Osmotically stable L forms ofHaemophilus influenzaeand their significance in testing sensitivity to pencillins

Abstract

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

Ampicillin killing curve patterns for ampicillin-susceptible nontypeable Haemophilus influenzae strains by the agar dilution plate count method

“…For such strains, Gram-stained preparations of the apparent surface growth on AD plates having concentrations above the MIC showed that essentially all of the cells had morphologic features of cell wall-defective forms. These observations were similar to those described previously (4,24,30) and are discussed below in relationship to the killing curve patterns for the 20 strains examined in this study.…”

Effects of Various Test Media on the Activities of 21 Antimicrobial Agents against Haemophilus influenzae

“…Examples of such agents include amoxicillin-clavulanate, cefuroxime, azithromycin, erythromycin, and clarithromycin. For the two ␤-lactam examples, the situation is further complicated by the fact that strains for which the MICs are at the high end of the distribution may have non-␤-lactamase-mediated resistance due to altered penicillin-binding proteins or may be spheroplast-producing strains (2,3,11,12,14,19,22,23,24).…”

Lower Respiratory Tract Infections

“…Modulation of the Band T-cell response in rats has been shown to influence the ability of P2 to protect them from challenge with a homologous NTHi strain (110). The interstrain heterogeneity (143) and antigenic drift (74,75) of the organism and its ability to survive in the absence of P2 (17,27,171) may preclude the use of P2 as a vaccine candidate except as a carrier of other proteins inserted into its hypervariable regions (136a).…”