One hundred and thirty eight penicillinase producing Neisseria gonorrhoeae (PPNG) and 239 non-PPNG strains were characterised serologically using a panel of seven monoclonal antibodies directed against protein 1A and seven against protein 1B. An association between serovar and susceptibility to antimicrobial agents, auxotype, and plasmid content was observed. Serogroup WI strains were more sensitive to penicillin, ampicillin, tetracycline, erythromycin, cefoxitin, and cefuroxime. Sixty five (82%) of the 79 WI strains were typed as being serovar Aedgkih, and 47 (72%) of these strains required arginine, uracil, and hypoxanthine for growth (AUH-). Seventy one (44%) of 160 WII/WIII strains were serovar Bacejk, and 42 (59%) of these required proline, citrulline, and uracil for growth (PCU-) and were plasmid free. Serovars Bcgk, Beghjk, Bacjk, and Bajk were associated with resistance to antimicrobial agents. Analysis of PPNG isolates showed a new serovar, Af, which was associated with strains imported from Malaysia and Singapore that required proline and ornithine for growth (Pro-Orn-) and carried the 24.5 megadalton transfer plasmid, the 2.6 megadalton cryptic plasmid, and the 4.5 megadalton penicillinase producing plasmid. Other associations between serovar and geographical location were noted.
The plasmid profiles of 160 strains of Neisseria gonorrhoeae isolated in Peninsular Malaysia, comprising 80 penicillinase-producing (PPNG) and 80 non-penicillinase-producing (non-PPNG) isolates, were determined. The 80 PPNG isolates were divided into two plasmid groups. All of them harbored two common plasmid species, a 4.4 megadalton (Md) R plasmid previously associated with beta-lactamase production in PPNG strains from the Far East and a 2.6 Md multicopy plasmid of unknown function. In addition to these two plasmids, 60 (75%) PPNG isolates also carried a large 24.5 Md conjugative plasmid. In contrast, the 80 non-PPNG strains were divided into three plasmid groups. All of them possessed the 2.6 Md cryptic plasmid, and 35 (44%) isolates also harbored the 24.5 Md transfer plasmid. Besides these two plasmids, one non-PPNG isolate carried an additional 7.8 Md cryptic plasmid.
The incidence of infections due to beta-lactamase-producing Neisseria gonorrhoeae is increasing in many parts of the world. An epidemiologic survey of infections caused by beta-lactamase-producing strains of N. gonorrhoeae at the University Hospital, Kuala Lumpur, from February 1977 to December 1985 (106 months) showed that the incidence rose from 4.8% (two cases) in 1977 to 49.4% (39 cases) by the end of 1985. The highest incidence of gonococcal infections was found to be in the group aged 20-39 years; the male-to-female ratio was 1.55:1. The mean inhibitory concentrations of benzylpenicillin were 0.12 microgram/ml for non-beta-lactamase-producing strains and 16 micrograms/ml for isolates of N. gonorrhoeae that produce beta-lactamase.
The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys-Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K.
The World Health Organization Western Pacific Region Gonococcal Antimicrobial Surveillance Programme (WHO WPR GASP) is a multicentric long term programme of continuous surveillance of the antibiotic susceptibility of Neisseria gonorrhoeae. In 1997 the programme examined the susceptibility of 8,594 isolates of gonococci to various antimicrobials in 15 focal points. The trend toward increased antimicrobial resistance noted in earlier years continued. The proportion of quinolone resistant gonococci reported from most centres was either maintained or else increased. More than half of the isolates tested in China-Hong Kong, China, Japan, Korea, and the Philippines had altered quinolone susceptibility and increases in the number and percentage of quinolone resistant strains were noted in most, but not all, of the other centres. Resistance to the penicillins was again widespread, and chromosomally mediated resistance was a significant factor. Penicillinase-producing Niesseria gonorrhoeae (PPNG) were present in all centres. All isolates were sensitive to the third generation cephalosporins and only a very few isolates in China were spectinomycin resistant. High level tetracycline resistance was concentrated in a number of centres including Singapore, Malaysia, the Philippines and Vietnam. The proportion of tetracycline resistant Neiserria gonorrhoeae (TRNG) in most of the remaining centres was less than 10 per cent.