Per/polyfluoroalkyl substances modulate plasmid transfer of antibiotic resistance genes: A balance between oxidative stress and energy support.

Emerging contaminants can accelerate the transmission of antibiotic resistance genes (ARGs) from environmental bacteria to human pathogens via plasmid conjugation, posing a great challenge to the public health. Although the toxic effects of per/polyfluoroalkyl substances (PFAS) as persistent organic pollutants have been understood, it is still unclear whether and how PFAS modulate the transmission of ARGs. In this study, we for the first time reported that perfluorooctanoic acid (PFOA), perfluorododecanoic acid (PFDoA) and ammonium perfluoro (2-methyl-3-oxahexanoate) (GenX) at relatively low concentrations (0.01, 0.1 mg/L) promoted the conjugative transfer of plasmid RP4 within Escherichia coli, while the plasmid conjugation was inhibited by PFOA, PFDoA and GenX at relatively high concentrations (1, 10 mg/L). The non-unidirectional conjugation result was ascribed to the co-regulation of ROS overproduction, enhanced cell membrane permeability, shortage of energy support as well as l-arginine pool depletion. Taking the well-known PFOA as an example, it significantly enhanced the conjugation frequency by 1.4 and 3.4 times at relatively low concentrations (0.01, 0.1 mg/L), respectively. Exposure to PFOA resulted in enhanced cell membrane permeability and ROS overproduction in donor cells. At high concentrations of PFOA (1, 10 mg/L), although enhanced oxidative stress and cell membrane permeability still occurred, the ATP contents in E. coli decreased, which contributed to the inhibited conjugation. Transcriptome analysis further showed that the expression levels of genes related to arginine biosynthesis (argA, argC, argF, argG, argI) and transport (artJ, artM, artQ) pathways were significantly increased. Intracellular l-arginine concentration deficiency were observed at high concentrations of PFOA. With the supplementary exogenous arginine, it was demonstrated that arginine upregulated conjugation transfer- related genes (trfAp, trbBp) and restores the cell number of transconjugants in PFOA-treated group. Therefore, the inhibited conjugation at high concentrations PFOA were attributed to the shortage of ATP and the depletion of L-arginine pool. These findings provide important insights into the effect environmental concentrations of PFAS on the conjugative transfer of ARGs, and update the regulation mechanism of plasmid conjugation, which is critical for the management of antibiotic resistance in aquatic environments.

Exposure to Al2O3 nanoparticles facilitates conjugative transfer of antibiotic resistance genes from Escherichia coli to Streptomyces.

The spread of antibiotic resistance genes (ARGs) has become a global environmental issue; it has been found that nanoparticles (NPs) can promote the transfer of ARGs between bacteria. However, it remains unclear whether NPs can affect this kind of conjugation in Streptomyces, which mainly conjugate with other bacteria via spores. In the present study, we demonstrated that Al2O3 NPs significantly promote the conjugative transfer of ARGs from Escherichia coli (E. coli) ET12567 to Streptomyces coelicolor (S. coelicolor) M145 without the use of heat shock method. The number of transconjugants induced by Al2O3 particles was associated with the size and concentration of Al2O3 particles, exposure time, and the ratio of E. coli and spores. When nanoparticle size was 30 nm at a concentration of 10 mg/L, the conjugation efficiency reached a peak value of 182 cfu/108 spores, which was more than 60-fold higher than that of the control. Compared with nanomaterials, bulk particles exhibited no significant effect on conjugation efficiency. We also explored the mechanisms by which NPs promote conjugative transfer. After the addition of NPs, the intracellular ROS content increased and the expression of the classical porin gene ompC was stimulated. In addition, ROS enhanced the mRNA expression levels of conjugative genes by inhibiting global regulation genes. Meanwhile, expression of the conjugation-related gene intA was also stimulated, ultimately increasing the number of transconjugants. Our results indicated that Al2O3 NPs significantly promoted the conjugative transfer of ARGs from bacteria to spores and aggravated the diffusion of resistance genes in the environment.

Negative effect of copper nanoparticles on the conjugation frequency of conjugative catabolic plasmids.

