Penicillinase Plasmid Research Articles

Evidence for transfer of CMY-2 AmpC beta-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans.

Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like beta-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans.

Antimicrobial susceptibilities and plasmid patterns of Neisseria gonorrhoeae in Bénin.

This study describes antimicrobial susceptibility patterns of Neisseria gonorrhoeae isolates obtained from female sex workers in Cotonou, Bénin. All isolates were susceptible to spectinomycin, ceftriaxone and ciprofloxacin, and susceptible to moderately susceptible to kanamycin; 9.8% of isolates were resistant to thiamphenicol; 9%, 87.5% and 3.5% were susceptible, moderately susceptible, resistant to trimethoprim-sulfamethoxazole, respectively; 94.4% and 99.3% were resistant to penicillin and tetracycline, respectively. All isolates with a minimal inhibitory concentration of tetracycline of >8 mg/l carried the 'American type' tetM plasmid; 94% and 6% of penicillinase-producing isolates possessed a 3.2 MDa and a 4.4MDa beta-lactamase plasmid, respectively. Surveillance of antimicrobial susceptibility of N. gonorrhoeae isolates to currently used drugs in Africa should become part of sexually transmitted diseases (STDs) control programmes.

Antimicrobial Chemotherapy in the Control of Surgical Infectious Complications

In spite of the progress in surgical technique and antibiotic prophylaxis, postoperative infection still accounts for both the commonest surgical complication and one of the most frequent nosocomial infections, also causing an increase in duration and costs of hospital stay. The choice of treatment for post-surgical infections requires an understanding of the usual infecting flora, available antimicrobial agents, and susceptibility patterns. The most common organisms in simple wound infection are Gram-positive cocci and mainly Staphylococcus aureus. Staphylococcus epidermidis and S. aureus (quite often methicillin-resistant strains) are the organisms which predominate in the infectious complications following clean surgical procedures with implantation of vascular grafts or prosthetic devices. Mixed aerobic and anaerobic flora are mainly responsible for cases of intra-abdominal and intra-pelvic postoperative infections: the most common aerobes are Enterobacteriaceae (Escherichia coli, Proteus spp., and others) and enterococci, and among anaerobes Bacteroides fragilis group prevails. Adequate drainage and surgical control of the source of infection, when needed, and adjunctive effective antimicrobial therapy are important factors in successful treatment of postoperative infections. Semisynthetic penicillinase-resistant penicillins and glycopeptides (vancomycin and teicoplanin) are the drugs of choice for the treatment of infections caused by penicillin-resistant methicillin-sensitive, and methicillin-resistant, respectively, S. aureus and S. epidermidis. For the treatment of intra-abdominal and intra-pelvic infections, animal and human studies support the recommendation that treatment should be directed against both Gram-negative enteric and anaerobic bacteria. Combinations of aminoglycosides with clindamycin or metronidazole have been widely used with great success; however adverse reactions such as nephrotoxicity and ototoxicity have been a problem in some patients. In recent years monotherapy with either a carbapenem (imipenem/cilastatin or meropenem) or a penicillin/β-lactamase inhibitor combination has been proposed. Among these combination antimicrobials, piperacillin combined with tazobactam is a very well designed formulation. Indeed, piperacillin is active against a broad range of Gram-negative and Grampositive pathogens, and tazobactam is a potent β-lactamase inhibitor which acts on a variety of clinically important plasmid and chromosomal beta-lactamases. This combination seems particularly attractive for the treatment of mixed polymicrobial anaerobic-aerobic infections such as intra-abdominal and intra-pelvic postoperative infections.

Subtyping of high-level plasmid-mediated tetracycline resistant Neisseria gonorrhoeae isolated in Scotland between 1992 and 1998.

Tetracycline resistant Neisseria gonorrhoeae (TRNG) contain a 25.2 MDa TetM plasmid encoding a 68 KDa cytoplasmic protein which confers high-level tetracycline resistance. The aim of this study was to subtype all TRNG isolated in Scotland between 1992 and 1998. Subtyping was performed by a polymerase chain reaction (PCR) assay which characterizes the TetM plasmid as either the Dutch variant (443 base pair product) or the American variant (777 base pair product). Of the 78 TRNG isolates, 35 were the American variant and 43 were the Dutch variant. TRNG were distributed amongst 30 serovar/auxotype classes, the most common being 1A6/NR (11.5%), 1A6/P (14.1%) and 1B4/NR (14.1%). The country where infection was acquired was known for 36 of the 46 TRNG strains isolated between 1996 and 1998. All infections acquired in Asia and South America were the Dutch variant whereas all infections acquired in Africa were the American variant. A penicillinase plasmid was present in 66% (23/35) of the American variant TRNG compared with 51% (22/43) of the Dutch variant: the 3.2 MDa penicillinase plasmid was found in 87% of the American variant TRNG whereas the 4.4 MDa penicillinase plasmid was found in 68% of the Dutch variant TRNG. We conclude that subtyping of TRNG by PCR is a useful tool in studying the epidemiology of gonococcal infection due to plasmid-mediated resistant isolates.

