Conjugative Transfer Of Resistance Research Articles

The use of cinnamon oil as antibacterial agent to eliminate some antibiotic-resistant bacteria isolated from water sources

The use of cinnamon oil as antibacterial agent to eliminate some antibiotic-resistant bacteria isolated from water sources

CTX-M-type ESBL-mediated resistance to third-generation cephalosporins and conjugative transfer of resistance in Gram-negative bacteria isolated from hospitals in Tamil Nadu, India.

Clinical pathogens, especially Gram-negative bacteria developing resistance to third-generation cephalosporins, are making clinical outcomes more complicated and serious. This study was undertaken to evaluate the distribution of CTX-M-type extended-spectrum β-lactamases (ESBLs) in Tamil Nadu, India. For this study, clinical samples were collected from five different hospitals located in Tamil Nadu and the ESBL-producing Gram-negative isolates were characterized. MIC was performed using cefotaxime and ceftazidime. The bla ESBL-producing genes were screened using multiplex PCR for the genes, CTX-M group-1, -2, -8, -9, -26. The conjugation studies were performed using Escherichia coli AB1157 as a recipient for the isolates harbouring plasmid-borne resistance following broth-mating experiment. In total, 1500 samples were collected and 599 Gram-negative bacteria were isolated that included E. coli (n=233), Klebsiella pneumoniae (n=182), Pseudomonas aeruginosa (n=79), Citrobacter spp. (n=30), Proteus mirabilis (n=28), Salmonella spp. (n=21), Acinetobacter baumannii (n=12), Serratia spp. (n=6), Shigella spp. (n=4), Morganella morganii (n=3) and Providencia spp. (n=1). MIC results showed that 358 isolates were resistant to cefotaxime and ceftazidime. Further, ESBL gene-amplification results showed that 19 isolates had CTX-M group-1 gene including E. coli (n=16), K. pneumoniae (n=2) and P. aeruginosa (n=1) whereas one M. morganii isolate had CTX-M group-9, which was plasmid-borne. Through conjugation studies, 12/20 isolates were found to be involved in the transformation of its plasmid-borne resistance gene. Our study highlighted the importance of horizontal gene transfer in the dissemination of plasmid-borne bla CTX-M-type resistance genes among the clinical isolates.

Marine Bivalve Mollusks As Possible Indicators of Multidrug-Resistant Escherichia coli and Other Species of the Enterobacteriaceae Family

The mechanisms for the development and spread of antibacterial resistance (ABR) in bacteria residing in environmental compartments, including the marine environment, are far from understood. The objective of this study was to examine the ABR rates in Escherichia coli and other Enterobacteriaceae isolates obtained from marine bivalve mollusks collected along the Norwegian coast during a period from October 2014 to November 2015. A total of 549 bivalve samples were examined by a five times three tube most probable number method for enumeration of E. coli in bivalves resulting in 199 isolates from the positive samples. These isolates were identified by biochemical reactions and matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, showing that 90% were E. coli, while the remaining were species within the genera Klebsiella, Citrobacter, and Enterobacter. All 199 isolates recovered were susceptibility tested following the European Committee on Antimicrobial Susceptibility Testing disk diffusion method. In total, 75 of 199 (38%) isolates showed resistance to at least one antibacterial agent, while multidrug-resistance were seen in 9 (5%) isolates. One isolate conferred resistance toward 15 antibacterial agents. Among the 75 resistant isolates, resistance toward extended-spectrum penicillins (83%), aminoglycosides (16%), trimethoprim (13%), sulfonamides (11%), tetracyclines (8%), third-generation cephalosporins (7%), amphenicols (5%), nitrofurans (5%), and quinolones (5%), were observed. Whole-genome sequencing on a selection of 10 E. coli isolates identified the genes responsible for resistance, including blaCTX-M genes. To indicate the potential for horizontal gene transfer, conjugation experiments were performed on the same selected isolates. Conjugative transfer of resistance was observed for six of the 10 E. coli isolates. In order to compare E. coli isolates from bivalves with clinical strains, multiple-locus variable number tandem repeats analysis (MLVA) was applied on a selection of 30 resistant E. coli isolates. The MLVA-profiles were associated with community-acquired E. coli strains causing bacteremia. Our study indicates that bivalves represent an important tool for monitoring antibacterial resistant E. coli and other members of the Enterobacteriaceae family in the coastal environment.

Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States

USA300 methicillin-resistant Staphylococcus aureus (MRSA) isolates are usually resistant only to oxacillin, erythromycin, and, increasingly, levofloxacin. Of these, oxacillin and levofloxacin resistances are chromosomally encoded. Plasmid-mediated clindamycin, mupirocin, and/or tetracycline resistance has been observed among USA300 isolates, but these descriptions were limited to specific patient populations or isolated occurrences. We examined the antimicrobial susceptibilities of invasive MRSA isolates from a national surveillance population in order to identify USA300 isolates with unusual, possibly emerging, plasmid-mediated antimicrobial resistance. DNA from these isolates was assayed for the presence of resistance determinants and the presence of a pSK41-like conjugative plasmid. Of 823 USA300 isolates, 72 (9%) were tetracycline resistant; 69 of these were doxycycline susceptible and tetK positive, and 3 were doxycycline resistant and tetM positive. Fifty-one (6.2%) isolates were clindamycin resistant and ermC positive; 22 (2.7%) isolates were high-level mupirocin resistant (mupA positive); 5 (0.6%) isolates were trimethoprim-sulfamethoxazole (TMP-SMZ) resistant, of which 4 were dfrA positive; and 7 (0.9%) isolates were gentamicin resistant and aac6'-aph2'' positive. Isolates with pSK41-like plasmids (n = 24) were positive for mupA (n = 19), dfrA (n = 6), aac6'-aph2'' (n = 6), tetM (n = 2), and ermC (n = 8); 20 pSK41-positive isolates were positive for two or more resistance genes. Conjugative transfer of resistance was demonstrated between four gentamicin- and mupirocin-resistant and three gentamicin- and TMP-SMZ-resistant USA300 isolates; transconjugants harbored a single pSK41-like plasmid, which was PCR positive for aac6'-aph2'' and either mupA and/or dfrA. USA300 and USA100 isolates from the same state with identical resistance profiles contained pSK41-like plasmids with indistinguishable restriction and Southern blot profiles, suggesting horizontal plasmid transfer between USA100 and USA300 isolates.

Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in Enterococcus italicus Strains of Dairy Origin

Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.

Dissemination of imipenem-resistant Acinetobacter baumannii strains carrying the ISAba1–bla OXA-23 genes in a Chinese hospital

An outbreak of 95 clinical infections with imipenem-resistant Acinetobacter baumannii in a Chinese hospital was investigated and the carbapenemase-encoding genes and their relationship with ISAba1 of these and a further 16 isolates recovered from the intensive care unit (ICU) environment were analysed. Almost all isolates were resistant to a wide range of antimicrobials; the lowest resistance rates were found for polymyxin E (17.1 %), cefoperazone/sulbactam (30.6 %) and ampicillin/sulbactam (67.6 %). Six pattern types defined by DNA macrorestriction patterns were distinguished among the clinical isolates with dissemination of pattern A (50 isolates) to patients in seven hospital units and pattern B (35 isolates) to eight units; the environmental isolates from ICUs were also of pattern A. All isolates were positive for the bla(OXA-66) and bla(OXA-23) genes. The OXA-23-encoding gene was located 34 bp downstream of ISAba1. No plasmids were detected and conjugal transfer of resistance was not demonstrated. The bla(OXA-23) probe hybridized with 200 and 220 kb ApaI chromosomal fragments for type patterns A and B, respectively.

Providencia stuartii with VIM-1 metallo-β-lactamase

Providencia stuartii with VIM-1 metallo-β-lactamase

Amoxycillin/clavulanate resistance as related to β-lactamase content in Escherichia coli strains from community-acquired urinary tract infections in Greece

Objective To determine the resistance rate to amoxycillin/clavulanate (AMC) in 100 Escherichia coli strains isolated from outpatients with urinary tract infection (UTI) in four Greek hospitals and assess the relationship between β-lactamase content and resistance to AMC. Methods Susceptibility to β-lactams was determined with the E-test. Sonic cell extracts were used as β-lactamase preparations. Conjugal transfer of resistance was performed in broth cultures. β-Lactamase quantities were evaluated by measuring nitrocefin hydrolysis. Isoelectric points (pls) of β-lactamases were determined by electrofocusing. The substrate specificity of the enzymes and the inhibitory activity of clavulanate were studied spectrophotometrically. Results Thirty-two isolates were resistant to ampicillin. Eight were resistant (MIC ≥ 32 mg/L) and 11 showed decreased susceptibility (MIC 4-16 mg/L) to AMC. The latter expressed at least four-fold higher amounts of TEM-1 β-lactamase compared with the TEM-1-producing AMC-susceptible isolates. Seven AMC-resistant isolates produced at least 16-fold higher amounts of TEM-1; in one isolate, resistance was attributed to an OXA-type β-lactamase. None of the AMC-resistant isolates was able to transfer resistance to AMC by conjugation. Clavulanate-resistant TEM variants were not detected. Conclusions Amoxycillin/clavulanate-resistant E. coli strains have become established in the Greek community. Resistance is mainly due to the production of large amounts of TEM-1 β-lactamase which is encoded from non-self-transmissible plasmids.

