Molecular epidemiology and novel combinations of auxotype, serovar, and plasmid content in tetracycline-resistant Neisseria gonorrhoeae isolated in Canada

Between October 1987 and June 1989, 84 isolates of Neisseria gonorrhoeae carrying the TetM resistance determinant (TRNG) were received at the Laboratory Centre for Disease Control, Ottawa, from six Canadian provinces and were characterized into classes based on auxotype, serovar and plasmid content. One-fifth (17/84) of the TRNG were also penicillinase producing (PPNG). The PPNG-TRNG isolates comprised six classes based on auxotype, serovar, and plasmid content. Most (16/17) PPNG-TRNG carried 3.2-MDa beta-lactamase plasmids and the 25.2-MDa TetM-containing plasmid. We report, for the first time, the association of a 4.5-MDa beta-lactamase plasmid with the 25.2-MDa plasmid in a clinical TRNG isolate. Non-PPNG TRNG isolates comprised 11 classes based on auxotype, serovar, and plasmid content, including two previously unreported auxotype-serovar classes, P/IB-26 and P/IB-20.     

Intrinsic penicillin resistance in penicillinase-producing Neisseria gonorrhoeae strains

The minimal inhibitory concentrations (MICs) of ampicillin for fifty strains of beta-lactamase-producing Neisseria gonorrhoeae (PPNG) isolated in Japan ranged from 1.56 to 200 micrograms/ml, and all the strains harbored a 4.5 megadalton plasmid. These strains were classified into two groups: dicloxacillin-susceptible (28%) and -resistant group (72%). A linear correlation was found in the dicloxacillin-susceptible strains between their beta-lactamase activity and the susceptibility to ampicillin, but not in the dicloxacillin-resistant strains. This suggests that the high ampicillin resistance in PPNG is due not only to acquiring the beta-lactamase producing plasmid, but also to some intrinsic resistance of the strains. To investigate a cause of the high ampicillin resistance, the beta-lactamase-producing plasmid, pTMS1, was transferred by conjugation to a penicillin-susceptible gonococcal strain as well as to its isogenic multiply antibiotic-resistant transformants, and the susceptibility of the transconjugants to ampicillin was determined. Acquisition of pTMS1 by a penicillin-susceptible strain resulted in a 32-fold increase in resistance to ampicillin, whereas the increase was 128-fold for its isogenic strains which contain some chromosomal mutations. These results suggest that reduced permeability of the outer membrane to ampicillin underlies the high ampicillin resistance of PPNG.     

Plasmid profiles of Neisseria gonorrhoeae in Peninsular Malaysia

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.     

A molecular characterization of H. influenzae plasmid pVe445: a comparison with N. gonorrhoeae plasmids pNG10 and pNG18

Haemophilus influenzae plasmid pVe445 (4.6 MDa) has been compared with Neisseria gonorrhoeae plasmids pNG18 (3.3 MDa) and pNG10 (4.6 MDa) by heteroduplex and restriction enzyme analysis. This study demonstrates that pVe445 differs from pNG18 only that it carries an additional 1.3-MDa DNA fragment between coordinates 0.9 and 2.2. Heteroduplex studies demonstrated that pNG10 and pVe445 are indistinguishable whereas restriction endonuclease analysis indicated minor differences in the distribution of AluI sites.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

不同时期分离的淋病奈瑟菌对5种抗生素的敏感性研究

目的：研究杭州市不同时期分离的林病奈瑟菌对5种抗生素的敏感性。方法：用琼脂烯释法对门诊1998年7月～2001年10月分离的285株淋病奈瑟菌进行青霉素、四环素、壮观霉素、氧氟沙星及头孢曲松的最小抑菌浓度（MIC）测定,并就PPNG株和non-PPNG株菌的MIC值进行了比较。结果：青霉素、四环毒等5种抗生素MIC值2001年～1998年两者之间比较,青霉素、四环素等5种抗生素MIC值2001年与1998年两者之间比较,除壮观霉素没有变化外,其余都有显著变化,而氧氟沙星变化最大,PP-NG菌株与非PPNG株菌MIC值除氧氟沙星外均存在差异。结论：表明了杭州市淋病奈瑟菌5种抗生素耐药性变迁,以便为临床选择用药提供依据。     

Penicillinase-producing Neisseria gonorrhoeae in Canada.

