- Volume 32, Issue 3, 1990

The type IV plasmid-mediated dihydrofolate reductase (DHFR), from a clinical strain of Escherichia coli isolated in South India, was prepared from a transconjugant containing the original clinical plasmid, E. coli J62-2 (pUK1123), and from E. coli C600 (pUK1150) containing a 2.6-kb Hin dIII fragment of pUK1123 cloned into plasmid pBR322. Both preparations were purified by methotrexate affinity chromatography. Automatic amino-acid sequencing of the N-terminal of the purified type IV enzyme from both sources gave an identical sequence which was clearly distinct from other plasmid-mediated trimethoprim-resistant DHFRs. The type IV DHFR showed most homology with the endogenous, chromosomally-encoded E. coli enzyme. Amino-acid sequence analysis also showed that the type IV enzyme preparation from E. coli J62-2 harbouring the original clinical plasmid, pUK1123, also contained the E. coli DNA-binding protein NS1. Analysis by polyacrylamide gel electrophoresis suggested that the type IV enzyme, in its native form, consists of a DHFR of Mr 33 000 coupled to a DNA-binding protein.

Of 190 isolates of Escherichia coli from children aged up to 5 years with diarrhoea, 72 (37-9%) were resistant to trimethoprim and of the 70 isolates tested, 38 transferred high level trimethoprim resistance (MIC > 1000 mg/L) into E. coli EC 1005. Of the enterotoxigenic isolates from which trimethoprim resistance was transferred, 41.2% also contained transmissible toxigenic characteristics. All the trimethoprim-resistant isolates were resistant to other antibiotics.

A comparison of the antigens of single representatives of five serotypes of Ureaplasma urealyticum, of three strains of U. diversum and of single ureaplasmal strains from four other animal hosts was performed by immunoblotting with monoclonal antibodies and a urease ‘enzyme-catch test’. The U. urealyticum serotype 8-specific, surface-expressed, 96-Kda antigen was not found in any of the strains of non-human origin. Differences in the distribution of 16- and 17-Kda antigens were also seen, not only between seroclusters A and B of U. urealyticum, but also with respect to animal strains. Five distinct epitopes were expressed on the urease from U. urealyticum and from chimpanzee ureaplasmal strains, but between one and three of these epitopes were either poorly expressed or not detected on the urease from the other animal strains. Apart from lacking the 96-Kda antigen of U. urealyticum serotype 8, chimpanzee strains gave results similar to those obtained with serocluster A of U. urealyticum. The results with the marmoset strain differed from those of all other non-human strains.

We sought evidence to determine if particular strains of Pseudomonas aeruginosa have a predilection for pulmonary colonisation in patients with cystic fibrosis (CF). The incidence of common pyocin types in non-CF isolates (74%) was similar to that noted in previous reports but differed significantly (χ2 = 16·7, p < 0·001) from the incidence of 40% observed in CF isolates. A retrospective analysis of respiratory isolates also indicated a relatively low incidence of common pyocin types (44%) in isolates from non-CF patients with chronic airways diseases and this incidence also differed significantly from that observed (73%) in other respiratory isolates from patients in the same hospital. These observations suggest that a subpopulation of P. aeruginosa exists which has a predilection for pulmonary colonisation in CF and other chronic pulmonary diseases and may assist in identification of factors affecting bacterial colonisation.

Bordetella pertussis produces several potential virulence factors. One of these is an adenylate cyclase which penetrates eukaryotic cells, is activated by calmodulin and generates high levels of intracellular cAMP. We have found that pertussis infection in man leads to production of high titres (2000-8000) of anti-B. pertussis adenylate cyclase antibodies. Such antibodies also are produced after pertussis vaccination. They persist into adulthood, cross the placenta and disappear a few months after birth. The anti-adenylate cyclase antibodies found in human serum during pertussis infection do not neutralise the catalytic and penetrative activities of the enzyme.