Susceptibility of gonococci isolated in London to therapeutic antibiotics: establishment of a London surveillance programme. London Gonococcal Working Group.

OBJECTIVES: To establish the in vitro susceptibility of gonococci isolated in the London area to antibiotics in current therapeutic use and to establish a sentinel surveillance system for monitoring trends...

Incidence, epidemiology and evolution of reduced susceptibility to ciprofloxacin in Neisseria gonorrhoeae in Korea

OBJECTIVE: To verify the decrease of susceptibility to ciprofloxacin in Neisseria gonorrhoeae, determine the size of the recently reported new beta-lactamase plasmid and explain the high prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG). METHODS: Gonococci were isolated from prostitutes in Korea. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. Plasmid was isolated by an alkaline lysis method. Patterns of Nhel-digested genomic DNA were compared after pulsed-field gel electrophoresis (PFGE). RESULTS: The minimum inhibitory concentration of ciprofloxacin for 50% of the isolates rose from 0.015 mg/L in 1993 to 0.12 mg/L in 1996. The proportion of PPNG remained at 70% or over during the 5-year period. The size of a novel beta-lactamase plasmid, first reported in 1994, was determined to be approximately 3.2 MDa, and 48% of the PPNG isolates contained it. Twelve of 50 isolates had the same PFGE pattern and nine others another pattern. CONCLUSION: The rapid decrease of fluoroquinolone-susceptible gonococci suggests that in the near future the drug may become less useful for gonorrhea treatment. The new 3.2-MDa plasmid may have been introduced as a result of the recent increase in overseas travel. The PFGE pattern suggests that high prevalence of PPNG may be due to dissemination of a few resistant clones among the high-risk groups.

Antimicrobial resistance of Neisseria gonorrhoeae in Liberia

The prevalence and molecular characteristics of penicillinase-producing Neisseria gonorrhoeae (PPNG) and tetracycline-resistant N. gonorrhoeae (TRNG) were determined in 10 clinics in Monrovia, Liberia, to assess the likely effectiveness of the current standard treatment with penicillin or tetracycline. One hundred gonococcal strains were isolated from 146 urethral swabs and 261 cervical swabs and screened for susceptibility to ceftriaxone, penicillin, spectinomycin and tetracycline by the disk diffusion method; 83% were resistant to penicillin and 63% to tetracycline. Twenty-one strains from 18 men and 3 women with uncomplicated gonorrhoea were subjected to more detailed characterization. These 21 strains belonged to 5 auxotype/serovar classes; 86% were PPNG/TRNG. Three PPNG harboured the 4·4 MDa penicillinase plasmid and 16 the 3·2 MDa plasmid. All TRNG harboured the 25·2 MDa plasmid and their MICs for tetracycline were >32 mg/L. They gave a PCR product which, according to its restriction pattern, corresponded to the American type tetM gene. By the agar dilution method, all strains exhibited intermediate resistance to sulphamethoxazole-trimethoprim (19:1) (co-trimoxazole) with MICs of 8–32 mg/L. All strains were susceptible to spectinomycin and ciprofloxacin. The MICs for gentamicin were 4–8 mg/L. The use of effective and affordable antimicrobial chemotherapy with either 500 mg ciprofloxacin or a single dose of gentamicin is discussed, with consideration of molecular biological, pharmacological and public health aspects.

Antimicrobial susceptibility and plasmid profile of Neisseria gonorrhoeae in India (New Delhi).

OBJECTIVES: To determine the antibiotic susceptibility and plasmid profile of all Neisseria gonorrhoeae strains (PPNG and non-PPNG) isolated from May 1995 to March 1996 in Lok Nayak Hospital, New Delhi,...

Antimicrobial susceptibilities of strains of Neisseria gonorrhoeae in Bangkok, Thailand: 1994-1995.

