Antibiotic susceptibilities and resistance genes of Ureaplasma parvum isolated in South Africa.

OBJECTIVES: There is only limited information on the antimicrobial susceptibilities and resistance genes of Ureaplasma parvum in South Africa. This study was designed to detect and characterize resistance genes in U. parvum. METHODS: Fifteen U. parvum isolates were investigated employing the broth microdilution method (tetracycline, doxycycline, ofloxacin, erythromycin, azithromycin and josamycin). Gene analyses were performed on target regions of: tet(M); gyrA, gyrB, parC and parE; erm(A), erm(B), erm(C) and erm(E); msr(A), msr(B), msr(C) and msr(D); 23S rRNA operons; and L4 and L22 ribosomal proteins. RESULTS: Seven of the U. parvum isolates were fully susceptible to the antibiotics tested. Five strains exhibited resistance to tetracycline (MICs 16-256 mg/L), one strain was resistant to ofloxacin (MIC 128 mg/L) and four strains were resistant to macrolides (MICs 128 mg/L); two strains showed dual resistance to tetracycline and erythromycin. The five tetracycline-resistant strains were found to have mosaic tet(M) genes, with one strain containing different specific regions to those previously described. Mutations in the L22 ribosomal protein were seen in three strains that were resistant to erythromycin (two strains) and erythromycin + azithromycin (one strain). For a further strain that was resistant to erythromycin and azithromycin, possible mechanisms of resistance remained elusive. CONCLUSIONS: This is the first report of quinolone, erythromycin and azithromycin resistance development in U. parvum from South Africa. A point mutation in parC (Pro-57 → Leu) and two novel mutations in parE (Ile-73 → Thr and a methionine insertion at codon 86) were found in an ofloxacin-resistant strain. The study reinforces the adaptability of U. parvum to develop resistance and acquire, modify and maintain transposon-located resistance genes.

Prevalence of genital mycoplasmas, ureaplasmas and chlamydia in pregnancy.

The study was designed to determine the prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with pre-term labour or HIV status. For pre-term labour (2003), 199 women were monitored for pre-term delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year age group from 2003. In women aged > or = 21 years, co-colonisation was 13%, although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma urealyticum in 2003, to other dual/triple combinations in 2005. Overall, major trends from both collection periods were that the prevalence of U. urealyticum tended to be higher in women > or = 26 years, while the prevalence of Chlamydia trachomatis and M. hominis lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, U. urealyticum or C. trachomatis. The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long-term aetiologies requires further investigations, certainly in relation to syndromic management regimens that fail to reduce colonisation rates.

Prevalence of Pneumocystis jirovecii and Mycoplasma pneumoniae in Patients Presenting with Pneumonia at Hospitals in Port Elizabeth

The study was conducted to determine the prevalence of Pneumocystis jirovecii and Mycoplasma pneumoniae in patients presenting with community-acquired pneumonia; in order to improve treatment management programmes. Sputum specimens from 45 patients presenting with pneumonia/symptoms of pneumonia admitted to hospitals in the Port Elizabeth region were assessed. Details of patient's gender; age; HIV and Mycobacterium tuberculosis status were provided by the hospitals. PCRs were performed employing primers directed at the following genes: P. jirovecii for detection of mitochondrial large subunit ribosomal RNA (mtLSUrRNA) and for cotrimoxazole resistance mutation analysis dihrdropteroate synthase (DHPS) and dihydrofolate reductase (DHFR); M. pneumoniae for detection of P1 adhesin and 16SrRNA. Women were seen to be at high risk for community-acquired P. jirovecii colonisation. Overall; prevalence of P. jirovecii was 73(33/45 patients). P. jirovecii was mainly associated with HIV (28/30 P. jirovecii-positive patients for which clinical data were available) and co-colonisation with M. tuberculosis was observed in 10 HIV cases and one HIV-negative patient. DHPS and DHFR primers seriously lacked sensitivity and on six and four PCR products obtained; respectively; no resistanceassociated mutations were found. M. pneumoniae was detected in one patient. The high prevalence of P. jirovecii and presence of M. pneumoniae in cases of pneumonia investigated emphasises that in the absence of definitive diagnoses; it is crucial to monitor treatment responses carefully; especially when first line antibiotic preferences are a-lactams or cephalosporins

Dihydropteroate synthase and novel dihydrofolate reductase gene mutations in strains of Pneumocystis jirovecii from South Africa.

