Urinary tract infection in a human male patient with Staphylococcus pseudintermedius transmission from the family dog.

Staphylococcus pseudintermedius is increasingly recognized as a human pathogen. We report the first case of an urinary tract infection in a male patient with this organism.

Persistent infection with Staphylococcus pseudintermedius in an adult oncology patient with transmission from a family dog.

Staphylococcus pseudintermedius is a well known commensal organism of dogs but also a canine opportunistic pathogen. Reports of this organism being recovered from specimens from humans might suggest an increase prevalence in human infections and/or improved diagnostic leading to more accurate identification. Here we report a case of persistent S. pseudintermedius infection in an adult female oncology patient including colonization of the tip of an indwelling catheter. Diligence by laboratories in correctly isolating and identifying this pathogen (including susceptibility testing) is essential for optimal patient care.

Real-time PCR detection of bacteria belonging to the Firmicutes Phylum.

Members of the bacterial Phylum Firmicutes occupy a wide range of habitats and can be either beneficial or detrimental in diverse settings, including food- and beverage-related industries. Firmicutes are responsible for the vast majority of beer-spoilage incidents and, as such, they have a substantial financial impact in the brewing industry. Rapid detection and identification of a bacterium as a Firmicutes is difficult due to widespread genetic transfer and genome reduction resulting in phenotypic diversity in these bacteria. Here we describe a real-time multiplex PCR to detect and differentiate Firmicutes associated with beer-spoilage from non-Firmicutes bacteria that may be present as benign environmental contaminants. A region of the 16S rRNA gene was identified and predicted to be highly conserved amongst, and essentially specific for, Firmicutes. A real-time PCR assay using a hydrolysis probe targeting this region of the 16S rRNA gene was experimentally shown to detect ten genera of Firmicutes known to be beer spoilers, but does not cross-react with eleven of twelve non-Firmicutes genera which can periodically appear in beer. Only one non-Firmicutes species, Zymomonas mobilis, weakly reacted with the Firmicutes probe. This rPCR assay has a standard curve that is linear over six orders of magnitude of DNA, with a quantitation limit of DNA from <10 bacteria. When used to detect bacteria present in beer, the assay was able to detect 50-100 colony forming units (CFU) of Firmicutes directly from 2.5 cm membranes used to filter 100 ml of contaminated beer. Through incorporation of a 4.7 cm filter and an overnight pre-enrichment incubation, the sensitivity was increased to 2.5-10 CFU per package of beer (341 ml). When multiplexed with a second hydrolysis probe targeting a universal region of the 16S rRNA gene, the assay reliably differentiates between Firmicutes and non-Firmicutes bacteria found in breweries.

Human papillomavirus DNA detection in sperm using polymerase chain reaction.

OBJECTIVE: To detect human papillomavirus (HPV) in semen and find if sperm washing removes HPV DNA. METHODS: Amplification by nested polymerase chain reaction (PCR) was used to detect viral DNA sequences in semen samples from 85 volunteers. Forty-five men had historical or clinical evidence of genital HPV infection (study group) and 40 were healthy, clinically HPV-negative semen donors. RESULTS: We detected HPV DNA in the sperm cells of 24 of 45 subjects (53%) with past or current HPV infections in contrast to three of 40 healthy subjects (8%) (P <.001). Overall, PCR detected HPV in 21 of 32 subjects (66%) with identifiable lesions and six of 53 (11%) without them (P <.001). Swim-up washings of all 27 prewash sperm cells with HPV reduced cellular HPV DNA below detectable levels in only two cases. CONCLUSION: HPV is present in sperm cells from infected and apparently healthy subjects, and sperm washing does not eliminate the risk of HPV transmission to recipients. We suggest that HPV DNA testing should be done on the semen of prospective donors, and those with positive tests should be excluded from donation.

Cost-effective algorithm for detection and identification of vancomycin-resistant enterococci in surveillance cultures.

A study was undertaken to develop an easy-to-use and cost-effective algorithm for the detection and identification of vancomycin-resistant enterococci (VRE) in surveillance cultures, because the incidence of VRE outbreaks in institutions across Canada has made continuous surveillance a necessity. Enterococcus faecium and Enterococcus faecalis carry transferable resistance genes and hence are a problem for infection control. In laboratory surveillance, however, Enterococcus gallinarum and Enterococcus casseliflavus, which exhibit low-level nontransferable resistance, are also encountered and often create confusion in identification. Included in this study were a total of 218 strains of enterococci and other streptococci isolated from surveillance cultures. Conventional methods were used to determine their biochemical activities, and speciation was attempted in 121 strains using a rapid multiplex polymerase chain reaction (PCR) method that utilized primers for the vanA, B, C1, C2/C3 and ddl genes. The results indicated that by using only a few tests (Gram stain, pyrrolidonyl arylamidase activity, motility, xylose and methyl-alpha-D-glucopyranoside utilization), Enterococcus faecium/faecalis strains could be accurately differentiated from Enterococcus gallinarum/casseliflavus strains. For the 121 strains on which PCR was performed, there was a 100% correlation with the biochemical identification, with the added advantage that the presence of van genes could be determined at the same time. The cost of identification using minimal biochemical testing and PCR was less than that of identification using automated systems or a battery of conventional biochemical methods. The algorithm presented here may be used in the microbiology laboratory.

Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost.

Control of Bordetella pertussis in the community is hampered by slow and insensitive diagnostic tests. We therefore examined the accuracy and cost of culture, direct fluorescent antibody (DFA) staining, and PCR in a routine clinical laboratory. Six hundred thirty seven nasopharyngeal swabs and aspirates in casamino acids transport medium were cultured, stained with polyclonal (Difco), and monoclonal (BL-5 and Accu-Mab) anti-B. pertussis reagents, and amplified by an IS481-specific PCR. PCR products were detected by a hybridization-enzyme immunoassay kit (Gen-eti-k DEIA, DiaSorin), with confirmation by a second PCR in a separate laboratory. Sensitivities and specificities of culture, polyclonal DFA, monoclonal DFA, and PCR were 36 and 100%, 11.4 and 94.6%, 8.3 and 98. 4%, and 95.0 and 99.3%, respectively, with a prevalence of 15.7%. The DFA tests were the most economical, and the PCR cost was 31% higher than culture. This study suggests that with minor improvements in economy, pertussis PCR can be implemented in a clinical laboratory with marked improvement in diagnostic accuracy.

Inhibition of human lymphocyte function by organic solvents.

UNLABELLED: We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers significantly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis. CONCLUSION: Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.

Primary pulmonary hypertension and human immunodeficiency virus infection.

This report details the case of a 42-year-old homosexual Caucasian male with infection due to human immunodeficiency virus type 1 (HIV-1) who presented with a four-month history of progressive dyspnea and was found to have clinical and hemodynamic evidence of severe pulmonary hypertension. He had had no opportunistic infections, and had a T helper lymphocyte count of 200×10(6)/L. Extensive clinical laboratory and radiological evaluations revealed no underlying cause. Microscopic examination of postmortem lung tissue revealed findings consistent with grade V pulmonary hypertension. Electron microscopic analysis and polyermase chain reaction detection of HIV-DNA from dissected pulmonary arterioles failed to provide any supportive evidence to suggest productive infection of the pulmonary arteriolar endothelial cells by HIV-1. Although HIV-1 likely plays a role in the pathogenesis of primary pulmonary hypertension, evidence for direct infection of pulmonary vessel endothelium was lacking in this case. The pathogenesis of primary pulmonary hypertension associated with HIV remains obscure.

The use of bacteriophages to differentiate serologically cross-reactive isolates of Klebsiella pneumoniae.

In serological typing of Klebsiella pneumoniae strains from human, equine and environmental sources, the capsular identity of many isolates could not be determined because of serological cross-reactivity. A panel of 91 bacteriophages able to lyse each of the 77 capsular serotypes of K. pneumoniae was isolated and tested for the ability to distinguish between strains in a collection of 17 clinical isolates of K. pneumoniae which exhibited cross-reactivity with two or more capsular type sera. Most isolates could be assigned a capsular type by performing a simple streak test with bacteriophage, although some required the application of an efficiency of plating analysis to discern capsular type. Bacteriophage typing was found to be an effective, inexpensive and clinically practical adjunct to serotyping in distinguishing serologically cross-reactive K. pneumoniae isolates, irrespective of their origin.

Expression of superoxide dismutase in Listeria monocytogenes.

The nature and expression of superoxide dismutase (SOD; EC 1.15.1.1) in the gram-positive food-borne pathogen Listeria monocytogenes were examined. Metal depletion and reconstitution studies and resistance to H2O2 and potassium cyanide inactivation indicated that L. monocytogenes has a single SOD which utilizes manganese as a metal cofactor. The specific activity of SOD was unchanged in cells exposed to a heat shock at 42 degrees C or grown in the presence of paraquat-generated superoxide anion or of metal chelators in the medium. SOD levels increased, however, as the cells progressed through the logarithmic phase of growth and into the stationary phase. Furthermore, SOD activity decreased with decreasing growth temperatures and declined concurrently with decreased growth when higher concentrations of sodium chloride were added to the medium. Cells grown anaerobically possessed relatively high levels of SOD, although these levels were about 10 to 30% lower than those of aerobically grown bacteria. Different isolates of L. monocytogenes were found to produce approximately equivalent levels of SOD, although greater differences in SOD expression were seen among other species of Listeria. When compared with L. monocytogenes, for example, Listeria welshimeri typically produced about 30% greater SOD activity, whereas Listeria murrayi produced about 60% less total SOD activity. Although all species of Listeria produced a single Mn-type SOD, differences in the relative electrophoretic mobility of the native enzymes were noted. These data suggest that the single L. monocytogenes SOD enzyme is constitutively produced in response to many environmental factors and may also be responsive to the cellular growth rate.