The incidence of infection with penicillinase-producing Neisseria gonorrhoeae (PPNG) reported to the Laboratory Centre for Disease Control in Ottawa has steadily increased since the first Canadian isolation of such a strain in 1976. As of September 1980 a total of 66 PPNG isolates had been referred for biological and genetic characterization as well as for central documentation of the epidemiologic aspects of each case. Over 90% of the infections were firmly traced to patients or contacts who had acquired the infection abroad; this indicates that Canada does not, as yet, have an epidemic focus of PPNG infection. This report includes a synopsis of the biological characteristics of these isolates and an analysis of the results of primary antibiotic treatment that illustrates the importance of considering spectinomycin as the antibiotic of choice for PPNG infections.     

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.     

Temperature-dependent plasmid integration into and excision from the chromosome of Bacillus stearothermophilus

A transformant of Bacillus stearothermophilus carrying a recombinant plasmid, pLP11 (9.5 MDa), on which the penicillinase gene (penP) and kanamycin resistance gene (kan) were located was subjected to mutagenesis, and a mutant plasmid (9.5 MDa; penP kan), designated pTRA117, was obtained. A transformant of B. stearothermophilus carrying pTRA117 could grow at 63 degrees C in medium containing kanamycin, whereas a transformant carrying pLP11 could not. Although pTRA117 was detected as covalently closed circular (ccc) DNA when it was extracted from transformants cultured at 48 degrees C, it was integrated into the host chromosome when the culture temperature was shifted up to 63 degrees C. If the culture temperature was lowered to 48 degrees C from 63 degrees C, a new plasmid (10.7 MDa; penP kan), designated pTRZ117, could be detected as ccc DNA; the size of this plasmid suggested that it was pTRA117 plus a 1.2 MDa DNA fragment of the host chromosome, and this was confirmed by Southern hybridization. pTRZ90 (7.9 MDa; kan) was constructed from pTRZ117 by the deletion of a 2.8 MDa DNA fragment that contained penP. Fresh transformants of B. stearothermophilus that carried either pTRZ117 or pTRZ90 could grow at 65 degrees C.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid.

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.     

杭州市淋病奈瑟菌质粒酶切图谱分析研究

目的 :了解杭州市淋病奈瑟菌质粒携带及质粒谱型分布情况。方法 :采用碱裂解法对门诊 2 0 0 0年 1月～ 2 0 0 1年 10月分离的 2 0 7株淋病奈瑟菌进行了质粒抽提及质粒谱分型研究 ,并对菌株的青霉素耐药现象和耐药性质粒的关系进行探讨。结果 :2 0 7株淋病奈瑟菌中 194株 (93 .7% )检见质粒带 ,其中含一条质粒带的 112株 (5 4.11% ) ,含两条质粒带的 12株 (5 .8% ) ,含三条带的 70株 (3 3 .82 % ) ,尚有 13株(6.2 8% )未检测到质粒。以 E.coli V5 17细菌质粒作分子量标准 ,测得这些分子量分别为 2 .6、4.5、和 2 4.5Md。质粒谱型以 2 .6 4.5 2 4.5 Md(3 3 .82 % )多见。结论 :杭州地区质粒酶切图谱的分析研究有助于淋病的治疗和防治 ,这将对该地区淋病奈瑟菌的分子流行病学调查和淋病监控提供依据     

Involvement of Azopirillum brasilense plasmid DNA in the productio of indole acetic acid

Indole acetic acid (IAA) production in Azospirillum brasilense strain Sp245 is controlled by a 85 MDa plasmid naturally present in this bacterium. In the presence of L-tryptophan, anthranilic acid production and almost no IAA production occurs in a derivative strain harbouring a Tn5-Mob insertion in the 85 MDa plasmid. Agrobacterium tumefaciens strain GM19023, upon transfer of Tn5-Mob labelled 85 MDa plasmid of A. brasilense Sp245, gains the ability to produce anthranilic acid.     

Virulence studies of Salmonella enteritidis phage types

Salmonella enteritidis phage types (PTs) 8, 13a and 24 could be distinguished by their virulence for BALB/c mice, their plasmid content and plasmid fingerprint. Virulent strains expressed long-chain lipopolysaccharide and carried a 38 MDa plasmid indistinguishable from that in Salm. enteritidis PT 4. Avirulent strains were smooth but did not carry the 38 MDa plasmid. Possession of antibiotic resistance factors by some strains of Salm. enteritidis PT 24 did not contribute to the virulence of their host strains.     