Failure of uncomplicated gonococcal infections acquired in the Far East to respond to doses of ciprofloxacin and ofloxacin recommended by the Centers for Disease Control and Prevention have been identified in Australia, the United Kingdom, and the United States. In the Republic of the Philippines, 54.3% of strains exhibited decreased susceptibility to fluoroquinolones; 12% of strains were resistant to ciprofloxacin. This study was undertaken to compare the antimicrobial susceptibilities of gonococcal isolates in Bangkok, Thailand, with those in the Republic of the Philippines. To determine the frequency and diversity of antimicrobial resistance, particularly to fluoroquinolones, in gonococcal strains in Bangkok, Thailand. Strains of Neisseria gonorrhoeae isolated from 101 patients with uncomplicated gonorrhea in Bangkok, Thailand, in July, 1994 (46 strains) and November, 1994 to July, 1995 (55 strains), were characterized by auxotype/serovar class, antimicrobial susceptibilities, and plasmid profile. Susceptibilities were determined to penicillin G, tetracycline, ceftriaxone, cefixime, cefoxitin, ciprofloxacin, ofloxacin, norfloxacin, erythromycin, kanamycin, and thiamphenicol. Of 101 strains, 89.1% (90/101) were resistant to penicillin or tetracycline. Plasmid-mediated resistance to penicillin or tetracycline was identified in 33.7% (34/101) of the isolates: penicillinase-producing Neisseria gonorrhoeae (17.8%; 18/101), tetracycline-resistant Neisseria gonorrhoeae (7.9%; 8/101), and penicillinase-producing/tetracycline-resistant Neisseria gonorrhoeae (7.9%; 8/101). Most penicillinase-producing strains (96.2%; 25/26) possessed the 4.4-megadalton (Md) beta-lactamase plasmid; one strain possessed the 3.2-Md beta-lactamase plasmid. Chromosomally mediated resistance to penicillin and tetracycline was exhibited by 51.5% (52/101) of strains, and 4.0% (4/101) were tetracycline resistant. All strains were susceptible to spectinomycin. Of 21.8% (22/101) strains exhibiting decreased susceptibility to ciprofloxacin (minimal inhibitory concentration [MIC] > or = 0.125 microgram/ml), one strain (ciprofloxacin MIC, 0.5 microgram/ml; ciprofloxacin inhibition zone diameter of 23 mm) had MICs of 2.0 and 8.0 micrograms/ml for ofloxacin and norfloxacin, respectively, indicating resistance to these agents. Decreased susceptibility to ciprofloxacin was identified in strains with chromosomally mediated resistance to penicillin or tetracycline and in penicillinase-producing strains. In Bangkok, Thailand, gonococcal isolates exhibit resistance to penicillin, tetracycline, kanamycin, and thiamphenicol. Decreased susceptibility to fluoroquinolones is emerging in a variety of strains of N. gonorrhoeae. Thus, all gonococcal infections should be treated with antimicrobial therapies known to be active against all gonococcal strains to reduce the spread of strains exhibiting decreased susceptibilities to fluoroquinolones.

Molecular epidemiology, in 1994, of Neisseria gonorrhoeae in Manila and Cebu City, Republic of the Philippines.

Failure of gonococcal infections to respond to 500 mg of ciprofloxacin or 400 mg of ofloxacin has been reported from Australia, the United Kingdom, and the United States. Recently, high rates of decreased susceptibility to the fluoroquinolones have been detected in penicillinase-producing Neisseria gonorrhoeae in the Republic of the Philippines. To assess the diversity of antimicrobial-resistant gonococcal strains isolated from female sex workers in Manila and Cebu City in the Republic of the Philippines in 1994. Isolates of N. gonorrhoeae isolated from 92 female sex workers in Manila (n = 28) and Cebu City (n = 64), respectively, were characterized by plasmid profile, auxotype/serovar class, and antimicrobial susceptibility profile. Plasmid-mediated resistance to penicillin or tetracycline was identified in 79.3% (73/92) of the isolates: penicillinase-producing N. gonorrhoeae (65/92; 70.7%), tetracycline-resistant N. gonorrhoeae (6/92; 6.5%), and penicillinase-producing/tetracycline-resistant N. gonorrhoeae (1/92; 1.1%). A beta-lactamase plasmid of 3.9 megadaltons was discovered. Of 54.3% (50/92) of strains resistant to nalidixic acid, 84% (42/50) of strains had minimum inhibitory concentrations of > or = 0.125 microgram/ml ciprofloxacin; penicillinase-producing N. gonorrhoeae (possessing the 3.05-, 3.2-, 3.9-, and 4.4-megadalton beta-lactamase plasmids, respectively) accounted for 68% (34/50) of these strains. In the Republic of the Philippines, gonococcal isolates resistant to penicillin or tetracycline accounted for 85.9% (79/92) of the isolates examined and included strains exhibiting resistance to fluoroquinolones. All gonococcal infections should be treated with antimicrobial therapies known to be active against all gonococcal strains to reduce the spread of strains exhibiting decreased susceptibilities to fluoroquinolones.