Further Studies of Transferable Antibiotic Resistance in Strains of Pseudomonas aeruginosa from Four Clinical Settings in Three Countries

This paper describes transferability of antibiotic resistance determinants in clinical isolates of Pseudomonas aeruginosa resistant to imipenem, cefotaxime and ceftazidime obtained from different clinical settings in three different countries. Two strains of Enterobacteriaceae (Escherichia coli K-12 and Proteus mirabilis P-38) and two strains of P. aeruginosa (PAO and ML) were used as recipient strains. The conjugative transfer of resistance was very specific, i.e. donor strains of P. aeruginosa transferred individual resistance determinants either to recipient strains E. coli K-12 and P. mirabilis P-38, or to P. aeruginosa PAO or ML. In a case when three different species (P. aeruginosa, Acinetobacter calcoaceticus, Stenotrophomonas maltophilia) were isolated from a single patient, a block of resistance determinants was transferred both to Enterobacteriaceae and P. aeruginosa recipients. A single plasmid with rather specific properties was suspected of being exchanged among strains of these different species. It was recommended to study the overall situation of transferability of individual resistance determinants in bacterial strains belonging to various species of nosocomial bacteria, especially of P. aeruginosa, by using a rather specific methodology.

Method to Determine Effect of Antibiotics at Residue Levels on R-Factor Transfer

An analytical system was developed which can assess the ability of antibiotic/antimicrobial residues (0.01-1.00 ppm) to affect the conjugal transfer of resistance among the Enterobacteriaceae. The donor strain, Escherichia coli RP-4 (Amr Tcr Nmr Kmr Lac+), and recipient strain, E. coli Sc-8632 (Smr Lac-), were incubated together in a 1:9 donor:recipient ratio for 18 h with gentle shaking (50 rpm) in brain heart infusion broth in the presence of residue levels of antibiotics. The mating cultures were serially diluted and spread-plated onto MacConkey agar containing 25 micrograms streptomycin/mL to select the total recipient population of sensitive E. coli Sc-8632 and transconjugants. After an 18 h incubation at 37 degrees C, the plates were replicated onto MacConkey agar containing 25 micrograms ampicillin/mL to select the ampicillin-resistant transconjugant population. Repeatability was good; the average transfer was 51.8%, with a coefficient of variation of 9.3%. Residue levels of tylosin (0.10 and 1.00 ppm) increased the transfer of the ampicillin marker beyond the 95% confidence limits. Oxytetracycline, bacitracin, streptomycin, penicillin, and virginiamycin did not increase the percent transfer. Oxytetracycline at 0.01 ppm decreased the percent transfer. In general, residue levels of antibiotics (0.01-1.00 ppm) did not affect the conjugal transfer of antibiotic resistance.

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.

Amikacin resistance associated with a plasmid-borne aminoglycoside phosphotransferase in Escherichia coli

Enzymatic phosphorylation of amikacin has not been reported previously in gram-negative bacteria. We found that extracts of MP1, a mutant of Escherichia coli JR66/W677 that is resistant to amikacin, were able to phosphorylate this aminoglycoside more rapidly than were extracts of the parental strain. Conjugal transfer of resistance from MP1 to a recipient strain was accompanied by acquisition in the transconjugants of amikacin phosphotransferase activity and of a 57-megadalton plasmid present in the donor. Partial purification of the phosphotransferase activity on amikacin-Sepharose 4B yielded an enzyme with a substrate spectrum similar to that of the 3'-neomycin-kanamycin phosphotransferase II found E. coli, except that it was also active against amikacin. A mutant of MP1, MP5, had increased susceptibility to amikacin and reduced phosphotransferase activity. MP9, a mutant MP5, was more resistant to amikacin and had increased phosphotransferase activity. The mutations leading to these alterations of amikacin susceptibility and amikacin phosphotransferase activity were transferable with the same plasmid that was associated with amikacin resistance and phosphotransferase activity in MP1. These studies demonstrate that resistance to amikacin in a laboratory strain of E. coli is due to an aminoglycoside phosphotransferase coded by a transferable plasmid-borne gene.

Analysis of Increasing Antibiotic Resistance of Klebsiella pneumoniae Relative to Changes in Chemotherapy

Three antibiotic-resistant strains of Klebsiella pneumoniae responsible for outbreaks of systemic infection in a hospital nursery were analyzed. Each organism emerged after prolonged use of a drug to which it was resistant. The first strain (RO16) produced neomycin phosphotransferase and contained three plasmids with molecular weights of 24, 25, and 30 X 10(6) daltons, respectively. Resistance to ampicillin, carbenicillin, neomycin, and kanamycin was transferred by conjugation at a frequency of 10(-6). The second strain of K. pneumoniae (RO106) produced gentamicin adenyltransferase and maintained aminoglycoside resistance only when propagated in antibiotic-containing medium. DNA analyses revealed eight species of plasmid DNA: one species with a molecular weight of 70 X 10(6) daltons apparently accounted for conjugal transfer of resistance to ampicillin, carbenicillin, chloramphenicol, and streptomycin. The third strain (RO180) was resistant primarily to colistin and lacked plasmids. Control of the outbreak due to this strain was achieved by aminoglycoside therapy.