Antimicrobial resistance markers as a monitoring index of gonorrhoea in Thailand

Multiplex PCR was applied to explore the antimicrobial-resistance profiles of 145 gonococci isolated from Bangrak Hospital, Thailand in 2007. All isolates were clearly identified for the plasmid-mediated resistant types of penicillin (Asia, Africa and Toronto) and tetracycline (American and Dutch). This method can also predict the decreased susceptibility to ciprofloxacin by detection of Ser-91 mutation. Prevalence rates of penicillinase-producing Neisseria gonorrhoeae (PPNG) and high-level tetracycline-resistance N. gonorrhoeae (TRNG) were shown to be high as 82.1% and 84.1%, respectively. Most PPNG carried the Africa-type (78.2%) while the American-type (61.8%) was harboured in most TRNG. Mono- and triple-resistance patterns were presented in 2.6% and 79.5% of male, 20.7% and 62.1% of men who have sex with men (MSM), 0% and 75.0% of female, and 10% and 70% of female sex workers (FSW). Additionally, the rate of the Dutch type was high in patients among the age of 35-44 years (57.1%) and female patients (43.8%). The changing types of plasmids have been noticed during the time period of study. The multi-resistance patterns of the gonococcal isolates can be used as an epidemiological index of gonorrhoea and human sexual behaviours. This information will support the management of individual patients as well as the public health surveillance.     

Genetic structure of natural populations of Nitrobacter in an aquatic environment

Nitrification has been essentially studied as a chemical process and the studies of the microbial population have been slightly worked out. This work constitutes a first attempt to study the diversity of Nitrobacter strains with the aim of working out the genetic structure of natural populations. The Nitrobacter population structure was compared between freshwater and sediments of the same lake. Nitrobacter isolates were identified and characterized by DNA/DNA hybridization, restriction pattern of rRNA genes, PCR/RFLP analysis of the ribosomal intergenic spacer and plasmid patterns. It was shown that this lake population was split into 3 subpopulations: a specific freshwater subpopulation, a specific sediment subpopulation and a nonspecific one. A 60 MDa plasmid was detected in 3,3% of freshwater isolates. A 37 MDa plasmid was detected in 59% of sediments isolates. This 37 MDa plasmid was not associated with a genomic group, but associated with a localization: the sediments. These results suggest that this 37 MDa plasmid could be implicated in the adaptation to sediment environment.     

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007 was examined. A plasmid of 88 MDa (pJS88) was identified in addition to the previously reported conjugally transferred plasmids of 32 (pKB32) and 4.8 MDa. Proteinase activity, reduced bacteriophage sensitivity, bacteriocin resistance, and conjugal transfer ability were encoded by pJS88. The ability to metabolize lactose (Lac+) was encoded by pKB32, and the 4.8-MDa plasmid was cryptic. When a strain containing both pKB32 and pJS88 was mated with a recipient deficient in host-mediated homologous recombination (Rec-), a plasmid of 40 MDa (pJS40) was observed in approximately 50% of the Lac+ transconjugants. DNA-DNA hybridization results indicated that pJS40 contained homology with both pKB32 and pJS88. These results indicated that pKB32 was conjugally transferred via conduction and suggested that pJS40 is a deletion derivative of a pKB32::pJS88 cointegrate. A Rec- strain containing pKB32 and pJS88 mediated Lac+ conjugal transfer, suggesting that the pKB32::pJS88 cointegrate could form via a rec-independent event. Resolution of the pKB32::pJS88 cointegrate was observed in both Rec- and Rec+ hosts. Cointegrate formation and resolution via rec-independent mechanisms suggest the involvement of a transposable element in the Tn3 family.     

Characterization of Neisseria gonorrhoeae strains isolated from patients with conjunctivitis

The conjunctivitis produced by Neisseria gonorrhoeae is the less frequently reported clinical form of gonococcal infection. We aim to phenotypically characterize N. gonorrhoeae isolated from conjunctivae sites. A total of six cases of this disease were notified in the Camagüey province, Cuba. All the strains isolated were penicillin-producing, showed the serogroup WI and exhibited the same antimicrobial susceptibility pattern and plasmid profile (2.6-3. 2-24.5). The results contribute to the characterization of N. gonorrhoeae strains circulating in our environment.