Coli Transconjugants Research Articles

Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand.

This study aimed to investigate the potential mechanisms associated with the persistence of chloramphenicol (CHP) resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans in Thailand. The CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates were tested for their CHP susceptibility in the presence and absence of phenylalanine arginine β-naphthylamide (PAβN). The potential co-selection of CHP resistance was investigated through conjugation experiments. Whole genome sequencing (WGS) was performed to analyze the E. coli (E329, E333, and E290) and Salmonella (SA448, SA461, and SA515) isolates with high CHP MIC (32-256 μg/mL) and predominant plasmid replicon types. The presence of PAβN significantly reduced the CHP MICs (≥4-fold) in most E. coli (67.9%) and Salmonella (64.9%). Ampicillin, tetracycline, and streptomycin co-selected for CHP-resistant Salmonella and E. coli-transconjugants carrying cmlA. IncF plasmids were mostly detected in cmlA carrying Salmonella (IncFIIAs) and E. coli (IncFIB and IncF) transconjugants. The WGS analysis revealed that class1 integrons with cmlA1 gene cassette flanked by IS26 and TnAs1 were located on IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids. IncFIA(HI1)/HI1B/Q1in SA448 contained catA flanked by IS1B and TnAs3. In conclusion, cross resistance through proton motive force-dependent mechanisms and co-selection by other antimicrobial agents involved the persistence of CHP-resistance in E. coli in this collection. Dissemination of CHP-resistance genes was potentially facilitated by mobilization via mobile genetic elements.

Investigation of multidrug-resistant plasmids from carbapenemase-producing Klebsiella pneumoniae clinical isolates from Pakistan.

The study aim was to investigate multidrug-resistant (MDR) plasmids from a collection of 10 carbapenemase-producing Klebsiella pneumoniae clinical isolates identified within the same healthcare institution in Pakistan. Full characterization of the MDR plasmids including structure, typing characteristics, and AMR content as well as determination of their plasmid-based antimicrobial susceptibility profiles were carried out. Plasmids were isolated from 10 clinical isolates of Klebsiella pneumoniae, and from a corresponding set of Escherichia coli transconjugants, then sequenced using Nanopore/Illumina technology to generate plasmid hybrid assemblies. Full characterization of MDR plasmids, including determination of next generation sequencing (NGS)-based AMR profiles, plasmid incompatibility groups, and types, was carried out. The structure of MDR plasmids was analyzed using the Galileo AMR platform. For E. coli transconjugants, the NGS-based AMR profiles were compared to NGS-predicted AMR phenotypes and conventional broth microdilution (BMD) antimicrobial susceptibility testing (AST) results. All carbapenemase-producing K. pneumoniae isolates (carrying either blaNDM-1, or/and blaOXA-48) carried multiple AMR plasmids encoding 34 antimicrobial resistance genes (ARGs) conferring resistance to antimicrobials from 6 different classes. The plasmid incompatibility groups and types identified were: IncC (types 1 and 3), IncFIA (type 26) IncFIB, IncFII (types K1, K2, K7, and K9), IncHI1B, and IncL. None of the blaNDM-1 and blaESBL-plasmids identified in this study were previously described. Most blaNDM-1-plasmids shared identical AMR regions suggesting potential genetic material/plasmid exchange between K. pneumoniae isolates of this collection. The majority of NGS-based AMR profiles from the E. coli transconjugants correlated well with both NGS-based predicted and conventional AST results. This study highlights the complexity and diversity of the plasmid-based genetic background of carbapenemase-producing clinical isolates from Pakistan. This study emphasizes the need for characterization of MDR plasmids to determine their complete molecular background and monitor AMR through plasmid transmission between multi-resistant bacterial pathogens.

Conjugative transfer of plasmid p_8N_qac(MN687830.1) carrying qacA gene from Staphylococcus aureus to Escherichia coli C600: potential mechanism for spreading chlorhexidine resistance.