Dihydropteroate synthase (DHPS) gene mutations have raised concerns about emerging sulfonamide resistance in Pneumocystis jirovecii. DHPS and dihydrofolate reductase (DHFR) gene products were amplified in clinical specimens from South African patients. One of 53 DHPS genes sequenced contained the double mutation Thr55Ala Pro57Ser. DHFR gene mutations detected were Ala67Val and the new mutations Arg59Gly and C278T.

Pneumocystis pneumonia.

Pneumocystis jiroveci is a common cause of pneumonia in South African patients with AIDS. Sulphonamide resistance may become a problem in South Africa, as patients are treated with prophylactic co-trimoxazole when their CD4 counts fall below 200 cells/microliter. Failure of prophylaxis and treatment has been observed, possibly due to infection with sulphonamide-resistant strains. Sulphonamide resistance has been reported elsewhere, and is due to point mutations at codons 55 and 57 of the dihydropteroate synthase gene. Strain typing is useful for molecular epidemiological purposes.

Comparative activity of eighteen antimicrobial agents against anaerobic bacteria isolated in South Africa.

The in vitro activity of 18 antimicrobial agents was determined against 378 anaerobic bacteria isolated in Bloemfontein, South Africa, during 1996/97. Against the gram-positive isolates, MICs of penicillin and cefoxitin were >0.5 microg/ml and >16 microg/ml, respectively, for five and three strains of non-perfringens Clostridium spp. Seventeen Peptostreptococcus anaerobius strains were resistant to penicillin (MIC > or = 2 microg/ml). All gram-positive anaerobes tested except one Peptostreptococcus sp. and one Clostridium sp. were susceptible to dalfopristin-quinupristin (MICs < or = 1 microg/ml). The carbapenems exhibited excellent activity against the gram-positive isolates and were effective against most gram-negative anaerobes, with the exception of the fusobacteria. Only seven strains exhibited decreased susceptibility to trovafloxacin (MICs > 2 microg/ml). In mixed anaerobic/aerobic infections, carbapenems and the fourth-generation quinolone trovafloxacin were the agents most suitable for us as broad-spectrum monotherapy.

Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa.

This study investigated the prevalence of nim genes (proposed to encode a 5-nitroimidazole resistance product) in 64 anaerobic/facultative anaerobic bacteria. Employing universal nim gene primers, 458-bp amplified fragments were recorded as presumptive positives in 22/64 strains at an annealing temperature of 52 degrees C and 15/64 strains at 62 degrees C, of which seven were propionibacteria. DNA sequencing confirmed the presence of nimA genes in Propionibacterium spp. (five strains), Actinomyces odontolyticus (one strain), Prevotella bivia (one strain) and Clostridium bifermentans (one strain) and nimB genes from five strains of Bacteroides fragilis. nimA genes were predominant in propionibacteria indicating a potential nimA gene source in anaerobic environments.

In vitro activities of 15 antimicrobial agents against clinical isolates of South African enterococci.

The activities of a panel of currently available antibiotics and the investigational agents LY 333328, linezolid, CL 331,002, CL 329,998, moxifloxacin (BAY 12-8039), trovafloxacin, and quinupristin-dalfopristin against 274 clinical isolates of enterococci were determined. No vancomycin resistance or beta-lactamase production was observed. Except for 12 isolates (all non-Enterococcus faecalis) showing reduced susceptibility to quinupristin-dalfopristin (MIC, >/=4 microg/ml), the new agents exhibited promising in vitro antienterococcal activity.

Enterococcal endocarditis--a case treated with teicoplanin and amoxycillin.

The study aimed to determine the antibacterial therapy effective in the cure of endocarditis caused by Enterococcus faecalis resistant to clinically achievable levels of vancomycin. Isolation of the causative enterococcus had been achieved by direct inoculation of the resected valve into the culture medium in theatre. The patient was known to have had an aortic valve defect since childhood and had recently undergone splenectomy following trauma. Blood cultures were negative prior to valve replacement. A perivalvular abscess was noted at operation. In vitro minimal bactericidal results and serum activity were the basis of the postoperative choice of drugs. The minimal bactericidal level of teicoplanin was 250 micrograms/ml and that of amoxycillin 64 micrograms/ml. Neither is achievable with the advocated dosage. A combination of these two cell-wall-active agents successfully eliminated the infection. Acting at two different sites in the synthesis of the bacterial cell wall, teicoplanin and amoxycillin were found to be bactericidal in vitro at the trough levels of the antibiotics in the serum. The patient recovered fully.