Due to their antimicrobial properties, copper nanoparticles (CuNPs) have been proposed to be used in agriculture for pest control. Pesticides removal is mainly done by microorganisms, whose genes usually are found in conjugative catabolic plasmids (CCP). The aim of this work was to evaluate if CuNPs at subinhibitory concentrations modify the conjugation frequency (CF) of two CCP (pJP4 and pADP1). CuNPs were characterized by scanning electron microscopy with an X-ray detector, dynamic light scattering and X-ray diffraction. Mating assays were done in LB broth supplemented with CuNPs (10, 20, 50 and 100 µg mL-1) or equivalent concentrations of CuSO4. Interestingly, we observed that in LB, Cu+2 release from CuNPs is fast as evaluated by atomic absorption spectrophotometry. Donor and recipient strains were able to grow in all copper concentrations assayed, but CF of mating pairs was reduced to 10% in the presence of copper at 20 or 50 µg Cu mL-1 compared to control. Thus, our results indicated that both copper forms, CuNPs or CuSO4, negatively affected the transfer of catabolic plasmids by conjugation. Since dissemination of degradative genes by conjugation contribute to degradation of pesticides by microorganisms, this work improves our understanding of the risks of using copper in agriculture soils, which could affect the biodegradative potential of microbial communities.

New secondary metabolites from an engineering mutant of endophytic Streptomyces sp. CS.

In previous work, a series of bioactive natural products had been isolated from the plant endophytic Streptomyces sp. CS, which was isolated from Maytenus hookeri. To mine new active metabolites, we describe introducing an alien carbamoyltransferase (asm21) gene into the strain CS by conjugal transfer. As a result, three recombinatorial mutants named CS/asm21-1, CS/asm21-2 and CS/asm21-4 were successfully constructed. Three mutants and wild type CS were cultured on solid medium, and the extracts were detected and analyzed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS profiles showed several unknown peaks that were present in the spectra of extracts of the CS/asm21-4 cultured on oatmeal solid medium. Then, three new naphthomycins O-Q (1-3), a new macrolide hookerolide (4) as well as nine known compounds were obtained from the solid cultured medium. Their structures were identified by spectra data. These new compounds showed moderate antimicrobial activities.

Investigation and Treatment of Fusidic Acid Resistance Among Methicillin-Resistant Staphylococcal Isolates from Egypt.

Methicillin resistance among staphylococci isolated from patients in northern Egypt has escalated alarmingly in the past decade. Data about the prevalence of fusidic acid (FA) resistance in Egyptian clinical isolates are limited. This work investigates the prevalence and mechanism of FA resistance among 81 methicillin-resistant staphylococcal isolates from major hospitals of Alexandria, Egypt. Some combinations for treating infections due to resistant isolates were studied. Twenty-six isolates (32.1%) were FA resistant (minimum inhibitory concentrations [MICs] = 2-1,024 µg/ml), and fusB and fusC genes coding for FA resistance were detected in 30.77% and 34.62% of the FA-resistant strains, respectively. One highly resistant isolate, S502 (MIC = 1,024 µg/ml), possessed both genes. Plasmid curing resulted in fusB loss and MIC decrease by 16-64 folds. Conjugation caused acquisition of FA resistance among susceptible isolates. Serial passages in subinhibitory FA concentrations produced mutants with increased MIC by 4-32 folds. The combination of FA with rifampin, gentamicin, or ampicillin/sulbactam, in a subinhibitory concentration, was synergistic against the isolates, including serial passage mutants, decreasing number of survivors by an average of 2-4 logs. A relatively moderate rate of FA resistance was detected in Alexandria hospitals. Combination therapy with gentamicin, rifampin, or ampicillin/sulbactam is crucial to preserve the effectiveness of FA.

Multidrug Resistance in Quinolone-Resistant Gram-Negative Bacteria Isolated from Hospital Effluent and the Municipal Wastewater Treatment Plant.