Intra-species restriction fragment length polymorphisms (RFLPs) of Yersinia enterocolitica were detected in assays with a cloned DNA fragment from Legionella pneumophila that included the 16S and 23S rRNA genes. By use of this method it was possible to identify different RFLP types within biogroups/serogroups which were indistinguishable by other means. Thus the 37 biogroup IV/serogroup O3 strains isolated worldwide from pig carcasses, pork and human patients, were subdivided into five different RFLP-types. Typing based on RFLP analysis was sensitive and independent of phenotypic characters. The method will be of value for the identification and evaluation of possible reservoirs and routes of infection.

The effect of various antigens of Leishmania major promastigotes on the function of human polymorphonuclear leucocytes (PMNLs) was examined. Different concentrations of L. major antigens were incubated with isolated PMNLs for various periods and the respiratory burst was assessed by Luminol-dependent chemilumi-nescence. All the Leishmania antigens employed inhibited the PMNL respiratory burst by 35-64%. PMNL viability was not affected either by the concentrations or type of the parasite antigen. Oxygen free radical scavengers enhanced the action of the antigens on the PMNL respiratory burst.

The adherence of epidemic methicillin-resistant Staphylococcus aureus-1 (EMRSA-1) to HEp2 cells and fibronectin and its survival on formica blocks were compared with the equivalent properties of methicillin-sensitive strains of S. aureus. EMRSA-1 bound to HEp2 cells and fibronectin significantly less than the other strains of S. aureus tested, but survival on formica blocks was similar for all strains except the 'Oxford' S. aureus, which survived poorly.

A small plasmid of 4.4 kb encoding resistance to streptomycin (Smr) was detected in a multiresistant Staphylococcus hyicus culture from a piglet with exudative epidermitis. The plasmid-encoded properties were determined by interspecies protoplast transformation experiments. This plasmid was further characterised by restriction endonuclease analysis and a preliminary restriction map was constructed. The plasmid from S. hyicus that conferred streptomycin resistance was designated as pSAI-1. It showed some structural homology with the streptomycin-chloramphenicol resistance plasmid pSK68 from S. aureus of human origin. The MIC of streptomycin in resistance mediated by pSAI-1 was about 10 times higher than the MICs in resistance mediated by Smr plasmids from human S. aureus strains.

Bacteria were grown from 63 (69%) of 91 specimens from necrotic tumours in 63 patients. Of the tumours, 14 were abdominal, 5 pelvic, 23 of the head and neck, 4 of the lungs, 4 mediastinal, 2 lymphatic, 3 of the breast, and 8 were miscellaneous. Aerobic or facultatively anaerobic bacteria only were present in 12 (19%) specimens, anaerobes only in 10 (16%), and mixed aerobic and anaerobic bacteria in 41 (65%). A total of 83 anaerobic and 47 aerobic and facultatively anaerobic bacteria were isolated. The predominant anaerobic bacteria were Bacteroides spp. (36 isolates), and anaerobic cocci (21) and Propionibacterium acnes (22). The aerobic and facultatively anaerobic bacteria most frequently isolated were Staphylococcus aureus, a-haemolytic streptococci, Escherichia coli (seven isolates each), S. epidermidis, Klebsiella pneumoniae and Pseudomonas aeruginosa (five isolates each). These data demonstrate that infection of tumours is usually polymicrobial.

The conditions required to kill Serratia marcescens with nalidixic acid, ciprofloxacin, norfloxacin or ofloxacin were determined in nutrient broth and in phosphate-buffered saline. They were found to be similar to the conditions required for these 4-quinolones to kill Escherichia coli. Bacterial RNA synthesis and bacterial cell division were essential for the bactericidal activity of nalidixic acid but all three fluoroquinolones were bactericidal against non-dividing S. marcescens. However, as with E. coli, bacterial RNA synthesis was essential for the bactericidal activity of norfloxacin though this was not required to kill S. marcescens with ciprofloxacin or ofloxacin.