Plasmid analysis of Neisseria gonorrhoeae isolates and dissemination of tetM genes in southern Africa 1993-1995.

One group (145 isolates) of Neisseria gonorrhoeae was collected from municipal clinics in Bloemfontein in 1994 and a second group (65 isolates) in 1995. Penicillin and tetracycline MICs were determined and plasmid analysis performed to monitor antimicrobial susceptibilities in conjunction with the occurrence of plasmids in these isolates. The prevalence of penicillin resistance caused by beta-lactamase plasmids remained constant at 9% during the study period. Three high-level tetracycline-resistant strains (MICs 16 mg/L), the first to be detected in South Africa, were isolated in 1994. Although there was a reduction in the percentage of isolates harbouring 24.5 MDa conjugative plasmids (from 79% in 1994 to 46% in 1995), this was partially counteracted by an increase in TetM-encoding conjugative plasmids (25.2 MDa) from 2% to 18.5%. The tetM genes of 13 isolates shown to exhibit high-level tetracycline resistance were characterized as the American type. The American-type tetracycline resistance plasmid was demonstrated in 11 isolates. Digestion with Bg/l showed that two isolates harboured tetM-encoding plasmids that differed from the American- and Dutch-type plasmids described previously: one isolate contained a plasmid that produced two fragments of different sizes from those of the American-type plasmid and the second isolate possessed an American/Dutch hybrid plasmid. Auxotyping/serotyping and random amplified polymorphic DNA analysis revealed a predominant tetracycline-resistant family (NR/IA-6, genomic group I) in Bloemfontein. As there is a high incidence of chlamydial infections in southern Africa requiring tetracycline therapy, selective pressures exist in the environment for the maintenance and rapid spread of high-level tetracycline-resistant N. gonorrhoeae. It is possible that tetM genes may have emanated from Botswana and/or Namibia to Bloemfontein. The establishment of high-level tetracycline-resistant N. gonorrhoeae in Bloemfontein was seen to be complex as a related group of strains was identified, plasmid dissemination was evident and two new TetM-encoding plasmids were demonstrated. The appearance of these TetM-encoding plasmids indicates either that the American- and Dutch-type plasmids are continuing to evolve or that tetM genes are being introduced into different families of 24.5 MDa conjugative plasmids.

Characterisation of penA and tetM resistance genes of Neisseria gonorrhoeae isolated in southern Africa--epidemiological monitoring and resistance development.

OBJECTIVE: To investigate penA and tetM resistance gene variation of Neisseria gonorrhoeae in order to define gene types for epidemiological monitoring and resistance development. DESIGN: Isolates of N. gonorrhoeae which were susceptible and resistant to penicillin and/or tetracycline were selected. Strains comprised South African isolates (22 from Bloemfontein, 13 from Transvaal, 20 from the Cape) and 15 Botswana and 4 Namibia isolates. The penA genes (2 kb) of all strains and tetM genes (765 bp) of 11 high-level tetracycline-resistant strains were amplified and restricted with HpaII. RESULTS AND CONCLUSIONS: Twelve different HpaII fingerprint patterns were obtained from the 74 isolates analysed for penicillin-binding protein (PBP) 2 gene (penA) alterations. Focusing on the transpeptidase domain, 25 isolates (3 whole gene patterns, minimal inhibitory concentrations (MICs) < or = 0.03-0.125 micrograms/ml) had restriction sites equivalent to those previously described for a susceptible strain. Of the remaining 9 PBP 2 gene groups, 25 strains fell into a designated group E. Penicillin/ penicillin + clavulanic acid MICs determined on these group E isolates gave a range of 0.125-2.0 micrograms/ml, although MICs against 4 strains were < or = 0.03 micrograms/ml. MICs of penicillin/penicillin + clavulanic acid for the 24 isolates that contained altered PBP 2 transpeptidase gene regions not designated group E were only < or = 0.03-0.125 micrograms/ml. The lack of a HpaII restriction site at nucleotide 1934 in the PBP 2 gene of group E strains was indicative of a small terminal region of N. cinerea DNA. This gene block, which was found in all the southern African areas studied, appears to predispose isolates to increased penicillin resistance. The 25.2 MDa conjugative plasmid carrying the tetM resistance determinant was readily demonstrated in 11 Botswana/Namibia isolates exhibiting high-level resistance to tetracycline (MICs > or = 16 micrograms/ml). The tetM gene was shown to be of the American type.

