Molecular mechanisms of cephalosporin resistance in Gram-negative bacteria of the Enterobacteriaceae family]

Extended spectrum beta lactamase genes were detected by the PCR in 87.6% of 231 Enterobacteriaceae strains isolated in medical institutions of Moscow, St. Petersburg, Tomsk and Nazran that showed a decrease in their susceptibility to 3rd generation cephalosporins. Alone or in various combinations TEM type beta lactamases were detected in 43.3% of the isolates, 46.8 and 51.2% of the isolates produced SHV type and CTX type beta lactamases respectively. Combinations of 2 and 3 different determinants were detected in 40 and 14% of the isolates respectively. Production of class C beta lactamases was suspected in 28% of the isolates by their resistance to cefoxitin. The gene of ACT type beta lactamase was detected in 1 strain of Klebsiella pneumoniae and the gene of CMY type beta lactamase was detected in 1 strain of Proteus mirabilis. By the NCCLS 100% of the isolates was susceptible to meropenem, 14% was susceptible to cefotaxime, 64% was susceptible to cefepime, 81% was susceptible to cefoperazone/sulbactam, 47% was susceptible to gentamicin, 57% was susceptible to amikacin and 36% was susceptible to ciprofloxacin.     

Genetic control of tetracycline resistance in bacteria]

Modern data on prevalence, structural and functional organization of the tetracycline resistance determinants in bacteria are reviewed. The three mechanisms of the antibiotic resistance are the tetracycline efflux, the ribosomal protection and the antibiotic modification. The problems of evolution of tetracycline resistance genes are discussed.     

Expression in Escherichia coli of cryptic tetracycline resistance genes from Bacteroides R plasmids

The putative clindamycin resistance region of the Bacteroides fragilis R plasmid pBF4 was cloned in the vector R300B in Escherichia coli. This 3.8-kb EcoRI D fragment from pBF4 expressed noninducible tetracycline resistance in E. coli under aerobic but not anaerobic growth conditions. The fragment does not express tetracycline resistance in Bacteroides, a strict anaerobe. The separate tetracycline resistance transfer system in the Bacteroides host strain V479-1 has no homology to the cryptic determinant on pBF4. In addition, this aerobic tetracycline resistance determinant is not homologous to the three major plasmid mediated tetracycline resistance regions found in facultative gram-negative bacteria, represented by R100, RK2, and pBR322. A similar cryptic tetracycline resistance fragment was cloned from pCP1, a separate clindamycin resistance plasmid from Bacteroides that shares homology with the EcoRI D fragment of pBF4. This study identifies cryptic drug resistance determinants in Bacteroides that are expressed when inserted into an aerobically growing organism.     

The role of R plasmids in the resistance of bacteria in air

The study of Escherichia coli J 53, used as a model, has revealed that some R plasmids isolated from Serratia marcescens and Klebsiella pneumoniae, found to be the cause of the outbreak of hospital infection, ensure, besides multiple drug resistance, also their viability in the air.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria

We have constructed a set of RP4 (NmS/TcS) and Tn5-Mob derivatives which have applications in experiments involving mobilization of replicons in many Gram-negative organisms. The different selection markers of the RP4 and Tn5-Mob derivatives include streptomycin, chloramphenicol, gentamicin, and spectinomycin resistance as well as mercury resistance, and a constitutively expressed lacZ gene. This choice of markers allows the use of these derivatives in bacteria which are naturally resistant to many antibiotics, and in strains which contain pre-existing resistance plasmids, transposons, or antibiotic cassette insertions. In addition, a RP4 derivative carrying the sacB gene of Bacillus subtilis was constructed. This allows the selection for the loss of RP4 after it has been used to mobilize other plasmids. A Tn5-Mob-sacB derivative with a new marker (Gm) was also developed, as were vectors which take advantage of the sacB gene to facilitate replacement of existing Tn5 inserts with other Tn5 derivatives. As an example of the use of these tools, three Rhizobium leguminosarum bv. viciae VF39 plasmids which have been shown to be involved in symbiosis were differentially tagged and mobilized (individually and in various combinations) to the plasmid-free Agrobacterium tumefaciens strain UBAPF2. None of the resultant Agrobacterium strains was able to fix nitrogen in symbiosis with peas.     