ABSTRACTThe methicillin resistant Staphylococcus aureus (MRSA) is recognized by its ability to acquire and transferring resistance genes through interspecies conjugative plasmids. However, transference of plasmids from Gram-positive cocci to Gram-negative bacilli is not well characterized. In this report, we describe the transfer of a conjugative plasmid carrying qacA from MRSA to Escherichia coli C600. We performed a conjugation experiment using a chlorhexidine resistant MRSA isolate (ST-105/SCCmec type III) carrying the gene qacA and qacC as the donor and a chlorhexidine susceptible E. coli C600 isolate as the receptor. Transconjugants were selected using MacConkey agar plates containing chlorhexidine in concentrations ranging from 0.25 to 16 g.L-1. To genotypically confirm the transfer of the resistance gene, the transconjugants were screened by Polymerase Chain Reaction (PCR) and submitted to Sanger’s sequencing. MRSA isolates successfully transferred the chlorhexidine resistance gene (qacA) to the recipient E. coli strain C600. The E. coli transconjugant exhibited an important reduction of chlorhexidine susceptibility, with MICs increasing from ≤ 0.25 to ≥ 16 g.L-1 after conjugation. The qacA gene was detected by PCR as well as in the Sanger’s sequencing analysis of DNA from transconjugant plasmids. To the best of our knowledge, this is the first report of the plasmid p_8N_qac(MN687830.1) carrying qacA and its transfer by conjugation from a MRSA to an E. coli. These findings increase concerns on the emergence of resistance dissemination across the genus and emphasizes the importance of continuous antiseptic stewardship.

A Klebsiella variicola Plasmid Confers Hypermucoviscosity-Like Phenotype and Alters Capsule Production and Virulence.

Hypermucoviscosity (hmv) is a capsule-associated phenotype usually linked with hypervirulent Klebsiella pneumoniae strains. The key components of this phenotype are the RmpADC proteins contained in non-transmissible plasmids identified and studied in K. pneumoniae. Klebsiella variicola is closely related to K. pneumoniae and recently has been identified as an emergent human pathogen. K. variicola normally contains plasmids, some of them carrying antibiotic resistance and virulence genes. Previously, we described a K. variicola clinical isolate showing an hmv-like phenotype that harbors a 343-kb pKV8917 plasmid. Here, we investigated whether pKV8917 plasmid carried by K. variicola 8917 is linked with the hmv-like phenotype and its contribution to virulence. We found that curing the 343-kb pKV8917 plasmid caused the loss of hmv, a reduction in capsular polysaccharide (P < 0.001) and virulence. In addition, pKV8917 was successfully transferred to Escherichia coli and K. variicola strains via conjugation. Notably, when pKV8917 was transferred to K. variicola, the transconjugants displayed an hmv-like phenotype, and capsule production and virulence increased; these phenotypes were not observed in the E. coli transconjugants. These data suggest that the pKV8917 plasmid carries novel hmv and capsule determinants. Whole-plasmid sequencing and analysis revealed that pKV8917 does not contain rmpADC/rmpA2 genes; thus, an alternative mechanism was searched. The 343-kb plasmid contains an IncFIB backbone and shares a region of ∼150 kb with a 99% identity and 49% coverage with a virulence plasmid from hypervirulent K. variicola and multidrug-resistant K. pneumoniae. The pKV8917-unique region harbors a cellulose biosynthesis cluster (bcs), fructose- and sucrose-specific (fru/scr) phosphotransferase systems, and the transcriptional regulators araC and iclR, respectively, involved in membrane permeability. The hmv-like phenotype has been identified more frequently, and recent evidence supports the existence of rmpADC/rmpA2-independent hmv-like pathways in this bacterial genus.

Cryptic prophages in a blaNDM-1-bearing plasmid increase bacterial survival against high NaCl concentration, high and low temperatures, and oxidative and immunological stressors.

In this study, we investigated the effect of cryptic prophage regions in a blaNDM-1-bearing plasmid, which was identified in a patient from South Korea, on the survival of bacteria against adverse environmental conditions. First, we conjugated the intact plasmid and plasmids with deleted cryptic prophages into Escherichia coli DH5α. The E. coli transconjugants carrying the plasmid with intact cryptic prophages showed increased survival during treatment with a high concentration of NaCl, high and low temperatures, an oxidative stressor (H2O2), and an immunological stressor (human serum). By contrast, the transconjugants carrying the plasmid with a single-cryptic prophage knockout did not show any change in survival rates. mRNA expression analyses revealed that the genes encoding sigma factor proteins were highly upregulated by the tested stressors and affected the expression of various proteins (antioxidant, cell osmosis-related, heat shock, cold shock, and universal stress proteins) associated with the specific defense against each stress. These findings indicate that a bacterial strain carrying a plasmid with intact carbapenemase gene and cryptic prophage regions exhibited an increased resistance against simulated environmental stresses, and cryptic prophages in the plasmid might contribute to this enhanced stress resistance. Our study indicated that the coselection of antibiotic resistance and resistance to other stresses may help bacteria to increase survival rates against adverse environments and disseminate.

Analysis of the Degradation of Broad-Spectrum Cephalosporins by OXA-48-Producing Enterobacteriaceae Using MALDI-TOF MS.

