A comparative genetic analysis of 42 clinical Klebsiella pneumoniae isolates, resistant to two or more antibiotics belonging to the broad-spectrum ␤-lactam group, sourced from Sydney, Australia, and three South American countries is presented. The study focuses on the genetic contexts of class 1 integrons, mobilizable genetic elements best known for their role in the rapid evolution of antibiotic resistance among Gram-negative pathogens. It was found that the class 1 integrons in this cohort were located in a number of different genetic contexts with clear regional differences. In Sydney, IS26-associated Tn21-like transposons on IncL/M plasmids contribute greatly to the dispersal of integron-associated multiple-drug-resistant (MDR) loci. In contrast, in the South American countries, Tn1696-like transposons on an IncA/C plasmid(s) appeared to be disseminating a characteristic MDR region. A range of mobile genetic elements is clearly being recruited by clinically important mobile class 1 integrons, and these elements appear to be becoming more common with time. This in turn is driving the evolution of complex and laterally mobile MDR units and may further complicate antibiotic therapy.

A class 2 integron was found in an Escherichia coli isolate mediating a urinary tract infection. Unlike other class 2 integrons from pathogens, the encoded IntI2 protein was functional. The integron possessed a dfrA14 cassette, and a second novel cassette in which a lipoprotein signal peptidase gene is predicted.

dEleven clinical class 1 integron-containing Pseudomonas aeruginosa isolates from Australia and Uruguay were investigated for the genomic locations of these elements. Several novel class 1 integrons/transposons were found in at least four distinct locations in the chromosome, including genomic islands. These elements seem to be undergoing successful dispersal by lateral gene transfer since integrons were identified across several lineages and more than one clonal line.

SYNOPSISThe sensitivity of Haemophilus influenzae to penicillins in vitro, determined either by serial antibiotic dilution in broth or by the disc method on agar, is apparently profoundly influenced by inoculum size if the results are read by macroscopic inspection. Microscopic inspection of the growth, however, reveals that the turbidity in heavily inoculated broth containing concentrations higher than the minimal inhibitory concentration is the product of L forms which have failed to succumb to osmotic lysis. Similarly, minute colonies appearing in the 'inhibition zone' of disc tests are composed of L forms. In both broth and agar tests reduction of the osmolality of the medium from 340 to 144 mOsm per kg failed to bring about lysis of organisms exposed either to ampicillin or amoxycillin. The significance of this remarkable osmotic stability of haemophilus L forms is discussed in relation both to testing of sensitivity of this organism to penicillins and to persistence of chronic haemophilus infections of the lower respiratory tract.It is generally accepted that inoculum size must be carefully regulated when testing the sensitivity of a bacterial species to an antibacterial drug, since too large an inoculum may give rise to apparent resistance of an organism which is in fact fully sensitive. The importance of the 'inoculum effect', however, varies in relation to different drugs, being minimal in the testing of the sensitivity of non-penicillinaseproducing organisms to penicillin. This is illustrated, for example, by the demonstration by Rolinson and Stevens (1961) that the minimal inhibitory concentration (MIC) of ampicillin for Salmonella typhi varied only between 0 6 ,ug per ml and 0 25 ,ug per ml when the inoculum size was varied between 1/10 and 1/100 000 of an overnight broth culture. In contrast, a similar variation of inoculum size caused a tenfold change in the MIC of tetracycline.In the course of extensive testing of the sensitivity of strains of Haemophilus influenzae to ampicillin and, more recently, amoxycillin, we have become aware that apparent resistance can readily result from the use of an inoculum only moderately in excess of that usually regarded as appropriate for general sensitivity testing, and microscopic inspection of the organisms surviving in drug concentrations higher than the MIC has shown them to be

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