Antibiotic susceptibility patterns and plasmid profiles of penicillinase-producing Neisseria gonorrhoeae strains in Durban, South Africa, 1990-1993.

The appearance of strains of Neisseria gonorrhoeae resistant, both chromosomally and plasmid-mediated, to penicillin and other antibiotics makes this versatile pathogen difficult to treat. There is, therefore, a need for surveillance of N. gonorrhoeae strains to determine the efficacy of current therapeutic measures. To survey the antibiotic susceptibilities and plasmid profiles of penicillinase-producing N. gonorrhoeae strains isolated over a 4-year period. Penicillinase-producing N. gonorrhoeae strains were detected by the chromogenic cephalosporin test. Minimum inhibitory concentrations to penicillin G, tetracycline, ceftriaxone, and ciprofloxacin were determined using the E-test. Plasmid DNA was obtained by the alkaline lysis method and profiles generated. Penicillinase-producing N. gonorrhoeae strains increased from 16.4% to 19.0% in the period from 1990 through 1993. Although all strains were resistant to penicillin, strains were susceptible to varying levels of ciprofloxacin, ceftriaxone, and even tetracycline. All penicillinase-producing N. gonorrhoeae strains possessed the 2.6-megadalton cryptic plasmid, and in addition 87.7% contained the 24.5-megadalton conjugative plasmid. Of the six known gonococcal beta-lactamase plasmids, the 4.4-megadalton Asian and 3.2-megadalton African plasmids were predominant. The most prevalent plasmid profile contained the 2.6-megadalton cryptic, 24.5-megadalton conjugative, and 4.4-megadalton Asian plasmids. To ensure effective treatment of gonorrhea, continued surveillance of the antimicrobial susceptibilities and plasmid profiles of penicillinase-producing N. gonorrhoeae strains is necessary.

Increased incidence of beta-lactamase-plasmid negative, high level methicillin-resistant Staphylococcus aureus (MRSA).

Recently isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) have high levels of resistance to the agent and produce beta-lactamase less frequently than methicillin-susceptible strains (MSSA). This phenomenon has been observed in Japan. The majority of MSSA strains (26 of 30) isolated in 1992 produced beta-lactamase while only 18 of 31 and 25 of 67 MRSA strains, isolated in 1991 and in 1992 respectively, produced the enzyme. We analyzed the beta-lactamase gene (blaZ) in 30 strains each of MRSA and MSSA isolated from our hospital using the polymerase chain reaction. We found that 28 MSSA strains but only 14 MRSA strains possessed the gene. In addition, the present study indicates that many recently isolated MRSA strains, which lacked blaZ gene and were negative for beta-lactamase, consistently produced coagulase of type II (coagulase typing is a marker of epidemiology for Staphylococcus aureus). These beta-lactamase negative, type II coagulase producing and highly methicillin-resistant MRSA strains are considered to have an increased ability to successfully colonize individuals and an increased epidemic potential, probably because of the saving of the energy otherwise expended in beta-lactamase production and of the loss of beta-lactamase plasmid coding the mecA gene repressor, resulting in constitutive PBP2' production. These factors may contribute to the wide spread of these strains of MRSA in a nosocomial environment.

Characterization of the plasmidic beta-lactamase CMY-2, which is responsible for cephamycin resistance.

The phenotype of Klebsiella pneumoniae HEL-1 indicates a plasmidic cephamycinase gene (blaCMY-2). Its sequence shows one open reading frame coding for a protein of 381 amino acids. CMY-2 is classified as class C beta-lactamase that is closely related to the plasmidic enzymes BIL-1 and LAT-1 and the chromosomal AmpC of Citrobacter freundii. The blaCMY-2 gene possibly was translocated onto a plasmid of C. freundii which spread to K. pneumoniae.

Nasopharyngeal carriage of Staphylococcus aureus and carriage of tetracycline-resistant strains associated with HIV-seropositivity.