Distribution of transferable trimethoprim and gentamicin resistance plasmids in Enterobacteriaceae

Following the employment of trimethoprim/sulfonamid and gentamicin in the general clinical praxis transferable plasmids with respective resistance function were identified at first in enteric bacteria from sewage before they could be detected in patient strains. Whereas the trimethoprim resistance plasmids represent different incompatibility groups (C, FII, I5, K, M, N, S, U) the gentamicin resistance plasmids are very similar IncM members of 62 MD in size. Therefore a clonal distribution of a particular gentamicin resistance plasmid has to be taken into consideration.     

Restriction endonuclease analyses of the incompatibility group P-1 plasmids RK2, RP1, RP4, R68, and R68.45

The P-group plasmids RP1, RP4, RK2, R68 and R68.45 were analyzed by the following restriction endonucleases:BamHI,BglII,EcoRI,HindIII,PstI,PvuII,SalI, andSmaI. No differences between RP1, RP4, and RK2 were found, and the plasmid R68.45 was found to contain a direct duplication of an existing DNA sequence in R68. Our map of RK2 differs from the published map of RK2 in the corresponding region of the R68 map that is duplicated in R68.45.     

Antibiotic resistance plasmids and bactericidal effect of chlorhexidine on Enterobacteriaceae

The effect of plasmid RP4 on the bactericidal effect of chlorhexidine on five Enterobacteriaceae has been tested. Escherichia coli, Serratia marcescens and Proteus mirabilis strains harbouring RP4 were more susceptible than R^- strains to this antiseptic. The role of 17 other plasmids harboured in the same bacterial host ( E. coli ) on the bactericidal effect of chlorhexidine has also been examined. Six plasmids (R751, R702, R144, RP4, pME206, S-a), of which four belonged to incompatibility group P, produced an increased bactericidal effect of chlorhexidine.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.     

Streptomycin and tetracycline resistance plasmids in Staphylococcus hyicus and other staphylococci

Plasmids associated with resistance to streptomycin, to streptomycin plus chloramphenicol or to tetracycline in Staphylococcus hyicus isolated from the skin of pigs have been compared, by restriction endonuclease digest patterns, with similar staphylococci from human sources and with published DNA base sequences. Several plasmids from Staph. hyicus have proved to have a very similar structure to those described from Staph. aureus but others appeared very dissimilar. This confirms the opinion that staphylococci from animal skin share a pool of plasmids with those from human skin but may also possess some of quite different structure.     

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

IncX plasmids are narrow host range plasmids of Enterobactericeae that have been isolated for over 50years. They are known to encode type IV fimbriae enabling their own conjugative transfer, and to provide accessory functions to their host bacteria such as resistance towards antimicrobial agents and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid diversity and prevalence is underappreciated. To address these possible shortcomings, we generated additional plasmid sequences of IncX plasmids of interest and compared them to the genomes of all sequenced IncX-like plasmids. These comparisons revealed that IncX plasmids possess a highly syntenic plasmid backbone, but that they are quite divergent with respect to nucleotide and amino acid similarity. Based on phylogenetic comparisons of the sequenced IncX plasmids, the IncX plasmid group has been expanded to include at least four subtypes, IncX1-IncX4. A revised IncX plasmid replicon typing procedure, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.     

Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage

A. FERNANDEZ-ASTORGA, A. FERNANDEZ DE ARANGUIZ, M. POCINO, A. UMARAN AND R. CISTERNA. 1992. The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37°C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6°C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower ( P < 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.     

Streptomycin and tetracycline resistance plasmids in Staphylococcus hyicus and other staphylococci.

Plasmids associated with resistance to streptomycin, to streptomycin plus chloramphenicol or to tetracycline in Staphylococcus hyicus isolated from the skin of pigs have been compared, by restriction endonuclease digest patterns, with similar staphylococci from human sources and with published DNA base sequences. Several plasmids from Staph. hyicus have proved to have a very similar structure to those described from Staph. aureus but others appeared very dissimilar. This confirms the opinion that staphylococci from animal skin share a pool of plasmids with those from human skin but may also possess some of quite different structure.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.