Relatedness among penicillin-binding protein 2b genes of Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae.

Penicillin-binding protein (PBP) 2b similarities among Streptococcus mitis, S. oralis, and S. pneumoniae using DNA fingerprinting and sequencing were investigated. The polymerase chain reaction (PCR) was performed on 41 penicillin-susceptible and -resistant clinical isolates of S. mitis and S. oralis using the susceptible S. pneumoniae R6 PBP 2b primers. PCR products were then analyzed using Hinf I and Sty I restriction enzymes. Of 41 S. mitis/S. oralis isolates studied 15 strains produced a PCR product of a similar size to that of S. pneumoniae R6. On fingerprinting these 15 strains, 11 different patterns were seen using Sty I restriction enzyme and 12 different patterns with Hinf I. The PBP 2b genes of the S. mitis and S. oralis isolates studied were found to be very heterogenous. The PBP 2b genes of two S. mitis isolates, MICs 0.5 and 2 micrograms/ml, were sequenced. These PBP 2b genes were found to possess a mosaic structure when compared to those of other S. pneumoniae and viridans streptococcal species. Analysis of these mosaic blocks indicates that both S. mitis strains contain areas that originated from S. pneumoniae as well as regions of unknown origin. PBP 2b sequence comparisons of a susceptible S. oralis with reported sequences of S. pneumoniae R6 and S. mitis NCTC 10712 revealed what appears at this stage to be nucleotide regions unique to S. oralis. A penicillin-resistant S. oralis strain contained a pneumococcal region of 272 bp that was flanked by S. oralis sequences. These specific S. oralis regions have been located in PBP 2b genes of penicillin-resistant S. oralis and S. pneumoniae isolates described from Europe and South Africa.

Antibiotic susceptibility of anaerobic bacteria isolated in Johannesburg.

In vitro susceptibilities of 198 anaerobic bacteria to seven antibiotics were evaluated by the agar dilution method. In addition to testing amoxycillin/clavulanic acid in a 2:1 ratio against the bacteria, the combination was also tested against 63 isolates using fixed concentrations of clavulanic acid and serial dilutions of amoxycillin. Penicillin and cefoxitin were not effective against beta-lactamase-producing Bacteroides isolates and only 50% of isolates were susceptible to the 2:1 amoxycillin/clavulanic acid combination. However, when varying concentrations of amoxycillin were used together with constant concentrations of clavulanic acid (4 micrograms/ml) only 9 of 55 amoxycillin-resistant Bacteroides were resistant to the combination. Two clostridia were found to produce beta-lactamases and as expected were resistant to penicillin. Of the non-beta-lactamase-producing clostridia 11% were resistant to penicillin and 5% resistant to cefoxitin. Imipenem was effective against the majority of anaerobes tested and only 5 Bacteroides isolates were resistant. All anaerobic strains were susceptible to chloramphenicol and only 6% of strains resistant to clindamycin. Eighty-five per cent and 51% of Bacteroides strains had minimum inhibitory concentrations within two dilutions of the breakpoints of chloramphenicol (16 micrograms/ml) and clindamycin (4 micrograms/ml) respectively. Three strains of Peptostreptococcus spp. were resistant to metronidazole.

In vitro antimicrobial susceptibility of viridans streptococci isolated from blood cultures.

The susceptibility of 211 viridans streptococci isolated from blood cultures to eight antimicrobial agents was determined. All the isolates were susceptible to cefotaxime, ceftriaxone, imipenem and vancomycin. Thirty eight percent of the isolates were resistant to penicillin (MICs greater than or equal to 0.25 micrograms/ml). Tetracycline resistance was found in 41% of the isolates and in 7% of these strains tetracycline resistance was combined with erythromycin resistance. Five Streptococcus mitis isolates exhibited increased (MIC 64 micrograms/ml and 128 micrograms/ml) or high-level (MIC greater than or equal to 500 micrograms/ml) resistance to gentamicin, kanamycin and tobramycin. Four of these isolates were also resistant to penicillin (MICs 16-32 micrograms/m). In vitro synergy was not demonstrated for combinations of penicillin and gentamicin against three Streptococcus mitis isolates with gentamicin MICs of 1000, 128 and 64 micrograms/ml. Results of this study indicate the importance of monitoring antibiotic resistance trends in viridans streptococci particularly with respect to penicillin and aminoglycoside resistance.