The aim of this prospective study was to investigate the relationship between carriage of antibiotic-resistant Staphylococcus aureus and infection with the human immunodeficiency virus (HIV). A total of 554 pernasal swabs was taken during a six-month period from 554 adult patients attending three outpatient clinics and from inpatients from a hospital in Nairobi, Kenya. Overall, 121 swabs (22%) yielded Staphylococcus aureus, there being significantly higher carriage in HIV-positive patients (71/264, 27%) than in HIV-negative patients (50/290, 17%); p = 0.008. Antimicrobial resistance rates were determined for 110 isolates and were high for penicillin (91%) and tetracycline (72%) and low for erythromycin (8%), methicillin (3%), gentamicin (5%) and chloramphenicol (0%). Genetic analysis showed plasmids in the range of 24-42 MDa to be associated with beta-lactamase production and plasmids in the range of 3-5 MDa to be associated with resistance to tetracycline, erythromycin and trimethoprim. All nine erythromycin-resistant strains were from HIV-positive patients (p = 0.02). There was a significant association of tetracycline resistance with HIV seropositivity (p = 0.002). The association of HIV seropositivity with Staphylococcus aureus carriage and carriage of antibiotic-resistant strains against the background of the HIV epidemic are of relevance in individual patient care and raise concern for public health.

Diversity of isolates of penicillinase-producing Neisseria gonorrhoeae (PPNG) in Honolulu, Hawaii: 1982-1991.

Gonococcal infections caused by penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates have increased in geographic distribution and prevalence. It was postulated that PPNG strains would become endemic in Honolulu and that, in turn, this city would serve as a reservoir for the introduction of PPNG strains into the continental United States. To assess the role of Honolulu as a reservoir for PPNG strains by assessing the diversity and persistence of PPNG strains between 1982 and 1991. A total of 432 PPNG strains were characterized by auxotype/serovar (A/S) class and plasmid content, and their distribution during the 10-year period was studied. Of 432 isolates, 373 (86.4%) possessed a 4.4-Mdal beta-lactamase plasmid; 39 (9.0%) possessed a 3.2-Mdal beta-lactamase plasmid; and 20 (4.6%) possessed a 3.05-Mdal beta-lactamase plasmid. A total of 53 A/S classes were identified. Asian, African, and Toronto PPNG strains belonged to 49 (92.5%), 15 (28.3%), and 11 (20.7%) A/S classes, respectively. Though all Toronto PPNG strains possessed a 24.5-Mdal conjugative plasmid, these plasmids could not be transferred by conjugation. Although some apparent microepidemics of PPNG strains were identified, most strains were isolated sporadically. A large number of different strains have been associated with PPNG infections in Honolulu, but there was no evidence that any strain persisted endemically during the study period. These observations have important implications for the design and assessment of community gonorrhea control strategies.

Cloning and expression of the penicillinase from a borderline methicillin-susceptible Staphylococcus aureus strain in Escherichia coli.

The blaZ gene contained in a single 17.2-kb beta-lactamase plasmid from a borderline methicillin-susceptible Staphylococcus aureus strain (a53) has been cloned in Escherichia coli. A Bluescript II derivative in which the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene was used as a multi-copy vector. One ampicillin-resistant colony was detected among 31 chloramphenicol-resistant transformants selected. This E. coli clone harbored a recombinant plasmid (pAH12) containing two different staphylococcal HindIII inserts (7.0 and 5.3 kb), of which only the former hybridized with a blaZ probe. The clone showed an ampicillin MIC of > 1024 micrograms ml-1, independently of the inoculum size used, and produced large amounts of beta-lactamase, which hydrolyzed nitrocefin and penicillin G but not methicillin of the beta-lactamase substrate, padac. In contrast, S. aureus a53 hydrolyzed all four substrates. The fact that high levels of staphylococcal penicillinase are unable to cause methicillin hydrolysis confirms that penicillinase hyperproduction is unlikely to be the true mechanism responsible for the borderline phenotype. These results also suggest that the two different beta-lactamases (penicillinase and methicillinase) associated with borderline S. aureus strains have a different genetic origin.

Antimicrobial susceptibilities of Neisseria gonorrhoeae strains representing five distinct resistance phenotypes.