This study is aimed to assess if hospital effluents represent an important supplier of multidrug-resistant (MDR) Gram-negative bacteria that, being discharged in the municipal collector, may be disseminated in the environment and bypassed in water quality control systems. From a set of 101 non-Escherichia coli Gram-negative bacteria with reduced susceptibility to quinolones, was selected a group of isolates comprised by those with the highest indices of MDR (defined as nonsusceptibility to at least one agent in six or more antimicrobial categories, MDR ≥6) or resistance to meropenem or ceftazidime (n = 25). The isolates were identified and characterized for antibiotic resistance phenotype, plasmid-mediated quinolone resistance (PMQR) genes, and other genetic elements and conjugative capacity. The isolates with highest MDR indices were mainly from hospital effluent and comprised ubiquitous bacterial groups of the class Gammaproteobacteria, of the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas, and of the class Flavobacteriia, of the genera Chryseobacterium and Myroides. In this group of 25 strains, 19 identified as Gammaproteobacteria harbored at least one PMQR gene (aac(6')-Ib-cr, qnrB, qnrS, or oqxAB) or a class 1 integron gene cassette encoding aminoglycoside, sulfonamide, or carbapenem resistance. Most of the E. coli J53 transconjugants with acquired antibiotic resistance resulted from conjugation with Enterobacteriaceae. These transconjugants demonstrated acquired resistance to a maximum of five classes of antibiotics, one or more PMQR genes and/or a class 1 integron gene cassette. This study shows that ubiquitous bacteria, other than those monitored in water quality controls, are important vectors of antibiotic resistance and can be disseminated from hospital effluent to aquatic environments. This information is relevant to support management options aiming at the control of this public health problem.

Risk factors and outcome for colistin-resistant Acinetobacter nosocomialis bacteraemia in patients without previous colistin exposure.

The clinical characteristics of patients with colistin-resistant Acinetobacter baumannii bacteraemia have been documented, but those of patients with bacteraemia caused by other Acinetobacter species remain unknown. Previous exposure to colistin has been shown to be associated with the emergence of colistin resistance, but may be not the only predisposing factor. In the current study, we highlight the risk and outcome of patients without previous exposure to colistin who acquired colistin-resistant Acinetobacter nosocomialis (ColRAN) bacteraemia. This 11-year single-centre retrospective study analysed 58 patients with ColRAN bacteraemia and 213 patients with colistin-susceptible A. nosocomialis (ColSAN) bacteraemia. Antimicrobial susceptibilities were determined with an agar dilution method. The clonal relationship of ColRAN isolates was determined with pulsed-field gel electrophoresis. A conjugation mating-out assay was conducted to delineate the potential transfer of colistin resistance genes. Multivariable analysis was performed to evaluate the risk factors for ColRAN bacteraemia. Chronic obstructive pulmonary disease (COPD) was independently associated with ColRAN bacteraemia (OR 3.04; 95% CI 1.45-6.37; p 0.003). Patients with ColRAN bacteraemia had higher APACHE II scores, but the two groups showed no significant differences in 14-day mortality (10.3% vs. 10.3%) or 28-day mortality (15.5% vs. 15.0%). ColRAN isolates had greater resistance than ColSAN isolates to all antimicrobial agents except for ciprofloxacin (0% vs. 6.6%). There were 16 different ColRAN pulsotypes, and two major clones were found. Colistin resistance did not transfer to colistin-susceptible A. baumannii or A. nosocomialis. These results show that COPD is an independent risk factor for acquisition of ColRAN bacteraemia. The mortality rates were similar between patients with ColRAN and ColSAN bacteraemia.

Detection of the classical G2576U mutation in linezolid resistant Staphylococcus aureus along with isolation of linezolid resistant Enterococcus faecium from a patient on short-term linezolid therapy: first report from India.

PURPOSE: Linezolid is an effective drug against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). We describe the emergence of linezolid resistance in MRSA and VRE from India. MATERIAL AND METHODS: One MRSA and two VRE strains were isolated from a patient on linezolid therapy of one week duration. All three isolates were resistant to linezolid with minimal inhibitory concentrations (MIC) ≥4 mg/L. The 746-bp region flanking the possible G2576U mutation on the corresponding DNA from the 23S rRNA was amplified by polymerase chain reaction (PCR) and amplicons were sequenced for all the three isolates. Conjugation experiments using the linezolid resistant MRSA (LRMRSA) and linezolid resistant VRE (LRVRE) isolates as donors and wild strains of corresponding genera as recipients were performed. RESULTS: The MRSA isolate had the classical G2576U mutation. High quality value scores in the sequencing software validated the mutation. Conjugation studies did not indicate presence of transferable resistance for linezolid. Sequencing did not indicate presence of any mutation in the two LRVRE isolates. CONCLUSIONS: This is the first report from India citing resistance in Staphylococcus and Enterococcus against Linezolid.

Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages.