Intra- and inter-specific transformation of Streptococcus pneumoniae to penicillin resistance.

Transformation studies were carried out with a penicillin susceptible (MIC 0.006 mg/l) laboratory strain of Streptococcus pneumoniae as recipient. Donor DNA was prepared from two clinical isolates of S. pneumoniae, four isolates of S. mitior and five isolates of S. sanguis. DNA from both isolates of S. pneumoniae generated penicillin-resistant transformants (MICs 0.03-2.0 mg/l). In addition, one isolate each of S. mitior and S. sanguis transformed the recipient to increased penicillin resistance, with MICs of 0.125 mg/l. DNA from the S. sanguis strain generated a transformant which showed a reduction in the penicillin-binding affinity of penicillin-binding protein (PBP) 2B. DNA from a second S. mitior strain generated intermediately resistant (MIC 0.5 mg/l) transformants in a single transformation round. The PBP profiles of these transformants showed an apparent molecular size alteration of PBP 2B, corresponding to a PBP found in the donor strain, and PBP 1B was no longer detected. Isolates of S. pneumoniae may apparently develop penicillin resistance either intrinsically, by intragenetic transfer, or by transfer of penicillin resistance determinants from viridans streptococci.

Comparative in vitro activity of several antimicrobial agents against selected clinical isolates.

A comparison between the in vitro activities of ceftazidime, cefotaxime, ceftriaxone, ampicillin, piperacillin, gentamicin, tobramycin and netilmicin was performed on selected clinical isolates. The activity of the agents was assessed by means of minimum inhibitory concentration determinations, and time-kill studies. The third generation cephalosporins were active against all members of the Enterobacteriaceae excluding some Enterobacter species. Their activity against these bacteria was generally comparable and greater than that of piperacillin. Netilmicin was the most active of the aminoglycosides tested against members of the Enterobacteriaceae; however, aminoglycoside-resistant strains were encountered. Ceftazidime was the most active of the third generation cephalosporins against the non-fermenters (e.g. Pseudomonas and Acinetobacter spp), its inhibitory activity also being greater than that of piperacillin. Using a time-kill study, ceftazidime also demonstrated greater bactericidal activity than piperacillin against an isolate of Pseudomonas aeruginosa. The aminoglycoside demonstrating the greatest activity against the non-fermenters was tobramycin.

Penicillin-binding proteins of Streptococcus pneumoniae.

Penicillin-binding protein (PBP) patterns of penicillin-resistant laboratory-constructed transformants were compared with the PBP profiles of 26 clinical isolates of Streptococcus pneumoniae. For transformation studies DNA from a penicillin-resistant clinical isolate was used to transform a susceptible laboratory strain. Penicillin resistance was achieved in two transformation cycles. The frequency of transformation appeared to be dependent on the genetic status of the recipient used for the second transformation cross. Penicillin resistance was also attained in a single transformation round when time was allowed for full expression of random multiple transformations. PBP 2b was the first PBP to show an alteration in penicillin-binding affinity. This PBP was not easily detected in those transformants for which penicillin MICs exceeded 0.2 mg/l. The PBP profiles of the clinical isolates were complex. In addition to previously-described PBPs, new intermediate classes were demonstrated. No correlation between PBP profile and susceptibility was observed with clinical isolates except that PBP-2b exhibited molecular weight changes in moderately susceptible strains.

In vitro selection of bacteria resistant to LY146032, a new cyclic lipopeptide.

Isolates of Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, and coagulase-positive and -negative staphylococci were investigated for their abilities, in vitro, to develop resistance to LY146032. Exposure of the organisms to incremental concentrations of LY146032 resulted in MICs 8- to 32-fold higher than those for the original isolates. After three passages on antibiotic-free medium, the high MICs were maintained for the coagulase-negative staphylococci and pneumococci, with a twofold decrease observed for the enterococci and a fourfold decrease observed for Staphylococcus aureus. The frequency of spontaneous emergence of resistance was highest with S. pneumoniae (1.2 X 10(-6) at 16 times the original MIC) and lowest with S. aureus (7.0 X 10(-10) at 8 times the original MIC). For bacteria For bacteria surviving time-kill studies MICs were also higher than were those for the original isolates. Exposure to LY146032 in vitro selected for strains with decreased susceptibilities to the antimicrobial agent. However, the emergence of resistance in vivo is unpredictable and can be evaluated only after prolonged clinical use of the drug.