Gonococcal resistance to beta-lactams and tetracycline involves mutation in loop 3 of the porin encoded at the penB locus.

penB is a chromosomal mutation that confers resistance to beta-lactams and tetracyclines and reduced susceptibility to quinolones in Neisseria gonorrhoeae. It is linked to the porin gene (por) and requires the increased expression of an efflux pump due to mtr. Transformation of a susceptible gonococcus (strain H1) with chromosomal DNA from strain FA140 (penA mtr penB; porin serovar IB1) and conjugal transfer of a beta-lactamase-expressing plasmid was used to produce isogenic strains for determination of equilibrium periplasmic penicillin concentrations by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). In transformants with the Mtr and PenB phenotypes, equilibrium concentrations of penicillin were reduced. DNA sequence analysis of por from isogenic penB and penB+ transformants revealed 14 sequence differences; nine of these differences resulted in amino acid changes. Three amino acid changes were found in the putative gonococcal equivalent of the pore-constricting loop 3 of Escherichia coli OmpF. Two of these changes (Gly-101-Ala-102-->Asp-Asp) result in an increased negative charge at this position in por loop 3. PCR products comprising the complete por gene from strain FA140 were transformed into strain H1-2 (penA mtr; porin serovar IB-3), with the resulting transformants having the antibiotic susceptibility phenotype associated with penB. penB-like mutations were found in loop 3 of clinical isolates of gonococci with chromosomally mediated resistance to penicillin. We conclude that penB is a mutation in loop 3 of por that reduces porin permeability to hydrophilic antibiotics and plays an important role in the development of chromosomally mediated resistance to penicillin and tetracycline in gonococci.

Use of the E test for methicillin sensitivity testing.

Performance of the E test for methicillin sensitivity testing was investigated using 21 methicillin-resistant and 28 methicillin-sensitive staphylococci. Thirty of the strains had previously given equivocal results with a strip diffusion method. The E test performed well with Mueller-Hinton agar plates incubated at 30 degrees C for 24 h but only after supplementation with 5% NaCl. Testing on agar without salt was necessary for the recognition of methicillin resistance in one strain. We recommend a breakpoint of 4 mg/L and heavy inocula. The E test can be controlled by testing a control strain on the same plate.

Continuing medical education: experience and opinions of consultants.

The right of consultants to study leave and expenses is not binding on trusts and has been eroded in directly managed units. Complaints led to a survey of consultants in North West Thames region. This showed that most consultants use their own time and money to maintain their knowledge by buying and consulting journals and textbooks, attending local meetings, and using their annual leave for study, but they feel the need for study leave to attend meetings of specialist societies and courses. Leave is usually granted readily but without cover provided by a locum and with a very limited contribution to expenses. To maintain quality of medical care both the right and obligation to take study leave should be contractual.

Gastro-intestinal involvement in Mycobacterium avium-intracellulare infection of patients with HIV.

In a study of 866 faecal specimens from 437 persons, Mycobacterium avium-intracellulare (MAI) was isolated from 14.8% patients with AIDS and 1.3% patients with symptomatic HIV infection but not from any HIV seronegative or asymptomatic HIV seropositive persons. These data support the hypothesis that the gastro-intestinal tract is the portal of entry for MAI and confirm that MAI infection is a manifestation of late-stage HIV disease. Positive faecal cultures correlated well with disseminated disease. The use of faecal cultures for early diagnosis is therefore recommended.

The dominance of a multiresistant strain of Neisseria gonorrhoeae among prostitutes and STD patients in The Gambia.

OBJECTIVE: To study the epidemiology of antibiotic resistant strains of Neisseria gonorrhoeae from sexually transmitted disease clinics in The Gambia. MATERIALS AND METHODS: One hundred and sixty five strains of N gonorrhoeae were tested for their antibiotic susceptibility, auxotype, serotype, and plasmid content. RESULTS: Of the total population 84 (51%) were non-penicillinase producing (nonPPNG) and 81 (49%) penicillinase-producing N gonorrhoeae (PPNG). There were 16 serovars, five auxotypes and 33 auxotype/serovar (A/S) classes in the total population and the nonPPNG. Among PPNG only five serovars, two auxotypes and nine A/S classes were found. One A/S class predominated, NR/IB-7 (86 isolates), of which 66 (77%) were PPNG and the remainder were chromosomally-mediated resistant N gonorrhoeae (CMRNG). These strains also showed reduced susceptibility to ciprofloxacin, ceftriaxone and tetracycline and were evenly distributed among patient groups. CONCLUSION: We have identified a relatively homogeneous gonococcal population with a core group of isolates exhibiting high levels of antibiotic resistance.