The objective of the study was to evaluate the activity of OXA-48 against different broad-spectrum cephalosporins and to identify the reaction products by MALDI-TOF MS. The action of OXA-48 on cefotaxime, ceftazidime, and ceftriaxone was assessed by this method, using an Escherichia coli J53 transconjugant carrying only the ~62 Kb IncL plasmid containing the blaOXA-48 gene, and the same strain without any plasmid was included as a negative control. In addition, a collection of 17 clinical OXA-48-producing Enterobacteriaceae, which were susceptible to broad-spectrum cephalosporins, was evaluated. MALDI-TOF MS-based analysis of the E. coli transconjugant carrying the blaOXA-48-harboring plasmid, and also the clinical isolates, showed degradation of cefotaxime into two inactive compounds—decarboxylated and deacetylated cefotaxime (~370 Da) and deacetyl cefotaxime (~414 Da), both with the hydrolyzed beta-lactam ring. Reaction products were not obtained when the experiment was performed with ceftriaxone or ceftazidime. From a clinical point of view, our study supports the idea that the efficacy of cefotaxime against OXA-48-producing Enterobacteriaceae is doubtful, in contrast to ceftazidime and ceftriaxone which could be valid choices for treating infections caused by these bacteria. However, further clinical studies confirming this hypothesis are required.

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla(TEM-52) -carrying plasmid (lactose-negative mutant of B1-54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Fresh faeces from a healthy volunteer, negative for cephalosporin-resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed-field gel electrophoresis profiles (PFGE). The avian extended-spectrum β-lactamase-positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1-54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla(TEM-52) -carrying plasmid. These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.

Providencia stuartii with VIM-1 metallo-β-lactamase

Providencia stuartii with VIM-1 metallo-β-lactamase

Plasmid-mediated carbapenem-hydrolysing β-lactamase KPC-2 in carbapenem-resistant Serratia marcescens isolates from Hangzhou, China

Sir, Carbapenems are considered to be one of the few drugs that are useful for the treatment of infections caused by multidrugresistant isolates with high levels of AmpC or extended-spectrum b-lactamase (ESBL) production. While carbapenem resistance has become a serious problem for non-fermenting bacteria, it has remained uncommon in Enterobacteriaceae. However, identification of carbapenem-resistant Enterobacteriaceae is increasingly common. Especially, reports of nosocomial infection due to carbapenem-resistant Serratia spp. have become significantly more common, which has mainly been attributed to production of b-lactamases that are capable of hydrolysing carbapenems such as IMP-1, IMP-6, VIM-2, and SME-1, 2 and 3. The KPC class A carbapenemases are capable of hydrolysing carbapenems, penicillins, cephalosporins and aztreonam, and are inhibited by clavulanic acid and tazobactam. The first report of this b-lactamase, KPC-1, was from a carbapenemresistant Klebsiella pneumoniae. KPC-2 was then found in isolates of K. pneumoniae, Salmonella enterica, Klebsiella oxytoca and Enterobacter spp. Soon afterward, KPC-3 was found in K. pneumoniae and Enterobacter cloacae from New York. Recently, KPCs were found in France, South America and Israel. We describe three clinical isolates of carbapenem-resistant Serratia marcescens that produce KPC-2. This is the first report of detection of a plasmid-encoded carbapenem-hydrolysing enzyme KPC-2 in S. marcescens. We collected three clinical isolates of carbapenem-resistant S. marcescens from our hospital in February 2006. The three isolates exhibited either resistance or reduced susceptibility to carbapenems with imipenem MICs of 8, 64 and 8 mg/L. The isolates were also resistant to penicillins, cephalosporins and aztreonam, but were susceptible to quinolones and aminoglycosides (Table 1). Following mating of the three S. marcescens isolates with an Escherichia coli (EC600) by conjugation, all of the resulting transconjugants exhibited a phenotype of resistance to b-lactams similar to that of the S. marcescens isolate used. The imipenem and meropenem MICs of transconjugants were 1 mg/L. They were also resistant to penicillins and aztreonam, and were resistant or intermediately resistant to cephalosporins, but were susceptible to quinolones and aminoglycosides (Table 1). The three S. marcescens isolates and three transconjugants, one derived from a mating with each parent strain, had identical plasmid profiles and restriction patterns (data not shown). Following the elimination of plasmids by repeated SDS treatment, the S. marcescens isolates became susceptible to all antibiotics except ampicillin. Isoelectric focusing analysis of crude enzyme extracts from the isolates (data not shown) demonstrated the production of two b-lactamases with apparent pIs of 6.5 and 6.7. The E. coli transconjugants produced the pI 6.7 b-lactamase only. Neither pI 6.7 nor pI 6.5 b-lactamase was detected in the three plasmid-eliminated S. marcescens strains. Given the pI of 6.7, we suspected the production of a KPC b-lactamase. To confirm the presence of a blaKPC gene, we conducted PCR amplification using the primers KPC-F and KPC-R. The reaction gave a positive result and DNA sequencing identified the gene as blaKPC-2. The data show that the carbapenem-hydrolysing enzyme in the three test S. marcescens isolates is KPC-2. The enzyme is encoded on a conjugative plasmid and mainly contributed to resistance to carbapenems. We presume that the unidentified pI 6.5 b-lactamase was either the degradation product or the precursor of KPC-2. Unidentified carbapenemase activity bands around pI 6.3–6.5 have also been detected from K. pneumoniae, which were presumed to be the degradation product of KPCs. The resistance against ampicillin remaining when the blaKPC-containing plasmid had been cured is probably due to the low activity of chromosomal AmpC in S. marcescens, which was not detected in IEF but confirmed through enzyme assay. The three S. marcescens isolates had identical plasmid profiles and restriction patterns, indicating that the same plasmid spread among these isolates. Most blaKPC-encoding plasmids could be conjugated into E. coli, except the blaKPC-1-encoding plasmid described by Yigit et al. Many blaKPC-encoding plasmids also encode blaTEM-1 and various ESBLs. However, only a blaKPC gene was identified from the plasmid in S. marcescens in this study. KPC family enzymes have been detected in a variety of Enterobacteriaceae but mainly in North America. The emergence of a KPC-2 type carbapenem-hydrolysing enzyme in China is alarming. It indicates that the spread of blaKPC genes is no longer a regional problem.

Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens.

The distribution of strains producing metallo-beta-lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a metallo-beta-lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the metallo-beta-lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to < or = 8 micrograms/ml), had no detectable blaIMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, > or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like metallo-beta-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

Bacterial conjugation between Escherichia coli and Pseudomonas spp. donor and recipient cells in soil.

Experiments conducted in microcosms containing loam soil samples inoculated with either E. coli or Pseudomonas spp. donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period. E. coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples. In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation. Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil. The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10(-3) to 10(-4]. Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.

Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants.

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon Tn501 into the Shigella chromosome through Tn501-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage P1-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stx- mutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a Tn10 derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stx- mutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycin-resistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx- mutants of S. dysenteriae 1 exhibited full virulence in the Serény test.

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP 2, WP 2hcr, WP 6 and WP 67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP 2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP 2, WP 2hcr; WP 6 and WP 67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 μg/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.

Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.

R-factor responsible for an outbreak of multiply antibiotic-resistant Klebsiella pneumoniae

Seven serotypes of Klebsiella pneumoniae isolated from different patients demonstrated resistance to the same eight antibiotics. A plasmid carrying resistance determinants to these antibiotics and mercury salts could be transferred in toto to a plasmidless strain of Escherichia coli. All E. coli transconjugants showed the same antibiotic resistance pattern. Digestion with restriction endonucleases yielded patterns that were identical for each of the R-factor transferred from the multiply resistant serotypes. Moreover, deoxyribonucleic acid-deoxyribonucleic acid hybridization demonstrated identity between the probe, pMAC20 (an R-factor from one serotype), and all R-factors isolated from the multiply resistant strains of K. pneumoniae and the E. coli transconjugants tested.

Mechanism of transferable resistance to chloramphenicol in Haemophilus parainfluenzae.

A clinical isolate of Haemophilus parainfluenzae resistant to chloramphenicol and tetracycline transferred both cam and tet determinants to Escherichia coli K-12 during mixed cultivation on solid media irrespective of the selection employed. The doubly resistant transconjugant was found to contain levels of the enzyme chloramphenicol acetyltransferase (CAT) comparable to those found in R plasmid-bearing chloramphenicol-resistant enteric bacteria. Purification of CAT from the transconjugant was achieved by affinity chromatography, and the electrophoretically homogeneous protein was compared with previously characterized CAT variants specified by R plasmids. Although the CAT associated with cam from H. parainfluenzae was found to be distinct from the three types described previously, its N-terminal peptide amino acid sequence was identical with that determined for a type II CAT. Attempts to demonstrate covalently closed circular deoxyribonucleic acid in the H. parainfluenzae donor and the E. coli transconjugant were unsuccessful. The cam and tet determinants were nontransmissible from E. coli but could be cotransferred following the introduction of a suitable conjugative plasmid.