OBJECTIVES: We studied the occurrence of diverse copper (Cu) tolerance genes from Gram-positive bacteria and their co-transfer with antibiotic resistance genes among Enterococcus from diverse sources. METHODS: Enterococcus (n = 922) of several species and from human, animal, environment and food samples were included. Antimicrobial and CuSO4 susceptibility and conjugation assays were performed by standard procedures, bacterial screening of Cu and antibiotic resistance genes by PCR, and clonality by PFGE/multilocus sequence typing. RESULTS: tcrB and cueO genes occurred in 15% (n = 137/922) and 14% (n = 128/922) of isolates, respectively, with the highest occurrence in piggeries (P < 0.05). They were more frequent among Enterococcus faecium (tcrB: 23% versus 8% in Enterococcus faecalis and 12% in other species; cueO: 25% versus 5% and 9%, respectively; P < 0.05). A correlation between phenotypic and genotypic assays was observed for most E. faecium (CuSO4 MIC50 = 24 mM in tcrB/cueO(+) versus CuSO4 MIC50 = 12 mM in tcrB/cueO(-)), but not for other species. Co-transfer of Cu tolerance (associated with tcrB, cueO or an unknown mechanism) with erythromycin, tetracycline, vancomycin, aminoglycosides or ampicillin resistance was demonstrated. A variety of PFGE types was detected among isolates carrying Cu tolerance mechanisms, some identified in sequence types (STs) often linked to human infections (E. faecium from ST18 and ST78 clonal lineages and E. faecalis clonal complex 2). CONCLUSIONS: Cu tolerance might contribute to the selection/maintenance of multidrug-resistant Enterococcus (including resistance to first-line antibiotics used to treat enterococcal infections) due to the use of Cu compounds (e.g. antiseptics/animal feed supplements). The distribution of the multicopper oxidase cueO and the co-transfer of ampicillin resistance along with Cu tolerance genes are described for the first time.

A treatment plant receiving waste water from multiple bulk drug manufacturers is a reservoir for highly multi-drug resistant integron-bearing bacteria.

The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.

Exogenous isolation of conjugative plasmids from pesticide contaminated soil.

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10 µg/ml) and tetracycline (20 µg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2 × 10(-6) and 9.2 × 10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.

[Effect of casamino acid on intergeneric conjugation in rapamycin-producing Streptomyces hygroscopicus ATCC29253].

OBJECTIVE: Streptomyces hygroscopicus ATCC29253 has attracted much interests due to its capacity of producing various secondary metabolites with strong bioactivities, including immunosuppressant rapamycin, nigericin, hexaenes, elaiophylin and hygrocins. OBJECTIVE: To investigate biosynthetic pathway of these metabolites and construct high-yield strains by genetic engineering, establishment of a highly efficient genetic manipulation system is critically required in this strain. METHODS: We tested the effects of conjugation media and donor strains on conjugal transfer from Escherichia coli to S. hygroscopicus ATCC29253 and other Streptomycetes. RESULTS: We found that both casamino acid and MgCl2 supplemented in conjugation media improved conjugation frequency in S. hygroscopicus ATCC29253. A random experiment led to the disclosure of an optimal combination of casamino acid and MgCl2 by which the conjugation frequency in S. hygroscopicus reached 1.5 x 10(-4). Meanwhile, we also found significant changes in conjugation frequencies of S. lividans, S. albus and S. avermitilis when casamino acid was supplemented in conjugation media. CONCLUSION: Casamino acid has significant influence on conjugation frequency in not only S. hygroscopicus ATCC29253 but also other Streptomyces such as S. lividans, S. albus and S. avermitilis.

Multiresistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant.

In this study, 1832 strains of the family Enterobacteriaceae were isolated from different stages of a municipal wastewater treatment plant, of which 221 (12.1%) were intI-positive. Among them 61.5% originated from raw sewage, 12.7% from aeration tank and 25.8% from the final effluent. All of the intI-positive strains were multiresistant, i.e. resistant to at least three unrelated antimicrobials. Although there were no significant differences in resistance range, defined as the number of antimicrobial classes to which an isolate was resistant, between strains isolated from different stages of wastewater treatment, for five ß-lactams the percentage of resistant isolates was the highest in final effluent, which may reflect a selective pressure the bacteria are exposed to, and the possible route of dissemination of ß-lactam resistant strains to the corresponding river. The sizes of the variable part of integrons ranged from 0.18 to 3.0 kbp and contained up to four incorporated gene cassettes. Sequence analysis identified over 30 different gene cassettes, including 24 conferring resistance to antibiotics. The highest number of different gene cassettes was found in bacteria isolated from the final effluent. The gene cassettes were arranged in 26 different resistance cassette arrays; the most often were dfrA1-aadA1, aadA1, dfrA17-aadA5 and dfrA12-orfF-aadA2. Regarding the diversity of resistance genes and the number of multiresistant bacteria in the final effluent, we concluded that municipal sewage may serve as a reservoir of integron-embedded antibiotic resistance genes.

Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera.

Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment.

Emergence of Proteus mirabilis harboring blaKPC-2 and qnrD in a Chinese Hospital.

Nineteen carbapenem-nonsusceptible Proteus mirabilis isolates were recovered from intensive care units in the Second Affiliated Hospital of Zhejiang University during a 3-month period. The isolates showed a high level of resistance against ciprofloxacin, in addition to their resistance against the carbapenems. Pulsed-field gel electrophoresis (PFGE) analysis showed that these isolates belonged to three clonal strains. PCRs and DNA sequence analysis of the carbapenemase and other ß-lactamase genes indicated that all the isolates harbored the bla(KPC-2) gene. Twelve of 19 isolates harbored the plasmid-mediated quinolone resistance (PMQR) genes, both the qnrD and aac(6')-Ib-cr genes. Eight representative isolates with high levels of quinolone resistance carried the similar mutation profiles of S83I in gyrA, E466D in gyrB, and S80I in parC. Reduced carbapenem susceptibility was transferred to Escherichia coli (EC600) in a conjugation experiment, while the quinolone resistance was not. DNA hybridization showed that qnrD was located on a plasmid of approximately 4.5 kb. In summary, large clonally related isolates of KPC-2-producing P. mirabilis emerged in a Chinese hospital, and qnrD was detected in KPC-producing P. mirabilis for the first time.

Cefoxitin as an alternative to carbapenems in a murine model of urinary tract infection due to Escherichia coli harboring CTX-M-15-type extended-spectrum ß-lactamase.

We investigated the efficiency of the cephamycin cefoxitin as an alternative to carbapenems for the treatment of urinary tract infections (UTIs) due to Escherichia coli producing CTX-M-type extended-spectrum ß-lactamases. The susceptible, UTI-inducing E. coli CFT073-RR strain and its transconjugant CFT073-RR Tc (pbla(CTX-M-15)), harboring a bla(CTX-M-15) carrying-plasmid, were used for all experiments. MICs of cefoxitin (FOX), ceftriaxone (CRO), imipenem (IMP), and ertapenem (ETP) for CFT073-RR and CFT073-RR Tc (pbla(CTX-M-15)) were 4 and 4, 0.125 and 512, 0.5 and 0.5, and 0.016 and 0.032 µg/ml, respectively. Bactericidal activity was similarly achieved in vitro against the two strains after 3 h of exposure to concentrations of FOX, IMI, and ETP that were 2 times the MIC, whereas CRO was not bactericidal against CFT073-RR Tc (pbla(CTX-M-15)). The frequencies of spontaneous mutants of the 2 strains were not higher for FOX than for IMP or ETP. In the murine model of UTIs, mice infected for 5 days were treated over 24 h. Therapeutic regimens in mice (200 mg/kg of body weight every 3 h or 4 h for FOX, 70 mg/kg every 6 h for CRO, 100 mg/kg every 2 h for IMP, and 100 mg/kg every 4 h for ETP) were chosen in order to reproduce the percentage of time that free-drug concentrations above the MIC are obtained in humans with standard regimens. All antibiotic regimens produced a significant reduction in bacterial counts (greater than 2 log(10) CFU) in kidneys and bladders for both strains (P < 0.001) without selecting resistant mutants in vivo, but the reduction obtained with CRO against CFT073-RR Tc (pbla(CTX-M-15)) in kidneys was significantly lower than that obtained with FOX. In conclusion, FOX appears to be an effective therapeutic alternative to carbapenems for the treatment of UTIs due to CTX-M-producing E. coli.

Rapid acquisition of decreased carbapenem susceptibility in a strain of Klebsiella pneumoniae arising during meropenem therapy.