Opsonophagocytosis of Pneumocystis carinii.

The interaction of Pneumocystis carinii purified from rat lungs with rat peritoneal macrophages and human circulating polymorphonuclear leucocytes was studied by amplified chemiluminescence and examination of stained cytospin preparations. A polyclonal rat antiserum to P. carinii was opsonic with both types of phagocyte. Complement had no opsonic properties alone but produced a synergic effect in combination with antibody.

The neutrophil chemiluminescence response to Pneumocystis carinii is stimulated by GM-CSF and gamma interferon.

The interaction of Pneumocystis carinii purified from rat lung with human polymorphonuclear leukocytes (PMN) was studied using amplified chemiluminescence. Pre-incubation of PMN with granulocyte-macrophage stimulating factor or gamma interferon resulted in phagocyte stimulation.

Epidemiology of penicillin resistant Neisseria gonorrhoeae.

OBJECTIVE: To study the epidemiology of Neisseria gonorrhoeae that exhibit both chromosomal and plasmid-mediated resistance to penicillin. MATERIALS AND METHODS: A total of 1589 strains of N gonorrhoeae isolated from patients attending St Mary's Hospital, London were tested for both their susceptibility to penicillin and for their auxotype and serotype. RESULTS: Of the 940 non-penicillinase producing N gonorrhoeae, 840 were considered penicillin sensitive (MIC less than or equal to 0.5 mg/l) and 100 were chromosomally-mediated resistant N gonorrhoeae (CMRNG), (MIC greater than or equal to 1.0 mg/l). Of the 649 penicillinase producing N gonorrhoeae (PPNG), 429 carried the 4.4 megadalton (MDa) penicillinase encoding plasmid and 220 carried the 3.2 MDa plasmid. CMRNG were predominantly serogroup IB (90%). PPNG with 3.2 MDa plasmid were the only group more often serogroup IA (58%) than IB (42%). Serovar IA-1/2 and requirement for arginine, hypoxanthine and uracil (AHU) were associated with increased susceptibility to penicillin whereas serovar IB-5/7 was associated with decreased susceptibility in nonPPNG. There was a significant difference in the distribution of the IA and IB serovars between PPNG carrying either the 4.4 MDa or 3.2 MDa plasmid. AHU and PAOU requiring strains were not found among PPNG and were uncommon among CMRNG. CONCLUSION: Some clear associations have been found but the pattern among PPNG appears more complex and in most instances could be related to clusters of a single strain over a short time span.

Chromosomal resistance to antibiotics in gonococci from Bahrain.

Ninety-one isolates of non-penicillinase-producing Neisseria gonorrhoeae from patients in Bahrain were tested for serotype, auxotype, and antibiotic susceptibility. Ten serovars and three auxotypes were found. Of the 91 isolates, 49 (54%) were serovar IB-5/7, 59 (65%) had a penicillin MIC greater than or equal to 1 mg/l, 39 (45%) had a cefuroxime MIC greater than or equal to 0.5 mg/l, and 63 (69%) had a tetracycline MIC of greater than or equal to 4 mg/l. No spectinomycin or high-level tetracycline resistance was seen. Seventy of the 91 isolates were tested against ciprofloxacin and ceftriaxone, and 40 (57%) and 26 (37%) had MICs greater than or equal to 0.03 mg/l, respectively. DNA from two penicillin-resistant isolates was capable of transforming recipient strain FA19 to donor level of penicillin and cephalosporin resistance in four steps. The first three steps were indicative of the acquisition of known resistance mutations. The existence of the fourth level transformants, with the ability of donor DNA to transform strain FA140 to higher levels of resistance, suggest the presence of another resistance mutation.

Plasmid analysis of Mycobacterium avium-intracellulare (MAI) isolated in the United Kingdom from patients with and without AIDS.

One hundred and forty-seven isolates (128 strains) of Mycobacterium avium-intracellulare (MAI) were screened by agarose gel electrophoresis for the presence of plasmids. Plasmids were characterised according to size and by Southern hybridisation analysis of intact and restriction endonuclease-digested DNA. Two cloned MAI plasmids, pLR7 and pLR20, were used as probes. There was no significant difference in the rate of plasmid carriage in MAI strains isolated from patients with the acquired immuno-deficiency syndrome (AIDS) and from non-AIDS patients in the UK, but a higher rate of plasmid carriage was observed in a panel of American strains from AIDS patients. Plasmids were grouped into two broad categories: small (mostly 14-30 kb) and large (greater than 150 kb). Southern blot analysis identified two distinct groups of small plasmids, the majority of which showed homology with pLR7. Plasmids from this group were significantly more common in strains of serotypes 4 and 8 which are particularly associated with AIDS.