Reduction of ferric iron by Listeria monocytogenes and other species of Listeria.

One mechanism by which Listeria monocytogenes is thought to obtain iron required for growth is through the extracellular reduction of a ferric iron source to the ferrous form. To better characterize this reductase activity we have developed a simple plate assay that allows detection of colonies of Listeria species able to reduce ferric iron. Cells are plated on an agar base medium containing a ferric iron source and ethylenediamine dihydroxyphenylacetic acid. Colonies are then overlain with soft agarose containing NADH, flavin mononucleotide, and Ferrozine, a chelator of ferrous iron. Colonies able to reduce the ferric iron source form a red-purple color as the ferrous iron is complexed with ferrozine. Using this qualitative assay we have shown that all species of Listeria are able to reduce ferric iron when presented as ferric ammonium citrate whereas most other species of Gram-positive and Gram-negative bacteria are not. Only Clostridium perfringens was able to reduce ferric iron to the same extent as Listeria. Listeria monocytogenes was further shown to be capable of reducing various ferric iron salts as well as iron bound to ferritin, transferrin, and 2,3-dihydroxybenzoic acid in the agar plate assay. The utility of this assay was demonstrated by using it to screen a bank of Tn916-derived mutants of L. monocytogenes for clones unable to reduce ferric iron. Four such mutants were identified and all were shown to have greatly decreased ferric reductase activity.

Species-specific detection of Listeria monocytogenes by DNA amplification.

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.

Identification of a maltose-inducible major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae.

The addition of maltose to the growth media of Actinobacillus pleuropneumonia (serotype 1) resulted in the induction of an outer membrane protein (OMP) with a molecular mass of 42 kDa. This protein had porin-like properties in that it was peptidoglycan-associated and was resistant to proteolysis by trypsin. A pleuropneumoniae expressing the 42 kDa OMP were unable to bind lambda phage. Similar proteins were also induced in A. pleuropneumoniae isolates representing serotypes 2 to 7 with the exception of serotype 4; however, not all isolates of any given serotype expressed a maltose-inducible OMP. Western immunoblotting using convalescent antiserae against the serotype 1 A. pleuropneumoniae indicated that the 42 kDa OMP was expressed in vivo and was cross-reactive with the maltose-inducible OMPs from other serotypes.

Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae.

The outer membrane protein profile of Actinobacillus (Haemophilus) pleuropneumoniae grown under iron-restricted and iron-replete conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A virulent serotype 1 isolate synthesized a novel protein with an apparent molecular weight of 105,000 (105K) and increased the synthesis of a 76K protein under iron-restricted conditions. Both proteins were synthesized within 15 min of establishment of iron-restricted conditions. Proteins of equivalent molecular weights could also be induced by iron restriction in serotype 2, 3, 4, 5, and 7 isolates of A. pleuropneumoniae. Convalescent-phase sera from serotype 1-infected pigs contained antibodies which recognized both the 105K and 76K proteins from all six serotypes examined, indicating that these proteins were expressed in vivo and were immunologically conserved. Cells expressing the 105K and 76K proteins also displayed an enhanced ability to bind Congo red and hemin, suggesting that one or both of these proteins functioned to acquire complexed iron during in vivo growth.

Characterization of a streptomycin-sulfonamide resistance plasmid from Actinobacillus pleuropneumoniae.

An Actinobacillus pleuropneumoniae strain contained a plasmid (pHD8.1) conferring resistance to streptomycin and sulfonamide. Restriction endonuclease mapping and DNA-DNA hybridization showed that pHD8.1 is related to RSF1010 from Salmonella panama, which also confers resistance to streptomycin and sulfonamide, and to pHD148 from Haemophilus ducreyi, which confers resistance only to sulfonamide.

Iron-repressible outer-membrane proteins of Pasteurella haemolytica.

The outer-membrane protein (OMP) profile of Pasteurella haemolytica grown under iron-replete and iron-restricted conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A serotype 1 isolate induced the synthesis of a new 77,000 Mr OMP in iron-restricted media while two other proteins of 100,000 Mr and 71,000 Mr were synthesized in increased amounts. None of these proteins were peptidoglycan-associated or heat-modifiable, and only the 100,000 Mr protein showed some degree of disulphide cross-linking. Kinetic analysis revealed that the iron-repressible proteins appeared in the outer membrane within 15 min of establishment of iron-restricted conditions. Analysis of P. haemolytica isolates representing serotypes 1 to 12 showed that iron-repressible OMPs of 77,000 Mr and 71,000 Mr could be induced in all 12 serotypes but that there was some variability in the expression of the 100,000 Mr protein. Immunoblotting of OMPs with convalescent sera from P. haemolytica-infected calves indicated that antibodies directed against all three iron-repressible OMPs were present, suggesting that these proteins were expressed in vivo.

Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.

Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclines limits the use of these agents for the treatment of chancroid.