A strain of Klebsiella pneumoniae (K1) was isolated from a catheterized patient with a urinary tract infection. The patient was subsequently treated with meropenem, after which K. pneumoniae (K2) was once again isolated from the patient's urine. Susceptibility testing showed that strain K1 was fully susceptible to carbapenem antibiotics with the exception of ertapenem, to which it exhibited intermediate resistance, whilst K2 was resistant to ertapenem and meropenem. From pulsed-field gel electrophoresis profiling both strains exhibited identical banding patterns. Both contained CTX-M-15, OXA-1, SHV-1 and TEM-1 ß-lactamase genes following PCR analyses. Outer membrane protein analysis demonstrated that K1 and K2 lacked an OMP of c. 40 kDa, with an additional OMP of c. 36 kDa missing from K2. Mutation studies showed that the K2 OMP phenotype could be selected by single-step carbapenem-resistant mutants of K1. Expression of transcriptional activator ramA and efflux pump component gene acrA were up-regulated in both strains by RT-PCR. Neither strain expressed ompK35, but ompK36 was found in both. Sequence analysis revealed gene sequences of ompK35, ompK36 and ramR in both strains; notably, ramR contained a mutation resulting in a premature stop codon. Transconjugation studies demonstrated transfer of a plasmid into E. coli encoding the CTX-M-15, TEM-1 and OXA-1 ß-lactamases. We concluded that the carbapenem-resistant phenotype observed from this patient was attributable to a combination of CTX-M-15 ß-lactamase, up-regulated efflux and altered outer membrane permeability, and that K2 arose from K1 as a direct result of meropenem therapy.

[Bioaugmentation of bioreactors with a pJP4 receiving transconjugant to enhance the removal of 2,4-D].

The paper first investigated horizontal transfer of a conjugative plasmid pJP4 to two pure strains of E. coli DH5alpha and Alcaligenes sp., and a mixed culture of aerobic granular sludge, respectively. With a pJP4 receiving transconjugant Alcaligenes sp. :: pJP4 as the bioaugmented bacteria, bioaugmentation experiments were conducted in an aerobic granular sludge reactor and a biofilm reactor, respectively, to enhance the removal of a recalcitrant compound 2,4-dichlorophenoxyacetic acid(2,4-D). Results showed that pJP4 successfully transferred to E. coli DH5alpha, Alcaligenes sp. and the mixed culture of aerobic granules. For the aerobic granular sludge reactor operated in semi-continuous mode and fed with 2,4-D sole carbon source wastewater, bioaugmentation with Alcaligenes sp. : : pJP4 increased 2,4-D average removal rate significantly with an enhancement of 12% -1 498%. For the biofilm reactor operated in sequence batch mode and fed with mixed carbon sources wastewater, supplementation of the transconjugant reduced system start-up time greatly from 16 d to 5 d. It is a feasible strategy to obtain special degradative transconjugants through gene augmentation and put them into bioreactor as bioaugmentation agent to enhance the removal of some specific pollutants.

Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper.

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.

Bioremediation of heavy metal toxicity from factory effluents by transconjugants bacteria.

The presence of heavy metals in aquatic environments is known to cause severe damage to aquatic life, beside the fact that these metals kill microorganisms during biological treatment of wastewater with a consequent delay of the process of water purification. Most of the heavy metal salts are soluble in water and form aqueous solutions and consequently cannot be separated by ordinary physical means of separation. Five bacterial strains were used in this study. Bacterial strains were marking using 10 antibiotics and 7 heavy metals to be use as a selectable markes in conjugation process. Mating were performed using five bacterial strains. These strains were genetically marking in relation to their tolerance to the different antibiotics and heavy metals. All matings between bacterial strains were successes. The biosorption capacities for all heavy metals determined were higher for some metals than others. The transconjugants strain Tr5 resulted from mating between the parental bacterial strains (B-6bs X B-21) was more efficient in molybdenum uptake than all bacterial strains when supplementednutrient media wi th wastewater. Bacterial strains (B-21) appeared a good uptake of heavy metal ions (copper, cadmium, iron, cadmium and Lead) than other bacterial strains. This work highlights the potential of bacterial strains B-21 in uptake of heavy metals. The transconjugant strain Tr3 resulted from mating between parental bacterial strains (B-1584 X B-287) was more efficient in chromium uptake than all bacterial strains.