli. The findings indicate that the vagina is a suitable site for colonization by P. pneumotropica in adult female rats, and that proliferation of P. pneumotropica may be due to increased affinity of the organism for cornified non-nucleated cells.     

Serotyping and Enzyme Characterization of Pasteurella haemolytica and Pasteurella multocida Isolates Recovered from Pneumonic Lungs of Stressed Feeder Calves

Ninety-one isolates of Pasteurella multocida (Pm) and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34 (27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories. Received: 29 August 1996 / Accepted: 15 October 1996     

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.     

Biofilm growth and detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.     

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.     

Morphotypic Conversion in Listeria monocytogenes Biofilm Formation: Biological Significance of Rough Colony Isolates

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.