Penicillin and cephalosporin resistance in gonococci.

Non-penicillinase producing Neisseria gonorrhoeae isolated at St Mary's Hospital, London were examined for the prevalence of resistance to penicillin and for decreased susceptibility to cefuroxime. Of the 941 non-PPNG tested 100 (10.6%) were resistant to penicillin (minimum inhibitory concentration, MIC, greater than or equal to 1 mg/l) and were considered to be chromosomally-resistant N gonorrhoeae (CMRNG). Decreased susceptibility to cefuroxime (MIC, greater than or equal to 0.5 mg/l) was detected in 79% of the CMRNG. The CMRNG were also more often prototrophic and of serogroup IB than the remaining non-PPNG. The correlation coefficient for resistance to penicillin and cefuroxime was high, 0.79. Transformation experiments with both genetically-defined strains and transformants obtained using DNA from clinical isolates, showed that increased resistance to cephalosporins was acquired in three steps in close association with penicillin. We think this suggests that the loci controlling resistance to the cephalosporins are identical or closely linked to those controlling penicillin resistance.

Separation of Pneumocystis carinii from the lung of the steroid-suppressed rat.

Pneumocystis carinii pneumonia was induced in rats by chronic corticosteroid immunosuppression. The parasite was separated from virtually all contaminating host cells using the technique of unit gravity sedimentation. Cellular contamination was routinely below 0.02%. The same technique allowed partial separation of the cyst from the trophozoite stage.

Pathogenesis of septicaemia.

This review concentrates on two aspects of the pathogenesis of septicaemia. Firstly, those factors that enable bacteria to resist or avoid host defence mechanisms, including opsonization, phagocytosis, intracellular killing and terminal complement lysis of bacterial cell walls, are described. Secondly, the complication of septicaemia by septic shock with reference to the role played by lipopolysaccharide and tumour necrosis factor is reviewed, as are the possible implications for therapy.

The production and characterisation of rabbit antiserum and murine monoclonal antibodies to Haemophilus ducreyi.

Rabbit antiserum and murine monoclonal antibodies were raised against a strain of Haemophilus ducreyi. The antiserum gave high immunofluorescence titres and strong dot blot reactions with all H. ducreyi strains tested and the only cross reaction was with Bordetella pertussis. Three monoclonal antibodies, all of isotype IgG2a, also gave high immunofluorescence titres with H. ducreyi but did not cross react with any other species tested. Immunoblotting showed the monoclonal antibodies to react with a single polypeptide band of mol. wt 29,000 in the outer-membrane fraction of H. ducreyi. These antibodies have potential for use as diagnostic reagents and for investigating the pathogenicity of H. ducreyi.

A comparison of specificity and biological activity of polyclonal and monoclonal antibodies raised against Salmonella minnesota R595 lipopolysaccharide.

Murine monoclonal antibodies (MAbs) and immune rabbit serum were raised against the rough mutant of Salmonella minnesota strain R595. These antibodies were tested for their ability to inhibit LPS-induced B-cell mitogenicity and neutralise LPS toxicity in chick embryos. Immune rabbit serum inhibited both mitogenicity and LPS lethality. None of the MAbs or a cocktail of antibodies were able to neutralise LPS lethality in chick embryos. However, they were able to inhibit mitogenicity by varying degrees.

Changes in penicillinase-producing Neisseria gonorrhoeae isolated in London.

To investigate changes in the gonococcal population over time, the plasmid content, serotype and auxotype have been determined for strains of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolated from patients attending the Praed Street Clinic for Sexually Transmitted Diseases between 1978 and 1987. Three distinct changes have been identified: (i) in the main plasmid type, from PPNG with the 3.2-Mda plasmid before 1982 to strains with the 4.4-Mda plasmid between 1982 and 1985; (ii) the decline during this time of strains also carrying the 24.5-Mda conjugal plasmid; (iii) the re-emergence of PPNG with the 3.2-Mda plasmid in 1986. The three typing methods used have identified eight clusters of strains which have been prevalent in different years between 1978 and 1987.