The susceptibilities of 109 strains of Neisseria gonorrhoeae to penicillin G, tetracycline, amoxicillin-clavulanic acid, cefotetan, cefoxitin, ceftriaxone, ciprofloxacin, and fleroxacin were determined. The activities of cefmetazole, cefuroxime, cefixime, and ofloxacin were also determined against 62 of these strains. Strains represented penicillin-susceptible (Pen(s)) N. gonorrhoeae; penicillinase-producing N. gonorrhoeae (PPNG) possessing 2.9-, 3.05-, 3.2-, or 4.4-MDa beta-lactamase plasmids; strains with high-level, plasmid-mediated tetracycline resistance (TRNG); strains with plasmid-mediated resistance to penicillin and tetracycline; and strains with chromosomally mediated resistance to penicillin and tetracycline (CMRNG). Ceftriaxone, cefixime, and ciprofloxacin were the most active agents tested against all strains. Pen(s), TRNG, and PPNG strains possessing a 3.2-MDa beta-lactamase plasmid were more susceptible to amoxicillin-clavulanic acid, extended- and broad-spectrum cephalosporins, and quinolones than were either PPNG strains possessing a 2.9-, a 3.05-, or a 4.4-MDa beta-lactamase plasmid or CMRNG strains.

Extended-spectrum beta-lactamase in Proteus mirabilis.

Over a period of one month, five isolates of Proteus mirabilis resistant to extended-spectrum cephalosporins colonized or infected five patients of the same intensive care unit. The antibiotic resistance phenotype and the metabolic profiles of the five strains were identical. All five produced a plasmid-mediated extended-spectrum beta-lactamase closely related to TEM-3/CTX-1 according to its pI (6.3), to its substrate profile and to the hybridization of the corresponding gene with blaTEM-3 specific oligonucleotide probes. Ability to produce this enzyme transferred by conjugation to Escherichia coli along with aminoglycoside and sulphonamide resistance. These resistance markers were encoded by a plasmid, (pAP1) which was identical in all five isolates but which was different from the TEM-3 beta-lactamase plasmids found in other Enterobacteriaceae. Plasmid pAP1 differed by its smaller size (47 kb), its EcoRI restriction pattern, its resistance co-markers (AAC(6') and sulphonamide only) and its much lower transfer frequency (2 x 10(-8)). Furthermore this plasmid did not belong to any known Enterobacteriaceae incompatibility group.

Isolation and Characterization of Membrane-Associated Form of Penicillase Plasmid (pI524) DNA in Staphylococcus aureus

Our studies on the association of penicillinase plasmid (pI524) DNA with its host bacterial (Staphylococcus aureus) membrane revealed that the membrane-associated forms of this plasmid could be isolated from exponentially grown cells lysed on neutral sucrose gradient. Analysis of putative plasmid-membrane complexes isolated from the clear lysates on sucrose gradients indicated that approximately 23% of plasmid (pI524) DNA was stably associated with the bacterial cell membrane fractions. This suggested that one of the three or four copies of this plasmid per cell was complexed to the cellular membrane. Examination of the effect of various enzymes, e.g., ribonuclease and protease, as well as antibiotics (rifampicin and chloramphenicol), on complexing have shown the possible involvement of protein(s) rather than RNA in mediating the complexing of this plasmid to the cell membrane. The specificity of plasmid pI524 to its host cell membrane was observed in an experiment where R6k was included in binding assay.

Genes involved in the regulation of beta-lactamase production in enterococci and staphylococci

In enterococci, the structural gene for beta-lactamase (blaZ) is identical to blaZ from Staphylococcus aureus. However, in the enterococci studied to date, beta-lactamase is produced constitutively, whereas in staphylococci it is often inducible. Recent reports have revealed the presence of two adjacent genes upstream of the staphylococcal blaZ thought to be the antirepressor (blaR1) and repressor (blaI) genes. In the present study, beta-lactamase expression mutants of the staphylococcal beta-lactamase plasmid pI524 were generated by transposon mutagenesis with the transposon Tn917. Tn917 insertions upstream of blaZ in either blaR1 or blaI resulted in constitutive beta-lactamase production, indicating that the repressor function is lost with insertion of Tn917 into either gene. This finding supports the concept that the staphylococcal beta-lactamase regulatory genes are encoded on a polycistronic mRNA. The corresponding region upstream of the enterococcal blaZ from Enterococcus faecalis HH22 was sequenced and compared with the staphylococcal blaR1 sequence. The two sequences were identical for 893 nucleotides, and then the sequences diverged completely. Therefore, in strain HH22, only 51% of the putative antirepressor gene is present and the repressor gene is also absent. In conclusion, constitutive beta-lactamase production in HH22 appears to be due to a lack of the regulatory genes blaR1 and blaI which regulate expression of blaZ in staphylococci.